Fluorescence Lifetime Imaging of p-tau Protein in Single Neuron with a Highly Selective Fluorescent Probe
Fluorescence Lifetime Imaging of p-tau Protein in Single Neuron with a Highly Selective Fluorescent Probe
复制标题
使用高选择性荧光探针对单个神经元中的 p-tau 蛋白进行荧光寿命成像
DOI：
10.1021/acs.analchem.8b03992
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发表时间：
2019-03-05
影响因子：
7.4
通讯作者：
Tian, Yang
中科院分区：
文献类型：
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作者：
Ge, Lihong;Tian, Yang
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，缺乏高选择性和准确性的p-tau蛋白分析方法是了解AD病理过程的瓶颈。本文合理设计和研制了一种高选择性荧光寿命成像显微探针（tau-p-tau），该探针以双核锌基为识别基，合成了改进的菁氨酸为染料基，用于检测p-tau蛋白。FLIM测量与荧光探针的浓度、照射光源和测量环境无关。同时，开发的tau-p-tau探针对p-tau蛋白具有较强的亲和力，从而提高了测定p-tau蛋白对A β、A β纤维、tau蛋白、蛋白激酶、ATP等的选择性。tau-p-tau探针的荧光寿命与p-tau蛋白浓度在1.0 ~ 5.0 μ M范围内呈良好的线性关系，最低检出限为85.15 +/- 0.03 nM。该探针对p-tau蛋白的响应时间估计小于6.2 s。此外，该探针具有低毒性和良好的生物相容性，已成功应用于单神经元水平的荧光成像监测p-tau蛋白的浓度波动。结果表明，氧化应激使活神经元内p-tau蛋白浓度发生明显变化。
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 (tau-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 tau-p-tau probe demonstrated strong affinity toward p-tau protein, thus enhancing the selectivity of determination of p-tau protein against A beta, A beta fibril, tau, protein kinases, ATP, and so on. The fluorescence lifetime of tau-p-tau probe showed a good linearity with the concentration of p-tau protein from 1.0 to 5.0 mu M 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.