The biology of highly reactive oxygen radical species is of great interest in many biomedical research disciplines, including neurodegeneration, aging, cancer, and infectious diseases.[1] There are a number of fluorescent reagents, such as 2,7-dichlorodihydrofluorescein (DCDHF), that can be used to detect free radicals, but they have significant limitations due to their facile oxidation by light and numerous non-radical oxidants such as hydrogen peroxide (H2O2). [2] HPF is a cell-permeable aromatic amino-fluorescein derivative that has little intrinsic fluorescence. [3] It undergoes oxidation only by highly reactive oxygen species (hROS) such as the hydroxyl radical, peroxynitrite, and hROS generated from a peroxidase/H2O2 system. It is inert to hypochlorite ion, nitric oxide, hydrogen peroxide (H2O2), superoxide, and other oxidants. Upon oxidation, HPF is converted to the highly fluorescent molecule fluorescein, with excitation/emission maxima of 490/515 nm, respectively, allowing the simple direct detection of highly reactive biological radicals.
Reference:
[1]. Matés, J.M., Pèrez-Gómez, C., and Nuñez de Castro, I. Antioxidant enzymes and human diseases. Clinical Biochemistry 32(8), 595-603 (1999).
[2]. Hempel, S.L., Buettner, G.R., O'Malley, Y.Q., et al. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: Comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radical Biology & Medicine 27(1), 146-159 (1999).
[3]. Setsukinai, K.i., Urano, Y., Kakinuma, K., et al. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J. Biol. Chem. 278(5), 3170-3175 (2003).
高度反应性氧自由基的生物学在许多生物医学研究领域，包括神经退行性疾病、衰老、癌症和传染病方面具有极大的研究价值[1]。有许多荧光探针，如2,7-二氯二氢荧光素（DCDHF），可用于检测自由基，但由于它们容易被光和许多非自由基氧化剂如过氧化氢（H2O2）氧化而具有重要局限性。[2] HPF是一种细胞渗透的芳香族氨基-荧光素衍生物，几乎没有固有荧光[3]。只有高度反应性氧自由基（如羟基自由基、过硝酸根离子和过氧化酶/H2O2系统产生的高度反应性氧自由基）能够使其氧化。它对次氯酸离子、一氧化氮、过氧化氢（H2O2）、超氧和其他氧化剂具有惰性。氧化后，HPF被转化为高度荧光的分子荧光素，其激发/发射最大值分别为490/515 nm，从而可以直接检测高度反应性生物自由基。