Electrochemically Controlled RAFT Polymerization for Highly Sensitive Electrochemical Biosensing of Protein Kinase Activity
Electrochemically Controlled RAFT Polymerization for Highly Sensitive Electrochemical Biosensing of Protein Kinase Activity
复制标题
电化学控制的 RAFT 聚合用于蛋白激酶活性的高灵敏电化学生物传感
DOI：
10.1021/acs.analchem.8b04221
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发表时间：
2019-02-05
影响因子：
7.4
通讯作者：
Niu, Li
中科院分区：
文献类型：
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作者：
Hu, Qiong;Kong, Jinming;Niu, Li
由蛋白激酶（PKs）催化的蛋白质磷酸化在许多生物过程中是必不可少的；因此，对PK活性的敏感检测和PK抑制剂的筛选对于疾病诊断和药物发现是不可或缺的。本研究利用电化学控制的可逆加成断裂链转移（eRAFT）聚合作为一种新的扩增策略，制备了一种高灵敏度的生物传感器，用于电化学检测PK活性。基于eRAFT聚合的电化学生物传感器的制造包括(1)通过金硫自组装将底物肽固定在金电极上，(2)PKs对底物肽的位点特异性磷酸化，(3)将含羧基链转移剂（cta）锚定在磷酸化位点上，以及(4)以二茂铁甲基丙烯酸甲酯（FcMMA）为单体，在恒电位条件下进行eRAFT聚合。通过eRAFT聚合，含有大量电活性Fc标签的长聚合物链可以从每个磷酸化位点重新接枝，从而显著放大电化学检测信号。在腺苷3′，5′-环单磷酸腺苷（cAMP）依赖性PK （PKA）存在的情况下，该生物传感器具有很高的选择性，检测限为1.02 μ mL（-1）。结果还表明，该方法可用于复杂血清样品和细胞裂解液中PK抑制剂的筛选和PK活性的检测。此外，它还具有制备简单、效率高、成本低等优点，是一种很有前途的PK活性检测和潜在PK抑制剂筛选工具。
Phosphorylation of proteins catalyzed by protein kinases (PKs) is essential to many biological processes; the sensitive detection of PK activity and the screening of PK inhibitors are thus integral to disease diagnosis and drug discovery. Herein, a highly sensitive biosensor has been fabricated for the electrochemical detection of PK activity by exploiting the electrochemically controlled reversible addition fragmentation chain transfer (eRAFT) polymerization as a novel amplification strategy. The fabrication of the eRAFT-polymerization-based electrochemical biosensor involves (1) the immobilization of substrate peptides onto a gold electrode by way of gold-sulfur self-assembly, (2) the site-specific phosphorylation of substrate peptides by PKs, (3) the anchoring of carboxyl-group-containing chain transfer agents (CTAs) to the phosphorylated sites, and (4) the eRAFT polymerization under a potentiostatic condition, using ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through the eRAFT polymerization, long polymer chains containing numerous electroactive Fc tags can be de novo grafted from each phosphorylated site, resulting in significant amplification of the electrochemical detection signal. The as fabricated biosensor is highly selective and features a very low detection limit of 1.02 mU mL(-1), in the presence of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent PK (PKA) as the model target. Results also demonstrate that it can be applied to the screening of PK inhibitors and the detection of PK activity in complex serum samples and cell lysates. Moreover, it holds the merits of easy fabrication, high efficiency, and low cost, which make it a promising tool for the detection of PK activity and the screening of potential PK inhibitors.