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Polymer Microbead-Based Surface Enhanced Raman Scattering Immunoassays Lijuan Wei,† Bo Jin,*,†,‡ and Sheng Dai*,‡ †

School of Earth and Environmental Sciences, The University of Adelaide, Adelaide, SA 5005, Australia School of Chemical Engineering, The University of Adelaide, Adelaide, SA 5005, Australia

‡

S Supporting Information *

ABSTRACT: The high sensitive surface enhanced Raman scattering (SERS) makes its broad utilization in biomolecule recognition. In this study, a highly specific polymer microbead-based Raman/SERS immunoassay system is developed and evaluated. Different analytical techniques such as UV−visible spectrophotometry, transmission electron microscopy, Raman spectrometry, and fluorescence microscopy have been employed to investigate the feasibility and effectiveness of gold nanoparticles (AuNPs) and polymer microbeads for immunoglobulin (IgG) recognition. The developed polymer microbead-based Raman/SERS immunoassay includes functional polystyrene (PS) microbeads, AuNPs, and SERS reporters, where the carboxylated PS microbeads serve as the immune-solid support, and the self-assembled monolayer (SAM) of 4-mercaptobenzoic acid (4-MBA) formed on the surface of 50 nm AuNPs is used as the SERS tags. Antibodies (donkey antigoat IgG) are bioconjugated to the PS microbeads, which are able to selectively recognize the 4-MBA/AuNP-conjugated antigens (goat antihuman IgG). The specific recognition of matched antibody and antigen can be confirmed by both fluorescence imaging and Raman/SERS analysis. By combining the Raman signals of polymer microbeads and SERS tags, the system could have promising application for simultaneous multiplex detection in homogeneous immunoassay systems.

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INTRODUCTION The specific molecular recognition (such as antibody−antigen, biotin−avidin, DNA−DNA, or RNA) is of great importance in biological analysis. Among them, the antibody−antigen specific interaction is most widely used for human disease detection. Human immune system can generate various antigens on the surface of abnormal cells. The detection of those antigens can be utilized for disease diagnosis. Current techniques for antigen detection such as serological test and enzyme-linked immunosorbent assay (ELISA) are usually tedious, timeconsuming, and less-sensitive at low antigen concentrations. Nowadays, human disease such as cancer tends to be complicated, which requires the multiplex diagnostics to simultaneously measure multiple analytes in one single sample. Additionally, the early stage disease diagnosis is crucial for the effective medical treatment and the decrease in therapy sideeffects. The development of a high-throughput, highly sensitive, and low-cost approach for accurate human disease detection in early stage is still a challenging topic. Until now, many approaches have been developed for multiplex detection, such as gold nanoparticles (AuNPs), 1,2 silver nanoparticles (AgNPs),3 quantum dots (QDs),4,5 optical encoded silica, and polymer beads.6−10 AuNPs or gold colloids have become an attractive material for biological applications, such as biosensing,11 bioimaging,12 photothermal therapy,13 and gene regulation.14 AuNPs could interact with some organic compounds and form self-assembled monolayers (SAM) on the surface. The localized surface plasmon resonance (LSPR) of the AuNPs enables to enhance the Raman signals of those organic molecules through the © 2012 American Chemical Society

formation of aggregates. This phenomenon is known as the surface enhanced Raman scattering (SERS). The advent of the SERS is able to enhance the Raman signals up to 1010 times, which significantly expands the applications of Raman scattering in molecular analysis due to its low detection limit. The effective enhancement factor of SERS is tough to control due to the difficulty in setting up the calibration curves. However, the utilization of SERS as a detection technique still offers unique advantages for multiplex diagnostics, such as (1) to enable ultrasensitive detection at single molecule level; (2) produce narrow vibration spectra; (3) obtain various vibrational fingerprints through the use of different molecules with unique spectra; and (4) label-free fluorescence. To date, SERS is becoming a powerful tool in the applications of single molecule detection, glucose sensors, amino acid, or nucleotide based sensors.15−20 AuNP-based immunoassays and SERS tags have become one of the most readout techniques developed for immunoassay analysis.21−25 In a typical SERS immunoassay, AuNPs are attached to a ligand via the thiol (−SH) group, while the other end of the ligand is connected to a protein or DNA. The formation of the SAM on nanostructured metal surface generates strong SERS signals of the ligand, while the attached protein or DNA allows specific recognition of other analytes. As a result, the target molecules and ligands can be detected qualitatively by analyzing the signals of the SERS tags. Ni et al. Received: March 20, 2012 Revised: July 13, 2012 Published: July 16, 2012 17174

