Cho Hyunju, Lee Chang-Seuk, Kim Tae Hyun
Department of ICT Environmental Health System, Graduate School, Soonchunhyang University, Asan 31538, Korea.
Department of Chemistry, Soonchunhyang University, Asan 31538, Korea.
Biomedicines. 2021 Apr 13;9(4):423. doi: 10.3390/biomedicines9040423.
We propose a simple label-free electrochemical biosensor for monitoring protein kinase activity and inhibition using a peptide-modified electrode. The biosensor employs cys-kemptide (CLRRASLG) as a substrate peptide which was immobilized on the surface of a gold electrode via the self-assembly of the thiol terminals in cysteine (C) residues. The interaction between protein kinase A (PKA) and adenosine 5'-triphosphate (ATP) on the cys-kemptide immobilized electrode can cause the transfer of ATP terminal phosphates to the peptide substrates at serine (S) residues, which alters the surface charge of the electrode, thus enabling monitoring of the PKA activity via measuring the interfacial electron transfer resistance with electrochemical impedance spectroscopy. The proposed sensor showed reliable, sensitive, and selective detection of PKA activity with a wide dynamic range of 0.1-100 U/mL and a detection limit of 56 mU/mL. The sensor also exhibited high selectivity, rendering it possible to screen PKA inhibitors. Moreover, the sensor can be employed to evaluate the activity and inhibition of PKA in real samples.
我们提出了一种简单的无标记电化学生物传感器，用于使用肽修饰电极监测蛋白激酶活性和抑制作用。该生物传感器采用半胱氨酸 - 肯普肽（CLRRASLG）作为底物肽，其通过半胱氨酸（C）残基中硫醇末端的自组装固定在金电极表面。蛋白激酶A（PKA）与固定在电极上的半胱氨酸 - 肯普肽上的腺苷5'-三磷酸（ATP）之间的相互作用可导致ATP末端磷酸基团转移至丝氨酸（S）残基处的肽底物上，这会改变电极的表面电荷，从而能够通过电化学阻抗谱测量界面电子转移电阻来监测PKA活性。所提出的传感器在0.1 - 100 U/mL的宽动态范围内对PKA活性进行了可靠、灵敏且选择性的检测，检测限为56 mU/mL。该传感器还表现出高选择性，使得筛选PKA抑制剂成为可能。此外，该传感器可用于评估实际样品中PKA的活性和抑制作用。
