Key Laboratory on Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
Key Laboratory on Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
Talanta. 2018 May 15;182:422-427. doi: 10.1016/j.talanta.2018.02.020. Epub 2018 Feb 7.
The peroxidase-like activity of 2',7'-difluorofluorescein (DFF), was investigated using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate in the presence of HO. DFF could catalyze oxidization of TMB by HO to produce a blue colored oxidation product. Effect of various reaction conditions, such as pH, temperature, HO concentration and reaction time on the catalytic activity of DFF was studied. The peroxidase-like activity of DFF was found to follow Michaelis-Menten kinetics, and its catalysis accorded with ping-pong mechanism. The calculated kinetic parameters (K) of DFF catalysis showed higher peroxidase-like activity than fluorescein and 2',7'-dichlorofluorescein (DCF). According to the radical capture and electron spin resonance (ESR) spectroscopy results, we confirmed that hydroxyl radical (•OH) is the active specie of catalytic process. It is known that the oxidation of galactose by galactose oxidase (GAOx) enzyme leads to the formation of HO, the HO released in this reaction was consequently quantified using DFF as peroxides mimics and TMB as the chromogen. Thus, a combination of above two reactions was exploited to establish a method for galactose detection. Under the optimum conditions, the linear range of this method was from 10 μM to 20 mM with the detection limit down to 3 μM. Moreover, the developed method was applied to detect galactose in urine samples. Our work will facilitate the utilization of DFF intrinsic peroxidase-like activity in medical diagnostics and biotechnology.
2',7'-二氟荧光素（DFF）的过氧化物酶样活性，使用 3,3',5,5'-四甲基联苯胺（TMB）作为显色底物，在 HO 的存在下进行了研究。DFF 可以催化 HO 氧化 TMB，产生蓝色氧化产物。研究了各种反应条件，如 pH 值、温度、HO 浓度和反应时间对 DFF 催化活性的影响。发现 DFF 的过氧化物酶样活性遵循米氏动力学，其催化符合乒乓机制。DFF 催化的动力学参数（K）的计算表明，其过氧化物酶样活性高于荧光素和 2',7'-二氯荧光素（DCF）。根据自由基捕获和电子自旋共振（ESR）光谱结果，我们证实了羟基自由基（•OH）是催化过程的活性物质。众所周知，半乳糖氧化酶（GAOx）酶氧化半乳糖会导致 HO 的形成，在这个反应中释放的 HO 被随后使用 DFF 作为过氧化物模拟物和 TMB 作为显色剂进行定量。因此，结合上述两种反应建立了一种用于检测半乳糖的方法。在最佳条件下，该方法的线性范围为 10 μM 至 20 mM，检测限低至 3 μM。此外，该方法还应用于检测尿液样品中的半乳糖。我们的工作将有助于利用 DFF 的内在过氧化物酶样活性在医学诊断和生物技术中。