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Acrylic acid (AA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were supplied by Acros. Polyvinyl alcohol (PVA 100 000) was purchased from Chem-Supply. Trisodium citrate dehydrate (Na3Ct) was obtained from Prolabo. Fluorescein (FITC)-conjugated AffiniPure donkey antigoat IgG (Ex/Em 492/520 nm), FITC-conjugated AffiniPure rabbit antihuman IgG, and DyLight649-conjugated AffiniPure goat antihuman IgG (Ex/Em 652/670 nm) were from Jackson ImmunoResearch (PA). Deionized water (DI water, 18.2 MΩ·cm−1) was from an EASY pure II ultrapure water purification system. Functional PS Microbead Synthesis. The poly(styreneco-AA) microbeads were synthesized using a modified suspension polymerization.8 In a typical experiment, BPO (0.2 g), EGDMA (0.233 g), styrene monomer (9.0 g), 1% PVA stock solution (18 mL), AA monomer (0.63 g), and DI water (52 g) were charged to a 250 mL three-neck flask equipped with nitrogen inlet/outlet, condenser, and mechanical stirrer. The mixture was stirred for 20 min, followed by room temperature (RT) deoxygenating for about 40 min. The flask was then merged into a preheated oil-bath at 85 °C. The stirrer speed was fixed at 340 rpm, and the reaction was allowed for 24 h. After the reaction, the obtained microbeads were washed three times with DI water and collected using the sieves between 130 and 600 μm. The microbeads were further purified by Soxhlet extraction in methanol for 24 h to remove any unreacted residuals and dried under vacuum. Bioconjugation of Antibody to Microbead Surface. Amounts of 1.5 mg of purified microbeads and 50 μL of EDC/ NHS/PBS stock solution (20 mg of EDC and 30 mg of NHS in 1 mL phosphate buffer saline (PBS, pH 7.4)) were mixed for 15 min at RT, followed by adding 10 μg of antibody (FITCconjugated donkey antigoat IgG). To eliminate photobleaching of fluorescence dyes, the eppendorf tube containing fluorescence-labeled antibody was wrapped and remained on a shake in dark for 2 h at RT. The conjugation mixture was quenched using 1 M hydroxylamine and kept the final hydroxylamine concentration of 100 mM in solution. The antibody-conjugated microbeads were washed with 1 mL phosphate buffer (0.01 M sodium phosphate, 0.25 M NaCl, pH 7.6) and centrifuged three times at 10 000 rpm for 1 min. The microbead suspension was further blocked using 1 mL blocking solution (50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0) for 30 min at RT and then washed four times using the wash solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0) by centrifuging at 10 000 rpm for 1 min. Finally, the antibodyconjugated microbeads were resuspended in 100 μL phosphate buffer. AuNP Preparation. Gold nanospheres were prepared by the reduction of gold(III) chloride trihydrate using sodium citrate.31 Briefly, 50 mL of 10−3 M HAuCl4·3H2O were brought to a boil, followed by rapidly adding 1% sodium citrate with different mixing ratios of HAuCl4/Na3Ct. After continuously stirring at the boiling temperature, red to purple color gold colloidal solutions were obtained. Self-Assembly Monolayer and Antibody Absorption on AuNP Surface. Antibody (DyLight649-conjugated AffiniPure goat antihuman IgG)-absorbed AuNPs were prepared according to a modified approach.26,32 An amount of 2.5 μL of 10−3 M 4-MBA in THF was added to 1 mL of gold aqueous colloidal solution and allowed to react for 2 min at RT. To remove any unreact 4-MBA, the solution was centrifuged at 10 000 rpm for 2 min and redispersed in 1 mL DI water. Then, 0.1

