Metal-Tagged CRISPR/Cas12a Bioassay Enables Ultrasensitive and Highly Selective Evaluation of Kanamycin Bioaccumulation in Fish Samples.

Abstract

The abuse of antibiotics in modern life and aquaculture has become a worldwide problem. Trace amounts of antibiotics discharged into natural water are increased in organisms through bioaccumulation and ultimately harm human health. Herein, we report a metal-tagged CRISPR/Cas12a bioassay and apply it to an ultrasensitive and highly selective evaluation of antibiotics bioaccumulation in wild fish samples. We integrated an element-tagging report probe and collateral cleavage activity of CRISPR/Cas12a. With the recognition and capture of target kanamycin by a "locked-activated" system, the activator strand was subsequently released to activate the collateral cleavage activity of Cas12a, followed by the cleavage of free Tm-Rep. After SA-MB capture, the biotin terminal was modified, and the uncleaved probe of Tm-Rep was removed. The acidized supernate containing the element tag fragment could be directly detected with 169Tm isotope monitoring by inductively coupled plasma mass spectrometry (ICPMS). With CRISPR/Cas12a biosensing and metal isotope detection by ICPMS, ultrasensitive and fast antibiotics analysis was realized with multiplex detection potential. Taking kanamycin as a modal analyte, a limit of detection as low as 4.06 pM was provided in a 30 min detection workflow. Besides, the bioaccumulation effect of kanamycin in a wild fish sample was also evaluated using the proposed strategy. We investigated the geographical distribution with Pseudorasbora parva samples collected in four different locations along a 600 km stretch of the Yangtze River. In addition, the bioaccumulation kinetics of antibiotics was evaluated in serum, muscle, and liver tissues of Pseudorasbora parva with 7 days of continuous feeding in a kanamycin-enriched environment.