Iron(Ⅱ) Chelates And Au/Ag Nanomaterials-based Study For Improving The Sensitivity Of Enzyme-linked Immunosorbent Assay

Enzyme-linked immunosorbent assay（ELISA）is an immune analytical method with rapidity,simplicity,low cost,and high accuracy,which has been widely applied to food safety,environmental monitoring,and medical diagnosis.However,the relatively low detection sensitivity of the traditional ELISA restricted its further extended application.The proper improvement strategies are put forward in this paper for solving the difficult problem.In chapter 1,we systematically review the research development of ELISA,including the fundamental principle,classifications,and key compositions of ELISA and its application in food safety,environmental monitoring,and clinical diagnosis.In addition,chelates and metal nanomaterials are summarized for the applications as the novel colorimetric signals in the detection field.The low detection sensitivity of the traditional ELISA is attributed to the low loading amount of enzyme and insufficient color brightness of oxidized 3,3’,5,5’-tetramethylbenzidine（TMB）.In Chapter 2,the stable colored chelates were quickly synthesized with Fe²⁺and phenanthroline（Phen）.Considering that the biotin-streptavidin system could increase the glucose oxidase（GOx）loading amount,we established the iron（Ⅱ）chelates-based colorimetric ELISA.Danofloxacin（DAN）,a type of widely-used quinolone antibiotics,poses a potential threat to human health owing to its excess residue.The practical applications of the novel ELISA were confirmed with DAN as the target.Under optimal conditions,the qualitative detection limits（LODs）of the novel ELISA in milk samples,chicken samples,beef samples,and pork samples were 2.5 ng m L^-1.The proposed immunoassay takes advantages of good specificity and high accuracy,which provides a quick solution for screening small-molecule hazardous materials in food samples such as milk.In Chapter 3,the enzyme loading amount is further enhanced to address the problem that the traditional ELISA has low sensitivity.We synthesized the Au/polydopamine/horseradish peroxidase composites（Au/PDA/HRP）with high enzyme loading amount inside and on the surface and established the Au/PDA/HRP-anti-E.coli O157:H7（m Ab）based ELISA.The optimal colorimetric signal was obtained by controlling the additive amount of HRP and m Ab.Under optimal conditions,the qualitative and quantitative LODs of the novel ELISA were 1.0×10³CFU m L^-1 and 2.8×10² CFU m L^-1,respectively.The evaluation of sensing performance indicates that Au/PDA/HRP-m Ab based ELISA has the excellent specificity and accuracy,which endows it with a broad application prospect in rapid detection of foodborne pathogens in milk.In chapter 4,to address the problem about the poor qualitative screening ability and quantitative detection sensitivity of the traditional ELISA,the cascade reaction is used for the significant enzyme signal amplification and meanwhile gold nanorod（Au NR）etching is used to increase the color change.Based on the enzyme cascade reaction-triggered Au NR etching,we developed a novel ELISA for sensitive detection of aflatoxin M1（AFM1）in milk.Initially,Au NRs with an aspect ratio of 2 were successfully synthesized.Biotin and streptavidin（SA）aimed at increasing the loading amount of glucose oxidase（GOx）.The cascade catalytic reaction of GOx and Mo O₄^2-could bring about oxidative etching of Au NRs,thus resulting in blue shift of the surface plasmonic resonance absorption peak with a remarkable color change from bule to pink.Under optimal conditions,the qualitative and quantitative LODs of the proposed immunoassay were 0.5 ng m L^-1 and 0.1 ng m L^-1,respectively,with a linear range of0.25-10 ng m L^-1.This method has a distinct visual qualitative LOD and a low quantitative LOD,which is appropriate for sensitive detection of varieties of mycotoxins in milk.In chapter 5,to address the problem that the traditional ELISA relying on 3,3’,5,5’-tetramethylbenzidine（TMB）oxidation has low quantitative detection sensitivity,we synthesized Ag-Au NCs with stronger fluorescence intensity than Au NCs.Ag⁰ existed in the core area and Ag⁺was located in the Au NC shell.This structure design made up for the surface defects of Au NCs,resulting in more excellent luminescent properties.Both I₂-induced oxidative etching of Ag-Au NCs and the strong binding force between I^-and Ag⁺could lead to fluorescence quenching of Ag-Au NCs.Based on I₂/I^--induced fluorescence quenching of Ag-Au NCs,we established a novel ELISA platform for detecting E.coli O157:H7 in milk.Under optimal conditions,the linear range of the proposed immunoassay was 3.3×10³–10⁶ CFU m L^-1 with LOD of 9.2×10² CFU m L^-1.The detection results indicates that the novel ELISA based on I₂/I^--induced fluorescence quenching of Ag-Au NCs is promising for rapid detection of varieties of foodborne pathogens in milk.In chapter 6,to address the problem that the traditional ELISA has low quantitative detection sensitivity because of insufficient color intensity of oxidized TMB,we synthesized the fluorescence-enhanced porous coordination network-224（FE-PCN-224）in alkaline conditions.In alkaline conditions,the chemical reaction between Zr⁴⁺and OH^-decreases the Zr⁴⁺content of PCN-224,leading to the decrease of their conjugative effects.The recovery of electron density of 4,4’,4′′,4′′′-（Porphine-5,10,15,20-tetrayl）tetrakis（benzoic acid）（TCPP）caused the fluorescence enhancement of PCN-224.Considering the large overlapping between the excitation spectrum of FE-PCN-224 and absorption spectrum of silver nanoparticles（Ag NPs）,we proved the feasibility of Ag⁺reduction-triggered fluorescence quenching of FE-PCN-224 and applied it to the novel ELISA for sensitive detection of E.coli O157:H7 in milk.The LOD of the novel ELISA was 3.3×10² CFU m L^-1,29.7 times more sensitive than that of the HRP-based conventional ELISA.This method takes advantages of high sensitivity,specificity,and accuracy,which provides a new solution for rapid detection of foodborne pathogens in milk.In chapter 7,we comprehensively summarize the research content of this work.In the two perspectives of enzyme signal amplification and the novel signal readout modes,a series of efficient strategies are proposed to enhance the detection sensitivity of ELISA.In chapter 2,the enzyme signal amplification of the biotin-streptavidin system and stable color of Fe²⁺-Phen chelates are used for sensitive qualitative detection of ELISA.The low coupling ratio between streptavidin and biotin still greatly restricts the enzyme loading.In chapter 3,the inner and surface of Au/PDA/HRP are used to increase enzyme loading,thus boosting the detection sensitivity of ELISA.The cascade reaction is a more efficient strategy of enzyme signal amplification.In addition,Au NR etching is a sensitive colorimetric signal output mode.In chapter 4,the combination between GOx/Mo O₄^2-cascade catalytic reaction and Au NR etching achieves the qualitative and quantitative sensitive detection of ELISA.The fluorescence biosensors are more sensitive than colorimetric ones.Besides,Ag is a more active metal element than Au.In chapter 5 and 6,fluorescence quenching triggered by Ag oxidative etching and Ag⁺reduction,respectively,addresses the problem of low detection sensitivity of ELISA.To sum up,the iron（Ⅱ）chelates and Au/Ag nanomaterials-based strategies put forward in this work can efficiently enhance the sensing performance of ELISA and greatly extend the application range in food safety.In the end,we discuss the existing defects and potential development directions.