Ratiometric Fluorescent Strategy for Localizing Alkaline Phosphatase Activity in Mitochondria Based on the ESIPT Process
Ratiometric Fluorescent Strategy for Localizing Alkaline Phosphatase Activity in Mitochondria Based on the ESIPT Process
复制标题
基于 ESIPT 过程定位线粒体碱性磷酸酶活性的比率荧光策略
DOI：
10.1021/acs.analchem.9b02917
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发表时间：
2019
影响因子：
7.4
通讯作者：
Ding Caifeng
中科院分区：
文献类型：
--
作者：
Zhang Peng;Fu Caixia;Zhang Qian;Li Shasha;Ding Caifeng
荧光探针是用于在体外和体内检测和定位感兴趣的物种的有力工具。虽然这些探针通常具有很高的选择性和灵敏度，但它们通常受到一些因素的影响，如检测条件和探针浓度。比率荧光法具有背景噪声低的优点，可以有效地解决这一问题，提高传感性能。因此，基于磷酸化的2-（2′-羟基苯基）-苯并噻唑衍生物开发了用于测定ALP活性的基于ESIPT的比率探针（HBTP-mito）。HBTP-mito是水溶性的，在TBS缓冲液中由于ESIPT的阻断而发射绿色荧光。在引入ALP后，HBTP-mito的磷酸酯被水解并且ESIPT过程被恢复。因此，514 nm处的荧光降低，而650 nm处的发射显示“开启”响应。在0 ~ 60 mU/mL范围内，随着ALP活性的增加，I514 nm/I650 nm的比值呈线性下降，检测限为0.072 mU/mL。该探针的良好性能使其不仅可用于生物样品中ALP活性的检测，而且可用于活细胞和体内ALP水平的定位。
Fluorescent probes are powerful tools for detecting and mapping the species of interest in vitro and in vivo. Although the probes always show high selectivity and sensitivity, they are usually affected by some factors, such as detecting conditions and the probe concentrations. Ratiometric fluorescent strategies, possessing advantage of low background noise, would solve the problem effectively and lead to a higher sensing performance. Thus, an ESIPT-based ratiometric probe (HBTP-mito) was developed on the basis of a phosphorylated 2-(2′-hydroxyphenyl)-benzothiazole derivative for the determination of ALP activity. HBTP-mito is water soluble and emits green fluorescence in TBS buffer due to the blockage of ESIPT. Upon the introduction of ALP, the phosphate ester of HBTP-mito was hydrolyzed and the ESIPT process was restored. Accordingly, the fluorescence at 514 nm decreases, while emission at 650 nm shows a “turn-on” response. The ratio of intensity (I514nm/I650nm) decreases linearly with ALP activity increasing from 0 to 60 mU/mL, obtained an LOD of 0.072 mU/mL. The favorable performance of the probe enables its application not only in the detection of ALP activity in biological samples, but also in the localization of the ALP levels in living cells and in vivo.