School of Environmental and Biological Engineering , Nanjing University of Science and Technology , Nanjing 210094 , P. R. China.
Center for Advanced Analytical Science, School of Chemistry and Chemical Engineering, MOE Key Laboratory for Water Quality and Conservation of the Pearl River Delta , Guangzhou University , Guangzhou 510006 , P. R. China.
Anal Chem. 2019 Feb 5;91(3):1936-1943. doi: 10.1021/acs.analchem.8b04221. Epub 2019 Jan 11.
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, 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.
蛋白质激酶（PKs）催化的蛋白质磷酸化对于许多生物过程至关重要；因此，PK 活性的灵敏检测和 PK 抑制剂的筛选是疾病诊断和药物发现的重要组成部分。本文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，构建了一种用于 PK 活性电化学检测的高灵敏生物传感器。eRAFT 聚合电化学生物传感器的制备涉及（1）通过金-硫自组装将底物肽固定在金电极上，（2）PK 对底物肽的特异性磷酸化，（3）将含羧基的链转移剂（CTA）锚定在磷酸化位点，以及（4）在恒电位条件下用二茂铁基甲基丙烯酸甲酯（FcMMA）作为单体进行 eRAFT 聚合。通过 eRAFT 聚合，每个磷酸化位点可以从头接枝含有大量电活性 Fc 标记的长聚合物链，从而显著放大电化学检测信号。所制备的生物传感器具有高度选择性，检测限低至 1.02 mU mL，在以环腺苷酸（cAMP）依赖性蛋白激酶（PKA）为模型靶标的情况下。结果还表明，它可用于 PK 抑制剂的筛选以及复杂血清样本和细胞裂解物中 PK 活性的检测。此外，它具有易于制造、效率高和成本低的优点，使其成为检测 PK 活性和筛选潜在 PK 抑制剂的有前途的工具。
