Study And Application Of Two Novel Alloy Nanomaterials For Improving The Detection Sensitivity Of Immunoassay

Immunoassay methods have received a lot of attention in the field of analytical testing because of their unique advantages.Enzyme-linked immunosorbent assay（ELISA）and Lateral flow immunoassay（LFIA）are two immunoassay methods that have been developed and are commonly used in clinical diagnosis,environmental monitoring and food safety detection due to their rapidity,simplicity,strong specificity,high throughput and low cost.The biggest limitation of these two immunoassay methods is their low sensitivities,which makes them difficult to accurately qualify and quantify the trace targets.In conventional ELISA methods,horseradish peroxidase（HRP）is commonly used to catalyze substrates to obtain the detection signal.However,the TMB signal catalyzed by HRP transduction system with a monochromic intensity change is unsuitable for naked eye detection because single color response is difficult to discriminate.Moreover,TMB with a low extinction coefficient(3.9×10⁴M^-1cm^-1)limits its sensitivity for detecting the contaminants with trace concentration.Among the conventional LFIA,gold nanoparticles（Au NPs）have become the classical label materials due to their simple synthesis,low cost and easy identification by naked eyes.However,the relatively low colorimetric signal intensity of the commonly used Au NPs with size of 20–40 nm leads to the low sensitivity of conventional LFIA.Therefore,to improve the sensitivity of these two major immunoassay methods has become an important issue that needs to be addressed urgently.In this study,the sensitivity of these two conventional immunoassay methods was improved by enhancing the detection signal intensity using novel alloy nanomaterials,thus expanding their application in high-sensitivity detection.In this study,two alloy nanomaterials were prepared as reporters to construct a novel ELISA and a novel LFIA,which were applied to actual detection to evaluate their performance.In the first chapter,conventional immunoassays and strategies to improve the sensitivity of conventional immunoassays were reviewed.And controlled synthesis methods of metal nanomaterials were introduced.Finally,the hazards and the detection research progress of the two target analytes,Amantadine（AMD）and Escherichia coli O157:H7（E coli O157:H7）were summarized.In the second chapter,gold and silver nanorings（Ag–Au nanorings）with highly tunable plasmonic properties were synthesized by galvanic replacement method.The growth of silver ions on Ag–Au nanorings mediated by alkaline phosphatase catalytic production of ascorbic acid（AA）was used as the plasmonic resonance signal output to establish an ultra-sensitive plasmonic ELISA（p ELISA）for qualitative and quantitative detection of AMD in four food samples（pork,chicken,duck and egg）.Under the optimal conditions,the linear ranges of this method for quantitative detection of AMD were 0.08–2 ng m L^-1and 4–12 ng m L^-1,the visible cut-off value of qualitative detection was 0.2 ng m L^-1,and limit of detection was 0.071 ng m L^-1,which were 10-fold and 4.7-fold lower than that of the conventional ELISA,respectively.The mean recoveries of the method for the detection of AMD in the four matrices were 84.97–108.22%,and the coefficients of variation were 2.15–14.55%.In addition,the results of the simultaneous analysis of 80 actual samples using p ELISA and the instrumental method were consistent,demonstrating the good accuracy of the p ELISA.In the second chapter,a bimetallic Ag–Au urchin-like hollow nanospheres（BUHNPs）based on the co-reduction and galvanic replacement reaction was synthesized.BUHNPs with hollow structure and spiky shell exhibited strong absorption ability in the visible region and excellent photothermal properties.The rough surface of nanoparticles increased the antibody binding sites and improved the coupling efficiency with antibodies.The BUHNPs with excellent colorimetric signal,remarkable photothermal performance and ultra-high antibody coupling efficiency were finally applied to the ultra-sensitive detection of E.coli O157:H7 by colorimetric immunochromatography（CM-LFIA）and photothermal immunochromatography（PT-LFIA）.The LOD of CM-LFIA and PT-LFIA were 2.48×10³and 5.5×10²CFU m L^-1,respectively,which were 4-fold and 18-fold lower than that of conventional Au NPs-LFIA(9.92×10³CFU m L^-1).The recoveries of CM-LFIA and PT-LFIA were88.32%–118.58%and 87.11%–109.22%,respectively.In addition,the results of the simultaneous analysis of actual samples using CM-LFIA,PT-LFIA and plate counting method were consistent,demonstrating the good accuracy of the LFIA.In the fourth chapter,a summary and outlook of the full work is presented.Two detection methods based on novel alloy nanomaterials established in this work were systematically summarized,and possible methods for improving the detection performance of ELISA and LFIA using novel alloy materials in the future were prospected.