Novel Enzyme-linked Immunosorbent Assay Based On Bifunctional Phage For Ochratoxin A Detection

Ochratoxin A（OTA）is one of the most compelling mycotoxins.Crops,coffee,and nut fruits contaminated by OTA is common worldwide,and resulting in a significant economic losses and serious threat to human health.Many countries and international organizations have set up the strict regulations to govern the OTA level in agricultural products.Various methods with high sensitivity and selectivity have been developed to prevent the OTA polluted crops and agricultural products transferring to the human food chain.Among them,enzyme-linked immunosorbent assays（ELISA）are one of the most popular analytical tools for mycotoxin routine screening test because of its unique advantages of simple operation,high-throughput,cost-effectiveness,and excellent specificity.However,conventional competitive ELISA based on HPR catalyzed tetramethylbenzidine（TMB）as signal output commonly suffers from two major shortcomings.First,the low extinction coefficients of TMB leads to a limited sensitivity,while the monochromic intensity change with target analyte concentration is unsuitable for on-site detection by naked-eye interpretation in resource-constrained regions.Second,the usage of toxic OTA and organic reagents for preparing enzyme or protein conjugates as competing antigens will lead to the potential environmental pollution and occupational hazard.M13 bacteriophage（M13 phage）is a filamentous virus,with a length and diameter of 900 nm and 6.5 nm,respectively.M13 consists of 2700 copies of the major p8 proteins and 3-5 copies of minor p3,p6,p7 and p9 proteins at the both ends.M13 phage is usually to mimic various target analytes,such as small molecules or proteins by the fusion expression of mimotopes at the N-terminal of p3 proteins.The abundant copies of p8 proteins can be extensively functionalized as a container for loading high-density of signal transducers or regulators（e.g.,fluorescent dyes and enzymes）,and thereby resulting in remarkably enhanced signal intensities of biosensors.Herein,a biotinylated M13 phage with OTA mimotopes was used as an alternative of OTA competing antigen,and also used as the“container”to load the numerous glucose oxidases（GOx）.In addition,the mercaptopropionic acid modified quantum dots（MPA-QDs）and gold nanoparticles were suggested as fluorescence signal,plasma colorimetric and photothermal dual-signals for the establishment of two novel ELISA methods for the sensitive detection of OTA.Firstly,the p8 protein of M13 phage carrying with OTA mimotope was modified with biotin molecules by the active ester method,and GOx was further conjugated on the surface of M13 phage via streptavidin-biotin system.MPA-QDs was chosen as fluorescent signal transducer because its fluorescent signal is extremely sensitive to hydrogen peroxide,while the hydrogen peroxide can be produced by the GOx catalyzed glucose.By the optimization of labeling conditions,each biotinylated M13phage was verified the great loading capacity with 269 streptavidin molecules,indicating the functionalized M13 phage can be used as the GOx container to enhance the sensitivity of ELISA.Under the optimal conditions,the proposed method demonstrated a good linear range for the quantitative detection of OTA from 4.8pg/mL to 625 pg/mL with a limit of detection（LOD）of 5.39 pg/mL.The LOD is approximately 26-fold lower than that of conventional HRP-based ELISA and 6-fold lower than that of GOx-OTA based fluorescent ELISA.The proposed method also showed good specificity and high accuracy for analyzing OTA in real corn samples.In addition,the results obtained from the proposed method were highly consistent with those obtained from the ultra-performance liquid chromatography method,indicating the high reliability of the proposed method for OTA quantitative detection.Secondly,a novel ELISA using colorimetric and photothermal signals as dual reporters was developed for OTA sensitive detection,in which the biotinylated M13phage was used as the competing antigen and the container of GOx,and the colorimetric and photothermal dual signals were used as the signal outputs to improve the sensitivity and accuracy of ELISA for OTA quantitative determination,in which the dual signals were generated from the gold nanoparticles aggregation by a horseradish peroxidase（HRP）/H₂O₂/tyramine（TYR）system.Under optimal conditions,the proposed ELISA using colorimetric and photothermal signals as dual reporters exhibited a good dynamic linearity ranging from 9.8 pg/mL to 312 pg/mL for OTA quantitative determination with the LOD value at 12.12 pg/mL and 8.58pg/mL,respectively.In addition,colloidal gold solution in the colorimetric format shows a vivid color change from deep blue to red with the OTA concentration increasement,which is suitable for use in the resource-limited regions,and the cutoff limit by a naked-eye interpretation result was 78 pg/mL.Moreover,the high recovery rates and low coefficient variations of proposed ELISA indicated the high accuracy and precision for OTA quantitative detection in real corn samples.Meanwhile,the results obtained from the developed method also showed a good agreement with those from HPLC method.