Eco-Friendly Fluorescent ELISA Based on Bifunctional Phage for Ultrasensitive Detection of Ochratoxin A in Corn

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.

Bifunctional M13 Phage as Enzyme Container for the Reinforced Colorimetric–Photothermal Dual-Modal Sensing of Ochratoxin A

“…Moreover, numerous biotin molecules were modified on the major p8 proteins. According to our previous report, the as-prepared bio-M13 OTA exhibits a high loading capacity of 269 streptavidin molecules [ 35 ]. This finding indicates that biotin-M13 OTA can be used as the enzyme container for carrying a mass of biotinylated GOx (biotin-GOx).…”

“…The abundant copies of p8 proteins can be extensively functionalized as a container for high-density loading of signal transducers or regulators (e.g., fluorescent dyes and enzymes), resulting in remarkably enhanced signal intensities of biosensors [ 34 ]. Previously, we adopted an OTA-mimicking M13 phage (M13 OTA ) integrated with an OTA mimotope at the p3 proteins as an enzyme container to improve the sensitivity of a fluorescent ELISA for OTA detection [ 35 ].…”

An Extremely Highly Sensitive ELISA in pg mL−1 Level Based on a Newly Produced Monoclonal Antibody for the Detection of Ochratoxin A in Food Samples

“…To the best of our knowledge, this is the most sensitive ELISA that has been achieved so far based on this newly produced mAb. In should be addressed that some aptamer (or Phage)-based ELISAs or sensors may obtain high sensitivity for OTA detection [37][38][39][40]. In the last decades, many ELISAs for the detection of OTA in food samples based on pAb or mAb have been developed [23][24][25][26][27][28][29][30][31][32][33][34][35][36].…”

A DNA tetrahedral nanomaterial-based dual-signal ratiometric electrochemical aptasensor for the detection of ochratoxin A in corn kernel samples

“…The current analytical methods to detect OTA include enzyme-linked immunosorbent assay (ELISA), 8 thin-layer chromatography (TLC), 9 surface-enhanced Raman scattering (SERS), 10 and fluorescence analysis. 11 Besides having advantages, these methods are limited by expensive and laborious processes, costly equipment, or proneness to false-positive results.…”