Studies On LSPR Sensing Film Of Colloidal Gold And Its Application For Immunoassay

Immunoassay is a powerful analytical tool for applications in the field of biochemical analysis and clinical diagnosis that based on the specific interaction between antigen and its complementary antibody. Several types of immunoassay techniques have been developed, such as enzyme-linked immunoassays, radioimmunoassay, immunoagglutination assays, and fluorescent immunoassays. These immunoassay techniques display important roles in clinical analyses, medical diagnostics, and environmental analyses. One common feature of these immunoassay technique listed above is the requirement of a label molecule for detecting bio-interactions. Great efforts have been made in the last two decades in development of several new types of optical biosensors, and their corresponding applications in the field of environment protection, biotechnology, medical diagnostics, drug screening, food safety, and security. Surface Plasmon Resonance (SPR), a phenomenon describes the interaction between the incident light wave and the surface electrons of metal film or particle that give rise to electromagnetic field (EF), is very sensitive to the changes of refractive index (RI). The potential of SPR for applications in the field of sensing was realized in the early 1980s by Liedberg et al., who were able to sense immunoglobulin antibodies by measuring the changes in critical angle when the antibodies bound selectively to a gold film. Generally, there are two types of SPR sensors: the propagating SPR (PSPR) sensor based on the response of EF to the changes of RI on a continuous metal film, and the localized SPR (LSPR) sensor based on sensing of the local changes of RI on the surface of metal nanoparticles. In these years, SPR technique, especially LSPR technique, has been applied in the label-free sensing on the detection of biomolecular interactions in real time. Self-assemble is a platform to extend the employment of LSPR for biosensing. Self-assemble was developed by G. Decher in 1991, who found a route to fabricate a multilayer structure by using the electrostatic interaction as the driving force. The self-assemble technique has been proven to be a rapid and experimentally simple way to produce complex multilayer structure with precise control over the composition and thickness. Although much work has been reported on self-assemble of monolayer gold nanoparticle films, few of them were used as the biosensing film. In this thesis, we fabricate the biosensor by self-assemble gold nanoparticles on polyelectrolyte films. All the relative studies are outlined as follows:1. Investigations on the preparation of colloidal gold nanoparticle monolayer film by electrostatic self-assembly.A serials of gold nanoparticle monolayer films were fabricated by self-assemble of gold nanparticles on the substrates which contained monolayer or trilayer of polyelectrolytes. Several types polyelectrolytes were chosen: poly(vinylbenzyl) trimethylammonium chloride (PVTC), poly(diallyl-dimethylammonium) (PDDA), and poly(allylamine) hydrochloride (PAH) as the cationic polyelectrolytes; polystyrenesulfonate (PSS) as the anionic polyelectrolyte. The assembled gold nanoparticle films were characterized by the UV-vis spectroscopy and SEM. The result showed that the PDDA/PSS/PVTC/AuNP assembly film owes good reproducibility, high intensity and excellent stability.We investigated the sensing performance of PDDA/PSS/PVTC/AuNP film by changing dielectric environment ("bulk"refractive index and"local"refractive index) around the assemble film. The result showed that the LSPR peak intensity, band intensity at 575nm, and band position of the LSPR peak of the gold assemble film were linear to the changes of RI of surrounding medium. It proved that the PDDA/PSS/PVTC/AuNP assembly film can be employed as a good LSPR sensing flim.2. Size optimization of the gold nanoparticles in the PDDA/PSS/PVTC/AuNP assembly filmThe effect of the assembled gold nanoparticle size on the response character of the sensing film was investigated. Several sizes of gold nanoparticles were chosen. The investigations were achieved by evaluating sensitivities of the LSPR peak intensity and LSPR band position of the assembly gold nanoparticles film to the changes in RI of surrounding medium. The result showed that the 41-nm assembly film exhibited maximum sensitivity on the response of LSPR band position to the changes of RI, and the 69-nm assembly film displayed maximum sensitivity on the LSPR band intensity.3. Biosensing by LSPR of nanoparticles on quartzBoth 41-nm and 69-nm gold nanoparticle assembly films were employed as the LSPR sensing film for biodetection. The changes inλ_max of LSPR_{41 nm} and Ext_{575 nm} of LSPR_{69 nm} were monitored during the adsoption of different concentrations of human IgG, and used to characterize the sensing properties of these films. The result showed that the LSPR_{69 nm} film was more sensitive, and it was applied in immunorecognition. The applicability of the biosensor in the presence of interference agents was verified by introducing rabbit IgG and bovine IgG in the detection of human IgG. The biosensing film exhibited a limit of detection of 1μg/mL for the human IgG interaction after immersed in goat-anti-human IgG for 30 min.