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.

miRNAs as Predictors of Barrier Integrity

“…In contrast to the signal amplification method based on DNA assembly, this is an enzyme-assisted method that can trigger the amplification reaction of DNA at a constant temperature with the assistance of different isothermal amplification enzymes, thus achieving exponential signal amplification. Both the rolling circle amplification (RCA) [ 148 , 149 , 150 , 151 ] and the exponential amplification reaction (EXPAR) [ 152 ] are examples of the isothermal signal amplification methods and have a detection limit mostly in the fM range.…”

Flow lithography for structured microparticles: fundamentals, methods and applications

“…42,127,134–136 Such barcoded particles have been used for multiplexed protein and nucleic acid detection from cell lysates, fixed tissues, and serum, as the non-fouling property of microparticles from certain materials, like PEG, avoids non-specific binding. 135,166–171 These graphically encoded particles can also be functionalized with magnetic beads to allow for orientation control and gentle separation/concentration of particles. 132…”

Recent Advances in Exosomal miRNA Biosensing for Liquid Biopsy

“…These tandem repeats can be functional sequences or combined with complementary sequences with motifs. Sulaiman et al developed a one-pot method for extracellular vesicle (EV) derived miRNA (EV-miRNA) detection, which is completed with EV lysis and miRNA capture inside encoded hydrogel microparticles ( Figure 3 A) [ 62 ]. The captured target miRNA is ligated to a universal linker that is complementary to the circular template amplified by RCA.…”

“…(d) The single hydrogel microparticle imaged by confocal fluorescence microscopy. Adapted with permission from [ 62 ]. Copyright 2022 Wiley-VCH GmbH.…”