Development of an ultra-sensitive ATP assay by an enzymatic ATP amplification reaction.
| Project/Area Number |
13650858
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
KURODA Akio Hiroshima University, Graduate school of advanced sciences of matter, Associate Professor, 大学院・先端物質科学研究科, 助教授 (50205241)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | polyphosphate / adenosine tri-phosphate / adenylate kinase / polyphosphate kinase / luciferase / detection of bacteria / ATP amplification / ルシフェリン |
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| Research Abstract |
ATP plays a central role in all aspects of metabolism and so any assays for ATP detection are of great importance in both pur and applied biochemistry. The bioluminescence assay of ATP using firefly luciferase is a well established technique. This assa is commercially used as a rapid hyginiene monitoring and recently as a technology against bioterrorism. However, this assay technique has a detection limit (for instance approximately 10^<th> colony-forming unit [CFU] of Escherichia coil per assay), which is not sensitive enough for some commercial applications. Here we designed an ultra-sensitive bioluminescence assay for ATP by employing adenylat kinase (ADK) as the first enzyme to convert AMP + ATP to two molecules of ADP and polyphosphate (polyp) kinase (PPK) as the second enzyme to convert ADP to ATP. In this reaction, excess AMP and polyp are used to drive ADK and PPX equilibrium toward ADP and AT formation, respectively. If one molecule of ATP present, the first ADK reaction starts and endogenous AMP is converted to ATP, producing a large bioluminescence signal compared to that obtained from the initial ATP present. This assay technique enabled us to detect bacterial contamination of one CFU per ml in a drinking water.
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Report
(3 results)
Research Products
(3 results)
