FEN1-assisted cascade enzymatic reaction (FACER) for one-step and washing-free detection of nucleic acid using personal glucose meter

Abstract

A novel one-step nucleic acid detection platform termed FACER (FEN1-assisted Cascade Enzymatic Reaction) has been developed, leveraging the structure-specific cleavage ability of the flap endonuclease (FEN1) enzyme for the sensitive detection of target DNA. FACER employs two specifically designed probes that hybridize with target DNA, forming a UP single flap structure cleaved by FEN1 to release adenosine monophosphate (AMP). The AMP serves as a substrate in a cascade enzymatic reaction (CER) involving myokinase, pyruvate kinase, and hexokinase, ultimately resulting in a decrease in glucose concentration measurable using a personal glucose meter (PGM). The method achieves a limit of detection as low as 4.8 pM under isothermal conditions, demonstrating high sensitivity and specificity. FACER was further validated in biological matrices, achieving precise detection of target DNA with high accuracy and reliability. This washing-free, isothermal platform eliminates the need for fluorescence labeling or complex instrumentation, offering a robust and cost-effective solution for point-of-care applications in resource-limited settings.