Poly‑L‑lysine-Coated Silver Nanoparticles as Positively ChargedSubstrates for Surface-Enhanced Raman ScatteringLucia Marsich,† Alois Bonifacio,*,† Subhra Mandal,‡ Silke Krol,‡,§ Claudia Beleites,† and Valter Sergo†
†Centre of Excellence for Nanostructured Materials (CENMAT) and Department of Industrial and Information Engineering,University of Trieste, Italy‡CBM (Cluster in Biomedicine), Trieste, Italy§Fondazione I.R.CCS Istituto Neurologico Carlo Besta, IFOM-IEO-Campus, Milan, Italy
ABSTRACT: Positively charged nanoparticles to be used assubstrates for surface-enhanced Raman scattering (SERS) wereprepared by coating citrate-reduced silver nanoparticles withthe cationic polymer poly-L-lysine. The average diameter of thecoated nanoparticles is 75 nm, and their zeta potential is +62.3± 1.7 mV. UV−vis spectrophotometry and dynamic lightscattering measurements show that no aggregation occursduring the coating process. As an example of their application,the so-obtained positively charged coated particles wereemployed to detect nanomolar concentrations of the anionicchromophore bilirubin using SERS. Because of their oppositecharge, bilirubin molecules interact with the coated nano-particles, allowing SERS detection. The SERS intensity increases linearly with concentration in a range from 10 to 200 nM,allowing quantitative analysis of bilirubin aqueous solutions.
■ INTRODUCTION
Raman spectroscopy is a nondestructive analytical techniquebased on the inelastic scattering of a monochromatic light(usually provided by a laser source) by vibrating molecules, andit gives qualitative and quantitative information on the chemicalconstituents of a given sample: a Raman spectrum is a uniqueand distinctive “molecular finger print”, and its intensity isproportional to the analyte concentration.1
Unfortunately, the applicability of Raman spectroscopy islimited by its poor sensitivity. To obtain an increase in theRaman intensity, analytes can be adsorbed on a nanostructuredmetal surface with adequate characteristics, yielding a surface-enhanced Raman scattering (SERS) spectrum.2 It is currentlywidely accepted that two mechanisms contribute to the SERSenhancement: electromagnetic and chemical. In the former, thelaser light excites the collective oscillations of the surfaceconduction electrons in a metal (surface plasmons). Thisexcitation results in the enhancement of the local electro-magnetic field intensity experienced by a molecule adsorbed orin close proximity to the metal surface. As a consequence of thepresence of this secondary electromagnetic field, the intensityof the Raman scattered light is enhanced. It should be stressedthat direct adsorption of an analyte on the surface is not neededfor this mechanism to operate. On the other hand, the chemicalmechanism enhances the Raman-scattering cross section of ananalyte directly adsorbed on the metal surface. The enhance-ment due to the latter mechanism depends on the chemicalnature of the analyte and on its adsorption geometry, and it isgenerally weaker than that due to the electromagnetic
mechanism.3 Besides these two mechanisms, if the analytehas an electronic transition in resonance with the wavelength ofthe exciting laser, a surface-enhanced resonance Ramanscattering (SERRS) spectrum is obtained,1 in which both theSERS and the resonance Raman (RR) effects contribute to theenhancement, yielding enhancing factors up to 15 orders ofmagnitude.4,5 Thus, SE(R)RS spectroscopy provides structure-specific vibrational spectra of adsorbates with extremely highsensitivity; moreover, the technique also ensures a highselectivity because only adsorbed species can be detected. InSERRS, additional selectivity is achieved upon tuning theexcitation laser in resonance with the analyte of interest, whoseRaman spectrum will selectively benefit from the resonanceenhancement. Because of these advantageous characteristics,SE(R)RS has become one of the most promising analyticalmethods for chemical and biochemical detection and analysis.6
