Advanced Ligase Chain Reaction Strategy to Generate a Circular DNA Walker for Electrochemiluminescent Detection of Single Nucleotide Polymorphism

Single nucleotide polymorphism (SNP) primarily refers to DNA sequence polymorphism caused by variations in a single nucleotide, which is closely associated with many diseases such as genetic disorders and tumors. However, trace DNA mutants typically exist in a large pool of wild-type DNA, making it challenging to establish accurate and sensitive approaches for SNP detection. Herein, we developed an advanced ligase chain reaction (LCR) strategy to output the circular DNA walker for signal amplification, which realized accuracy and sensitive SNP detection based on the electrochemiluminescent (ECL) platform. Unlike the general LCR system that utilizes two sets of short single-stranded DNA (ssDNA) primers to generate double-stranded DNA amplification products, we ingeniously designed a long single-stranded DNA primer to replace one set of short ssDNA primers, allowing for the generation of circular DNA products upon complementing the target. Noticeably, the circular DNA serves as a DNA walker that can be easily purified by nucleases to eliminate unreacted primers and byproducts, significantly improving accuracy and sensitivity. Then, the circular DNA walker moved along a linearly ordered DNA quenching probe track modified on the ECL sensing interface, restoring the ECL signals by cleaving the quenching probes labeled on the DNA track with the help of apurinic/apyrimidinic endonuclease 1. Employing the p53 gene as a model, we realized the sensitive detection of mutant p53 in the range from 10 aM to 10 pM, with a detection limit of 6 aM, providing a promising platform for SNP detection.