In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy)) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy)/AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy)/AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy)/AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy)due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis. 3 3 3 3 3 2+ 2+ 2+ 2+ 2+
