Automated Microchannel Resistance Readout of Immunorecognition via Programmable Nuclease Mediation for On-Site Biosensing

Achieving fully automated transduction and signal amplification of small molecule target recognition events into ultrasensitive, detectable electrical signals remains challenging, owing to the need for seamless integration of cascade biochemical reactions. Here, we present an integrated and portable biosensing platform that couples immunorecognition with programmable nuclease-mediated electrical transduction, in which sequential current pulse signals generated by multiple microspheres flowing through the microchannel are recorded and quantified. In this assay, target competes with an antigen-guide DNA conjugate for antibody binding, and the residual guide DNA activates <i>Clostridium butyricum</i> Argonaute (CbAgo) to cleave biotinylated target DNA on magnetic nanoparticles. This cleavage modulates the ability of magnetic nanoparticles to capture polystyrene microspheres, and the resulting change in microsphere concentration is directly quantified as a microchannel resistance variation, enabling amplification-free, quantitative detection. This streamlined architecture minimizes manual processing and supports field deployment. As proof of concept, the platform achieved on-site detection of enrofloxacin in milk with a limit of detection of 4.59 pg/mL. The integration of immunological specificity, programmable nuclease catalysis, and portable electrical readout establishes a generalizable framework for automated, point-of-need biosensing of diverse small molecules and biomarkers.