Collective Signal Colorimetric Multiplexing for Rapid RNA Detection

Colorimetric biosensors remain a cornerstone of point-of-need diagnostics because they are inexpensive, instrument-light, and visually intuitive. However, conventional formats struggle to combine true multiplexing with the sensitivity required for early-stage detection, particularly in complex clinical matrices. We report a multiplex colorimetric RNA assay that exploits a collective hybridization mechanism: multiple sequence-specific targets contribute to a single amplified ensemble signal rather than being segregated into separate channels. This architecture enables rapid analysis in under one hour, with limits of detection on the order of 10⁴ copies/mL and quantitative performance across a broad 100 aM–1 nM dynamic range in both buffer and undiluted serum. Thermodynamic evaluation of probe–target interactions supports the proposed collective hybridization model and explains the observed signal gain relative to conventional one-probe/one-signal designs. Because the readout is consolidated into a single colorimetric channel, panel expansion primarily involves adding new capture probes without increasing workflow complexity, preserving the operational simplicity that is critical for decentralized testing environments. These results define a general analytical framework for collective signal nucleic acid assays and point toward next-generation diagnostic tools that deliver rapid, sensitive, and multiplexed RNA detection while remaining compatible with low-resource clinical and public health settings.