Faculty of Chemical Engineering, Industrial University of Ho Chi Minh City, 12 Nguyen Van Bao St. Go Vap, Ho Chi Minh City, 70000, Vietnam.
Protein J. 2021 Oct;40(5):682-688. doi: 10.1007/s10930-021-09985-6. Epub 2021 Apr 15.
In this study, a fluorescence detection strategy is reported for the peptidase activity assay, which is based on fluorescence resonance energy transfer (FRET) from a fluorophore-labelled peptide to graphene oxide (GO). By the hydrolysis of the peptide, the fluorophore-labelled peptide releases the fluorophore 5-carboxyfluorescein, which can avoid quenching from GO. Thus, the increased intensity of the obtained fluorescence signal in the assay is directly dependent on the peptidase activity. As a model case of the developed strategy, the activity determination of pancreatic elastase (PE) is performed. Under the optimal experimental conditions at an excitation wavelength of 494 nm, the activity of PE can be determined in the range from 0.003 to 0.10 U/mL, with a detection limit of 0.001 U/mL at the emission wavelength of 518 nm. This is ultra-sensitive for the determination of PE. The specificity of the method is demonstrated by the analysis of PE under complex conditions using fetal bovine serum as the substrate. Hence, the developed method might provide an intrinsically convenient, sensitive platform for the PE activity assay and related biochemical studies due to its homogeneous, and fluorescence-based detection strategy.
在这项研究中，我们报道了一种基于荧光共振能量转移（FRET）的荧光检测策略，用于肽酶活性测定，该策略基于荧光团标记肽向氧化石墨烯（GO）的荧光共振能量转移。通过肽的水解，荧光团标记的肽释放荧光团 5-羧基荧光素，从而避免了来自 GO 的淬灭。因此，测定中获得的荧光信号强度的增加直接取决于肽酶的活性。作为该策略的一个模型案例，进行了胰蛋白酶（PE）活性的测定。在最佳实验条件下，激发波长为 494nm 时，PE 的活性可在 0.003 至 0.10U/mL 的范围内测定，在发射波长为 518nm 时检测限为 0.001U/mL。这对于 PE 的测定是超灵敏的。该方法使用胎牛血清作为底物在复杂条件下分析 PE，证明了该方法的特异性。因此，由于其均相和基于荧光的检测策略，该方法可能为 PE 活性测定和相关生化研究提供了一个内在方便、灵敏的平台。
