The structure of N-linked glycans is dependent upon enzymes that add or trim saccharide monomers. A new technology is described that integrates enzymes into capillary electrophoresis analytical systems to evaluate enzyme function or to leverage enzymes for structural identification. These capillary separations utilize nanogels which have a thermally dependent viscosity. At temperatures below ~22 °C nanogels have a liquid-like viscosity. At higher temperatures nanogels have a gel-like viscosity. This property makes it easy to fill and pattern nanogels in narrow microfluidic channels or narrow-bore capillaries at low temperature. Once the nanogel is loaded in the capillary, the fluids are then locked in place by raising the temperature to gel the material. In addition to this thermally dependent viscosity, nanogels maintain enzyme function and are biocompatible. As a result, these matrices are modified to contain enzymes and then are introduced and patterned in a separation capillary. This enables the precise placement of 2-5 nanoliter enzyme reaction zones at the beginning of a capillary with a total liquid volume less than 1 microliter. Enzyme reactors of this low volume incorporate mixing and are operated with precise temperature control. Following enzymatic conversion, the reaction products are separated and detected. When the reaction is complete the capillary contents are expelled and refilled with a fresh enzyme zone. Moreover, the entire process of capillary patterning is automated. This approach reduces the time for enzymatic conversion to minutes. In addition to creating flexible and reprogrammable sample processing, nanogels are a tunable microscale bioseparation. The analyte resolution of biomolecules separated in nanogel yields separation efficiencies up to 2 million theoretical plates. A range of applications will be discussed to demonstrate this new approach to translate enzyme-based separations into a microscale format. This is <u>significant</u> to chemical separations because generic microscale channels generate a sophisticated multifunctional device that can be programmed, erased, and run repeatedly.

Capillary electrophoresis separations with integrated with enzymes: New tools to advance glycobiology

ACS Spring 2022

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