Development of an ultra-sensitive ATP assay by an enzymatic ATP amplification reaction.
通过酶促 ATP 扩增反应开发超灵敏 ATP 测定。
基本信息
- 批准号:13650858
- 负责人:
- 金额:$ 2.18万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (C)
- 财政年份:2001
- 资助国家:日本
- 起止时间:2001 至 2002
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
ATP在代谢的各个方面都起着核心作用,因此ATP检测的任何分析在纯生物化学和应用生物化学中都非常重要。使用萤火虫荧光素酶的ATP的生物发光测定是一种成熟的技术。这种assa在商业上被用作快速卫生监测,最近被用作对抗生物恐怖主义的技术。然而,该测定技术具有检测极限(例如每次测定约10 μ<th>菌落形成单位[CFU]的大肠杆菌),这对于某些商业应用不够灵敏。我们设计了一种超灵敏的ATP生物发光检测方法,采用腺苷酸激酶(ADK)作为第一个将AMP + ATP转化为两分子ADP的酶,多磷酸(聚)激酶(PPK)作为第二个将ADP转化为ATP的酶。在该反应中,过量的AMP和息肉分别用于驱动ADK和PPX平衡朝向ADP和AT形成。如果存在一个ATP分子,则第一个ADK反应开始,内源性AMP转化为ATP,产生与从存在的初始ATP获得的生物发光信号相比大的生物发光信号。这种测定技术使我们能够检测到饮用水中每毫升一个CFU的细菌污染。
项目成果
期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
S.Tanaka et al.: "A sensitive method for detecting AMP by utilizing polyphosphate-dependent ATP regeneration and bioluminescence reactions"Biochemical Engineering Journal. 9. 193-197 (2001)
S.Tanaka 等人:“利用多磷酸盐依赖性 ATP 再生和生物发光反应检测 AMP 的灵敏方法”《生物化学工程杂志》。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
S. Tanaka et al.: "A sensitive method for detecting AMP by utilizing polyphosphate-dependent ATP regeneration and bioluminescence reactions"Biochemical Engineering Journal. 9. 193-197 (2001)
S. Tanaka 等人:“利用多磷酸盐依赖性 ATP 再生和生物发光反应检测 AMP 的灵敏方法”《生物化学工程杂志》。
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- 影响因子:0
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KURODA Akio其他文献
KURODA Akio的其他文献
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{{ truncateString('KURODA Akio', 18)}}的其他基金
Novel method utilizing bisulfite conversion with dual amplification-refractory mutation system polymerase chain reaction to detect circulating pancreatic beta cell cfDNA.
利用亚硫酸氢盐转化和双重扩增-难治性突变系统聚合酶链反应检测循环胰腺 β 细胞 cfDNA 的新方法。
- 批准号:
18K07448 - 财政年份:2018
- 资助金额:
$ 2.18万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Detection of pancreatic beta cell DNA in the circulation utilizing epigenetic modification
利用表观遗传修饰检测循环中的胰腺 β 细胞 DNA
- 批准号:
15K06910 - 财政年份:2015
- 资助金额:
$ 2.18万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Application of a transgenic plant conferred with phosphite-oxidizing ability to efficient utilization of phosphite waste
具有亚磷酸盐氧化能力的转基因植物在亚磷酸盐废物高效利用中的应用
- 批准号:
25630382 - 财政年份:2013
- 资助金额:
$ 2.18万 - 项目类别:
Grant-in-Aid for Challenging Exploratory Research
ATP amplification reaction in pico-liter chamber and its application to bacteria detection.
皮升室中ATP扩增反应及其在细菌检测中的应用
- 批准号:
23656529 - 财政年份:2011
- 资助金额:
$ 2.18万 - 项目类别:
Grant-in-Aid for Challenging Exploratory Research
Development of NADH regeneration system by thermostable phosphite dehydrogenase
耐热亚磷酸脱氢酶NADH再生系统的开发
- 批准号:
22360346 - 财政年份:2010
- 资助金额:
$ 2.18万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Analysis of an enzymatic domain that is involved in specificity of phosphoryl donors, polyphosphate and ATP
参与磷酰基供体、多磷酸盐和 ATP 特异性的酶结构域分析
- 批准号:
15560676 - 财政年份:2003
- 资助金额:
$ 2.18万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Study on microbial proteiolipid involved in haydroxyapatite formation.
微生物蛋白脂参与羟基磷灰石形成的研究。
- 批准号:
11650819 - 财政年份:1999
- 资助金额:
$ 2.18万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
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