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Title: High-Throughput Analysis of Enzyme Activities

Abstract

High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surfacemore » of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different detection modes. The detection limits are 22, 15, and 3.6 μM ADP for using fluorometer, ICCD and PMT respectively. The Michealis constant K m(ATP) for protein kinases ranges from 5 to 100 μM. For inhibitor screening, in order to get the most accurate result, ATP concentration should be closed to K m. In this case, further lower the detection limit of ADP is needed before the direct detection of ADP can be actually used in kinase inhibitor screening.« less

Authors:
 [1]
  1. Iowa State Univ., Ames, IA (United States)
Publication Date:
Research Org.:
Ames Lab., Ames, IA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
933034
Report Number(s):
IS-T 2294
TRN: US200814%%285
DOE Contract Number:
AC02-07CH11358
Resource Type:
Thesis/Dissertation
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; AUTOMATION; CAPILLARIES; CATALYSTS; CHARGE-COUPLED DEVICES; DETECTION; ELECTROPHORESIS; EMISSION SPECTRA; ENZYME ACTIVITY; ENZYMES; FLUORESCENCE; IMMOBILIZED ENZYMES; KINETICS; OPTICAL PROPERTIES; PHOSPHOTRANSFERASES; PROTEINS; REACTION KINETICS; ROBOTS; SENSITIVITY; SUBSTRATES

Citation Formats

Lu, Guoxin. High-Throughput Analysis of Enzyme Activities. United States: N. p., 2007. Web. doi:10.2172/933034.
Lu, Guoxin. High-Throughput Analysis of Enzyme Activities. United States. doi:10.2172/933034.
Lu, Guoxin. Mon . "High-Throughput Analysis of Enzyme Activities". United States. doi:10.2172/933034. https://www.osti.gov/servlets/purl/933034.
@article{osti_933034,
title = {High-Throughput Analysis of Enzyme Activities},
author = {Lu, Guoxin},
abstractNote = {High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different detection modes. The detection limits are 22, 15, and 3.6 μM ADP for using fluorometer, ICCD and PMT respectively. The Michealis constant K m(ATP) for protein kinases ranges from 5 to 100 μM. For inhibitor screening, in order to get the most accurate result, ATP concentration should be closed to Km. In this case, further lower the detection limit of ADP is needed before the direct detection of ADP can be actually used in kinase inhibitor screening.},
doi = {10.2172/933034},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}

Thesis/Dissertation:
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