Rolling Circle Amplification-Enabled Ultrasensitive Point-of-Care Test Method for Aflatoxin B1 in the Environment and Food

Aflatoxin B1 (AFB1) contamination poses a fatal risk to human beings and urgently needs highly sensitive detection for environmental monitoring and food safety. However, the existing challenges are the unsatisfied sensitivity of the immunoassay methods and the complex matrix effect. Rolling circle amplification (RCA) is a promising method for nucleic acid isothermal amplification due to its high specificity and sensitivity. Herein, we constructed a general RCA-based point-of-care test method (RCA−POCT). With biotinylated antibodies, streptavidin, and biotinylated RCA primers, we realized the signal transduction and preliminary signal amplification. In this way, the fluorescent signal of the immunocomplex on the microwells was greatly enhanced. Under optimal conditions, we recorded sensitive detection limits for aflatoxin B1 (AFB1) of 1.94, 16.3, and 37.7 fg/mL (femtogram per microliter), and wide linear ranges with 5 × 10⁻⁶ to 5, 5 × 10⁻⁵ to 5, and 5 × 10⁻⁵ to 5 ng/mL in the irrigation water, field soil, and peanut samples, respectively. Satisfactory recovery, specificity, repeatability, and reproducibility were observed. The RCA−POCT was validated by comparing it to the HPLC method. This work provides a general RCA-assisted detection method for AFB1 in the environment and food.aflatoxin