Mixed Monolayers on Gold Nanoparticle Labels for Multiplexed

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Anal. Chem. 2009, 81, 9643–9650

Mixed Monolayers on Gold Nanoparticle Labels for Multiplexed Surface-Enhanced Raman Scattering Based Immunoassays Gufeng Wang, Hye-Young Park,† and Robert J. Lipert* Institute for Physical Research and Technology, Ames LaboratorysU.S. Department of Energy, and Department of Chemistry, Iowa State University, Ames, Iowa 50011 Marc D. Porter Departments of Chemistry, Chemical Engineering, Bioengineering, and Pathology, University of Utah, 383 Colorow Drive, Salt Lake City, Utah 84108-1201 This paper describes a new approach, based on selfassembled mixed monolayers, to the design and preparation of extrinsic Raman labels (ERLs). ERLs function as spectroscopic tags for the readout of sandwich-type immunoassays using surface-enhanced Raman scattering (SERS). They are created by coating gold nanoparticles with Raman reporter molecules and antibodies specific for the target analyte. Mixed-monolayer ERLs are formed by covering gold nanoparticles with a mixture of two different thiolates. One thiolate serves to covalently bind antibodies to the particles, imparting biospecificity to the ERLs, while the other thiolate produces a strong Raman signal. Mixed-monolayer ERLs can be prepared in a few relatively simple steps using readily available materials. The SERS intensity of each type of ERL can be tuned to match other ERLs by adjusting the mixed monolayer composition, greatly facilitating the generation of sets of ERLs for multiplexed applications. The work herein not only describes the new pathway for ERL production, but also demonstrates the simultaneous qualitative and quantitative multiplexed detection using a set of four mixedmonolayer ERLs. Recent developments in nanometric materials and spectroscopic instrumentation have rekindled widespread interest in both the fundamental underpinnings1-4 and applications of surfaceenhanced Raman scattering (SERS).5-9 With respect to the latter, several laboratories have explored the use of SERS as a readout * Corresponding author. E-mail: [email protected]. Phone: 515-294-8837. Fax: 515-294-0062. † Current address: Institut Pasteur Korea, Sampyeong-dong 696, Bundanggu, Seongnam-si, Gyeonggi-do, Korea 463-400. (1) Otto, A.; Futamata, M. Top. Appl. Phys. 2006, 103, 147–182. (2) Stockman, M. I. Top. Appl. Phys. 2006, 103, 47–66. (3) Schatz, G. C.; Young, M. A.; Van Duyne, R. P. Top. Appl. Phys. 2006, 103, 19–46. (4) Moskovits, M. Top. Appl. Phys. 2006, 103, 1–18. (5) Smith, W. E.; Faulds, K.; Graham, D. Top. Appl. Phys. 2006, 103, 381– 396. (6) Jarvis, R.; Clarke, S.; Goodacre, R. Top. Appl. Phys. 2006, 103, 397–408. (7) Vo-Dinh, T.; Yan, F.; Wabuyele, M. B. Top. Appl. Phys. 2006, 103, 409– 426. 10.1021/ac901711f CCC: $40.75  2009 American Chemical Society Published on Web 10/29/2009

technique in bioassays.10-21 Our laboratory has mainly focused on sandwich-style immunoadsorbent assays using labels based on chemically modified gold nanoparticles, which we refer to as extrinsic Raman labels (ERLs). ERLs are designed to serve two proposes: (1) to bind selectively to a captured antigen and (2) to yield a strong Raman spectroscopic signature through the SERS effect. With these labels, the identity of each antigen is determined from the characteristic SERS spectrum of the nanoparticle-bound reporter species, whereas the amount of antigen is quantified by the spectral intensity. The advantages of this detection method derive largely from three features of this labeling strategy. First, the intensity of the response for the immobilized reporters rivals that of fluorescent dyes.22 In some cases, SERS has proven sufficiently sensitive to detect single molecules.23-32 Second, the widths of Raman spectral (8) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev. 1999, 99, 2957–2975. (9) Park, H. Y.; Driskell, J. D.; Kwarta, K. M.; Lipert, R. J.; Porter, M. D.; Schoen, C.; Neill, J. D.; Ridpath, J. F. Top. Appl. Phys. 2006, 103, 427–446. (10) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science (Washington, D.C.) 2002, 297, 1536–1540. (11) Docherty, F. T.; Clark, M.; McNay, G.; Graham, D.; Smith, W. E. Faraday Discuss. 2004, 126, 281–288. (12) Graham, D.; Goodacre, R. Chem. Soc. Rev. 2008, 37, 883–884. (13) Graham, D.; Mallinder, B. J.; Smith, W. E. Biopolymers 2000, 57, 85–91. (14) Graham, D.; Mallinder, B. J.; Whitcombe, D.; Smith, W. E. ChemPhysChem 2001, 2, 746–748. (15) Graham, D.; Mallinder, B. J.; Whitcombe, D.; Watson, N. D.; Smith, W. E. Anal. Chem. 2002, 74, 1069–1074. (16) Kneipp, K.; Flemming, J. J. Mol. Struct. 1986, 145, 173–179. (17) Kneipp, K.; Kneipp, H.; Kartha, V. B.; Manoharan, R.; Deinum, G.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 1998, 57, R6281–R6284. (18) Kneipp, K.; Pohle, W.; Fabian, H. J. Mol. Struct. 1991, 244, 183–192. (19) McHugh, C. J.; Docherty, F. T.; Graham, D.; Smith, W. E. Analyst 2004, 129, 69–72. (20) Sun, L.; Yu, C. X.; Irudayaraj, J. Anal. Chem. 2007, 79, 3981–3988. (21) Sun, L.; Yu, C. X.; Irudayaraj, J. Anal. Chem. 2008, 80, 3342–3349. (22) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. J. Phys.: Condens. Matter 2002, 14, R597–R624. (23) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667–1670. (24) Le Ru, E. C.; Meyer, M.; Etchegoin, P. G. J. Phys. Chem. B 2006, 110, 1944–1948. (25) Le Ru, E. C.; Etchegoin, P. G.; Meyer, M. J. Chem. Phys. 2006, 125. (26) Kneipp, K.; Kneipp, H. Appl. Spectrosc. 2006, 60, 322A.

