Primer exchange reaction activation of CRISPR/Cas12a system to construct a versatile and label-free electrochemical sensing platform

The detection of antibiotics and pesticide residues is extremely important to maintain food safety. In this work, a versatile and label-free electrochemical biosensing platform was developed to sensitively detect antibiotics and pesticide residues using primer exchange reaction (PER) signal amplification technology integrated with CRISPR/Cas12a system. Initially, single-stranded DNA (ssDNA, S1) rich in guanine was pre-modified on the electrode surface to capture hemin and form G-quadruplex/hemin complexes, achieving strong electrochemical signal output. When the target was available, it could bind to the aptamer and release the hairpin template of PER, and with the help of DNA polymerase, the short primers pre-modified on the electrode surface were extended into long ssDNA through two PER cycles. The long ssDNA generated by PER circuit contained multiple binding sites of CRISPR/Cas12a, which activated CRISPR/Cas12a system and rapidly cleaved S1 on the electrode surface, preventing the formation of G-quadruplex/hemin complexes, resulting in significantly weakened electrochemical signal and achieving label-free and sensitive detection of the target. Benefiting from the programmability of DNA, universal detection of ampicillin, kanamycin, and acetamiprid was achieved by simply changing the hairpin substrate of PER, with detection limits of 0.33 pM, 0.38 pM, and 0.78 pM (S/N = 3), respectively. Meaningfully, the sensing platform successfully performed the detection of ampicillin in milk and livestock wastewater samples, supplying a new electrochemical analysis method with respect to accurate and reliable detection of actual samples.