Label-free electrochemical measurement of protein tyrosine kinase activity and inhibition based on electro-catalyzed tyrosine signaling.
Label-free electrochemical measurement of protein tyrosine kinase activity and inhibition based on electro-catalyzed tyrosine signaling.
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基于电催化酪氨酸信号传导的蛋白酪氨酸激酶活性和抑制的无标记电化学测量。
DOI：
10.1016/j.bios.2011.07.033
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发表时间：
2011-10
影响因子：
12.6
通讯作者：
中科院分区：
文献类型：
--
作者：
建立了一种新的无标记电化学法测定蛋白酪氨酸激酶(PTK)活性。表皮生长因子受体(EGFR)是一种典型的PTK，在所有实体瘤中占很大比例，它被用作模型激酶。以聚(谷氨酸，酪氨酸)(4：1)多肽为底物，采用硅烷化学法将其固定在氧化铟锡(ITO)电极表面。多肽中的酪氨酸(Tyr)残基作为电化学信号报告。其伏安电流由溶解电子介体Os(Bpy)32+(bpy=2，2‘-联吡啶)催化，提高了灵敏度。Tyr的磷酸化导致其电化学电流的损失，从而为PTK活性提供了一种传感机制。详细研究了ITO表面硅烷化和多肽固定化的实验条件，以利于Tyr电化学信号的产生。该生物传感器具有较高的灵敏度和良好的稳定性。该传感器的检测下限为1um L−-1，并可用于对酶抑制的定量分析。根据抑制剂浓度依赖的电化学信号，估算了PD-153035、OSI-774和ZD-1839三种EGFR抑制剂的半最大抑制值IC50及其相应的抑制常数Ki值，与传统的激酶法测定结果一致。这种电化学生物传感器可以以阵列的形式实现，用于体外PTK活性的高通量分析和PTK抑制剂的筛选，以用于实际诊断应用和药物开发。
A novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, as a substrate of EGFR, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)32+(bpy=2,2′-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 1UmL−1. Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC50of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants Kiwere estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.
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DOI：
10.1016/s0021-9673(99)00484-7
发表时间：
1999-07
期刊：
Journal of chromatography. A
影响因子：
--
作者：
Jeffrey B. Wheatley;Donald E. Schmidt
通讯作者：
Jeffrey B. Wheatley;Donald E. Schmidt
影响因子：
7.4
作者：
Guo, Liang-Hong;Qu, Na
通讯作者：
Qu, Na
DOI：
10.1007/978-1-60761-670-2_8
发表时间：
2010-01-01
期刊：
SURFACE PLASMON RESONANCE: METHODS AND PROTOCOLS
影响因子：
--
作者：
Takeda, Hiroyuki;Goshima, Naoki;Nomura, Nobuo
通讯作者：
Nomura, Nobuo
影响因子：
7.3
作者：
Pawar, Vijaykumar G.;Sos, Martin L.;Rauh, Daniel
通讯作者：
Rauh, Daniel
DOI：
10.1002/chin.200805268
发表时间：
2008-01
期刊：
ChemInform
影响因子：
--
作者：
E. Goldsmith;Radha Akella;Xiaoshan Min;Tianjun Zhou;J. Humphreys
通讯作者：
E. Goldsmith;Radha Akella;Xiaoshan Min;Tianjun Zhou;J. Humphreys