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Scheme 1. Representation of ERLs Prepared with (A) Coimmobilization, (B) Bifunctional Reporter, and (C) Mixed Monolayer

features for most organic compounds are typically 10-100 times narrower than those of fluorescence. This characteristic minimizes the potential for overlap in the response from different labels. Third, unlike fluorescent labels, Raman labels are not susceptible to photobleaching and quenching. We have employed differing versions of this strategy in the low-level detection of proteins [e.g., immunoglobulin G (IgG)33,34 and prostate-specific antigen (PSA)35,36], viruses (feline calicivirus37 and porcine parvovirus9), and bacteria (Escherichia coli O157: H7,9 Mycobacterium avium subsp. paratuberculosis,38,39 and Erwinia herbicola,9 a simulant for the bacterium Yersinia pestis, a potential biowarfare agent). In our earlier investigations, ERLs were prepared following two different pathways. In the first approach, the ERL was formed by coimmobilization of both the antibody and Raman label directly onto the surface of the gold nanoparticle,33 as depicted in Scheme 1A. Note that the antibody was simply adsorbed onto the particle surface, while the reporter molecule was coupled to the gold surface by the chemisorption of an aromatic thiol or disulfide moiety. While successfully applied to the concurrent qualitative analysis of two biolytes (i.e., rat and rabbit IgG), questions remained regarding the cause of the observed apparent nonspecific adsorption of ERLs. If ERLs were exchanging weakly adsorbed antibodies, specific antibody-antigen binding could result in the capture of the wrong reporter molecule, which would appear to be nonspecific binding. Thus, the simple adsorption of antibodies to the nanoparticles could lead to “cross-talk” between different ERLs present in the same suspension, resulting in false positive readings. (27) Kneipp, K.; Kneipp, H.; Bohr, H. G. Top. Appl. Phys. 2006, 103, 261–278. (28) Jiang, J.; Bosnick, K.; Maillard, M.; Brus, L. J. Phys. Chem. B 2003, 107, 9964–9972. (29) Xu, H. X.; Aizpurua, J.; Kall, M.; Apell, P. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 2000, 62, 4318–4324. (30) Xu, H. X.; Bjerneld, E. J.; Kall, M.; Borjesson, L. Phys. Rev. Lett. 1999, 83, 4357–4360. (31) Michaels, A. M.; Nirmal, M.; Brus, L. E. J. Am. Chem. Soc. 1999, 121, 9932–9939. (32) Nie, S.; Emory, S. R. Science (Washington, D.C.) 1997, 275, 1102–1106. (33) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, 4903–4908. (34) Driskell, J. D.; Uhlenkamp, J. M.; Lipert, R. J.; Porter, M. D. Anal. Chem. 2007, 79, 4141–4148. (35) Grubisha, D. S.; Lipert, R. J.; Park, H.-Y.; Driskell, J.; Porter, M. D. Anal. Chem. 2003, 75, 5936–5943. (36) Park, H.-Y.; Lipert, R. J.; Porter, M. D. Proc. SPIE 2004, 464–477. (37) Driskell, J. D.; Kwarta, K. M.; Lipert, R. J.; Porter, M. D.; Neill, J. D.; Ridpath, J. F. Anal. Chem. 2005, 77, 6147–6154. (38) Yakes, B. J.; Lipert, R. J.; Bannantine, J. P.; Porter, M. D. Clin. Vaccine Immunol. 2008, 15, 227–234. (39) Yakes, B. J.; Lipert, R. J.; Bannantine, J. P.; Porter, M. D. Clin. Vaccine Immunol. 2008, 15, 235–242.

