This paper reports a novel immunoassay based on surface-enhanced Raman scattering (SERS) and immunogold labelling with silver staining enhancement. Immunoreactions between immunogold colloids modified by a Raman-active probe molecule (e.g., 4-mercaptobenzoic acid) and antigens, which were captured by antibody-assembled chips such as silicon or quartz, were detected via SERS signals of Raman-active probe molecule. All the self-assembled steps were subjected to the measurements of ultraviolet-visible (UV-vis) spectra to monitor the formation of a sandwich structure onto a substrate. The immunoassay was performed by a sandwich structure consisting of three layers. The first layer was 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 was the complementary Hepatitis B virus surface antigen (Antigen) molecules captured by PAb on the substrate. The third layer was composed of the probe-labelling immunogold nanoparticles, which were modified by mouse monoclonal antibody against Hepatitis B virus surface antigen (MAb) and 4-mercaptobenzoic acid (MBA) as the Raman-active probe on the surface of gold colloids. After silver 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 [small nu](8a) aromatic ring vibration of MBA versus the concentration of analyte (Antigen) was obtained and the non-optimized detection limit for the Hepatitis B virus surface antigen was found to be as low as 0.5 [micro sign]g mL(-1).

In this paper, we propose two new approaches for preparing active substrates for surface-enhanced Raman scattering (SERS). In the first approach (method 1), one transfers AgI nanoparticles capped by negatively charged mercaptoacetic acid from a AgI colloid solution onto a quartz slide and then deoxidizes AgI to Ag nanoparticles on the substrate. The second approach (method 2) deoxidizes AgI to Ag nanoparticles in a colloid solution and then transfers the Ag nanoparticles capped by negatively charged mercaptoacetic acid onto a quartz slide. By transfer of the AgI/Ag nanoparticles from the colloid solutions to the solid substrates, the problem of instability of the colloid solutions can largely be overcome. The films thus prepared by both approaches retain the merits of metal colloid solutions while they discharge their shortcomings. Accordingly, the obtained Ag particle films are very suitable as SERS active substrates. SERS active substrates with different coverages can be formed in a layer-by-layer electrostatic assembly by exposing positively charged surfaces to the colloid solutions containing oppositely charged AgI/Ag nanoparticles. The SERS active substrates fabricated by the two novel methods have been characterized by means of atomic force microscopy (AFM) and ultraviolet−visible (UV−vis) spectroscopy. The results of AFM and UV−vis spectroscopy show that the Ag nanoparticles grow with the increase in the number of coverage and that most of them remain isolated even at high coverages. Consequently, the surface optical properties are dominated by the absorption due to the isolated Ag nanoparticles. The relationship between SERS intensity and surface morphology of the new active substrates has been investigated for Rhodamine 6G (R6G) adsorbed on them. It has been found that the SERS enhancement depends on the size and aggregation of the Ag particles on the substrates. Especially, we can obtain a stronger SERS signal from the substrate prepared by method 1, implying that for the metal nanoparticles capped with stabilizer molecules such as mercaptoacetic acid, the in situ deoxidization in the film is of great use in preparing SERS active substrates. Furthermore, we have found that the addition of Cl- into the AgI colloid solution changes the surface morphology of the SERS active substrates and favors stronger SERS enhancement.

Mercaptoacetic acid-capped spherical silver nanoparticles with a diameter of about 17 nm were prepared by a simple chemical reaction. The formation process of the silver nanoparticles was investigated by UV-visible (UV-vis) spectroscopy and transmission electron microscopy. The results show that the spherical and rodlike particles were formed at the beginning of the reaction, and then the rodlike particles were gradually converted into spherical particles with the reaction continuing. Finally, the content of the rodlike particles was less than 3% in the silver colloid. Thus, the final obtained silver nanoparticles were uniform in their shapes and showed little variation in their sizes. This silver colloid can remain stable for several weeks, which makes it convenient for use in practice. We also investigated the effect of Clon the rate of changes in the optical properties of the silver colloid by UV-vis absorption. The results indicate that Claccelerates the aggregation of the colloidal particles by effectively screening the repulsive electrostatic interactions between the negatively charged silver particles. We used the self-assembled technique to transfer the silver nanoparticles onto solid substrates from the colloid with and without Clin the solution. The UV-vis spectra show that the absorption band red shifts and a new band appears at a longer wavelength when the silver nanoparticles are transferred onto the substrate from the colloid with Clin the solution, indicating that the silver nanoparticles can grow and aggregate on the substrate. This was further confirmed by the atomic force microscopy measurements. Both the silver colloid and the substrates prepared by transferring the silver nanoparticles from the silver colloidal solution onto the quartz slides can serve as surface-enhanced Raman spectroscopy (SERS)-active substrates. It was found that the SERS enhancement depends on the sizes and aggregation of the silver particles, and the addition of Clgenerates much stronger SERS signals.

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