developed a SERS−immunogold system, where various Raman reporter-labeled immunogold colloids for antigen detection have been developed.26 Their results revealed that different antigens could be detected simultaneously by choosing different Raman reporters. The detection limit of SERS immunogold colloids for prostate-specific antigen could reach femtomolar level.22 The platform for pathogenic virus detection using the SERS immunoassay system showed a detection limit of 1 × 106 viruses/mL.27 The pegylated SERS nanotags have been developed for in vivo tumor targeting and spectroscopic detection by Qian and co-workers.28 The human carcinoma cells were detected by strong SERS signals after incubating the single-chain variable fragment (ScFv) antibody conjugated pegylated SERS nanotags with the cells. Guven et al. presented a selective SERS sandwich immunoassay for Escherichia coli enumeration using gold nanorod-coated magnetic nanoparticles. The system allowed rapid detection of E. coli with high sensitivity within 70 min.29 These breakthroughs have led to significant interests in exploring SERS immunoassays as a promising protocol for disease diagnostics. In comparison, the bead-based SERS immunoassay is not well studied. To date, bead-based fluorescence immunoassays have been well-established in modern bioanalysis, for example, flow cytometer.30 However, photobleaching, broad emission spectra and different excitation wavelengths of fluorophores limit the application of the bead-based fluorescence immunoassays for multiplex analysis.7 The bead-based SERS immunoassays have obvious advantages over the fluorescence assays, such as fluorescence label-free, narrow vibration spectra, and high sensitivity. Raez et al. developed a platform for biomolecule sensing using Raman or IR encoded polymer beads.8 The beads were used not only as the support for the antibody−antigen recognition but also as the fingerprint vibration spectrum reporter. The antigen detection concentration could be as low as 150 ng/mL. Jun and co-workers embedded AgNP labels into sulfonated PS beads for multiplex protein (streptavidin and p53) analysis,7 where the ligands could be examined by both fluorescence and SERS. Considering the end-use applications, various sized polymer beads can be synthesized using different polymerization techniques. The chemical composition, structures, and functionalities of the polymer beads can be easily tailored, which gives rise to the different bead Raman spectra for multiplex analysis. This study combines AuNPs, SERS tags, and polymer microbeads to build bead-based Raman/SERS homogeneous immunoassay. Carboxylated PS microbeads were surface conjugated with FITC-labeled donkey antigoat IgG. The SAMs on AuNP surface were further bound with DyLight649-labeled goat antihuman IgG. In the study, goat antihuman IgG can be considered as the antigen for donkey antigoat IgG. We demonstrate the feasibility of the specific antibody−antigen recognition on the surface of PS microbeads based on Raman/SERS and fluorescence analysis. This new approach combines the structure properties of polymer microbeads and the SERS signals of the SAM for IgG detection, which has potential applications for high-throughput fluorescence label-free bead-based multiplex diagnostics.

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EXPERIMENTAL SECTION Materials. Gold(III) chloride trihydrate (HAuCl4·3H2O), 4-mercaptobenzoic acid (4-MBA), styrene, benzoyl peroxide (BPO), ethylene glycol dimethacrylate (EGDMA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. 17175

