Multivalent aptamer–mediated competitive control of CRISPR/Cas12a enables amplification-free electrochemical detection of Salmonella Typhimurium

Nucleic acid amplification-free detection of Salmonella Typhimurium ( S. Typhimurium ) remains challenging for decentralized food safety monitoring. Herein, we report a multivalent aptamer–mediated competitive electrochemical CRISPR platform for amplification-free detection of S. Typhimurium . A hybridization chain reaction (HCR) was employed to construct HCR-multiApt, a multivalent aptamer scaffold containing repeated crRNA-complementary activator regions. In the absence of bacteria, HCR-multiApt interacts with the Cas12a–crRNA complex and activates trans -cleavage of surface-immobilized electrochemical reporters. Upon target binding, S. Typhimurium competitively occupies the aptamer domains and reduces the accessibility of the crRNA-complementary activator regions, thereby suppressing Cas12a activation and modulating the electrochemical signal. A three-dimensional reduced graphene oxide/gold nanoparticle (3D-rGO/AuNPs) interface was used to enhance probe immobilization and electron transfer, while Hg 2 + -mediated T–Hg²⁺–T coordination enabled conformational regulation of the methylene blue-labeled reporter for improved signal transduction. Mechanistic studies, including microscale thermophoresis, enzyme kinetic analysis, and electrophoretic mobility shift assay, supported the target-dependent competitive regulation mechanism and indicated that HCR-multiApt enhanced the apparent Cas12a activation–cleavage efficiency mainly by increasing productive activation rather than directly altering the intrinsic catalytic activity of activated Cas12a. Under optimized conditions, the platform exhibited a linear detection range of 10 0 –10 8 CFU mL −1 with a detection limit of 3.75 CFU mL −1 . Selective discrimination against non-target bacteria and acceptable recoveries of 83.0–105.7% in spiked milk samples were achieved, with results consistent with conventional plate counting. This competitive multivalent EC-CRISPR strategy provides a programmable framework for amplification-free electrochemical pathogen detection.