CRISPR-Cas12a-mediated colorimetric aptasensor of Netilmicin based on enzymes-assisted signal amplification and nanozyme employing a rationally truncated aptamer

Netilmicin (NET) is a typical veterinary antibiotic used in aquaculture and animal husbandry, but its excessive accumulation poses a serious threat to the ecological environment and human health. Therefore, a novel and highly sensitive Crispr-Cas12a-mediated colorimetric aptasensor was developed by combining the enzyme-assisted signal amplification strategy and magnetic NMOF-Pt nanozyme composite material (NPSM) for the visual detection of veterinary antibiotic residues in real samples. A high-affinity truncated aptamer (N-20) targeting NET was evolved from a parental aptamer sequence with the help of comprehensive recognition mechanism studies. Subsequently, N-20 was elaborately designed into the isothermal enzyme-assisted strand displacement amplification reaction. The amplified products (Act-DNAs) activated the trans-cleavage activity in the Crispr Cas12a system. The NMOF-Pt nanozyme was released from NPSM after cleaving the single chain of DNA (ssDNA). The quantity of released nanozyme was positively correlated with the amount of NET to quantitatively achieve the detection with the linear range and limits of detection (LOD) of about 0.003-30 ng/mL and 1.15 pg/mL, respectively. This method was successfully applied to detect NET in Lake water, milk and pork samples with satisfactory recoveries ranging from 89.9-104.3%. The constructed aptasensor demonstrated superior analytical performance and exhibited enormous application prospects in food supervision, environment monitoring, and medical laboratory science.