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To address this potential limitation, we developed the process in Scheme 1B, which used a bifunctional Raman reporter molecule, 5,5′-dithiobis(succinimidyl-2-nitrobenzoate) (DSNB), to covalently couple the antibody to the particles and thus prevent antibody exchange between ERLs.35 We synthesized DSNB to take advantage of its following properties. First, the symmetric nitro stretching [νs(NO2)] of DSNB has a very large intrinsic Raman cross section. Second, the disulfide group of DSNB reacts with gold to form a thiolate adlayer. Third, the resulting structure of the DSNB-derived adlayer ensures that the nitro group is covalently immobilized in close proximity to the particle surface, which is important for maximizing Raman scattering.40 Fourth, DSNB contains succinimidyl groups that react with amine groups for the covalent attachment of antibodies to the particles. Scheme 1B improved particle stability and reduced the limit of detection via a lower level of nonspecific ERL adsorption.33,35 Using this type of ERL, we recently reported on the femtomolar detection of PSA directly in human serum.35 This approach, while working with a high level of effectiveness, nevertheless required the synthesis of the bifunctional reporter. This paper introduces an alternative design for ERLs that has a comparable level of performance but features a much more facile route to fabrication. Scheme 1C introduces the new design, which incorporates the attributes of a bifunctional reporter, while eliminating its synthesis. In this scheme, the surface of gold nanoparticles is modified with two different thiolates, each formed by using commercially available thiols. One thiolate is derived from the bifunctional compound dithiobis(succinimidyl propionate) (DSP), which has both disulfide and succinimidyl functionalities for the respective chemisorption onto gold and the facile covalent coupling of antibodies to the particle. DSP, however, is an intrinsically weak Raman scatterer. Consequently, the second thiolate component consists of molecules that have large Raman cross sections and serve as the reporter species. This scheme therefore facilitates the production of distinctive ERLs, hereafter referred to as mixed-monolayer ERLs, for multianalyte assays by using different antibodies paired with a unique thiol reporter molecule. As mentioned, the narrow line widths of Raman spectral bands make markers based on Raman labels attractive for multiplexed applications. Recently, there has been considerable interest in exploiting this potential,14,15,20,41-53 and several approaches have (40) Kennedy, B. J.; Spaeth, S.; Dickey, M.; Carron, K. T. J. Phys. Chem. B 1999, 103, 3640–3646. (41) Graham, D.; Smith, W. E.; Linacre, A. M. T.; Munro, C. H.; Watson, N. D.; White, P. C. Anal. Chem. 1997, 69, 4703–4707. (42) Kim, K.; Lee, H. S.; Kim, N. H. Anal. Bioanal. Chem. 2007, 388, 81–88. (43) Jun, B. H.; Kim, J. H.; Park, H.; Kim, J. S.; Yu, K. N.; Lee, S. M.; Choi, H.; Kwak, S. Y.; Kim, Y. K.; Jeong, D. H.; Cho, M. H.; Lee, Y. S. J. Comb. Chem. 2007, 9, 237–244. (44) Chen, J. W.; Jiang, J. H.; Gao, X.; Gong, J. L.; Shen, G. L.; Yu, R. Q. Colloids Surf., A 2007, 294, 80–85. (45) McCabe, A. F.; Eliasson, C.; Prasath, R. A.; Hernandez-Santana, A.; Stevenson, L.; Apple, I.; Cormack, P. A. G.; Graham, D.; Smith, W. E.; Corish, P.; Lipscomb, S. J.; Holland, E. R.; Prince, P. D. Faraday Discuss. 2006, 132, 303–308. (46) Kim, J. H.; Kim, J. S.; Choi, H.; Lee, S. M.; Jun, B. H.; Yu, K. N.; Kuk, E.; Kim, Y. K.; Jeong, D. H.; Cho, M. H.; Lee, Y. S. Anal. Chem. 2006, 78, 6967–6973. (47) Vo-Dinh, T.; Yan, F.; Wabuyele, M. B. J. Raman Spectrosc. 2005, 36, 640– 647. (48) Su, X.; Zhang, J.; Sun, L.; Koo, T. W.; Chan, S.; Sundararajan, N.; Yamakawa, M.; Berlin, A. A. Nano Lett. 2005, 5, 49–54.

been used to construct labels. These labels are often composed of metallic nanoparticles, dye-based Raman reporter molecules for use in resonance enhanced Raman scattering, and coatings made up of polymers or silica.54 The coatings are then functionalized to impart analyte specificity. The design introduced herein simplifies the generation of ERLs for multiplexed applications. These labels do not require a polymeric or silica coating or resonance-enhanced reporter molecules. Our use of structurally simpler compounds reduces the possibility of spectral overlap. This approach facilitates the identification of sets of ERLs with minimal spectral overlap. We will show that these ERLs have a level of performance, with respect to sample handling and detection, comparable to ERLs based on DSNB and can be readily applied to multianalyte assays for different IgGs. EXPERIMENTAL SECTION Reagents. Gold nanoparticles (60 nm in diameter, 2.6 × 1010 particles/mL) were purchased from Ted Pella. Polyclonal goat anti-human IgG, goat anti-mouse IgG, goat anti-rabbit IgG, human IgG, mouse IgG, rabbit IgG, StartingBlock, and borate buffer packs (50 mM) were acquired from Pierce Biotechnology. Polyclonal goat anti-rat IgG and rat IgG were obtained from USBiological. Polyclonal goat-hosted antibodies were chosen to have minimal cross-reactivities with as many other analytes as possible. Octadecanethiol (ODT), sodium chloride, DSP, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS) packs (10 mM), and bovine serum albumin (BSA) were procured from Sigma Aldrich. Two-part epoxy was purchased from Epotek. Raman reporter molecules 4-nitrobenzenethiol (4-NBT), 2-methoxybenzenethiol (2-MeOBT), 3-methoxybenzenethiol (3-MeOBT), 4-methoxybenzenethiol (4-MeOBT), and 2-naphthalenethiol (NT) were obtained from Sigma Aldrich at the highest purity available. DSNB was synthesized according to a recent literature procedure35 and stored in acetonitrile with a concentration of 1.0 mM. All buffers were prepared in deionized water and then passed through a membrane filter (Costar) with a 0.22-µm pore size. Preparation of ERLs. ERLs were prepared from gold nanoparticles by first depositing a monolayer of functional thiol molecules by spontaneous adsorption. The purpose of the monolayer is 2-fold: (1) to produce a strong Raman signal and (2) to provide an amide linkage to antibodies. In the bifunctional reporter approach, a single compound, DSNB, serves both purposes, while in the mixed monolayer approach, two thiols, DSP and a Raman reporter, are added to the gold nanoparticle surface simultaneously. The method for preparing DSNB-coated ERLs has been described earlier.37 Briefly, a 1.0 mL gold nanoparticle colloidal suspension was first adjusted to pH 8.5 by adding 40 µL of 50 mM borate buffer. Next, 10 µL of 1.0 mM DSNB in acetonitrile was added to the gold colloidal suspension and mixed for 8 h. Excess DSNB was then removed from the suspension by centrifuging at 2000g for 10 min and carefully removing the (49) Doering, W. E.; Nie, S. M. Anal. Chem. 2003, 75, 6171–6176. (50) Faulds, K.; Smith, W. E.; Graham, D. Anal. Chem. 2004, 76, 412–417. (51) Faulds, K.; McKenzie, F.; Smith, W. E.; Graham, D. Angew. Chem., Int. Ed. 2007, 46, 1829–1831. (52) Brown, L. O.; Doorn, S. K. Langmuir 2008, 24, 2277–2280. (53) Wang, C. G.; Chen, Y.; Wang, T. T.; Ma, Z. F.; Su, Z. M. Adv. Funct. Mater. 2008, 18, 355–361. (54) Brown, L. O.; Doorn, S. K. Langmuir 2008, 24, 2178–2185.

