Nanopipette Sensing of APE1 Activity via Enzymatic Recycling and DNA Tetrahedron−Streptavidin Reporting: Profiling in Cancer Diagnostics, Oxidative Stress, and Cell Cycle

Abstract

Abstract Apurinic/apyrimidinic endonuclease 1 (APE1) is a clinical biomarker whose small molecular size hinders direct detection by conventional nanopores (30 nm). Here, we report a nanopipette platform integrating APE1-driven enzymatic recycling amplification with DNA tetrahedron−streptavidin (TDN−SA) complex-based resistive-pulse reporting to overcome this size limitation. APE1 specifically cleaves hairpin DNA probes, generating output strands that displace biotin-modified DNA oligo from the TDN−SA complex, reducing translocation event frequency in 30 nm nanopipettes. This signal modulation enables APE1 quantification with a detection limit of 10−5 U/mL and a linear range spanning 7 orders of magnitude (10−5 to 10 U/mL). The method discriminates APE1 activity between normal and cancer cell lines (HeLa, MCF-7, A549 vs. 293T, MCF-10A), tracks oxidative stress-induced elevation (0−4 h H2O2 treatment), and reveals cell cycle-dependent regulation (lowest in G0/G1, peaking in S phase). Furthermore, the system evaluates APE1 inhibition by 7-nitroindole-2-carboxylic acid in inflammatory macrophages, demonstrating its utility for drug efficacy assessment. This label-free platform overcomes the protein size constraint of traditional nanopore methods, offering a versatile tool for profiling low-abundance protein biomarkers in native cellular contexts.