Sensitive and Amplification-Free Electrochemiluminescence Biosensor for HPV-16 Detection Based on CRISPR/Cas12a and DNA Tetrahedron Nanostructures

Rapid and accurate detection of biomarkers was very important for early screening and treatment of diseases. Herein, a sensitive and amplification-free electrochemiluminescence (ECL) biosensor based on CRISPR/Cas12a and DNA tetrahedron nanostructures (TDNs) was constructed. Briefly, 3D TDN was self-assembled on the Au nanoparticle-deposited glassy carbon electrode surface to construct the biosensing interface. The presence of the target would activate the trans-cleavage activity of Cas12a-crRNA duplex to cleave the single-stranded DNA signal probe on the vertex of TDN, causing the Ru(bpy)₃²⁺ to fall from the electrode surface and weakened the ECL signal. Thus, the CRISPR/Cas12a system transduced the change of target concentration into an ECL signal enabling the detection of HPV-16. The specific recognition of CRISPR/Cas12a to HPV-16 made the biosensor have good selectivity, while the TDN-modified sensing interface could reduce the cleaving steric resistance and improve the cleaving performance of CRISPR/Cas12a. In addition, the pretreated biosensor could complete sample detection within 100 min with a detection limit of 8.86 fM, indicating that the developed biosensor possesses the potential application prospect for fast and sensitive nucleic acid detection.