Rapid, on-site and quantitative paper-based immunoassay platform for concurrent determination of pesticide residues and mycotoxins

Highlights: • A paper sensor to detect pesticides and mycotoxins: simultaneity from sample pretreatment to detection. • Sensitive and specific monoclonal antibodies against carbaryl and carbofuran were developed, respectively. • A cost and resource-efficient microscope-based subcloning method was developed in the hybridomas screening. • The on-site paper sensor demonstrated simultaneous quantification of six hazard chemicals rapidly and accurately. -- Abstract: Mycotoxins and pesticides are prevalent in cereal food. It is difficult to detect these two kinds of hazard factors simultaneously in rapid assay. In order to find a solution to the problem, carbamates and aflatoxins were selected in this study to establish a rapid, on-site, and quantitative paper sensor. Two novel monoclonal antibodies (mAbs) against carbaryl and carbofuran (1D2 and G11) were developed. The IC50 values (half maximal inhibitory concentration) were 0.8 ng/mL and 217.6 ng/mL for carbaryl and carbofuran, respectively. Based on the sensitive and specific mAbs, a multi-TRFICA (time-resolved fluorescence) paper sensor was developed, which simultaneously detected six types of hazardous chemicals, including AFB₁, AFB₂, AFG₁, AFG₂, carbaryl, and carbofuran. A universal sample pretreatment method for mycotoxins and pesticides was explored to apply on established competitive indirect enzyme-linked immunosorbent assay (icELISA) and multi-TRFICA-paper sensor. The established paper sensor can be easily observed with naked eyes, qualitatively under a UV lamp, and quantitated using a home-made device. It exhibited a calculated limit of quantity for AFTs, carbaryl, and carbofuran of 0.03, 0.02, and 60.2 ng/mL in corn samples, respectively. The spiking-recoveries and real sample studies proved that multi-TRFICA-paper sensor is an accurate, sensitive, and high throughput detection method for simple and low-cost analysis in corn samples.