Introduction

For the last three decades Raman spectroscopy has been extensively employed to investigate biological molecules and materials, because it can provide rich structural information as well as quantitative and qualitative information about them, and moreover, it can be applied to aqueous samples and samples under physiological conditions in a nondestructive manner.^{1 2 3} However, Raman spectroscopy has one serious disadvantage; the sensitivity of Raman spectroscopy is not enough for various biological or biomedical applications. Therefore, many trials have been made to improve or enhance the sensitivity of Raman spectroscopy. The use of resonance Raman effect is one of them. Recent marked progress in Raman instrumentation has improved largely the sensitivity of Raman spectroscopy. However, still the sensitivity of Raman spectroscopy is often insufficient particularly for the quantitative analysis, and microanalysis of biological molecules with the low concentration.

Surface-enhanced Raman scattering (SERS) has recently been a matter of keen interest because it can readily enhance Raman signals by a factor of 10^3–14. ^{4 5 6 7 8 9 10} Since the success of Raman measurements of single molecules by SERS,^{11 12} SERS has attracted much greater attention than before from the points of both basic science and applications. Recent remarkable progress in the studies of the mechanism of SERS and its experimental techniques has broadened and strengthened the potential of Raman spectroscopy in the applications of biology and medicine.

SERS has three major advantages for bioanalytical applications.^{13 14 15 16 17 18 19 20} One is the enormous enhancement of the Raman cross section of adsorbed molecules by a factor of 10³–10¹⁴. ^{6 7 8 9 10 11 12} When SERS is applied to a quantitative assay, one can achieve higher sensitivity and lower detection limit. The SERS effect becomes even more remarkable if the frequency of the excitation light is in resonance with a major absorption band of the adsorbed molecules being illuminated surface-enhance resonance Raman scattering (SERRS). Second, in SERS spectra there is a marked reduction in the fluorescence background that often interferes Raman scattering from biological molecules. The third advantage is the surface selectivity that the SERS effect provides. This means that only molecules or molecular segments on or very close to a metal surface can yield SERS signals.

Recently, a great number of SERS studies have been carried out by using noble metal colloid nanoparticles, which are small in size by comparison with the wavelength of the incident light. The shape and size of single metal nanoaggregates govern the overall enhancement.^{21 22} SERS of molecules adsorbed on colloidal Ag and Ag nanoaggregates in a solution offers new and interesting possibilities as an analytical tool for detecting various types of molecules at extremely low concentrations.

SERS holds great possibility for the investigations of biological materials from small molecules to tissues. SERS-based bioanalytical applications include the following: (1) Microanalysis or trace analysis of simple biological compounds such as amino acids, nucleotides, and biological pigments.^{23 24 25 26 27 28 29 30 31 32 33 34 35 36} Because of the very high sensitivity of SERS, one can obtain Raman spectra of biological molecules at concentrations down to ∼10⁻¹³ mol/L. (2) DNA gene probes, gene diagnosis, quantitative assay of double-stranded DNA, and studies of antitumor drug target complexes.^{37 38 39 40 41 42 43 44 45 46 47 48} (3) Assay of thiol groups.⁴⁹ (4) Enzyme immunoassay employing SERS.^{50 51 52 53 54 55 56 57 58 59} (5) The SERS microprobe approach in the determination of the distribution of biological species and drug within the living cell.^{60 61 62}

Immunoassay, which is based on a specific interaction between an antigen and a complementary antibody, is a powerful analytical tool for biochemical analyses, clinical diagnosis, and environmental monitoring, and is one of the most promising fields in the applications of SERS. The purpose of this review paper is to discuss the potential of SERS in immunoassay. Many analysis methods, such as surface plasmon resonance,^{63 64 65 66} atomic force microscopy^{67 68 69 70 71 72} (AFM), and quartz crystal microbalance,^{73 74} electrochemical detection,⁷⁵ have been developed for a direct measurement of the antigens binding to antibody molecules immobilized on a substrate. To increase the detection sensitivity of analytes, many kinds of conventional labelling immunoassay techniques, e.g., enzyme-linked immunosorbent assay^{76 77} (ELISA), fluorescence,^{78 79} and chemiluminescence,^{80 81 82 83} have widely been applied. Recently, metallic colloid nanoparticles have also been successfully applied to the label techniques in immunoassay because of their easily controllable-size distribution, long-term stability, and friendly biocompatibility with antibodies, antigen proteins, DNA, and RNA.^{84 85 86 87 88 89 90 91 92 93 94} Many novel methods using metallic colloid nanoparticles have been developed, such as colloidal Au labeling immunoassay systems detected with transmission electron microscopy⁸⁸ or scanning electron microscopy,⁸⁹ even by nakedeye,^{90 91 92 93} imaging of gold colloidal particles by conjugating the immune complexes on conductive substrates with scanning tunneling microscope,⁹⁴ and so on.