supernatant with a syringe. The gold particles were resuspended in 2.0 mM borate buffer and incubated with 20 µg of antibody for 16 h, followed by exposure to 100 µL of 10% BSA for 8 h to block nonspecific binding sites. The suspension was then centrifuged, the clear supernatant removed, and the loose red sediment resuspended in 1.0 mL of 2.0 mM borate buffer containing 1% BSA. The centrifugation and resuspension process was repeated two additional times, and the particles were subsequently resuspended in 0.5 mL of 2.0 mM borate buffer containing 1% BSA. An appropriate volume of 10% NaCl solution was added to yield a final NaCl concentration of 150 mM to promote antibody-antigen binding. As a final step, the labeled nanoparticles were passed through a 0.22-µm syringe filter (Costar) to remove any large aggregates. The preparation of mixed monolayer ERLs used a protocol modified from the above procedure. After adjusting the pH of the gold colloidal suspension, a total of 2.26 nmol of mixed thiols (i.e., the linker molecule DSP and a Raman reporter) was added and mixed for 10 min. The DSP:reporter mole ratio was 1:4 unless otherwise specified. After thiolation, 20 µg of antibody was added to the suspension and the remainder of the process followed that described above for DSNB ERLs. Preparation of Capture Substrates and Assay Procedures. The method for preparing the antigen-capture substrate was modified from the protocol described earlier.37 Briefly, templatestripped gold (TSG)55 served as the substrate. To make TSG, silicon wafers (test grade, University wafers) were cleaned in ethanol and dried with a stream of high-purity nitrogen gas. The cleaned wafers were coated with a 250-nm gold film using an Edward 306A resistive evaporator. Glass microscope slides were cut into 1 × 1 cm squares and sonicated in diluted Contrad 70 (Micro, Cole-Parmer), deionized water four times, and ethanol twice, each for 30 min, and dried under a nitrogen stream. The clean glass chips were affixed to the gold-coated wafer with twopart epoxy (Epoxy Technology) and cured at 150 °C for 1.75 h. The glass chips were then gently detached from the silicon wafer. The sandwiched gold film remains on the topside of the glass chip to yield a smooth TSG surface. Next, an ODT monolayer was stamped onto the TSG substrate to define the assay address on the chip. In this process, a polydimethylsiloxane (PDMS, Dow Corning) stamp with a centered, 3-mm hole was immersed in 2.0 mM ODT for 1 min and dried with a stream of high-purity nitrogen gas. The dried PDMS stamp was then gently pressed onto a TSG substrate for 30 s. This process leaves an uncoated gold area (3-mm diameter) that is surrounded by a hydrophobic ODT monolayer that acts as a well to confine droplets of aqueous fluids. A 10-µL drop of 3.0 mM DSP in DMSO was added to the address for 25 min to create a DSP monolayer on the gold surface. The chip was then rinsed with ethanol and dried with high-purity nitrogen gas. Chips prepared in this manner can be used immediately or stored in a desiccator for several weeks. The primary capture antibody (20 µL, 100 µg/mL), diluted in 50 mM borate buffer (pH 8.5), was applied to the sample area and allowed to react for 8 h in a humidity chamber. This step forms a layer of capture antibodies that is coupled to the DSP (55) Stamou, D.; Gourdon, D.; Liley, M.; Burnham, N. A.; Kuik, A.; Vogel, H.; Duschl, C. Langmuir 1997, 13, 2425–2428.