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M NaOH was used to adjust the pH of the colloidal solution to 9, followed by adding 20 μg of antibodies and reacting for 1 h by rotation in dark at RT. After the reaction, the mixture was blocked using 100 μL of 10% BSA solution for 30 min. The colloidal solution was centrifuged and washed three times using water and finally redispersed in 100 μL of DI water. Immunoassays. Approximately 20 antibody-conjugated PS microbeads were added to 100 μL of antibody-conjugated AuNP solution and mixed for 1 h in dark at RT under gentle rotation. The solution was then centrifuged at 5000 rpm (1 min) for several times and redispersed in 100 μL DI water. The final microbeads were subjected to fluorescence image and Raman spectrum analysis. Equipment. The UV−vis absorption spectra of AuNPs, AuNP-SAMs, and antibody conjugation were recorded using a Shimadzu UV-1601 UV−vis spectrophotometer. Transmission electron micrographs of various AuNPs were taken using a Phillips CM200 Transmission electron microscope (TEM) operating at an accelerating voltage of 160 kV. The SERS spectrum of 4-MBA self-assembled AuNPs was monitored using a Peak Seeker Pro Raman spectrometers equipped with a 200 mW 785 nm laser. A Raman microscope equipped with a digital camera was employed to measure the Raman spectra of solid samples such as polymer microbeads. The integration time for all SERS and Raman measurements was 3 s. Fluorescence images were taken using an inverted Nikon Eclipse TE300 microscope equipped with a Nikon DXM1200 digital camera. Fluorescence spectra of various supernatant solutions during the washing process were monitored using a Shimadzu RF-530/PC Spectrofluorometer.

Figure 1. Comparison on the absorption spectra of AuNPs prepared at the mixing HAuCl4/Na3Ct ratio of 1:2 and 1:1.5, AuNPs (mixing ratio of 1:1.5) with 4-MBA SAMs before and after centrifuge, and AuNPs (mixing ratio of 1:1.5) after goat antihuman IgG bioconjugation.

interact with the −SH group. The 4-MBA used in this study is an aromatic compound with both carboxyl group (−COOH) and −SH group. While adding 4-MBA to gold colloids, 4-MBA self-assembles on the surface of AuNPs via the formation of an Au−S bond. The formed SAMs could enhance the Raman signals of 4-MBA, known as the SERS. After the formation of SAMs, these AuNPs tend to aggregate due to the increase in surface hydrophobicity.37 The AuNP aggregation further facilitates the enhancement in SERS signals. Both AuNPs prepared at the mixing ratio of 1:2 and 1:1.5 were reacted with 4-MBA to prepare their SAMs, followed by examining the SERS spectra of 4-MBA using a Raman spectrometer and the SPR of AuNPs using a UV−visible spectrophotometer. From the UV−vis examination, the formation of SAMs does not alter the LSPR spectra for AuNPs, and no absorption is observed at high wavelengths (Figure 1). The identical absorption of the AuNPs with and without SAMs identifies no significant AuNP aggregation in solution. After centrifugation to remove free 4-MBA in solution, the AuNPs with SAMs can be easily redispersed into aqueous media after centrifugation. The LSPR of the washed SAMs is similar to that before washing, indicating that the SAMs are stable in aqueous medium. However, the absorption of the SAMs after washing drops compared with those of AuNPs and the SAM before washing. That is attributed to the mass loss associated with washing process, where a small amount of self-assembled gold colloids are sticked onto the wall of a plastic centrifuge tube through hydrophobic interaction. The surface bound 4-MBA results in an increase in the hydrophobicity of AuNPs, which facilitates the physical adsorption of SAMs onto the hydrophobic surfaces. TEM was used to examine the SAM formation on gold surface (Figure 2C). The morphology of the SAM appears to be similar to the AuNPs but has a thin layer on the surface. Figure 3 shows the SERS spectrum of 4-MBA in the presence of AuNPs prepared at the mixing ratio 1:1.5. The SERS spectrum of 4-MBA is dominated by two peaks, at 1074 cm−1 and 1583 cm−1, which can be assigned to the υ8a and υ12 aromatic ring vibrations, respectively.38,39 It was also found that the SERS signals of 4-MBA in the presence of the AuNPs prepared at the mixing ratio of 1:2 are much weaker than that of 1:1.5. That might be attributed to the small particle size and