Raman spectroscopy has been thought to hold considerable promise for enzyme immunoassay because, showing abundant, yet sharp and well-resolved bands, it contains much chemical information useful for enzyme immunoassay.^{50 51 52 53 54 55 56 57 58 59} However, in general, the sensitivity of Raman spectroscopy is not enough for immunoassay. In order to overcome this difficulty, Cotton etal.⁵⁰ utilized SERRS effect for Raman enzyme immunoassay. In their system, resonance dye, p-dimethylaminoazobenzene was covalently attached to an antibody directed against human thyroid simulating hormone (TSH), and the resultant conjugate was used as the reported molecule in a sandwich immunoassay for TSH antigen. The intensity of the resultant SERRS signals showed a good correlation with TSH antigen concentration over a range of from 4 to 60 μIU/mL⁵⁰ (equal to about 10⁻⁶ mg/mL) .

The SERS method has several advantages for enzyme immunoassay over other spectroscopic techniques. First, a SERS spectrum shows very specific and narrow Raman lines, minimizing the spectroscopic overlap of different labels. Second, unlike fluorescence probes SERS reporter groups do not self-quench, so that the intensity of the signal can be enhanced by increasing the number of SERS reporter groups.⁵⁰ Dou etal.⁵¹ developed a new enzyme immunoassay based on SERS. In their system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with peroxidase (POD). If this immunocomplex is subjected to the reaction with orthophenylenediamine and hydrogen peroxide, azoaniline is generated.⁵¹ This azocompound is adsorbed on a Ag colloid, giving strong SERS signals. Porter etal.^{54 55} proposed an immunoassay readout method based on SERS in a dual analyte sandwich assay. This method exploits SERS-derived signals from extrinsic three different reporter molecules that are coimmobilized with biospecific species on Au colloids.

We recently proposed a novel immunoassay based on SERS and immunogold labeling with Ag staining enhancement.⁵⁹ Immunoreactions between immunogold colloids modified by Raman-active probe molecules [e.g., 4-mercaptobenzoic acid (MBA)] and antigens, which were captured by antibody-assembled chips, were detected via SERS signals of Raman-active probe molecules. The immunoassay was performed by a sandwich structure. After Ag staining enhancement, the antigen is identified by a SERS spectrum of MBA. A working curve of the intensity of a SERS signal at 1585 cm⁻¹ due to the ν_8a aromatic ring vibration of MBA versus the concentration of antigen was obtained and the nonoptimized detecting limit for the Hepatitis B virus surface antigen (Antigen) was found to be as low as 5×10⁻⁴ mg/mL. ⁵⁹

This review paper consists of four parts. The first part is concerned with the enzyme immunoassay based on the indirect SERS method proposed by Dou etal.⁵¹ In this immunoassay, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with POD.⁵¹ The second part reports a near-infrared (NIR) SERS technique that directly detects the immune reaction on the Au colloidal nanoparticles without any procedure for bound/free (B/F) separation.⁵³ The third part describes the new immunoassay using probe-labeling immunogold with Ag staining enhancement via SERS technique, which has been used for the quantitative detection of Antigen by means of a sort of self-assembled sandwich structure immobilized on a silicon or quartz substrate.⁵⁹ The last part discusses the improvement of the immunogold nanolabeling and Ag staining enhancement method described in the third part.^{95 96} In the improved method, the Au/Ag immunocoreshell nanoparticles instead of the immunogold nanoparticles are used as the labels in this sandwich immunoassay system and the procedure of Ag staining enhancement is avoided.

