Colorimetric ELISA based on glucose oxidase-regulated the color of acid–base indicator for sensitive detection of aflatoxin B1 in corn samples

Abstract

This study described a sensitive direct competitive enzyme-linked immunosorbent assay (ELISA) for naked-eye detection of aflatoxin B1 (AFB1) by using glucose oxidase (GOx)-regulated bromocresol purple (BCP) color change. GOx was used as an alternative to horseradish peroxidase (HRP) for oxidization of glucose into hydrogen peroxide and gluconic acid. BCP, whose color is significantly sensitive to pH variation, was used as a signal output. Under optimal conditions, the developed method exhibited a considerably high sensitivity for AFB1 detection with a cutoff limit of 100 pg/mL by the naked eye. The reliability of the developed colorimetric ELISA using naked-eye detection showed no false negative and false positive results among 70 AFB1 spiked tests. Furthermore, the developed method showed a good linear range of 25–200 pg/mL for AFB1 quantitative detection with a half-maximal inhibitory concentration at 66.72 pg/mL, which was approximately 10-fold lower than that of conventional HRP-based ELISA (IC50 = 707 pg/mL). The recoveries from four kinds of AFB1-spiked concentrations in corn extract solutions ranged within 80.56%–108.53%, with a coefficient of variation range of 1.69%–11.86%. These results exhibited good agreement with those of LC–MS/MS method indicating an acceptable accuracy and precision for AFB1 quantitative detection in actual corn samples. In brief, this study was the first to use a GOx-mediated color change of BCP in immunoassay for naked-eye detection of AFB1. This study also provided a new method for high-throughput screening detection of other small molecular chemicals using naked eye in resource-constrained countries.