Amplification-Free CRISPR/Cas12a-Based Electrochemical Biosensor with Enhanced Sensitivity for Viral Detection

To detect contagious viral nucleic acids, traditional biosensors often require target amplification steps or use fluorescence and Raman probes tagged on nucleic acids, which are time-consuming, complex, and expensive. Recently, the CRISPR/Cas12a has received the attraction for development of nucleic acid biosensors, beyond its conventional role-like gene editing, but the enhancement of the sensitivity of CRISPR/Cas-based biosensors is still required to simplify the biosensing steps. Here, we develop a CRISPR/Cas12a-based electrochemical biosensor for the detection of viral nucleic acids in a simple manner. The novel mismatch Ag probe (MAP), as a sensing probe, and the highly conductive gold electrode on indium tin oxide with a nano array (GELITION) are introduced that enable the amplification-free and ultrasensitive detection of nucleic acids using a CRISPR/Cas12a system. The biosensing ability of the developed biosensor is validated using human papillomavirus type 16 and 18 viral DNAs (HPV16 and HPV18), achieving a limit of detection (LOD) of 1 fM without amplification and complex steps. Our developed biosensor is expected to be applicable in detecting various viruses and could contribute to the early detection of future pandemics.