A novel sensitization strategy for uncertain single stranded probe on electrode surface based on terminal deoxynucleotidyl transferase and G-rich spherical nucleic acids

In recent years, RPA-CRISPR/Cas12a-based electrochemical biosensors have emerged as highly effective tools for nucleic acid detection. However, the random trans -cleavage characteristics of Cas12a on single-stranded nucleic acid sequences can result in the presence of uncertain nucleic acid residues on the electrode surface post-reaction. This situation complicates subsequent sensitization strategies. In this study, we devised a universal sensitization protocol mediated by terminal deoxynucleotidyl transferase (TdT) , a template-independent DNA polymerase and G-rich spherical nucleic acids (G-rich SNAs) with superior performance. Initially, Cas12a was activated by the specific RPA amplification product and cleaved the closed single stranded DNA (C-ssDNA) on the electrode surface. Then, the TdT enzyme extended residual single stranded DNA (R-ssDNA) on the electrode surface, forming G-Quadruplex structures. Subsequently, G-rich SNAs were immobilized on the electrode surface via π-π stacking interactions with the G-quadruplexes. Consequently, a substantial accumulation of hemin occurred within the G-quadruplex cluster structures, generating significant electrochemical signals. We applied this method to the trace detection of Staphylococcus aureus ( S. aureus ) and achieved a good linear calibration curve with concentrations ranging from 0 CFU/mL to 10 4 CFU/mL. Notably, the limit of detection (LOD) was as low as 1.29 CFU/mL, surpassing that of most similar sensors. These results demonstrate that our proposed universal sensitization method effectively addresses the limitations associated with the random trans -cleavage characteristics of Cas12a.