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

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.