A "Space-for-Time" Strategy Based on Single-Cell Dual-Isotope ICP-MS Analysis Enabling High-Throughput and Sensitive...

miRNAs regulate cancer progression and serve as both biomarkers and therapeutic targets in chemotherapy and gene therapy. Current analytical platforms lack the capacity to concurrently satisfy single-cell resolution and target specificity while maintaining high-throughput performance and cost-effectiveness. This limitation underscores the critical demand for innovative precision detection technologies. In this study, we focused on two key regulatory miRNAs in breast cancer therapeutic assessment and developed a "space-for-time" strategy using dual-isotope ICP-MS for single-cell miRNAs quantification. The spatial expansion of analytes via single hydrogel microbead encapsulation and collision gas utilization significantly prolonged single-particle ion cloud duration, overcoming the limitations of traditional quadrupole mass spectrometry in the precise dual-isotope quantification. A custom-designed microfluidic chip integrating droplet generation and inertial focusing achieved a single-cell encapsulation efficiency of 57%, thereby enabling a high throughput of ∼1000 cells/min in ICP-MS. In-bead rolling circle amplification combined with nanoprobe labeling enabled femtomolar-level sensitivity for miR-21 and miR-10b detection. This platform facilitated high-throughput, quantitative profiling of miRNA expression across various breast cancer subtypes and offered enhanced resolution of therapeutic responses. In chemotherapy monitoring, our approach revealed early molecular signatures of apoptosis/invasion, outperforming traditional viability and invasion assays in sensitivity and timeliness. For gene therapy evaluation, the platform uncovered subtype-specific differences in miRNA inhibition at single-cell resolution, highlighting tumor heterogeneity thus may potentially guiding precision treatment strategies. Overall, our platform represents a powerful tool for miRNA quantification and therapeutic evaluation, offering robust support for personalized oncology and preclinical drug screening.