A bienzyme electrochemical probe for flow injection analysis of okadaic acid based on protein phosphatase-2A inhibition: an optimization study.
A bienzyme electrochemical probe for flow injection analysis of okadaic acid based on protein phosphatase-2A inhibition: an optimization study.
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DOI：
10.1016/j.ab.2008.10.027
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发表时间：
2009-02
影响因子：
2.9
通讯作者：
G. Volpe;E. Cotroneo;D. Moscone;L. Croci;L. Cozzi;G. Ciccaglioni;G. Palleschi
中科院分区：
文献类型：
--
作者：
G. Volpe;E. Cotroneo;D. Moscone;L. Croci;L. Cozzi;G. Ciccaglioni;G. Palleschi
已组装的双酶电化学探针，并用于监测抑制酶蛋白磷酸酶-2A（PP 2A）的冈田酸（OA），利用的生化途径，其中PP 2A参与的特定特征。该酶对糖原磷酸化酶a（PHOS a）具有显著活性，其进而催化糖原转化为葡萄糖-1-磷酸（G-1-P）。此外，OA及其衍生物强烈抑制PP 2A。由于这种性质的组合，PP 2A被用于开发涉及离线酶孵育的初步阶段的测定系统（OA/PP 2A、PP 2A/PHOS a、PHOS a/糖原+磷酸盐）。该离线步骤之后是H2 O2的电化学检测，H2 O2是两个连续酶促反应的最终产物：G-1-P与碱性磷酸酶（AP）产生葡萄糖，然后葡萄糖与葡萄糖氧化酶（GOD）产生过氧化氢。将这两种酶共固定在尼龙网膜上，该尼龙网膜置于插入流动注射分析（FIA）系统的H2 O2铂探针上。在研究的第一阶段，优化了所有分析参数。在随后的阶段中，评价OA对PP 2A酶的抑制。该系统的校准显示，OA检测的工作范围在30至250 pg ml-1之间。总分析时间为离线酶孵育的50分钟和生物传感器响应的4分钟之和。
A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen+phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml−1. The total analysis time is the sum of 50min for the off-line enzymatic incubations and 4min for the biosensor response.