A high affinity of the analyte for the metal substrate of choiceis an essential requirement for obtaining a SERS signal. In manycases, the interaction between the analyte and the metal surfaceis mediated by electrostatic forces operating between oppositecharges, in addition to more specific interactions. Colloidaldispersions of metal nanoparticles are widely used as efficientSERS substrates, and a careful control over their surface chargeis very important to tune their affinity toward an analyte.7 Oneof the most used protocols to prepare Ag nanoparticles for
Received: June 12, 2012Revised: July 30, 2012Published: August 29, 2012
SERS is the reduction of Ag+ ions with sodium citrate.8,9
Citrate-reduced colloids are very easily prepared, are stable formonths, and yield very intense SERS spectra.10 However, thenanoparticles have a negative surface charge due to theadsorbed citrate anions,10 so that this substrate provides apoor SERS enhancement when used with negatively chargedanalytes. The same problem is encountered when usinghydroxylamine as reducing agent, which yields negativelycharged colloids.9−11
To overcome this problem, other synthetic methods havebeen developed in which different reducing agents are used.9−12
Reduction with NaBH4 yields positively charged Ag nano-particles, but these colloids are less stable than citrate-reducedones.9,10,13 Another strategy consists of coating Ag nano-particles with a layer of positively charged molecules, such assilanes,14 cetyltrimethylammonium bromide (CTAB),15 andpolyethyleneimine.16
The aim of this work is to develop a SE(R)RS-substrateconsisting of Ag nanoparticles with a positive surface charge tobe used for the analysis of organic anions; these can adsorb onthe SERS substrate via electrostatic interaction and can thus bedetected using SE(R)RS. Such a positively charged SE(R)RSsubstrate is obtained upon coating citrate-reduced Ag nano-particles (Cit-AgNPs), a widely used substrate because of itsease of preparation and efficient enhancement of Ramanspectra,10 with poly-L-lysine (PLL). PLL is a small naturalhomopolymer belonging to the group of cationic poly aminoacids, and even in moderately alkaline media it containspositively charged hydrophilic amino groups (pKa = 10,53). Inthis Article, we report the preparation of PLL-coated Agnanoparticles (PLL-AgNPs) and their characterization usingUV−vis spectrophotometry, transmission electron microscopy,dynamic light scattering (DLS), zeta (ζ) potential measure-ments, and SERS spectroscopy. A possible application of thePLL-AgNPs as SERRS sensors for bilirubin quantification isalso presented.Bilirubin is a yellow organic molecule of clinical interest
formed as a metabolic waste product of heme breakdown.17−20
At physiological pH, in aqueous solutions bilirubin is thoughtto be mainly present as a dianion.19 Despite several worksreporting RR spectra of bilirubin solutions in a variety ofsolvents and conditions,21−25 there is a lack of published studiesabout the application of SERRS to bilirubin26,27 and inparticular to bilirubin detection.28 In our opinion, this scarcitycould be possibly due to the lack of a reliable positively chargedSERS-active metal colloid having a high affinity for the bilirubinanions. The PLL-AgNPs described in this Article were shownto be an efficient SERS substrate for bilirubin quantification inaqueous solutions.
sodium citrate tribasic dehydrate, poly-L-lysine hydro-bromide (PLL)(mol wt 15 000−30 000), bilirubin (mixture of isomers (IIIA, VIIIA,and IXA)), and sodium hydroxide were purchased from Sigma-Aldrich.Phosphate buffer solution (20 mM, pH 8.00) was prepared usingK2HPO4 and KH2PO4, both purchased from Sigma-Aldrich. Allchemicals were used without further purification. Ultra pure Milli-Qwater (Merck Millipore, Billerica, MA) was used for the preparation ofall solutions.Cit-AgNPs Preparation. Cit-AgNPs were prepared according to
the procedure described by Lee and Meisel.8 45 mg of AgNO3 wasdissolved in 250 mL of Milli-Q water. The solution was heated toboiling under reflux, and then 5 mL of freshly prepared trisodium
citrate aqueous solution (1% w/v) was added dropwise under vigorousstirring. The mixture was kept boiling under reflux and stirring for 1 hand then slowly cooled to room temperature. All glassware wasthoroughly cleaned using concentrated HNO3 and then chromic acid(2.77 g of K2Cr2O7 in 100 mL of H2SO4), and carefully rinsed withMilli-Q water.
