采用高通量、均相、基于荧光强度的方法，以纳摩尔灵敏度检测腺苷二磷酸和其他核糖核苷二磷酸。

High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other ribonucleoside diphosphates with nanomolar sensitivity.

机构信息

Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451, USA.

出版信息

Anal Biochem. 2011 Aug 15;415(2):190-6. doi: 10.1016/j.ab.2011.04.027. Epub 2011 Apr 24.

DOI:10.1016/j.ab.2011.04.027

Abstract

A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.

摘要

一种新的、均相、高通量兼容的荧光分析方法，用于定量检测纳摩尔浓度的核糖核苷二磷酸（rNDPs）。该方法的原理是通过多核苷酸磷酸化酶（EC 2.7.7.8）将 rNDPs 转化为 RNA，然后通过商业核酸检测染料的荧光增强来检测 RNA。包含与 RNA 产物互补的商业 RNA 均聚物以提高对 ADP 和 UDP 的灵敏度。显示了纳摩尔浓度的 ADP、UDP、GDP 和 CDP 的标准曲线。该测定法检测到由丙酮酸激酶催化丙酮酸磷酸化生成的 75 nM ADP，其信号与基线的比值为 2.8。该测定法可用于连续或不连续模式。