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monolayer through carboxamide bonds formed between the amine groups on the protein and the succinimidyl ester of the DSP. The substrate was rinsed with 10 mM PBS, followed by exposure to 20 µL of StartingBlock blocking buffer for 16 h. The capture substrate was then exposed to 20-µL aliquots of analytes diluted in PBS buffer for 8 h in the humidity chamber. After rinsing with 2 mM borate buffer containing 150 mM NaCl, the captured analytes were exposed to 20 µL of the corresponding ERL suspension for 16 h. Finally, the substrates were rinsed with 2 mM borate buffer containing 150 mM NaCl before drying gently with a stream of high-purity nitrogen gas. SERS Measurement. The Raman spectra were collected with a NanoRaman I fiber-optic-based Raman system (Concurrent Analytical). The light source was a 30-mW, 632.8-nm He-Ne laser. The spectrograph consisted of an f/2.0 Czerny-Turner imaging spectrometer (6-8-cm-1 resolution) and a Kodak 0401E CCD thermoelectrically cooled to 0 °C. The incident laser light was focused to a 25-µm diameter spot on the substrate at normal incidence using an objective with a numerical aperture of 0.68. The power at the sample was ∼3 mW. The same objective was used to collect the scattered radiation. Signal integration times were varied according to the scattering intensity of the sample chip. The peak intensity used to quantify the analyte concentration was determined from an average of 20 spectra collected at 20 different spots in the assay address. RESULTS AND DISCUSSION Thiols as Raman Reporter Molecules for ERL Assembly. This study examines the feasibility of using mixed thiolate monolayers to prepare ERLs for multiplexed sandwich-type immunoassays. One thiolate serves to covalently bind antibodies to the particles, imparting biospecificity to the ERLs, while the other thiolate (e.g., a benzyl or naphthyl thiolate) produces a strong Raman signal. This approach, as noted earlier, serves as a simple pathway to produce ERLs with spectrally distinct Raman reporters, greatly facilitating multiplexed applications. We tested 14 thiol compounds for potential Raman reporter use and display the SERS spectra of a set of four reporters in Figure 1. The spectra were obtained following the chemisorption of the Raman reporters on 60-nm gold particles. The four reporters and the spectral feature used to identify each label are 4-NBT (1336 cm-1), 3-MeOBT (992 cm-1), 2-MeOBT (1037 cm-1), and NT (1384 cm-1). This set is suitable for a tetraplexed application. Tunable ERL Intensity. One major difficulty in developing multiplexed detection is to find reporters with distinct spectral features having comparable intensities. If the intensities differ significantly, the response of one reporter may dominate that of a mixture of labels and make it difficult to identify and quantify the signal from weaker reporters. The mixed monolayer approach provides a facile process for tuning the scattering intensity of the Raman labels in a mixture by changing the surface concentration of reporter molecules on the gold particles. In practice, this can be readily achieved by varying the linker (DSP) to reporter ratio added to the gold colloidal suspension during the monolayer formation step. To demonstrate this capability, ERL suspensions were prepared with different fractions of 4-NBT coverage on the gold nanoparticle surface. Each suspension was incubated with the same total number of moles of thiols (2.26 nmol) but with varying DSP:49646

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Figure 1. SERS spectra of a set of four Raman reporters for multiplexing. The reporter molecules 4-NBT (4-nitrobenzenethiol, black), 3-MeOBT (3-methoxybenzenethiol, red), 2-MeOBT (2-methoxybenzenethiol, green), and NT (naphthalenethiol, blue) were chemisorbed through a gold-sulfur bond on 60-nm gold nanoparticles. The particles were then deposited on a flat gold surface for spectrum collection. The peaks used for quantification were labeled with an asterisk. Spectra were normalized and shifted for illustration.

Figure 2. SERS intensity of ERLs as a function of the mole fraction of Raman reporter molecule (4-NBT) in the thiol mixture used to coat the gold nanoparticle surface. After monolayer formation, the ERLs were coated with goat anti-rabbit IgG and immobilized onto a flat gold surface coated with rabbit IgG. Intensities are of the 1336 cm-1 vibrational band.

NBT mole ratios. The ERLs were then coated with goat anti-rabbit IgG, incubated on a rabbit IgG-coated gold substrate for 16 h, and read out by SERS. Figure 2 displays the SERS intensity as a function of 4-NBT mole fraction added to gold colloidal suspension. It is clear that the SERS intensity is closely related to the DSP: 4-NBT mole fraction. The solid line is a least-squares fit of the data to a Langmuir isotherm with lateral interactions.56 The good agreement suggests that competitive chemisorption of the two types of thiols to the gold nanoparticle surface is a reasonable description of the coating process. When the 4-NBT mole fraction is below 0.1, the ERL intensity undergoes a near-linear increase with the mole fraction of 4-NBT. Note that over a wide range of 4-NBT mole fractions, the number of antibodies immobilized on the nanoparticles will remain contant because of the much larger size of the antibodies (200-500 times larger) relative to DSP. The (56) Adamson, A. W. Physical Chemistry of Surfaces, 5th ed.; John Wiley & Sons: New York, 1990; pp 770.

