Department of Chemistry, School of Chemistry and Molecular Engineering , East China Normal University , Dongchuan Road 500 , Shanghai 200241 , China.
Anal Chem. 2019 Mar 5;91(5):3294-3301. doi: 10.1021/acs.analchem.8b03992. Epub 2019 Feb 14.
Hyperphosphorylated tau (p-tau) protein is one of the key markers of Alzheimer's disease (AD). However, a lack of analytical methods for p-tau protein with high selectivity and accuracy is the bottleneck to understand the pathological processes of AD. In the present work, a highly selective fluorescence lifetime imaging microscopic (FLIM) probe (τ-p-tau) was rationally designed and developed for determination of p-tau protein, in which binuclear zinc units were designed as the recognition groups, and an improved cyanine was synthesized as the dye unit. The FLIM measurement is independent of the concentration of fluorescent probes, irradiation light sources, and measurement environments. Meanwhile, the developed τ-p-tau probe demonstrated strong affinity toward p-tau protein, thus enhancing the selectivity of determination of p-tau protein against Aβ, Aβ fibril, tau, protein kinases, ATP, and so on. The fluorescence lifetime of τ-p-tau probe showed a good linearity with the concentration of p-tau protein from 1.0 to 5.0 μM with a low detection limit of 85.15 ± 0.03 nM. The response time of the present probe for p-tau protein was estimated to be less than 6.2 s. Furthermore, taking the advantages of low toxicity and good biocompatibility, the developed probe was successfully applied to monitor the fluctuation of concentration of p-tau protein by FLIM imaging at single neuron level. It was found that the concentration of p-tau protein inside live neurons obviously changed upon oxidative stress.
过度磷酸化的 tau（p-tau）蛋白是阿尔茨海默病（AD）的关键标志物之一。然而，缺乏具有高选择性和准确性的 p-tau 蛋白分析方法是理解 AD 病理过程的瓶颈。在本工作中，我们合理设计并开发了一种高选择性的荧光寿命成像显微镜（FLIM）探针（τ-p-tau），用于测定 p-tau 蛋白，其中双核锌单元被设计为识别基团，并且合成了一种改进的菁染料作为荧光基团。FLIM 测量与荧光探针的浓度、辐照光源和测量环境无关。同时，所开发的 τ-p-tau 探针对 p-tau 蛋白具有很强的亲和力，从而提高了测定 p-tau 蛋白对 Aβ、Aβ 纤维、tau、蛋白激酶、ATP 等物质的选择性。τ-p-tau 探针的荧光寿命与 p-tau 蛋白的浓度在 1.0 到 5.0 μM 范围内呈现出良好的线性关系，检测限低至 85.15 ± 0.03 nM。本探针对 p-tau 蛋白的响应时间估计小于 6.2 s。此外，利用低毒性和良好的生物相容性的优势，该探针成功地应用于通过 FLIM 成像在单个神经元水平监测 p-tau 蛋白浓度的波动。结果发现，活神经元内 p-tau 蛋白的浓度在氧化应激下明显发生变化。