Part 1—Enzyme Immunoassay by Indirect Surface-Enhanced Raman Scattering Method

Dou etal.⁵¹ proposed an enzyme immunoassay utilizing indirect SERS method. The detection limit of this system was found to be about 10⁻⁷ mg/mL, which was lower by one-order than that of the system employed by Cotton etal.⁵⁰ The proposed system is illustrated in Fig. 1.⁵¹ In this system, antibody immobilized on a solid substrate reacts with antigen which binds with another antibody labeled with POD. When this immunocomplex is reacted with orthophenylenediamine and hydrogen peroxide, azoaniline is generated as a reaction product (Fig. 1). SERS signals from azoaniline absorbed on Ag colloid are measured to estimate the concentration of antigen [mouse-Immunoglobulin G (IgG)]. The SERS reporter group of this system is a simple and stable dye that show very strong Raman bands due to the N=N and C=C stretching modes. Moreover, the selectivity is extremely high because only the dye yields SERS signals. Of note in this system is that the concentration of antigen is determined indirectly via the SERS signals of the reaction product. Therefore, the sensitivity of the method is free from the Raman scattering intensity of the label directly attached to antibody. This method was named the indirect SERS method.⁵¹

Figure 1

(a) Enzyme immunoassay based upon indirect SERS method and (b) enzyme reaction investigated.

Figure 2(a) shows a normal Raman spectrum of the reaction mixture of 10⁻⁶ mol/L orthophenylenediamine, 0.1% POD, and 0.136% hydrogen peroxide after the enzyme reaction.⁵¹ Note that the spectrum shows strong fluorescent background. A SERS spectrum of the reaction mixture of the above three compounds is shown in Fig. 2(b).⁵¹ Before we applied the SERS method to the enzyme reaction mixture, SERS spectra had been measured for 10⁻⁶ mol/L orthophenylenediamine, 0.1% POD, and 0.136% hydrogen peroxide, separately,⁵¹ and we had confirmed that no peak was observed in the obtained spectra expect for a weak feature around 1650 cm⁻¹ due to water. Therefore, there is little doubt that the SERS signals in the spectrum of Fig. 2(b) arise from the enzyme product generated by the oxidation-condensation reaction of orthophenylenediamine. Of particular importance is that only the enzyme reaction product yields strong SERS signals and that the enzyme or substrate itself does not show any detectable SERS peak. In other words, the concentration of the product can be monitored selectively without any interference. It is also noted that the strong fluorescent background is markedly reduced in the SERS spectrum [compare Figs. 2(a) and 2(b)]. Bands at 1582 and 1442 cm⁻¹ are due to the C=C and N=N stretching modes, respectively.

Figure 2

(a) A normal Raman spectrum of the reaction product (azoaniline) of the enzyme reaction shown in Fig. 1. (b) A SERS spectrum of the enzyme-substrate mixture after the reaction.

SERS spectra of azoaniline produced by the enzyme reaction of orthophenylenediamine with peroxide and the immunocomplex labeled by POD are displayed in Fig. 3.⁵¹ The concentration of the antigen was changed from 1.575×10⁻⁷ mg/mL [Fig. 3(d)] to 2.5×10⁻⁶ mg/mL [Fig. 3(a)]. A good straight line was obtained between the intensity of the band at 1442 cm⁻¹ versus the concentration of antigen. The correlation coefficient (R) between them was calculated to be 0.999 for the concentration range from 1.58×10⁻⁷ to 2.5×10⁻⁶ mg/mL. ⁵¹ The detection limit of this SERS enzyme immunoassay method was found to be about 10⁻⁷ mg/mL, which was lower by one order of magnitude than that found for a previously reported method employing SERS.⁵¹ Even higher sensitivity might be expected if one could find more proper enzyme reaction system containing immunocomplexes because the sensitivity of this method is not controlled by Raman intensity of reporter molecules covalently bounded with antibody.

Figure 3

SERS spectra of the reaction product of orthophenylenediamine with the immunocomplex labeled with POD. The concentration of antigen was 2.50 (a), 0.625 (b), 0.315 (c), and 0.1575 (d) ng/mL. The trace (e) is the spectrum of water.

Part 2—Detection of Immune Reaction Without Bound/Free Antigen Separation by Near-Infrared Surface-Enhanced Raman Scattering

