CRISPR/Cas12a-Assisted Chemiluminescence Sensor for Aflatoxin B₁ Detection in Cereal Based on Functional Nucleic Acid and In-Pipet Rolling Circle Amplification

Abstract

Herein, we report a CRISPR/Cas12a-assisted chemiluminescence sensor for aflatoxin B 1 (AFB 1 ) detection based on functional nucleic-acid-mediated target recognition and in-pipet rolling circle amplification-mediated signal amplification. In this sensor, we performed rolling circle amplification on the inside of the pipet to enrich horseradish peroxidase (pipet-poly-HRP). When AFB 1 is present, it interacts with functional nucleic acids and results in the release of the activator. The activator is designed to activate the CRISPR/Cas12a system, which cleaves the pipet-poly-HRP to liberate HRP. The freed HRP can then be measured by chemiluminescence to quantify AFB 1 . This CRISPR/Cas12a-assisted chemiluminescence sensor enables facile, highly sensitive, and specific detection of AFB 1 , with a linear range from 50 pg/mL to 100 ng/mL and a detection limit of 5.2 pg/mL. Furthermore, it exhibits satisfactory recovery and has successfully challenged AFB 1 detection in cereal samples. The proposed sensor offers a novel rapid screening approach that holds great promise for food security monitoring.

Isothermal Reciprocal Catalytic DNA Circuit for Sensitive Analysis of Kanamycin

“…The ingenious integration of multiple amplicons could compensate for the limitations of individual amplicons and fully utilize their strengths, thus substantially improving the amplification efficiency. Some multilayered DNAzyme-involved CHR systems have been constructed, while the cofactor-dependent feature and the vulnerable substrate limited their widespread biosensing applications. , RCA involves the continuous replication of the complementary sequence of the circular DNA template through the successive lengthening of a primer strand in the presence of polymerase and dNTPs, resulting in the generation of long DNA nanowires. − With its simple design, low signal leakage, and high amplification efficiency, RCA becomes an ideal candidate for integration with the CHR amplicon to construct a robust reciprocal catalytic DNA circuit. Therefore, it is expected that a universal and powerful reciprocal catalytic circuit could enable high-performance biosensing in complex food matrices.…”

“…Programmable DNA amplification circuits have emerged as a powerful tool for monitoring food contaminants. − These circuits usually operate at a constant temperature, including catalytic DNA assembly (CDA), − cascade hybridization reaction (CHR), − DNAzyme-catalytic reactions, − and rolling circle amplification (RCA). − CHR mediates the cross-opening of hairpin reactants in response to a trigger, resulting in the formation of long copolymers . It exhibits moderate amplification capability, low cross-talk, and easy integration with FRET transduction for providing a readout signal.…”

Space-Confined Electrochemical Aptasensing with Conductive Hydrogels for Enhanced Applicability to Aflatoxin B1 Detection

“…Aspergillus flavus commonly exists in soil and contaminates a variety of crops, such as peanuts and maize . Food sources infected by this fungus produce aflatoxins, causing significant health risks and damage to humans and animals. , Aflatoxin B1 (AFB1), in particular, is regarded as the most harmful aflatoxin substance and has been classified as a group I carcinogen by the International Agency for Research on Cancer (IARC) .…”

Rapid and sensitive point-of-care PTS-CRISPR assay for food safety monitoring of aflatoxin B1

“…28,29 Despite great success, CRISPR-based detection techniques have barely been applied to food safety. 30 Herein, with the excellent trans-cleavage capability of Cas12a, we constructed a PTS-CRISPR method capable of quantitative and sensitive analysis of AFB1. The analytical principle of this work is shown in Scheme 1.…”

“…28,29 Despite great success, CRISPR-based detection techniques have barely been applied to food safety. 30…”