Figure 3. Immunoassay of mouse IgG using mixed-monolayer ERLs. (A) SERS spectra of the reporter molecule 4-NBT at analyte concentrations of 1000, 100, 10, 3, and 1 ng/mL and blank, from top to bottom. Spectra were shifted for illustration. (B) Dose-response curve quantified with the 1336 cm-1 peak of 4-NBT in a semilog scale. (C) Dose-response curve at low analyte concentration (below 10 ng/ mL) on a linear scale.

increase in SERS intensity appears to approach a plateau at high 4-NBT mole fractions, indicating surface saturation. The important conclusion is that the scattering intensity of the ERLs can be tuned by varying the reporter fraction in the mixed monolayer, imparting a high level of flexibility in the selection of Raman reporters for multiplexed applications. Biospecificity of ERLs Prepared with Mixed Thiol Monolayer. Biospecificity is achieved by covalently attaching antibody molecules to the gold nanoparticles using the bifunctional linker molecule DSP. Covalent linking is crucial in multiplexed detection, since it minimizes the possibility of crosstalk that could arise from the exchange of antibodies between different ERLs. We first examined the bioactivity of the antibodies attached to mixed-monolayer ERLs by running a standard sandwich-assay calibration curve using serially diluted solutions of mouse IgG as the analyte. Both the capture substrate and the ERLs were coated with goat anti-mouse IgG antibodies. Figure 3A shows the representative SERS spectra of the captured ERLs on the gold

substrate at different mouse IgG concentrations. The Raman reporter was 4-NBT, a simple molecule with the same vibrational band as DSNB. The spectral intensity increases in response to increasing analyte concentration, clearly demonstrating that ERLs prepared in this manner retain the binding affinity of the antibodies and are suitable for immunoassays. The presence of the mouse IgG was quantified by the strongest vibration peak, νs(NO2) at 1336 cm-1. The dose-response curve of this assay is presented in Figure 3B,C. Figure 3B shows the typical sigmoidal dose-response curve in a semilog scale for these sandwich assays. Figure 3C presents an expanded view on a linear scale of the dose-response curve at low concentrations. The curve is linear in this range with a slope (i.e., sensitivity) of 790 cps/ (ng/mL). The limit of detection (LOD), defined as the concentration that results in a signal equal to 3 times the standard deviation of the blank, is 0.7 ng/mL. As a comparison, Figure S-1A-C (Supporting Information) shows the SERS spectra and dose-response curves of the same mouse IgG assay using ERLs prepared using the bifunctional linker DSNB. The sensitivity of this assay is 310 cps/(ng/mL) and the LOD is 0.6 ng/mL. Compared to Figure 3A-C, the shapes of the dose-response curves using ERLs prepared with the two different methods are very similar and the detection limits are almost identical. This result demonstrates that ERLs prepared with the mixed monolayer approach did not lose any bioactivity or analytical sensitivity. We also examined the specificity of the mixed monolayer ERL binding. Anti-mouse IgG was used to coat the capture substrates and ERLs. The capture substrates were then incubated with either mouse IgG or human IgG and then labeled with the ERLs. The response of the capture substrate incubated at the very high concentration of 1.0 µg/mL human IgG was only 2300 cps. In comparison, the substrate incubated with 1.0 µg/mL mouse IgG produces a strong reading of 88 000 cps, ∼40 times higher than that of the human IgG. This shows that the antibodies on the mixed monolayer ERLs have retained their specificity. These ERLs are usually stable at 4 °C for at least 4 weeks without visible aggregation or detectable loss of biospecificity. With excellent stability, simple preparation, tunable intensity and biospecificity, mixed monolayer ERLs show good potential for multiplexed analysis. Qualitative Multianalyte Detection. In our multianalyte detection approach, the capture substrate is coated with a mixture of antibodies, each specific to a possible analyte in the sample. Correspondingly, ERLs specific to each antigen, each tagged with a unique Raman reporter, are prepared separately and mixed together. For analysis, the substrates are first exposed to the sample solution and then to the ERL mixture. Specific ERLs are retained and detected only if the corresponding antigen is present in the sample. Ideally, the set of Raman reporters should be selected so that each type of reporter can be detected and quantified without interference from other reporters. Figure 4 displays the results of a simultaneous, dual-analyte assay. Two kinds of antibodies, goat anti-rabbit and goat antimouse IgG, served as capture antibodies, and two sets of ERLs, one specific for rabbit IgG and one for mouse IgG, were prepared and combined. The mouse IgG-specific ERLs were tagged with 4-NBT (identification peak at 1336 cm-1) and rabbit IgG-specific Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

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Figure 4. Simultaneous detection two analytes: rabbit IgG (labeled as R) and mouse IgG (labeled as M). The reporter molecules are 4-MeOBT and 4-NBT, respectively.

