Ratiometric paper-based microfluidic chip with cascaded RCA and dendritic mesoporous silica amplification for smartphone-based quantification of miRNA-21

A low-background paper platform enables cascaded RCA-based ratiometric sensing. • Blue CQDs provide self-calibrated and reliable miRNA-21 quantification. • Smartphone-assisted portable POCT enables noninvasive urinary miRNA analysis. The non-invasive detection of microRNA-21 (miRNA-21), a promising biomarker for bladder cancer, is challenged by its low abundance in urine and the lack of portable, sensitive analytical methods. Herein, we report an integrated smartphone-based paper microfluidic platform that combines rolling circle amplification (RCA), DMSN-based fluorescence enhancement, carbon quantum dots (CQD) -assisted ratiometric fluorescence detection, and smartphone imaging into a portable analytical system for quantitative miRNA-21 detection. A ratiometric fluorescence strategy is employed using blue-emitting CQDs covalently immobilized on glass fiber filter paper as an internal reference, while the RCA product is detected by a DMSN-coated QD probe. Under optimal conditions, the method achieves a limit of detection (LOD) of 1.06 nM for miRNA-21 with a linear range of 0.25–200 nM (R 2  = 0.9891). The assay exhibits excellent specificity against mismatched sequences and other miRNAs, and spike-recovery tests in artificial urine yield recoveries of 88.5–120.3% with relative standard deviations below 15%. The platform shows good batch-to-batch reproducibility (RSD < 7%). Spike recovery tests using clinical urine samples from healthy individuals yielded recoveries of 86.97%–108.5%, confirming the method's high accuracy and reliability in complex urine matrices. Integrated with a 3D-printed portable device and a custom smartphone application, this paper-based sensing platform provides a practical, low-cost, and portable analytical platform for point-of-care testing (POCT) of miRNA-21. The graphical abstract was prepared using SolidWorks, Cinema 4D (C4D), and Microsoft PowerPoint. No AI-assisted tools were used in the generation, preparation, or editing of the figures or graphical abstract. Download: Download high-res image (158KB) Download: Download full-size image