Structural Encoding-decoupled Universal Recognition Enables Amplification-free Multiplex miRNA Detection

Abstract Accurate multiplex analysis of microRNAs (miRNAs) remains constrained by enzymatic amplification, spectral crosstalk, and instrumental complexity. Herein, we report a conceptually distinct PhC-SQ platform that integrates photonic crystal (PhC) structural encoding with S9.6-mediated universal recognition for amplification-free multiplex miRNA detection. PhC microspheres act as structural color barcodes for target encoding, while quantum dots (QDs) enriched at the microsphere interface serve as fluorescent reporters. Target miRNAs hybridize with surface-bound DNA probes to form DNA/RNA duplexes, which are recognized by the S9.6 antibody. Subsequent biotin–streptavidin assembly recruits QDs, enabling interfacial fluorescence enrichment and enhanced sensitivity without enzymatic amplification. By decoupling encoding from readout, the platform eliminates spectral crosstalk and enables accurate multiplexing. A dual-mode readout, combining confocal quantification with direct UV visualization, further supports point-of-care applicability. Validation in cellular samples shows good agreement with RT-PCR. Overall, this work establishes a structural encoding-universal recognition-interfacial signal enrichment strategy for robust and field-deployable multiplex miRNA detection.