Induced Core-Shell Transformation in Agarose-Chitosan Nanoreactor Beads Enable Single Channel Coding for dPCR Multiplexing.

Hybrid nanoreactor beads composed of agarose and chitosan exhibit a previously unreported structural transformation when transferred into a non-aqueous medium and subjected to thermocycling. We were able to define which pH and temperature conditions lead to spontaneous reorganization into a stable core-shell architecture in beads, driven by pH-dependent chitosan protonation and thermally induced network restructuring. We elucidate the mechanistic basis of this transformation and show that the resulting shell layer and core structure provide distinct optical signatures that can be tuned through the processing conditions. Leveraging this intrinsic material response, we introduce a single-channel bead coding strategy that enables simultaneous digital PCR amplification and bead identification. As a proof of concept, we demonstrate multiplexed digital PCR of multiple samples in a single run, illustrating how a fundamental structure-forming mechanism in hybrid hydrogels can be translated into a powerful functional capability for bioanalytical assays.