Electrochemiluminescence Biosensing Platform Based on CRISPR/Cas12a and DNA Nanotweezer-Mediated Catalytic Hairpin Assembly Amplification

The detection of microRNAs (miRNAs) biomarkers has great potential in the early diagnosis of acute myocardial infarction (AMI). Herein, we constructed an electrochemiluminescence biosensing platform based on DNA nanotweezer (DNT)-mediated catalytic hairpin assembly (CHA) and CRISPR/Cas12a system for detecting potential AMI biomarker miRNA-133a. DNT, as a programmable molecular scaffold, can precisely organize molecules at the nanoscale and output high signal-to-background ratio detection signals, which is introduced into the construction of sensing platforms. When the target miRNA was presented, the hairpin in DNT was opened, which altered the DNT structure from a closed state to an open state and exposed the catalytic sequence for CHA. Subsequently, a large number of F/A-F duplexes were generated after the addition of fuel strands (F) and antifuel strands (A-F), which served as the target for activating the CRISPR/Cas12a system. The activated Cas12a collaterally cleaved the signal probe (H1) on the electrode surface, causing the labeled Ru(bpy)₃²⁺ to detach from the electrode surface, resulting in a weakened ECL signal. We found that compared with the general CHA reaction, the DNT-mediated CHA reaction significantly lowers the leakage of the circuit; thus, a high signal-to-background ratio and detection sensitivity can be obtained. Therefore, we developed a highly sensitive biosensing platform for detecting miRNA-133a with a detection limit of 0.12 fM. This sensing strategy provides a new approach for nucleic acid detection and disease diagnosis.