ERLs were tagged with 4-MeOBT (1038 cm-1). There is no detectable overlap in the spectral response of the two labels. We note that the SERS response of 4-NBT is much stronger than that of 4-MeOBT, and so the mole fraction of 4-NBT used in the coating step was lowered by a factor of 60 (keeping the total number of moles of thiols constant) to realize comparable SERS intensities from both sets of ERLs. The substrates were exposed to solutions containing 100 ng/mL rabbit IgG and 100 ng/mL mouse IgG, 100 ng/mL mouse IgG only, 100 ng/mL rabbit IgG only, and PBS buffer (blank). After rinsing, the chips were incubated with the ERL mixture for 16 h. In Figure 4, the SERS spectra of the four samples are displayed. The top spectrum contains peaks from both 4-MeOBT at 1038 cm-1 and 4-NBT at 1336 cm-1, indicating the presence of both rabbit IgG and mouse IgG. The second spectrum has a strong peak at 1336 cm-1 and a negligible peak at 1038 cm-1, signaling the presence of mouse IgG. Similarly, the third curve has a strong peak at 1038 cm-1 and a clean background at 1336 cm-1, indicating the presence of mouse IgG and the absence of rabbit IgG. The fourth curve (the blank) shows the absence of both IgGs. These data begin to demonstrate the effectiveness of using mixed monolayers in the design of ERLs for a multiplexed assay. Figure 5 displays the simultaneous detection of three analytes using a different set of reporters: 3-MeOBT (992 cm-1), 2-MeOBT (1022 cm-1), and 4-NBT (1336 cm-1). The corresponding antigens are rabbit, human, and mouse IgG. As before, the 4-NBT coverage was adjusted to match the intensity of the other two reporters. Shown in Figure 5, from top to bottom, are the SERS spectra for a mixture of all three analytes, combinations of two analytes, individual analytes, and a blank. Thus, the SERS spectra qualitatively indicate the presence of the analytes, demonstrating successful multiplexing. In these multiplexed assays, we observed differences in antibody specificity. For example, ERLs specific to mouse IgG always give a clean background when the relevant antigen is absent. On the contrary, ERLs coated with anti-rabbit and antihuman IgGs often showed a small signal (below 500 cps) when the corresponding antigen is absent. Through repeated experiments involving testing different antibody lot numbers and sources 9648

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Figure 5. Simultaneous detection of three analytes: rabbit IgG (labeled as R), human IgG (labeled as H), and mouse IgG (labeled as M). The reporter molecules are 3-MeOBT, 2-MeOBT, and 4-NBT, respectively.

and varying the antibody-reporter pairings, the unwanted signals could not be eliminated and we concluded they were caused by antibody cross-reactivity. Moreover, the clean blank background for the anti-mouse IgG ERLs in all the spectra is a clear indication that the ERLs in a mixed suspension do not exchange antibodies or Raman reporters. As seen in Figure 5, nonspecific binding and weak crossreactions result in an increased background and the presence of features of nontargeted ERLs in all of the spectra. The cumulative result can be pronounced. See, for example, the peak at 1060 cm-1, which is common to all of the ERLs. The result is that the LODs in antibody-based multiplexed applications will be adversely affected. However, in most cases, we can set an appropriate threshold (e.g., 500 cps in Figure 5) as a criterion for positive identification of a specific antigen. We add that the mixed monolayer method can be applied to other multiplexing systems that use other molecular recognition mechanisms (e.g., aptamers or oligonucleotides). The above two examples show that we can qualitatively detect multiple analytes in a single assay by using the characteristic peaks of simple reporter molecules. It is worth noting that the SERS spectra of these reporters are relatively simple so that the multiplexed spectra are not as congested as those of resonance Raman dyes. Simultaneous Quantitative Detection of Four Analytes. In multiplexed quantitative detections, standard dose-response curves need to be established for each of the possible analytes, with and without the other analytes present. This section demonstrates the simultaneous quantitative detection of four analytes in a single assay on one address using mixed-monolayer ERLs. The assay chips were first coated with four antibodies, goat anti-mouse IgG, goat anti-human IgG, goat anti-rabbit IgG, and goat anti-rat IgG, in the same address using a solution containing all antibodies at an equal mole fraction. Corresponding ERLs were prepared separately and mixed at equal volumes to have a final gold particle concentration 4 times that of a single analyte assay. The Raman reporters used for goat anti-mouse IgG, goat antihuman IgG, goat anti-rabbit IgG, and goat anti-rat IgG were 4-NBT, 2-MeOBT, 3-MeOBT and NT, respectively. Again, the surface

Figure 6. Quantitative detection of four analytes using single analyte chips and a tetraplexed chip. Dose-response curves of (A) mouse IgG, black squares; (B) human IgG, green circles; (C) rabbit IgG, red triangle up; and (D) rat IgG, blue triangle down. The open symbols and dashed lines are results obtained from a single analyte chip, and the solid symbols and lines were measured on the tetraplexed chip. The reporter molecules are 4-NBT, 3-MeOBT, 2-MeOBT, and NT for mouse IgG, human IgG, rabbit IgG, and rat IgG, respectively. Also shown in each graph are the signals in the three nonanalyte channels as the analyte concentration was varied. Table 1. Tetraplexed Detection of Four IgG’s analyte

Raman reporter

Raman peak (cm-1)

mouse IgG

4-NBT

1336

human IgG

2-MeOBT

1037

rabbit IgG

3-MeOBT

992

rat IgG

NT

1384

single analyte tetraplexed single analyte tetraplexed single analyte tetraplexed single analyte tetraplexed

mole fraction of 4-NBT was reduced by a factor of 60 to match the intensity of the ERLs having other Raman reporters. The chips were exposed to a series of solutions of a single analyte at varying concentrations and then to the ERL mixture. The spectral intensities of all reporters were measured simultaneously and corresponding dose-response curves were constructed. Crossreactivity of antibodies and antigens will show up as signal in the wrong analyte channel, e.g., increasing rat signal when the mouse IgG concentration is increased. Figure 6 shows the individual dose-response curves of each analyte collected from the tetraplexed chip. For comparison, the dose-response curve of each analyte of interest was collected on single-analyte chips in a parallel experiment and is also displayed. Note first that the background (i.e., sample blanks) of all multiplexed dose-response curves has increased compared to the single analyte assays, but to different extents (Table 1). For example, the single analyte assay for mouse IgG assay has background signal of 23 cps but 894 cps (∼40× higher) for the tetraplexed format. Similarly, human IgG, rabbit IgG, and rat IgG assays display an increase in the background level of 6×, 16×,

sensitivity [cps/(ng/mL)]

background level (cps)

