Establishment And Evaluation Of Nanogold And Nanosilver-based Novel Immunoassays

Objective: Establish convenient, rapid and high sensitive immunoassays based on nanogold and nanosilver, and explore industrialization production process. Provide methodologyical basis for development of novel rapid detection methods. Methods: A novel fluorescence quenching immunochromatographic strip(FQ-ICS) for detecting heavy metal cadmium was developed, and the producing process conditions were optimized. Meanwhile, a low-cost portable reading equipment based on smartphone was constructed and evaluated. By using higher sensitive Raman optical signals as the readout signal of ICS, we established a novel Surface Enhanced Raman Spectroscopy-based ICS(SERS-ICS) for heavy metal chromium detection in real water samples. The results were compared with inductively coupled plasma mass spectrometry(ICP-MS). The specificity and repeatability of this SERS-ICS were evaluated. Based on the oxidization phenomenon of aggregated silver nanoparticles(AgNPs) found in the construction process of SERS-ICS, we established an ultrasensitive Surface Enhanced Raman Spectroscopy-based plasmonic ELISA(SERS-plasmonic ELISA) for Prostatic sepcific antigen(PSA) and Ractopamine(Rac) detecting. The principle and sensitivity of this assay were determinated. Clinical serum samples were detected and evaluated by comparing with Time Resolved Fluorescence Immunoassays(TRIFAs). In order to reduce the cost and get convenient naked-eye observation, an etching silver nanoprisms-based plasmonic ELISA(Triangular AgNPRs-plasmonic ELISA) for high sensitive detecting PSA was established. The feasibility, the sensitivity and specificity were verified. Clinical serum samples were detected and evaluated by comparing with TRIFAs. A simple colorimetric aptamer sensors for PSA detection based on the localized surface plasmon resonance(LSPR) of AuNPs was established. The reaction conditions were optimized and the sensitivity and specificity were evaluated by detecting clinical serum samples. The results were compared with TRIFAs. At the same time a low-cost colorimetric equipment based on smartphone was constructed and virified. Results: A FQ-ICS for Cd2+ detection in water was established, the fluorescence signal was positively correlated with Cd2+ concentration. The sensitivity of the FQ-ICS is 15-fold higher than AuNPs-ICS, and it can detect real water samples. After optimizing producing process, the FQ-ICS could be stored about one year. The stability of strip reading equipment we made is good, it can be used to read both AuNPs-ICS and fluorescence ICS. The sensitivity of this SERS-ICS is 10-5ng/m L and the range of linearity is 10-6ng/m L to 10-2ng/mL. The specificity and repeatability were good, there are no significant difference between the results of SERS-ICS and the ICP-MS in real water samples detection. The sensitivity of SERS-pELISA for PSA and Rac detection were 10-9ng/mL and 10-6ng/m L respectively. There are no significant difference between the results of SERS-pELISA and time-resolved fluoroimmunoassays(TRFIAs) in clinical serum samples detection. The sensitivity of the silver nanoprism etching-based plasmonic ELISA for PSA detection was 4.1 fg/mL and the range of linearity is 1 fg/mL-10-6 fg/mL. The specificity and repeatability were excellent, no significant difference was found between the results of silver nanoprism etching-based plasmonic ELISA and time-resolved fluoroimmunoassays(TRFIAs) in clinical serum samples detection. The sensitivity of colorimetric aptamer sensor for PSA detection was 2ng/mL and the range of linearity is 5ng/mLto 20ng/m L. The specificity was good, no significant difference found between the results of colorimetric aptamer sensor and time-resolved fluoroimmunoassays(TRFIAs) in clinical serum samples detection. The usability of portable colorimetric equipment we constructed is good. Conclusions: A high sensitive and positive readout FQ-ICS and its production process have been established and optimized in this study. The expiration date is one year and no significant differences found between groups. The stability and usability of prepared low-cost portable strip reading equipment is good, which is suitable for on-site detection and point-of-care test. Three immunologic detection methods and one immunologic-like detection method have been developed. These methods have advantages of high sensitivity, rapid, easy-to operate, and can be used as quick and efficient detection means.