One-Pot Simultaneous Detection of Multiple DNA and MicroRNA by Integrating the Cationic-Conjugated Polymer and Nuclease-Assisted Cyclic Amplification

A simple biosensor based on the cationic polymers and nuclease-assisted cyclic amplification has been constructed for the one-pot multiple detections of DNA and microRNA. In this work, we designed and synthesized a broad-spectrum polymer quencher, diazobenzene-containing poly(p-phenylethynylene) (azo-PPE(+)), to efficiently quench multiple fluorophores. Based on this concept, we prepared three DNA probes modified with three fluorophores (FAM, TAMRA, and Cy5), which entirely complement the target nucleic acids. In the presence of the target nucleic acid, the DNA probe hybridizes with the target and forms a duplex chain that is subsequently cleaved by nuclease into oligonucleotide fragments. Meanwhile, the target nucleic acid is released and rehybridized with other DNA probes, resulting in the cyclic degradation of the DNA probe. Due to the weak electrostatic interaction between the oligonucleotide fragment and azo-PPE(+), the fluorescence signal is restored and the target could be specifically detected. The combination of nuclease-assisted cyclic amplification and azo-PPE(+) enables a one-pot multiple nucleic acid detection with high sensitivity and specificity, even for the single-base-mismatched target. Moreover, the biosensor performs well in real biological samples and can distinguish miRNA expression in diverse cell lines, demonstrating its potential clinical diagnostic prospects.