PLL-AgNPs Preparation. A 0.1% w/v poly-L-lysine aqueoussolution was prepared. Next, 50 mL of this PLL solution was added to50 mL of Cit-AgNPs previously diluted 1:25 with Milli-Q water. Toremove PLL excess, the mixture was centrifuged in 1.5 mL ofpolypropylene Eppendorf tubes at 6708g for 20 min, the supernatantwas removed, and fresh Milli-Q water was added. This washingprocedure was repeated three times. The resulting coated nano-particles dispersion is stable against aggregation for up 24 h. Before itsuse as SERS substrate, the stock dispersion of coated nanoparticles waspelleted by centrifugation, and from each Eppendorf tube wasremoved 1.47 mL of the supernatant (corresponding to 98% of theinitial volume) with a micropipet, resulting in a concentrated aqueousdispersion of coated nanoparticles.
Nanoparticles Characterization. UV−vis spectra were acquiredwith a Perkin-Elmer Lambda bio20 spectrophotometer, between 350and 700 nm. Samples were prepared by diluting 10× the citrate-reduced silver nanoparticles stock solution with Milli-Q water in a 3mL cuvette.
DLS and the ζ-potential measurements were performed using aMalvern Instruments’ Zetasizer (Nano-ZS Nanoseries, UK).
Transmission electron microscopy (TEM) was performed with aPhilips EM208 scanning electron microscope using a carbon-coatednickel grid (Carbon Film 200 Mesh, Ni, 50/bx produced by ElectronMicroscopy Sciences). Uranyl acetate (UO2(CH3COO)2·2H2O) wasadded to the dried PLL-AgNPs on the TEM grids as contrast agent, tovisualize the positively charged organic layer around the NPs, asreported in the literature for the chitosan.29
Raman Instrumentation. Spectroscopic measurements weredone using an inVia Raman system (Renishaw, Wotton-under-Edge,UK). The laser sources (514.5 nm argon-ion laser and 785 nm diodelaser) were focused on the sample, consisting of a 50 μL dropdeposited on a UV-grade CaF2 slide, through a 10× objective (0.25N.A.).
The 514.5 and 785 nm laser power at the sample was 7 and 150mW, respectively, except for bilirubin SERRS measurement, in whichcase the power of the 514.5 nm laser was 650 μW to avoid bilirubinphotodegradation. To avoid interference from anomalous bandsarising from Ag colloids when green excitation is used,30 SERSspectra of PLL-AgNPs were obtained using the 785 nm laser.
Bilirubin Solutions. A bilirubin stock solution was prepareddissolving bilirubin in anhydrous dimethyl sulfoxide to obtain a finalconcentration of 5 mM. 100 μM bilirubin solutions were prepareddiluting 50 times the stock solution with a 10 mM NaOH aqueoussolution. Solutions with a bilirubin concentration lower than 100 μMwere prepared using a 20 mM buffer phosphate solution (pH 8) fordilution. Extreme care was taken to avoid bilirubin photodegradationupon light exposure: all dilutions were done in a dark room with redsafelight illumination.
Normal-Raman spectrum of citrate aqueous solution with 1 Mconcentration was acquired using the 514.5 nm laser with anacquisition time of 50 s. Cit-AgNPs SERS spectrum was collectedusing the 514.5 nm with a total acquisition time of 30 s. The normalRaman spectrum of solid PLL was acquired with a 785 nm laser, andthe acquisition time was 10 s. 100 μM bilirubin RR spectra werecollected using the 514.5 nm laser with an acquisition time of 30 s.SERRS spectra of bilirubin were collected with the 514.5 nm laseradding 2.5 μL of the PLL-AgNPs to 50 μL of bilirubin solution with anacquisition time of 50 s. For each bilirubin concentration, threedifferent bilirubin solutions were prepared and measured independ-ently. Extreme care was taken to avoid bilirubin photodegradationupon laser exposure.
