Ligation initiated self-priming isothermal polymerization enabled nano-signal amplification for APE1 sensing and logical unlocking

Abstract

In this work, we developed a label-free and one-tube method for apurinic/apyrimidinic endonuclease 1 (APE1) sensing based on ligation initiated self-priming isothermal polymerization (SPIP) and signal amplification of fluorescent copper nanoparticles (CuNPs). This method was rationally proposed with modular design. In target recognition module, APE1 could specifically cleave substrate to release a linker DNA that served the ligation of an adenine/thymine (AT)-rich hairpin and a phosphorothioate-modified hairpin with the assistance of ligase. Then in SPIP amplification and signal transduction modules, once driven by polymerase, enzymatic extension and self-folding alternately occurred on the primary DNA originated from the ligation reaction, which resulted in accumulation of numerous AT-rich templates repeatedly included in the elongated dsDNA products, and ultimately enabled rapid formation of fluorescent CuNPs to output amplified signal. The accurate target identification and efficient signal amplification facilitated the highly selective and sensitive detection of APE1 with limit of detection of 8.6 × 10−4 U/mL. Benefiting from the robustness of this method, the normal cells and tumor cells could be distinguished unambiguously with this method according to the test results of cellular APE1. Moreover, the sensing strategy was employed in the concatenated AND logical operation and molecular keypad unlocking, which further displayed its extensibility and versatility.