Electrochemiluminescence Biosensor for Human Papillomavirus DNA Based on Nanoparticle Charge Density Regulation through a Target Activated CRISPR/Cas12a System.

The negative charge of nanoparticles affects their diffusion toward the negatively charged indium tin oxide (ITO) electrode, and this had been coupled with target-activated CRISPR/Cas12a to develop a homogeneous electrochemiluminescence (ECL) biosensor for human papillomavirus 16 (HPV 16) DNA (chosen as model target) detection. Tris-(2,2′-bipyridyl) ruthenium-(II) chloride hexahydrate-doped SiO₂ nanoparticles (Ru@SiO₂ NPs) with a diameter of about 50 nm was selected as the ECL probe.They were formed by encased Ru-(bpy)₃²⁺ in SiO₂ nanoparticles, and this can effectively prevent the leakage of Ru-(bpy)₃²⁺ molecules and improve the stability of the probe. A hairpin probe (HP) was carefully designed and modified on the Ru@SiO₂ NPs surface and resulted in the generation of a large negative charge on the surface of the nanoparticles. The strong electrostatic repulsion prevented the nanoparticles from reaching the negatively charged ITO electrode, leading to the weakness of the ECL signal detected. In the presence of a target, Cas12a is activated and the single-stranded region of HP is cleaved and separated from the Ru@SiO₂ surface, resulting in less negative charge of the particles and enhanced ECL signal of the system. The ECL signal was linearly correlated with the logarithm of the HPV 16 DNA concentration in the range of 10 fM–10 nM with a limit of 3.4 fM (S/N = 3) under optimal conditions. The biosensor shows exceptional performance in the detection of HPV16 DNA in real samples. [ABSTRACT FROM AUTHOR]