FRET - Förster Resonance Energy Transfer || Semiconductor Quantum Dots and FRET

12Semiconductor Quantum Dots and FRETW. Russ Algar, Melissa Massey, and Ulrich J. Krull

12.1Introduction

Quantum dots (QDs) are one of the most interesting new materials that haveemerged over the last 20 years. These brightly luminescent nanoparticles havegarnered significant attention from researchers working in the fields of biology,chemistry, physics, engineering, and at the interface between these fields. Apowerful spectroscopic tool that also spans these disciplines is F€orster resonanceenergy transfer (FRET). In this chapter, the compelling partnership betweenquantum dots and FRET is considered at both the fundamental and applied levels,emphasizing utility in biological applications. The text begins with a short review ofFRET in Section 12.2, which reiterates the basic equations that relate physicalparameters to experimental observables. Conventional FRET with a well-defineddonor–acceptor pair serves as a reference point for comparison with quantum dots.Next, Section 12.3 describes the structure, chemistry, and optical properties ofquantum dots. The purpose of this section is threefold: (i) impart the reasons for thewidespread interest in quantum dots; (ii) outline the methods for using quantumdots as functional materials in biological applications; and (iii) provide a primer onquantum dot photophysics, which clearly have an important role in energy transfer.Cumulatively, Sections 12.2–12.3 provide sufficient background to fully appreciatethe utility, advantages, and disadvantages of FRET with quantum dots, which arediscussed in Section 12.4. Adherence and potential departures from the F€orsterformalism are also discussed. Section 12.5 provides a comprehensive review of theuse of quantum dots as FRET donors in biological applications. These applicationsinclude assays, biosensing and chemosensing, distance measurements/conforma-tional studies, and photodynamic therapy. Ensemble, single-pair, and solid-phaseFRET configurations are considered. Section 12.6 reviews similar applications ofquantum dot acceptors with chemiluminescent, bioluminescent, or lanthanidedonors. Energy transfer between quantum dots and other nanomaterials is reviewedin Section 12.7; these mechanisms are similar to the F€orster mechanism, butnot strictly equivalent. Several examples of biological applications are given.

FRET – Förster Resonance Energy Transfer: From Theory to Applications, First Edition.Edited by Igor Medintz and Niko Hildebrandt.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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For completeness, a few examples of nonbiological applications of FRET withquantum dots are described in Section 12.8, although this topic is not thefocus of the chapter. While every effort has been made to be comprehensive inSections 12.5–12.7, there are undoubtedly omissions. Apologies are extended to anyauthors whose work has been overlooked.

12.2A Quick Review of FRET

The details of FRET have been described elsewhere in this book and a thoroughreview is not needed here. Knowledgeable readers may wish to skip this section.Briefly, FRET is a nonradiative, through-space excitation energy transfer process.The electronic excitation energy of a donor (D) is transferred to a groundstate acceptor (A) via dipole–dipole interactions. The dipoles of interest are thetransition moments associated with donor relaxation and acceptor excitation.Molecular contact between the donor and acceptor is not required, and no photonis involved. Energy must be conserved in the process, requiring that the donor andacceptor share resonant transitions for relaxation and excitation, respectively.Furthermore, as a dipole–dipole interaction, close proximity and suitable alignmentbetween the donor and acceptor are required for efficient energy transfer. Theseconcepts were reduced to an elegant formalism by F€orster [1], and can be summa-rized by Equations 12.1–12.3.

kFRET ¼ 1tD

R0

r

� �6

: ð12:1Þ

R60 ¼

9 ln 10ð ÞWDk2JðlÞ

128p5Nn4: ð12:2Þ

JðlÞ ¼ÐFDðlÞeAðlÞl4dlÐ

FDðlÞdl : ð12:3Þ

The rate of energy transfer, kFRET, is given by Equation 12.1, where k0¼krþ knr¼ t�1

D is the sum of the radiative and nonradiative decay rates for the donor,R0 is the F€orster distance, and r is the separation distance between donor andacceptor. The F€orster distance is the separation at which the rate of energy transfer isequal to the intrinsic relaxation rate of the donor, k0, and thus is where theprobability of energy transfer is 50%. Equation 12.1 is deceptively simple becausemuch of the complexity is contained within the R0 parameter, Equation 12.2, whereWD is the quantum yield of the donor, k2 is the orientation factor, J(l) is the spectraloverlap integral, N is Avogadro’s number, and n is the refractive index of thesurrounding medium. The orientation factor takes on a value of k2¼ 2/3 for adynamic and isotropic distribution of donor and acceptor orientations [2]. Thespectral overlap integral, which describes the degree of resonance between donorand acceptor, is defined by Equation 12.3, where FD(l) is the donor fluorescence

476j 12 Semiconductor Quantum Dots and FRET

emission spectrum, and eA(l) is the molar absorption coefficient of the acceptor.Both parameters are a function of wavelength, l, and integrated over the relevantrange. The FRET efficiency (i.e., quantum yield of energy transfer) is defined byEquation 12.4 and tends toward 100% at r< 0.5R0 and 0% at r> 1.5R0.

E ¼ kFRETk0 þ kFRET

¼ R60

r6 þ R60

: ð12:4Þ

E ¼ 1� IDA=IDð Þ ¼ 1� tDA=tDð Þ: ð12:5ÞIADIDA

¼ EWA

WD 1� Eð Þ ¼WA

WD

� �R0

r

� �6

: ð12:6Þ

Experimentally, the FRET efficiency can be measured from changes in donoremission intensity or lifetime using Equation 12.5. The subscripts D and A denoteintrinsic values for the donor and acceptor, respectively, while DA and AD denotevalues for donor in the presence of acceptor and vice versa; I is an emission intensity;and t is an amplitude-weighted emission lifetime. When the acceptor is emissive,and with proper controls (e.g., correction for direct excitation of acceptor), similarinformation can be derived from the intensity ratio for acceptor and donoremissions, Equation 12.6. These standard parameters and equations provide abasis for evaluating FRET in the context of quantum dot donors or acceptors.

12.3Quantum Dots

Quantum dots are zero-dimensional systems wherein charge carriers (i.e., electronsand holes) are confined in all three dimensions. The confinement of the carriersresults in the quantization of their allowed energies and the emergence of discretestates. The term quantum dot was coined by Reed et al. to describe a zero-dimensional semiconductor nanostructure embedded in a bulk solid matrix com-posed of a different semiconductor material [3,4]. The terminology is an extension ofquantum wire and quantum well that are used to describe one-dimensional and two-dimensional systems that have carrier confinement in two dimensions and onedimension, respectively. The emergence of discrete electronic states at the nanoscaleis in contrast to bulk semiconductor materials, which exhibit electronic bandstructures [i.e., conduction bands (CBs) and valence bands (VBs)]. The discreteelectronic states bring about many new size-dependent properties that are bothphysically interesting and useful in many different applications.In this chapter, the interest is in colloidal semiconductor nanocrystal quantum dots.

In contrast to the epitaxial quantum dots, first described by Reed et al. [3,4], colloidalquantum dots are prepared as a dispersion in a solvent via bottom-up chemicalsynthesis. It is also important to emphasize that high-quality quantum dots arecrystalline rather than amorphous in structure. Finally, this chapter deals exclusivelywith semiconductor materials, although clusters of metal atoms can also be zero-dimensional systems and have occasionally been referred to as “quantum dots” [5].

12.3 Quantum Dots j477

However, in the context of biological applications, the impact and excitementgenerated by colloidal semiconductor quantum dots have resulted in the terminol-ogy quantum dot (QD) being generally associated with the colloidal semiconductorvariety, and no further distinction is made in this chapter.

12.3.1A Brief History

Brus and coworkers at Bell Laboratories reported the first colloidal QDs in 1983 [6].In contrast to most other researchers who were interested in fabricating quantumnanostructures, Brus et al. developed a bottom-up chemical synthesis rather than atop-down epitaxial approach [7]. It was not until the mid-1990s, however, thatBawendi, Guyot-Sionnest, and others pioneered refined synthetic methodsthat yielded nearly monodisperse, nanocrystalline QDs [8–10]. Although it wasanticipated that QDs would be important for further miniaturization of electronics(e.g., nanoscale transistors), it was largely the special photophysical properties ofhigh-quality QDs that rapidly catalyzed scientific interest across several fields.Considering biological applications, the watershed moment was arguably thepublication of two seminal studies in 1998: Chan and Nie [11], and Bruchezet al. [12] prepared water-soluble, photoluminescent CdSe/ZnS core/shell QDsand used them for cellular imaging. The total citations of all the papers notedabove [6–12] grew from 1031 at the close of 1999 to nearly 16 000 at the close of 2011[13]. Almost half of these citations are credited to Refs [11,12], demonstrating thewidespread interest that developed for using QDs in biological applications.Alivisatos, another QD pioneer who worked with Brus, recently noted how remark-able the transition was from early skepticism about QDs to fanfare after the turn ofthe millennium [14].

12.3.2The Structure of Quantum Dots: The Core

QDs are composed of 102–104 atoms, are roughly spherical in shape, and their sizetypically ranges from 1–10 nm in diameter [15–17]. An illustration and electronmicroscope image of a QD are shown in Figure 12.1. Although a large variety ofsemiconductor materials have been used to fabricate QDs, the most popularmaterial choices tend to be CdS, CdSe, and CdTe due to their well-known propertiesand established synthetic methods [8,18,19]. However, concern over the use of heavymetals has, in part, driven the development of QDs composed of other materials, forexample, InP [20,21]. In general, binary semiconductors (e.g., II–VI, III–V semi-conductors) with direct band gaps have been the most popular materials; however,ternary alloy semiconductor materials are also of interest [22,23]. The opticalproperties of QDs (vide infra) composed of ternary alloys can be tuned by bothsize and relative composition. In the case of binary QDs, the material is stillimportant in determining optical properties, but only size enables tuning of thoseproperties.


Due to their crystalline nature, QDs are faceted and therefore neither trulyspherical nor isotropic [24]. For example, CdS and CdSe QDs generally adopt thewurtzite or zinc blende crystal structure depending on the synthetic method,although the former appears to be more common and is the more stable bulk form[8,18,25–29]. The crystal structure is important in determining the shape of theQD: wurtzite CdSe nanocrystals are hexagonal prisms and zinc blende CdSnanocrystals are tetrahedrons [25]. Nonetheless, QDs are synthesized to havesimilar dimensions along each axis and are conveniently approximated as spheri-cal. This is in contrast to, for example, quantum rods that are synthesized to have anontrivial aspect ratio [26–28], or other more exotic shapes such as tetrapods [29].It should also be noted that the surface area-to-volume ratio of a QD is anotherimportant aspect of its structure. For example, a 5 nm CdS QD has approximately15% of its 3300 atoms at the nanocrystal surface [30] and a 4 nm CdSe QD hasapproximately 33% of its 1500 atoms at the surface [31]. The surface of a QD is nota trivial interface and will be seen to be critical to utilizing QDs and FRET inbiological applications.

Figure 12.1 (a) Illustration showing that theelectronic structure of a QD nanocrystal isintermediate to the bulk and molecular regimes.(b) Cartoon of a colloidal QD showingstabilizing ligands and crystal facets. (c)Transmission electron microscope image of a

single QD with visible lattice fringes. Scale baris 5 nm. (d) Absorption and emission spectrafor various sizes of CdSe QDs. (Adapted withpermission from Ref. [79]. Copyright 2010,American Chemical Society.)


12.3.3The Optical Properties of Quantum Dots

The most compelling aspect of QDs is their size-dependent optical and electronicproperties. Changes in these properties depend on the total number of atoms, bothat the surface and within the interior [25]. In bulkmaterials, the optical excitation of asemiconductor results in the formation of a loosely bound electron-hole pair orexciton. The electron occupies the quasi-continuum of energy levels, that is, theconduction band and the hole occupies the valence band. However, in the case ofQDs, the density of electronic states is not sufficient to form complete bandstructures and discrete energy levels exist at the band edges [16]. These twosituations are depicted in Figure 12.1 along with conventional molecular orbitals.The QD is an example of a quantum mechanical particle-in-a-box or, moreaccurately, the three-dimensional analogue, a particle-in-a-sphere [17]. Conse-quently, the position of each energy level is determined by the size of the nano-crystal. This phenomenon, which is known as quantum confinement, is discussed inmore detail in Section 12.3.6. The essential point is that the relative positions of thehighest occupied state and lowest unoccupied state – equivalent to the highestoccupied molecular orbital (HOMO) and lowest unoccupied molecular orbital(LUMO) for molecular dyes, and roughly corresponding to the top of the valenceband and the bottom of the conduction band of a bulk material – are determined bythe size of the QD. In other words, the band gap energy of the semiconductormaterial becomes size-dependent at the nanometer scale. QD photoluminescence(PL) results from the radiative recombination of the exciton across that energy gap,and is thus characterized by the size-tunable absorption and emission across a broadrange of wavelengths [17,25,32], as shown in Figure 12.1. This is perhaps the mostrenowned property of QDs.The wavelength range over which QD PL can be tuned is determined by selection

of the semiconductor material. If the QD size becomes too large, then the quantumconfinement effect vanishes. This upper threshold is determined by the Bohrexciton radius of the material, which represents the preferred separation distancebetween the electron and hole in the bulk material [24,32]. As an example, the Bohrexciton radius for CdSe is 5.6 nm [33]. The lower size threshold is often limited bythe difficulty of synthesizing good quality QDs at small size scales (e.g., CdSe<2 nm), as well as decreased optical absorption [34]. Figure 12.2 illustrates theapproximate size and PL wavelength tuning ranges for various QD materials. Inparticular, note that CdSe spans the visible region of the spectrum, contributing toits widespread popularity. The shift in CdSe QD emission across the visiblespectrum corresponds to changing the nanocrystal size from 2 to 6 nm [35].In addition to its ability to be size-tuned, QD PL emission is also narrow and

symmetric, approximating a Gaussian profile with full width at half maximum(FWHM) typically in the range of 25–35 nm [17,36]. The FWHM and other typicalfeatures of QD absorption and PL spectra are shown in Figure 12.3. The quantumyields of high quality QDs are often in excess of 40% [9,18,24] and have beenreported to reach as high as 85% [37]. QD PL lifetimes generally exceed 10 ns


[32,38,39] and typically exhibit multiexponential decay kinetics [24,40,41]. However,it should be noted that both the quantum yield and lifetime depend to a large degreeon the quality of the nanocrystal, its coating, and its environment – lower quantumyields and shorter lifetimes can potentially be observed.In contrast to their PL spectra, the linear absorption spectra of QDs are very broad,

extending, and generally increasing in intensity at wavelengths shorter than the size-tuned band gap energy, as shown in Figure 12.1. This potentiates a large effectiveStokes shift between the excitation wavelength and QD PL emission that is typicallyon the order of many tens of nanometers, and can potentially exceed 100–200 nm.

Figure 12.2 Approximate PL emission ranges and core sizes for different QD materials.Reprinted with permission from Ref. [121]. Copyright 2011, American Chemical Society.

Figure 12.3 Typical features in absorption and emission spectra of QDs, which are oftencharacterized by their first exciton peak and/or band edge PL maximum. In high-quality samples,band gap PL is absent.


The actual Stokes shift, defined by the difference between the lowest energyabsorption band (i.e., the first exciton peak) and PL emission, is similar tofluorescent dyes and �10–20 nm [42]. Typical absorption coefficients for QDs arein the range of 105–106M�1 cm�1 and increase with larger nanocrystal sizes andshorter excitation wavelengths [24,38,43,44]. At worst, the value tends toward104M�1 cm�1 for small nanocrystals at their first exciton peak. Cumulatively, thesevalues are comparable with the maximum absorption coefficients of the moststrongly absorbing fluorescent dyes and, depending on the selection of excitationwavelength, are often 101–102-fold larger [36,38]. Considering two-photonabsorption, QDs provide a similar enhancement with cross sections on the orderof 103–104GM and potentially reaching 47 000GM [45–47]. This is approximately101–102-fold larger than the best organic dyes. The two-photon absorption crosssection increases with increasing nanocrystal size.

12.3.4Overcoming the Limitations of Molecular Fluorophores

The optical properties of QDs are much better suited to multiplexed analyses andimaging than those of molecular fluorophores [24,36,38,48]. In general, N differentexcitation wavelengths are needed to efficiently excite N different fluorescent dyeswith well-resolved emissions. Due to the comparatively narrow absorption bands ofdyes, this typically requires the use of multiple excitation sources in combinationwith complex optical filtering or the serial acquisition of each signal. In contrast, theefficient excitation of N different QDs in the visible region of the spectrum (e.g.,CdSe QDs) is possible using a single excitation wavelength in the UV–blue region ofthe spectrum. In this case, only a single excitation source and relatively simple opticsare required, and the parallel acquisition of multiple PL signals (i.e., multicolorimaging or measurements) is greatly facilitated.The challenge of multiplexing with molecular fluorophores also extends to their

emission spectra. Compared to QDs, dye emission is broad due to the red tailassociated with relaxation to an excited vibrational state, often approaching orexceeding 75 nm in spectral width. The narrower QD PL allows more signals tobe fit and resolved within a given wavelength range. Moreover, the symmetric andapproximately Gaussian emission profile simplifies the identification and decon-volution of overlapping signals [49]. For example, as many as eight overlapping QDemission profiles have been deconvolved within a 200 nm wavelength range [50].The emission color of a molecular dye, which is a function of the relative energies

of its HOMO and LUMO, is determined by its chemical structure and bonding.Therefore, a shift of the emission wavelength strictly requires synthesis of a new dyemolecule. Although a series of structurally similar molecular dyes (e.g., cyanine dyeslike Cy3, Cy5, and Cy7) can sometimes span a spectral range, this does not alwaysreduce synthetic complexity and tends to yield only discrete shifts of the PLemission. In contrast, a continuum of different QD sizes and (thus) emissioncolors can generally be synthesized using a common protocol where only thereaction time and/or temperature is varied. This is not to suggest that it is


necessarily easier to synthesize high quality QD materials, but that it is moreconvenient to synthesize a series of QDs compared to a series of dyes, therebysimplifying multiplexing from a materials standpoint as well.Another well-documented advantage of QDs is their superior resistance to photo-

bleaching compared to most molecular fluorophores [11,24,38,51–53]. Similar to itsquantum yield, the resistance of a QD toward photobleaching is a function of thequality of the nanocrystal and the conditions of its local microenvironment. None-theless, high-quality QDs clearly offer enhanced photostability, and this property ismost likely a consequence of the confinement of the electron and the hole – the latterof which hinders photooxidation [18]. QDs are thus highly advantageous in experi-ments that require optical tracking over long time periods, or the use of higherexcitation rates to increase signal-to-noise ratio. A particularly prominent example ofthis capability is the use of QDs in single-molecule (particle) spectroscopy experi-ments, which benefits from both their photostability and brightness.Another feature of QD PL that has attracted considerable attention is the PL

intermittency, or “blinking,” of single QDs under continuous illumination [54]. Thatis, a single QD transiently switches between emissive and nonemissive states.Although this phenomenon can also be observed with molecular fluorophores, it ismore prominently associated with QDs. Since blinking is asynchronous across theensemble, it is only visible at the single QD level. Originally seen as only detrimen-tal, blinking is now recognized as a useful confirmation of single QD tracking.Nonetheless, structural modifications of QDs that reduce blinking have beendeveloped (vide infra).

12.3.5The Structure of Quantum Dots: The Shell

There are two basic structural motifs associated with quantum dots: core QDs thatconsist of a single nanocrystalline material and core/shell QDs that consist of a corenanocrystal coated with a layer of a different semiconductor material, as shown inFigure 12.4. Themost common example of the latter is a CdSe/ZnSQD, where CdSeis the core material and ZnS is the shell material. Core/shell QDs have morefavorable optical properties than core-only QDs, including larger quantum yields,and are thus used in most applications.The superior optical properties of core/shell QDs are rooted in the structural

defects that are inherent to core nanocrystals. The termination of a core

Figure 12.4 Cartoon illustrations of core, core/shell, and core/shell/shell QDs.


nanocrystal yields unsatisfied bonding capacity at its surface [25]. Dangling bonds,vacancies, surface reconstructions, impurities, as well as interactions with adsor-bates can potentially cause the formation of localized states that fall within theotherwise forbidden band gap. These are referred to as trap states, and generallyinhibit radiative recombination of the exciton by spatially separating the electronand the hole [25,32,55,56]. Nonradiative relaxation mechanisms may be concomi-tantly enhanced by carrier trapping or by introduction of new mechanisms such asthe chemical reactivity of a surface-trapped electron or hole. The overall effect onthe optical properties of the QD is a reduction in quantum yield. Characteristicallybroad and long wavelength band gap PL sometimes emerges and is associated withradiative recombination of the exciton through trap states at the QD surface (seeFigure 12.3). This PL is undesirable and, in contrast to the narrow size-tunableband-edge PL associated with radiative recombination within the core nanocrystal,does not depend on the nanocrystal size. To minimize trap states, the corenanocrystal can be coated with a layer of a second semiconductor material.This shell structure is able to passivate the surface of the core nanocrystal andsignificantly increases the quantum yield of the QD [24,25,32,36]. In organicsolvents, core QDs typically have quantum yields �10% compared to �30% formany core/shell QDs [18,24,39]. The surface of the core may also be prone tooxidation, as in the case of Se in CdSe QDs [57,58], such that the shell providesprotection against oxidation and other chemical reactivity [32]. As a consequenceof the deleterious effects of an aqueous environment and bioconjugation on thePL of core nanocrystals, core/shell QDs are used almost exclusively in biologicalapplications. The few exceptions tend to be analytical methods that draw infor-mation from electrochemical or compositional properties rather than opticalproperties.The shell material for a core/shell QD is chosen to have a larger band gap and be

structurally compatible with the core material [25]. The larger band gap helpsconfine the exciton to the core nanocrystal. Typically, there is a small bathochromicshift in the PL emission wavelength upon growth of the shell that is due to theleakage of the exciton wave function into the shell [8,9]. The magnitude of the shiftdepends on band-edge offsets between the core and shell, as well as the thickness ofthe shell. Considering structural compatibility, the lattice mismatch between the twomaterials should be minimal. For example, the lattice mismatch is relatively large(10.6%) between CdSe and ZnS [37]. It is for this reason that the quantum yield of

CdSe/ZnS QDs is maximized with 1–2 monolayers of ZnS shell (�3A�thickness per

monolayer). As the thickness increases further, dislocations and other defects beginto form due to the lattice mismatch, reducing the quantum yield [8]. In the absenceof these effects, however, thicker shells will better passivate and protect the corenanocrystal [36]. It is possible to grow thicker shells using CdS or ZnSe rather thanZnS, since the lattice mismatches are reduced to 6.3% and 3.9% for CdSe/ZnSe andCdSe/CdS, respectively [37]. In the case of the latter, shell thicknesses >5 nm havebeen grown [59]. Although the limited ability to grow thick, defect-free ZnS shells onCdSe cores is a disadvantage, a ZnS shell is advantageous in that the material islargely nontoxic and chemically stable. Furthermore, in so much as the shell is


intended to isolate the core nanocrystal from its surrounding environment, it alsoisolates the environment from the core nanocrystal. Indeed, experiments havesuggested that a ZnS shell around a Cd-based core material greatly reduces potentialcytotoxicity associated with leaching of Cd2þ ions [60,61]. The synthesis of core/shell/shell QDs (see Figure 12.4) such as CdSe/CdS/ZnS and CdSe/ZnSe/ZnSprovides the opportunity to capitalize on the advantages of a ZnS shell whileavoiding defect-inducing strain on the CdSe core via an intermediate “wettinglayer” of lower lattice mismatch [57]. In addition to thicker shells, these QDs haveimproved crystallinity and monodispersity following shell growth, resulting inquantum yields of 70–80% (cf. 40–60% for CdSe/ZnS). While discrete multishellstructures are one of the architectures that ease lattice strain, gradient transitionsbetween core and shell materials, for example, CdSe/CdS-CdxZnyS-ZnS, are alsopossible [59]. Overall, thicker and higher-quality shells with less core strain andfewer defects yield QDs with higher quantum yield, even greater photostability, andminimal blinking (see, for example, Ref. [59]). In the context of FRET, however, it isimportant to note that thicker shells also increase the minimum separation betweenthe optically active QD core and a suitable donor/acceptor partner. That is, QDs withthicker shells offer inherently lower FRET efficiencies; the increase in F€orsterdistance with improved quantum yield generally will not offset the thicker shell.Finally, it is important to note that a shell material for a core/shell QD is selected

on the basis of not only its structural compatibility with the core material, but also itsrelative band gap energy and offsets. For example, CdSe/ZnS is a type-I QDmaterial.That is, the relative positions of the ZnS bands confine both the electron and the holeto the CdSe core: the shell conduction band-edge state is higher in energy than thecorresponding core state, and the shell valence band-edge state is lower in energythan the corresponding core state. In contrast, the energy offsets between the coreand shell bands in type-II QDs (e.g., CdTe/CdSe, CdSe/ZnTe) are such that thecarriers tend to be spatially separated: the conduction and valence band-edge statesof the shell are either lower or higher in energy than the corresponding core states[62]. This carrier separation – an electron/hole in the shell and hole/electron in thecore – can be advantageous in some applications (e.g., energy conversion/photo-voltaics), but can be detrimental to QDPL. The spatial separation reduces the overlapbetween the electron and hole wave functions, decreasing the rate of radiativerecombination, whichmust now occur across the core/shell interface. The result is alower quantum yield and longer PL lifetime [62,63]. Although a type-II architecturecan be used to generate near-infrared (NIR) PL from QD core materials with visibleband gaps, type-I confinement is generally preferred in applications where thebrightness of the QD is paramount. These include the use of QDs as luminescentprobes for FRET-based sensing.

12.3.6Quantum Confinement

The semiconductor materials used to fabricate QDs are crystalline solids built upfrom their constituent atoms. In the bulk, their electronic structure is characterized


by a lower energy valence band (VB) that is filled with electrons, and a conductionband (CB) that is empty. These bands arise from the linear combination of atomicorbitals associated with each constituent atom, and are typically separated by anenergy gap of 0.5–3.5 eV [56]. The VB and CB comprise a quasi-continuum ofdiscrete states that are separated by very small energies – a large density of states.The magnitude of this spacing is inversely proportional to the number of atoms inthe semiconductor crystal, which is large for bulk materials. Quantum confinementoccurs when the number of atoms in a semiconductor crystal becomes small, theenergy between states increases, and the density of states becomes small. That is, atthe size scale of a QD.The development of quantum confinement in QDs can be conceptualized

from either bottom-up or top-down. In the latter, a QD is imagined to form as abulk crystal is shrunk down to a small number of unit cells at the nanometerscale. Since the bulk CB and VB edges are the last states to form as the numberof atoms becomes large [16,25,31], the allowed states shrink away from theband-edges and decrease in density as the crystal shrinks to a small number ofatoms. The band gap energy thus increases as the QD becomes smaller,resulting in the observed hypsochromic shift in QD PL. From the bottom-up perspective, too few atoms are added to the nanocrystal for the linearcombinations of atomic orbitals to form complete bands, thus leaving discretestates that occupy a smaller range of energies than the bands of the bulkmaterial. Since the VB and CB are centered on the energy of the originatingatomic orbitals (e.g., the top of the CdSe VB is approximated by a linearcombination of Se 4p orbitals and the bottom of the CB is approximated by alinear combination of Cd 5s orbitals [25]), this yields the bathochromic shift inQD PL with increasing core size. Both concepts are equivalent, and aresummarized in Figure 12.5.It is also instructive to consider the formation of H€uckel orbitals for linear

polyenes. This system is a well-known molecular analogue for a one-dimensionalsemiconductor, and the allowed wave functions for an electron are those withnodes at the termini of a linear polyene. This principle extends to a semi-conductor, where nodes must occur at the boundary conditions of the crystal [31].The situation is similar for a QD, where the exciton takes on the character of aparticle-in-a-sphere: the nanocrystal dimensions determine the allowed wavefunctions with the requirement of nodes at the surface of the nanocrystal [64].From a particle-in-a-sphere model, the QD energy gap is expected to scale as R�2,where R is the radius of the QD [65]; however, as recognized by Brusand coworkers [6], the band gap energy depends on two competing effects:(i) increased kinetic energies associated with confinement of the electron andhole, causing a shift to higher energies and (ii) increased electrostatic attraction,causing a shift to lower energies. Brus estimated the size-dependent band gapenergy of QDs using an effective mass approximation for the kinetic energy, ahydrogenic Hamiltonian, and particle-in-a-sphere basis wave functions[30,31,56,66,67]. The result is shown in Equation 12.7, where EB is the bulkband gap energy, me and mh are the effective masses for electrons and holes, e is


the semiconductor dielectric constant, and ERy� is the Rydberg energy for theelectron–hole pair.

