Clinically applicable assay of cardiac troponin I by CRISPR/Cas12a-amplified single-particle collision electrochemistry

Owing to its rapid response and ultra-high sensitivity at single-entity level, single-particle collision electrochemistry (SPCE) shows great application prospects in sensitive detection of biomolecules. However, its widespread use in actual samples is constrained by the poor signal conversion efficiency and heavy interference from complicated samples. Here, a new SPCE biosensor for clinical analysis of cardiac troponin I (cTnI) was developed based on the effective signal amplification of CRISPR/Cas12a as well as the good separation and enrichment capabilities of magnetic nanobeads (MBs). The trans-cleavage capability of CRISPR/Cas12a can only be triggered in the presence of target cTnI to effectively cleave ssDNA2 on the surface of Au@Pt nanoparticles (Au@Pt NPs) within 30 min. Each cTnI molecule could cause the release of 4.68×104 Au@Pt NPs by utilizing the superior cleaving activity of CRISPR/Cas12a, significantly amplifying the signal and improving the sensitivity of analysis. The developed SPCE biosensor for cTnI had a detection limit as low as 0.35 fg/mL, which was 2–6 orders in magnitude less than existing cTnI detection techniques. Additionally, MBs allow the biosensor to detect cTnI in patient samples with excellent sensitivity and specificity by directly sampling from the complex matrix.