Dual-Channel Suspended Microfluidic Biosensor with In-Channel Alkyne–Azide Click Chemistry for Enzyme Immobilization

Dual-channel suspended microfluidic biosensor using in-channel alkyne–azide “click” chemistry for covalent enzyme immobilization inside sealed SU-8 microchannels. Two parallel channels with integrated metal electrodes act as sensing and reference paths, enabling differential thermal detection. A chitosan underlayer with tunable thickness$(\sim 2-10 \mu ~\mathrm{m})$is electrodeposited to maximize enzyme loading without clogging, then functionalized via$\text{Cu}(\mathrm{I})$-catalyzed click chemistry to form a uniform enzyme coating. A brief thermal anneals yields$\sim 20 \%$higher conductance and a more symmetric I-V response, enhancing sensor stability. In catalase-$\mathrm{H}_{2} \mathrm{O}_{2}$assays, the dual-channel design produces a clear thermal signal with minimal baseline drift. The immobilized enzyme layer retains over 90 % of its activity after repeated cycles, and the same click-chemistry process scales seamlessly to a five-channel array for multiplexed sensing.'”