MOE Key Laboratory of Laser Life Science, Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
Biosens Bioelectron. 2012 Jan 15;31(1):299-304. doi: 10.1016/j.bios.2011.10.035. Epub 2011 Oct 25.
Herein, we describe a novel electrochemiluminescence (ECL) biosensor for protein kinase activities and inhibition monitoring based on the magnetic beads (MB) technology and signal enhancement of gold nanoparticles (GNP). In this design, ECL nanoprobes were prepared by conjugating GNP with phosphorylated DNA capture probes and tris-(2,2'-bipyridyl) ruthenium (TBR)-cysteamine. Zirconium cations, a specific bridging agent, mediate the linkage between biotin modified phosphorylated peptides and ECL nanoprobes. The complexes were then captured and enriched on the electrode surface by streptavidin-coated MB for ECL reaction. To confirm the feasibility of this biosensor, we employed protein kinase A (PKA) as the model kinase to validate the assay and a satisfactory detection limit of 0.005 U/mL was achieved. The combination of ECL and GNP lays a solid foundation for highly sensitive assay, meanwhile, the coupling of MB surfaces used for separation and capture with unmodified ECL electrode detection results in a greatly simplified and reusable protocol. Thus, our biosensor offers great promise for a highly sensitive and simple assay for protein kinase activity. Furthermore, the inhibition of PKA activity was monitored on the basis of the ECL signals change in response to the concentration of PKA inhibitor.
在此，我们描述了一种基于磁珠（MB）技术和金纳米粒子（GNP）信号增强的新型用于蛋白质激酶活性和抑制监测的电化学发光（ECL）生物传感器。在该设计中，通过将 GNP 与磷酸化 DNA 捕获探针和三（2,2'-联吡啶）钌（TBR）-半胱氨酸缀合来制备 ECL 纳米探针。锆离子作为特定的桥联剂，介导生物素修饰的磷酸化肽与 ECL 纳米探针之间的连接。然后，通过链霉亲和素包被的 MB 将复合物捕获并富集在电极表面上，以进行 ECL 反应。为了证实该生物传感器的可行性，我们采用蛋白激酶 A（PKA）作为模型激酶来验证该测定法，实现了令人满意的检测限为 0.005 U/mL。ECL 和 GNP 的结合为高灵敏度测定奠定了坚实的基础，同时，用于分离和捕获的 MB 表面与未修饰的 ECL 电极检测结果的结合导致了大大简化和可重复使用的方案。因此，我们的生物传感器为蛋白质激酶活性的高灵敏度和简单测定提供了很大的前景。此外，根据 ECL 信号的变化监测 PKA 活性的抑制，以响应 PKA 抑制剂的浓度。