EQDðRÞ ¼ EB þ p2�h2

2R2

1me

þ 1mh

� �� 1:786

e2

eR� 0:248E �

Ry : ð12:7Þ

The second term in the right-hand side of Equation 12.7 is the energy of a particle-in-a-sphere, and the third term is the Coulomb energy between the electron andhole. The latter neglects a small correction term for the dielectric discontinuity atthe nanocrystal interface, which would make the Coulomb energy slightly lessshielded [31]. The electron and hole are loosely bound and thus tend to reside nearone another to maximize their Coulombic interaction; however, semiconductordielectric constants are large and screening is significant, resulting in an additionaltendency for the electron and hole to reside near the center of the QD to maximizedielectric stabilization. This spatial correlation is considered in the fourth term andis generally small compared to the second term, the latter dominating the energychange with a strong dependence on size. Equation 12.7 is thus often truncatedafter the first three terms on the right-hand side.Quantum confinement allows the band gap energy of a QD to be tuned over a

range>1 eV with changes in nanocrystal size [68]. In the case of CdSe, the bulk band

Figure 12.5 Quantum confinement results insize-tunable QD PL. (a) Photograph of PL fromCdSe/ZnS QDs of increasing size (left to right)illuminated under UV light. (b) Qualitativechanges in the QD band gap energies, Eg, anddensity of states for CdSe QD510 (2.7 nmdiameter), QD530 (2.9 nm), QD555 (3.5 nm),QD570 (3.8 nm), QD590 (4.3 nm), and QD610

(4.8 nm). The conduction (CB) and valence(VB) bands of bulk CdSe are shown forcomparison. The energy scale is expanded as10E for clarity. Transitions for absorption andband-edge emission are shown. (Adapted withpermission from Ref. [121]. Copyright 2011,American Chemical Society.)


gap energy is 1.7 eV and the energy can be tuned over the range 1.9–2.8 eV (PL from�650–450 nm) as the particle size is decreased from approximately 7 nm to 2 nm[8,16,17]. Although qualitatively useful, Equation 12.7 provides only reasonablequantitative agreement at the upper limit of quantum confinement. At small or evenmoderate QD dimensions, the proportionality to R�2 breaks down and moresophisticated models are needed [25,30].In addition to the size-dependent shift in the band gap energy, quantum

confinement has a second consequence: an increase in oscillator strength.This is responsible, in part, for the emergence of strong peaks in the absorptionspectra of QDs compared to the featureless spectra of the corresponding bulkmaterial [6,25,30]. From Fermi’s golden rule, the oscillator strength for a transi-tion depends on the matrix element mixing the initial and final states, and also theoverlap between the two states. The important overlap for light absorption by asemiconductor is that between the electron and hole state; the oscillator strengththerefore increases as the electron–hole overlap increases due to quantumconfinement [30,67]. A second effect arises from Heisenberg’s uncertaintyprinciple. In a bulk crystal, the momentum and energy of an electron are welldefined, but its position is uncertain. However, due to the particle-in-a-spherecharacter of a QD, it is the position and energy of the electron that are well knownand the momentum that is uncertain. The bands of the bulk semiconductorcondense into the discrete states of the QD and the energy of these states can bethought of as a superposition of closely spaced states from the bulk crystal [16,25].Both energy and momentum must be conserved in an optical transition, anduncertainty in the former enhances the probability that a given transition willsatisfy these conditions. Thus, closely spaced transitions that would normallyoccur at slightly different energies in the bulk are compressed into a single intensetransition in a QD [16,25].

12.3.7Quantum Dot Photophysics

The detailed photophysics of QDs are well beyond the scope of this chapter; however,we provide a superficial overview of several processes that have analogues com-monly known for molecular fluorophores. The reader is referred to the citedreferences for more in-depth descriptions, and should also recognize that severalaspects of QD photophysics, though reasonably understood, are not yet definitive intheir details. Although some of the ensuing discussion may seem esoteric, many ofthese phenomena will be revisited in discussing QDs as donors in FRET.From the standpoint of their most utilized property, PL, the electronic structure of

QDs is most conveniently described using a two-level model. When an exciton isformed upon excitation, an electron occupies the 1Se state and the correspondinghole occupies the 1Sh state. These quantum-confined states are analogues of themolecular LUMO and HOMO, and are derived from the bulk CB and VB,respectively. The intervening energy gap is dependent on the size of the corenanocrystal, as described earlier. Another important property of QDs is their broad


absorption spectra and, in practice, QDs with visible PL (e.g., CdSe/ZnS) are usuallyexcited in the UV–blue region of the spectrum. Such transitions are higher in energythan the 1Sh–1Se transition and the excess energy above the energy gap is dividedbetween the electron and hole [69]. States higher in energy than the 1Se state andlower in energy than the 1Sh state are generally implied in a two-level model,although not considered explicitly, since PL is dominated by the former states. Thisis a consequence of very efficient nonradiative intraband transitions to the 1Se and1Sh states, which yields band-edge PL regardless of the initial excitationwavelength –an analogue of Kasha’s law for molecular fluorophores. The energy associated withintraband transitions is converted into kinetic energy through a phonon-drivenmechanism, which, interestingly, was not intuitive given the lower density of statesin QDs compared to bulk semiconductors, resulting in the expectation of a “phononbottleneck.” The full details of intraband transition mechanisms in QDs, and howthe phonon bottleneck is overcome, can be found elsewhere [65,68,70–79].The FWHM of QD PL spectra can also be understood in terms of a basic two-level

model with quantum confinement. In a perfect ensemble of QDs, with exactly Natoms per QD, the emission line width would be very narrow – similar to atomicemission – and show satellite lines for coupling to phonons in the nanocrystal. Infact, this has been observed experimentally at the single-QD level [80]. However, inreal ensembles there are always N�DN atoms per QD, that is, a finite sizedistribution. The Gaussian profile for QD PL is thus largely due to inhomogeneousbroadening that results from a distribution of nanocrystal sizes, and corresponds toa size-dependent Gaussian distribution of 1Se–1Sh transitions [80,81].PL emission from a QD results from interband radiative recombination across the

1Se–1Sh energy gap. In the case of a structurally perfect QD, nonradiativerecombination of the exciton should not occur and the QD should have a quantumyield near unity [33]. Again, real samples are not perfect and quantum yields can varyfrom a few percent to more than 80% depending on the preparation. One school ofthought is that nonradiative interband transitions are not negligible and assisted byphonons and/or surface states [17,41,56,70,82]. The latter can exist at energieswithin the band gap and localize carriers, potentially providing circuitous pathwaysfor nonradiative recombination. These pathways can sometimes be observed asbroad PL emission at wavelengths longer than the band-edge emission [83]. This isreferred to as band gap emission (cf. band edge), and originates from radiativeexciton recombination between deep trap states (i.e., a large energy differencerelative to 1Se or 1Sh) located within the band gap. Interestingly, blinking dynamicshave been shown to vary with the surface properties and ensemble quantum yieldsof QDs [84–88]. This has raised the question of whether ensemble quantum yieldsare a function of the frequency of “on/off” cycles as individual QDs blink. A furtherquestion is the extent to which the ensemble quantum yield is determined by a “darkfraction” of nonluminescent QDs. Studies have addressed these issues and foundthat the bright fraction of QDs correlates with the ensemble quantum yield, and thatblinking is not coupled to the dark fraction [89,90]. Furthermore, an increase inensemble quantum yield is frequently observed upon the adsorption or conjugationof large biomolecules such as proteins [91,92]. Typically, this has been attributed to


greater surface passivation across the ensemble of QDs; however, this phenomenonhas now been correlated to an increase in the fraction of bright QDs rather thanchanges in their individual brightness [90]. This indicates a role for surface/trapstates in determining whether a QD is bright or dark, and that suitable passivation ofdeleterious surface states can interconvert QDs from dark to bright. Overall, the netquantum yield of an ensemble of QDs is likely determined by both the size of thedark fraction and the relative nonradiative and radiative interband transition rateswithin the bright fraction.An obvious failing of using a simple two-level model to describe band-edge QD PL

is its inability to account for the 10–20 nm Stokes shift between the lowest energy1Sh–1Se absorption band (i.e., first exciton peak) and the peak PL emission wave-length. In the case of CdSe and CdS QDs, the Stokes shift is a consequence ofsplitting of the 1Sh–1Se exciton state into a set of closely spaced states. One ofthese states, located at the band edge, is forbidden and cannot be populated byoptical excitation [31,33,68,78,93–96]. Nonetheless, this dark state can be occupiedby intraband relaxation following optical excitation to allowed exciton states ofhigher energy, and the Stokes shift is attributed to PL emission from this state[42,80,93,96–100]. This state has been also implicated in explaining the long lifetimeof the QD excited state [55,79,80,94,98,101]. The frequent observation of biexpo-nential QD PL decays has been attributed to the formation of an exciton while acarrier is trapped at the surface of the QD nanocrystal [41].In addition to PL emission intensity, quantum yield, and lifetime, another

useful photophysical property is the polarization of PL. Unlike molecular fluo-rophores that exhibit a “bright axis” parallel to their transition moment, QDsexhibit a dark axis parallel to their c-axis [80,102]. As a consequence, the PLemission of CdSe QDs is either circularly polarized or unpolarized [24]. Althoughlinearly polarized emission is not obtained from QDs, it can be obtained fromelongated quantum rods [103].Returning to the blinking of QDs, this behavior is largely attributed to cycles of

photoionization and neutralization of the nanocrystal. The result is “off/on”switching between dark and bright states, on time scales ranging from milli-seconds to minutes, where the probability distributions of the on/off times followan inverse power-law [54,84,104,105]. A “gray” state with intermediate brightnessis also observable [105]. The photoionization process is potentially associatedwith Auger ionization and/or carrier trapping by surface states. The blinkingkinetics depend on excitation intensity, and the quantum yield of photoionizationhas been estimated to be 10�6 for CdSe QDs [104]. Significantly decreasedblinking has been observed in the presence of thiol ligands [106], with QDs thathave shell thicknesses of 6–8 nm [59], and in alloyed QDs that have a smoothrather than abrupt confinement potential [107]. The putative ionization mecha-nism suggests that electron or hole transfer to a neutral QD should efficientlyquench its PL and, indeed, this has been observed experimentally [108,109].While blinking can only be directly observed in PL measurements on single QDs,it is nonetheless present in ensembles, as noted previously in the discussion onthe QD quantum yield.


12.3.8Quantum Dot Synthesis

The highest quality colloidal QDs are typically synthesized by the pyrolysis oforganometallic and chalcogen precursors, where rapid nucleation is followed byslower and steady growth [8–10,110]. The important components of all QD syntheticrecipes are the monomers/precursors, a coordinating solvent, other coordinatingligands, the temperature, and the growth time. A generic synthesis scheme is shownin Figure 12.6.In the synthesis of CdSe QDs, the Cd precursor is usually dimethyl cadmium,

Me2Cd [8–10], or cadmium oxide, CdO [37,111]. The selenium precursor is usuallytrioctylphosphine selenide, TOPSe, or bis(trimethylsilyl)selenide, (TMS)2Se [8–10].

Figure 12.6 Schematic of (a) typical CdSe core synthesis and (b) ZnS shell growth. Recipes andconditions vary.


TOPSe can be easily prepared by dissolving selenium in trioctylphosphine (TOP).The use of CdO and TOPSe precursors is often preferred due to their greater stabilityand ease of preparation [10,112,113]. The most common coordinating solvent istrioctylphosphine oxide (TOPO) and/or TOP, while other ligands such as long-chainalkyl acids [114,115], alkyl amines [37,114–116], alkyl phosphines [8,18,111], andalkyl phosphonic acids [29,111] are also used in QD synthesis.A typical synthetic protocol would involve heating TOPO to a high temperature

(e.g., >300 �C) under an inert gas such as Ar or N2, injecting a solution containingthe precursors to initiate rapid homogeneous nucleation, followed by crystal growthfor some time at a lower temperature (e.g., 230–300 �C). Typically, the growth timecan vary from a few seconds to minutes depending on the size of QD desired. Thegrowth of the QDs is conveniently monitored via the absorption or PL spectra ofaliquots that are taken from the reaction mixture at different time intervals.Cadmium-rich QDs are almost always grown to avoid the formation of deep surfacetraps that are associated with selenium-rich facets, as well as poor binding of basicligands to selenium sites [79].The role of the coordinating solvent and other ligands is to stabilize the growth of

the nanocrystal. Themolecules dynamically adsorb and desorb from the QD surface,and can help add, remove, or rearrange atoms at the nanocrystal surface [26,29,117].This helps ensure that good crystallinity is obtained. Despite the dynamic exchange,a monolayer of ligands remains bound to the QD on average and preventsaggregation [117]. The monomer concentration is also very important since efficientgrowth requires supersaturation and allows smaller nanocrystals to grow faster thanlarger ones at sufficiently high monomer concentrations [26]. This is referred to as a“focusing” regime and yields narrow nanocrystal size distributions. When mono-mer concentrations are below this threshold, Ostwald ripening occurs and the sizedistribution broadens [26]. Ostwald ripening is a process in which smaller particlesdissolve to “feed” the growth of larger particles, and is driven by the decrease in thesurface area-to-volume ratio of the particle population. The rate of QD growth isimportant in determining not only the size distribution, but also the nanocrystalshape. As the rate of growth increases, different facets of the nanocrystal grow atdifferent rates and nonspherical shapes result [26,29]. Overall, the synthesis of high-quality QDs represents a controlled precipitation reaction in which sufficient energyis provided to ensure that the nanocrystals anneal, and where control over growth isrooted in kinetics rather than thermodynamics [118].In the case of core/shell QDs such as CdSe/ZnS, the shell synthesis is carried out

similarly to the core synthesis. Typically, a solution of bis(trimethylsilyl)sulfide,(TMS)2S [8,9] and either dimethyl or diethyl zinc, Me2Zn or Et2Zn [8,9], is added tothe core nanocrystals in TOPO. The temperature for the shell growth is usually inthe range of 100–150 �C, and it has been reported that different temperatures areoptimal for QDs of different sizes [8]. One challenge in shell growth is avoidingnucleation of the shell precursors. The shell growth may be done as an immediatesecond step in the same flask as the core [9], or as a separate and subsequent step in asecond flask after collection and clean up of the grown core QDs [8,18]. A variationon the shell growth procedure is to inject shell precursor solutions alternately, rather


than as a mixture of both precursors, in a successive ion layer adsorption andreaction (SILAR) method [116].The QDs collected from organometallic syntheses are coated with mixtures of

TOPO and other ligands (e.g., hexadecylamine), and can be dispersed in nonpolarsolvents such as toluene, chloroform, and hexanes. These native QDs are notsoluble in polar solvents. Of the commonly used QD materials, CdTe seems to besomewhat unique in its ability to yield bright QDs in aqueous syntheses [119],albeit that these QDs are not as monodisperse as those prepared using organo-metallic methods.

12.3.9Quantum Dot Coatings

In contrast to molecular fluorophores, QDs do not have intrinsic solubility andmustbe coated with surfactants, small bifunctional organic molecules, or macromole-cules to provide solubility and dispersion of individual QDs. In biological applica-tions, aqueous solubility is necessary and the coatings useful in this regard can bebroadly classified as ligand-based or polymer-based, as well as neutral, charged, orzwitterionic. Overviews of coatings for aqueous QDs can be found in recent reviews[120,121]. Figure 12.7 illustrates the two interfaces of the QD coating that can bephysicochemically tailored, as discussed next for different coating types.

Figure 12.7 Two interfaces of a QD coating:(a) solution interface, which can be tailored tobe (i) cationic, (ii) zwiterrionic, (iii) anionic, or(iv) neutral (e.g., PEGylated); and (b) inorganic/organic interface, where the coating assembles

to the QD through either (i) interdigitation ofpendant alkyl groups on a polymer, (ii) dativebinding of pendant coordinating groups on apolymer, or (iii) dative binding of individualcoordinating ligands.


Ligand coatings comprise a monolayer of small, bifunctional molecules thatcoordinate directly to the inorganic surface of the QD. These interactions can besuperficially understood on the basis of hard/soft acid–base interactions [73], andare fundamentally similar to the chemistry of transition metal ion complexes. Forexample, Cd2þ is a soft metal ion and coordinates favorably with a soft thiol ligand.Indeed, the most widely used ligands have been monodentate (e.g., mercaptopro-pionic acid, MPA) or bidentate (e.g., dihydrolipoic acid, DHLA) thiols with distalcarboxyl groups [36]. Ligand coatings are usually prepared by exchange with TOP/TOPO ligands used in QD synthesis. This process is driven by the greater affinity ofthe thiol for the QD surface, as well as mass-action through an excess of the newthiol ligand. QDs coated with MPA, DHLA, or similar ligands are compact andcharged; colloidal stability is maintained via electrostatic repulsion among QDs.Aggregation often occurs in low pH or high ionic strength solutions [122–124].Zwitterionic thiol terminated ligands such as penicillamine [125], cysteine [126], orderivatives of DHLA [127] can improve stability over a range of pH values and alsoreduce nonselective binding compared to purely anionic or cationic coatings. Inaddition, a diverse array of polyethylene glycol (PEG) appended thiol ligands havebeen used to coat QDs [128–132]. These ligands do not depend on charge forsolubility and offer superior colloidal stability across a large range of pH and ionicstrengths, low nonselective binding, biocompatibility, and even terminal functionalgroups (e.g., carboxyl, amine, biotin, see Figure 12.8) for bioconjugation. Mixedfilms of different ligands can also be prepared and offer some tuning of interfacialproperties [131]. Analogous to thiols, dithiocarbamate ligands derived from aminoacids have also been found to coordinate to the QD surface, but their use has beenmore limited [133–135].Instability and aggregation is often associated with the desorption of thiol ligands

from the surface of the QD over time. After the coating procedure, the ligands areassociated with the QDs, but not at equilibrium, since the solution concentration ofthe ligand is approximately nil. Desorption occurs as equilibrium is re-established.To this end, it has been reported that the use of bidentate thiol ligands such as DHLAincrease the shelf life of ligand-coated QDs to periods ranging from several monthsto a year [129]. Effectively, multidentate ligand coatings can also be prepared by crosslinking monodentate ligands at the QD surface [136]. Further, studies suggest thatthe thiolate form of the ligand binds strongly to the QD [137], such that low pH canprotonate the thiolate and drive desorption [123]. Coating instability is also associ-ated with ultraviolet (254 nm) photooxidation of thiol ligands to disulphides, whichmay be catalyzed by the QD surface [138]. QDs coated with bidentate ligands are lessstable toward photooxidation than QDs coated with monodentate thiols due to thestrong tendency of bidentate thiol ligands to form intramolecular disulfides[138,139]. Finally, a common side effect of ligand exchange is a tendency towardmuch lower quantum yields in the aqueous phase compared to QDs capped withTOP/TOPO/hexadecylamine (HDA) in the organic phase [82,138,140]. Some meth-ods to circumvent this behavior have been reported [137,141]. Although the lowerquantum yields of ligand-coated QDs are not optimal for FRET, the compact size is adistinct advantage.


Polymeric coatings have been traditionally based on amphiphilic polymers, whereassembly with the QD is driven by the hydrophobic interactions and interdigitationof pendant alkyl chains with the native ligands (e.g., TOPO) of the QD [142,143]. Thehydrophilic component of amphiphilic polymers usually incorporates carboxylgroups, amine groups, PEG chains, or combinations thereof. The amphiphilicstructure is typically synthesized through the copolymerization of different func-tional monomers or grafting of alkyl chains and/or PEG chains to polymer back-bones. Polymer coatings often give much brighter QDs than ligand coatings, withonly small decreases in quantum yield; however, this comes at the expense of largerhydrodynamic size and coating thickness [144,145]. The latter is not necessarilyoptimal for FRET applications, since it could limit the maximum energy transferefficiency. Recently, polymer coatings have been developed with pendant imidazole

Figure 12.8 Illustration of selected surfacechemistries and conjugation strategies appliedto QDs. The gray periphery around the QDrepresents a general coating. This coating canbe associated with the surface of the QDthrough either (a) coordinated monodentate orbidentate thiols, (b) coordinated imidazole orpolyimidazole (e.g., polyhistidine) groups, or(e) hydrophobic interactions with native TOPOligands. The exterior of the coating mediates

solubility through the display of (c) amine orcarboxyl groups, or (d) functionalized PEG.Common strategies for bioconjugation include(a) thiol modifications; (b) polyhistidine tags;(f) electrostatic association with the QDcoating; (g) mutual chelation of Ni2þ;(h) maleimide activation and coupling; (i) activeester formation and coupling; and (j) biotin-labeling and SA-QD conjugates. Figure not toscale.


or dithiol groups that coordinate directly to the QD surface and at least partiallydisplace the native ligands [146,147]. These coordinating polymer coatings offersmaller hydrodynamic dimensions. Since different monomers can be copolymer-ized, or pendant groups/chains grafted to a polymer backbone, separate functional-ities can be introduced for aqueous solubility and bioconjugation.

12.3.10Quantum Dot Bioconjugation

The conjugation of aqueous QDs with proteins, peptides, nucleic acids, and otherbiomolecules is of significant research interest and unavoidably critical to thebiological applications of QDs. Several common bioconjugation strategies areillustrated in Figure 12.8, and are often borrowed or adapted from methods ofprotein labeling. The different strategies can be broadly classified into three groups:(i) covalent conjugation, which utilizes the formation of new bonds betweenbiomolecular functional groups and the QD coating; (ii) physisorption, which relieson the spontaneous association of biomolecules with QD coatings due to electro-static, polar, or hydrophobic interactions; and (iii) coordination, which utilizes adative interaction between biomolecular functional groups and the inorganicsurface of the QD or, alternatively, mutual chelation of a metal ion by both theQD coating and a biomolecule. Enzymes [148,149], antibodies [150–152], small-molecule-binding proteins [91,92,153], peptides [154–157], oligonucleotides[158–161], and carbohydrates [162–164] are among the many biomolecules thathave been coupled to QDs. While a brief a description of QD bioconjugation will begiven here, the reader is referred to a recent review on nanoparticle bioconjugationfor a much more comprehensive treatment of the subject [165]. The followingdiscussion provides the background needed to appreciate the role of bioconjugatechemistry in setting up QDs in FRET configurations.Many biological probes, such as synthetic oligonucleotides and peptides, can

readily incorporate nucleophilic linkers (e.g., alkyl amines and thiols) or amino acidresidues (e.g., N-terminus, lysine, and cysteine) at defined positions for conjugationto QDs. In the case of native proteins, the most common targets for coupling are theamine, carboxyl, and thiol groups associated with accessible lysine, glutamic acid/aspartic acid, and cysteine residues within the protein structure. All of these samefunctional groups are frequently associated with QD coatings and can be availablefor reaction. Conjugation via carbodiimide activation and zero-length couplingbetween carboxyl groups associated with QDs/biomolecules and amine groupsassociated with biomolecules/QDs are widely practiced, and the intermediateformation of succinimidyl esters is often used to increase coupling efficiency[166,167]. However, methods of this type tend to require significant optimizationand are prone to irreproducibility. The number of proteins per QD (i.e., conjugatevalence) is difficult to control and does not translate directly from the relativestoichiometry of the reaction. Here, the challenge is twofold: (i) amine and carboxylgroups are ubiquitous in proteins; and (ii) QDs cannot be prepared with an arbitrarynumber of chemical handles and rather have a plurality of reactive sites. These


factors are exacerbated by competing hydrolysis, which typically necessitates the useof excess activating reagents, thus rendering the final conjugate valence a complexfunction of both reaction stoichiometry and reaction conditions. Moreover, given thepolyvalent reactivity of both proteins and QDs, the protein orientation at the QD isoften poorly controlled and nonstoichiometric cross-linking can occur. This canresult in conjugate heterogeneity and even sample aggregation [165]. Alternatively,thiol groups derived from cysteine residues have relatively low abundance in nativeproteins, albeit that they tend to form unreactive and structurally important disulfidebridges in native proteins. These bridges may be reduced to reactive thiols at the riskof altered biological activity and this is a common strategy with antibodies. In someinstances, uniquely reactive cysteine residues can be introduced at specific sitesthrough protein mutagenesis. Regardless of origin, cysteine residues are oftencoupled to QDs using heterobifunctional cross-linkers with a maleimide group[152,168]. Targeting the thiol groups of biomolecules tends to provide more controlover conjugate reactions than carbodiimide chemistry.There are several criteria for an ideal bioconjugate reaction with a QD: control over

the average number of biomolecules per QD; minimal polydispersity in the former;a predictable, defined, and reproducible orientation of the biomolecule on the QD;stable conjugation under mild conditions with low reactant concentrations andwithout competing hydrolysis; chemoselectivity and (potentially) bioorthogonality;retention of QD PL and colloidal stability; and preservation of biomolecular activity[165]. These criteria are not trivially satisfied. Although conjugation with syntheticpeptides and oligonucleotides is typically quite tractable, controlled protein conju-gation can be challenging. As such, considerable effort has been directed towarddeveloping and applying novel bioconjugate chemistries that approach the aboveideals. It is now widely realized that the bioconjugate chemistry is a key determinantfor the efficacy of the final QD-bioconjugate in applications. Emerging bioconjugatechemistries that have been adopted include the strain-promoted azide–alkynecycloaddition [169], tetrazine ligation [170], hydrazone ligation [171,172], andexpressed protein ligation [173], as well as enzyme-mediated bioconjugationsuch as that using biotin ligase [174] or the recombinantly modified haloalkanedehalogenase (Halo-Tag) system [175]. Since these chemistries tend to rely onnonbiological functional groups, proteins and other biomolecules must be prela-beled to introduce one of the reactive moieties. This is often done throughheterobifunctional molecules with a succinimidyl ester or maleimide function,although unnatural amino acid incorporation (proteins) and synthetic derivatives(e.g., oligonucleotides and peptides) are also possibilities [165]. Similarly, the QD canbe modified with the cognate functional group for bioconjugation using a hetero-bifunctional cross-linker. In some cases, a ligand or polymer coating might besynthesized with these groups initially. Despite the dependence on traditionalchemistries for introducing nonbiological functional groups at the outset, thenewfound chemoselectivity still permits better control over bioconjugate reactionswith the QDs.A QD-bioconjugate strategy that deserves special attention is self-assembly.

Perhaps the most prominent example is the spontaneous coordination of


polyhistidine tags to the ZnS shell of CdSe/ZnS QDs. This rapid and stableassociation has been used to assemble QD-bioconjugates with polyhistidine-taggedrecombinant proteins, synthetic peptides, and modified oligonucleotides[154,157,165,171,176–180]. The single point of attachment offered by polyhistidinetags helps control biomolecule orientation. Dissociation constants are typically onthe order of 10�9M in bulk solution, and equilibrium binding is reached within afew minutes at room temperature [179]. As a consequence, polyhistidine self-assembly enables control over QD-bioconjugate valence through an approximatelyone-to-one correlation with stoichiometry, albeit still subject to a Poisson distribu-tion of valences – particularly at lower average ratios [181]. The distribution ofvalences has an effect on the determination of FRETefficiencies, which is discussedin Section 12.5.1.5. Nonetheless, self-assembly generally provides the best controlover conjugate valence, as well as a defined point of attachment to the QDwith somecontrol over the orientation of the biomolecule [165]. Other motifs, such as cysteine-rich metallothionein domains [182] and thiol-terminated oligonucleotides [183,184]will also spontaneously self-assemble to QDs. These strategies are generally –

although not universally – applicable with ligand-coated QDs. The biomoleculetag (e.g., polyhistidine and thiol) must have access to the ZnS shell and equal orgreater affinity for that inorganic interface than the ligands. Alternatively, the need toaccess the shell material can be obviated by modifying the QD ligand or polymercoating with Ni2þ-nitrilotriacetic acid (NTA) derivatives [185–188]. The Ni2þ ismutually chelated by the NTA and a polyhistidine tag, with a dissociation constanton the order of 10�13M [165]. Some carboxylated polymer coatings can also be usedto coordinate Ni2þ directly – without NTA – and assemble polyhistidine-appendedproteins [189,190].Electrostatic self-assembly is compatible with both ligand and polymer coatings.

Through the control of pH, the native pI of a protein or charge associated with anengineered tag can drive association with an oppositely charged QD [92]. Cationicpolymers have also been used to mediate the assembly of oligonucleotides aroundnegatively charged QDs [191–193]. These strategies can be effective and convenient,but typically do not offer the ability to engineer any control over the bioconjugateproperties.Finally, the well-known biotin–streptavidin (SA) binding interaction is perhaps the

most widely used method for preparing QD-bioconjugates, including those withantibodies [194], peptides [195], proteins [196], oligonucleotides [159], and aptamers[160]. Assuming a relatively narrow distribution of available binding sites across apopulation of QD-SA conjugates, this method also provides good control overconjugate valence. However, a recent report has suggested that these conjugatescan be heterogeneous and fail to offer good control over biomolecular orientation[197]. The commercial availability of QD-SA conjugates, as well as biotinylatedbiomolecules or biotinylation kits, contributes greatly to the popularity of thismethod. Conversely, biotinylated ligands have been developed for QDs [198] andpotentiate conjugation using Avidin bridges with biotinylated biomolecules [199] ordirectly with Avidin fusion proteins.


12.3.11Quantum Dot Nomenclature in This Chapter

In the following sections of this chapter, QDs are referred to by their compositionand peak emission wavelength. For example, CdTe/ZnS core/shell QDs with peakemission at 600 nm are denoted as “CdTe/ZnS QD600,” and CdS QDs (core only)with peak emission at 490 nm are denoted as “CdS QD490.” Given the overwhelm-ing popularity of CdSe/ZnS core/shell QDs, this material is not generally specifiedin the shorthand notation and should be assumed if no other material is indicated.That is, “QD525” and “QD620” refer to CdSe/ZnS QDs with peak emissionwavelengths of 525 nm and 620 nm, respectively. All values are rounded to thenearest multiple of 5 nm.

12.4Quantum Dots and FRET

While it has already been noted that QDs overcome the limitations of molecularfluorophores in experiments utilizing the direct excitation of fluorescence, QDs alsooffer new opportunities and unique advantages in FRET configurations such asthose shown in Figure 12.9. The properties of QDs can enhance energy transferefficiencies, facilitate the design of donor–acceptor systems, simplify quantitativemeasurements, and enable multiplexing. This section describes the motivationand underpinnings for utilizing FRET with QDs (QD-FRET), both as donors andacceptors, and further addresses the applicability of the F€orster formalism andimportance of bioconjugate chemistry in designing these systems.