Almost all immunoassays currently being employed are so-called heterogeneous immunoassays that request a procedure for the separation of B/F antigens. However, the B/F separation is a rather cumbersome procedure and reagent consuming. We showed that NIR SERS spectroscopy holds considerable promise in detecting the immune reaction on the Au colloid particles without any procedure for the B/F separation.⁵³ The procedure for this method is illustrated in Figs. 4(a) and 4(b).⁵³ Antibody and immune complex are adsorbed on Au colloid particles. In the system free antigens cannot be adsorbed on the Au colloid surface, because the surface of the Au colloid particles is blocked by bovine serum albumin (BSA). A SERS spectrum of antimouse IgG at 2.2×10⁻⁸ mol/L adsorbed on the Au colloid particles shows a number of Raman bands due to amide groups and aromatic acid residues of antimouse IgG. A SERS spectrum of the same system at 1.9×10⁻¹⁰ mol/L does not give any SERS signal. However, Raman bands again appeared after the reaction of antimouse IgG at 1.9×10⁻¹⁰ mol/L with the antigen on the Au colloid particles.⁵³

Figure 4

(a) Antibody adsorbed on Au colloid particle. (b) Immune complex on Au colloid particle.

Figures 5(a) and 5(b) show NIR–SERS spectra of the Au colloid solution and antimouse IgG of 2.2×10⁻⁸ mol/L adsorbed on the Au colloid particles, respectively.⁵³ The spectrum of antimouse IgG has an appearance fairly different from that of a typical protein Raman spectrum.⁹⁷ However, bands due to the amide groups (1645 and 1261 cm⁻¹) and those assignable to tryptophan (Trp) residues (1467, 1112, and 880 cm⁻¹) are clearly identified in the SERS spectrum of Fig. 5(b). Table 1 summarizes the frequencies and assignments of observed Raman bands. It is noted that the bands due to Trp are enhanced largely. According to previous SERS studies of proteins without a prosthetic group,^{30 98 99 100} bands due to amide I and III are relatively weak in the SERS spectra of proteins, but those due to Trp and tyrosine residues (Tyr) appear strongly. It is also important to point out that the SERS spectra of proteins vary markedly with experimental conditions. For example, a SERS spectrum of BSA adsorbed on a Ag electrode with the potential corresponding to zero charge for Ag is different from that of BSA adsorbed on colloidal Ag at pH 8.0.^{98 100} In this study, a 0.01 mol/L phosphate-crtrate buffer (pH=7.0) containing a 0.005 mol/L NaCl solution has employed throughout the experiments, and all the SERS spectra were obtained under such experimental conditions.

Figure 5

(a) A NIR SERS spectrum of the Au colloid solution. (b) A NIR SERS spectrum of antimouse IgG of 2.2×10⁻⁸ mol/L adsorbed on Au colloid particles. Radiation of 1064 nm from an Nd:yttrium–aluminum–garnet laser was used as the excitation source, and the power at the sample point was typically 100 mW.

Table 1

The spectrum in Fig. 5(b) provides very interesting information about the adsorption of the protein on the Au colloids.⁵³ The amide I and III bands appear at 1645 and 1261 cm⁻¹, respectively, which are typical frequencies for α-helix structure of a protein.⁹⁷ However, IgG has β sheet-rich structure, and, in fact, their amide I and III bands are observed at 1673 and 1239 cm⁻¹ in the normal Raman spectra.^{101 102} Therefore, it seems that the bands due to the α-helix parts of the antimouse IgG are particularly enhanced in the SERS spectrum. This indicates that the α-helix parts are closer to the surface of Au colloid particles.⁵³

Figure 6(a) shows a NIR SERS spectrum of antimouse IgG of 1.9×10⁻¹⁰ mol/L adsorbed on the Au colloid particles.⁵³ The Raman bands observed in the SERS spectrum of Fig. 5(b) disappear completely probably because the concentration of antimouse IgG was diluted by 100 times. Figure 6(b) depicts a NIR SERS spectrum of IgG–antimouse IgG complex on the Au colloid particles.⁵³ Raman bands again emerge, although they are weak. Note that the frequencies of those bands are very close of those of the Raman bands in the spectrum shown in Fig. 5(b). It seems that all the observed bands arise from the antibody part of IgG–antimouse IgG complexes adsorbed on the Au colloid particles.⁵³ In the system shown in Fig. 4 the only antibody part can be adsorbed directly on the Au surface, giving the SERS signals. Bound and free antigens do not show significant Raman bands since free antigen molecules are blocked by BSA molecules and bound antigen molecules are adsorbed indirectly on the Au surface. The proposed method allows one to detect a trace amount of immune complex on Au colloid particles without any need for the B/F separation. It was also concluded from the earlier study that the configuration of antimouse IgG is modified significantly upon the reaction of antigen with antimouse IgG on the colloid particles, emerging intense SERS signals.⁵³

Figure 6

(a) A NIR SERS spectrum of antimouse IgG of 1.9×10⁻¹⁰ mol/L adsorbed on Au colloid particles. (b) A NIR SERS spectrum of IgG–antimouse IgG complex on Au colloid particles.

