Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays.
Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays.
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DOI：
10.1016/j.bios.2014.01.002
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发表时间：
2014-06-15
影响因子：
12.6
通讯作者：
Li, Jun
中科院分区：
文献类型：
--
作者：
Swisher, Luxi Z.;Prior, Allan M.;Shishido, Stephanie;Nguyen, Thu A.;Hua, Duy H.;Li, Jun
用交流伏安法（ACV）测量癌症相关酶组织蛋白酶B的蛋白水解活性，所述交流伏安法（ACV）使用附着到用垂直排列的碳纳米纤维（VACNF）制造的纳米电极阵列（NEA）的二茂铁（Fc）标记的四肽。这种组合使得能够使用具有增强的电化学信号的高AC频率（~1 kHz）。组织蛋白酶B对Fc-肽的特异性蛋白水解产生ACV峰电流相对于反应时间的衰减。可以提取原始数据的指数分量并定义为“提取的蛋白水解信号”，其允许使用异质Michaelis-Menten模型进行一致的定量分析。对纯化的组织蛋白酶B的“特异性常数”kcat/KM =（3.68 ± 0.50）× 104 M− 1 s −1。对不同浓度的人乳腺组织全组织裂解液、加标不同浓度组织蛋白酶B的组织裂解液以及免疫沉淀后的组织裂解液中组织蛋白酶B活性的检测显示，29.1 μg·mL-1全组织裂解液中组织蛋白酶B浓度比免疫沉淀样品高约13.4 nM。通过电子束光刻得到的定义明确的规则VACNF NEA显示了组织蛋白酶B蛋白水解的更快动力学，kcat/KM = 9.2 × 104 M− 1 s −1。这些结果说明了这种技术作为一种便携式多路电子系统的潜力，用于癌症诊断的血清或血液样品的快速蛋白酶谱。
The proteolytic activity of a cancer-related enzyme cathepsin B is measured with alternating current voltammetry (ACV) using ferrocene (Fc) labeled tetrapeptides attached to nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). This combination enables the use of high AC frequencies (~1 kHz) with enhanced electrochemical signals. The specific proteolysis of the Fc-peptide by cathepsin B produces decay in the ACV peak current versus the reaction time. The exponential component of the raw data can be extracted and defined as the “extracted proteolytic signal” which allows consistent quantitative analyses using a heterogeneous Michaelis-Menten model. A “specificity constant” kcat/KM = (3.68 ± 0.50) × 104 M−1s−1 for purified cathepsin B was obtained. The detections of cathepsin B activity in different concentrations of whole lysate of human breast tissue, tissue lysate spiked with varied concentrations of cathepsin B, and the tissue lysate after immunoprecipitation showed that there are ~13.4 nM higher cathepsin B concentration in 29.1 μg·mL−1 of whole tissue lysate than the immunoprecipitated sample. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis with kcat/KM = 9.2 × 104 M−1s−1. These results illustrate the potential of this technique as a portable multiplex electronic system for cancer diagnosis by rapid protease profiling of serum or blood samples.
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影响因子：
2.9
作者：
Nagler, DK;Storer, AC;Menard, R
通讯作者：
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影响因子：
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DOI：
10.1067/moe.2002.122834
发表时间：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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