Data Processing. All data preprocessing and analysis wasperformed using several packages of the R software for statisticalanalysis.31 The position of the bands was estimated with msProcess,32
a package providing tools for spectra processing. Raman and SERSspectra were analyzed with hyperSpec,33 an R package to handlehyperspectral data sets, in this case spectra plus concentrations. Forbilirubin quantification, the region between 600 and 800 cm−1 of thespectra was considered, because in this region of the spectrum the PLLdoes not have any interfering band. A linear baseline correction wasperformed. The intensity at the maximum of the 680 cm−1 band of thebilirubin SERRS spectrum was chosen for calibration purposes.The calibration curve, the 95% confidence interval for the
calibration, the limit of detection (LOD), and the limit ofquantification (LOQ) were calculated using chemCal,34 an R packagefor calibration data in analytical chemistry. The use of weightedregression for calibration was considered and checked, but it was foundnot necessary, as the standard deviation of SERS intensity was notincreasing with concentration in the interval used for calibration.
■ RESULTS AND DISCUSSIONNanoparticles Characterization. The Cit-AgNPs have an
absorption maximum at 405 nm (Figure 1), indicating that the
average diameter of the nanoparticles is between 70 and 80 nm,as reported in the literature.1,2,8 The PLL-AgNPs have anabsorption maximum at 410 nm (Figure 1). This red shift ofthe plasmonic frequency from 405 to 410 nm can be attributedto the adsorption of the PLL on the nanoparticles surface,indicating that the coating succeeded. The relatively small 5 nmred shift rules out the formation of large nanoparticlesaggregates upon PLL coating TEM images of the PLL-AgNPs(Figure 1, insets) and confirms the size of the NPs asdetermined with UV−vis spectrophotometry, and the negativestain with uranyl acetate clearly shows the PLL layer around thenanoparticles.The ζ-potentials of the Cit-AgNPs and of the PLL-AgNPs
are −35.0 ± 1.2 and +62.3 ± 1.64 mV, respectively, confirmingthe modification of the nanoparticles surface charge fromnegative to positive upon PLL coating. Furthermore, ζ-potential absolute values are greater than 30 mV, indicatingthat the suspension of Cit-AgNPs and PLL-AgNPs in water isstable.35 The ζ-potential for the PLL-AgNPs is more positivethan those reported in literature for other SERS-active coated
nanoparticles,12,36 indicating a greater charge density andtherefore potentially electrostatic interactions with negativelycharged analytes.The values of the average hydrodynamic diameter, measured
with DLS, are 92.9 ± 1.7 nm for the Cit-AgNPs and 86.4 ± 1.1nm for the PLL-AgNPs. The negligible difference betweenthese two values, possibly caused by the different charge densityat the surface, confirms that no aggregation occurs during thecoating process.The normal-Raman spectrum of citrate aqueous solution and
the SERS spectrum of the Cit-AgNPs (Figure 2a and b) shows
intense vibrational bands between 800 and 1050 cm−1 andbetween 1300 and 1450 cm−1, in agreement with the datapreviously reported in the literature.10 The presence of thesame spectral features in both Raman and SERS spectrademonstrates that citrate ions are adsorbed on the silvernanoparticles. According to the literature,10 the bands between1300 and 1500 cm−1 are due to symmetric carboxylatestretching mode, and those between 800 and 1000 cm−1 arecaused by C−COO and C−OH stretching modes. The relativeband intensities in SERS spectra are governed by theorientation of the molecules adsorbed on the metal surface(i.e., SERS selection rules),37 and they suggest that citrate ionsadsorb to the Ag surface through their carboxylic groups, asproposed by Munro et al.10 On the other hand, the SERSspectrum of the PLL-AgNPs (Figure 2d) does not show thecitrate spectral features, but exhibits several bands that are alsofound in the Raman spectrum of the PLL (Figure 2c). Inparticular, the bands at 1678 and 1240 cm−1 in the SERSspectrum of PLL-AgNPs were assigned to the amide I andamide III modes, respectively, observed at similar Raman shiftsin the PLL Raman spectrum. Such values for these amidemodes indicate that the “random coil” structure of the PLL in
Figure 1. UV−vis extinction spectra of Cit-AgNPs (- - -) and PLL-AgNPs (−); TEM images (insets A, B) of PLL-AgNPs treated withuranyl acetate (UO2(CH3COO)2·2H2O) to enhance the contrast dueto the PLL layer around the metal nanoparticles.