Part 3—Immunoassay Using Probe-Labeling Immunogold Nanoparticles With Ag Staining Enhancement via Surface-Enhanced Raman Scattering

We recently proposed a novel immunoassay based on SERS and immunogold labeling with Ag staining enhancement.⁵⁹ This is also an indirect SERS method. Immunoreactions between immunogold colloids modified by a Raman-active probe molecule (e.g., MBA) and antigens, which were captured by antibody-assembled chips such as silicon or quartz, were detected via SERS signals of Raman-active probe molecules.⁵⁹ It was found that the nonoptimized detection limit for Antigen is as low as 5×10⁻⁴ mg/mL. ⁵⁹

Figure 7 illustrates the proposed system. The immunoassay is performed by a sandwich structure consisting of three layers. The first layer is composed of immobilized antibody molecules of mouse polyclonal antibody against Hepatitis B virus surface antigen (PAb) on a silicon or quartz substrate. The second layer is the complementary Antigen molecules captured by PAb on the substrate. The third layer consists of the probe-labeling immunogold nanoparticles, which have been modified by mouse monoclonal antibody against Hepatitis B virus surface antigen (MAb) and MBA as the Raman-active probe on the surface of Au colloids. After Ag staining enhancement, Antigen is identified by a SERS spectrum of MBA.

Figure 7

The process of self-assembled sandwich structure immobilized on a silicon or quartz substrate using MBA-labeling immunogold nanoparticles with the Ag staining enhancement method.

In this system, all the self-assembled steps were subjected to the measurements of AFM to monitor the formation of a sandwich structure onto a substrate.¹⁰³ Figure 8 shows AFM height images of each immobilized step on silicon microchips.⁵⁹ These AFM images suggest that the Antigen molecules execute the immuno-identification with the PAb molecules and are firmly captured by the (3-amino-propyl)trimethoxysilane (APTMS)-glutaraldehyde (GA)-PAb-Antigen substrate. The immunogold nanoparticles are also strongly bound to the surface through the immuno-identification. After Ag staining, a layer of Ag film covers the surface of the sandwich self-assembled multilayer.

Figure 8

AFM height images of one by one immobilized steps on silicon microchips. (a) an APTMS-GA surface; (b) an APTMS-GA-PAb surface; (c) an APTMS-GA-PAb-Antigen surface, where the concentration of the Antigen was 100 μg/mL; (d) an APTMS-GA-PAb-Antigen-Immunogold surface, where the concentration of the Antigen was 0.1 mg/mL; (e), the same as (d), but after Ag staining enhancement; (a) 1.5 μm×1.5 μm; (b)–(e) 2 μm×2 μm.

The spectral features of MBA in SERS spectra can confirm the selective immunoassays. Figure 9 shows SERS spectra of MBA adsorbed on the immunogold nanoparticles after the Ag staining enhancement.⁵⁹ The Antigen solutions with different concentrations of 0, 0.5, 1, 2, 10, 20, 40, and 50×10⁻³ mg/mL were examined by the method depicted in Fig. 7, and their SERS spectra are shown in Figs. 9(a)–9(h), respectively. Strong SERS bands at 1585 and 1076 cm⁻¹ are assigned to ν_8a and ν₁₂ aromatic ring vibrations, respectively.¹⁰⁴ Figure 10 illustrates the relationship between the intensity of the peak at 1585 cm⁻¹ and the concentration of Antigen. We developed a calibration model that predicts the concentration of Antigen in the range of 1–40×10⁻³ mg/mL. The inset of Fig. 10 depicts the model; the R and standard deviation are 0.98 (n=5) and 214, respectively, and the unoptimized detecting limit of Antigen is as low as 5×10⁻⁴ mg/mL.

Figure 9

SERS immunoassay using MBA-labeling nanoparticles with the Ag staining enhancement method. Antigen with concentrations of 0, 0.5, 1, 2, 10, 20, 40, and 50 ×10⁻³ mg/mL was detected by this method, and their SERS spectra from 1800 to 400 cm⁻¹ are shown in (a), (b), (c), (d), (e), (f), (g), and (h), respectively. All the spectra were dealt with the normalized operations assuming the intensity of the peak at 520 cm⁻¹ due to silicon as the intensity of 10 000.