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RESULTS AND DISCUSSION AuNPs and Their SAMs. Aqueous gold colloidal solutions were prepared using the trisodium citrate reduction method. In this study, different sizes of AuNPs were prepared by varying the feed molar ratios of HAuCl4·3H2O and Na3Ct from 1:1.0, 1:1.5, to 1:4.0. Our results show that it is difficult to obtain stable gold colloidal particles at a HAuCl4·3H2O:Na3Ct ratio of 1:1, where phase separation can be observed after standing the samples at room temperature for few days. However, stable redcolor AuNPs with small particle size can be synthesized at the mixing molar ratio beyond 1:2. While changing the mixing ratio to 1:1.5, purple-color AuNP suspension can be obtained, which can be stable at room temperature for several months. According to the chemical reaction of Na3Ct and HAuCl4, the stoichiometric ratio of HAuCl4·3H2O/Na3Ct is 2:3. As such, while the mixing ratio is less than 1, not enough citrate ions can be found on the surface of AuNPs, which gives rise to the unstable suspensions. Beyond that ratio, stable AuNPs can be formed. The LSPR of various AuNPs were characterized by UV−vis absorption spectrophotometer with their morphologies being examined by TEM. For the AuNPs prepared at the mixing HAuCl4/Na3Ct ratio of 1:2, the LSPR is observed at 526 nm. However, the maximum absorption peak shifts to 543 nm for the gold colloids prepared at the mixing ratio of 1:1.5 (Figure 1). Figure 2A,B compares the TEM images of the above synthesized AuNPs. It is evident that the average size of AuNPs is around 20 nm at the mixing ratio of 1:2, while these AuNPs at the mixing ratio of 1:1.5 are nonspherical with an average size of about 50 nm. The SAM of molecules on gold surface has been intensively studied.33−36 It has been well established that AuNPs prefer to 17176

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Figure 3. Comparison on the Raman and SERS spectra for the beadbased Raman/SERS immunoassays. The vibration bands at 1074 and 1583 cm−1 for the SERS of 4-MBA on AuNP surface, while the bands at 999 and 1029 cm−1 for the Raman of PS microbeads. The laser wavelength is 785 nm associated with a 3 s integration time.

The magnitude of the enhancement in SERS can be expressed using the effective enhancement factor (EEF). The estimation of the EEF can be described using EEF =

ISERSNbulk IbulkNSERS

Where ISERS and Ibulk are the intensities of the same band for the SERS and bulk spectra, Nbulk is the number of molecules for a bulk samples, and NSERS is the number of molecules in SERS.40,41 Using the band of 1074 cm−1, the EEF was calculated to be 5.4 × 105, which agrees with those EEF values for other SERS tags. In this study, we aim to explore SERS reporters in beadbased immunoassays. Antibodies (goat-anti human IgG) were introduced to the surface of self-assembled AuNPs. Because of the hydrophilic characteristic of proteins, the presence of antibodies could enhance the stability of gold colloids in aqueous solution. Thus, the conjugated AuNP solutions can be easily redispersed during the washing process. Figure 1 compares the LSPR absorptions of the self-assembled gold colloids before and after antibody conjugation. It has been documented that the aggregation of gold colloids associate with the spectral change of surface plasmon bands (red-shift) due to the particle intermolecular interaction.42,43 Our results from Figure 1 clearly demonstrate that there is no red-shift or aggregation after antibody conjugation. The identical maximum plasmon bands of gold colloidal solution with and without SAM and antibody absorption indicate the possible core−shell− corona structure of the system, which might be ready for further immunoassay applications as a SERS reporter. In order to avoid nonspecific binding during the biological applications of SERS reporters, the antibody and 4-MBA-modified gold

