Direct multiplex detection of full length viral nucleic acids across multi-demics

Abstract

Diagnosis of infectious diseases caused by mutagenic pathogens often relies on nucleic acid-based detection, and CRISPR-based diagnostic technology has emerged as a promising point-of-care strategy. However, most current CRISPR diagnostics depend on the preamplification of the target prior to the CRISPR reaction to improve sensitivity, which carries the risk of amplification bias and affects quantification capabilities. In this study, we present a Cas12a-mediated field-effect transistor (FET) biosensor for amplification-free detection of viral nucleic acids associated with infectious diseases, targeting pathogens such as SARS-CoV-2, respiratory syncytial virus, and influenza A. The FET system, connected to a single-strand DNA-immobilized extended gate (EG) electrode, exhibits a synergistic effect of high-sensitivity sensing from the FET and amplification through the collateral cleavage activity of Cas12a. This FET sensor system demonstrated a sensitivity that is 10,000 times greater than that of fluorescence assays. The successful simultaneous detection of three full sequences of viral DNA on a single 8-well EG suggests practical feasibility for multimarker detection of infectious diseases, which can lead to more severe outcomes in cases of co-infection. By utilizing the same EG regardless of the target, the system enables mass production and cost-effectiveness. Additionally, a sensitivity-based EG selection approach minimizes measurement errors due to device-to-device variation.