Figure 10

The relationship between the intensity of the SERS signal at 1585 cm⁻¹ and the concentration of Antigen. Inset: a calibration model that predicts the concentration of Antigen in the range from 1 to 40 ×10⁻³ mg/mL.

One must notice in Fig. 9(h) that the SERS signals of MBA show unusually strong when the Antigen concentration is 5×10⁻⁴ mg/mL. This is probably because at a high concentration, there are more immunogold nanoparticles on the slide surface due to the immunoreaction. Therefore, many immunogold nanoparticles lead to a great deal of Ag aggregates onto the surface after the Ag staining. The large aggregates can remarkably enhance the SERS signals much more strongly than a single nanoparticle according to the electronic field theory of SERS.¹⁰⁵ Therefore, the SERS signal increase remarkably in Figs. 9(h) and 10.

Figure 11 shows SERS spectra of MBA on the APTMS-GA-PAb-Antigen-immunogold substrate where the concentration of Antigen is as low as 5×10⁻⁴ mg/mL before (a) and after (b) the Ag staining enhancement.⁵⁹ After the Ag staining enhancement, the Raman signals are increased by 10–100 times, thereby improving the detection sensitivity of this immunoassay method.

Figure 11

SERS spectra of MBA in immunoassay using the MBA-labeling immunogold nanoparticles. (a) before and (b) after the Ag staining enhancement. The detection limit of Antigen was as low as 0.5 ×10⁻³ mg/mL. The spectra were dealt with the normalized operations assuming the intensity of the peak at 520 cm⁻¹ due to silicon as the intensity of 10 000.

This method has combined the advantages of the SERS technique with those of the nanolabeling method.⁵⁹ The advantages of the immunogold nanoparticles, such as their easily controllable-size distribution, long-term stability, and friendly biocompatibility confirm the reproducibility of the immunoassay.

Part 4—Immunoassay Using Probe-Labeling Au/Ag Immunocoreshell Nanoparticles via Surface-Enhanced Raman Scattering

In the immunoassay described in Part 3, the Ag staining method plays an important role; it can remarkably enhance Raman signals by several decuples, improving the detecting sensitivity of the immunoassay. However, the reduced Ag film covers the surface of the APTMS-GA-PAb-Antigen-immunogold substrate, and thus the bioactivity of the antibodies and antigens may be destroyed significantly. Therefore, we developed a new label that possesses both SERS enhancing ability and good biocompatibility. We used the Au/Ag immunocoreshell nanoparticles instead of the immunogold nanoparticles as the labels in the above sandwich immunoassay system.^{95 96}

The experimental procedure in Part 4 is mostly the same as that mentioned in Part 3. The immunoassay was also performed by a sandwich structure consisting of three layers. It should be pointed out that the third layer is composed of the MBA-labeling Au/Ag immunocoreshell nanoparticles. The MBA-labeling Au/Ag immunocoreshell nanoparticles were prepared by the following four steps. First, the Au/Ag coreshell nanoparticles were prepared according the literature.¹⁰⁶ In a typical process, 5 mL of 10⁻³ mol/L Au colloids was diluted to 95 mL, and then 1.0 mL of a 1% trisodium citrate solution was added to the earlier solution. After heating the solution to boil temperature, 5.0 mL of 0.01 mol/L AgNO ₃ was added under continuous stirring to produce the desired final bimetallic colloids. To prevent the formation of separate Ag particles, 0.5 mL AgNO ₃ solution was added each 5 min. The coreshell nanoparticles used have a 13-nm-diameter Au core that is coated by an 8-nm-thick Ag shell. Second, 4 μL of MBA in methanol (1×10⁻³ mol/L) was added as the Raman-active probe to 1.0 mL of Au/Ag core-shell colloids. After 12 h standing under stirring, the MBA modified Au/Ag core-shell colloids were purified by centrifugation and resuspended with 1.0 mL borate buffer (2 mM, pH=9) . As the third step, 5 μL of MAb (2.0 mg/mL) PBS buffer solution (a PBS buffer solution; KH ₂ PO ₄/K ₂ HPO ₄, pH=7.4) was added to 1.0 mL MBA-labeling Au/Ag coreshell colloid. The amount of MAb we added into the MBA-labeling Au/Ag coreshell colloid was 50% more than the minimum amount for coating the unmodified portion of the colloid surface. Finally, to assure that no space around the surface of coreshell colloids was left, 10 μL of BSA (2% m/m) solution was added to the mentioned MBA-labeling Au/Ag coreshell colloid, to occupy the uncoated place.

