Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences (CAS) , Beijing 100190, China.
Anal Chem. 2015 Mar 3;87(5):3080-6. doi: 10.1021/ac504773n. Epub 2015 Feb 10.
This study demonstrates a novel ratiometric fluorescent method for real-time alkaline phosphatase (ALP) activity assay with stimulus responsive infinite coordination polymer (ICP) nanoparticles as the probe. The ICP nanoparticles used in this study are composed of two components; one is the supramolecular ICP network formed with guanine monophosphate (GMP) as the ligand and Tb(3+) as the central metal ion, and the other is a fluorescent dye, i.e., 7-amino-4-methyl coumarin (coumarin) encapsulated into the ICP network. Upon being excited at 315 nm, the ICP network itself emits green fluorescence at 552 nm. Coumarin dye encapsulated in the ICP network emits weak fluorescence at 450 nm upon excitation at the same wavelength (315 nm), and this fluorescence emission becomes strong when the encapsulated dye is released from the network into the solution phase. Hence, we develop a ratiometric fluorescent assay based on the ALP-induced destruction of the supramolecular ICP network and the release of coumarin. This mechanism can be used for real-time ratiometric fluorescent monitoring of ALP activity by continuously measuring the ratio of fluorescent intensity at the wavelength of 552 nm (F552) to that at 450 nm (F450) (F552/F450) in the time-dependent fluorescent spectra of the coumarin@Tb-GMP suspension containing ALP with different activities. Under the experimental conditions employed here, the F552/F450 value is linear with the ALP activity within a range from 0.025 U/mL to 0.2 U/mL. The detection limit is down to 0.010 U/mL (S/N = 3). Moreover, the assay developed here is employed for ALP inhibitor evaluation. This study offers a simple yet sensitive method for real-time ALP activity assay.
本研究展示了一种新颖的比率荧光法，用于实时碱性磷酸酶（ALP）活性测定，使用对刺激响应的无限配位聚合物（ICP）纳米粒子作为探针。本研究中使用的 ICP 纳米粒子由两部分组成；一部分是由鸟嘌呤单磷酸（GMP）作为配体和 Tb(3+)作为中心金属离子形成的超分子 ICP 网络，另一部分是荧光染料，即包封在 ICP 网络中的 7-氨基-4-甲基香豆素（香豆素）。在 315nm 激发下，ICP 网络本身发射 552nm 的绿光。包封在 ICP 网络中的香豆素染料在相同波长（315nm）激发下发射弱荧光在 450nm，当包封的染料从网络释放到溶液相中时，这种荧光发射变得强烈。因此，我们开发了一种基于 ALP 诱导的超分子 ICP 网络破坏和香豆素释放的比率荧光测定法。这种机制可用于通过连续测量含不同活性 ALP 的香豆素@Tb-GMP 悬浮液的时间依赖性荧光光谱中 552nm（F552）处的荧光强度与 450nm（F450）处的荧光强度之比（F552/F450），实时比率荧光监测 ALP 活性。在本实验条件下，F552/F450 值与 0.025U/mL 至 0.2U/mL 范围内的 ALP 活性呈线性关系。检测限低至 0.010U/mL（S/N=3）。此外，本研究中开发的测定法用于 ALP 抑制剂评价。本研究提供了一种简单而灵敏的实时 ALP 活性测定方法。
