Microfluidics Featuring Multilayered Hydrogel Assemblies Enable Real-Time NMR-Monitoring of Enzyme Cascade Reactions

Compartmentalized chemical reactions at the microscale are interesting from many perspectives including (multi)functional surfaces and biotechnology. Monitoring the molecular content as a measure of functional performance at these small scales is challenging. As a means to address this challenge, we leverage microtechnology and biocompatible materials to integrate a compact, reconfigurable reaction cell featuring electrochemical functionality with high-resolution nuclear magnetic resonance spectroscopy (NMR). We demonstrate the operation of this system by monitoring the activity of enzymes immobilized in chemically distinct layers within a multi-layered chitosan hydrogel assembly. As a benchmark, we observed the parallel activities of urease (Urs), catalase (Cat), and glucose oxidase (GOx) by recording NMR spectra to extract reagent and product concentrations in real-time. As a result, simultaneous monitoring of a cooperative enzymatic process (GOx + Cat) together with an independent process (Urs) is achieved. Using Michaelis-Menten progress curve analysis of the NMR data, kinetic data is extracted: in the case of GOx, the Michaelis constants (K_M) are consistent with previous reports, while for Urs, deviations are observed, attributed to an inhibitory effect under our reaction conditions. The system therefore enables the construction of complex reaction cascades with spatial control, as would be interesting in, for example, metabolic engineering and multiplexed sensing applications.