LOD (ng/mL)

121 102 102 94 99 24 88 72

23 894 14 77 79 1255 32 129

0.3 3.3 0.3 1.2 1.3 9.0 0.8 2.9

and 4×, respectively. We add that the background level is fairly constant in the presence or absence of other analytes, indicating it is dominated by the cross-reactivity between the antibodies on the ERLs and the antibodies on the capture surface and not by cross-reaction between antibodies and antigens. Because nontarget analytes have minimal effect on background levels, the generation of standard curves is simplified, but the higher background levels have an adverse impact on multiplexed detection limits. Figure 6A shows the single analyte and tetraplexed mouse IgG assays. As found in Table 1, the background of the tetraplexed IgG assay has significantly increased, but the sensitivity undergoes only a slight decrease. The higher background thus degrades the LOD from 0.3 to 3.3 ng/mL. Figure 6A also shows the background responses for other analytes as a function of mouse IgG concentration. Again, the strengths of the backgrounds are fairly constant as the mouse IgG varies over a wide concentration range, which further demonstrates that the observed nonspecific adsorption results from the interaction between the antibodies on the ERLs and on the capture substrate. Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

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Figure 6B shows the dose-response curves for human IgG assays using 2-MeOBT as the reporter. The background level increased slightly from 14 to 77 cps for the tetraplexed format. The sensitivities are similar for the single analyte and tetraplexed formats. Therefore, the observed LOD increase from 0.3 to 1.2 ng/mL is again due to the increased background. Figure 6C shows the dose-response curves for rabbit assays using 3-MeOBT as the reporter. The background level increased significantly for the tetraplexed format, resulting in an increase in LOD by almost an order of magnitude from 1.3 to 9.0 ng/mL. Figure 6D shows the dose-response curves for rat assays using NT as the reporter. The background level increased from 32 to 129 cps, but the sensitivity remained about the same. This led to an increased LOD from 0.8 to 2.9 ng/mL when changing from a single analyte assay to tetraplexed format. We noticed in these assays that anti-mouse IgG antibody displayed a significant background signal (∼900 cps) due to crossreactivity, which is contrary to the earlier examples (Figures 4 and 5), where anti-mouse showed negligible cross-reactivity background. This resulted from the use of different batches of antibodies in these experiments. The differences in batch-to-batch quality in antibodies introduced variability in the experiments and had an impact on the LODs in the multiplexed detection. However, this variability should not affect quantification once the calibration curves are established for a specific batch of antibodies. The single analyte mouse IgG assay was performed with reduced 4-NBT surface coverage in Figure 6A. It is informative to compare it with the single analyte mouse IgG assay without reducing 4-NBT coverage (Figure 3B). The shapes of the two dose-response curves are basically the same except the reduced 4-NBT assay resulting in a reduced intensity level. Expectedly, a reduced analytical sensitivity was observed [121 cps/(ng/mL) at the lower 4-NBT coverage compared to 789 cps/(ng/mL) without reduced the 4-NBT coverage]. Interestingly, the LODs from the two assays are not appreciably different. The specific assay presented here shows an LOD of 0.3 ng/mL, compared to 0.7 ng/mL for the single analyte assay presented in Figure 3. We believe this is because our assay platform is sufficiently sensitive that the LOD is not determined by the readout sensitivity but rather by the nonspecific binding background. Thus, reducing

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the brightness of the Raman reporters does not affect the assay detection limit. CONCLUSIONS A new labeling design based on mixed monolayer coated gold nanoparticles was introduced and demonstrated with IgG assays. The mixed monolayer ERLs have proven to perform as well as ERLs based on a bifunctional reporter molecule. The illustrated examples clearly demonstrate that it is simple to use thiols to prepare multiple ERLs that can be qualitatively and quantitatively detected in a single assay at a single address. This is because the mixed monolayer approach provides a facile route to the generation of distinct spectroscopic tags with tunable intensity. While these examples show success in multiplexing a small number of analytes, the major obstacle to expanding this multiplexing capability to a larger number of analytes currently results from the cross-reactivity of the antibodies. While the antibodies we are using are of the highest quality that can be obtained from the industrial and research sectors, they still show significant levels of cross-reactivity at low detection levels (sub-ng/mL to ng/ mL of IgG molecules). With better antibodies, it will not be difficult to expand the multiplex number further. It is also anticipated that this mixed monolayer method would be very successful in bioassays using biorecognition elements such as oligonucleotides, which usually have a higher binding constant and a lower crossreactivity. Ongoing work is seeking to take advantage of this new type of ERL. ACKNOWLEDGMENT This work was supported by Concurrent Analytical, Inc. through a grant from the DARPA CEROS program and by the Institute for Combinatorial Discovery of Iowa Sate University. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract DE-AC02-07CH11358. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 30, 2009. Accepted October 10, 2009. AC901711F