No abstract

Conventional enzyme-linked immunosorbent assay (ELISA) is commonly used for Ochratoxin A (OTA) screening, but it is limited by low sensitivity and harmful competing antigens of enzyme-OTA conjugates. Herein, a bifunctional M13 bacteriophage with OTA mimotopes fused on the p3 protein and biotin modified on major p8 proteins was introduced as an eco-friendly competing antigen and enzyme container for enhanced sensitivity. Mercaptopropionic acid-modified quantum dots (MPA-QDs), which are extremely sensitive to hydrogen peroxide, were chosen as fluorescent signal transducers that could manifest glucose oxidase-induced fluorescence quenching in the presence of glucose. On these bases, a highly sensitive and eco-friendly fluorescent immunoassay for OTA sensing was developed. Under optimized conditions, the proposed method demonstrates a good linear detection of OTA from 4.8 to 625 pg/mL and a limit of detection (LOD) of 5.39 pg/mL. The LOD is approximately 26-fold lower than that of a conventional horse radish peroxidase (HRP) based ELISA and six-fold lower than that of a GOx-OTA conjugate-based fluorescent ELISA. The proposed method also shows great specificity and accepted accuracy for analyzing OTA in real corn samples. The detection results are highly consistent with those obtained using the ultra-performance liquid chromatography-fluorescence detection method, indicating the high reliability of the proposed method for OTA detection. In conclusion, the proposed method is an excellent OTA screening platform over a conventional ELISA and can be easily extended for sensing other analytes by altering specific mimic peptide sequences in phages.

“Point of care” (POC) methods without expensive instruments and special technicians are greatly needed for high-throughput analysis of mycotoxins. In comparison, the most widely used screening method of the conventional enzyme-linked immunosorbent assay (ELISA) confronts low sensitivity and harmful competing antigens. Herein, we develop a plasmonic-photothermal ELISA that allows precise readout by color-temperature dual-modal signals based on enzymatic reaction-induced AuNP aggregation for highly sensitive detection of ochratoxin A (OTA). The bifunctional M13 phage carrying OTA that mimics the mimotope on the end of p3 proteins and abundant biotin molecules on the major p8 proteins is adopted as an eco-friendly competing antigen and enzyme container for amplifying the signal intensity. Under optimal conditions, both colorimetric and photothermal signals enable good dynamic linearity for quantitative OTA detection with the limits of detection at 12.1 and 8.6 pg mL−1, respectively. Additionally, the proposed ELISA was adapted to visual determination with a cutoff limit of 78 pg mL−1 according to a vivid color change from deep blue to red. The recoveries of OTA-spiked corn samples indicate the high accuracy and robustness of the proposed method. In conclusion, our proposed strategy provides a promising method for eco-friendly and sensitive POC screening of OTA. Moreover, it can be easily applied to other analytes by changing the involved specific mimotope sequence.