Integrated microfluidic platform coupling with hydrogel microparticle carriers for multiplexed MiRNA detection via cascaded enzyme-free amplification and smartphone-based fluorescence readout

A sensitive miRNA detection method was developed by combining shape-encoded hydrogel microparticles with a cascaded enzyme-free isothermal amplification strategy, enabling highly specific and low-background analysis. Detection limits of 0.2 pM for miRNA 21 and 7.5 pM for miRNA let-7a were obtained, with excellent specificity and minimal matrix interference. Integration with a microfluidic chip further improved detection throughput and operational efficiency. To enable automated on-chip analysis, a multi-channel microfluidic control chip was designed, incorporating pneumatically actuated microvalves and dual syringe pumps for precise fluid routing. Thus, a compact and portable platform was constructed by integrating this control chip with an online fluorescence detection chip, achieving seamless liquid handling, signal acquisition, and analysis. The platform was further applied to automated detection of miRNA 21 and let-7a expression patterns under inflammatory stimulation of a neuro-peripheral immune interaction model. This demonstrated the practical utility of the proposed integrated microfluidic platform for physiological studies, which also possessed potential for point-of-care diagnostics involving inflammation-associated miRNAs.