Self-Powered FEN1 Biosensor Based on Accelerated CRISPR/Cas Trans-Cleavage around Porous Fe₃O₄ Nanoparticles

Flap endonuclease 1 (FEN1) is a structure-specific endonuclease that plays a critical role in the maintenance of genome integrity. In this work, we demonstrate a novel self-powered electrochemical FEN1 biosensor for potential applications in molecular diagnosis. Porous Fe₃O₄ nanoparticles are first prepared, and single-strand DNA probes are absorbed on the surface of the nanoparticles. Thus, electrochemical species of [Fe(CN)₆]^3- can be encapsulated inside the porous nanoparticles with the molecular gate of negatively charged DNA. On the other hand, a dumbbell structured DNA probe with 5' flap is designed. FEN1 is able to cleave the flap and activate the CRISPR/Cas system for the digestion of single-stranded DNA around Fe₃O₄ nanoparticles. As a result, the leakage of [Fe(CN)₆]^3- contributes to an enhanced electrochemical response, which can be used to reveal the level of FEN1. The high sensitivity of this biosensor is due to the application of porous nanomaterials and Mn²⁺ accelerated CRISPR/Cas cleavage. It succeeds in detection of biological samples and screening of FEN1 inhibitors. Therefore, this proposed method has potential applications in the early diagnosis of diseases and drug discovery.