Quantitative and Multiplex Detection of Extracellular Vesicle‐Derived MicroRNA via Rolling Circle Amplification within Encoded Hydrogel Microparticles

Abstract Extracellular vesicle‐derived microRNA (EV‐miRNA) represent a promising cancer biomarker for disease diagnosis and monitoring. However, existing techniques to detect EV‐miRNA rely on complex, bias‐prone strategies, and preprocessing steps, making absolute quantification highly challenging. This work demonstrates the development and application of a method for quantitative and multiplex detection of EV‐miRNA, via rolling circle amplification within encoded hydrogel particles. By a one‐pot extracellular vesicle lysis and microRNA capture step, the bias and losses associated with standard RNA extraction techniques is avoided. The system offers a large dynamic range (3 orders of magnitude), ease of multiplexing, and a limit of detection down to 2.3 zmol (46 × 10 −18 m ), demonstrating its utility in clinical applications based on liquid biopsy tests. Furthermore, orthogonal measurements of EV concentrations coupled with the direct, absolute quantification of miRNA in biological samples results in quantitative measurements of miRNA copy numbers per volume sample, and per extracellular vesicle.