Figure 2. (a) Raman spectrum of 1 M citrate solution; (b) SERSspectrum of Cit-AgNPs; (c) Raman spectrum of PLL (10% w/v aqsolution); and (d) SERS spectrum of PLL-AgNPs. λex is 514.5 nm for(a,b) and 785 nm for (c,d).
aqueous solution has been retained upon adsorption on themetal surface.38 Beside the amide I and III, other bands in theSERS spectrum of Figure 2d can be attributed to PLL, mostnotably the ones at 1440, 1132, and around 890 cm−1. Theobservation of such bands is consistent with the displacementof citrate by PLL on the nanoparticles surface, corroboratingthe conclusions inferred from UV−vis, TEM, and ζ-potentialmeasurements.The interpretation of the PLL-AgNPs in terms of detailed
adsorption geometry is not trivial, and it is further complicatedby the presence of other bands apparently absent in the PLLRaman spectrum. For instance, the sharp band at 1003 cm−1 isprobably due to residual aromatic protecting group used in thePLL synthesis (as described by PLL purveyor). Amino groupsare known to strongly interact with Ag,39 and such aninteraction in the case of PLL on Ag is supported by thepresence of the bands at 1132 and 1034 cm−1 (Figure 2d), bothof which have been associated with vibrations of the aminogroup in SERS spectra of amines.40
SERRS Spectra of Bilirubin. According to previousstudies,23,25,41 the bands at 1580 and 1617 cm−1 of thebilirubin RR spectrum (Figures 3 and 4a) originate from CC,
CO, and C−N stretching modes of the lactam rings, whereasthe band at 1271 cm−1 is due to a lactam ring C−C stretchingand N−H bending modes. On the other hand, the assignmentof many other bands, such as those at 960 and at 677 cm−1, isstill uncertain.The bilirubin SERRS spectrum is very similar to the RR
spectrum (Figure 4), and therefore its bands can be readilyassigned according to the same scheme. This similarity in therelative intensity pattern between RR and SERRS spectra isexpected, and it is due to a relaxation of the SERS surfaceselection rules in the resonant conditions present in SERRS.1,42
The small shifts (<3−4 cm−1) observed between most bands ofRR and SERRS spectra suggest that the bilirubin intramolecularhydrogen-bonding pattern between carboxylates and pyrrolichydrogens is, at least in part, still present,21,23 although altered
upon adsorption. Similar changes in some of the hydrogen-bonding sensitive bands, such the small downshift for the 1617cm−1 and the 1271 cm−1 bands, and the upshift of the 1577cm−1 band, were also observed in the RR spectrum ofbilirubin−albumin complex,21 suggesting a parallel with PLL−bilirubin. When complexed with albumin, bilirubin forms salt-bridges between its carboxylates and two charged amino-acidicamino-groups of the protein, while maintaining one intra-molecular hydrogen bond between a carboxylate and twopyrrolic hydrogens.43 At the same time, one of the lactamcarbonyls becomes loosely hydrogen bonded to the protein.A similar interaction could reasonably take place also
between bilirubin and PLL-AgNPs, because PLL has positivelycharged amino groups and can act as a hydrogen-bond donor tothe bilirubin lactam carbonyls. Unfortunately, a direct proofabout salt-bridges formation from RR/SERRS spectra is notpossible, because propionates carboxylates are not conjugatedwith the extended chromophoric π-systems of bilirubin, andtherefore do not benefit from the RR enhancement, remainingundetected in resonant spectra. However, indirect effects ofsuch interaction can be observed. A loosening of theintramolecular hydrogen bonds due to the formation of salt-bridges between carboxylates could explain the downshift of theN−H bending mode, whereas the formation of a weakhydrogen bond between PLL and one of the lactam carbonylscould explain the upshift of the mode at 1617 cm−1, associatedwith a lactam carbonyl stretching. The upshift the other lactammode at 1577 cm−1, and the upshift and dramatic broadening ofthe sharp and intense unassigned band at 677 cm−1, are moredifficult to explain. In general, a more detailed description ofthe PLL−bilirubin interaction, which is beyond the scope ofthis Article, would require a more comprehensive andconsistent bilirubin normal-mode analysis.The dominant contribution of the resonance effect to the
