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 H₂O₂. DFF could catalyze oxidization of TMB by H₂O₂ to produce a blue colored oxidation product. Effect of various reaction conditions, such as pH, temperature, H₂O₂ 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_cat) 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 H₂O₂, the H₂O₂ 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.

在H ₂ O ₂存在下，使用3,3'，5,5'-四甲基联苯胺（TMB）作为生色底物，研究了2'，7'-二氟荧光素（DFF）的过氧化物酶样活性。DFF可以催化H ₂ O ₂氧化TMB，产生蓝色的氧化产物。研究了pH，温度，H ₂ O ₂浓度和反应时间等各种反应条件对DFF催化活性的影响。发现DFF的过氧化物酶样活性遵循Michaelis-Menten动力学，其催化符合乒乓机制。计算的动力学参数（K _cat）的DFF催化显示出比荧光素和2'，7'-二氯荧光素（DCF）更高的过氧化物酶样活性。根据自由基捕获和电子自旋共振（ESR）光谱结果，我们证实羟基自由基（•OH）是催化过程的活性物种。已知半乳糖氧化酶（GAOx）氧化半乳糖会导致H ₂ O ₂，H ₂ O _{2的形成。}因此，使用DFF作为过氧化物模拟物和使用TMB作为发色剂对在该反应中释放的二甲基甲酰胺进行了定量。因此，利用以上两个反应的组合来建立半乳糖检测方法。在最佳条件下，该方法的线性范围为10μM至20 mM，检测限降至3μM。此外，将开发的方法用于检测尿液样品中的半乳糖。我们的工作将促进DFF内在过氧化物酶样活性在医学诊断和生物技术中的利用。