Real‐Time NMR Monitoring of Spatially Segregated Enzymatic Reactions in Multilayered Hydrogel Assemblies**

Abstract

Compartmentalized chemical reactions at the microscale are important in biotechnology, yet monitoring the molecular content at these small scales is challenging. To address this challenge, we integrate a compact, reconfigurable reaction cell featuring electrochemical functionality with high‐resolution NMR spectroscopy. 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 monitoring reagent and product concentrations in real‐time. Simultaneous monitoring of an independent enzymatic process (Urs) together with a cooperative process (GOx + Cat) was achieved, with chemical conversion modulation of the GOx + Cat process demonstrated by varying the order in which the hydrogel was assembled.

Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility–Mass Spectrometry

“…High-throughput approaches have been developed to monitor biocatalytic transformations in real time; among them are implementations of benchtop NMR and/or IR/UV spectrometers coupled to flow reactors. − Yet, probing multistep reaction cascades often requires sophisticated analysis to resolve, assign, and quantify overlapping signals. ,, Moreover, the sensitivity of currently reported NMR approaches precludes the detection of low-concentration species, meaning that these approaches fall short of our requirements.…”