A hydrogel microsphere-based catalytic hairpin assembly for simple miRNA detection

MicroRNAs (miRNAs) are endogenous non-protein-coding single-stranded RNAs that play a pivotal role in gene regulation and hold great biomarker potential for cancer diagnosis. However, the detection of miRNAs in blood samples still faces challenges due to their short chain length, low abundance, high sequence homology, severe matrix interference, and susceptibility to degradation. Although the RT-qPCR method is sensitive, it is optimized for long-stranded RNA amplification and requires reverse transcription and temperature cycling, which increases the complexity of detection. Enzyme-based isothermal amplification techniques such as RCA eliminate the need for reverse transcription and temperature cycling, but they rely on expensive enzymes and require strict maintenance of enzyme activity during operation, which limits their practical applications. Catalytic hairpin assembly is a sensitive enzyme-free isothermal amplification strategy with low cost and simple operation, yet the background interference is unavoidable for homogeneous environments and the non-specific adsorption of proteins in blood samples is also a practical problem that CHA must face. This paper reports a hydrogel microsphere-based CHA method that integrates CHA signal amplification with the solid-phase carrier of hydrogel microspheres for the detection of miRNAs in blood samples. As the solid-phase carrier for CHA, hydrogel microspheres allow free entry of target miRNAs while effectively blocking large biomolecules such as proteins in the matrix (block rate of 64% for BSA). The target-triggered CHA reactions occur exclusively within the microspheres, resulting in the concentration of target molecules and their effective separation from the sample matrix. In addition, natural dehydration of microspheres after CHA reduces the volume of microspheres to one-eighth of its original size, which can further enhance the signal intensity. Microfluidic droplets serve as templates for microsphere fabrication, ensuring excellent uniformity of the hydrogel microspheres (RSD of the diameter is less than 2%.). Under optimized conditions, this method achieves a limit of detection (LOD) as low as 200 pmol L^-1 for miRNA-21. Notably, this represents a 3-fold LOD enhancement that is uniquely enabled by the dehydration treatment, in addition to the inherent signal amplification effect of the CHA reaction. The assay also exhibits a wide linear detection range spanning from 500 pmol L^-1 to 100 nmol L^-1, and retains excellent single-base mismatch discrimination capability. The recovery rates of miRNA-21 in human serum samples range from 99.5% to 104%. This proof-of-concept method exhibits favorable application potential for the detection of miRNA biomarkers in complex biological samples with a simple, low-cost, enzyme-free mode.