Controlling the Synthesis and Assembly of Silver Nanocrystals for Single-Molecule Detection by SERS

Christine H. Morana, Younan Xia*a

aDepartment of Biomedical Engineering, Georgia Institute of Technology and Emory University, 901 Atlantic Drive, Atlanta, GA 30332 USA

ABSTRACT

Detecting toxic chemical or biological agents in low concentrations requires a highly specific sensing technique, such as surface-enhanced Raman spectroscopy (SERS). The controlled synthesis of metallic nanocrystals has provided a new class of substrates for more reliable and sensitive SERS applications. The nanocrystal shape plays a major role in designing SERS substrates for maximizing the SERS enhancement factor (EF). Assembling nanocrystals into dimers can further amplify the EF, opening the door to the possibility of single-molecule detection. Here, we briefly discuss our recent work on the synthesis of silver (Ag) nanocrystals and their assembly into dimers and other reliable techniques to form hot spots with sufficiently high EF for single-molecule detection by SERS.

Keywords: silver nanocrystals, surface-enhanced Raman spectroscopy, single-molecule detection, dimers, plasmon resonance

1. INTRODUCTION Single-molecule (SM) detection is the ultimate goal of many sensing techniques. It is extremely difficult to detect an analyte at a single-molecule level because its signals are typically weak and often obscured by the background noise. In recent years, the use of surface-enhanced Raman spectroscopy (SERS) has made incredible progress toward the detection of single molecules due to two factors.1-3 First, the Raman signal from a molecule, while intrinsically weak, arises from its unique vibrational modes, so it can be used like a finger-print to reveal the identity of an analyte. Second, when the molecule is placed in the strong electric field generated at the surface of a plasmonic metal nanocrystal, the Raman signal can be enormously enhanced by many orders of magnitude to greatly boost up the sensitivity. In SERS, the enhancement is critically dependent on the localized surface plasmon resonance (LSPR) of the metal nanocrystal.4

The LSPR can be described as the collective oscillation of the conduction electrons in a metal nanocrystal via excitation at a particular wavelength of electromagnetic radiation. The LSPR of a metal nanocrystal can be tuned by manipulating its size, shape, and composition, as well as its interactions (i.e., plasmonic coupling) with neighboring particles.5-6 In addition to the selective absorption and scattering of light, the LSPR generates an electric field at the surface of the nanocrystal. Significantly, this electric field is much stronger at the sharp corners of a nanocube, or in the gap between two nanocrystals, than other regions of the particle(s). Such areas containing extraordinarily strong surface electric fields are therefore referred to as “hot spots”. The field enhancement in a hot spot can be sufficiently strong to surpass the threshold for SM detection.7 The possibility of SM detection has been proposed and modeled theoretically, but it has been exceedingly difficult to prove experimentally.8

1.1 Conventional approaches to single-molecule detection

The most commonly used approach to SM detection experimentally is to use an extremely low concentration of SERS active molecules and a system consisting of random aggregates of metal nanoparticles.1-3 Although low concentrations are used to approximate that only one molecule is present for each particle or aggregate, this approach involves too many uncontrolled parameters, including the size of the aggregates and number of molecules per aggregate. As a result, though these experiments seem promising, they cannot guarantee SM sensitivity. Another approach which has become more common is the bianalyte approach.8 This technique uses two different molecules at relatively high concentrations and analyzes the ratio of intensities between the SERS signal from these two types of molecules. The expected result is a ratio of signal intensities similar to the ratio of molecules in the initial mixture. However, occasionally the signal of only one molecule dominates and it is assumed that a pure group of very few molecules located in a hot spot are being probed. Again, these experiments cannot guarantee that the signals measured indeed come from a single molecule, but they

Invited Paper

Micro- and Nanotechnology Sensors, Systems, and Applications IV, edited by Thomas George, M. Saif Islam, Achyut Dutta, Proc. of SPIE Vol. 8373, 837321

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provide more concrete support for the role of hot spots in significantly enhancing signal from a very small number of molecules.

1.2 Designing and measuring hot spots

Designing nanocrystal systems with well-defined and controllable hot spots is the ultimate goal of SERS and related applications. In our group, we have not only synthesized Ag nanocubes and nanobars with sharp corners which are well-suited for SERS applications, but also fabricated dimers composed of Ag nanocrystals.9-11 These dimers have hot spots in the gap region between the particles, and molecules trapped in the gap contribute the greatest amount to the SERS signal. Dimers are advantageous to other aggregated nanoparticle systems in that the orientation of particles, size of particles, and number of particles in an aggregate can all be readily controlled.