Figure 2. TEM images of AuNPs at the mixing HAuCl4/Na3Ct ratio of 1:2 (A) and 1:1.5 (B) synthesized using the citric reduction method, and the AuNPs (mixing ratio 1:1.5) with 4-MBA SAMs (C). The scale bar is 20 nm.

excess citrate ions on AuNP surface, which decrease the opportunity of aggregation. Therefore, the AuNPs prepared at the mixing molar ratio of 1:1.5 for HAuCl4·3H2O and Na3Ct is more suitable for the SERS-related investigation. Fifty nanometer gold colloids tend to aggregate after the SAM formation rather than 20 nm AuNPs. At moderate pH, 4-MBA is hydrophobic. The SAM formation results in the increase in hydrophobicity. On the basis of the Derjaguin, Landau, Verwey, and Overbeek theory (DLVO theory), strong but short-ranged van der Waals attractions can influence the stability of colloidal dispersions, so those AuNPs prefer to aggregate, especially during the centrifugation process.26 However, the aggregation of AuNPs gives rise to the increase in SERS signals. Under the experimental condition, 2.5 μL 10−3 M 4-MBA in THF was charged to 1 mL of 10−3 M AuNP aqueous solution to prepare the SAM. The calculated AuNP concentration is 1.56 × 1011 particles/mL with an average AuNP surface area of 7850 nm2. The molar ratio of 4-MBA to AuNP is 9.6 × 103 or about 1.22 4-MBA per nm2 of AuNP surface. 17177

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images can be detected under blue or green light illumination for the synthesized functional PS microbeads before bioconjugation (Figure 5B,C). Accompanying with the conjugation of FITC labeled donkey antigoat IgG antibody to the surface of microbeads, the fluorescence image can be observed under blue light but not the green light illumination (Figure 5E,F). The above experimental results demonstrate that the existing carboxyl groups on the surface of microbeads are able to conjugate IgG using EDC/NHS chemistry. Additionally, there is no stray fluorescence interference to the system. The Raman spectrum of the functional PS microbeads is shown in Figure 3. The strongest vibration peak is located at 999 cm−1, which is assigned to υ1 symmetrical ring of PS. The other peak related to the υ18A vibrations is located at 1029 cm−1.47 Although carboxyl groups are functionalized on the surface of PS microbeads, there is no Raman spectrum representation for the carboxyl group. The reason is that the concentration of functional monomer AA is 10 times less than the styrene monomer (86.4 mM). After antibody conjugation, no difference on the Raman spectrum is observed due to the fact that the conjugated antibody on microbead surface is very small. Immunoassays. We aim to develop a novel approach for IgG detection using bead-based Raman/SERS immunoassay. The details on the bead-based Raman/SERS immunoassays are shown in Scheme 1. Both functionalized PS microbeads and 4MBA self-assembled AuNPs are conjugated with different antibodies. In this study, FITC-labeled donkey antigoat IgG is considered as the antibody, while DyLight649-labeled goat antihuman IgG can be considered as antigen in the system. Because of the specific recognition of antibody and antigen, the AuNPs can be attached to the polymer microbeads for the matched antibody−antigen pair. Therefore, the Raman bands of PS and the SERS signals of 4-MBA can be measured simultaneously. The immunoassays examined using Raman/SERS spectra are shown in Figure 3. As mentioned above, the typical Raman spectrum of polymer microbeads displays two vibrational bands at 999 cm−1 and 1029 cm−1, and the vibrational bands of 4MBA SAM are located at 1074 cm−1 and 1583 cm−1 for the SAM. While mixing the matched IgG functionalized polymer microbeads (donkey antigoat IgG) and AuNPs (goat antihuman IgG), both the Raman signals of PS microbeads and the SERS signals of 4-MBA can be observed. The Raman shifts are reproducible in our repeating experiments (Supporting Information Figure S3), where the vibration bands at 1074 and 1583 cm−1 for the SERS of 4-MBA and 999 and 1029 cm−1 for the Raman of PS beads can be observed for all repeats, while the antibody coated polystyrene microbeads are mixed with the matched antigen conjugated with 4-MBA-AuNP. However, the Raman intensity ratios of the 4-MBA and PS are difficult to repeat since the effective enhancement factor of SERS is not a controlled parameter. The possible reasons might be attributed to the polydispersed PS microbead distribution and the inhomogeneous 4-MBA reporter distribution on microbead surface. The result confirms the specific recognition of antibody and antigen. Furthermore, in order to examine the nonspecific reaction between antibody and antigen, PS microbeads, conjugated with FITC-labeled rabbit antihuman IgG, were mixed with DyLight649-labeled goat antihuman IgG (unmatched IgG) conjugated AuNPs. The Raman spectrum is measured as shown in Figure 3, where the SERS peaks of 4MBA at 1074 cm−1 and 1583 cm−1 cannot be observed. The

