Bridging Target Recognition with Direct Molecular Counting: A DNA Framework Converter-Mediated nFCM Assay Platform for Quantifying Diverse Biochemical Analytes

Molecular counting technologies have greatly advanced biochemical analysis with ultrahigh sensitivity, excellent accuracy, and intuitive readouts. However, current methods remain limited by complex and time-consuming procedures, complicated data acquisition or analysis, or restricted multiplexing capabilities. Herein, we report an icosahedral DNA nanoprobe (IDNP)-mediated nanoflow cytometry (nFCM) assay platform that enables simple and rapid (approximately 30 min per sample) quantification of diverse biochemical analytes by bridging target recognition with direct molecular counting. The IDNP functioned as a "nano-signal converter", transforming nFCM-undetectable small-sized analytes into detectable IDNP-derived nanosignals, thereby allowing the molecular counting of these target analytes using nFCM. This developed IDNP-mediated nFCM assay platform successfully quantified multiple analytes, including miR-21, thrombin, and Pb²⁺, with results highly consistent with conventional methods (qPCR, ELISA, and ICP-MS, respectively). Furthermore, the platform was extended to achieve site-specific m⁶A quantification, revealing upregulated methylation at specific sites of CDCP1 mRNA in bladder cancer cells and let-7a-5p miRNA in colorectal cancer cells compared with normal epithelial cells. Overall, this IDNP-mediated nFCM assay platform provides a powerful and versatile approach for rapid, simple, and multiplexed biochemical analysis, showing broad potential in biomedical and clinical applications.