Label-Free Homogeneous Electrochemical Sensing Platform for Protein Kinase Assay Based on Carboxypeptidase Y-Assisted Peptide Cleavage and Vertically Ordered Mesoporous Silica Films
Label-Free Homogeneous Electrochemical Sensing Platform for Protein Kinase Assay Based on Carboxypeptidase Y-Assisted Peptide Cleavage and Vertically Ordered Mesoporous Silica Films
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DOI：
10.1021/acs.analchem.7b01739
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发表时间：
2017-09-05
影响因子：
7.4
通讯作者：
Yang, Xudong
中科院分区：
文献类型：
--
作者：
Liu, Jinquan;Cheng, Hong;Yang, Xudong
本文提出了一种简单、稳健且无标记的均相电化学传感平台，其通过整合羧肽酶Y（CPY）辅助的肽切割反应和垂直有序的介孔二氧化硅膜（MSFs）而构建用于检测蛋白激酶活性和抑制。在该传感平台中，巧妙地设计了由激酶特异性识别序列和多个带正电荷的精氨酸（R）残基组成的底物肽。在蛋白激酶的存在下，底物肽被磷酸化，然后立即抵抗CPY切割。磷酸化的多肽通过非共价静电吸附作用能有效地吸附在带负电的MSFs修饰的ITO电极表面。吸附的肽随后用作阻碍物以防止电活性探针（FcMeOH）通过垂直排列的纳米孔扩散到电极表面，导致电化学信号的可检测的降低。为验证该传感平台的可行性和通用性，选择蛋白激酶A（PKA）和酪蛋白激酶II（CK 2）作为模型，检测限分别为0.083和0.095 μ mL（-1）。该传感平台具有结构简单、操作方便、磷酸化和切割效率高的优点，其优点是无需复杂的修饰或固定化过程即可进行均相溶液反应。此外，由于抑制和蛋白激酶活性检测的细胞裂解物中的关键作用，该传感平台在激酶相关的生物分析和临床生物医学中显示出巨大的潜力。
Presented herein is a simple, robust, and label free homogeneous electrochemical sensing platform constructed for the detection of protein kinase activity and inhibition by integration of carboxypeptidase Y (CPY)-assisted peptide cleavage reaction and vertically ordered mesoporous silica films (MSFs). In this sensing platform, the substrate peptide composed of kinase-specific recognized sequence and multiple positively charged arginine (R) residues was ingeniously designed. In the presence of protein kinase, the substrate peptide was phosphorylated and then immediately resisted CPY cleavage. The phosphorylated peptide could be effectively adsorbed on the negatively charged surface of MSFs modified indium-tin oxide (ITO) electrode (MSFs/ITO) by noncovalent electrostatic attraction. The adsorbed peptide was subsequently used as a hamper to prevent the diffusion of electroactive probe (FcMeOH) to the electrode surface through the vertically aligned nanopores, resulting in a detectable reduction of electrochemical signal. As demonstrated for the feasibility and universality of the sensing platform, both protein kinase A (PKA) and casein kinase II (CK2) were selected as the models, and the detection limits were determined to be 0.083 and 0.095 UmL(-1), respectively. This sensing platform had the merits of simplicity, easy manipulation, and improved phosphorylation and cleavage efficiency, which benefited from homogeneous solution reactions without sophisticated modification or immobilization procedures. In addition, given the key role of inhibition and protein kinase activity detection in cell lysates, this proposed sensing platform showed great potential in kinase-related bioanalysis and clinical biomedicine.