12.4.1Quantum Dots as Donors

There are three properties of QDs that are particularly advantageous from thestandpoint of a donor for FRET: (i) their broad absorption spectra and large molarabsorption coefficients; (ii) their narrow, size-tunable PL with good quantum yields;and (iii) their nontrivial and (bio)chemically accessible surface area. These and otherbenefits of pairing QDs as donors with dye acceptors for FRET are elaborated uponhere, as are the few liabilities.First, consider an arbitrary FRET pair and recall that a nominal acceptor can only

function as an acceptor when in its ground state. Tomeet this criterion across the fullensemble, the donor must be excited at a wavelength where the acceptor hasnegligible absorption. If the donor and acceptor are both dyes, the characteristicallysmall Stokes shift of each fluorophore typically requires that the former is excited atthe hypsochromic edge of its absorption spectrum. The unfortunate caveat is thatthe donor dye is less strongly absorbing at such a wavelength, causing its excitationrate and signal-to-noise ratios to decrease. Thus, a compromise must generally be

12.4 Quantum Dots and FRET j499

sought in practice. In the case of fluorescent acceptors, a background of directlyexcited acceptor fluorescence must be accounted for in any analysis of FRET-sensitized acceptor fluorescence. In contrast, QD donors are not subject to thislimitation: the QD absorption becomes stronger as the excitation is moved toprogressively shorter wavelengths that are further away from the absorption band ofthe acceptor dye. The excitation wavelength can be selected to coincide with the local

Figure 12.9 (a) QDs are good FRET donors forfluorescent protein (FP), dye, and goldnanoparticle (Au NP) acceptors. The dashedcircle represents an arbitrary R0 measured fromthe QD center. The scale at the right indicateshow R0 proportionally increases as the numberof proximal acceptors, a, increases. In addition,QDs are good acceptors for luminescent

terbium complexes and bioluminescentluciferase donors. (b) Qualitative spectraloverlap (shaded regions) for a CdSe/ZnSQD625 as (i) a donor for a fluorescent dye,AF647, and (ii) an acceptor for a luminescentterbium complex donor. (Adapted withpermission from Ref. [121]. Copyright 2011,American Chemical Society.)


minimum in the acceptor’s absorption/excitation spectrum. Alternatively,the comparatively large two-photon absorption cross sections of QDs alsopermit efficient nonlinear excitation with minimal background excitation of anacceptor dye.Next, consider the QD PL and its impact on both the F€orster distance and the

experimental observation of FRET. The sixth power of the F€orster distance scaleslinearly with both the spectral overlap integral and the donor quantum yield, seeEquation 12.2. The favorable quantum yield of QDs thus increases the rate of FRETand its overall efficiency. The bigger advantage, however, is arguably the size-tunablenature of that PL and its narrow, symmetric spectral profile. FRETpairs comprisingtwo molecular fluorophores generally suffer from increased direct excitation of theacceptor (vide supra) and increased overlap between their emission spectra as thespectral overlap integral increases – a clearly antagonistic effect. This limitationarises from the small Stokes shift and broad, red-tailed emission of the donor dye.Tuning of any of these parameters must also occur in discrete steps by selectingdifferent donor and/or acceptor dyes. In contrast, the ability to tune the wavelengthof QD PL over a continuous range offers the parallel ability to tune the spectraloverlap integral continuously. The narrow PL is such that the spectral overlapintegral can approach its maximum value with minimal overlap between the QD PLand the fluorescence of the acceptor dye, maximizing resolution of FRET-sensitizedPL from the latter. This is particularly important in experiments that collect emissionin discrete channels using bandpass filters (e.g., microscopes) rather than acquiringfull emission spectra. Even if full spectra are measured, the Gaussian PL profile ofthe QDs greatly facilitates deconvolution of donor and acceptor PL in instances ofemission overlap.These advantages of QD donors are particularly useful in multiplexed FRET

configurations – that is, the simultaneous interrogation of two or more donor–acceptor pairs in the same system. Generally, spectrally resolved FRET pairscomprising only molecular fluorophores require a different excitation source (orwavelength selection) to interrogate each pair. Measurements must be done eitherserially or by integrating two excitation sources for simultaneous use. Indeed, thistechnical challenge spawned, at least in part, the commercial availability of multi-pass excitation filters and dichroics for microscopy. In contrast, the ability tosimultaneously excite different colors of QD donors at a common wavelengthcan potentiate the technically straightforward, one-shot acquisition of multicolorPL from multiple FRET pairs. The use of an approximately monochromaticexcitation source can also help avoid potential chromatic aberrations and tediousalignment of multiple excitation beams in imaging experiments. A further advan-tage in multiplexing is that the narrow PL of QD donors helps to minimize theoverlap between (i) FRET-sensitized acceptor emission from the short wavelengthFRET pair and (ii) the donor emission from the long-wavelength FRET pair.Compared to dyes, a greater number of QDs can be fit within a given spectralwindow and their PL contributions more readily deconvolved. Thus, the use of darkquenchers (i.e., non-fluorescent dyes) as acceptors for QD donors can provide theopportunity for highly multiplexed FRET [200].


The third advantage of QD donors is their scaffold capability, wherein multipleacceptors can be concentrically arrayed around a single QD donor. While this doesnot change the rate of FRET between the QD and a particular acceptor, the net rate ofFRET from the QD donor to any acceptor increases according to Equation 12.8,where a is the number of acceptors at a common donor–acceptor separationdistance, r. The corresponding expressions for FRET efficiency and the acceptor/donor emission ratio are given by Equations 12.9 and 12.10.

kFRET ¼ atD

R0

r

� �6

: ð12:8Þ

E ¼ aR60

r6 þ aR60

: ð12:9Þ

IADIDA

¼ aWA

WD

� �R0

r

� �6

: ð12:10Þ

The FRETefficiency can thus be optimized somewhat independent of both r and theF€orster distance, R0. Importantly, these centrosymmetric QD-FRET configurationsare readily accessible through bioconjugation. Molecular fluorophores do not offerusable surface area, and multiacceptor geometries with dye donors must rely onsecondary macromolecular scaffolds with discretely reactive sites that may have anasymmetric arrangement (cf. equivalent, contiguous sites at a QD surface). Further,labeling a macromolecular scaffold with only one donor at a specific and unique siteis not necessarily trivial.Despite the many advantages of QD donors, there is a potential caveat: the radius

of a QD adds a fixed amount to the donor–acceptor separation. Therefore, in somuch as the QD size can tune the spectral overlap integral and FRET efficiency, italso imposes an inherent minimum value for r, which corresponds to the radius ofthe QD and its coating. The latter should not be neglected, since, for example,polymer coatings can add significant distance between the QD and acceptor.Nonetheless, polymer coatings remain advantageous from the standpoint of offer-ing the highest quantum yield and increased rates of FRET. The net effect of theseconflicting factors must generally be evaluated within the context of a particularexperiment. Regardless, the imposedminimum donor–acceptor distance highlightsthe important advantage of being able to array multiple acceptors to increase theFRET efficiency, potentially mitigating this limitation.

12.4.2Applicability of the F€orster Formalism

The discussion in the previous section tacitly assumed that the F€orster formalismwas applicable to QDs. While studies have suggested this to be true, it is not obviousthat it should be the case. The first point of interest is the dipole–dipole approxima-tion: the F€orster formalism models energy transfer between two point dipoles, butthe size of a QD is comparable to the typical donor–acceptor distance in FRET


experiments. A priori, the dipole–dipole approximation should break down; however,using detailed theoretical calculations, Allan and Delerue [201] and Curutchet et al.[202] determined that it is valid to use the F€orster formalism with direct band-gapsemiconductor nanocrystals. The CdSe QD transition density effectively behaves aspositive and negative charges that are localized at the center of the nanocrystal andseparated by a distance of 0.7 nm – an approximate point dipole [202]. This resultalso confirmed that donor–acceptor distances should be calculated from the centerof the QD. Further, and quite importantly, the minimum donor–acceptor separationimposed by the radius of the QD ensures the applicability of the F€orster formalism,even when the QD and acceptor are in physical contact. In contrast, the dipole–dipole approximation breaks down when two molecular fluorophores are in contact.In addition to theoretical studies, the results of several experimental studies are in

agreement with the expectations of the F€orster formalism. FRET efficiencies havebeen found to scale with the value of the spectral overlap integral, the number ofproximal acceptors, and the minimum donor–acceptor separation imposed by theQD [203]. Moreover, FRET efficiencies measured from both steady-state and time-resolved PLs have shown generally good agreement with predictions derived fromgeometric models. For example, using a rigid polypeptide as a variable length spacer,Pons et al. [204] and Medintz et al. [205] observed excellent agreement betweenmeasured and predicted FRET efficiencies as a function of changes in the QDdonor–dye acceptor separation. Good correspondence between ensemble andsingle-pair FRET-derived distance measurements [181] further suggested the appli-cability of the F€orster formalism. Some experiments have indicated that deviationsfrom the F€orster formalism can be observed when acceptors are in contact with theQD surface, resulting from interactions with surface states; however, passivation ofthese states restores adherence to the F€orster model [206]. Charge transfer as acompeting pathway with FRET may also cause deviations from the F€orsterformalism.A majority of studies with QDs assume a value of k2¼ 2/3 for the orientation

factor in FRET calculations. This value is appropriate for dynamic and randomorientations of both the donor and acceptor transition moments. However, CdSeQDs have been shown to have a dark axis with a doubly degenerate transition dipole(i.e., circular emitter) in the orthogonal plane [207]. Therefore, the assumption thatk2¼ 2/3 is not strictly valid. Further, given that a QD can be conjugated withmultipleacceptors, different values of k2 may strictly apply to acceptors at different positionsrelative to the c-axis. Nonetheless, the partially random orientation of the QDtransition dipole, the (usually) random and dynamic orientation of the acceptortransition dipole, and the likely distribution of acceptor positions relative to the QDacross an ensemble appear to render k2¼ 2/3 a useful approximation.Finally, the inhomogeneous properties of QDs warrant some discussion in the

context of FRET. A dark fraction of QDs, being nonemissive, is invisible in a FRETexperiment. However, the effect of a dark fraction on the ensemble quantum yieldmay be significant if the result is an underestimation of the calculated F€orsterdistance. This is particularly true if the bright fraction has a unity quantum yieldrather than a nonunity quantum yield, which is always measured for an ensemble.


Elucidation of the real quantum yield for a bright fraction of QDs, and a convenientmethod of experimental measurement thereof, will enable more accurate distancecalculations with QDs as components of FRET-based spectroscopic rulers. Asdiscussed in Section 12.5.3, however, this is not the primary interest in the useof QDs as donors in FRET. At present, FRET-based assays and sensing configura-tions have that distinction, and approximations of the QD quantum yield seem tosuffice.Another consideration is the effect of the inhomogeneously broadened QD PL

spectrum on the spectral overlap integral. The ensemble PL spectrum of a QD,which is measured and used to calculate the spectral overlap integral with anacceptor, does not coincide to that of an individual QD, which participates in energytransfer. The homogeneous linewidth of individual QDs is narrower than theheterogeneously broadened ensemble PL spectrum, potentially resulting in anoverestimate of the spectral overlap integral and F€orster distance. Reformulationsof the spectral overlap integral have been developed to address this effect ofinhomogeneous broadening, and can account for temperature dependent changesin homogeneous linewidth [208]. Nevertheless, at ambient temperatures and withrelatively monodisperse samples, the spectral overlap calculated from the ensemblePL seems to provide a good approximation for QD donors in FRET. Nonetheless, theeffect of polydispersity can be readily observed as a small hypsochromic shift(typically not more than a few nanometers) in the QD PL between samples withand without efficient FRET. This results from the different energy transfer rates forQDs associated with the hypsochromic and bathochromic edges of the ensembleQD PL spectrum, which is a function of the slope of the acceptor absorptionspectrum [209].Overall, the evidence to date suggests that the F€orster formalism is valid for FRET

with QD donors, although there are often necessary approximations and simplifi-cations in the reduction of that formalism to practice.

12.4.3QDs as Acceptors

While QDs are excellent donors in FRET, they are poor acceptors when combinedwith molecular fluorophores as donors [210]. Although the broad absorption of QDscan result in very large spectral overlap integrals and thus F€orster distances, it alsoresults in very efficient and unavoidable direct excitation – regardless of excitationwavelength. This is compounded by the typical lifetime mismatch between QDs(often> 10 ns) and dye donors (usually< 10 ns). As a consequence, a QD is rarely inits ground state at the same time a proximal molecular fluorophore is in its excitedstate; thus, the number of available QD acceptors is negligible. The use of QDs asacceptors has been demonstrated using other QDs [211] or luminescent lanthanidecomplexes as donors [212–217]. In these systems, the donor lifetime is comparableto, or longer than, the QD acceptor lifetime. The use of lanthanide donors withmillisecond excited-state lifetimes is particularly promising: time-gating betweenflash/pulsed excitation and the acquisition of emission signals allows excited-state


QDs to return to the ground state while a majority of lanthanide donors remain inthe excited state. After a suitable delay, the real number of ground-state QDacceptors is essentially the nominal value, and the number of excited-state lantha-nide donors remains virtually unchanged. Recently, lanthanide-based upconvertingnanoparticles (UCNPs) have also been used as donors for QDs due to their ability tobe excited through the sequential absorption of two IR photons [218]. An IRwavelength is too long for excitation of the QD acceptors, which do not exhibitupconversion, and thus remain in the ground state. Note that the photon density inthese experiments is not sufficient for two-photon excitation of the QDs. Finally,QDs are also excellent acceptors in both chemiluminescence resonance energytransfer (CRET) and bioluminescence resonance energy transfer (BRET), whereoptical excitation is not required [173,189,219–224]. Instead, a chemical reactionproduces an excited-state fluorophore that is a suitable donor (i.e., blue–greenemission) for QD acceptors. Energy transfer can occur if this reaction is localizedwithin several nanometers of a QD.Considering the F€orster formalism, the dipole–dipole approximation holds as well

for QD acceptors as it does for QD donors. However, a dark fraction of QDs, and/or aheterogeneous quantum yield across the ensemble will not affect the rate of FRET,since only the donor quantum yield contributes to the F€orster distance. Thesuitability of using a spectral overlap integral calculated from the ensembleabsorption spectrum for the QD acceptor has not yet been formally studied.A priori, the effect of inhomogeneous broadening is expected to be similar tothat for QD donors when the spectral overlap is greatest near the first exciton peak inthe absorption spectrum of the QD acceptor – a hitherto infrequent situation. Incontrast, when spectral overlap is greatest at shorter wavelengths, as typical of mostreports with QD acceptors, the effect should be reduced due to the higher density ofstates for individual QDs at these energies.Aside from potentially large spectral overlap integrals and F€orster distances, a

major advantage of the use of QDs as acceptors in FRET is multiplexing capability.Analogous to the ability to excite multiple colors of QD donors using a commonexcitation source, a common donor can transfer energy to multiple colors of QDacceptors. The narrow QD PL and the ability to use Gaussian deconvolution can, inprinciple, be used to maximize multiplexing capacity. In practice, this is somewhatcomplicated by the multiple emission lines of most lanthanide donors and the red-tailed emission of chemiluminescent or bioluminescent donors, but not prohibi-tively so. In complex sample matrices, a further advantage is the ability to forgooptical excitation (CRET/BRETdonor), introduce time-gating (lanthanide donors), oruse IR excitation (UCNP donors) to minimize challenges associated with scattering,autofluorescence, and other background signals. Large effective Stokes shifts canalso be obtained since most lanthanide complexes are efficiently excited in theultraviolet and UCNPs are excited in the IR.Previously, it was noted that the assembly of multiple acceptors per QD enhanced

the net rate of FRET. This is not strictly true when a QD acceptor is surrounded by anarray of multiple donors. The rate of energy transfer from any one particular donorto the QD acceptor is fixed by the separation distance and corresponding F€orster


distance; however, there is an increase in the net rate of sensitization of the QDacceptor via energy transfer. A caveat, however, is that the donor PL also increases asmore donor is introduced into the system. The physical situation is considerablymore complex in its details than the QD donor–multiple acceptor configuration. Toillustrate, we consider interrogating multiple and equivalent lanthanide donors perQD with flash/pulsed excitation. At sufficiently low excitation intensities, theassembly of multiple donors only has an antennae effect, increasing the fractionof total QD assemblies with a single quantum of excitation energy. In this regime,the presence ofmultiple donors has no effect on energy transfer. At higher excitationintensities, multiple quanta of excitation energy may be absorbed by a single QDassembly (i.e., more than one excited-state donor per QD acceptor), and enhanceacceptor sensitization by providing “multiple attempts” at energy transfer. Forexample, if the FRET efficiency between an arbitrary lanthanide donor and theQD is E, the probability that the QD acceptor will be sensitized, PA, is given byEquation 12.11, where d is the number of donors (assumed to behave indepen-dently):

PA ¼ 1� 1� Eð Þd: ð12:11Þ

Equation 12.11 is a simplification for illustrative purposes; it does not provide thefull description of a QD acceptor interacting with multiple lanthanide donors. Giventhe typical 104–106-fold mismatch in excited-state decay times between the lantha-nide donors and QD acceptor (millisecond versus nanosecond), the QD is, to a firstapproximation, always in its ground state and a good acceptor from the perspectiveof the excited lanthanides. Thus, multiple quanta of energy can be transferred to theQD, enhancing the FRET-sensitized acceptor PL. For a general multiple donor–QDacceptor configuration, the average number of quanta transferred to the QDacceptor will depend on the donor decay rate, the energy transfer rate, and theQD decay rate. A CRET/BRET configuration with a QD acceptor is even morecomplex due to the added reaction dynamics and diffusion rates of the donor.Complex multidonor/acceptor FRET systems can be modeled; however, characteri-zation of the donor, acceptor, and their geometry is essential. The details of suchmodeling are beyond the scope of this chapter.

12.4.4The Importance of Bioconjugate Chemistry

In biological applications of QDs and FRET, the QD is almost exclusively utilized as abioconjugate. Biomolecules serve as a bridge between the QD and a paired acceptor/donor (e.g., dye molecule and lanthanide complex), either directly or throughintrinsic biochemical activity (e.g., ligand–receptor binding), and provide theproximity needed for FRET. Two bioconjugate reactions are therefore important:modifying a QD with a biomolecule, as discussed in Section 12.3.10, and labelingthat biomolecule with an acceptor/donor, as discussed here. Since the FRET-inducedquenching or sensitization depends on the coupling between acceptors/donors and


the QD, the details of each bioconjugate reaction can have a significant impact onenergy transfer. The most pertinent considerations are therefore the number andposition(s) of acceptor/donor-labeled sites per biomolecule, the number and orien-tation(s) of biomolecules per QD, and any heterogeneity across the ensemble.Analogous to conjugating biomolecules to QDs, control over the labeling of a

biomolecule with an acceptor/donor is very much a function of the type ofbiomolecule. For example, oligonucleotides and peptides can be synthesizedwith nucleophilic linkers (e.g., alkyl amines and thiols) or amino acid residues(e.g., N-terminus, lysine, and cysteine) at predetermined positions for labeling withsuccinimidyl ester, isothiocyanate, and maleimide derivatives of an acceptor/donor.Many dye modifications are available commercially when ordering a peptide oroligonucleotide sequence. In contrast, proteins are more difficult to label at uniquepositions with succinimidyl esters or isothiocyanates due to a multitude of accessi-ble lysine residues. This challenge equally applies to cadaverine or ethylenediaminederivatives of acceptors/donors due to the multitude of glutamic and aspartic acidresidues. Cysteine residues, which typically have much lower abundance, are thusmore amenable to acceptor/donor labeling at specific sites. Alternatively, somefluorescent dyes are now available as bioorthogonal derivatives (e.g., azide or alkynefor “click” reactions) and may provide better control over labeling reactions. While itis not strictly necessary to have a single acceptor/donor per biomolecule, it oftenfacilitates the design and analysis of a QD-FRET system. If a biomolecule is labeledat multiple sites with an acceptor/donor and conjugated to a QD, each acceptor/donor will be at a different distance away from the QD and yield a distribution ofenergy transfer rates. Application of Equation 12.9 will yield an average separationdistance that is more heavily weighted by those acceptors/donors closest to the QD.This average value will also depend on whether the conjugated proteins have arandom or specific orientation when bound to the QD. Interpretation of “mixed”ensemble data of this nature is not straightforward without prior knowledge ofconjugate orientation, well-defined labeling sites, and their relative positions.As noted earlier, another challenge is tightly controlling the number of biomo-

lecules per QD. In a QD-FRET experiment, this control generally translates intoknowledge of the total number of acceptors/donors per QD. High-affinity self-assembly methods – typified by the aforementioned coordination of alkylthiol- [184],metallothionein- [182] and polyhistidine-appended biomolecules [179,180] to QDs –generally provide the best control over conjugate valence [165]. Further, thesemethods provide a defined point of attachment to the QD that ideally sets a fixedorientation for the biomolecule and, by extension, a fixed position for an acceptor/donor. However, when using self-assembly methods at low concentrations, themagnitude of the dissociation constant must be kept in mind since assembly maynot be stoichiometric. Failing to do so may result in an erroneous conclusion of lowFRETefficiency. Regardless of the bioconjugate method, the extraction of physicallymeaningful data from energy transfer rates and efficiencies requires reliableknowledge of the number of acceptors per QD. Separation of QD conjugatesfrom unbound acceptor/donors and experimental determination of acceptor/donorvalences (e.g., through deconvolution of composite absorption spectra) is often an


important quality control measure. Sapsford et al. have recently reviewed analyticalmethods for the characterization of QD and other nanoparticle bioconjugates [225].The necessary degree of control over the QD-bioconjugate architecture is deter-

mined by the FRET application of interest. Experiments that aim to elucidate thephotophysics of energy transfer or extract physical parameters (e.g., distances) fromspectroscopic measurements require precise control over the number and relativeposition of acceptors/donors per QD. Architectures designed for assays and sensingapplications typically require reproducible and well-defined changes in FRETefficiency as the number of acceptors/donors per QD changes. Here, preciseknowledge of the number of acceptors per QD is not as important as the abilityto rationally tune and optimize function; however, another important criterion isretention of the native activity of the biomolecule. Biochemical utility is not possiblewithout the latter.

12.5Quantum Dots as Donors in Biological Applications

This section summarizes several examples of the use of QDs as FRET donors inbiological applications. Although every effort has been made to be thorough, thissection is not intended to be an exhaustive review of the research to date – a nearimpossibility given its fervent pace – but rather a conceptual and illustrativeoverview of different configurations, their properties, and applications. Much ofthe focus is on three basic mechanisms used to put QDs and FRET to work forbioanalysis: the modulation of FRET efficiency through either association/dissocia-tion, changes in donor–acceptor distance, or changes in the spectral overlap integral.Conformational insights from FRET, single-pair FRET, solid-phase FRET, andphotodynamic therapy with FRET are also discussed in the context of QD donors.

12.5.1Association and Dissociation to Modulate QD-FRET

The association of acceptors with QD donors or, conversely, the dissociation ofacceptors from QD donors, can modulate FRET efficiency by changing the numberof proximal acceptors, that is, the a term in Equation 12.9. Binding events betweenligand and receptor, antibody and antigen/hapten, enzyme and substrate, aptamerand target, or nucleic acid and complement are some of the many biorecognitionmotifs that can be interrogated through the associative/dissociative modulation ofFRET. The general strategy is for a QD donor to be conjugated with one half of abinding pair while the cognate half of that pair is labeled with an acceptor dye. Thenumber of binding events determines the number of proximal acceptors per QD,and the corresponding changes in FRET efficiency provide an analytical signal forbinding. This signal can be correlated to analyte concentration, biological activity, orother relevant parameters. The FRETefficiencymay bemeasured directly or througha proxy such as FRET-sensitized acceptor fluorescence, or the acceptor/donor PL


ratio. The following subsections describe several applications of associative anddissociative modulation of FRET.

12.5.1.1 Bioanalysis of CarbohydratesMedintz et al. were the first to demonstrate FRETwith QD donors in the context ofbiosensing [91]. Conjugates of DHLA-coated QDs with maltose-binding protein(MBP) were prepared using polyhistidine self-assembly. Acceptor-labeled b-cyclo-dextrin (b-CD) was bound by the MBP to quench the QD PL via FRET. In oneconfiguration, shown in Figure 12.10, the donor was QD560 and the acceptor was adark quencher, QSY-9. The addition of increasing amounts of maltose resulted inthe progressive recovery of the QD PL. A second configuration used QD530 withCy3-labeled MBP and Cy3.5-labeled b-CD. This QD530-to-Cy3-to-Cy3.5 energytransfer relay was designed to improve the efficiency of energy transfer from theQD, which was limited by both the radius of the QD and the distance to the MBPbinding site in the configuration with QSY-9. The assembly of 10 MBP-Cy3 per QDresulted in >90% PL quenching, whereas 10 MBP with bound QSY-9-labeled b-CDquenched the QDPL by only 65%. Both configurations hadmicromolar affinities formaltose.Another sugar-sensing strategy was developed by Freeman et al. using QD570

coated with boronic acid ligands [226]. These ligands form boronic esters withglucose, galactose, dopamine, and other diols. Galactose or dopamine was labeledwith Atto-590 dye and bound to the QDs to generate at FRET-“ON” state in a

Figure 12.10 (a) Schematic of QD-FRETmaltose sensor. Maltose displaces QSY-9labeled b-cyclodextrin from the binding site ofMBP assembled to QD560, disengaging FRET.(b) Absorption (pink) and PL (green) spectra

for the QD560; absorption spectrum for theQSY-9 (blue). (c) Recovery of QD PL as afunction of maltose concentration. (Adaptedwith permission from Ref. [91]. Copyright 2003,Macmillan Publishers Ltd.: Nature Materials.)

12.5 Quantum Dots as Donors in Biological Applications j509

competitive binding format where the acceptor was displaced by unlabeled analytein the sample. The acceptor/donor PL ratio provided the analytical signal, decreasingwith increasing concentrations of analyte. The detection limits for glucose, galac-tose, and dopamine were 1, 50, and 100 mM, respectively. Since boronic acids formesters with almost any cis diol, the disadvantage of this strategy was poor selectivity.

12.5.1.2 Homogeneous ImmunoassaysHomogeneous immunoassays have also been developed using QD-FRET. Forexample, Wei et al. developed a sandwich immunoassay for estrogen receptor b

(ER-b) using monoclonal anti-ER-b-QD565 conjugates in combination with Alexa-Fluor 568 (AF568)-labeled polyclonal anti-ER-b [151]. The antibodies bound todifferent epitopes of the ER-b to generate the proximity for a FRET-“ON” analyticalsignal capable of detection to 0.05 nM. It is important to note that there was anaverage of seven AF568 dye molecules per polyclonal antibody. The large size of animmunoglobulin tends to yield large donor–acceptor separations such that multipleacceptor dyes are critical for maximizing FRET efficiency. As an alternative to asandwich format, a competitive binding format is also possible with QDs and FRET.As shown in Figure 12.11, Goldman et al. demonstrated the use of QD-antibodyfragment conjugates for the detection of 2,4,6-trinitrotoluene (TNT) using FRET[150]. Black Hole quencher-10 (BHQ-10)-labeled trinitrobenzene (TNB), a TNTanalogue, was bound by anti-TNTsingle-chain variable fragments (scFv) conjugatedto QD530/555/570, resulting in FRET. There were�17 scFv per QD. TNTcompetedfor the scFv binding sites and displaced the BHQ-10-TNB; the recovery of the QD PLwas used as the analytical signal [150]. Nikiforov and Beechem reported a similarcompetitive binding immunoassay for cortisol [167]. QD605 were conjugated with�12–15 cortisol molecules per QD and anticortisol was labeled with Cy5 as anacceptor. In the absence of cortisol, binding between the QD-cortisol conjugates andCy5-labeled anti-cortisol provided the proximity needed for FRET. Increasingamounts of cortisol in a sample decreased the amount of FRET by competingwith the QD conjugates for the antibody binding sites [167]. Compared to asandwich immunoassay, the advantage of the competitive format is that it offersa shorter donor–acceptor distance by eliminating one of the antibodies and/orattaching the antigen or hapten directly to the QD. Nonetheless, it still remainsbeneficial to utilize antibodies with high degrees of labeling.Another notable application of FRET between fluorescently labeled antibodies and

QDs is probing phosphorylation by protein kinases. For example, Lowe et al.measured the activity of human epidermal growth factor receptor 2 (Her2), atyrosine kinase, using QD-FRET [227]. Peptide substrate was incubated withHer2 and phosphorylated at a NEYFYV recognition site adjacent to the C-terminus.This site was N-terminally flanked by successive polyglycine (spacer) and poly-histidine tracts. MPA-coated QD655 were added and self-assembled with thepeptides. AlexaFluor 660 (AF660)-labeled antiphosphotyrosine was added andbound to the phosphotyrosine residue, resulting in efficient FRET. The AF660/QD PL ratio provided an analytical signal proportional to the concentration of Her2.Interestingly, this assay was deployed in combination with an analogous, but


orthogonal assay for urokinase-type plasminogen activator (uPA), a protease, asillustrated in Figure 12.12. The uPA peptide substrate was biotinylated for assemblyto SA-QDs and labeled with a gold nanoparticle as a quencher (see Section 12.7). Thelimits of detection (LODs) for Her2 and uPAwere 7.5 nM and 50 ng/ml, respectively[227]. In an earlier report, Freeman et al. used Atto-590-labeled antiphosphoserinewith QD560-peptide conjugates to measure the phosphorylation of serine residuesby casein kinase 2 (CK2) [228]. Quenching of the QD PL via FRET provided theanalytical signal. These researchers also utilized an antibody-free format whereadenosine triphosphate (ATP) was labeled with Atto-590 instead. Both methodspermitted the detection of �0.1 unit of CK2 activity [228].

