Persistent Chemiluminescence-Enabled Digital Bead Counting for Quantifying Attomolar MicroRNAs

作者：Chao Lei, Wenjiao Fan, Jingjing Shi, Zhaowei Tian, Xinrui Duan, Chenghui Liu · 发表于：Nano Letters · 年份：2025 · DOI：10.1021/acs.nanolett.5c00838 · 被引用次数：7 · 研究领域：MicroRNA in disease regulation、Advanced biosensing and bioanalysis techniques、RNA Interference and Gene Delivery

Digital biosensing is the state-of-the-art technique for precisely quantifying low-abundance biomarkers but heavily limited to sophisticated fabrication of sealed microchambers and fluorescence signal readout. Herein, a novel persistent, enhanced-chemiluminescence (E-CL)-enabled microchamber-free digital counting strategy is proposed for miRNA analysis by using fully open microbeads (MBs) as independent microreactors and signaling units. The employment of a phenothiazine derivative enhancer efficiently transfers the flash-type CL of horseradish peroxidase (HRP)-H 2 O 2 -luminol into persistent and stable E-CL with more than 10 3 -fold signal enhancement. More importantly, by leveraging single miRNA molecule-activated deposition of HRP, the driving power of E-CL, on the miRNA-loaded MBs, the long-lasting E-CL can be finely sustained on the MBs’ surface, achieving CL-based binary MB counting for digital miRNA quantification at the aM level. This persistent E-CL-powered microchamber-free digital design may well complement prevalent fluorescence-based digital bioassays by effectively addressing their inherent drawbacks (photobleaching/quenching and scattering light background), thus expanding the digital biosensing toolbox.