Stereolithographic Immobilized Enzyme Reactors

Enzymes are naturally occurring catalysts that are used for a variety of laboratory applications, including many applications in analytical chemistry. Whereas some assays require the direct quantitation of enzymes in a particular matrix, most applications only require enzymes to catalyze one step in the analytical procedure. Examples of such applications include the conversion of proteins to peptide fragments for mass spectrometric analysis, or the enzymatic conversion of polysaccharides such as starch to smaller oligo-, di-, and monosaccharides in the analysis of food carbohydrate contents. The enzymes that perform this conversion step may interfere with the final assay. They are therefore removed from the samples by some form of sample preparation, which requires additional laboratory steps and more chemicals. 

We have devised an immobilized enzyme reactor for the conversion of starch to smaller oligosaccharides. This modular system comprised multiple components produced by stereolithography, a 3D-printing technique that creates sub-millimeter channels in the printed material. The printed material (FormLabs printer and proprietary resins) chemically resembles poly(methyl methacrylate) (PMMA), an optically transparent plastic that allows for visual inspection and is resistant to both aqueous buffers over a wide pH range and organic solvents like hexane. All printed fluidic components were designed to be part of a modular fluidic ‘toolbox’ that can be used in different configurations for a variety of applications. These components include a static mixer, sample extractors, enzyme cartridges, and easy-to-use fluidic connectors (Grajewski et al., SLAS Europe, 2019, Barcelona).

An immobilization procedure was developed to covalently attach the enzyme, α-amylase, onto the channel walls. Starch was dissolved in aqueous buffers and flowed through these channels with immobilized enzymes to catalyze conversion of starch to smaller oligosaccharides. The enzymes remained in the column during the catalysis, which eliminates the need for further sample purification before analysis. The activity of the enzymes was monitored by measuring enzyme kinetic parameters with fluorescence read-out. The immobilized enzymes were still highly active, with their activity depending on starch concentration. Negative control experiments employing immobilized, catalytically inactive proteins (in this case, bovine serum albumin), confirmed that starch conversion was catalyzed by the immobilized enzyme.

This conversion of starch to smaller oligosaccharides is an important first step for a future miniaturized total analysis system for starch and other polysaccharides in food. Further development of the microsystem may include the quantification of smaller oligosaccharides by subsequent enzymatic conversion to glucose, which can be detected either colorimetrically or electrochemically by small, portable, analytical systems. These systems may find application in the food industry, or, when equipped with different enzymes, the diagnostic and pharmaceutical industries.