12.5.1.3 Hybridization AssaysThe QD-FRETdetection of specific nucleic acid sequences relies on the conjugationof QD donors with probe oligonucleotides and subsequent hybridization with targetsequences. At least five different strategies have been used to generate a FRETsignal

Figure 12.11 (a) Schematic of a QD-FRETcompetitive immunoassay for TNT (analyte). PLis quenched with binding of BHQ-10-TNB(analogue-quencher) and recovered with theaddition of TNT. (b) Response to TNT and

potential interferents Tetryl, 2,6-dinitrotoluene(DNT), and 2-A,4,6-DNT. (Adapted withpermission from Ref. [150]. Copyright 2005,American Chemical Society.)


from these biorecognition events. The simplest strategy is the use of target nucleicacid labeled with an acceptor fluorophore. When the acceptor is brought in closeproximity to the QD donor upon hybridization, the FRET-sensitization of the formerand quenching of the latter provide an analytical signal. As more target hybridizesacross the ensemble, greater apparent FRET is observed. Beyond the researchlaboratory, this is a viable strategy for clinical bioanalyses that use polymerase chain

Figure 12.12 Schematic of a simultaneousimmunoassay and protease assay. Her2phosphorylates its polyhistidine-appendedpeptide substrate and uPA hydrolyzes itsbiotinylated, gold nanoparticle-labeled peptidesubstrate. The former assembles with MPA-coated QD655 and dye-labeled

antiphosphotyrosine; the latter binds to SA-coated QD525. As shown (arrows), the result ofHer2 and/or uPA activity is quenching of theappropriate QD PL via energy transfer.(Reprinted with permission from Ref. [227].Copyright 2012, American Chemical Society.)


reaction (PCR) amplification as part of in vitro sample preparation. In many cases,however, methods without target labeling are preferred. A derivative strategy of theabove that avoids target labeling is a competitive displacement format where QD-probe conjugates are hybridized with acceptor-labeled proxies for the target. Anytarget in the sample competes with these proxies for the probe hybridization sites;the result is a progressive decrease in FRET as the concentration of target increasesand displaces more of the acceptor-labeled proxy. Unfortunately, these competitiveformats tend to be relatively insensitive to target at concentrations less than theproxy. A third strategy, which avoids both target labeling and a labeled proxysequence, is to use an acceptor dye that spontaneously binds to double-strandedDNA (dsDNA) with a concomitant enhancement in its fluorescence, for example, anintercalator or groove binder. Although many of these special dyes also have someaffinity for single-stranded material and concomitant background fluorescence, thegreater affinity and/or larger fluorescence enhancement with duplex nucleic acidtypically yields an increase in acceptor/QD PL ratio as the target concentrationincreases. A frequent drawback of this strategy is that the absorption and emissionproperties of the dyes are generally less favorable for FRET than conventional dyes;however, the association of multiple dyes along the length of double-strandednucleic acid can potentially compensate for this shortcoming. A fourth strategyis the use of a sandwich assay format where the target is indirectly labeled via thehybridization of a short, acceptor-labeled reporter oligonucleotide. The reporterhybridizes along the target sequence at a segment adjacent to that hybridized withthe probe oligonucleotide. In the context of FRET, this strategy offers the advantagesof direct target labeling without the actual requirement of target labeling. Severalexamples of these general strategies are described next. A fifth strategy, design of amolecular beacon, does not rely on the associative/dissociative modulation of FRETand is discussed in Section 12.5.2.Some of the earliest research in this area was by Bakalova et al., who developed a

hybridization assay for screening potential small interfering RNA (siRNA)sequences against target messenger RNA (mRNA) [229]. Target mRNA was ampli-fied and labeled via PCR using a Cy5-labeled nucleotide. Hybridization with QD580-probe conjugates provided an analytical signal via FRET. The study found that theaccessibility of the sense mRNA to the QD-conjugated antisense probe siRNA wasimportant for hybridization efficiency. Optimization of linker length was necessaryfor good function, since the size of the QD hindered mRNA hybridization at shortlinker lengths [229].Algar and Krull developed a multiplexed hybridization assay using QD525-Cy3

and QD605-AlexaFluor 647 (AF647) FRET-pairs [230]. As shown in Figure 12.13,each color of QD was conjugated with probe oligonucleotides complementary to agiven target sequence. Targets were directly labeled with acceptor dye. The Cy3/QD525 and AF647/QD605 PL ratios were used as the analytical signals andincreased as the concentration of target increased. The LODs for the two FRETpairs were 40 and 12 nM, respectively [230]. This strategy provides the opportunityfor multiplexed detection in an ensemble using basic spectrofluorimetry, wheresingle-molecule spectroscopy, multiple excitation sources, spatial registration, and/


or sorting technology are not required. Furthermore, the use of fluorescent accept-ors permits robust, ratiometric analysis that does not strictly require a reference statewithout FRET, and which is much more tolerant of variations on concentration,excitation intensity, matrix effects, and drift/noise between experiments. Thechallenge in the above format was the nonspecific adsorption of oligonucleotideson the mercaptoacetic acid (MAA)-coated QDs [230]. Adsorption resulted in a highbackground signal from noncomplementary sequences, long hybridization times,changes in the thermodynamic stability of probe–target hybrids, and potential false-positive signals [230,231]. These challenges were overcome by pairing QD525 withethidium bromide (EB) as an acceptor dye. Since the EB fluorescence was strongestwhen intercalated in dsDNA, adsorption did not strongly affect the EB/QD PL ratio,which increased upon hybridization. The trade-off for better selectivity was back-ground fluorescence from nonintercalated EB, resulting in a slightly higher LOD of80 nM. Nonetheless, this disadvantage was offset by the ability to detect target evenin the presence of a sixfold excess of noncomplementary oligonucleotides, or a 10-fold excess of genomic DNA (Deoxyribonucleic acid).Zhou et al. also adopted the use of EB as an acceptor dye with QD555 donors in a

hybridization assay [232]. In this case, the use of EB was not motivated by

Figure 12.13 (a) Schematic of a QD-FRETstrategy for a two-plex hybridization assay.QD525 is paired with Cy3 and a particular probeoligonucleotide sequence; QD605 is paired withAF647 and a different probe sequence.(b) FRET-sensitized acceptor PL and the Cy3/

QD525 and AF647/QD605 PL ratios areproportional to the concentration of thecorresponding target. (Reprinted from Ref.[230]. Copyright 2007, with permission fromElsevier.)


nonspecific adsorption, but rather to avoid direct labeling of target with AlexaFluor594 (AF594), see Figure 12.14 [161]. The QDs were coated with thioalkyl-PEGligands to avoid difficulties from nonspecific adsorption. Wu et al. reported a similarDNA hybridization assay with low nonspecific binding using QD535 coated withhydroxyl ligands. The target DNA was labeled with Rhodamine Red (RhR) as afluorescent acceptor dye [233].Interestingly, the adsorption of DNA on QDs has also been exploited for the

design of QD-FRET hybridization assays. Jiang et al. used a blue fluorescent cationicpolymer to mediate the assembly of infrared dye-labeled probe–target hybrids onMAA-coated CdTe QD615 [234]. The fluorescent polymer also enhanced the PL of

Figure 12.14 (a) Schematic of a QD-FREThybridization assay using either labeled targetor unlabeled target with an intercalating dyeacceptor, ethidium bromide. Hybridizationbrings the acceptor dye in sufficientlyclose proximity to the QD for FRET to occur.

(b) FRET-induced quenching of QD555 andsensitization of AF594 with increasing amountsof hybridized labeled target. (Adapted withpermission from Ref. [232]. Copyright 2008,American Chemical Society.)


the QD at excitation wavelengths shorter than 400 nm, presumably due to FRET,despite the generally poor acceptor character of QDs. The difference in the strengthof electrostatic attraction between dsDNA and single-stranded DNA (ssDNA)enabled differentiation of target from noncomplementary sequences, with theformer providing larger FRET-sensitized infrared dye emission.In other work, Xu et al. developed a QD-FRET strategy for DNA detection that

incorporated nicking endonuclease (NEase) amplification [235]. An unlabeled probeoligonucleotide was designed to be complementary to the target of interest, whichincorporated a recognition site for NEase. When the target was present, the probeand target hybridized and became a substrate for NEase. The activity of NEasenicked the probe strand and the two resulting fragments dissociated from the target.The target was then available to bind additional probe strands, resulting incyclic nicking that amplified the amount of probe consumed. To complete theassay, NEase was denatured and two reporter strands were added to form a sandwichhybrid with any remaining probe sequences. One reporter was labeled with a CdTeQD540 donor and the other was labeled with a Rhodamine B (RhB) acceptor. TheFRET-sensitized RhB–QD PL ratio served as the analytical signal and was inverselyproportional to the amount of target. The dynamic range of the assay was 0.1–50 nMtarget [235].

12.5.1.4 Bioanalyses Using Aptamers and DNAzymesNucleic acid aptamers are frequently conjugated to QDs as probes for analytes otherthan DNA or RNA. FRET-based detection strategies typically rely on the structure-switching properties of aptamers, which are initially and reversibly hybridized withtheir complements, then preferentially bind their cognate, nonnucleic acid targetthrough complex secondary structures, releasing the complement strand [236]. Ifthe complement is labeled with a suitable acceptor dye, then modulation of FRETcan occur. Levy et al. were the first to demonstrate this strategy with QD-FRET for thedetection of thrombin [160]. The strategy is shown in Figure 12.15. Thrombin-binding aptamer (TBA) was conjugated to QD525 and hybridized with an acceptor-labeled oligonucleotide (in this case, a dark quencher) resulting in efficient FRET.This oligonucleotide was displaced upon the addition of thrombin and binding bythe aptamer, restoring QD PL and providing an analytical signal. Several otherresearchers have used analogous strategies with a variety of other aptamers conju-gated to QDs. For example, Kim et al. paired QD600-aptamer conjugates with a darkquencher for assaying platelet-derived growth factor (PDGF) at concentrationsbetween 0.4–1.6 nM [237]. Similarly, Chen et al. paired QD605 with Cy5 for assayingATP binding [238]. The decrease in the Cy5/QD PL ratio with added ATP providedan analytical signal with linear response from 0.1 to 1.0mM and an LOD of 24mM.In both these examples, acceptor-labeled oligonucleotides were initially hybridizedwith the appropriate aptamer and displaced by binding of the aptamer’s cognatetarget, resulting in the loss of FRET.In a particularly impressive demonstration of the utility of combining aptamers

with QDs and FRET (Figure 12.16), Bagalkot et al. developed a conjugate capable oftargeting cancer cells and signaling drug delivery [239]. QD490 were conjugated


with prostate-specific membrane antigen (PSMA)-binding aptamer and then incu-bated with fluorescent doxorubicin (Dox) to yield a FRET-“ON” state with efficientquenching of the QD PL. The Dox, in addition to being a chemotherapeutic, servedas an acceptor dye for the QD and was itself quenched upon association with theaptamer. The conjugates selectively targeted prostate cancer cells and were taken upvia endocytosis stimulated by binding of extracellular PSMA by the aptamer. BothQD and Dox PL were restored after 1.5 h within the cells, indicating release of theDox, possibly through biodegradation of the aptamer. The cytotoxicity of the Doxtoward the prostate cancer cells was retained using the QD-aptamer conjugate as adelivery vector while, importantly, the cytotoxicity toward healthy cells was subs-tantially decreased [239]. A somewhat similar strategy was adopted by Chi et al. forthe detection of thrombin in vitro [240]. QD565 were conjugated with a stem–loopvariant of TBA and incubated with BOBO-3, a dye that undergoes a progressivefluorescence enhancement between bulk solution, binding with ssDNA, and

Figure 12.15 (a) Design of two-piece aptamerbeacon construct with TBA (black) and quencholigonucleotide (gray). (b) Schematic forrecovery of QD PL when quencher (opencircles) is displaced by thrombin (�). (c) Kinetic

trace of recovery of QD PL with the addition ofthrombin or lysozyme at t¼ 10min. (Adaptedwith permission from Ref. [160]. Copyright2005, Wiley-VCH Verlag GmbH, Weinheim.)


intercalation in dsDNA. FREToccurred between the QD and BOBO-3 intercalated inthe stem of the aptamer. When the aptamer bound thrombin, the stem–loop wasopened and the BOBO-3 was no longer intercalated. The analytical signal was adecrease in BOBO-3 fluorescence, which resulted from weak association with theaptamer–thrombin complex, loss of proximity to the QD, and subsequent loss ofFRET. The effects of any BOBO-3 still bound to the aptamer–thrombin complexwere mitigated by its diminished quantum yield when not intercalated. The LOD ofthe assay was 1 nM [240].In addition to oligonucleotides and aptamers, DNAzymes have also found utility

as probes for QD-FRETsensing configurations. As shown in Figure 12.17, Wu et al.conjugated oligonucleotide substrates for Pb2þ-specific DNAzyme and Cu2þ-spe-cific DNAzyme to QD530 and QD625, respectively, for multiplexed heavy metaldetection [241]. These substrates were hybridized with the corresponding

Figure 12.16 Schematic for (a) FRET-inducedquenching of QD490 PL by intercalation of Doxinto conjugated PSMA-binding aptamer, and(b) cellular uptake and intracellular drug releasewith recovery of QD PL. (c) PL images of PSMA-expressing cells following incubation with QD-

PSMA-binding aptamer–Dox conjugates: (i) 0 hand (ii) 1.5 h. QD PL is indicated in green; DoxPL is indicated in red. The scale bars are 20mm.(Adapted with permission from Ref. [239].Copyright 2007, American Chemical Society.)


apo-DNAzymes and distally labeled with a dark quencher – Iowa Black FQ or RQ forQD530 and QD625 donors, respectively. The apo-DNAzymes were similarly labeledat their proximal termini. This dual acceptor-labeling provided efficient quenchingof QD PL via FRET. In the presence of either Pb2þ or Cu2þ, the hydrolytic activity ofthe cognate metallo-DNAzyme disengaged FRET, restoring the QD PL as ananalytical signal. Both one-plex and two-plex assays were possible with LODs of0.2 nM for Pb2þ and 0.5 nM for Cu2þ [241]. The key to the success of this assay wassuitably coating the QDs that tend to be strongly and irreversibly quenched by Cu2þ

and some othermetal ions. Here, the QDs were coated with�3 nm thick silica shellsprior to conjugation of the substrate/apo-DNAzyme hybrids, effectively protectingthe QD from metal ions in the sample solutions.

12.5.1.5 Bioanalysis of Hydrolytic EnzymesThe detection of protease and nuclease activity using QD-FRET has also been anapplication of interest. In contrast to the aforementioned applications, which havetypically relied on equilibrium binding for the detection of analytes, most examplesof protease or nuclease detection are driven by their catalytic activity – the hydrolysisof peptide or phosphodiester bonds. This response to catalytic activity rather thanconcentration is an important advantage. Proteases, for example, are biosynthesizedas inactive zymogen precursors and/or controlled in vivo by endogenous inhibitors[242]. Analysis methods that respond to protease concentration rather than activitymay not adequately discriminate between active and inactive enzymes. There is acommon design for QD-FRET probes sensitive to proteolysis: peptides incorporat-ing amino acid sequences selectively cleaved by a target protease are labeled with anacceptor and assembled around a central QD donor to yield a FRET-ON state with

Figure 12.17 (a) Schematic of DNAzymestrategy for heavy metal detection using QD-FRET. Each apo-DNAzyme is dual labeled withIowa Black quencher. The addition of Cu2þ orPb2þ induces the hydrolytic activity of thecorresponding DNAzyme, disengages FRET

and restores QD PL. (b) Example of multiplexeddetection using QD530 for Pb2þ and QD625for Cu2þ. (Adapted with permission fromRef. [241]. Copyright 2010, American ChemicalSociety.)


quenched QD PL. Proteolytic activity disengages FRET by cleaving the peptidebetween the distal acceptor and proximal attachment to the QD. The recovery of QDPL and/or loss of FRET-sensitized acceptor PL provide an analytical signal. Some ofthe proteases that have been targeted by QD-FRET probes include caspase-1 [154],caspase-3 [243], chymotrypsin [154], collagenase [154,156], thrombin [154], trypsin[244,245], and botulinum neurotoxin A [246], among others. The design for nucleaseprobes is analogous, utilizing a nucleic acid substrate instead of a peptide.In one seminal report, Medintz et al. assembled a series of dye-labeled peptides to

DHLA-coated QDs and tracked the activity of various proteases [154]. A polyhisti-dine-appended peptide substrate for caspase-1 was labeled with Cy3 and self-assembled to QD540, while similar substrates for thrombin, collagenase, andchymotrypsin were labeled with QXL-520, a dark quencher, and conjugated toQD510/520. The general format is shown in Figure 12.18. In each case, proteaseactivity was signaled by the loss of FRET and quantitative information on velocitieswas extracted through prior calibration of FRET efficiency against the valence ofassembled peptide. This procedure was greatly facilitated, if not uniquely enabled,by the efficiency of polyhistidine-driven self-assembly. Michaelis–Menten kineticparameters – the Michaelis constant, Km, and the turnover number, kcat – were

Figure 12.18 (a) Schematic for modulardesign of peptide substrates in QD-FRET-basedsensing of protease activity. (b) Changes in QDPL intensity as a function of the number of QXLdark quencher-labeled thrombin substrates per

QD520. (c) Plot of quenching and FRETefficiencies for the data in panel B. (Adaptedwith permission from Ref. [154]. Copyright2006, Macmillan Publishers Ltd.: NatureMaterials.)


determined by plotting proteolytic velocities as a function of substrate/enzymeconcentration in excess substrate/enzyme assays. Inhibition constants could bequantitatively measured in analogous assay formats, and the QD-peptide conjugateswere further applied to the screening of potential thrombin inhibitors in amicrotiterplate assay [154]. Almost concurrently, Shi et al. reported peptide-coated QD545 asFRET-based probes for collagenase activity [156]. In contrast to the previous work byMedintz et al. where a maximum of 5–20 peptides were assembled per QD [154], Shiet al. wholesale exchanged hydrophobic TOPO ligands on native QDs with shortRGDC peptides to yield water-soluble QD conjugates that were also proteasesubstrates [156]. The peptides were then labeled with Rhodamine Red (RhR) asa fluorescent acceptor dye. Collagenase activity could be tracked both in vitro and incell culture (see Figure 12.19) through changes in the ratio of QD/RhR PL.In later work, fluorescent proteins were adopted as acceptors for QDs. Suzuki et al.

utilized a green fluorescent protein (GFP) mutant with a trypsin substrate insertionand C-terminal polyhistidine sequence as an acceptor for QD495 [244]. The QDswere modified with a Ni2þ-NTA linker to bind the GFP mutant and enable FRET.This FRET pair was nonideal since the GFP had an emission maximum (510 nm)poorly resolved from the QD PL, and was also nontrivially excited (directly) byexcitation at 390 nm. Trypsin activity was indicated by a subtle �15 nm bath-ochromic shift in the PL spectra rather than an overt change in intensity [244].

Figure 12.19 (a) Schematic of proteasedetection using acceptor (Rh)-labeled, peptide-coated QD545 and FRET. (b) Two color PLimages of HTP126 (collagenase negative) and

HTP125 (collagenase positive) cells at 0 and15min of incubation with the QD probes.(Adapted with permission from Ref. [156].Copyright 2006, American Chemical Society.)


As shown in Figure 12.20, Boeneman et al. prepared an mCherry mutant with acaspase-3 substrate insert and terminal polyhistidine sequence that self-assembledto DHLA-coated QD550 [243]. For this FRETpair, however, there was clear recoveryof QD PL and loss of FRET sensitized mCherry PL with proteolysis. Initial reaction

Figure 12.20 (a) Schematic of fluorescentprotein (mCherry)-peptide linker construct forsensing protease activity (Caspase-3). FRET isdisengaged and QD PL is recovered withincreasing activity. (b) Deconvolved PL spectrashowing quenching of QD PL and increasing

FRET-sensitization of mCherry PL as moremCherry are assembled per QD. (c) Michaelis–Menten plot for caspase-3 activity toward thepeptide-linker substrate (DEVD cleavage site).(Adapted with permission from Ref. [243].Copyright 2009, American Chemical Society.)


velocities in caspase-3 assays were derived from prior calibration of the mCherry/QD PL ratio against the number of mCherry assembled per QD. The obtainedvalues for Km and kcat were comparable to those previously reported for a similarpeptide substrate. Compared to other assay formats, the QD-mCherry conjugaterequired almost an order of magnitude less substrate and three orders ofmagnitude less enzyme. The activity of as little as 20 pM of capase-3 (3.3 units)was detectable [243].Sapsford et al. developed QD-FRET probes for botulinum neurotoxin serotype A

light chain protease (LcA), a potential biothreat agent and food contaminant [246]. ACy3-labeled peptide substrate was designed and optimized starting from therecognition sequence of synaptosomal-associated protein 25 (SNAP-25) that iscleaved by LcA. Single-step and two-step assay formats were explored. In the former,substrate peptides were preassembled, via polyhistidine tails, to QD525 coated withNi2þ-supplemented carboxylated polymer. The conjugates were incubated with LcA,where the loss of QD-Cy3 FRET and recovery of the QD PL intensity were used tomeasure proteolysis. In the two-step format, the substrate peptide was first incu-bated with LcA and then mixed with DHLA or DHLA-PEG-coated QD550. Here,only undigested peptide bound to the inorganic interface of the QD550 and createdthe necessary proximity for FRET. Two steps were needed because the LcA wasunable to hydrolyze the substrate peptide when directly coordinated to the QD550.This result was attributed to steric hindrance between the QD550 and LcA. Incontrast, the success of the one-step assay with QD525 was attributed to goodavailability for the substrate when bound to the periphery of the polymer coating. Aslittle as 0.35 nM of LcA was detectable [246]. Sapsford et al. also applied QD-FRETprobes for protease activity within a microchip-based analytical platform andcompared the results to those obtained in conventional microtiter plate assays[245]. In this study, trypsin was used as the protease. Values for Km and kcatmeasured from the microchip were in general agreement with those measuredusing microtiter plates and had the added benefit of a 12-fold reduction in assayvolume [245].In these examples, the initial number of acceptors per QD was an important

design consideration. Mathematically, the largest change in FRET efficiency occurswhen one and only one acceptor is cleaved from the QD; however, this potentiallylimits the dynamic range of an assay and may be ineffective if the pairwise FRETefficiency is low. In the latter case, multiple acceptors are needed to augment theFRET efficiency and provide good analytical signal. Medintz and coworkers initiallysuggested that, in endpoint assays, the optimum acceptor valence is just prior to theonset of the plateau in a FRET efficiency-acceptor valence curve [154,243,245]. Thischoice represents a compromise that can offer good overall FRET efficiencies,reasonable sensitivities per proteolytic event, and a useful dynamic range. Morerecently, however, it has become clear that better analytical figures of merit can beobtained in kinetic assays that track full progress curves for protease activity and usethe acceptor/donor PL ratio as the analytical signal [247]. The latter parameterplateaus at a much larger number of acceptors per QD and often exhibits somewhatlinear behavior until FRET efficiencies become very high.


Assays for nuclease activity have been developed using strategies analogous tothose for protease activity. Acceptor-labeled, double-stranded oligonucleotides areconjugated to a QD donor and the loss of FRET ismeasured as a function of nucleaseactivity. In two examples demonstrating the detection of DNase activity, Gill et al.paired QD580 donors with Texas Red acceptors [184], while Suzuki et al. [244] pairedeither QD530 or QD565 donors with AlexaFluor 532 (AF532) or AF568 acceptors,respectively. In the case of the latter, it was possible to combine the DNase probewith the aforementioned QD490-GFP probe for trypsin, allowing the simultaneousone-pot detection of nuclease and protease activity [244].

12.5.1.6 Gene DeliveryIn yet another set of applications, the groups of Wang and Leong have used QDs andFRET to investigate the condensation of DNA into polyplexes with cationic poly-mers, as well as the subsequent unpacking of such complexes [191,193,248,249].Polyplexes, which are potential vectors for gene therapy, require careful optimizationso that the complexes neither dissociate prematurely nor exhibit tight binding thatprevents unpacking and gene delivery. To this end, plasmid DNA was biotinylatedand conjugated with SA-QD605. Cationic polymers were labeled with Cy5 so thatFRETwas engaged upon association with the QD-conjugated DNA. Polyplexes thatincorporated chitosan, polyethyleneimine, and polyphosphoramidates were charac-terized in this manner. It was possible to observe intracellular delivery andtrafficking of the polyplexes, and quantitatively measure their unpacking ratesusing optical microscopy, spectroscopy, and single-molecule detection [191,193].In a further study, a FRETrelay was elegantly used to probe two rate-limiting steps ingene delivery: the unpacking of polyplexes and degradation by nuclease activity [249].Plasmid DNA was labeled with Cy3, conjugated with QD525, and then assembledwith Cy5-labeled cationic polymer. The energy transfer relay within the polyplexeswas from the QD (initial donor) to Cy3 (intermediary acceptor/donor) to Cy5 (finalacceptor). Unpacking of the DNA polyplex was indicated by the loss of Cy5 PL andnuclease activity was indicated by the loss of Cy3 PL [249]. This configuration wasapplied intracellularly as shown in Figure 12.21.

12.5.2Changes in Distance to Modulate QD-FRET

The sensitivity of FRET to the donor–acceptor separation is such that conforma-tional changes induced in QD-bioconjugates are often sufficient to change theenergy transfer efficiency. Potential stimuli for conformational changes includeligand binding or other biorecognition events, changes in local environment, anddenaturation/unfolding. In parallel, the r term in Equation 12.9 can be made tochange if the donor and/or acceptor are suitably positioned. Molecular beacons(MBs) are a classic example of this type of design. An MB comprises an oligo-nucleotide hairpin that is labeled at its opposing termini with a donor and acceptor,where the stem–loop structure provides the proximity for FRET. Hybridization ofthe hairpin with its complement yields linear dsDNA and separation of the donor


Figure 12.21 (a) Schematic of two-step QD-FRET for monitoring polyplex unpacking andDNA degradation. E12 and E23 are FRETefficiencies from QD525 to nuclear dye (Cy3),and Cy3 to Cy5. (b) Intracellular analysis ofpolyplex unpacking. The combined imageshows QD, Cy3, and Cy5 PL at 4 hposttransfection (scale bar is 10 mm). The

nucleus is stained blue. FRET efficiencies perpixel were calculated from the individual imagechannels. The purple arrow indicatescondensed DNA; the yellow arrow indicatesunpacked and intact DNA. (Adapted fromRef. [249]. Copyright 2009, with permissionfrom Elsevier.)


and acceptor. The decrease in FRET efficiency and concomitant increase in donoremission provides an analytical signal. Several studies have investigated the effectsof replacing a molecular fluorophore donor with a QD donor. In one example, MBswith 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL) as a quencher wereprepared by coupling 30 DABCYL-modified hairpins to MAA-coated QD490 at their50 terminus [158]. The QD PL intensity increased sixfold with the addition of nucleicacid target and increased minimally with the addition of noncomplementary nucleicacid. For comparison, emission intensity changes close to 10-fold are typical of MBswithmolecular dyes; the smaller change with the QD-MBwas attributed to the largerdonor–acceptor separation imposed in the hairpin by the radius of the QD [158].Nonetheless, the QD-MBs had the advantage of improved photostability, showing nodecrease in PL over 10min of continuous illumination compared to a 15% decreasefor an analogous MB with a 6-carboxyfluorescein (6-FAM) donor. QD-MBs have alsobeen developed using SA-QD515/565/605 and biotinylated Black Hole Quencher-2(BHQ2)-labeled hairpins [250]. There were an estimated 12–15 hairpins per QD andFRET efficiencies were in the range 35–61%. An 80% increase in QD PL intensitywas observed upon the addition of complementary target. Some single nucleotidepolymorphism discrimination was also achieved [250].Highlighting the importance of bioconjugate chemistry, Cady et al. found that the

QD-oligonucleotide linkage strategy influenced the signal-to-background ratioassociated with QD-MBs [251]. Hairpins coupled to carboxylated polymer-coatedQDs through an amide bond offered a 57% larger increase in QD PL upon targethybridization compared to biotinylated hairpins bound to SA-QDs, likely due to thelarger donor–acceptor separation imposed by the SA layer. In another interestingstudy, Chen et al. found that QD800-MB conjugates were much less susceptible tointracellular nuclease degradation [252]. The nuclease degradation of MBs (or othernonspecific interactions) that resulted in false-positive signals was found to occur inthe cell nucleus. In contrast to conventional MBs, the QD-MB conjugates wereexcluded from the nucleus and retained in the cytoplasm where minimal false-positive signals were observed [252]. This allowed the measurement of oncogeneexpression in breast cancer cells. The QDs, however, were not used as part of theFRETpair; they were rather used for their size-based exclusion from the nucleus andfor tracking the fate of conjugated dye-labeled MBs.Although the above studies used dark quenchers as acceptors, QD-MBs with

fluorescent acceptors have also been demonstrated. Medintz et al. used two FRETpairs for this purpose: QD510 with carboxytetramethylrhodamine (TAMRA) orQD590 with Cy5 [177]. The dye-labeled oligonucleotide hairpins were conjugated tothe DHLA-coated QDs through self-assembly following chemical ligation to poly-histidine peptides. Figure 12.22 illustrates the design. Compared to the aforemen-tioned bioconjugate strategies, this chemistry permitted excellent control over thenumber and orientation of hairpins per QD, minimized the donor–acceptorseparation in the hairpin, and did not require purification from unconjugatedQDs/hairpins.Another strategy with nucleic acids is to exploit differences in the rigidity between

ssDNA and dsDNA to modulate donor–acceptor distances. In one example,


Peng et al. used cationic poly(diallyldimethylammonium chloride) (PDDA) to drivethe assembly of Cy3-labeled probe oligonucleotides on MAA-coated CdTe QD500[253]; in another example, Lee et al. modified QD530 with a mixture of cationic andneutral PEG ligands to drive the assembly of TAMRA-labeled oligonucleotides [254].In both cases, hybridization was signaled by a decrease in FRET efficiency, whichwas attributed to the increased rigidity of the probe–target duplex and consequentincrease in donor–acceptor distance.Beyond nucleic acid assays, a unimolecular immunoassay strategy was reported

by Stringer et al. and relied on conformational changes to modulate QD-FRET [255].Human cardiac troponin I (cTnI) was the analyte of interest and QD545 wereconjugated with anti-cTnI using a protein A bridge. The protein A ensured theproper orientation of the anti-cTnI through self-assembly to the Fc region of theantibody. The anti-cTnI was labeled with AlexaFluor 546 (AF546) dye as a FRETacceptor. Upon binding of cTnI, a large conformational change associated with theanti-cTnI significantly decreased the QD-AF546 separation distance. The resultingincrease in FRET-sensitized AF546 emission served as the analytical signal. In cleansamples, cTnI detection in the range of 32–500 nMwas possible; the LODwas 55 nMwith blood plasma as a sample matrix [255].Finally, it is also worth noting that Medintz et al. reported a reagentless, FRET-

based biosensing assembly based on QD-MBP conjugates [256]. The MBP was

Figure 12.22 (a) Schematic showing theassembly and signaling of a QD-FRETmolecular beacon. (b) PL spectra showing theprogressive quenching of QD590 and FRET-sensitization of Cy5 as more hairpins are

assembled per QD. (c) PL spectra showing theresponse of the QD-FRET molecular beacon tocomplementary and noncomplementary DNA.(Adapted with permission from Ref. [177].Copyright 2007, American Chemical Society.)


labeled at an allosteric site with Cy3 and self-assembled to QD510/530 through apolyhistidine tag. The MBP underwent a scissoring-like conformational changeupon binding maltose, changing the local environment of the Cy3 and resulting inquenching. The FRET efficiency and QD PL did not change upon maltose binding;rather, the analytical signal from the conformational change was only associatedwith changes in the quantum yield of the Cy3. Although the MBP-Cy3 wouldfunction in sensing without the QDs, the use with FRETprovided greater flexibilityin the choice of excitation wavelength and, putatively, would permit multiplexingwith a single excitation source if analogous constructs were prepared for otheranalytes using different colors of QDs.

