Direct Potentiometric Detection of Multiple Viral Nucleic Acids via Cas12a-Integrated Field-Effect Transistor Biosensors

Abstract

The rapid diagnosis of high-risk viral pathogens is critical for public health, yet current nucleic acid-based methods often necessitate pre-amplification steps. These processes can introduce amplification bias and hinder accurate quantification. To address these limitations, we report a Cas12a-mediated Field-Effect Transistor (FET) biosensor designed for the amplification-free detection of viral nucleic acids, including SARS-CoV-2, Respiratory Syncytial Virus (RSV), and Influenza A. Our system utilizes an extended gate (EG) electrode functionalized with single-stranded DNA (ssDNA). By leveraging the synergistic combination of the FET’s high-sensitivity transduction and the signal amplification provided by Cas12a’s collateral cleavage activity, we achieved a detection sensitivity 10,000-fold higher than traditional fluorescence-based assays. Furthermore, the platform demonstrates high practical utility through the simultaneous detection of multiple viral sequences on a single 8-well EG chip, facilitating the identification of co-infections. The use of a universal EG design allows for target-independent mass production and improved cost-efficiency. Finally, we implemented a sensitivity-based EG selection strategy to mitigate measurement errors stemming from device-to-device variation, ensuring robust and reproducible performance for point-of-care applications. Part of this presentation is based on our previously reported study on Cas12a-mediated FET biosensing systems (Sens Act B: Chemical, 443 (2025) 138212).