Preparation Of Horseradish Peroxidase Labeled Silica Nanoparticles And Their Application In Enzyme-Linked Immunosorbant Assay

This dissertation focuses on the development of a new strategy for preparing enzyme conjugates using nanoparticles as carriers and their application in Enzyme-linked immunosorbant assay (ELISA). ELISA is the most popular technique used for immunoassays, and the immunoassay based on nanoparticles as labels is a new technique that bears a lot of advantages. In our r.esearch, we combined these two techniques and developed a novel technique.In chapter one, we reviewed the development of enzyme-linked immunosorbant assay, especially focused on signal enhancement labeling strategy. The conventional and newly developed techniques for enzyme conjugates preparation and signal enhancement were summarized.In chapter two, we described the preparation of HRP labeled silica nanoparticles and their application in sandwich ELISA. Silica nanoparticles with a diameter 50±5 nm were synthesized by W/O microemulsion method and used as carriers for preparation of antibody-enzyme-nanoparticle complex. First of all, these silica nanoparticles were modified by amino silane to generate amino groups on the surface, and then HRP were coupled to the surface of aminated silica nanoparticles by peroxidation method, finally the antibody were bridged on the enzyme labeled silica nanoparticles by oxidated dextran. We compared the effect of HRP labeling by three different kinds of amino silane, and we also investigated the effect of different dose of HRP and antibody used for labeling. In optimized condition, the detection limit of HBsAg by our method is 0.06 ng/mL, the linear range is 0.15-12.5 ng/mL and the detection of 260 serum samples by our method and conventional ELISA gave completely consistent results for all samples. The corelation between our method and conventional ELISA for determination of 30 positive serum is excellent with a correlation factor r = 0.992.In chapter three, we described the application of HRP labeled silica nanoparticles for determination of chloramphenicol. We investigated the feasibility of HRP labeled silica nanoparticles used in competitive ELISA. We used three different immunoassay strategies and two different enzyme conjugates prepared by different amino silane for determination of chloramphenicol. The sensitivity of nanoparticle-based competetive ELISA for determination of chloramphenicol by different methods ranged from 0.012 ng/mL to 0.29 ng/mL, and the linearity were excellent. We chose the strategy and enzyme conjugates which showed best performance according to our research results to carry out the recovery experiment, the recovery was 85.01-101.23% and CV was 6.89-25.66%, which indicated that our method was feasible and effective. We also used the optimized strategy for determination of CAP samples and found that the correlationship between conventional ELISA and our method was excellent.In conclusion, compared with conventional method that enzyme molecules were directly coupled to antibody or antigen molecules, the preparation of enzyme conjugates by using silica nanoparticles as carriers had several advantages. First of all, it was easy to handle and the labeling process was controllable; secondly, it has high repeatability and easy to purify; thirdly, it could enhanced the signal by several times.