A novel method for quantifying enzyme immobilization in porous carriers using simple NMR relaxometry

Enzyme immobilization plays a crucial role in enhancing the stability and recyclability of enzymes for industrial applications. However, traditional methods for quantifying enzyme loading within porous carriers are limited by time-consuming workflows, cumulative errors, and the inability to probe enzymes adsorbed inside the pores. In this study, we introduce Time-Domain Nuclear Magnetic Resonance (TD-NMR) relaxometry as a novel, non-invasive technique for directly quantifying enzyme adsorption within porous carriers. Focusing on epoxy methyl acrylate carriers, commonly used in biocatalysis, we correlate changes in T 2 relaxation times with enzyme concentration, leading to the development of an NMR-based pore-filling ratio that quantifies enzyme loading. Validation experiments demonstrate that TD-NMR-derived adsorption curves align closely with traditional photometric measurements, offering a reliable and reproducible alternative for enzyme quantification. The accessibility of tabletop TD-NMR spectrometers makes this technique a practical and cost-effective tool for optimizing biocatalytic processes. Furthermore, the method holds promise for real-time monitoring of adsorption dynamics and could be adapted for a wider range of carrier materials and enzymes. • Time-Domain NMR relaxometry is a reliable method for detecting enzyme adsorption in porous carriers. • T2 relaxation times in porous carriers are directly correlated with enzyme concentration. • An NMR pore-filling model was developed to describe the relationship between T2 relaxation times and enzyme concentration. • The method was validated using epoxy methyl acrylate carriers, with results showing strong agreement with traditional photometric data across different pore sizes.