The ability of a nanocrystal or an aggregate to enhance the Raman signal is measured by the SERS enhancement factor (EF). The EF is calculated using the formula (ISERS × Nbulk)/(Ibulk × NSERS), where the intensity of a particular vibrational band of the molecule is compared under SERS conditions (ISERS) and ordinary Raman conditions (Ibulk).7,10,11 The signal intensity is normalized to the number of molecules probed under each condition (Nbulk and NSERS). Therefore, the EF represents the signal enhancement which is provided by the nanocrystals (including hot spots), and can be used to quantify the effectiveness of a particular substrate for SERS. The minimum EF for SM detection is estimated to be on the order of 107, and so this figure is used as a benchmark for designing substrates for SM-SERS.7

Most recently, we have demonstrated the capability for SM detection by using the bianalyte approach and the hot spots formed between a Ag nanocube and a Ag substrate. Given that the SERS EFs calculated for Ag dimers and Ag nanocube-substrate hot-spots are in the same order of magnitude, Ag dimers are promising for use in the detection of molecules close to the single molecule level.

2. SILVER NANOCRYSTALS AS SERS SUBSTRATES 2.1 Individual silver nanocrystals

Individual Ag nanocrystals are excellent SERS substrates albeit their EFs may not be sufficiently high for SM detection. However, SERS measurements on individual nanocrystals with well-defined sizes and shapes can provide useful information with regard to SERS and the hot-spot phenomenon. To this end, our group has synthesized single-crystal Ag nanocubes, nanospheres, and nanobars, to name a few, using a solution-phase method based on polyol reduction.5,12-13 The control and fine tuning over both the shape and size have proven critical for using these Ag nanocrystals as SERS substrates.9,13,14

Because of the formation of hot spots at the corners of a Ag nanocube, the SERS enhancements of a nanocube were shown to be about three orders of magnitude higher than those from a Ag nanosphere of a similar size (Figure 1).14 However the SERS EF of a Ag nanocube was only on the order of 105, which is still not high enough on its own to detect single molecules. The minimum EF for SM detection is estimated to be on the order of 107, just two orders of magnitude larger.7 So, while individual nanocrystals are interesting on their own, it should be more interesting to use them as building blocks to generate assemblies such as dimers in an effort to create more powerful hot spots and increase their likelihood for SM detection.

Figure 1. Comparison of the SERS spectra of 1,4-benzenedithiol (1,4-BDT) adsorbed onto Ag nanocrystals of two different shapes: (a) cube (38 nm edge length) and (b) sphere (35 nm diameter), as well as the ordinary Raman spectrum (c) of 1,4-BDT itself. The SERS intensity from 1,4-BDT on a cube is much greater than on a sphere, with an EF of 5.8 × 105 and 5.7 × 102, respectively. Adapted with permission from Ref. 14. Copyright 2009 American Chemical Society.

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2.2 Dimers of silver nanocrystals

Our group has demonstrated several approaches to the fabrication of dimers from Ag nanocrystals. A common approach for forming dimers from individual nanoparticles is to manipulate the stability of the colloidal suspension. Capillary forces and hydrophobic interactions can also be used to drive dimer formation, and are particularly useful for forming dimers with Ag nanocubes by taking advantage of their inherent asymmetry.

Manipulating colloidal stability can be accomplished by disrupting the balance of attractive and repulsive forces between particles, as described by the Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory.15 In short, the DLVO theory describes the overall potential energy of a colloidal system, in which the sum of the attractive van der Waals forces and the repulsive electrostatic forces regulates the interparticle interactions.11,15 The simplest way to disrupt this stability is to decrease the repulsive electrostatic forces, which is typically accomplished by increasing the electrolyte concentration in the suspension medium.

In one approach, we added Fe(NO3)3 to a suspension of Ag nanocubes to increase the electrolyte concentration of the solution while simultaneously etching the corners of the cubes. This resulted in a suspension of nearly 60% dimers of Ag nanospheres (Figure 2).10 When used as SERS substrates, these dimers exhibited EFs of about 108.

Figure 2. (A) Dimers of Ag nanospheres synthesized by etching Ag nanocubes with Fe(NO3)3. (B) SERS spectra of 4-methylbenzenethiol collected from the dimers with two different orientations, and comparing the SERS to an individual Ag nanosphere. The arrow indicates the direction of the excitation laser polarization. Adapted with permission from Ref. 10. Copyright 2010 Wiley.

