Electrochemical Activity Assay for Protease Analysis Using Carbon Nanofiber Nanoelectrode Arrays
Electrochemical Activity Assay for Protease Analysis Using Carbon Nanofiber Nanoelectrode Arrays
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DOI：
10.1021/acs.analchem.8b05189
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发表时间：
2019-03-19
影响因子：
7.4
通讯作者：
Li, Jun
中科院分区：
文献类型：
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作者：
Song, Yang;Fan, Huafang;Li, Jun
人们强烈需要生物分析技术来快速检测具有高灵敏度和高特异性的蛋白酶活性。本研究报告了一种基于活性的电化学方法实现这一目标。纳米电极阵列（NEAs）与嵌入垂直排列的碳纳米纤维（VACNF）制造的功能与特定的肽底物含有二茂铁（Fc）标签。的动力学蛋白质水解曲线测量与连续重复交流伏安法，从其中的催化活性是作为指数衰减时间常数的倒数的基础上的非均相Michaelis-Menten模型。比较三种不同长度的肽底物，发现六肽H2 N-（CH 2）（4）CO-Pro-Leu-Arg-Phe-Gly-Ala-NH-CH 2-Fc是组织蛋白酶B的最佳探针。该活动强烈依赖于温度，并且在体温附近最高。在优化的肽底物和测定条件下，组织蛋白酶B活性和浓度的检测限分别达到2.49 × 10（-4）s（-1）和0.32 nM。肽底物对同源蛋白酶显示出高特异性，三种癌症相关蛋白酶组织蛋白酶B、ADAM 10和ADAM 17之间的交叉反应可忽略不计。这种电化学方法可以开发成多路芯片，用于癌症诊断和治疗监测中的蛋白酶活性的快速分析。
There is a strong demand for bioanalytical techniques to rapidly detect protease activities with high sensitivity and high specificity. This study reports an activity based electrochemical method toward this goal. Nano electrode arrays (NEAs) fabricated with embedded vertically aligned carbon nanofibers (VACNFs) are functionalized with specific peptide substrates containing a ferrocene (Fc) tag. The kinetic proteolysis curves are measured with continuously repeated ac voltammetry, from which the catalytic activity is derived as the inverse of the exponential decay time constant based on a heterogeneous Michaelis-Menten model. Comparison of three peptide substrates with different lengths reveals that the hexapeptide H2N-(CH2)(4)CO-Pro-Leu-Arg-Phe-Gly-Ala-NH-CH2-Fc is the optimal probe for cathepsin B. The activity strongly depends on temperature and is the highest around the body temperature. With the optimized peptide substrate and measuring conditions, the limit of detection of cathepsin B activity and concentration can reach 2.49 x 10(-4) s(-1) and 0.32 nM, respectively. The peptide substrates show high specificity to the cognate proteases, with negligible cross-reactions among three cancer-related proteases cathepsin B, ADAM10, and ADAM17. This electrochemical method can be developed into multiplex chips for rapid profiling of protease activities in cancer diagnosis and treatment monitoring.