Enzyme reactions in nanoporous, picoliter volume containers

Document Type

Article

Publication Date

1-17-2012

Department

Department of Mechanical Engineering-Engineering Mechanics

Abstract

Advancements in nanoscale fabrication allow creation of small-volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ∼19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Assessment of small-molecule and green fluorescent protein diffusion from the vessels indicates that pore sizes on the order of 10 nm can be obtained, allowing capture of proteins and diffusive exchange of small molecules. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate amplex red through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme (K m and V max) were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics, and high-throughput screening.

Publisher's Statement

© 2011 American Chemical Society. Publisher’s version of record: https://doi.org/10.1021/ac202726n

Publication Title

Analytical Chemistry

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