12.5.3Conformational Insights from QD-FRET

Given the well-known “molecular ruler” capability of FRET [257], nanoscale distancemeasurements using QDs were a natural development following their establish-ment as excellent FRETdonors. The first such study was done by Medintz et al., whodetermined the structure of a QD-MBP conjugate through a series of FRETmeasurements [258]. A series of six polyhistidine-appended MBP mutants withcysteine residues at different sites were labeled with RhR-maleimide. TheMBP-RhRwas then assembled to DHLA-coated QD555 at different ratios. The QD donor–RhRacceptor separation distances derived from FRETefficiencies were correlated with insilico structural models based on crystallographic data and used to determine theorientation of the MBP: its C-terminal polyhistidine tag was bound to the surface ofthe QD and its binding site was accessible to bulk solution [258]. This “triangulation”strategy, shown in Figure 12.23, is expected to be applicable to biomolecules otherthan proteins, with the provision that site-specific labeling is feasible. Indeed,changing the location of an acceptor dye label on a biomolecule is a definingfeature of other studies on QD-bioconjugate architecture described in this section.To study the effects of QD conjugation on oligonucleotides, Algar and Krull

modified MPA-coated QD525 with an average of 1–2 probe oligonucleotides usingcarbodiimide coupling [231]. The oligonucleotides were then hybridized with Cy3-labeled complements to generate FRET. The effect of the surface of the QD onoligonucleotide conformation was investigated bymeasuring the FRETefficiency foroligonucleotide probes with two different linker lengths, and by switching the Cy3label between the proximal and distal terminus of the complement. Minimalchanges in FRET efficiency were observed, indicating that the oligonucleotideshad a tendency to adsorb to the surface of the QD. Additional experiments and FRETstudies suggested that the adsorptive interactions were driven by hydrogen bondingbetween the neutral MPA ligands and nucleobases; the extent of adsorption wasaffected by changes in pH, ionic strength, base sequence, and the presence ofchaotropic agents [231,259]. At the opposite extreme, Han et al. developed a methodfor directly functionalizing QD550 with a dense layer of oligonucleotide ligands(�40 per QD) or, alternatively, SA-His6 for modification with biotinylated oligonu-cleotides [260]. To gain insight into the structure of the oligonucleotide layers, the


QDs were hybridized with 5 equivalents of complementary, carboxy-X-rhodamine(ROX)-labeled oligonucleotide. The ROX served as an acceptor for the QD donor andQD-ROX separation distances were determined from the observed FRET efficien-cies. Both sets of results were counterintuitive. For the QD directly functionalizedwith DNA, the calculated QD-ROX distance was longer than expected, based ongeometric estimates, and this result was attributed to repulsion between the densely-packed oligonucleotides, resulting in a fully extended conformation. Conversely, thecalculated QD-ROX separation with the SA-His6 bridge was shorter than bothgeometric predictions and the distance measured for direct functionalization. Thisresult was attributed to a lower density of oligonucleotides, permitting an averageconformation that more closely approached the QD [260]. It is unclear if there may

Figure 12.23 (a) Refined FRET-derivedstructure of MBP assembled to a CdSe/ZnSQD555 via a polyhistidine tag. The various RhRacceptor dye positions used to determine theMBP orientation are highlighted. (b) Space-filling model of the MBP assembled to the QD(Zn atoms in pink; sulfur atoms in teal). The redshell indicates the radius of the DHLA ligands.No visualized structure includes thepolyhistidine tail. (Reprinted with permission

from Ref. [258]. Copyright 2004, NationalAcademy of Sciences, USA) (c) Structuralmodel for one orientation of a biotinylatedoligonucleotide bound to a SA-coated QD605.The purple spheres indicate acceptor dyepositions and flexibility. The inset shows aschematic of the configuration. (Reprinted withpermission from Ref. [197]. Copyright 2010,American Chemical Society.)


have also been a contribution from heterogeneous attachment of the oligonucleo-tides due to the multiple binding sites presented by SA-His6.In another study with DNA, Boeneman et al. compared the architecture of QD-

oligonucleotide conjugates prepared through (i) polyhistidine-oligonucleotidechimera self-assembly to DHLA-PEG-coated QD530, and (ii) binding of biotiny-lated oligonucleotides to commercially available SA-QD605 [197]. Insight into theorientation of the conjugated oligonucleotides was gleaned through a combina-tion of in silico structure modeling and FRET experiments where the position of aCy3 or Cy5 acceptor dye was varied along the length of oligonucleotide hybrids,see Figure 12.23. The polyhistidine conjugation method was found to yieldconjugates where the oligonucleotides were, on average, radially extendedfrom the QD. In contrast, a wide range of possible orientations was determinedfor the biotin-SA conjugation method, and was attributed to heterogeneousattachment of the SA to the QDs [197].It should be noted that all of these studies were done using ensemble measure-

ments. FRET-based molecular ruler experiments at the single molecule or, rather,single-pair FRET level (spFRET), are well known with molecular fluorophores andused to probe conformational dynamics [257]. The utilization of QD donors inspFRET and the potential for measuring conformation dynamics are discussed inSection 12.5.5.

12.5.4Dynamic Modulation of the Spectral Overlap Integral and QD-FRET

While the previous sections have highlighted the use of changes in donor–acceptorproximity and stoichiometry to modulate FRET, it is also possible to modulate FRETwithout altering these parameters. This capability arises from use of an acceptorchromophore that is sensitive to certain changes in microenvironment andresponds by shifting its absorption in and out of resonance with the QD donor.That is, modulating FRET by switching between states of strong and weak spectraloverlap. The insensitivity of high-quality core/shell QDs to changes in micro-environment is often an advantage; however, it also frequently precludes the useof a QD as an optical probe for physicochemical changes in microenvironment. Incontrast, several molecular dyes are known to be good probes for various propertiesof a microenvironment. Although these fluorescent dyes can be used on their own,coupling to a QD via FRET offers several potential advantages. The sensitization ofthe dye fluorescence through FRET from a QD donor widens the spectral range overwhich the dye can be optically interrogated, and further adds the possibility ofefficient two-photon excitation. The latter is particularly appealing in cellular and invivo imaging. Further, FRET sensitization also potentiates ratiometric detection byreferencing the changes in the dye fluorescence intensity to the donor QD PLintensity. As noted previously, ratiometric measurements are quite robust and aresometimes referred to as “self-calibrating,” since the correlation between the PLratio and a physicochemical parameter is largely independent of instrumentalparameters. This is not the case whenmeasuring the intensity of only one PL signal.


One application that is well suited to the modulation of QD-FRET via changes inspectral overlap is pH sensing. Colorimetric pH indicator dyes exhibit a large shift intheir absorption spectrum between conjugate acid and base forms. In a FRETexperiment with fixed donor emission, this shift is often of sufficient magnitude toswitch between strong and weak spectral overlap. For example, Snee et al. directlyconjugated a squaraine (SQR dye; fluorescence peak at 650 nm) to the amphiphilicpolymer coating of QD615 to create a ratiometric pH sensor [261]. The absorption ofthe squaraine dye exhibited a small hypsochromic shift, decrease in absorptioncoefficient, and decrease in fluorescence quantum yield with increases in pH. Incontrast, the QD PL was negligibly affected by the changes in pH. This resulted in aprogressively lower SQR–QD PL ratio as the solution pH became more basic in therange of pH 6–10, see Figure 12.24. Importantly, the pH-dependent ratiometricresponse was insensitive to a decade variation in excitation intensity and changes inexcitation wavelength. Similar designs have used fluorescein conjugated to QD455and QD490 for pH sensing [244,262]. In these cases, the fluorescein exhibited aconcurrent increase in its absorption coefficient and quantum yield as pH becamemore basic in the range of pH 5–8, yielding a FRETenhancement and increase in thefluorescein–QD PL ratio. It is important to recognize that the surface of the QD isnot the same environment as bulk solution – particularly when the QD is stabilizedas a polyelectrolyte (i.e., charged coating). For example, Krooswyk et al. recentlyfound that the pH-dependent interfacial properties of a carboxylated QD contributedto the ionization of fluorescein, as a QD conjugate, over a broader pH range than inbulk solution [263]. This is not necessarily a detriment, but is an importantconsideration in design and optimization.In another example of pH sensing, Dennis et al. assembled pH-sensitive mOrange

fluorescent proteins to QD520 [264]. The absorption spectrumof themOrangewas inresonance with the QD donor at basic pH and out of resonance at acidic pH. ThemOrange/QDPL ratio changed>12-fold between pH 6–8 and 20-fold between pH 5–10. In addition to nativemOrange, aM163Kmutant with elevated pKa (7.9 versus 6.9)

Figure 12.24 (a) Schematic of a CdSe/ZnSQD615 coated with an amphiphilic polymerthat has been modified with a pH-sensitivesquaraine dye. The dye absorption shifts in andout of resonance with the QD PL as a function

of pH. (b) Composite PL spectra showingincreasing FRET as the pH decreases. (Adaptedwith permission from Ref. [261]. Copyright2006, American Chemical Society.)


was also used in QD assemblies to provide better response at higher pH, with theM163K/QDPLratio changing 16-fold betweenpH5–10. The experimentally observedpKa values for the mOrange and M163K QD-FRET sensors were 7.0 and 7.4,respectively. Importantly, the QD-mOrange conjugate exhibited greatly improvedphotostability compared to mOrange alone. In an elegant application, the QD-mOrange probe was modified with a polyarginine sequence to facilitate endosomaluptakebyHeLa cells.As shown inFigure12.25, themOrange/QDPLratiowas tracked

Figure 12.25 (a) Schematic of color changesfor a QD-mOrange pH sensor as it progressesthrough the endocytic pathway. (b) PL images(left to right: QD520 PL, directly excitedmOrange, FRET-sensitized mOrange) ofsensors taken immediately after (t0) and 2 h

postdelivery to cells. There is a decrease inFRET over time as the endosome matures andacidifies. (Reprinted with permission fromRef. [264]. Copyright 2012, American ChemicalSociety.)


over time and permitted observation of the acidification of endocytic vesicles as theyprogressed to early endosomes and late endosomes [264].Beyond pH changes, the absorption (and fluorescence) properties of a dye can also

be changed by redox activity. In one example, Freeman et al. functionalized CdSe/CdS/ZnSQD635 with Nile blue, a nonfluorescent dye that served as a dark quencherfor the QD via FRET [265]. Reduction of the Nile blue caused a shift in its absorptionspectrum and the loss of spectral overlap with the QD donor, disrupting FRET asshown in Figure 12.26. Nicotinamide adenine dinucleotide (NADH) was a suitablereducing agent and this provided the opportunity to monitor the activity of alcoholdehydrogenase, a NADþ/NADH dependent enzyme, in vitro through FRET-modu-lation of the QD PL intensity. The introduction of the QD-Nile blue conjugates to thecellular cytosol via electroporation enabled the measurement of changes in metabo-lism upon stimulation with D-glucose (increase) or taxol (decrease). Although thisformat did not offer the advantage of ratiometric detection, it remained highlyadvantageous in that it adapted the NADþ/NADH sensitivity of Nile blue tofluorescence detection, which is generally the method of choice in cellular studies.

Figure 12.26 (a) Schematic for NADH-sensingusing Nile blue conjugated to QD635. (b) PLimage of HeLa cells with internalized QDs.The scale bar is 5 mm. (c) Time-dependentrecovery of QD PL following the introduction of

(i) D-glucose and (ii) L-glucose. The insets showPL intensity images of cells before and afterD-glucose exposure. (Adapted with permissionfrom Ref. [265]. Copyright 2009, Wiley-VCHVerlag GmbH, Weinheim.)


This capability would not be possible without the use of the QD as a FRET donor.Another advantage of the FRETcoupling was that the favorable optical properties ofthe QD were being interrogated even though the Nile blue functioned as the sensor,thus potentiating facile multiplexing and two-photon excitation in futureapplications.Medintz et al. demonstrated FRET between a QD555 donor and a photochromic

dye acceptor, BIPS [(10,3-dihydro-10-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro-[2H-1-benzopyran-2,20-(2H)-indoline])] [266]. A polyhistidine-mutant of MBP waslabeled with BIPS and assembled to the QD at a ratio of 20 MBP per QD, andfive BIPS per MBP. Under UV illumination, the colorless, UV-absorbing spiropyran(SP) form of BIPS converts to a colored merocyanine (MC) form that has a broadabsorption band centered at�650 nm. The reverse transition, fromMC to SP, can beinduced by illumination with white light (or any wavelength absorbed by the MC).Only the MC form of BIPS has spectral overlap with the QD555 donor and, in thismanner, FRET can be photoswitched between “ON” and “OFF” states. FRETbetween the QD and MC can be measured by excitation at 440 nm, where neitherthe MC form nor SP form absorbs. This flexibility is uniquely enabled by QD-FRET,albeit that energy transfer to the MC form does drive conversion to the SP form[266]. While this study was an excellent proof-of-concept, biological applications ofthis photochromic capability have not emerged. Nonetheless, there may be long-term applications in optical logic and computing.A final study worth noting is a pO2 sensor that comprised a CdxZn1�xSe/

CdyZn1�yS QD548 conjugated with a phosphorescent osmium(II) polypyridylcomplex (OsPP) [267]. The role of the QD was that of a FRET sensitizer, withtransduction via the quenching of the OsPP phosphorescence by dissolved oxygen.Although there was no modulation of the spectral overlap integral, the application isnonetheless similar to those described earlier. The primary advantage of the QDwasthat it provided a 60-fold larger two-photon absorption cross-section than the OsPP.This is another example of how FRET with a QD donor, even when not directlyinvolved in sensing, can improvemeasurement techniques by combining the opticalproperties of QDs with the chemical sensing capability of certain luminescentprobes.

12.5.5Single-Pair QD-FRET

Compared to ensemble FRET measurements, one of the advantages of spFRET isthe ability to detect energy transfer at low ensemble FRETefficiencies. That is, whenthe number of acceptors across an ensemble of donors is less than one per QD. Thiseffect becomes important in QD-FRET assays as the LOD is approached: ensemblemeasurements begin to show negligible quenching of the QD PL and scantsensitization of acceptor PL – a low apparent FRET efficiency. In spFRET, however,each QD is interrogated individually and data from the fraction of FRET-“ON” QDscan be separated from the background of the FRET-“OFF” QDs. The latter fractionmay also include QDs that have an acceptor, but are dark due to blinking.


Consequently, rare and stochastic events, or subpopulations of QDs with heteroge-neous physical or spectroscopic properties, can be observed when they would beotherwise unresolved in the ensemble.While QD-FRET benefits from the use of spFRETmeasurements, QDs synergis-

tically offer several advantages over molecular dyes when used as donors in spFRETexperiments. For example, their brightness improves signal-to-background ratiosand their greater photostability can permit tracking over longer time periods.Blinking can complicate data analysis, but confirms that a single QD is beingmeasured. Further, the narrow PL spectra of QDs can minimize donor cross talk inthe acceptor detection channel, and their broad absorption allows direct excitation ofthe acceptor to be avoided. These advantages notwithstanding, it should be notedthat one of the most prominent applications of spFRET – tracking the stochastic anddynamic conformational changes of single biomolecules – is not widely reportedwith QDs. The preference for this type of experiment is a system with a 1 : 1 : 1 ratioof donor, biomolecule, and acceptor, where the donor and acceptor are labeled atspecific sites on the biomolecule. Such configurations are very difficult to achievewith QD bioconjugates. The large size of a QD is also likely to perturb the nativedynamics of a biomolecule to some degree. Further, full use of FRET as aspectroscopic ruler between 0.5–1.5R0 may be hindered since the radii of mostQDs approach 1R0. Nonetheless, many other spFRETexperiments can benefit fromthe use of QDs.Analogous to other spFRETsystems [268,269], experiments with QD donors tend

to utilize either confocal or total internal reflection fluorescence (TIRF) microscopyfor detection. One strategy is to track the passage of QD-FRETpairs through the focalvolume of a laser excitation source, via either their Brownian motion [181] or flow ofa dilute sample through a capillary [159]. Analytical information is obtained bycomparing the temporal distributions of photon bursts associated with QD donorand dye acceptor PL. These methods minimally perturb bioconjugates andallow high-throughput observation of large numbers of FRET pairs; subpopula-tions and other heterogeneity within macroscopically homogeneous samples canbe resolved [181]. A second strategy is to sparsely immobilize QD-FRETpairs at aninterface to be interrogated in series [270]. At the risk of surface-induced artifacts,this approach permits observation of dynamics that are asynchronous and thusinvisible in larger populations. Analytical information is derived by comparingthe PL intensity trajectories between donor and acceptor channels for singleQD-FRET pairs.One of the first spFRET studies with QD donors was reported by Hohng and Ha

[270]. SA-QD585 were mixed with biotinylated and Cy5-labeled DNA Hollidayjunctions, and then immobilized on a glass substrate treated with biotinylatedbovine serum albumin (biotin-BSA). A broad range of FRET efficiencies wasextracted from the measurement of many immobilized QD–DNA-Cy5 pairs. Lessthan 1% of the QDs yielded analyzable traces due to the large size of SA-QDs (i.e.,low FRET efficiency) and the need to work at substoichiometric QD :DNA ratios toensure only one Holliday junction per QD. Nonetheless, it was possible to observeanticorrelation between the QD and Cy5 PL trajectories for many of the immobilized


Figure 12.27 (a) Schematic of QD540-MBP-RhR (acceptor) conjugates. (b) spFRETdetection setup. (c) Example of QD and RhRbursts. Coincident signals with a combinedintensity above the threshold level are selectedfor analysis (arrows). Experimental emissionratio distributions compared with Poisson

distribution predictions: (d) N¼ 0.5 MBP-RhRper QD, (e) N¼ 4 MBP-RhR per QD, and(f) N¼ 4 MBP-Cy5 per QD. The Cy5 is a pooracceptor for the QD, as reflected in theemission ratio distribution. (Adapted withpermission from Ref. [181]. Copyright 2006,American Chemical Society.)


QDs, including the Holliday junction switching between two different conforma-tions [270]. The data were validated by repeating the experiment with a moreclassical Cy3–Cy5 FRET pair.In another early study, Pons et al. utilized solution-phase measurements to

investigate spFRET with QD donors [181]. An analysis of QD540 assembled withdifferent numbers of RhR-labeled MBP demonstrated that FRET efficiencies anddonor–acceptor separations derived from ensemble and spFRET measurementswere consistent with one another. The spFRET efficiency was derived from theemission ratio, g, according to Equation 12.12, where E(n) is the FRETefficiency forn acceptors, IA and ID are the burst signals for the donor (D) and acceptor (A),WD(A)

is a donor (acceptor) quantum yield, and sD(A) is the excitation cross-section fordonor (acceptor) at the laser wavelength. The s terms accounted for any directexcitation of the acceptor. Importantly, the spFRET experiments also resolveddifferent subpopulations for the QD-MBP conjugates, (see Figure 12.27 for exam-ples). It was confirmed that macroscopically homogeneous samples prepared withNMBP per QD actually comprised a heterogeneous system where the real number ofMBP per QD, n, followed a Poisson probability (p) distribution according toEquation 12.13. Furthermore, when a reagentless, allosteric QD-MBP-Cy3 sensorfor maltose (see Section 12.5.2, Ref. [256]) was tested with spFRET, it was possible todifferentiate the fractions of QD-MBP conjugates with and without bound maltoseusing the emission ratio [181].

g ¼ IAIA þ ID

¼ EðnÞ þ nsA=sD

WD=WAð Þ 1� EðnÞ½ þ EðnÞ þ nsA=sD: ð12:12Þ

pðN; nÞ ¼ Nn

n!exp ð�NÞ: ð12:13Þ

Given the above results, FRET efficiencies derived from ensemble measurementscan be corrected for the effects of the Poisson distribution using Equation 12.14 [181].Unfortunately, this equation cannot be solved analytically for n, and ensemble FRETefficiencies plotted as a functionofN,E(N), cannot bedirectlyfitted to return values forr or E(n). The correctionmust usually be derived using numerical methods or customfitting algorithms. IfR0 is known, the predicted ensemble E(N) curve can be calculatedfor arbitrary values of r using Equations 12.9 and 12.14 (a¼ n) and compared to theexperimental data. The residuals between the predicted values and experimentalresults can be minimized by using an iterative algorithm to scan through differentvalues of r. The best fit provides Poisson corrected values for r and E(n). Figure 12.28shows an example of the difference between measured and corrected values of E(n).Without the correction, themeasured efficiencies are biased to lower values due to thefraction of QDs with n¼ 0 in the Poisson distribution. As a consequence, r would beoverestimated if Equation 12.9 were applied.

Eðn NÞ ¼XNmax

n¼0

pðN; nÞEðnÞ: ð12:14Þ


Considering sensing applications, the seminal contribution for spFRET was agenetic analysis reported by Zhang et al. [159]. This report was also the first exampleof DNA detection using QDs and FRET. SA-QD605 were used as a scaffold toassemble sandwiched DNA hybrids that comprised a biotinylated capture probe, aCy5-labeled reporter probe, and the target nucleic acid sequence. In this design, theproximity needed for FRET between the QD and Cy5 was mediated by probe/target/reporter hybridization, which occurred prior to the addition of the SA-QDs.Analytical information was derived from PL bursts recorded during capillaryflow of the sample solution through the detection volume, as shown in Figure 12.29.Coinciding bursts of QD and Cy5 PL above minimum threshold values wereindicative of a QD with bound target DNA. An LOD of 4.8 fM was obtained andwas �100-fold better than dye-based MBs. The utility of the assay was furtherdemonstrated in an oligonucleotide ligation assay to detect point mutations inclinical samples [159]. This same group later used QD-probe bioconjugates andFRET as a quantitative detection method for methylation-specific polymerase chainreaction (MSP) assays [271]. In this case, Cy5-labeled primers were used to generatedirectly labeled targets through MSP. Compared to conventional methods, QD-FRET offered single-step detection and only required as few as eight cycles of MSP(cf. 40 cycles with other methods). The simultaneous detection of two methylatedsequences was possible using two different FRET pairs with a common donor:QD585 with either AF594 or Cy5. Each acceptor dye was associated with differentprobe and primer sequences.Following the above work, Zhang and Johnson continued to develop capillary flow

spFRETmethods for bioanalysis with QD donors [272–275]. For example, a cocainesensor was developed using SA-QD605 conjugated with cocaine-binding aptamer[273]. In the absence of cocaine, a short Cy5-labeled oligonucleotide was hybridizedto the aptamer sequence and provided the proximity needed for FRET. Analogous tothe structure switching described previously, the Cy5 was displaced when theaptamer preferentially bound cocaine present in the samples. The loss of FRET

Figure 12.28 Representative experimental data showing the difference between the measuredensemble FRET efficiency and the values corrected for heterogeneity due to a Poisson distributionof acceptors per QD.


afforded an analytical signal and could be used to detect cocaine with a LOD of0.5mM. However, rather than working with only a FRET-“OFF” configuration, aFRET-“ON” configuration was also demonstrated. The FRET-“ON” configurationwas tripartite and comprised a QD donor, Cy5, and a dark quencher. The latter hadbroad absorption and served as an acceptor for the QD, either via direct energytransfer or an intermediate FRET relay through the Cy5. Cocaine binding displacedthe dark quencher, restoring FRET-sensitized Cy5 PL as the analytical signal. Inother work, Zhang et al. developed a model assay for measuring the interactionbetween the human immunodeficiency virus (HIV) regulatory protein, Rev, and theRev responsive element (RRE) within the env gene of the HIV-1 RNA genome[274,275]. The in vitro model comprised a Cy5-labeled peptide sequence derivedfrom the Rev protein, and a biotinylated stem–loop IIB ribonucleotide sequence ofRRE conjugated with SA-QD605. These peptide and ribonucleotide sequences areresponsible for binding between the corresponding native biomolecules. FRET-sensitized Cy5 PL provided an analytical signal proportional to Rev-peptide binding,enabling the measurement of dissociation constants and inhibition of Rev-RRE

Figure 12.29 QD-spFRET as a DNAhybridization assay. (a) Positive for target DNA:(i) nanoassembly; (ii) QD605 bursts; and(iii) Cy5 bursts. (b) Negative for target DNA:

(i) unassembled components; (ii) QD605bursts; and (iii) Cy5 bursts. (Adapted withpermission from Ref. [159]. Copyright 2005,Macmillan Publishers Ltd.: Nature Materials.)


binding by proflavin or neomycin B. Measurement via spFRETpermitted resolutionof the QD-Cy5 FRET against a large background of proflavin fluorescence, withoutthe spectral deconvolution that would have been necessary in ensemblemeasurements.Recently, Zhang et al. revisited the sandwich hybridization assay format originally

used for DNA detection with QD-spFRETand developed new capabilities [276,277].In one study, the capillary flow spFRETmethod was adapted for two-plex detection[276]. Two different biotinylated probe oligonucleotide sequences were used incombination with two different reporters: one reporter was labeled with AlexaFluor488 (AF488) and completed a sandwich hybrid with a target DNA sequence derivedfrom the HIV-1 gene; the other reporter was labeled with AF647 and completed asandwich hybrid with a target sequence derived from the HIV-2 gene. Afterassembly of the hybrids to QD605, coinciding bursts of QD PL with directly excitedAF488 PL and/or FRET-sensitized AF647 PL provided the two analytical signals. Theexcitation wavelength was 488 nm. In a second study, the capillary QD-spFRETmethod was applied to address the challenge posed by the short length of microRNA(miRNA) for PCR amplification [277]. Cy5-labeled reporter oligonucleotides werepaired with SA-QD605 for FRET, and biotinylated capture probes were used tocomplete a sandwich hybrid with DNA targets generated from themiRNA sequenceof interest through isothermal exponential amplification reaction (EXPAR). TheLOD (with amplification) was 0.1 aM and single-nucleotide differences betweensequences in the let-7 miRNA family were discriminated.A final example of interest is the observation of enzyme turnover using QD-

spFRET. Enzymes typically have in vitro Km values in the micromolar to millimolarrange; however, fluorescently labeled enzyme substrates at this concentration tendto yield prohibitively high background PL for single-molecule fluorescence spec-troscopy. As shown in Figure 12.30, Sugawa et al. addressed this limitation byadopting QD-spFRET [278]. Recombinant humanmyosin Va (an ATPase) with a Lys-to-Cys mutation was first labeled at that site with peptide-coated QD520 using aheterobifunctional cross-linker and then nonspecifically immobilized on a glasssubstrate. Cy3-labeled ATP was introduced and its binding to a single QD-ATPaseconjugate was observed through FRET-sensitized Cy3 PL. The flexibility in excitationwavelength for the QD permitted almost complete suppression of directly excitedfluorescence from the excess Cy3-ATP. Furthermore, the photostability of the QDpermitted the reaction kinetics at an individual enzyme to be tracked over severalminutes. Off-rates were derived from the anticorrelation of the QD and Cy3 PLtrajectories, and reflected both substrate turnover and dissociation from the enzymewithout catalysis [278]. The experiments were done at millimolar levels of dithio-threitol (DTT) to reduce blinking of the QDs [106,278].

12.5.6Solid-Phase QD-FRET

In a solid-phase QD-FRET system, QD donors are immobilized at an interface andassembled with dye acceptors. At low densities of QDs, these systems largely mimic


solution-phase QD-FRET pairs. At higher densities, it becomes difficult to definediscrete FRETpairs since each acceptor dye potentially interacts with more than oneimmobilized QD donor. These latter systems are more complex than FRETpairs inbulk solution, and require more sophisticated models for their photophysicalbehavior and FRET efficiency. Nonetheless, high-density solid-phase QD-FRETconfigurations are amenable to biosensing and other applications. Overall, solid-phase QD-FRET systems are much less commonly employed than solution-phaseQD-FRET systems, likely due to the extensive interest in using QDs as intracellularprobes, as well as the additional chemistry required. The latter includes developingan effective method for the immobilization of QDs. Given the strong distance

Figure 12.30 (a) Recombinant human myosinVa (an ATPase) immobilized on a glass slidewith FRET-sensitization of bound Cy3-ATP (pinkcircle) against a background of diffusing Cy3-ATP (gray circles). (b) Histogram of off-rates forATPase-Cy3-ATP binding. The pink curverepresents binding; the blue curve is

background. Representative trajectories for QDand Cy3 PL (c) without and (d) with ATPase (S1-K270C). Note the switching between FRET-“ON” and “OFF” states in panel (d). (Adaptedwith permission from Ref. [278]. Copyright2010, Wiley-VCH Verlag GmbH, Weinheim.)


dependence of FRET, the immobilization of a multilayer of QDs is nonideal. Sincetypical F€orster distances are comparable to the diameter of a QD and its coating, onlythe topmost layer of QDs in amultilayer structure can efficiently participate in FRET.Subsurface layers of QDs add background donor PL to the system without signifi-cantly enhancing energy transfer rates. An approximatemonolayer or submonolayerof immobilized QDs is therefore desirable, and three primary methods have beenreported for the assembly of these thin films: biomolecular tethers, chemicalconjugation reactions between an interface and the coating on the QD, andinterfacial ligand exchange. These strategies are listed in the order from least tomost intimate contact between the bulk interface and QD. Electrostatic binding ofQDs to an interface is the fourth strategy for immobilization; however, thistechnique is more commonly used to generate multilayer structures throughlayer-by-layer assembly. Examples of applications for each of these immobilizationmethods are described in the following subsections.

