Protein kinases play a pivotal role in cellular regulation and signal transduction, the detection of protein kinase activity and inhibition is therefore of great importance to clinical diagnosis and drug discovery. In this work, a novel electrochemical platform using the electrochemically mediated polymerization as an efficient and cost-effective signal amplification strategy is described for the highly sensitive detection of protein kinase activity. This platform involves 1) the phosphorylation of substrate peptide by protein kinase, 2) the attachment of alkyl halide to the phosphorylated sites via the carboxylate-Zr⁴⁺-phosphate chemistry, and 3) the in situ grafting of electroactive polymers from the phosphorylated sites through the electrochemically mediated atom transfer radical polymerization (eATRP) at a negative potential, in the presence of the surface-attached alkyl halide as the initiator and the electroactive tag-conjugated acrylate as the monomer, respectively. Due to the electrochemically mediated polymerization, a large number of electroactive tags can be linked to each phosphorylated site, thereby greatly improving the detection sensitivity. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase (PKA) with a detection limit down to 1.63 mU mL⁻¹. Results also demonstrate that it is highly selective and can be used for the screening of protein kinase inhibitors. The potential application of our platform for protein kinase activity detection in complex biological samples has been further verified using normal human serum and HepG2 cell lysate. Moreover, our platform is operationally simple, highly efficient and cost-effective, thus holding great potential in protein kinase detection and inhibitor screening.

蛋白激酶在细胞调节和信号转导中起着关键作用，因此蛋白激酶活性和抑制的检测对于临床诊断和药物发现非常重要。在这项工作中，描述了一种新的电化学平台，该平台使用电化学介导的聚合作为一种高效且具有成本效益的信号放大策略，可高度敏感地检测蛋白激酶活性。该平台涉及1）蛋白激酶将底物肽磷酸化，2）卤代烷通过羧酸盐-Zr ⁴⁺与磷酸化位点的连接-磷酸盐化学，以及3）在表面附着的烷基卤化物作为引发剂和电活性剂存在下，通过负离子电势通过电化学介导的原子转移自由基聚合（eATRP）从磷酸化位点原位接枝电活性聚合物标记共轭丙烯酸酯分别作为单体。由于电化学介导的聚合作用，大量电活性标签可以连接到每个磷酸化位点，从而大大提高了检测灵敏度。该平台已成功应用于检测cAMP依赖性蛋白激酶（PKA）的活性，检出限低至1.63 mU mL ^-1。结果还表明，它具有很高的选择性，可用于筛选蛋白激酶抑制剂。我们的平台在复杂生物样品中检测蛋白激酶活性的潜在应用已使用正常人血清和HepG2细胞裂解液进一步验证。此外，我们的平台操作简单，高效且具有成本效益，因此在蛋白激酶检测和抑制剂筛选方面具有巨大潜力。