A signal-switchable electrochemiluminescence biosensor based on the integration of spherical nucleic acid and CRISPR/Cas12a for multiplex detection of HIV/HPV DNAs

Herein, we report a "on-off" signal-switchable electrochemiluminescence (ECL) biosensor for simple, sensitive and quantitative detection of two kinds of virus genes, human immunodeficiency virus (HIV) and human papilloma virus (HPV-16) DNAs, by the integration of spherical nucleic acid (SNA) with CRISPR/Cas12a. The concept behind this approach is "on" signal based on a sandwich type assay after ECL luminophores of carbon dots (CDs) labeled SNA incubated with the biosensor in the presence of HIV DNA; and "off" signal upon the biosensor incubation with assistant DNA (HIV DNA sequence), CDs-labeled SNA and Cas12a/crRNA/HPV-16 DNA ternary complex relying on the collaterally cleavage of Cas12a. The cross-interference between HIV and HPV-16 DNAs can be avoided by adding Cas12a effector to the system or not. Using this strategy, HIV and HPV16 DNAs are successfully quantitatively detection with detection limits of 30 fM and 0.32 pM (S/N = 3), respectively. Importantly, this system does not rely on multiplex signal reporting molecules, thus simplifying the detection system and avoiding the likelihood of cross interferences presented in the assay. Furthermore, multiplex detection of HIV/HPV-16 DNAs in human serum samples are successfully achieved within 2 h without additional nucleic acid amplification step. As demonstrated, the proposed biosensor has the potential for multiplex detection in clinical diagnosis.