Introducing NaCl during the early stages of a polyol synthesis could also induce the formation of Ag nanosphere dimers.11 This approach, however, has size limitations because large nanocrystals tend to develop facets on the surface. If the nanocrystals grow much larger than 30 nm, for example, they develop into cubes. As the shape of the monomer increased in complexity from a sphere to a cube, the number of different possible conformations between the two monomers increased. Two cubes can interact face-to-face, for example, or edge-to-edge. As a result, the dimers of cubes were in uncontrolled orientations with respect to one another, yielding a non-uniform product. This is not desirable due to the sensitivity of the LSPR and resulting hot spots on shape and orientation of nanocrystals within the dimer. Due to the smaller size of the nanosphere dimers, the EF was slightly lower, about 107, however, this is still in the range for SM detection.

Capillary forces during drying or template-assisted self-assembly (TASA) can both be used to form dimers that are supported on a solid substrate.9,16 During drying, dimers of Ag nanocubes form randomly on substrates, and the majority of these dimers tend to form face-to-face conformations. These structures have allowed us to probe the hot spot SERS EF apart from the other regions on the dimer.9 After functionalizing dimers of cubes with SERS active molecules, the molecules outside of the hot-spot were removed by plasma cleaning. The EF from the hot spot was calculated to be 108, demonstrating further that, though small, the hot spot can produce fields sufficiently high for SM detection.

By using hydrophobic interactions to drive dimer formation, individual facets of Ag nanocubes can be selectively functionalized with hydrophobic, self-assembled monolayers (SAMs).17 The hydrophobic interactions can drive dimer formation in a more highly controlled manner, since the dimers of cubes formed using this approach were all interacting face-to-face.

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The simplest approach of all, however, is to create a hot spot in the gap between an individual metal nanocrystal and a metal surface. When Ag nanocubes are simply deposited onto Au or Ag surfaces, the spaces between the corners of the nanocube and the metal surface naturally become hot spots.18 Recently, we have used hot spots generated in this manner to demonstrate SM detection.

3. SINGLE-MOLECULE DETECTION When Ag nanocubes were deposited onto Au or Ag substrates, very small hot spots were generated at the corners of the cubes in contact with the metal surface, as shown in Figure 3. The substrate material is important, since the strength of the hot spot depended on the ability of the substrate to support a surface plasmon. Like the dimers formed between two metallic nanocrystals, the LSPR coupling between the two surfaces generates much higher electric fields than the nanocrystal alone. The EF in these hot spots was calculated to be about 108, the same as the dimers of larger Ag nanospheres and Ag nanocubes described above.18

Figure 3. (A) The orientation of a Ag nanocube on the Au substrate. The green line indicates the direction of laser polarization. (B) The E-field enhancement in the gray region from (A) calculated using the DDA method. The cube itself is 2 nm above the surface, and the plane plotted is located 1 nm above the surface. Adapted with permission from Ref. 18. Copyright 2011 Wiley.

Figure 4. (A) Dark field image of Ag nanocubes, with an overlay indicating the relative SERS intensities of crystal violet (CV) (red) and rhodamine 6G (R6G) (green). (B) A histogram demonstrating the distribution of the ratio of SERS intensities between the two molecules. The extreme events (%R6G <20 % or > 80%) indicate hot spot phenomena, where the signal is dominated by a small number of one type of molecule. Adapted with permission from Ref. 18. Copyright 2011 Wiley.

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To demonstrate SM detection capability, we used the bianalyte technique, in which two SERS-active dyes were mixed with cubes before deposition on the substrate. We mapped out the SERS active particles using a color overlay to indicate relative intensity of the two dye molecules, shown in the dark-field image in Figure 4A. Most particles should exhibit equal signal intensity from each dye since equal concentrations of each dye were used in the sample preparation. However, we found that the signals measured from many particles were dominated by just one of the dyes (Figure 4). In particular, when the concentration of each dye was 100 nM, the effect was more pronounced than when the concentration was slightly higher. As shown in the histogram in Figure 4B, there was a distribution of relative SERS intensities. About 10 nanocubes demonstrated signal from mostly crystal violet (CV), and several nanocubes exhibited signal dominated by rhodamine 6G (R6G). This pattern indicates that the signal was obtained from just a few molecules, and possibly just one, properly positioned in the hot spot.

4. CONCLUSIONS Controlling the hot spots of Ag nanocrystals can be a very powerful tool for detection of analyte molecules present in extremely low concentrations, which is of great interest to many communities, especially defense and medicine. Because SERS is a highly specific technique, it has become the focus of many molecular detection approaches. For SERS to become practical and reliable, the signal enhancement needs to be well controlled and understood. Dimers composed of Ag nanospheres and Ag nanocubes can reproducibly fabricated, and make the study of hot spot EFs more reliable. The gaps present in Ag nanocrystal dimers provide an area of high signal enhancement, which have shown to be great enough for single-molecule detection. These dimers, therefore, have great potential for practical sensing applications.

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