An immobilization-free electrochemical biosensor based on CRISPR/Cas13a and FAM-RNA-MB for simultaneous detection of multiple pathogens.

To better respond to biosecurity issues, we need to build good technology and material reserves for pathogenic microorganism screening. Here, we designed an electrochemical/optical signal probe with a common fluorophore and an electrochemically active group, breaking the previous perception that the signal probe is composed of a fluorophore and a quenching group and realizing the response of three signals: electrochemistry, fluorescence, and direct observation. Then, we proposed a homogeneous electrochemical nucleic acid detection system based on CRISPR/Cas named "HELEN-CR" by integrating free electrochemical/optical signal probes and Cas13a cleavage, achieving a limit of detection of 1 pM within 25 min. To improve the detection sensitivity, we applied recombinase polymerase amplification to amplify the target nucleic acid, achieving a limit of detection of 30 zM within 45 min. Complemented by our self-developed multi-chamber microfluidic chip and portable electrochemical instrument, simultaneous detection of multiple pathogens can be achieved within 50 min, facilitating minimally trained personnel to obtain detection results quickly in a difficult environment. This study proposes a simple, scalable, and general idea and solution for the rapid detection of pathogenic microorganisms and biosecurity monitoring.