Establishment And Evaluation Of Analysis Methods For Bpa And HCV-Ab Based On SERS And Its Combination With Immunoassays

With the widely application of SRES in the field of highly sensitive analysis and based on the excellent SERS enhancement effect of gold and silver nanoparticles, a series of novel, rapid and sensitive BPA and HCV-Ab detection methods were fabricated, combining with the traditional immunoassay. All the studies were shown in the following steps.In the first part, the label-free SERS technology for sensitive detection of BPA was studied.(1) The silver nanoparticle was prepared and aggregated by adjusting the pH value and adding the flocculating agent of NaCl, which was served as the SERS substrate. Under the experimental conditions of pH=3, 0.4 mol/L NaCl added, the volume ratio of silver sol and BPA solution was 1:1, the limit of detection (LOD) for BPA was 0.5 μg/mL. This method was operated easily, and can be used for qualitative detection of BPA rapidly.(2) The AgNPs-decorated filter paper substrate was fabricated in this section,which supported favorable stability, uniformity and sensitivity. Then, BPA was detected by the AgNPs-decorated filter paper assisted with Cys. In aqueous solution BPA is caught by Cys through the electrostatic interaction between positively charged groups of -NH3+ and hydroxy of BPA. Then, BPA-tailed Cys can self-assemble to the surface of SERS substrate. The results showed that 10 mM Cys adsorbed BPA to the surface of AgNPs at pH value of 3, an ultra sensitivity of 0.005 ng/mL was achieved,and the spiked recovery was 90.2 %-121.1% in the range of 1-10 ng/mL. This solid substrate was cost lowly and easy to carry,therefore,which exhibits a promising prospect in the high throughput analysis of BPA.In the second part, two novel methods for BPA were established using SERS-combined immunoassay.(1) Firstly, a "sandwich" structure based on SERS labeled competitive immunoassay for detection of BPA was fabricated. Au/Ag core-shell nanoparticles was synthesized and double-labeled with Raman report molecule and anti-BPA. The results indicated that the analysis for BPA was in the linear range of 10 μg/mL～1 ng/mL, with the LOD of 1 ng/mL. No cross-reaction existed with the analogues, and the spiked recovery was 78%-123%.(2) Next, SERS combined with colloidal gold immunochromatography(SERS-GICA) method was established for rapid, highly sensitive and quantitative detection of BPA. The gold colloid of 20 nm size was labeled with 4MBA and anti-BPA for preparation of SERS-immunoprobe. The test strip was firstly assembled and showed high sensitivity, specificity and excellent uniformity and stability. After the immunochromatography process within 15 min, the test strip of SERS-GICA for BPA was measured on the T line. The SERS signal of 4MBA at T line is inversely proportional to the BPA concentration. The LOD of this method for BPA is 0.1 ng/mL and is about 300 fold higher than the traditional GICA by visual observation, which is especially applicable for small molecule pollutant analysis in the field test.In the third part, the SERS combined ELISA method is applied for analysis of HCV-Ab with ultrasensitivity. On the principle of double antigen sandwich ELISA,the SERS signal of TMB+ is in direct proportion to the HCV-Ab content. TMB+ is the product of enzymatic hydrolysis reaction of HRP and TMB. The result is that the method for HCV-Ab has good linear relationship in the range of 0.625～10 pg/mL with the LOD of 0.625 pg/mL, which is obviously higher than the common ELISA for HCV-Ab. This analysis is ultrasensitive, easy operation, environmentally friendly, and without introducing the other Raman reporter, will have the great potential in the immunoassay field in the future.In conclusion, this paper is combined SERS technology with immunoassay,which supported their respective advantages for analysis of BPA and HCV-Ab, and these methods shows a broad application prospect in the area of environmental monitoring and medical diagnosis.