Figure 12 shows a SERS spectrum of MBA measured when MBA was added into the Au/Ag coreshell colloid. From Fig. 12, one can see that the Au/Ag coreshell nanoparticles have strong SERS activity.¹⁰⁶ We again used AFM to confirm the self-assembled step (see Fig. 13). It can be seen from the section analysis of AFM images in Fig. 13 that the biomolecules and the Au/Ag immunocoreshell nanoparticles form many compacted islands on the silica substrate. The height and diameter of the islands change obviously at each immobilized step. It is clear that the Au/Ag immunocoreshell nanoparticles have been successfully immobilized onto the surface of APTMS-GA-PAb-Antigen by the immunoreaction.

Figure 12

A SERS spectrum of MBA added into the Au/Ag coreshell colloid.

Figure 13

AFM height images of one by one immobilized steps on silicon microchips. (a) A surface after the PAb immobilized on the APTMS-GA substrate, with the horizontal distance between two triangle symbol is 43.0 nm and the vertical distance is 2.3 nm; (b) a surface after the Antigen captured by PAb of the substrate, with the horizontal distance is 39.0 nm and the vertical distance is 5.7 nm; (c) a sandwich structure composed of the PAb, Antigen, and the Au/Ag immuno-coreshell nanoparticles with MBA-labeling, with the horizonal distance is 70.3 nm and the vertical distance is 6.1 nm. (a)–(c) 2 μm×2 μm.

Figure 14(a) depicts SERS signals of MBA in this sandwich immunoassay system where the concentration of Antigen is 0.02 mg/mL. Most of strong SERS bands are assigned to MBA. Figure 14(b) shows a SERS spectrum of MBA measured by using the method mentioned in Part 3 with the same concentration of Antigen (0.02 mg/mL). Comparison of both spectra in Figs. 14(a) and 14(b) reveals that the MBA-labeling Au/Ag immunocoreshell nanoparticles used as labels are as useful as the Ag staining method in terms of the SERS enhancement. Note that the procedure method is simpler than the method described in Part 3.

Figure 14

SERS immunoassay with two kinds of the labeled methods. (a) using MBA-labeling Au/Ag immunocoreshell nanoparticles, (b) using the MBA-labeling immunogold nanoparticles with Ag staining enhancement.

The major difference between these two methods is that the Au/Ag coreshell nanoparticles are used to prepare the immunolabels instead of the Au nanoparticles. The SERS signals of MBA indicate that this Au/Ag coreshell nanoparticles have strong Raman band enhancing ability. Another important difference is that the Ag staining enhancement is avoided in the experimental procedure in Part 4. The advantages of the procedure without the Ag staining enhancement step are the simplification of the experiment procedure as well as the prolongation of bioactivity of the biomolecules after the SERS detection.

Conclusion

The application of SERS to immunoassay is one of the most promising topics among the SERS applications and has attracted a number of researchers. In this review paper, we present three immunoassay methods and one improvement via SERS. Table 2 compares the three SERS immunoassay methods investigated. Compared with the ELISA method having the detection limit of ng/mL to pg/mL,⁷⁶ the methods by Dou etal.^{51 53} described in Parts 1 and 2 do not show any superiority on the detection limit. However, both methods have provided new immunoassay methods by SERS. Their advantages are mainly based on the characteristics of SERS. For example, the detection of the Part 1 method does rely on the concentration of the azodye, which is not covalently bound with the antibody. In other words, the concentration of antigen is determined indirectly via the SERS signals of the reaction product, the azodye. Therefore, the sensitivity of the method is free from the Raman scattering intensity of the label directly attached to antibody. The Part 2 method can provide the evidence for a slight modification of the configuration of antimouse IgG on the formation of an immune complex. This NIR SERS method holds considerable promise in detecting the immune reaction without any procedure for the B/F separation. These are the useful complements for the ELISA method. However, none of the SERS immunoassay methods proposed has been realized yet. The immunoassay methods using SERS reported in this review have been carried out only in laboratories. Further development of the studies on SERS mechanism, Raman instrumentation, nanotechnology, and novel ideas should make SERS real practical techniques not only in immunoassay but also various bioassay, clinical diagnosis, and environmental monitoring.

Table 2