Integrating CRISPR-Cas12a with catalytic hairpin assembly as a logic gate biosensing platform for the detection of polychlorinated biphenyls in water samples

Polychlorinated biphenyls (PCBs) are ubiquitous persistent organic pollutants that cause harmful effects on environ-mental safety and human health. There is an urgent need to develop an intelligent method for PCBs sensing. In this work, we proposed a logic gate biosensing platform for simultaneous detection of multiple PCBs. 2,3 ',5,5 '-tetrachlorobiphenyl (PCB72) and 3,3 ',4,4 '-tetrachlorobiphenyl (PCB77) were used as the two inputs to construct biocomputing logic gates. We used 0 and 1 to encode the inputs and outputs. The aptamer was used to recognize the inputs and release the trigger DNA. A catalytic hairpin assembly (CHA) module is designed to convert and amplify each trigger DNA into multiple programmable DNA duplexes, which initiate the trans-cleavage activity of CRISPR/ Cas12a for the signal output. The activated Cas12 cleaves the BHQ-Cy5 modified single-stranded DNA (ssDNA) to yield the fluorescence reporting signals. In the YES logic gate, PCB72 was used as the only input to carry out the logic operation. In the OR, AND, and INHIBIT logic gates, PCB72 and PCB77 were used as the two inputs. The output signals can be visualized by the naked eye under UV light transilluminators or quantified by a microplate reader. Our constructed biosensing platform possesses the merits of multiple combinations of inputs, intuitive digital output, and high flexibility and scalability, which holds great promise for the intelligent detection of different PCBs.