miRNA Cell Tracer: Multifunctional Microgels for Spatially Resolved and Wide-Range Detection of Intracellular miRNA at Single-Cell Level

Sensitive and spatially resolved detection of intracellular microRNAs (miRNAs) is essential for understanding gene regulatory networks at the single-cell level, yet low abundance and heterogeneous distribution challenge conventional methods. Despite the availability of a variety of particle biosensors, their intracellular use for live-cell miRNA detection remains challenging. Here, we demonstrate an enabling amplification-free detection and spatial mapping of miRNAs in single cells. We employ large (∼600 nm) biocompatible microgels, which provide high local probe density, protect molecular beacons from nuclease degradation, and enhance fluorescence signals upon hybridization with target miRNAs. Microfluidic mechanoporation efficiently delivers the microgels directly into the cytosol without perturbing endogenous miRNA levels or cell viability. Functionalized microgels generate robust fluorescence signals upon hybridization with target miRNAs. In intracellular sensing, the system achieves a low-picomolar limit of detection (5.6 pM) and a dynamic range spanning three orders of magnitude (pM-nM). The biosensor also exhibits excellent specificity; negligible fluorescence was observed both with nontarget and precursor miRNAs. Application in healthy (MCF10A) and cancer (MCF7) breast cells enabled single-cell quantification of endogenous miR-191-5p and miR-363-5p, revealing variability both among individual cells and between cell types, along with distinct intracellular localization patterns reflecting multiple concentration levels. The approach combines high sensitivity, wide dynamic range, quantitative precision, and spatial resolution, allowing amplification-free monitoring of miRNA expression in live cells. This versatile platform provides a powerful tool for intracellular biosensing, with potential applications in live-cell diagnostics, therapeutic response profiling, and studies of single-cell gene regulation.