Two Novel Enzyme-linked Immunosorbent Assay For The Detection Of HBsAg And Cronobacter

Enzyme-Linked Immunosorbent Assays?ELISA?is a high-throughput heterogeneous immunoassay,which combines the specific recognition between antigen and antibody with the high catalytic capacity of enzymes.Traditional ELISA based on colorimetric signal generated by horseradish peroxidase?HRP?catalyzed substrate tetramethylbenzidine,suffers from low detection sensitivity and is not suitable for naked eye detection.Recently,highly sensitive ELISA methods based on novel signal transition including fluorescence,plasma resonance,electrochemistry and chemiluminescence have attracted much attentions,and have been widely used in environmental monitoring,clinical diagnosis,food safety and other fields.In this study,a fluorescence ELISA and a plasmonic ELISA?pELISA?based on novel signal output were developed for highly sensitive detection of hepatitis B surface antigen?HBsAg?and Cronobacter,respectively.Mercaptopropionic acid capped CdTe quantum dots?MPA-QDs?is highly sensitive to hydrogen peroxide?H₂O₂?,whose fluorescence can be quenched largely by trace amount of H₂O₂.Thus,a fluorescence ELISA for highly sensitive detection of HBsAg was developed,where glucose oxidase was used as a label which can generate H₂O₂via catalytic oxidation of glucose,and H₂O₂ sensitive MPA-QDs was used as a signal output.Under optimized conditions,the proposed fluorescence ELISA method showed a limit of detection of 1.16 pg/mL,which was approximately 430-fold lower than that of HRP-based conventional ELISA.The average recoveries of the proposed fluorescence ELISA dealing with HBsAg-spiked serum samples ranged from 98.0%to 126.8%with the relative standard derivation below 10%,indicating good reproducibility and good accuracy of the method.Results obtained by detecting 35real HBsAg-negative and 31 HBsAg-positive serum samples exhibited excellent agreement with those obtained using commercial time-resolved fluorescence immunoassay kit?R²=0.9907?.The above results indicate that the proposed method based on fluorescence quenching of CdTe quantum dots is used for quantitative detection of HBsAg in serum samples with ultrahigh sensitivity and good reliability.The traditional gold seeds growth-based plasmonic enzyme-linked immunosorbent assay?pELISA?possess higher sensitivity,but the size and morphology of gold nanoparticles?AuNP?during the growth process is strictly controlled by the reducing reagents content,and reducing agent is fragile toward surrounding environment,resulting in poor stability of the method.Single-stranded DNA?ssDNA?could adsorbed on the surface of AuNP due to strong affinities between nitrogen of DNA bases and noble atom,the morphology of AuNP adsorbed by ssDNA during the growth process is related to the adsorbed amount of ssDNA,but ssDNA is extremely sensitive to·OH.Based on the above principles,this study established a novel pELISA based on ssDNA-directed AuNP growth.This assay was developed for Cronobacter detection in powder infant formula?PIF?sample,catalase was used to replace HRP in the conventional ELISA to regulate the H₂O₂ in the system,H₂O₂ is converted to·OH by Fenton Reaction to mediate the growth morphology of AuNP.Under optimized conditions,the proposed pELISA realized naked-eye detection of Cronobater?C.muytjensii ATCC 51329?with a cut off concentration of 3×10⁵ cfu/mL.Meanwhile,a highly sensitive quantitative detection for the of C.muytjensii ATCC51329 was realized by using UV-Vis with a good linear range?3×10² cfu/mL to 3×10⁷cfu/mL?,with a limit of detection of 1.6×10² cfu/mL,which were approximately162.5-folds lower than that of conventional ELISA.Moreover,the proposed method showed a good accuracy and precision detecting PIF samples spiked with different concentrations of C.muytjensii ATCC 51329,with average recoveries ranged from90.79%to 119.09%and coefficient of variation ranged from 4.24%to 9.55%.