Beijing National Laboratory for Molecular Sciences, Organic Solids Laboratory, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
ACS Appl Mater Interfaces. 2011 Apr;3(4):1175-9. doi: 10.1021/am2000104. Epub 2011 Mar 21.
In this paper, we describe a new continuous fluorescence turn-on method for trypsin assay and inhibitor screening in situ. This assay is designed based on the following assumptions: (1) It is expected that the fluorescein-labeled peptide composed of six arginine residues (Arg(6)-FAM) with positive charges will interact with the negatively charged edge of water-soluble graphene oxide (GO) because of electrostatic interactions to form a GO/Arg(6)-FAM complex. As a result, the fluorescence of fluorescein will be quenched because of the energy transfer from fluorescein to GO. (2) Arg(6)-FAM can be hydrolyzed into small fragments in the presence of trypsin, and accordingly, the GO/Arg(6)-FAM complex will be dissociated, gradually leading to fluorescence recovery for the solution. In this way, the trypsin activity can be easily assayed with the ensemble of Arg(6)-FAM and GO. Additionally, the ensemble can be employed for screening of the inhibitors of trypsin.
在本文中，我们描述了一种新的用于原位胰蛋白酶检测和抑制剂筛选的连续荧光开启方法。该检测方法基于以下假设：（1）预计带正电荷的由六个精氨酸残基组成的荧光素标记肽（Arg(6)-FAM）将与带负电荷的水溶性氧化石墨烯（GO）边缘因静电相互作用而发生相互作用，从而形成 GO/Arg(6)-FAM 复合物。结果，由于荧光素向 GO 的能量转移，荧光素的荧光将被猝灭。（2）Arg(6)-FAM 在存在胰蛋白酶的情况下可以被水解成小片段，因此，GO/Arg(6)-FAM 复合物将被解离，导致溶液的荧光逐渐恢复。通过这种方式，可以用 Arg(6)-FAM 和 GO 的混合物轻松测定胰蛋白酶的活性。此外，该混合物可用于筛选胰蛋白酶的抑制剂。
