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Hydrogels are polymer-based materials with a high water content. Due to their biocompatible and cell-friendly nature, they play a major role in a variety of biotechnological applications. For many of these applications, diffusibility is an essential property influencing the choice of material. We present an approach to estimate diffusion coefficients in hydrogels based on absorbance measurements of a UV area imaging system. A microfluidic chip with a y-junction was employed to generate a fluid-hydrogel interface and the diffusion of lysozyme from the fluid into the hydrogel phase was monitored. Employing automated image and data processing, analyte concentration profiles were generated from the absorbance measurements and fits with an analytical solution of Fick’s second law of diffusion were applied to estimate diffusion coefficients. As a case study, the diffusion of lysozyme in hydrogels made from different concentrations (0.5–1.5% (w/w)) of an unmodified and a low-melt agarose was investigated. The estimated diffusion coefficients for lysozyme were between 0.80 ± 0.04×10$^{-10}$ m$^{2}$ s$^{-1}$ for 1.5% (w/w) low-melt agarose and 1.14 ± 0.02×10$^{-10}$ m$^{2}$ s$^{-1}$ for 0.5% (w/w) unmodified agarose. The method proved sensitive enough to resolve significant differences between the diffusion coefficients in different concentrations and types of agarose. The microfluidic approach offers low consumption of analyte and hydrogel and requires only relatively simple instrumentation.

The immobilization of enzymes in biocatalytic flow reactors is a common strategy to increase enzyme reusability and improve biocatalytic performance. Extrusion-based 3D bioprinting has recently emerged as a versatile tool for the fabrication of perfusable hydrogel grids containing entrapped enzymes for the use in such reactors. This study demonstrates the suitability of water-in-oil high internal phase emulsions (HIPEs) as 3D-printable bioinks for the fabrication of composite materials with a porous polymeric scaffold (polyHIPE) filled with enzyme-laden hydrogel. The prepared HIPEs exhibited excellent printability and are shown to be suitable for the printing of complex three-dimensional structures without the need for sacrificial support material. An automated activity assay method for the systematic screening of different material compositions in small-scale batch experiments is presented. The monomer mass fraction in the aqueous phase and the thickness of printed objects were found to be the most important parameters determining the apparent activity of the immobilized enzyme. Mass transfer limitations and enzyme inactivation were identified as probable factors reducing the apparent activity. The presented HIPE-based bioinks enable the fabrication of flow-optimized and more efficient biocatalytic reactors while the automated activity assay method allows the rapid screening of materials to optimize the biocatalytic efficiency further without time-consuming flow-through experiments involving whole printed reactors.