Fluorescence Event Counting for miRNA Detection Enabled by Surface-Confined Hybridization Chain Reaction with Integrated Optical Gain

Le résumé fourni par la source

MicroRNAs (miRNAs) are key biomarkers for disease diagnosis and biological research, yet their reliable quantification in complex samples remains challenging. Fluorescence-based detection is often limited by heterogeneous amplification efficiency and the inherent variability of intensity-based readouts, making measurements vulnerable to both biochemical and optical fluctuations. Although digital approaches such as digital PCR can mitigate some of this variability, they pose a significant barrier to access due to their reliance on specialized micro-partitioning devices and instrumentation. To address this, we developed an accessible, microscopy-based event-counting strategy. Our design integrates a microlens array for optical signal enhancement with a surface-confined hybridization chain reaction (HCR). This configuration transduces miRNA targets into spatially discrete, countable fluorescence events, enabling reproducible quantification under a unified imaging and analysis framework on a standard widefield microscope. The assay demonstrates log-log linearity from 10 fM to 1 nM and achieves detection limits of 2.36-3.34 fM for three model miRNAs (miR-21, miR-122, and miR-155). In a 50% serum matrix, it maintains accurate quantification with recoveries of 93-112%. By shifting the readout from error-prone analog intensity measurements to robust event counting, this work provides a scalable and accessible platform for miRNA profiling.

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