colloids were blocked with 10% BSA, and any unbounded antigens in the solution were removed by centrifugation and redispersion washing cycles in water until very weak fluorescence intensity was observed in the washed supernatant solution (Figure S1, Supporting Information). The SERS spectrum of the 4-MBA on AuNP surface after antibody absorption is shown in Figure 3. The identical SERS spectrum as the SAM is observed, which indicates that the protein binding and washing procedure cannot change the SAMs of AuNPs. In addition, antibody on the corona layer has no contribution to the SERS signals between the core−shell SAMs. Polymer Microbeads and Surface Bioconjugation. PS microbeads are widely employed in bioanalytical fields, especially in setting up immunoassays for IgG detection as the immune-solid support.7,44,45 Size distributions of the microbeads can be controlled during the preparation using different polymerization techniques. In this study, suspension polymerization is applied to produce the large size polymer microbeads, where small amounts of AA are used as the comonomer to generate carboxyl groups on the surface of PS microbeads. The main purpose of introducing AA functional segments is to modify the surface chemistry of PS microbeads so as to enhance the stability and to facilitate further surface conjugation. In our synthesis formula, AA is the sole compound that has the functional group. Becuase of its hydrophilic characteristics, these functional groups prefer to stay on the microbead surface, which facilitates further surface modification. The characterization of surface functional AA groups was examined through potentiometric and conductometric titrations of microbead suspensions (Figure 4). To eliminate the

Figure 4. Potentiometric and conductometric back-titrations of synthesized carboxyl functionalized PS microbeads using HCl standard solution at room temperature.

influence of CO2 during the titration process, DI water was also titrated at identical conditions and used as the blank. The transition of the two break points in the conductometric titration curve can be used to quantify the amount of functional AA on microbead surface. From the curve, it is calculated that about 3% AA are located on the surface of polymer microbeads, while other AA segments are encapsulated inside the microbeads.46 The fluorescence labeled antibody of donkey antigoat IgG were covalently conjugated to the surface of functional PS microbeads via EDC/NHS coupling chemistry. The comparison on the surface antibody conjugation can be observed in the fluorescence microscopic images in Figure 5. No fluorescence 17178

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Figure 5. Comparison on the fluorescence images of polymer microbeads before and after conjugation with antibody in the presence of matched or unmatched antigen-labeled AuNPs. The scale bar is 100 μm.

Scheme 1. Schematic Representation of the Bead-Based Raman/SERS Immunoassays for IgG Recognition, Where the Core− Shell−Corona Structured SERS Reporter Is Used to Recognize the Matched IgG Conjugated on the Surface of Raman-Active Polymer Microbeads

examine the immunoassays. In this study, fluorescence-labeled antibodies were conjugated to polymer microbeads and gold SAMs with their fluorescence images monitored using a fluorescence microscope. In order to eliminate nonspecific absorption, both polymer microbeads and SAM AuNPs were blocked using an excess amount of BSA before the fluorescence

results demonstrate that bioconjugated AuNPs can only attach to the surface of PS microbeads in the presence of matched IgG, which indicates that the system has high sensitivity and selectivity. In order to reinforce our experimental results from Raman/ SERS study, fluorescence imaging analysis was also used to 17179