12.5.6.1 Biomolecular Surface TethersSapsford et al. were the first to use biomolecular tethers for the assembly of solid-phase QD-FRET systems [205,279]. In one study, a NeutrAvidin (NA)-modifiedsurface was prepared by chemically cross-linking the protein to a glass slide thatwas first modified with a thin film of mercaptosilane. Either biotinylated MBP orbiotinylated IgG (>20 biotins per protein) was bound to this layer as a tether,followed by QD555 conjugated with both MBP and Avidin as a platform forpotential QD-FRET applications. The estimated distances between the bulkinterface and the QD were 33 nm and 40 nm for the biotinylated MBP and theIgG surface tethers, respectively [279]. Subsequent work, shown in Figure 12.31,replaced the protein tethers with a more robust, rod-like ß-strand peptide [205].The C-terminus of the peptide was biotinylated to permit immobilization onto aNA-modified glass slide, and a distal N-terminal hexahistidine motif was used tobind and immobilize DHLA-coated QDs. Polyhistidine-tagged MBP with a Cy3label at an allosteric site was then assembled to immobilized QD510/530/590,enabling a reagentless, solid-phase FRETassay for maltose [205], albeit with lowersensitivity than the analogous solution-phase assay [256]. An important differencebetween the discussed immobilization strategies was that the biotinylated proteintether required conjugation of the QD with Avidin and MBP prior toimmobilization, whereas the polyhistidine-peptide tether allowed theimmobilization of unconjugated QDs with subsequent attachment of MBP.The capacity for solid-phase bioconjugation, such as in the latter strategy, cangreatly facilitate purification from excess biomolecules. The potential dis-advantage, from the standpoint of FRET, is that the conjugate valence is neithereasily controlled nor conveniently measured.Beyond proteins and peptides, Chen et al. used dsDNA as a surface tether for the

immobilization of QDs [280]. Amine-terminated oligonucleotides were immobi-lized on an epoxysilane-modified glass slide and were complementary to a thiol-terminated oligonucleotide sequence that was self-assembled to MPA-coatedQD525. A second thiol-terminated oligonucleotide sequence was similarly


conjugated to the QDs and served as a probe for a nucleic acid target of interest. Thetarget was labeled with a Cy3 acceptor, and FRET provided the means for detectionupon hybridization at the DNA-tethered QD film. Both the immobilization of theQD-oligonucleotide conjugates and subsequent hybridization assay were donewithin a microfluidic channel. This format enabled a 15-fold decrease in assaytime and 1000-fold decrease in sample volume compared to a similar assay on anoptical fiber platform, albeit at the expense of lower sensitivity [280]. Further,exploiting the selectivity of Watson–Crick base-pairing in this immobilizationstrategy could potentially offer programmable immobilization of QDs to differentsites. Chen et al. also demonstrated that the surface could be regenerated and usedfor multiple cycles of QD immobilization by denaturing the dsDNA tether usingchaotropic reagents [280]. The same group later developed another microfluidic,solid-phase QD-FRET hybridization assay; this time using a biotinylated surface forthe immobilization of QDs [281]. SA-QD520 were immobilized by injection into themicrofluidic channel and then further conjugated with biotinylated probe oligonu-cleotides in channel. In assays with Cy3-labeled target sequences, the hybridizationkinetics were so fast that the solution-phase plug of injected targets was depletedbefore reaching the end of the microfluidic channel. As shown in Figure 12.32, theresult was two discrete regions along the channel length: an initial region thatexhibited strong FRET-sensitized Cy3 PL, and another that exhibited unquenchedQD PL. The length of the former region was proportional to the amount of targetdetected and allowed quantitation on the basis of a length measurement rather thanonly the Cy3/QD PL ratio [281].

Figure 12.31 Schematic of surface-immobilized, QD-FRET maltose sensor. Thelinker is a biotinylated YEHK peptide (Bt-YEHK7). The MBP is labeled at an allosteric site

with Cy3. Due to a conformational change, theCy3 is quenched when MBP binds maltose.(Adapted with permission from Ref. [205].Copyright 2006, American Chemical Society.)


12.5.6.2 Chemical Conjugation to an InterfaceIn one example of covalent conjugation to an interface, Qi et al. devised a solid-phasesystem that used both FRET and photocurrents to detect nucleic acid hybridization[282]. Using carbodiimide chemistry, carboxyl-coated QD590 were covalentlycoupled to a 3-aminopropyltriethoxysilane (APTES) film between two electrodeson a SiO2 substrate. The immobilized QDs were cross-linked with Cy5-labeleddsDNA (each strand having an amine-terminated linker) to generate two analyticalsignals: (i) FRET-sensitized Cy5 PL and (ii) a significant photocurrent between thetwo electrodes. An increase in the photocurrent was characteristic of a fullycomplementary dsDNA cross-linker, while no significant photocurrent wasobserved for a mismatched dsDNA cross-linker. Although not fully developedinto an assay, this study highlighted the potential advantage of multimodal detectionin identifying false-positive signals from nonspecific adsorption, or discriminatingbetween target andmismatched sequences without the need for thermal or chemicaldestabilization.Solid-phase QD-FRET assays for protease activity have also been developed using

covalent conjugationofQDs to an interface.Kim et al. immobilizedspotsofSA-QD525

Figure 12.32 Microfluidic solid-phase QD-FRET hybridization assay. (a) PL images ofFRET-sensitized Cy3 PL, QD PL, PL merge, andcorresponding Cy3 and QD channel intensityprofiles (Cy3: orange, QD: green) for theinjection of (i) 28.5, (ii) 19.0, and (iii) 9.5 fmol

of Cy3-labeled target DNA. (b) In-channel Cy3/QD PL ratio profiles for the data from panels(a)–(c). (c) Channel length covered by FRET-sensitized Cy3 PL as a function of the amountof target injected. (Reprinted from Ref. [281].Copyright 2012, American Chemical Society.)


on an N-hydroxysuccinimide-activated hydrogel glass slide [283,284]. The QDs werethen conjugated with biotinylated peptide substrates for matrix metalloproteinase-7(MMP-7) [284]. The peptides were labeled with TAMRA as a fluorescent acceptor dyeand the analytical parameter was the QD/TAMRA PL ratio, which increased withgreater proteolytic activity. The analysis time was 1–2h, the dynamic range wasapproximately three orders of magnitude, and the LOD was 100ng/ml.

12.5.6.3 Interfacial Ligand ExchangeInterfacial ligand exchange was developed by Algar and Krull for the facile andreproducible immobilization of QDs for solid-phase FRET applications [285,286].This chemistry relied on modification of a glass or silica substrate with a highdensity of bidentate or tetradentate thiol linkers through grafting onto an amino-silane film. Ligand-coated CdSe/ZnS QDs were spontaneously immobilized at thisinterface, via the thiol interaction with the ZnS shell, as an approximate monolayer.These thin films formed the basis for a series of nucleic acid hybridization assays onoptical fibers or small glass beads. In one assay, a QD530 monolayer was modifiedwith thiol-terminated oligonucleotide probes [287], while in another a QD620monolayer was modified with dithiol-terminated probes [286]. The target nucleicacid sequences, which were directly labeled with Cy3 or indirectly labeled withAF647 as a sandwich hybrid, were detected through the Cy3/QD530 and AF647/QD620 PL ratios, respectively. The LODs for these assays were in the range of2–5 nM without any PCR amplification of target.In another set of hybridization assays on optical fibers, illustrated in Fig-

ure 12.33, the QD monolayer was coated with an adsorbed layer of NA, modifiedwith biotinylated oligonucleotide probes, and further passivated with BSA.Importantly, this interfacial chemistry was developed for multiplexed assays.In one configuration, a mixed film of QD530 and QD620 was immobilizedand conjugated with two biotinylated probe oligonucleotide sequences [288].The corresponding nucleic acid targets were labeled with Cy3 and AF647, whichserved as acceptors for the QD520 and QD620, respectively. The contributions ofthe individual emitters to the composite PL spectrum were deconvolved using asimple algorithm that fit the data with a linear combination of the spectra for eachemitter. In a second configuration, a thin film of only QD530 was modified withtwo different oligonucleotide probes [289]. The two target nucleic acids werelabeled with either Cy3 or RhR through sandwich hybridization. FRET-sensitizationof these dyes provided the analytical signals, which were again measured as PLratios after spectral deconvolution. In this case, the emissionof one acceptor (e.g., Cy3)was the numerator and the combined emission of the QD donor and other acceptor(e.g., QD and RhR) was the denominator. The latter provided an empiricallyuseful, albeit partial, correction for the two energy transfer pathways with acommon donor. The one-donor–two-acceptor configuration was enabled by thesimilar absorption spectra of the Cy3 and RhR (both provided good spectral overlapwith the QD donor) and their partially resolved fluorescence spectra. The LODs forthese assays were between 1–10nM and the discrimination of single base-pairmismatches was possible.


Both of the aforementioned two-plex assay configurations were extended to three-plex assays by incorporating a detection channel based on the direct excitation offluorescence from Pacific Blue (PB) [290]. This dye, which was efficiently excited atthe same wavelength used to excite the QDs (405 nm), had blue fluorescence atshorter wavelengths and resolved from that of the QD530. The ability to resolve asingle base-pair mismatch was retained even in the three-plex format. A FRET-only

Figure 12.33 (a) Schematic of a multiplexed,solid-phase QD-FRET nucleic acid hybridizationassay utilizing an optical fiber and evanescentwave excitation. Different DNA targets andacceptor dyes are indicated in different colors:blue¼Pacific Blue (PB; direct excitation);yellow¼Cy3; orange¼RhR; and red¼AF647.The labeled gQD and rQD correspond toQD530 and QD620, respectively. (b) Differentspectroscopic configurations: (i) two-plex,

QD520-Cy3, QD620-AF647; (ii) two-plex,QD520-Cy3/RhR; (iii) three-plex, PB, QD520-Cy3, QD620-AF647; (iv) three-plex, PB, QD520-Cy3/RhR; (v) three-plex, QD520-Cy3/RhR,QD620-AF647; (vi) four-plex, PB, QD520-Cy3/RhR, QD620-AF647. (Reprinted from Ref. [291]with kind permission from SpringerScienceþBusiness Media. Copyright 2010,Springer-Verlag.)


three-plex configuration was subsequently demonstrated using a mixed film ofQD530 and QD620 donors with Cy3, RhR, and AF647 acceptors; the addition ofPacific Blue then potentiated a four-plex hybridization assay [291]. These and theaforementioned multiplexed configurations are summarized in Figure 12.33.These studies by Algar and Krull [286–291] demonstrated that it was possible to

build multiplexed assays around thin films of coimmobilized QD donors and anoverlying layer of acceptor dyes, without discrete and well-defined FRET pairs. Thespectral properties and resonance of donor–acceptor pairs was relied upon to sortout the analytical information. The drawback of the approach was the fixed numberof hybridization sites, which were divided among all the oligonucleotide probesequences, decreasing the maximum number of a given acceptor at the interface asthe degree of multiplexing increased. This effect decreased themaximum interfacialFRETsignal for a given target, although the signal per bound acceptor was expectedto be unchanged. Further, at low target concentrations, FRET-sensitized acceptorshad to be resolved on a large background of donor PL that resulted from themultitude of immobilized QD donors. This challenge equally applies to solution-phase QD-FRET assays, but can be easily avoided by lowering the concentration ofQD donors. In the solid-phase format, this background had to be mitigated byminimizing the active area of the solid-phase assay [286]. A strategy shown to beeffective for addressing both these limitations was the association of multipleacceptor dyes with each hybridization event, for example, by dual labeling a reporteroligonucleotide in a sandwich assay [286]. This approach was suggested to createadditional energy transfer pathways for each donor, and also increase the fraction ofdonors that were able to participate in FRET by reducing the competition foravailable acceptors. Cumulatively, the strategy of using mixed films of immobilizedQD donors and FRET-based detection offered several advantages. In particular, one-pot preparation of a multiplexed assay that uses a single excitation source, withoutthe need for discrete sensor elements or spatial registration of probes, is highlyamenable to developingminiaturized and chip-based diagnostics. The advantages ofratiometric detection are also inherent in this strategy, and the ensemble compati-bility and simple spectral readout ensure technical simplicity.

12.5.6.4 Electrostatic ImmobilizationTran et al. reported one of the earliest examples of immobilized QDs for FRET-basedassay development [292]. Negatively charged DHLA-coated QDs were immobilizedon positively charged, poly-L-lysine-coated glass slides using electrostatic assembly.Dark quencher-labeled antibodies were conjugated to the immobilized QDs throughelectrostatic attraction mediated by a dimeric and bifunctional protein G-basiczipper molecular adapter that had high affinity for the Fc region of IgG. Theimmobilized QD PL decreased with increasing immobilization of the quencher-labeled IgG. A full immunoassay was not developed in this work although there isclearly such potential.Feng et al. immobilized multiple colors of QD using a layer-by-layer strategy

to develop a solid-phase hybridization assay [293,294]. A series of anionicQD560/595/615 were immobilized inside a porous anodic aluminum oxide


template using intervening polycationic dendrimer layers. This arrangement cre-ated an energy transfer relay where the excitation energy of the outer layer of QD560was transferred along the band-gap gradient to the inner layer of QD615. This innerlayer was functionalized with oligonucleotide probes, and hybridization with Cy5-labeled target sequences added an additional step to the energy transfer relay. TheFRET-sensitized Cy5 PL provided an analytical signal with a subnanomolar LOD. Agradient of QD colors/sizes is perhaps the only multilayer architecture that isamenable to efficient solid-phase QD-FRET with a terminal dye acceptor boundthrough biomolecular components.

12.5.6.5 Advantages of Immobilized QDsThere are several benefits inherent to utilizing a solid-phase QD-FRET system.Analytically, the immobilization of QDs at an interface permits access to near-fieldinterrogation techniques such as evanescent wave spectroscopy [287,288] andwhispering gallery modes [295], as well as access to plasmonic [296,297] or photoniccrystal enhancements of QD PL [298]. Synthetically, immobilization affords the useof smaller amounts of material, provides the ability to wash away excess reagentsfrom bioconjugate reactions (cf. dialysis, gel permeation or affinity chromatography,centrifugation, or electrophoresis), and can expand the range of reaction conditionssuitable for bioconjugation. Considering the latter, thioalkyl acid ligands are themost widely available and easily implemented coatings for QDs, but are prone toaggregation at low pH, high ionic strength, or in nonaqueous solvents. Theimmobilization of QDs largely negates these limitations and thus provides newpossibilities for optimizing bioconjugate chemistry. In assays, complex samplematrices can be washed away prior to measurements, there is potential forregeneration and reuse of the immobilized QD-bioconjugates over multiple cyclesof use [286,287], and high-stringency conditions can be used when they wouldotherwise compromise the colloidal stability of solution-phase QDs.A final consideration is the impact of a high-density array of QD donors and/or

acceptors on the net rate of energy transfer. The rate of energy transfer betweendonor and acceptor in a discrete pair is proportional to the inverse sixth power oftheir separation distance, r�6. If the acceptor is replaced with an infinite, one-dimensional line of acceptors, the net rate of energy transfer is proportional to theinverse fifth power of the perpendicular distance between the donor and acceptorarray, h�5. Similarly, the net energy transfer rates scale with the inverse fourth orthird powers of distance, h�4 and h�3, with an infinite plane or infinite crystal ofacceptors, respectively [211]. Pragmatically, “infinite” simply refers to dimensional-ity that is much larger than the range of dipole–dipole interactions; a useful estimateis approaching 102 nm. These changes in the net rate of energy transfer are notmodifications of the F€orster formalism, but rather the result of integrating over avery large number of acceptors. Section 12.6 will describe the use of QDs asacceptors, and multidimensional acceptor architectures of the type in Figure 12.34lend themselves to the interfacial monolayers andmultilayers of QDs. Of course, thereal distance scaling for the energy transfer rate will be less favorable than d�4, sincethe QDs will not be point acceptors (i.e., d is comparable to the distance between


close-packed QD centers); however, an enhancement of energy transfer rates is stillexpected. Unfortunately, a biologically relevant solid-phase FRET system with QDacceptors has yet to be reported.The solid-phase QD-FRET systems reported to date frequently utilize a two-

dimensional array of QD donors with an overlying array of acceptors. The simplestmodel for this type of configuration is a single acceptor at perpendicular distance habove a plane of hexagonally packed QD donors. As discussed in Section 12.4.3,multiple donors per acceptor are expected to change the net range of energy transferto the acceptor. Figure 12.35 shows simple simulations for PA, the probability ofenergy transfer to the acceptor, based on Equation 12.11 with close-packed arrays ofdifferently sized QD donors and three different F€orster distances. The simulationdoes not account for the possibility of multiple energy transfer events for the sameacceptor. Clearly, PA increases and energy transfer extends to larger values of h as thedensity of QD donors increases. The enhancement above a single donor–singleacceptor FRET pair is most dramatic when the F€orster distance is larger than thediameter of QDs. In addition to the advantages described earlier, solid-phase QD-FRET configurations should be considered for developing assays involving energytransfer over long distances, such as when the biomolecular probe is very large (e.g.,antibody).

12.5.7Photodynamic Therapy

Photodynamic therapy (PDT) involves irradiation of a photosensitizer (PS), whichhas accumulated within malignant tissues, to generate a sensitized cyotoxic effectthat results in cell death. In the type I mechanism, excitation of the PS produceshydroxyl radicals (�OH), superoxide (�O2

�), hydrogen peroxide (H2O2), or other

Figure 12.34 (a) Discrete FRET pair consistingof a single donor, D, and a single acceptor, A,separated by distance, d. The energy transferrate, kFRET, scales as the inverse sixth powerof d. (b) Multiple FRET interactions in a systemwith a single donor at a distance h above a two-dimensional plane of acceptors (only one

dimension is shown). The energy transfer ratescales as the inverse fourth power of h.(c) Multiple FRET interactions in a system witha single donor at a distance h above a three-dimensional array of acceptors (only twodimensions shown). The energy transfer ratescales as the inverse third power of h.


damaging free radicals by electron transfer or hydrogen abstraction [299,300]. In themore common type II mechanism, energy transfer from the PS (excited triplet state)to ground state triplet oxygen (3O2) yields singlet oxygen (1O2) [299,300]. Bothmechanisms cause irreversible damage to biomolecules, cellular structures, andorganelles via the free radicals or singlet oxygen produced. The singular advantage ofPDT is that both accumulation of the PS and subsequent irradiation are needed toinduce toxicity, thereby permitting a therapeutic effect in tumor tissue whileminimizing harmful systemic effects. The most common PS agents are porphyrins,chlorins, and phthalocyanines, although some other nonmacrocyclic chromophoresare also useful [299,301,302]. One example of the former is Photofrin1 (porfimersodium) – a mixture of oligomeric hematoporphyrin derivatives that is clinically

Figure 12.35 (a) Top view of simulatedmonolayers of QD donors of different sizes in aclose-packed arrangement, and the acceptorcentered above the monolayer at aperpendicular distance h. The monolayerdimensions are approximately 60� 60 nm.

(b) Calculated PA, Equation 12.11, versus hcurves (solid lines) for the monolayers in panelA, at different values of R0: 3 nm (blue), 4 nm(green), and 5 nm (red). The correspondingcurves for a discrete donor–acceptor pair areshown for reference (dashed lines).


approved to treat certain types of cancers and which is activated by �630 nmlaser light [299]. In general, the ideal PS will have several characteristic features:(i) a well-defined chemical composition; (ii) no toxicity in the absence of irradiation;(iii) targeted and selective delivery to tumor tissue; (iv) rapid clearance from the body;(v) water solubility; (vi) efficient energy transfer to molecular oxygen; (vii) efficientexcitation in the near-infrared therapeutic window (�700–900 nm); and(viii) excellent photostability (in a type II mechanism) [299,300,303]. Unfortunately,no current PS satisfies all of these criteria. It has been suggested that the physicaland optical properties of QDsmay be able to address some of the current limitationsof PDT.One limitation of many macrocyclic PS drugs is low solubility and a tendency

toward aggregation with a reduction in photochemical activity and cell penetration[299]. Limited photostability, a typically narrow band of optimal excitation in thevisible region of the spectrum (> 700 nm), and limited mechanisms for targeteddelivery are also potential hindrances to the greater efficacy of many PS candidates.Given that the surface coating of QDs can be chemically tailored to support goodaqueous solubility, targeting ligands, and the conjugation of diverse molecules, QDsare potentially an ideal carrier for a PS – unresolved issues associated with thepotential toxicity and clearance of QDs notwithstanding [302]. Compared to amolecular PS, the broad absorption of QDs provides greater flexibility in selectingan excitation wavelength [304], as well as larger one- and two-photon absorptioncross sections [300,305]. The latter is particularly important since the NIR thera-peutic window can be accessed using the highly efficient two-photon excitation ofQDs. The QD PL can also be tuned through its size and composition for optimalspectral overlap with existing PS candidates that absorb light in the visible spectrum.FRET is the critical link needed to simultaneously harness the favorable propertiesof QDs and those of molecular PS agents that efficiently generate singlet oxygen.Although there have been reports of direct free radical or singlet oxygen generationfromQDs, themore promising strategy appears to be an energy transfer relay from aphotoexcited QD to a conjugated PS that subsequently sensitizes triplet oxygen tosinglet oxygen, as shown in Figure 12.36. The QD PL is also more favorable fortracking and imaging purposes than phosphorescence from the PS.In one study, Tsay et al. labeled phytochelatin-related peptides with either Rose

Bengal or chlorin e6 as PS/acceptors for CdSe/CdS/ZnS QD540 and QD620,respectively [300]. The QDs were rendered water-soluble by coating with a mixtureof labeled and unlabeled peptides that had a cysteine-rich terminus for assembly tothe QDs. The F€orster distances were 4.5 and 4.4 nm for the QD-Rose Bengal andQD-chlorin e6 FRET pairs, respectively. Although efficient FRET was observed forthe QD-Rose Bengal, a lower intrinsic efficiency was observed for the QD-chlorin e6and a conjugate valence of 26 was required to reach 50% FRETefficiency. However,the QDs were advantageous in that their first exciton absorption at 610 nm wasapproximately 10-fold larger than that of the chlorin e6 peak at 654 nm. The QD-PSconjugates had singlet oxygen quantum yields between 9–31%, depending on thePS, conjugate valence, and excitation wavelength. Singlet oxygen could be producedby direct excitation of the PS, or by excitation of the QDs and energy transfer to the


PS. For unknown reasons, the PS agents had lower singlet oxygen quantum yieldsas QD-conjugates; however, this was potentially offset by the stronger absorption ofthe QDs and their good photostability. More recently, Qi et al. covalently coupleda porphyrin to water-soluble QD595 coated with PEGylated poly(maleic anhydride-alt-1-octadecene) [305]. The QD two-photon absorption cross section was800–2400 GM units between 800–1100 nm. With an estimated F€orster distanceof 2.7 nm, FRET efficiencies increased up to 60% when more than 100 porphyrinmolecules were conjugated per QD. Despite the moderate FRETefficiencies, and asa consequence of the large two-photon absorption cross section of the QD, theQD-porphyrin conjugates produced twofold more singlet oxygen than the porphyrinalone under two-photon excitation. Several other examples of QD-PS conjugates forPDT can be found in recent reviews [299,301,302].

12.6Quantum Dots as Acceptors in Biological Applications

The ideal FRETacceptor has (i) a large molar absorption coefficient across the entirerange of donor PL to yield a large spectral overlap integral, (ii) a large Stokes shift sothat FRET-sensitized acceptor PL is well resolved from the donor PL, and (iii)negligible absorption at the excitation wavelength of the donor. QDs satisfy the firstand second criteria, but critically fail to satisfy the third, being characterized bystrong absorption at all UV-visible wavelengths shorter than their PL emission.Consequently, any QD that has spectral overlap with a potential donor will be very

Figure 12.36 (a) Sensitization of triplet oxygen to singlet oxygen via FRET from a QD donor to anintermediated photosensitizer (PS). (b) Examples of PS agents: (i) water-soluble porphyrinderivative, (ii) chlorin e6 derivative, and (iii) Rose Bengal.


efficiently excited in parallel with the donor. If that donor is a fluorescent dye, thiseffect is problematic due to the typically longer lifetimes of QDs (>10 ns versus<10 ns for dyes) [210]. Promptly after excitation of the dye, both the dye and QD in anominal FRET pair will be in an excited state; however, an excited-state QD cannotserve as an energy acceptor. As the dye returns to its ground state via its intrinsicpathways, a much greater proportion of the QDs remain in their excited state,precluding an efficient energy transfer. Therefore, QDs are poor acceptors formolecular dye donors. Nonetheless, the challenge of direct optical excitation of QDshas been overcome by using chemiluminescent donors, bioluminescent donors,lanthanide donors, or other QDs as donors. These configurations are discussed inthe following subsections.

12.6.1Chemiluminescence Resonance Energy Transfer (CRET)

A chemiluminescent reaction is one in which a product is formed in an excited stateand emits luminescence to return to its ground state. Perhaps the best knownexample is the chemiluminescent oxidation reaction between luminol and hydrogenperoxide (H2O2), in the presence of a suitable catalyst, to yield an a-hydroxyperoxideintermediate that decomposes into excited state 3-aminophthalate (3APA) [306]. The3APA has its peak luminescence at �420–430 nm, which is strongly resonant withthe broad absorption of many QDs emitting in the visible and NIR regions of thespectrum. If the 3APA is localized near a QD (< 1.5R0), then energy transfer canoccur and is referred to as chemiluminescence resonance energy transfer (CRET).The distinction between CRETand FRET is that, in the former, the donor originatesfrom a chemical reaction rather than optical excitation. Importantly, QDs remain inthe ground state by eliminating the need for optical excitation, and are thus excellentacceptors in CRET.The first example of CRET with a QD acceptor was reported in 2006 by Huang

et al., who conjugated MPA-coated CdTe QDs with horseradish peroxidase(HRP) to drive the p-iodophenol-enhanced chemiluminescent reaction betweenluminol and H2O2 [307]. Conjugation of the HRP to the QD served to localizethe excited-state 3APA product around the QD, thereby enhancing the CRETefficiency compared to an analogous system with unconjugated HRP that onlyweakly adsorbed to the QDs. CRET was demonstrated with QD555/590/620/660, including a two-plex configuration with QD555 and QD660. Wang et al.subsequently demonstrated three-plex CRET using the luminol/H2O2/p-iodophenol system and HRP-conjugated CdSe/ZnS QD545, QD600, andQD675 [308]. As an alternative to conjugating QDs with HRP, Li et al. directlyconjugated seven colors of MAA-coated CdTe QD with luminol, and observedCRET in the presence of H2O2 and sodium hypochlorite [309]. These experi-ments typically use QD conjugate concentrations on the order of 10�6M andluminol, p-iodophenol, and H2O2 concentrations on the order of 10�4M.However, it is worth noting that too much H2O2 can cause undesirablequenching of QD luminescence [307].

12.6 Quantum Dots as Acceptors in Biological Applications j553

In another example of QD-CRET, Du et al. acryloylated and encapsulated HRPwithin an acrylamide nanocapsule to catalyze the chemiluminescent reaction ofluminol [310]. The outer polymer layer was conjugated with one of six differentcolors of CdTe QD. The observation of CRET-sensitized QD luminescence wasdependent on HRP activity in the presence of H2O2 and luminol. By increasing theQD/HRP ratio from 1.2 to 9.6, it was possible to increase the effective CRET rationearly 18-fold [310]. In contrast to other studies where multiple HRP or luciferases(vide infra) are assembled around a central QD, this format comprised multiple QDacceptors around a central HRP, providing greater opportunity to maximize energytransfer rates.Interestingly, Zhao et al. observed CRET without conjugation of HRP or luminol

to QD acceptors [223,224]. Relatively high reagent concentrations – CdTe QDs at50–100 mM, 0.15–1.0mM luminol, and 0.15–1.0mM sodium hypobromite – wereneeded to compensate for the absence of specific localization between the QDs andluminol. Encounter-limited CRET was observed between the 3APA product ofluminol oxidation and five different colors of QD. Two-plex CRETwas also observedwith QD570 and QD660 [223], although multiplexed CRET is not analytically usefulwithout the localization of chemiluminescent reactions to specific colors of QD.Zhao et al. applied CRET with a single color of QD as a detection strategy formicrochip-based electrophoretic separations [224]. Luminol and CdTe QDs wereconstituents of the running buffer, and sodium hypobromite was introduced at theoutlet of the separation channel. Several amino acids (Ala, Arg, Asp, Asn, Cys, Glu,Gly, Lys, Ser, and Trp), N-acetylcysteine, glycylcysteine, glutathione, epinephrine,isoprenaline, and dopamine were found to inhibit the chemiluminescent reaction atthe channel outlet, providing an indirect signal for detecting the elution of thesecompounds [224].Willner and coworkers developed a set of QD-CRET sensors based on HRP-

mimicking DNAzyme-catalyzed reactions between luminol and H2O2. The simplestconfiguration was based on G-quadruplex complexes of either TBA or ATP-bindingaptamer (ATPBA) with hemin and the respective substrate, thrombin or ATP [222].Glutathione-coated QD615 were conjugated with approximately 10 copies ofaptamer, incubated with either ATP or thrombin target, further incubated withhemin, and the chemiluminescence measured after the addition of H2O2 andluminol. The CRET-sensitized QD luminescence increased with increasingamounts of thrombin or ATP. The LOD for thrombin was 1.4 nM. F€orster distanceswere estimated to be 2.7 nm and 4.6 nm for the thrombin and ATP systems,respectively. The dissociation constants were 25 nM and 6 mM for the TBA- andATPBA-conjugates, respectively, which were comparable to those for the aptamersalone. In another study, a more complex design for an ATP sensor using QD-CRETwas reported [221]. Glutathione-coated QDs were conjugated with 10 copies of anoligonucleotide that comprised two subunits: one subunit was part of a catalytic G-rich DNAzyme and the other subunit was part of an ATP-binding aptamer. Asecond, unconjugated oligonucleotide comprised cognate domains for the firstoligonucleotide; however, the two oligonucleotides only associated to form a catalytichemin/G-quadruplex in the presence of ATP, see Figure 12.37. Thus, the production


of chemiluminescence and CRET-sensitized QD luminescence was a function of theATP concentration and provided a LOD of 100 nM. Measurement of the distance-dependent CRET-sensitized QD luminescence circumvented the background signalassociated with the slight catalytic activity of unbound hemin. In another format,QDs were conjugated with approximately 10 copies of a DNA hairpin that incorpo-rated a target recognition sequence within the loop and hid the DNAzyme sequencein the stem [221]. Upon hybridization with target, opening of the stem–looppermitted formation of the hemin/G-quadruplex and recovery of peroxidase-likecatalytic activity. QD490, QD560, and QD620 were conjugated with different probehairpins to enable a three-plex assay. The CRET-sensitization of different colors ofQD luminescence provided resolved analytical signals, as shown in Figure 12.38.

