A Modular Chemo-Responsive Fluorescent DNA Probe-Based Platform for Multiplex Reactive Biomolecular Analysis Using Fluorescence-Encoded Beads

The multiplex analysis of reactive biomolecules is crucial in diagnostics and life science research. However, conventional methods using small-molecule-based fluorescent probes are limited in the number of simultaneously detectable targets, owing to the spectral overlap of their fluorescence wavelengths. Consequently, we developed a multiplex analysis platform using chemo-responsive DNA-based fluorescent probes. A target-responsive moiety was installed in the phosphate backbone of the probe to strategically destabilize the DNA duplex. Reaction with a target molecule such as hydrogen peroxide or nitroreductase cleaves this moiety, restoring duplex stability and thereby triggering the accumulation of fluorescent DNA probes on beads functionalized with a complementary sequence. By encoding beads with distinct fluorescence intensity ratios and sizes, we achieved the simultaneous and specific detection of multiple targets in a single sample. The system performed well even with complex biological samples. This modular “reaction-to-accumulation” strategy offers a generalizable approach for developing DNA probe-based multiplex detection systems tailored to different target molecules.