Enzyme-based Capillary Separations with Biocompatible Gels

Biocompatible gels advance microscale separations by enabling new separation modalities critical to evaluating the activity of biomolecules. Phospholipid nanogels are thermally reversible media that maintain the biological function of complex biomolecules and provide a means to create and embed multifunctional assays in capillary separations. In this presentation nanogel technologies are described to create nanoliter reactions zones to interrogate biomolecules in seconds. To date, as many as 7 sequential processing steps have been performed in series in microscale channels. This is possible because the viscosity of nanogel is thermally dependent and thermally reversible. At temperatures below ~22°C nanogels have liquid-like viscosity. At higher temperatures nanogels have a gel-like viscosity. This property makes it easy to fill and pattern nanogels in narrow-bore capillaries at low temperatures using an automated capillary electrophoresis instrument. Once the nanogel is loaded into the capillary, the fluids are then locked in place by raising the temperature to gel the material. 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. In this way, a series of discreate reaction zones is created and integrated with a separation step. Enzyme reactors of this low volume are mixed electrophoretically and then the substrate and products, or products, are separated, detected, and quantified. This approach is automated and reduces the time for enzymatic conversion from hours to minutes. The analyte resolution of biomolecules separated in nanogel yields efficient separation. Applications with hydrolase and transferase enzymes are demonstrated. This work is significant to separations because it transforms standard electrophoresis methods into sophisticated multifunctional separations that are programmed, erased, and repeatedly run.