12.6.2Bioluminescence Resonance Energy Transfer (BRET)

Bioluminescence is the biological analogue of chemiluminescence. That is, chemi-cal energy is converted into light energy within a biological system [311,312]. Thisreaction is typified by the action of luciferase enzymes on luciferin substrates,generally yielding blue or yellow light depending on the substrate. Luciferases areone type of bioluminescent protein and light is emitted in proportion to the turnoverof substrate. The so-called photoproteins are the other type of bioluminescentprotein and exhibit luminescence proportional to their concentration by virtue ofbeing stabilized luciferase-substrate intermediates that lack only a cofactor neces-sary for reaction and emission [311,312]. To date, only luciferases – and Renilla

Figure 12.37 (a) QD-CRET assay for ATP.Luminol is oxidized by the assembled hemin-G-quadruplex. The different subunits of the DNAare highlighted in purple, blue, and red.(b) Emission spectra as a function of ATP

concentration 0–0.1mM (1–7). The CRET-sensitized QD luminescence increases at620 nm. (Figure adapted with permission fromRef. [221]. Copyright 2011, American ChemicalSociety.)


reniformis (the sea pansy) luciferase in particular – have been utilized as donors forQD acceptors in bioluminescence resonance energy transfer (BRET). Analogous toCRET, the absence of optical excitation enables the use of QDs as excellent acceptorsin BRET.Many examples of the use of QDs as BRETacceptors have been given by Rao and

coworkers [173,175,189,220,313,314]. The first and most prominent example, givenby So et al., was the development of self-illuminating QDs and their application to invivo imaging [220]. Despite the favorable properties of QDs with respect to in vivoimaging, significant limitations are the scattering, tissue autofluorescence, smalldepth of penetration, and potential photodamage associated with near UV-blueexcitation. While the two-photon excitation of QDs in the NIR ameliorates theseissues, it is not particularly suited to in vivo imaging over large areas. To this end, Soet al. conjugated a Renilla luciferase mutant (Luc8) to QDs and, upon introduction ofcoelenterazine substrate, observed bright QD luminescence, as illustrated inFigure 12.39 [220]. The bioluminescence was most intense at 480 nm, yieldingexcellent spectral overlap with nearly all colors of QD in the visible and NIR region.

Figure 12.38 Multiplexed DNA analysis usingQD-CRET. (a) Three different colors of QD(QD620, QD560, and QD490) are conjugatedwith three different hairpin inactivatedDNAzymes. (b) Luminescence spectra inresponse to different combinations of targetDNA (TGT): (1) no target, (2) TGT560,

(3) TGT620, (4) TGT490, and (5) all threetargets. (c) Luminescence spectra in responseto different combinations of target DNA (TGT):(1) TGT490þ TGT620, (2) TGT490þ TGT560,(3) TGT560þ TGT620. (Adapted withpermission from Ref. [221]. Copyright 2011,American Chemical Society.)


Substrate turnover by the QD-conjugated Luc8 produced excited-state coelentera-mide in close proximity to the QD, resulting in efficient BRET. Since the donoremission intensity in BRET is determined by a reaction rate, the efficiency of energytransfer cannot be readily determined and is qualitatively assessed using the BRETacceptor/donor luminescence intensity ratio. CdSe/ZnS QD605 and QD655, andCdTe/ZnS QD705 and QD800 were used as acceptors. In application, three colors ofQD-Luc8 conjugates were intramuscularly injected into nudemice (see Figure 12.39)and visualized after intravenous injection of coelenterazine substrate, providing asignal-to-background ratio in excess of 103. The QDs were also coconjugated withLuc8 and a polycationic arginine nonamer for cellular delivery and imaging, both invitro and in vivo [220]. Similarly, Kosaka et al. have emulated Rao and coworkers andprepared QD655-Luc8 conjugates for real-time, semiquantitative in vivo lymphaticimaging of mice without any image processing [315].Rao and coworkers have also worked to improve the quality of QD-Luc8

conjugates [175,314]. Initially, the Luc8 was conjugated to carboxyl-coated QDs

Figure 12.39 (a) Schematic of a QD-Luc8(luciferase) conjugate for BRET. (b) Multiplexedin vivo imaging with a mouse model injected atfour sites: (i) QD800-Luc8, (ii) QD705-Luc8,(iii) QD665-Luc8þQD705-Luc8þQD800-

Luc8, and (iv) QD655-Luc8. The emission filtertransmission range is shown below each image.(Adapted with permission from Ref. [220].Copyright 2006, Macmillan Publishers Ltd.:Nature Biotechnology.)


using poorly controlled carbodiimide chemistry that resulted in a distribution ofLuc8 attachment points and orientations on the QD [220]. To address thislimitation, Zhang et al. adopted the use of an enzyme-catalyzed ligation strategyto couple Luc8 to QDs in a site-specific manner [175]. The HaloTag protein (HTP)is a recombinant haloalkane dehalogenase that has lost its ability to cleavesubstrate carbon–halogen bonds due to mutation of its His272 residue toPhe272. This change prevents hydrolysis of an ester bond formed in the HTP-haloalkane intermediate, creating a conjugate linkage to biomolecules modifiedwith an HTP substrate or “tag.” Here, QDs were chemically modified with achloroalkane linker and ligated to an HTP–Luc8 fusion protein, resulting inefficient BRET [175]. This conjugation strategy was expected to be amenable toQD-Luc8 conjugation within living cells and animals. In contrast, Xing et al.continued to use carbodiimide coupling to prepare QD-Luc8 conjugates, butmodified the process by further acryloylating Luc8 for subsequent encapsulationwithin an acrylamide nanogel [314]. The unmodified QD-Luc8 conjugates werefound to have only moderate stability in blood or serum, losing bioluminescence/BRET activity within 48 h in vitro and within 24 h in vivo; however, the nanogel-encapsulated conjugates retained significant activity for up to 5 days in vitro and 4days in vivo [314]. Interestingly, Ma et al. did away with bioconjugation altogetherand used Luc8 to directly template the aqueous biomineralization of NIR emittingPbS QDs, thereby producing an efficient BRET system [313].Another interest of Rao and coworkers has been protease sensing using QD-Luc8

conjugates [173,189]. In one iteration, Yao et al. genetically fused Luc8 with aGGPLGVRGGH6 peptide tail that served two functions: (i) the hexahistidine tractchelated Ni2þ in concert with carboxylate-coated QD655 to effectively conjugate theLuc8 to the QDs via a peptide bridge; and (ii) the PLGVR tract served as a substratefor matrix metalloproteinase-2 (MMP-2) [189]. In the absence of MMP-2, Luc8 wasbound to the QD, resulting in efficient BRET. MMP-2 activity cleaved the peptidelinker at the site indicated above and disengaged BRET. Shi et al. reported that theresulting 5 ng/ml LOD forMMP-2 [189] was two orders ofmagnitude better than theFRET-based method [156]. In a second iteration, Xia et al. coupled peptide fusions ofLuc8 to QDs using site-specific intein-mediated ligation chemistry [173]. Thepeptide fusions were selected to be substrates for MMP-7, MMP-2, and uPA(Figure 12.40). MMP-7 could be detected in serum with an LOD of 5 ng/ml usingQD655. A two-plex assay for MMP-2 and uPA was also demonstrated, using theBRET-sensitization of QD655 and QD705, respectively, as the analytical signal. Thecorresponding LODs were 1 ng/ml and 500 ng/ml.Beyond protease detection, Cissell et al. developed a QD-BRET method for the

detection of nucleic acid sequences in a competitive assay format shown inFigure 12.41 [219]. Renilla luciferase was conjugated to “sense” probe oligonucleo-tides and QD710 were conjugated to “antisense” probe oligonucleotides. In theabsence of target, the sense and antisense probes hybridized, yielding efficientBRET. Any nucleic acid target present competed to hybridize with the QD-antisenseprobe conjugates, such that the BRET ratio decreased as the concentration of targetincreased. The LOD was 4 pmol (20 nM) of target [219].


Figure 12.40 (a) Schematic of a QD-Luc8BRET probe for protease activity. (b) Detectionof MMP-7 activity using a QD655-Luc8conjugate. (c) Multiplexed detection of MMP-2

and uPA using QD655 and QD705 conjugatesof Luc8. (Adapted with permission fromRef. [173]. Copyright 2008, American ChemicalSociety.)

Figure 12.41 Competitive QD-BRET assay forDNA analysis using luciferase (Rluc)- and QD-conjugated oligonucleotides: (a) absence oftarget and (b) presence of target. (Reprinted

from Ref. [219] with kind permission fromSpringer ScienceþBusiness Media. Copyright2008, Springer-Verlag.)


12.6.3Lanthanide Donors

CRETand BRETaddress the challenge of undesirable excitation of QD acceptors byeliminating optical excitation. Another solution is to create a lifetime mismatchbetween the QD acceptor and a potential donor so that optical excitation can still beused. Luminescent lanthanide ions have excited-state lifetimes �1000-fold longerthan QDs, and are effective FRET donors for QD acceptors when the experimentalsystem is properly configured and optimized.The luminescence of lanthanide ions arises from f–f electronic transitions

within their partially filled 4f orbitals, which are shielded from the surroundingmicroenvironment by 5s and 5p orbitals, resulting in narrow band emission [212].Considering the visible region of the spectrum, Tb3þ and Eu3þ are the most usefulby virtue of their green and red PL. These colors result from 3 to 4 prominentemission bands across a broad spectral range (>100 nm). The lanthanide f–ftransitions are spin and Laporte forbidden, resulting in low transition probabilit-ies. As a consequence, trivalent lanthanide ions tend to have long excited-statelifetimes that are typically on the order hundreds of microseconds to milliseconds,as is the case for Tb3þ and Eu3þ [212]. Since most molecular fluorophores haveexcited-state lifetimes on the order of nanoseconds [316], the long lifetime ofluminescent lanthanide ions provides a distinct advantage in terms of signal-to-noise [317]. Measurements taken under steady-state illumination can often becontaminated by large amounts of scattering and background PL that masksignals of interest – especially if the sample is a complex biological matrixwith many endogenous fluorophores. Lanthanide ions overcome this limitationwhen combined with pulsed/flash excitation and time-gating. A flash of lightsimultaneously excites both the lanthanides and background fluorophores. Emis-sion for the latter decays away within tens of nanoseconds; however, more than99% of the lanthanide ions remain in the excited state, decaying over a muchlonger time scale. By introducing a delay or “time-gate” on the order of 100–101 msbetween excitation and acquisition of PL signals, the lanthanide PL can bemeasured on a dark background with complete suppression of unwanted back-ground. Detector integration times are typically 102–103 ms in time-gated experi-ments with luminescent lanthanide ions.A less advantageous consequence of the forbidden nature of f–f transitions is that

lanthanide ions have molar absorption coefficients that can be more than 104–105-fold smaller than organic dyes. This limitation has been overcome by utilizing a so-called antenna effect where an organic, chromophoric ligand is bound to thelanthanide ion, absorbs incident light, and transfers its excitation energy to thelanthanide ion, which then luminesces. These complexes include both chelates andcryptates. In general, the ligand is excited by UV light to a singlet state andundergoes intersystem crossing to the lowest triplet state, which then excites thebound lanthanide ion by energy transfer [316]. When combined with the intrinsicelectronic structure of Tb3þ or Eu3þ, ligand-to-lanthanide energy transfer yields avery large effective Stokes shift (>100 nm). Importantly, the ligandmolar absorption


coefficient is comparable to many fluorescent dyes (�103–104M�1 cm�1) andprovides comparable brightness for the lanthanide complex.In the context of FRET with lanthanide donors, the quantum yield needed to

calculate F€orster distances is that of the lanthanide ion. The quantum yield observedin experiments is actually the product of the ligand antenna-to-lanthanide energytransfer efficiency and the lanthanide ion quantum yield, and this must beaccounted for appropriately [213]. QDs are good acceptors for lanthanide ionsdue to their strong and broad absorption that is resonant with multiple lanthanideemission lines and results in very large spectral overlap integrals. It is ideal to choosea QD with a PL maximum that falls either between the various lanthanide emissionlines, or beyond the longest wavelength emission line. In practice, flash excitationand time-gating is necessary to suppress directly excited QD PL and permitacquisition of lanthanide PL and FRET-sensitized QD PL. Note that the QD is stilla poor acceptor immediately after flash excitation; pairing with a lanthanide donorsimply allows the experiment to wait until the QD returns to its ground state andbecomes a good acceptor.Hildebrandt and coworkers were the first to investigate the use of QDs as

acceptors for lanthanide donors, and did so in a series of studies where the well-known biotin–SA interaction was used as a model system [213,215,216]. Thelanthanide donors included Tb3þ or Eu3þ complexed with a glutamate ligandwith two 6-carboxybipyridyl arms, and a Eu3þ macrobicyclic tris(bipyridine)(TBP) cryptate, see Figure 12.42. All three lanthanide complexes were preparedas SA conjugates and paired with biotinylated QD655. Due to large overlap integrals,the estimated F€orster distances were as large as �10–11 nm. In time-gated mea-surements, shown in Figure 12.43, the addition of an increasing quantity of QD to afixed amount of lanthanide resulted in progressively greater FRET-sensitized QD

Figure 12.42 Examples of luminescentlanthanide complexes: (a) Eu3þ tris(bipyridine);(b) glutamate skeleton N-functionalized withtwo anionic bipyridyl chromophoric units, plusa pendant carboxylic acid. (c) Octadentate cage

based on 2-hydroxyisophthalamide. (Adaptedwith permission from Refs [213] and [321].Copyrights 2006 and 2011, American ChemicalSociety.)


PL. Saturation in the time-gated QD/lanthanide PL ratio was observed when eachbiotin molecule per QD had bound a lanthanide-SA conjugate. In the model assays,the lanthanide-to-QD energy transfer provided a limit of detection that was 1–2orders of magnitude lower than the classic Er3þ(TBP)-allophycocyanin (APC) time-gated FRET pair [213].The time-gating parameters for the aforementioned studies [213,215] were a

250 ms delay gate after UVpulsed/flash excitation at 308 nm, and a 750 ms integrationtime. The delay prevented the PL signal from being overwhelmed by the intense,directly excited QD PL signal that typically decayed to negligible levels withinmicroseconds. This decay time scale was a convolution of the actual decay time forthe QD PL, <1ms, and the decay in the response of the detector/electronics to theprompt burst of directly excited QD PL.In addition to the time-gated sensitization of QD PL after flash excitation and a

microsecond delay, evidence of lanthanide-to-QD FRET can be found in PL decaymeasurements [215,216,318]. Energy transfer from the lanthanide to the QDintroduces a new, shorter lifetime component in the lanthanide decay; this compo-nent reflects the rate of energy transfer to the QD. Furthermore, the QD PLwill takeon a lifetime that is commensurate with the rate of energy transfer – typically on theorder 102–103 ms. Energy transfer rates can be extracted from both of thesemeasurements and should reasonably agree with one another [247,318].Hildebrandt and coworkers further developed lanthanide-to-QD FRET into a

highly multiplexed format that used a SA-luminescent terbium complex (SA-LTC)conjugate as a common donor for biotinylated CdSe/ZnS QD530, QD565, QD605,and QD655 and CdSeTe/ZnSQD712 [214]. Representative time-gated PL spectra areshown in Figure 12.44. The Tb3þ ligand was based on an isophthalamide structure

Figure 12.43 Increase in time-gated, FRET-sensitized QD PL as more Tb3þ-complexlabeled SA binds to biotinyalted QD655. Theinset shows the QD/Tb3þ PL intensity ratio as a

function of the ratio of biotinylated QD and SA-Tb3þ-complex. (Adapted with permission fromRef. [213]. Copyright 2006, American ChemicalSociety.)


[319–321]. Starting with 1–2 nM of SA-LTC, the addition of increasing amounts ofbiotinylated QD increased the FRET-sensitized QD/LTC ratio until saturation [214].The LODs were between 0.1–1 pM depending on the assay format and QD selection,and >40-fold better than a Eu(TBP)–APC FRET pair in a direct comparison. Time-gated acquisition was between 50–450 ms after pulsed excitation. Interestingly, LTC-to-QD energy transfer was also used as a molecular ruler and applied to measuringthe dimensions of the series of QDsmentioned earlier [318]. Careful analysis of LTCand QD PL decays revealed that while the smallest QD530 were approximatelyspherical, the larger QDs were “cigar-shaped” ellipsoids with two short axes and onelong axis.Recently, Algar et al. used CdSe/ZnS QD620 as an intermediary in a time-gated

FRET relay with a lanthanide donor [247]. In time-gated measurements, the QDserved as an acceptor for a luminescent Tb3þ donor (FRET1), resulting in an excitedstate that subsequently served as a donor for an AF647 acceptor (FRET2). In promptmeasurements (i.e., no time-gating), the QD served directly as a donor for the

Figure 12.44 (a) Schematic of multiplexed,time-gated QD-FRET with a common Tb3þ

donor. The QDs are biotinylated and SA islabeled with a Tb3þ-complex. (b) PL spectrameasured at different ratios of biotinylated QDper SA-Tb3þ-complex. (c) Close-up of the data

from panel (b), showing the deconvolvedcontributions of QD530, QD565, QD605,QD655, and QD715. (Adapted with permissionfrom Ref. [214]. Copyright 2010 Wiley-VCHVerlag GmbH, Weinheim.)


AF647. The system is illustrated in Figure 12.45, and was assembled using labeledpeptides and/or oligonucleotides as bridges between the Tb/AF647 and the QD.Polyhistidine tags were used to control the conjugate valences. The first step of therelay, FRET1, comprised a multiple donor–single acceptor configuration. As moreTb-labeled peptide was assembled per QD, the time-gated FRET1-sensitized QD PLincreased. This increase was due to an increase in the net rate of energy transfer tothe QD; the rate of energy transfer from an arbitrary Tb donor was fixed andcorresponded to a FRET1 efficiency of �95%. Considering the second step of therelay, the FRET2-sensitized AF647 PL increased as the number of AF647-labeledpeptides per QD increased. This increase was due to an increase in the net rate ofenergy transfer from the QD; the rate of energy transfer to an arbitrary AF647 dyewas fixed and corresponded to an efficiency of 36% for a single acceptor. As a singledonor–multiple acceptor configuration, however, the FRET2 efficiency increased as

Figure 12.45 (a) (i) Time-gated FRET-sensitization of QD PL via energy transfer froma Tb3þ-complex (FRET1). Both the Tb3þ-complex and QD are initially excited by a flashof UV light. The QD relaxes to its ground stateafter a 55 ms delay and becomes a good FRETacceptor for the Tb3þ-complex. (ii) Time-gatedsensitization of AF647 PL via energy transfer

from the QD (FRET2), initiated by FRET1.(b) The QD serves as a nanoscaffold for theassembly of biomolecules labeled with Tb andAF647: (i) peptides, (ii) oligonucleotides, and(iii) both peptides and oligonucleotides.(Reprinted with permission from Ref. [247].Copyright 2012, American Chemical Society.)


the number of AF647 per QD increased (�80% at 5 AF647 per QD). The FRET2

pathway was observed in both prompt and time-gated measurements. Importantly,Algar et al. found that the QD-to-AF647 FRET2 pathway was independent of the Tb-to-QD FRET1 pathway. As a consequence, it was possible to extend the prompt utilityof the QD-AF647 FRET2 pair – for example, sensing protease activity or nucleic acidhybridization – into a time-gated mode [247]. Tb-labeled peptides were coassembledto QDs that were also conjugated with AF647-labeled substrates for trypsin or,alternatively, probe oligonucleotides complementary to AF647-labeled targets. Bothassays demonstrated analytical performance that was comparable to similar config-urations without time-gated FRET1 sensitization. The potential benefit of time-gating QD-FRET assays is improved signal-to-noise ratios with samples prone tointerference from autofluorescence or scattering. In principle, all the assays withQD donors and fluorescent dye acceptors from Section 12.5 could be adapted totime-gated measurements using FRET-sensitization from a lanthanide.Exploring another concept for sensing, Algar et al. further demonstrated that their

time-gated FRETrelay could be used to generate two analytical signals from a singlecolor/population of QDs, see Figure 12.46 [247]. The FRET2-sensitized AF647/QDPL ratio that depended on the number of AF647 acceptors per QD was determinedin prompt PL measurements and provided one analytical signal. The secondanalytical signal was the magnitude of the time-gated FRET1-sensitized QD PL,which was a function of the number of Tb donors per QD. This signal wasdetermined from time-gated PL measurements; however, due to quenching ofthe time-gated QD PL by FRET2, its value was determined from the sum of the time-gated QD and AF647 PL, with a correction for the nonunity quantum yield of thelatter. These two signals enabled a model two-plex nucleic acid hybridization assaywith only QD620-oligonucleotide conjugates, where one target sequence was labeledwith Tb and the other was labeled with AF647. All measurements were made with acommercial fluorescence plate reader and thus feasible in many laboratories. Thecapacity for a two-plex assay using a single color of QD is a break from theconvention of using N different colors of QD for multiplexing to the Nth degree,and provides multifunctionality in a single sensing vector.Overall, the advantages of lanthanide donor–QD acceptor FRETpairs are fourfold:

(i) spectral overlap integrals and F€orster distances 50–100% larger than most QD–dye FRETpairs due to the broad and strong absorption of QDs; (ii) higher sensitivitythan lanthanide–dye FRET pairs due to the larger F€orster distances; (iii) superiormultiplexing capability compared to lanthanide–dye FRET pairs due to (a) thenarrow size-tunable QD PL and (b) the ability to use a common lanthanide donorfor multiple colors of QD; and (iv) superior signal-to-noise ratios in measurementsof complex samples by virtue of time-gating to suppress background PL.

12.6.4Quantum Dot Donors (for Quantum Dot Acceptors)

In addition to their compatibility with chemiluminescent, bioluminescent, andlanthanide donors, QD acceptors (QDA) can also be paired with QD donors (QDD).


Here, the similar PL lifetime between the QDA and QDD is able to partiallycompensate for the efficient direct excitation of the former. That is, there is anontrivial statistical probability that the QDA will return to its ground state prior tothe QDD in a doubly excited QDD–QDA assembly (for equal QDD andQDA lifetimes,t, approximately 23% of these assemblies will be a viable FRET pair at time t afterflash excitation). Nonetheless, these configurations almost invariably retain sub-stantial background from the direct excitation of the QDA, which can be exacerbated

Figure 12.46 Time-gated, two-plex DNAhybridization assay using a two-step QD-FRETrelay. (a) Prompt (0ms) and time-gated (55ms)PL spectra characteristic of the assembly ofdifferent amounts of Tb3þ-complex (Tb) and/orAF647 to QDs through selective DNAhybridization (omitted for clarity): (i) no AF647,no Tb; (ii) 10 AF647, 0 Tb; (iii) 0 AF647, 16 Tb;and (iv) 10 AF647, 16 Tb. The solid black lines

show scaling of the prompt PL spectrum to fitthe time-gated PL spectrum (via numericaldeconvolution). (b) Orthogonal calibrationcurves based on measurement of (i) the promptAF647/QD PL ratio and (ii) time-gated totalQDþAF647 PL sensitization. (Reprinted withpermission from Ref. [247]. Copyright 2012,American Chemical Society.)


by the typically stronger absorption of the QDA compared to QDD due to its largersize. Lower excitation intensities may help increase the apparent FRET efficiency: asthe probability of concurrently exciting QDD and QDA within the same assembly isreduced, the subpopulation of FRET assemblies with only excited QDD increases,thus increasing the rate of FRET across the ensemble. Increased quenching of QDD

PL and increased FRET-sensitization of QDA PL above its directly excited backgroundare the expected results. However, there is a parallel increase in the subpopulation ofassemblies with only excited QDA, such that the relative magnitude of directly excitedQDA PL is not necessarily suppressed (using the unquenched QDD PL as a referencepoint). The trade-off for lower excitation intensities is the reduced signal-to-noise inmeasurements. Further, this approach becomes less effective as the fraction ofproximal QDD–QDA FRET pairs decreases, that is, the FRET pairs are not fullyassembled. Despite the potential shortcomings, several bioanalytical applicationshave been developed on the basis of FRET between two different colors of QD.Li et al. reported a model sandwich immunoassay using QD–QD FRET for

detection and an ancillary technique, capillary electrophoresis (CE), to addresspoor signal-to-noise that resulted from a heterogeneous mixture of QDD, QDA, andQDD–QDA pairs across an ensemble [322]. The mixed PL signals negatively biasedthe measured FRET efficiency in ensemble measurements. Glutathione-coatedCdTe QD530 were used as donors and conjugated with mouse IgG; MAA-coatedCdSe/ZnS QD630 were conjugated with goat antimouse IgG and used as acceptors.Electropherograms were measured in both donor and acceptor PL channels and,through a simple adaptation of Equation 12.5, returned FRET efficiencies between39–70%. In contrast, the efficiency values derived from ensemble measurementswere between 13–52%. The range of FRET efficiencies reported were for experi-ments where the concentration of acceptors was increased at constant donorconcentration [322]. Curiously, in the case of 1 : 1 donor–acceptor complexes, theFRET efficiency measured from CE should have been constant. The changes inFRETefficiency as a function of acceptor concentration were thus likely an effect ofmultivalent complexes.Hu et al. used QD–QD FRET pairs to develop a glucose assay, as shown in

Figure 12.47 [323].MAA-coatedCdTeQD560donorsweremodifiedwith concanavalinA (ConA) and CdTe QD610 acceptors were modified with D-(þ)-glucosamine. TheConA-QD560 were then incubated with samples. At low concentrations of glucose inthe sample, subsequent addition of the glucosamine-QD610 resulted in binding to theConA-QD560 with FRET-induced quenching of the latter. At higher concentrations ofglucose, the ConA binding sites were predominantly occupied and the FRETpair wasunable to form. The QD560 PL intensity relative to a control sample with no glucosewas used as the analytical parameter. FRET-sensitization of the QD610 PL wasobserved, but was small compared to the directly excited component. The assayresponse was linear for glucose concentrations between 0.1–2.0mM, with an LOD of0.03mM. Visual detection, shown in Figure 12.47, was also possible in the rangefrom 0.1 to 4.0mM glucose as the ensemble PL changed between red andgreen. The analysis was effective with both serum and urine samples without anypretreatment [323].


Liu et al. investigated FRET between two QDs bound to the cell membrane ofHeLa cells displaying CD71 antigen [324]. QD615 acceptors were conjugated withantiCD71 monoclonal antibody and QD545 donors were conjugated with goatantimouse IgG. The QD615 conjugates bound to the cell membrane and werethen bound themselves by the QD545 conjugates. The latter process was tracked intime using fluorescence microscopy and spectral measurements, revealing aprogressive decrease in QD545 PL (55%) and sensitization of additional QD615PL (32%). Control experiments using QD545 conjugated with human IgG (cf. goatantimouse IgG) showed no FRET-sensitized signal changes when added to cellsstained with the QD615-antiCD71 conjugates [324]. This work was primarilyinteresting because it demonstrated FRET between QDs in a biological matrix.In a solid-phase FRETconfiguration, Seker et al. prepared a film with CdTe QD560

donors and QD640 acceptors using layer-by-layer assembly with poly-L-lysine (PLK)and poly-L-glutamic acid (PLE) [325]. The hierarchy of the film, coated onto a glasssubstrate, was a layer of QD560, 3–9 alternating layers of PLK and PLE, and a finallayer of QD640. Different lengths of PLK and PLE yielded films with differentmorphologies, many of which were very heterogeneous and had poor surfacecoverage. The highest quality film utilized PLK100 and PLE25 (the number ofmonomers is indicated) and achieved 80% surface coverage. FRET betweenthe QD560 and QD640 manifested as an increase in QD640 PL and decrease in

Figure 12.47 (a) Schematic for glucoseanalysis using FRET between two QDs:(i) sample without glucose and (ii) sample withglucose. (b) Effect of the relative amounts of

QD560 and QD610 (G:R ratio) on the visualdetection of glucose. (Adapted from Ref. [323].Copyright 2011, with permission from Elsevier.)


QD560 PL. Films with only one or the other QD were used for comparison. In apreliminary application, FRET-sensitization of the QD640 was diminished throughthe activity of a bovine pancreatic protease cocktail, and was tracked through adecrease in the PL lifetime of the QD640 acceptor [325].A number of physical studies have examined FRET with monolayers of QDs

[326–330]. The most common FRET pair for these studies is a green-emitting QDD

and red-emitting QDA. Energy transfer is observed between the two QDs whenmixed together in the same thin film. This is characterized by a decrease in PLintensity and lifetime for the QDD, and an increase in these parameters for the QDA.Interestingly, the same effect can be observed between differently sized QDs withinthe same population. That is, when a nominally single color/size of QD isimmobilized as a close-packed monolayer, the PL intensity and lifetime onthe hypsochromic edge of the ensemble spectrum are diminished, while thoseparameters for the bathochromic edge are increased. FRET between QDs inmonolayer or multilayer structures is primarily of interest for light-harvestingapplications where the process can efficiently funnel absorbed energy to a commonendpoint (vide infra).

12.7Energy Transfer between Quantum Dots and Other Nanomaterials

This section provides an overview of energy transfer between QDs and goldnanoparticles, graphene oxide (GO), and carbon nanotubes (CNTs). Mechanistically,these processes are similar to FRET, but are not strictly equivalent in the details.There is already a large array of biological applications for energy transfer betweenQDs and gold nanoparticles, and a growing list with carbon nanomaterials. Severalrepresentative examples are summarized herein.

