Ionic Liquid-Modified Leakage-Suppressed Catalytic Hairpin Assembly with Time-Resolved Fluorescence for Direct miRNA Detection in Serum: Application to Esophageal Cancer Screening

Catalytic hairpin assembly (CHA) has emerged as a powerful enzyme-free nucleic acid amplification technique for point-of-care diagnostics, particularly in miRNA-based cancer screening. Despite its potential, clinical translation of CHA is hindered by two critical challenges: background leakage caused by spontaneous probe hybridization and serum matrix interference in fluorescence-based detection. This study presents an innovative solution by developing an ionic liquid-modified CHA system integrated with time-resolved fluorescence technology. Through systematic screening, 1,1,3,3-tetramethylguanidinium acetate was identified as the optimal ionic liquid that reduces leakage by 90% via DNA minor groove binding, without compromising catalytic efficiency. Although CHA can be performed directly in serum and other complex biological samples, background fluorescence originating from biological matrices (e.g., serum) frequently compromises the detection sensitivity and accuracy. In this research, we utilized a time-resolved fluorescence technology, which efficiently curbed serum autofluorescence, leading to a remarkable 1000-fold improvement in sensitivity compared to conventional fluorescence technology. This dual-optimized system was successfully applied to both high-throughput microarray analysis and rapid lateral flow strip detection. A clinical evaluation using 100 serum samples revealed excellent diagnostic accuracy for esophageal cancer, representing a significant improvement over conventional CHA methods in terms of clinical reliability and practical utility.