title: Enzyme reactions in nanoporous, picoliter volume containers.
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.
authors.affiliation: Graduate School of Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory, Knoxville, Tennessee 37996, United States.
authors.forename: Piro
authors.initials: P
authors.lastname: Siuti
authors.forename: Scott T
authors.initials: ST
authors.lastname: Retterer
authors.forename: Chang-Kyoung
authors.initials: CK
authors.lastname: Choi
authors.forename: Mitchel J
authors.initials: MJ
authors.lastname: Doktycz
mesh_terms: D002384:Catalysis; D002468:Cell Physiological Phenomena; D004800:Enzymes, Immobilized; D005947:Glucose / Q000378:metabolism*; D005949:Glucose Oxidase / Q000737:chemistry / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008856:Microscopy, Fluorescence; D049329:Nanostructures; D036103:Nanotechnology; D016062:Porosity
chemical_list: D004800:Enzymes, Immobilized; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D005947:Glucose