12.7.1Gold Nanoparticles

Gold nanoparticles are crystallites that can vary between �1–100 nm in size and aresynthesized in a variety of shapes, including spheres, cylinders, and hollow shells. Ingeneral, the latter are referred to as gold nanorods (Au NRs) or gold nanoshells,respectively, while approximately spherical particles are referred to simply as goldnanoparticles (Au NPs). Similar to QDs, gold nanomaterials derive special opticalproperties from their small size and several review articles have discussed theseproperties in detail [331–336]. Themost important property of Au NPs in the contextof energy transfer is their light absorption and interactions with dipole emitters. AuNPs are well known for their characteristic red color that arises from a localizedsurface plasmon resonance (LSPR) at approximately 520–530 nm for NP diametersbetween 5–50 nm [333]. In contrast to the band gap energy of QDs, the plasmonresonance of Au NPs is only weakly dependent on Au NP size. Nonetheless, as thediameter of an Au NP increases toward 100 nm, the LSPR resonance gradually shiftscloser toward 600 nm, broadens, and becomes more intense. In general, for noble

12.7 Energy Transfer between Quantum Dots and Other Nanomaterials j569

metal nanoparticles at sizes below or comparable to the mean free path of electronsin the material (�50 nm for Au), light induces a coherent oscillation of electronsbetween opposite surfaces of a NP – a localized surface plasmon – that is responsiblefor the intense absorption peak [332,334]. When the Au NP diameter decreasesbelow 5nm, the LSPR becomes strongly damped and becomes featureless atdiameters< 2 nm [331,333]. In terms of tunable nanoscale properties, the highsensitivity of the plasmon resonance to nanoparticle shape and anisotropy has beenof greater utility than simple size-tuning of spherical particles. For example, Au NRsthat have a nonunity aspect ratio exhibit two resonances: one associated withtransverse oscillations of electrons and another associated with longitudinal oscilla-tions. As the aspect ratio increases, the latter resonance shifts to longer wavelengths(�700–1100 nm), broadens, and becomes more strongly absorbing [332,334]. AuNPs and Au NRs have molar extinction coefficients that are several orders ofmagnitude larger than molecular chromophores, and the absorbed light energy israpidly thermalized [331,332].Qualitatively, and similar to bulk gold surfaces, Au NPs are known to be good

quenchers of molecular fluorescence at NP sizes< 40 nm and distances> 2 nm[337,338]. In the case of Au NPs less than 2 nm in diameter, Strouse and coworkersquantitatively studied the quenching of fluorescent dyes to develop, validate, andapply a theoretical framework for nanometal surface energy transfer (NSET) thatwas analogous to the theory developed for a bulk metal interface [338–341]. Theessential findings were that a characteristic 50% energy transfer distance could bederived, that there was a dependence on the LSPR spectral overlap, and that the rateof energy transfer had an inverse fourth-power dependence on the relative donor–Au NP distance. This latter property is in contrast to the inverse sixth-powerdependence of classical FRET. Overall, the primary advantage of AuNPs as acceptorsis efficient quenching that can extend the useful range of energy transfer from<10 nm (FRET) to between 15–20 nm (NSET). Importantly, Pons et al. demon-strated that NSET was also the best model to describe the quenching of CdSe/ZnSQDs by 1.4 nm Au NPs, and similarly found an extended distance range for energytransfer, see Figure 12.48 [204]. A critical aspect of the study was the use of rigid,variable-length peptides and polyhistidine self-assembly to exercise good controlover the energy transfer configuration. In many reports of applications for the use ofAu NPs to quench QDs, a FRET mechanism is casually or phenomenologicallysuggested without good control over the QD–Au NP separation or relative stoichi-ometry. Pragmatically, the details of the quenching mechanism are most importantin studies that seek to use a long-range molecular ruler, whereas the design andapplication of assay formats and sensing configurations can generally be accom-plished with a largely qualitative model.Several assays using QDs as donors for Au NP acceptors have been designed

around a competitive format. For example, early work by Oh et al. demonstrated thatbinding of biotinylated 2–3 nm Au NPs to SA-QD615 resulted in efficient QD PLquenching; however, progressively diminished quenching was observed whenAvidin was introduced as a competitive binder of the biotinylated Au NPs [342].Oh et al. then adapted this format to detecting protein glycosylation by coating 3 nm


Au NPs with ConA and coating QD605 with dextran (Dex), relying on the affinity ofConA toward mannosyl and glucosyl groups [343]. The strategy is shown inFigure 12.49. In the absence of a glycosylated protein, the ConA-Au NPs wereable to bind the Dex-QDs to yield PL quenching via energy transfer. Otherwise,glycosylated proteins in samples competed to bind with the ConA-NPs resulting inPL recovery that was proportional to the amount of glycosylated protein. In additionto a proof-of-concept assay with Avidin and its nonglycosylated counterpart, Neu-trAvidin, the system was used to measure the degree of chemical glycosylation ofBSA and differentiate between hyperglycosylated and hypoglycosylated recombinantglucose oxidase from yeast strains. This assay was then inverted and adapted to asolid-phase format by immobilizing ConA-QD525/605 conjugates onto an NHS-activated hydrogel-coated glass slide [344]. In this case, Dex-Au NP conjugates werebound to the immobilized QDs, resulting in efficient PL quenching. The QDs

Figure 12.48 (a) Schematic of QD-YEHKpeptide-Au NP conjugates. The QD-acceptorseparation is controlled by varying, m, thenumber of YEHK repeats. (b) Plots ofquenching efficiency versus separation distancefor three different systems: QD-YEHK peptide-

Au NP, QD-dsDNA-Au NP, and QD-YEHK-Cy3.Fits for different energy transfer models areshown, including FRET, NSET, and DMPET(dipole-to-metal-particle-energy transfer).(Adapted with permission from Ref. [204].Copyright 2007, American Chemical Society.)


partially recovered their PL in the presence of mannosylated protein. Tang et al.developed a solution phase method for glucose detection using a similar format,where ConA-QD525 conjugates were paired and preassembled with b-CD modifiedAu NPs [153]. In this case, glucose displaced the b-CD from the ConA binding sitesto recover the QD PL in a manner linearly dependent on the glucose concentrationbetween 50 nM–15mM. Good selectivity was observed for glucose over a variety ofsugars and other potential interferences, and detection in serum was possible. Inanother application, Liang et al. combined Mn-doped CdTe QD655/720 goat antihu-man IgG conjugates with Au NR-rabbit antihuman IgG conjugates to create asandwich immunoassay for IgG with detection between 50 nM–2.5mM [345]. Theadvantage of the Au nanorod is that it is an efficient acceptor for NIR emitters, whichhas been difficult to achieve with molecular dyes. Further, the extended range ofenergy transfer with Au NRs is particularly suited to large biomolecules such asimmunoglobulins.Using a dissociative FRET-signaling mechanism, Chang et al. developed a

protease assay on the basis of energy transfer between a central QD625 donorand 1.4 nm Au NP acceptors conjugated via peptide linkers [155]. The peptideincorporated an LGPA amino acid sequence that was cleaved by collagenase,resulting in loss of the proximity necessary for energy transfer and partial recoveryof the QD PL. Physical characterization of the conjugates was limited, but the resultssuggested a significant degree of steric hindrance with respect to proteolysis. Thequenching efficiency decreased from 70% to 50% after 47 h with 0.2mg/mlcollagenase. Other protease assays using QDs and FRET with molecular dyeacceptors have typically required< 30min [154,156,243]. In a more recent study,

Figure 12.49 Schematic of protein glycosylation assay using dextran (Dex)-conjugated QDs,ConA-coated Au NPs, and energy transfer. (Reprinted with permission from Ref. [343]. Copyright2006, Wiley-VCH Verlag GmbH, Weinheim.)


Lowe et al. used 3 h incubation times in an assay for uPA activity [227]. Peptidesubstrates for uPAwere labeled with 1.4 nm Au NPs and biotinylated at the oppositeterminus. After incubation with uPA, SA-QD525 were added to assemble with thebiotinylated peptides. As uPA activity increased, the amount of hydrolyzed peptideincreased, the number of Au NPs that bound to QDs decreased, and the QD PLincreased. As noted earlier, this assay was also used in parallel with one forphosphorylation (see Section 12.5.1) [227]. The contrast between the aforemen-tioned studies [155,227], like the previously discussed LcA assay [246], highlights theutility of two-step protease assays with (i) hydrolysis of peptide substrate off the QDand (ii) subsequent peptide assembly to the QD. These assays are a viable alternativeto one-step methods when the protease-catalyzed hydrolysis of peptide substrates ishindered in close proximity to a nanoparticle.A solid-phase assay format has also been developed for protease detection using

immobilized QDs as donors for Au NP acceptors. As shown in Figure 12.50, Kimet al. immobilized SA-QDs as spots on a glass slide and conjugated biotinylated

Figure 12.50 Multiplexed assay for proteaseactivity with three colors of SA-QD immobilizedon a glass slide and conjugated with Au NPsvia different biotinylated peptide substratelinkers. QD525, QD605, and QD655 werepaired with substrates for MMP-7, caspase-3,and thrombin. Assay results are shown: (a) SA-QDs only;

(b) SA-QD-peptide-Au NP conjugates;(c)þMMP-7; (d)þ caspase-3; (e)þ thrombin;and (f) mixture of proteases and inhibitors. Theblack background of the image has beenremoved for reproduction purposes. (Adaptedwith permission from Ref. [283]. Copyright 2008,American Chemical Society.)


peptides that were labeled with a distal 1.4 nm Au NP [283]. The resulting proximityto the Au NPs resulted in �80% quenching of the QD PL. In a multiplexedconfiguration, QD525, QD605, and QD655 were immobilized as discrete spotsand the corresponding peptide linkers were selected to be substrates for MMP-7,caspase-3, and thrombin, respectively. QD PLwas recovered with specific cleavage ofthe peptide substrates by the proteases, with intensity proportional to the concen-tration of protease. The LODs were 10 ng/ml, 20 ng/ml, and 1 U/ml for MMP-7,caspase-3, and thrombin, respectively [283]. The LOD for MMP-7 was morefavorable than an analogous assay with a TAMRA acceptor for the QD525 donor[284]. Despite the spectrally resolved QD PL, discrete spots were necessary formultiplexing due to the fabrication of all three QD-peptide conjugates via the biotin–SA interaction and the use of a common quencher for all three detection channels.Nonetheless, with the use of different QD emitters it should be possible to resolvethe activity at each spot spectrally, that is, without imaging. Furthermore, the use of acommon Au NP acceptor for all three colors of QD facilitated multiplexingcompared to the previous format that paired QD525 with a TAMRA acceptor[284]. If this strategy were to be extended to multiplexing using the colors of QDdiscussed earlier, the TAMRA emission would significantly overlap with the QD605PL and, in turn, the PL from the acceptor for QD605 would overlap with the QD655PL. The QD-dye format is less convenient than the QD-Au NP format due to theneed for spectral deconvolution, and also less efficient due to the shorter range ofFRETcompared to NSET. The trade-off, however, is the loss of ratiometric detection.Considering small molecule analytes, Liu et al. developed a method for the

simultaneous detection of cocaine and adenosine that was based on the aggregationand dispersion of QDs and 13 nm Au NPs [346]. The QDs and Au NPs wereconjugated with different oligonucleotide probes that hybridized to different regionsof an adenosine- or cocaine-binding aptamer. In this state, the QDs and Au NPsassociated to form aggregates, resulting in quenching of the QD PL, as shown inFigure 12.51. In the presence of adenosine or cocaine, the hybridization linkagebetween QDs and Au NPs was disrupted due to analyte binding by the aptamer andthe subsequent change in secondary structure. QD PLwas recovered with the loss ofproximity between the AuNPs andQDs.Multiplexing was achieved with QD525 andQD585 donors, and by taking advantage of the inherent selectivity of DNAhybridization to assemble a particular aptamer to a given color of QD. TheLODs for cocaine and adenosine were 120 mM and 50 mM, respectively.Energy transfer between QDs and Au nanomaterials has also been exploited for

DNA detection. Li et al. developed a method starting from the adsorption of targetDNA on positively charged Au NRs [347]. Subsequent hybridization of the adsorbedtarget with QD655-labeled probe oligonucleotides resulted in quenching of the QDPL. As part of a larger study comparing MB designs, Cady et al. demonstrated a QD-MB using 1.4 nm Au NPs as the quencher [251]. Surprisingly, the MBs with Au NPquenchers offered a slightly smaller signal change with hybridization than ananalogous design with a dye quencher, Iowa Black FQ. Note that this result maynot reflect a direct comparison between FRET and NSET, since biophysical factorsmay also influence opening of the hairpin.


12.7.2Carbon Nanomaterials

Carbon nanomaterials such as graphene, graphene oxide, carbon nanotubes, andbuckminsterfullerene (C60) have emerged as effective quenchers of luminescence.Although there is still some debate over the precise mechanism(s), it has beensuggested that dipolar resonance energy transfer and/or electron transfer areresponsible for PL quenching in proximity to carbon nanomaterials [348]. Thissection describes examples from the literature where FRET has either beensuggested as the putative mechanism or not ruled out.

12.7.2.1 Graphene and Graphene OxideGraphene is a two-dimensional hexagonal lattice of sp2-hybridized carbon withsingle-atom thickness [349,350]. Graphene oxide (GO) is similar, but incorporatesoxygen and hydrogen at both sp2- and sp3-hybridized carbon centers and, as a

Figure 12.51 (a) Schematic of aptamer-linkedQD (Q1)-Au NP (1, 2) nanostructures anddetection of adenosine. (b) PL spectra for theQD525-adenosine-binding aptamer–Au NPcombination with and without adenosine, andcorresponding saturation curve. (c) PL spectrafor QD585-cocaine-binding aptamer–Au NP

combination with and without cocaine, andcorresponding saturation curve. (d) Multiplexedconfiguration and PL spectra showing responseto different samples. (Adapted with permissionfrom Ref. [346]. Copyright 2007, AmericanChemical Society.)


consequence, has much greater aqueous solubility and can be readily functionalizedwith biomolecules [351]. GO has recently been shown to deliver DNA to a cell,protect it from enzymatic degradation, and have low toxicity [352]. Interestingly, GOalso exhibits UV, visible, and NIR luminescence that originates from electron–holerecombination in localized electronic states, rather than from band-edge transitionsas typical of semiconductor materials [349]. Although further elucidation of themechanism is needed, a prevailing idea is that these localized states are sp2 clustersof finite size, bound by an sp3 matrix, where the size of the former determines thewavelength of luminescence through the confinement of p-electrons. EachGO sheetcan have many of these clusters, resulting in broad and featureless emission bands[349,350]. GO oxide is also a good quencher of PL [350], which is currently its mainutility in combination with QDs. Morales-Narv�aez et al. found that GO was thestrongest quencher of QD PL among several carbon materials, exceeding graphite,nanotubes, and nanofibers [353].Liu et al. developed a sandwich immunoassay for a-fetoprotein (AFP), a prospec-

tive hepatocellular carcinoma biomarker, using GO-capture antibody conjugates andCdTe QD510-reporter antibody conjugates [351]. In the presence of AFP, theimmunocomplex brought the QDs and GO in close proximity, resulting in 42%PL quenching of the former. The addition of 2% BSA was needed to reducenonspecific adsorption. The assay had an LOD of 0.15 ng/ml AFP. Quenching ofthe QD PL was marked by a decrease in its lifetime and, interestingly, occurred at adistance potentially in excess of 20 nm (assuming the antibodies were orientatedperpendicular to the nanomaterial interfaces) [351]. While the authors did notsuggest a specific quenching mechanism (e.g., FRET, charge transfer), the long-range quenching is suggestive of a surface energy transfer (SET)-type mechanism.Chen et al. used an alternative approach with GO and QDs in a two-color, multi-plexed homogeneous immunoassay for Human Enterovirus 71 (EV71) and Cox-sackievirus B3 (CVB3) [354]. SA-coated QD525 and QD605 were conjugated withbiotinylated antibodies for EV71 and CVB3, respectively, and then mixed with GO.The QD-antibody conjugates adsorbed to the GO resulting in 70% quenching.Added viruses were able to selectively disrupt the corresponding QD-antibody–GOcomplex, leading to a partial recovery of QD PL. The latter was used as the analyticalsignal and provided LODs of 0.42 and 0.39 ng/ml of EV71 and CVB3 [354]. Althoughnot explicitly noted by the authors, FRET/SETwas the likely quenching mechanism;the amphiphilic polymer and SA coating on the QD are expected to be unfavorablefor charge-transfer quenching.Dong et al. recently reported a molecular beacon that comprised a QD donor and

1� 1mm2 GO acceptor [355]. Oligonucleotide hairpins were covalently coupled toMPA-coated CdTe QD590, subsequently incubated with samples of interest, andthen mixed with GO. In the absence of target nucleic acid, the QD-hairpinconjugates adsorbed to the GO, resulting in 90–97% PL quenching. Adsorptionof the hairpins to the GO was attributed to favorable p–p stacking and hydrogen-bonding interactions. Adsorption was less favorable when the hairpins wereconverted to duplex DNA upon hybridization with target, decreasing the numberof QDs adsorbed to the GO and concomitantly reducing quenching efficiency (35%).


Putatively, the more rigid, extended conformation of the duplex DNA also limitedthe distance of closest approach between the QDs and GO, resulting in a decrease inenergy transfer efficiency [355]. The authors derived QD–GO separation distances of3.2 and 9.4 nm without and with target, respectively. Quenching was attributed toFRET at separations< 7 nm, and surface energy transfer (SET) at separations> 7nm. The detection of target DNAwas strongly dependent on the amount of QD-MBand GO used in the experiment, requiring optimization of concentrations for goodanalytical performance. The assay had a linear range between 50–1500 nM targetand a LOD of 12 nM. Contrast of approximately 3 : 1 between fully complementarytarget and a single-base pair mismatch was obtained. Interestingly, the limit of thedynamic range was significantly larger than the number of hairpin oligonucleotides(�300 nM), suggesting poor hybridization efficiency. The strategy was extended tothrombin detection using TBA in place of hairpin probes [355].

12.7.2.2 Carbon NanotubesCarbon nanotubes (CNTs) were discovered by Iijima in 1991 and comprise one ormore sheets of graphene rolled into a seamless tube [356,357]. Depending on itschirality, a CNT can be either metallic or semiconducting. Single-walled CNTs(SWNTs) are formed from a single graphene layer, whereas multiwalled CNTs(MWNTs) are formed from a concentric arrangement of SWNTs of increasingdiameter. Semiconducting SWNTs exhibit photostable NIR PL (900–1600 nm) thatcan be tuned as a function of chirality, whilemetallic SWNTs are excellent quenchersof PL [357,358]. The diameters of SWNTs are typically in the range of 0.3–3.0 nm,whereas the diameter of MWNTs may approach 100 nm. Tube lengths can spanfrom nanometers to micrometers. Unfortunately, CNTs can be challenging to workwith due to difficulties in purification, batch-to-batch variability, and, in biologicalapplications, their hydrophobicity [357]. Oxidative methods for purification, how-ever, have led to CNTs with carbonyl and carboxylic acid groups at open ends anddefect sites. At the cost of altering electronic properties, these groups improvesolubility and facilitate bioconjugation. In these respects, other tools of the tradeinclude cycloaddition reactions, the coordination of ligands through p–stackinginteractions, and complexation by hydrophilic macromolecules [165].Similar to graphene and GO, the qualitative ability of CNTs to quench QD PL is

much better known than the quantitative mechanism. In one early study, Pan et al.mixed MAA-coated QD550 with CNTs functionalized with either cationic amine,neutral hydroxyl, or anionic carboxylate functional groups [359]. The degree of QDPL quenching was dependent on the CNT functional group: quenching wasstrongest with the amine-CNTs and weakest with the carboxylate-CNTs, whichwas consistent with electrostatic assembly between the QDs and CNTs. Thequenching mechanism, however, was not elucidated. Biju et al. conjugated SA-coated QD585 and QD605 to biotinylated SWNTs and observed similar quenchingof the QD PL intensity and a decrease in PL lifetime [360]. This was attributed toFRET, but was not unambiguous spectroscopically; however, given the SAmoleculesand amphiphilic polymer coating surrounding the QDs, FRETwas more likely thancharge-transfer quenching.


In a much-needed study, Shafran et al. specifically addressed whether chargetransfer or resonance energy transfer occurred between QD600 and SWNTs [361].Individual SWNTs were attached to an AFM tip and brought in close proximity to asingle, immobilized QD600, where photon count rate histograms were recorded as afunction of tip height. The blinking properties of the QD were exploited to helpelucidate the quenching mechanism. As noted earlier, blinking of QD PL is thoughtto arise from intermittent charging of the QD core by carrier trapping at thenanocrystal interface. Charge transfer was, therefore, expected to alter blinkingstatistics, but no such changes were observed experimentally [361]. The PL quench-ing data was rather in agreement with a dipole–dipole coupling mechanismfor energy transfer. Characteristic energy transfer distances, R0, were between12–40 nm – considerably larger than conventional FRET. This enhancement wasdue to the one-dimensional nature (cf. zero-dimensional) of the SWNTacceptor andcorresponding ability to generate excitons along its length. The data and energytransfer model suggested that peak energy transfer efficiency was independent ofSWNTchirality [361]. This latter conclusion is particularly important for applicationsutilizing PL quenching, since it is difficult to obtain pure SWNT samples with asingle chirality.In application, Cui et al. exploited the quenching of QD PL by MWNTs for a

sandwich immunoassay and a three-color, multiplexed sandwich hybridizationassay [362]. The general formats are shown in Figure 12.52. In each case, thecapture antibody/oligonucleotide probe was conjugated to the MWNT and thereporter antibody/reporter oligonucleotide was conjugated to the QD. The targetof interest induced a sandwich structure, bringing the MWNTs and QDs into closeproximity and quenching the QD PL. The degree of quenching was proportional tothe amount of target in the sample. The target of interest in the sandwichimmunoassay was breast cancer-associated antigen 1 (BRCAA1) and the methodafforded a LOD of � 0.4 nM. A three-plex hybridization assay made use of QD510,QD555, and QD600 reporters in the same fashion, permitting detection of eachtarget in the range of 0.2–200 pM [362]. In another report, Tian et al. utilized anadsorption-based strategy for DNA detection through the SWNT-induced quenchingof QD PL [363]. CdTe QD575-probe oligonucleotide conjugates were adsorbed toSWNTs via p–stacking interactions, resulting in quenching of QD PL. The probesequence was complementary to the PB2 gene diagnostic of the H5N1 strain ofinfluenza A. The hybridization of target DNA with the probe oligonucleotides wasmore favorable than the p–stacking interactions with the SWNTs; as a consequence,the QD-probe/target hybrids were desorbed from the SWNTs with a recovery of QDPL. The dynamic range of the assay was 10 nM–20 mM [363].

12.8Nonbiological Applications of Quantum Dots and FRET

A few illustrative examples of nonbiological applications of QD-FRET are describedin this section. However, much like this chapter, the overall interest in QD-FRET has


Figure 12.52 Schematic for sandwich hybridization assays (System 1, System 2) or sandwichimmunoassays (System 3) using QD and CNT conjugates of oligonucleotides or antibodies.(Reprinted with permission from Ref. [362]. Copyright 2008, American Chemical Society.)

12.8 Nonbiological Applications of Quantum Dots and FRET j579

been largely dominated by biological applications. The intention here is to brieflyhighlight the broader utility and potential impact of QD-FRET, which the interestedreader can pursue through other resources.

12.8.1Photovoltaic Cells

Ironically, this nonbiological application of QD-FRET is inspired by nature. Thephotosystems critical to photosynthesis in plants and other organisms use FRETas a key cog in their machinery for converting sunlight into chemical energy.Researchers have long endeavored to match Nature’s efficiency in developingphotovoltaic devices that convert sunlight into electrical energy [364–366]. Thisgoal is quickly becoming a critical need, since the demand for energy will soonoutstrip the supply. QDs are of interest in this research due to their strong andbroad absorption, chemical robustness, photostability, and potential capacity formultiexciton generation (i.e., more usable charge carriers per photoexcitation)[367]. Although several studies have looked at using QDs for the direct photo-injection of charge into an electrode, a few studies have combined QDs and FRETfor this purpose.In the hopes of improving solar cell efficiency and stability, Buhbut et al. recently

evaluated the incorporation of QDs and FRET into dye-sensitized solar cells (DSSCs)[368,369]. Here, both the broad absorption and narrow, tunable emission of QDswere exploited. CdSe/CdS/ZnS QD680 acted as light-harvesting antennae andtransferred their excitation energy to a charge separating dye molecule, anunsymmetrical squaraine dye with a large extinction coefficient (319 000M�1 cm�1).In turn, the FRET-sensitized squaraine dyes injected electrons into the conductionband of a nanocrystalline TiO2 electrode as part of a photovoltaic cell. Figure 12.53illustrates the design. The QDs were incorporated within the TiO2 to provideisolation from the redox active electrolyte and avoid degradation or PL quenching.The multishell structure was chosen to help inhibit direct charge injection from theQD to the electrode. Time-resolved PL measurements confirmed FRET from QDs todye, at �44% efficiency, through a decrease in the QD PL lifetime and increase inthe squaraine lifetime. The incident-photon-to-current-efficiency (IPCE) of the cellswas 5–10% in the range between 400–675nm and, compared to the squaraine alone,offeredasubstantial increase inIPCEbetween400–550nm[368].Overall, theIPCEwaslower than anticipated due to inefficient charge injection from the squaraine; however,the advantage of theQD-FRETstrategy is that light absorption and charge injection areeffectively decoupled, permitting separate optimization of each component [368].The challenge in this design is maximizing FRET. Although QD emission can bereabsorbed by the squaraine dye and have the same photovoltaic effect, this innerfiltering is less efficient than energy transfer [369]. Charging of the QDs must alsobe avoided since this strongly quenches PL and FRET.Ruland et al. constructed “rainbow solar cells” using CdTe QD520, QD550,

QD580, and QD620 [370]. The MAA-coated QDs were assembled layer-by-layerand color-by-color on a quartz-supported indium tin oxide (ITO) electrode using


intervening layers of cationic PDDA. The photoelectrochemical cell was completedwith a Cu2S counter electrode and polysulfide electrolyte. The composite QD filmonly showed PL emission from the QD620, confirming an efficient FRET cascadealong the band gap gradient of the layered QDs. Three configurations wereinvestigated: a forward rainbow configuration with QD520 adjacent to the ITO; areverse rainbow configuration with QD620 adjacent to the ITO; and a referenceconfiguration with only QD620. The forward configuration provided the highestIPCE as a result of more efficient energy transfer to the electrolyte interface wherecharge separation occurred through oxidation of the QDs. Reduced electrolyte was

Figure 12.53 (a) Schematic of a photovoltaicsystem. Nanocrystalline (Nc) TiO2 is grown ona transparent conducting oxide (TCO) and QDsare bound through a MPA linker. The QDs arethen coated with a thin layer of amorphousTiO2, which is further modified with dyemolecules and immersed in an I�/I3� liquid

redox electrolyte. (b) IPCE curves for cells withonly QDs (red line), only dye molecules (blueline), and both QDs and dyes (black line). TheQD–dye combination enhances IPCE between400–550 nm. (Adapted with permission fromRef. [368]. Copyright 2010, American ChemicalSociety.)

12.8 Nonbiological Applications of Quantum Dots and FRET j581

neutralized at the anode and holes migrated through the CdTe layer to the ITOcathode. Although the power conversion efficiency of the CdTe QD rainbow-modified photocathodes was not comparable to those observed in DSSCs andorganic solar cells, this work showed the potential for development of QD-basedlight-harvesting devices that could potentially increase the usable range of the solarspectrum [370].

12.8.2Light-Emitting Diodes (LEDs)

QDs are attractive materials for solid-state lighting and light conversion due to theirunique optical properties. While lighting contributes greatly to the quality of life inthe developed world, it also contributes significantly to total energy consumption[371]. It is therefore important to develop high quality and energy-efficient lighting.Photometric performance is evaluated on the basis of color rendering index (CRI),luminous efficacy of optical radiation (LER), and correlated color temperature (CCT)criteria. Light-emitting diodes (LEDs) are more energy efficient than incandescentand fluorescent light sources, but appealing white light emission is not an inherentproperty. The integration of QDs and FRET is one manner in which white lightemission can be engineered from LED sources. For example, QDs can offer higherLER values than conventional phosphors since their narrow PL avoids deep redemission. The QD-FRET-based LEDs can be divided into two classes: FRET-converted and FRET-enhanced [371].Achermann et al. demonstrated a QD-FRET-converted LED where an electrically

pumped quantum well efficiently transferred energy to QDs through FRET [372].This strategy can overcome limitations associated with low carrier mobility andphotooxidation in electrically pumped hybrid QD/organic LEDs where there isformal charge transfer to QDs [372]. It also bypasses the insulating capping layers onQDs. An InGaN quantumwell with emission at 400 nm acted as a FRETdonor whena close-packed layer of TOP/TOPO-coated CdSe/ZnS QD575 was assembled on top.While such a system can be pumped electrically, energy transfer was studied usingpulsed optical excitation of the quantum well at 266 nm. Energy transfer was 50%efficient and occurred at transfer rates that, beyond use for solid-state lighting, werefast enough for application in optical amplifiers and lasers [372]. Further advancesderived from this seminal work were recently reviewed by Demir et al. [371],including improvements in FRETefficiencies through optimization of temperature[373], incorporation of a cyan-emitting wafer to improve luminescence from the QDlayer [374,375], an inverted design (n-i-p diode geometry versus p-i-n geometry) toprovide improved color conversion efficiency [376], and etching of multiple wellsinto the LED so that QDs are proximal to multiple quantum wells for even greaterimprovement in color conversion efficiency [377].FRET-enhanced LEDs have been constructed using cascaded FRET within multi-

layers of multiple colors of QD, which funnel energy from smaller, blue-emittingQDs to larger, red-emitting QDs [378–380]. For example, relative to a film of onlyred-emitting QDs, these cascade designs are able to enhance emission and color


conversion from the red-emitting QDs, despite there being a lower percentage of thered-emitting QDs. This is attributed to recycling of trapped excitons by virtue ofefficient energy transfer [371].

12.9Summary

The combination of QDs and FRET is compelling in both basic and applied researchfor many reasons. As nanomaterials, QDs have several favorable characteristics:

� Small size and nontrivial surface area� Core/shell structure that protects and enhances optical properties� Physicochemical properties that can be tailored through ligand and polymer

coatings� Diverse bioconjugate techniques such as passive adsorption, electrostatic bind-

ing, covalent coupling, biochemical ligation, and dative bonding� Potential for cellular/in vivo delivery.

As luminescent materials, QDs have many advantageous optical properties:

� Strong and broad absorption spectra� Large two-photon absorption cross sections� Narrow, size-tunable PL that results from quantum confinement� Additional tuning of PL through material selection� Favorable quantum yields and good photostability� Superior multiplexing capability.

As donors in FRET, QDs offer the following benefits:

� Generally good theoretical and experimental agreement with F€orster theory� Ability to maximize the spectral overlap integral while minimizing donor/

acceptor emission cross talk� Ability to tune FRET efficiency by arraying multiple acceptors per QD� Ability to minimize direct excitation of acceptors.

As acceptors in FRET, QDs offer the following benefits:

� Exceptionally large spectral overlap integrals� Ability to increase FRET-sensitization rates by arraying multiple donors per QD� Ability to use a common donor for multiplexed FRET.

As donors/acceptors in FRET, or similar mechanisms with other nanomaterials,QDs can be utilized in several applications:

� Ensemble, single-pair, and solid-phase configurations� Immunoassays� Hybridization assays� Enzyme assays

12.9 Summary j583

� Probes for small molecule analytes, for example, metabolites and drugs� pH sensing� Tracking therapeutic delivery� Conformational analysis of biomolecules as QD conjugates� Photodynamic therapy� Cellular imaging� Light harvesting and solid-state lighting.

In the aforementioned context, this chapter has expounded the fundamentalspectroscopy of QD-FRET, pragmatic considerations for experiments, and thecurrent state of the art in utilizing QDs and FRET for biological applications.Many more exciting advances are surely on the horizon for this powerful tandem.

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FRET - Förster Resonance Energy Transfer || Semiconductor Quantum Dots and FRET

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