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immunoassay study. After bioconjugation, excessive fluorescence-labeled antibodies can be observed in the supernatants after centrifugation. To remove the contribution from these absorbed fluorescence antibodies, the antibody bioconjugated PS microbeads and SAM AuNPs were washed three to five times until weak fluorescence intensities of these dyes were observed in the washed supernatant solution (Figure S1 and S2, Supporting Information). It can be found from Figure 5B,C that no fluorescence images can be detected for the PS microbeads, and thus there should be no contribution from the microbeads after further fluorescence label conjugation. After conjugating the FITC-labeled donkey antigoat IgG to the PS microbeads, the images can show up under blue light illumination (Figure 5E), but there is no fluorescence image under green light illumination (Figure 5F). The size of the DyLight 649 IgG conjugated SAM AuNPs is too small to be detected by the fluorescence microscope. While mixing the antigen conjugated SAM AuNPs with antibody conjugated polymer microbeads, the images can be observed using both blue light and green light illumination (Figure 5H,I), which implies the attachment of the surface-modified AuNPs to the surface-modified polymer beads via the antibody/antigen specific interaction. For comparison purposes, FITC-labeled rabbit antihuman IgG to the PS beads were also mixed with 649 labeled goat antihuman IgG conjugated with AuNPs. The fluorescence image under green light cannot be observed (Figure 5L). Fluorescence image reveals that there is no fluorescence signal under green light illumination, and the unmatched IgG conjugated AuNPs cannot be recognized by antibody labeled PS microbeads. Therefore, the fluorescence image analysis further reinforces the immunoassay results from the Raman/SERS study. The immunoassay interaction is highly selective and highly specific. Both Raman and fluorescence analysis indicate the specific recognition between antibodies and antigens. In clinical applications, fluorescence labels are always used for bioanalysis, but the autofluorescence, photobleaching, and peak overlapping hinders its end-use applications. Our system combines the Raman signals of polymer microbeads (support) and SERS signals of the 4-MBA (reporter) for biomolecular diagnosis. Various polymer microbeads can be easily synthesized, which gives rise to different Raman spectra. The formation of SAM on the gold surface results in significant enhancement of reporter SERS signals. The Raman/SERS assays combines the vibrational information of various microbead supports and SERS reporters to identify IgG, and thus, the microbeads and reporters are fluorescence label-free with no photobleaching and fluorescence interference. On the basis of different Raman spectra of polymer microbeads and the large effective enhancement factors of SERS reporter, we are able to explore the applications of such systems for multiplex analysis. Polymer microbeads together with various SERS tags can offer infinite Raman vibrational information with high accuracy, which can be used to identify different biomolecules simultaneously from one homogeneous immunoassay.

demonstrate that the core−shell−corona structured SERS reporters can be successfully readout on the polymer microbeads through the specific recognition between donkey antigoat IgG and goat antihuman IgG. Compared with traditional bead-based fluorescence immunoassays, the developed polymer microbead-based Raman/SERS immunoassays have the following advantages: label-free, no photobleaching, high selectivity, and highly multiplexed. It has potential applications for multiplex analyte detection from one homogeneous immunoassay.

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ASSOCIATED CONTENT

S Supporting Information *

Fluorescence and Raman spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (S.D.); [email protected] (B.J.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful for the research grants from the Australia Research Council (ARC DP110112927 and LP0562076) and scholarships for L.W. provided by the China Scholarship Council (CSC).

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CONCLUSIONS A novel technique for IgG detection using polymer microbeadbased Raman/SERS immunoassay has been developed. We find that 50 nm AuNPs is more suitable for producing significant SERS signals of the SAMs and being used as the SERS reporters in the Raman/SERS immunoassays. Both fluorescence microscope and Raman spectrometer characterization 17180

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