A developed cascade DNA recycling amplification integrating with microchip electrophoresis for ultrasensitive...

Cascade isothermal nucleic acid amplification techniques (cascade INAATs) are more sensitive than single INAATs and have been applied in bioanalysis in recent years. In this paper, a developed cascade DNA recycling amplification strategy comprising apurinic/apyrimidinic endonuclease 1 (APE1)-assisted target recycling amplification (AATRA) and structure-gated strand displacement amplification (SGSDA) was first investigated. The main improvement lies in a specifically designed closed-lock hairpin probe (cHP), which establishes a cascade amplification system featuring a unique "two-step conformational switching" mechanism. First, this cHP is converted into a transformable hairpin probe (tHP) via APE1-mediated cleavage during the AATRA. Second, tHP undergoes a critical structural transformation catalyzed by Klenow Fragment (KF) polymerase to generate a self-priming template, thereby initiating the SDA. This is a new type of SDA and can be called SGSDA. The stringent, enzyme-triggered "two-step conformational switching" mechanism serves as the critical driver and control point for the entire cascade amplification process, representing a significant advancement in enhancing amplification specificity and reducing cross-reactivity and background signal. This developed cascade recycling amplification was then integrated with microchip electrophoresis (MCE) and applied to a quantitative detection of C. sakazakii. Under the optimal conditions, the AATRA-SGSDA-MCE biosensing method achieved a broad linear range from 10¹ to 10⁶ CFU/mL and an outstanding limit of detection of 3 CFU/mL for C. sakazakii. Moreover, the proposed strategy was successfully applied to the detection of C. sakazakii in artificially contaminated milk powder, demonstrating the advantages of rapid testing speed, high sensitivity and strong specificity.