Exponential strand displacement amplification-assisted microchip electrophoresis for rapid and simultaneous detection of base excision repair enzymes.

DNA is inherently susceptible to damage from endogenous and exogenous sources. Base excision repair (BER) maintains genomic integrity, with its core enzymes Flap endonuclease 1 (FEN1) and Apurinic/apyrimidinic endonuclease 1 (APE1) implicated in various malignancies. Herein, an integrated microchip electrophoresis (MCE) and exponential strand displacement amplification (ESDA) method is presented for the simultaneous and sensitive detection of FEN1 and APE1. To achieve the simultaneous specific recognition of two enzymes and their ESDA cyclic amplification, two target-specific probes with distinct structures were specially designed: a dumbbell probe M1 for FEN1 and a double-stranded probe M2 for APE1. Other two template strands P1 and P2 were also carefully designed for the ESDA cyclic amplification. Because there were only four probes participating in this ESDA reaction, the background interference was significantly reduced and the detection sensitivity was increased corresponding. This approach achieves the limits of detection as low as 4 × 10⁻8 U/μL and 5 × 10⁻8 U/μL for FEN1 and APE1 respectively (S/N = 3) under the optimal conditions. Both enzymes were successfully quantified in cancer cell lysates and spiked serum samples with high specificity and sensitivity, indicating that this ESDA-MCE method is promising for the early diagnosis and effective treatment of cancer and related diseases.