Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China.
Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China; Shanghai Key Laboratory of Bio-Energy Crops, Shanghai University, Shanghai 200444, PR China.
Biosens Bioelectron. 2015 Dec 15;74:666-72. doi: 10.1016/j.bios.2015.07.023. Epub 2015 Jul 11.
An electrochemical method is established in this work for the assay of α-glucosidase activity and the inhibitor screening through one-step displacement reaction, which can be directly used in cell medium. The displacement reaction can be achieved via strong binding of 4-aminophenyl-α-D-glucopyranoside (pAPG)/magnetic nanoparticles (MNPs) to pyrene boric acid (PBA) immobilized on the surface of graphite electrode (GE), compared to that of dopamine (DA)/sliver nanoparticles (AgNPs). Since α-glucosidase can specifically catalyze MNPs/pAPG into MNPs/pAP which has no binding capacity with PBA, the activity of both isolated and membrane bound enzyme can be well evaluated by using this proposed method. Meanwhile, signal amplification can be accomplished via the immobilization of DA at the outer layer of AgNPs, and the accuracy can be strengthened through magnetic separation. Moreover, this method can also be utilized for inhibitor screening not only in the medium containing the enzyme but also in cell medium. With good precision and accuracy, it may be extended to other proteases and their inhibitors as well.
本工作建立了一种电化学方法，通过一步置换反应测定α-葡萄糖苷酶活性和抑制剂筛选，可直接用于细胞培养基。与多巴胺（DA）/银纳米颗粒（AgNPs）相比，4-氨基苯-α-D-吡喃葡萄糖苷（pAPG）/磁性纳米颗粒（MNPs）可以与固定在石墨电极（GE）表面的芘硼酸（PBA）发生强烈结合，从而实现置换反应。由于α-葡萄糖苷酶可以特异性地将 MNPs/pAPG 催化为与 PBA 没有结合能力的 MNPs/pAP，因此可以使用这种方法很好地评估游离酶和膜结合酶的活性。同时，可以通过在 AgNPs 的外层固定 DA 来实现信号放大，并且通过磁分离可以增强准确性。此外，该方法不仅可以用于含酶的培养基中，还可以用于细胞培养基中的抑制剂筛选。该方法具有良好的精密度和准确性，可能会扩展到其他蛋白酶及其抑制剂。
