Two Novel Enzyme-linked Immunosorbent Assays For The Detection Of Ochratoxin A

Enzyme-linked immunosorbent assay?ELISA?is a commonly used technique in clinical diagnosis,environmental monitoring,and food quality control because of its simplicity,low cost,and high throughput.Conventional ELISA using horseradish peroxidase?HRP?-catalyzed tetramethylbenzidine?TMB?as signal output,commonly detects the analytes by recording the absorbance value of the substrate.However,this method cannot be used for the detection of trace pollutants or biomarkers in food or clinical samples because of its relative low sensitivity,and this method is unsuitable for on-site detection in resource-constrained regions because the generated yellow solution with different intensities is difficult for naked-eye discrimination.This research is aimed to develop a fluoresence ELISA and a plasmonic ELISA based on the traditional direct competitive ELISA platform for sensitive and naked-eye detection of ochratoxin A?OTA?in corn samples.Quantum dots?QDs?have emerged as promising fluorescent markers due to their excellent photophysical properties such as high resistance to photobleaching and chemical degradation,high quantum yield,broad absorption spectra with narrow and symmetric emission bands,and so on.QDs-based fluorescence immunoassays have been widely used for the detection of various analytes,such as small molecules,metal ions,organic compounds,and biomolecules,and attracted much attention in recent years.Most of these methods are limited by the decay of activity of biomolecules?such as antibody?and fluorescent properties of QDs during the conjugation of antibody and QDs.So,it is necessary to develop ELISA using label-free QDs as signal output.In this propose,the novel ELISA based on fluorescence quenching of label-free QDs has been developed,in which QDs were used as a fluorescent signal output,and glucose oxidase?GOx?was used as an enzyme tracer.The fluorescence of QDs can be quenched by hydrogen peroxide?H₂O₂?produced in GOx-mediated glucose oxidation reaction.This proposed fluorescence ELISA demonstrated high sensitivity for the detection of OTA in corn extract.The detail contents are described as follows:first,mercaptopropionic acid?MPA?caped CdTe QDs?MPA–QDs?were synthesized,whose fluorescence variation was extremely sensitive to the presence of hydrogen peroxide?H₂O₂?and hydrogen ions;secondly,the GOx–OTA conjugates were synthesized according to the DCC/NHS method,in which the labeled amount of OTA molecule on per GOx protein was calculated at 2.27:1 according to the UV-vis spectrum.The optimized concentrations of anti-OTA monoclonal antibody?mAb?and GOx–OTA were 0.08?g/mL and 0.31?g/mL according to the results of checkerboard titration.The immunoreaction between mAb and GOx–OTA was performed at 37°C for 60 min in 0.02 M PB buffer containing 5%?v/v?methanol?pH 6.5?;the GOx-mediated glucose oxidation reaction was performed at 37°C for 60 min.Under the optimized conditions,the proposed method exhibited a good linear ranging from2.4 pg/mL to 625 pg/mL for OTA quantitative determination with a reliable correlation coefficient?R²=0.9932?.The regression equation could be represented by y=0.6247ln?x?+4.8543.The limit of detection?LOD?was calculated at 2.2 pg/mL,which is 15-fold lower than that of conventional HRP-based ELISA.Recovery studies were conducted to evaluate the accuracy and precision of the proposed method by analyzing OTA-spiked corn samples with different OTA concentrations.The average recoveries of the intra-assay ranged from 86.6%to 106%with a coefficient of variation?CV?ranging from 4.22%to 13.8%,and those for the inter-assay ranged from 84.4%to 107%and 7.13%to 14.5%,respectively.The reliability of the proposed fluorescence ELISA was compared with that of a commercial ELISA kit evaluated by analyzing 20 OTA-spiked real corn samples.The results of the proposed method demonstrated a good performance in recovery and reproducibility.Meanwhile,a direct competitive plasmonic ELISA?dc-pELISA?was developed for naked eyes detection of OTA,where HRP+H₂O₂+tyramine?TYR?-induced gold nanoparticles?AuNPs?aggregation was used as signal output,and the aggregation mechanism was explored.The optimized experimental concentrations for AuNPs aggregation were stated as follows:HRP,H₂O₂,and TYR concentrations are 10?g/mL,10?M and 1 mM,respectively;the optimal OTA–CAT and mAb concentration were 0.78?g/mL and 0.31?g/mL.Under the optimized conditions,the proposed method showed a good dynamic linear ranging from 12.5 pg/mL to 150pg/mL for OTA quantitative determination with a reliable correlation coefficient?R²=0.992?.The regression equation could be represented by y=-1.06ln?x?+7.581 with a half-maximal inhibitory(IC₅₀)concentration of 84.75 pg/mL,and a LOD of 17.8pg/mL,respectively.It was also demonstrated to have a low cut-off limit value at 150pg/mL by naked eyes.These values were approximately 2.9-,2.7-,and 13-folds lower than that of conventional ELISA.The proposed immunoassay also showed excellent selectivity,and no obvious cross-reaction with other common mycotoxins.The average recoveries of the intra-and inter-assays ranged from 82%to 109.5%,and the coefficient of variation for all the assays was less than 15%,indicating acceptable levels of precision for OTA quantitative analysis.There were no false-negative and false-positive results when dealing with 80 OTA spiked corn tests,indicating good reliability of the developed naked-eye readout dc-pELISA for OTA qualitative detection.