A Triple-Signal Output Lateral Flow Platform Leveraging CRISPR/Cas12a and Nanozyme Cascades for Ultra-Sensitive Aflatoxin B1 Detection

Abstract

The presence of the potent carcinogen aflatoxin B1 (AFB1) in agricultural products poses a grave threat to food safety and human health, underscoring the imperative development of highly sensitive detection methodologies. Despite the considerable efforts that have been dedicated to aptamer-based biosensors, the sensitivity of typical nucleic acid-integrated aptasensors is still limited by inefficient strand displacement. To address this limitation, a “three-in-one” multireadout lateral flow assay for AFB1 was developed, integrating CRISPR/Cas12a single-stranded DNase activity with PtPdRu nanozyme-mediated colorimetric signal amplification. The detection process initiates with a “locked activator” DNA configuration, wherein aptamer-specific recognition of AFB1 activates the nonspecific ssDNA cleavage activity of CRISPR/Cas12a, resulting in a fluorescence output. The integrated PtPdRu functions as both a colorimetric probe and a peroxidase mimic, allowing for catalytic amplification based on 3,3′,5,5′-tetramethylbenzidine for improved colorimetric detection. The signal-amplified aptasensor achieves ultrasensitive detection with a limit of detection of 0.22 pg/mL, which is 13-fold and 2.7-fold lower than those of the fluorescence mode and the intrinsic colorimetric mode, respectively. Coupled with a smartphone app, the “samples‑in, signal‑out” strategy exhibits high specificity in moldy corn and peanut samples. The triple-modal outputs provide a versatile tool for sensitive detection of AFB1 in agricultural products.