SERRS spectrum allows the detection of the bilirubin even if itis not directly adsorbed on the metal surface, but on the top of
Figure 3. (a) Raman spectrum of 100 nM bilirubin with Cit-AgNPs;(b) Raman spectrum of 100 nM bilirubin; and (c) SERRS spectrum of100 nM bilirubin with PLL-AgNPs; all of the spectra are shown on thesame intensity scale. Because of its low intensity, spectrum (a)magnified 25 times (×25) is also reported; λex = 514.5 nm.
the PLL coating. On the other hand, the PLL coating does notbenefit from the resonance enhancement, and its SERSspectrum is less intense than the SERRS spectrum of bilirubin,but still observable in the absence of bilirubin (Figure 2d).The calibration curve for bilirubin quantification with SERRS
is presented in Figure 5, using the intensity of the bilirubin
band at 680 cm−1 to avoid interference with the bands due toimpurities in the metallic substrate (see the ExperimentalSection). The lowest concentration measured was 10 nM,which is 3 orders of magnitude lower than the lowestconcentration previously reported, as detected withSERRS.26,28 For concentrations between 10 and 200 nM, thecorrelation between the spectrum intensity and the bilirubinconcentration is linear (Figure 5b), with a coefficient ofdetermination (R2) of 0.9979. For concentrations lower than 10nM, the spectrum does not show bilirubin vibrational bands.On the contrary, for concentrations higher than 200 nM, thesignal does not increase, likely because the surface of the PLL-AgNPs is saturated by bilirubin molecules. The limit ofdetection (LOD) and the limit of quantification (LOQ)calculated from the calibration curve are 24 and 45 nM,respectively.
■ CONCLUSIONSThe preparation of PLL-coated silver nanoparticles wasreported, and consistent results from UV−vis, TEM, z-potential, DLS, and SERS microscopy prove that the PLLforms a positively charged polymer layer around the nano-particles, without inducing their aggregation. This studydemonstrates that the PLL-coated nanoparticles can be usedas stable, reproducible, and efficient SERS substrates fordetection and quantification of anionic chromophores such asbilirubin. Because of its characteristics, such substrate could bealso applied for the SERRS-based quantification of othernegatively charged analytes, such as dye-labeled DNA.44
■ ACKNOWLEDGMENTSWe would like to thank Sabina Passamonti (Life ScienceDepartment, University of Trieste) as well as Claudio Tiribelli,Cristina Bellarosa, Pablo Giraudi, and Silvia Gazzin (Centre forLiver Studies, Trieste) for sharing their protocols andknowledge about bilirubin. Many thanks also to RichardWennberg (University of Washington School of Medicine) forthe many stimulating discussions.
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Figure 5. (a) Bilirubin SERRS intensities at 690 cm−1 as a function ofbilirubin concentration. (b) Intensity average value of the band at 690cm−1 (+), calibration curve in the concentration range 10−200 nM(· · ·), and the 95% confidence interval for the calibration (−−−); theequation for the calculated calibration curve is y = 3431 + 146x, wherey is Raman intensity and x is bilirubin concentration (nM).
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