School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, Guangdong, 510006, PR China; Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China.
Department of Physiology, Shandong University School of Medicine, Jinan, Shandong, 250012, PR China.
Biomaterials. 2017 Sep;140:220-229. doi: 10.1016/j.biomaterials.2017.06.032. Epub 2017 Jun 23.
Alkaline phosphatase (ALP) is a family of enzymes involved in the regulation of important biological processes such as cell differentiation and bone mineralization. Monitoring the activity of ALP in serum can help diagnose a variety of diseases including bone and liver diseases. There has been growing interest in developing new chemical tools for monitoring ALP activity in living systems. Such tools will help further delineate the roles of ALP in biological and pathological processes. Previously reported fluorescent probes has a number of disadvantages that limit their application, such as poor selectivity and short-wavelength excitation. In this work, we report a new two-photon fluorescent probe (TP-Phos) to selectively detect ALP activity. The probe is composed of a two-photon fluorophore, a phosphate recognition moiety, and a self-cleavable adaptor. It offers a number of advantages over previously reported probes, such as fast reaction kinetics, high sensitivity and low cytotoxicity. Experimental results also showed that TP-Phos displayed improved selectivity over DIFMUP, a commonly utilized ALP probe. The selectivity is attributed to the utilization of an ortho-functionalised phenyl phosphate group, which increases the steric hindrance of the probe and the active site of phosphatases. Moreover, the two-photon nature of the probe confers enhanced imaging properties such as increased penetration depth and lower tissue autofluorescence. TP-Phos was successfully used to image the endogenous ALP activity of hippocampus, kidney and liver tissues from rat.
碱性磷酸酶（ALP）是一类参与细胞分化和骨矿化等重要生物学过程调节的酶。监测血清中 ALP 的活性有助于诊断包括骨骼和肝脏疾病在内的多种疾病。人们对开发用于监测活体内 ALP 活性的新型化学工具越来越感兴趣。这些工具将有助于进一步阐明 ALP 在生物学和病理学过程中的作用。先前报道的荧光探针存在一些限制其应用的缺点，例如选择性差和短波长激发。在这项工作中，我们报告了一种新的双光子荧光探针（TP-Phos），用于选择性检测 ALP 活性。该探针由双光子荧光团、磷酸识别部分和自切割接头组成。与先前报道的探针相比，它具有许多优势，如快速反应动力学、高灵敏度和低细胞毒性。实验结果还表明，TP-Phos 对 DIFMUP（一种常用的 ALP 探针）表现出更高的选择性。这种选择性归因于利用邻位官能化的苯膦酸酯基团，增加了探针和磷酸酶活性部位的空间位阻。此外，探针的双光子性质赋予了增强的成像特性，例如增加的穿透深度和更低的组织自发荧光。TP-Phos 成功地用于成像大鼠海马、肾脏和肝脏组织中的内源性 ALP 活性。
