Molecular Encoded Beads for Multiplexed Nucleic Acid Quantitative Detection.

Here, we present an improved version of our previously proposed DNA coding method, incorporating three key modifications: (1) modification of the hybridization region in the coding probe, (2) replacement bead type, and (3) introduction of a third fluorescent dye that overcomes previous limitations in quantitative nucleic acid detection. To demonstrate practical utility, we developed a trifluorophore system capable of simultaneously quantifying eight distinct nucleic acids using flow cytometry with just two excitation channels and three emission channels. This plug-and-play strategy enables concurrent encoding and target detection, significantly simplifying the workflow. The platform achieves detection limits in the pM level, meeting sensitivity requirements for multiplex nucleic acid analysis applications. Overall, these advancements establish a versatile quantitative detection platform that enables rapid, robust, and high-throughput multiplex nucleic acid detection.