Sun Yanyan, Sun Yunlong, Peng Yufei, Xu Danke
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, No 163, Xianlin Avenue, Nanjing, 210023, PR China.
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, No 163, Xianlin Avenue, Nanjing, 210023, PR China.
Anal Chim Acta. 2025 Oct 1;1369:344341. doi: 10.1016/j.aca.2025.344341. Epub 2025 Jun 18.
The aberrant expression of microRNAs (miRNAs) is closely associated with the onset and progression of many diseases, particularly in malignant tumors, where it usually involves coordinated changes in the expression levels of multiple miRNAs. However, existing miRNA detection techniques still face many technical challenges in achieving multiplex detection within a single tube. Therefore, there remains a critical need to develop multiplex miRNA detection methods with high specificity, simplicity, and sensitivity.
A non-gel sieving capillary electrophoresis (NGCE) detection strategy based on enzyme-free strand displacement was proposed to address this issue, incorporating magnetic bead purification and drag tags for efficient multiplex miRNA detection. This strategy successfully overcomes the challenge of separating multiple amplification products and unreacted probes in NGCE detection by integrating drag tags on substrates and a magnetic separation-assisted system. Enzyme-free entropy-driven circuit (EDC) amplification enhances the sensitivity. Seven miRNAs were selected as targets, with varying thymine tail lengths attached to EDC substrate probes. Following EDC amplification and magnetic bead purification, baseline separation of the seven miRNA products was successfully achieved using NGCE technology. The detection limits ranged from 154.6 amol to 933.8 amol, with recovery rates in human serum between 88.36 % and 115.14 %, and the relative standard deviations (RSD) under 10 %. This detection strategy shows no cross-reactivity and can distinguish single-nucleotide mismatched miRNAs at different sites with high specificity.
By adjusting the length of the drag tag, this strategy can be further expanded to simultaneously detect more than ten miRNAs. Furthermore, it significantly reduces detection costs by eliminating the need for fluorescent labeling or enzymatic participation. This strategy has great potential in the multiplex miRNA detection and broad application in the early diagnosis and prognostic management of malignant tumors.
