水溶性聚噻吩比率探针的设计合成及传感性质研究

发布时间：2018-11-19 08:48

【摘要】：三磷酸腺苷(ATP)是生物体内一种重要的生物小分子,在许多生命过程中起关键作用。ATP的浓度异常,往往会引发许多疾病,因此建立简单快速、灵敏度高、选择性好的检测方法不仅对生命过程的基础研究,而且对临床诊断,药物分析等领域也有着十分重要的意义。荧光技术具有操作简便,灵敏度高,选择性好,易于原位成像等优点,已经被广泛应用于生物活性物质的检测。目前报道的ATP荧光探针,其荧光信号通常基于单波长发射强度的变化,该方法容易受到外界环境的影响,信噪比低。而比率型荧光探针,利用探针与底物结合后,出现激发或发射光谱的移位,体系中出现两个荧光发射强度随底物的变化而变化,利用这两个不同波长处荧光强度的比值而进行的检测。该方法可以提高检测的动态响应范围,建立起内标从而极大地削弱其它因素的干扰,是提高荧光探针灵敏度的一个方便而有效的途径。近年来,基于共轭聚合物的荧光探针因为其良好的光电性质和信号放大效应受到了广泛关注。其中,聚噻吩衍生物由于其构象和聚集形态对外界刺激十分敏感,在分析物的诱导下,其荧光光谱会发生显著变化,同时伴随着溶液颜色的明显改变,因此作为一种荧光及比色双模式检测的探针材料受到众多科研工作者的追捧。本论文设计合成了一种比率型ATP荧光探针(Cou-PT),将香豆素荧光团引入到聚噻吩(PT)的侧链上,香豆素荧光团作为FRET能量给体,共轭聚噻吩主链作为FRET能量受体。其检测原理如下:未结合的Cou-PT采用无规线团构象,一旦加入ATP后,Cou-PT与ATP形成复合物,从而导致聚噻吩主链从无规线团构象转变成平面构象并且发生链间的进一步聚集,体系荧光淬灭,同时紫外光谱明显红移。利用加入ATP前后,Cou-PT吸收光谱与香豆素荧光团发射光谱重叠的变化,来调控香豆素荧光团到聚噻吩主链的FRET效率,偶联聚集诱导的荧光淬灭(ACQ)两种机制,实现对ATP的比率检测。本论文利用荧光光谱,紫外吸收光谱,圆二色光谱,动态光散射光谱等分析手段,系统研究了比率探针Cou-PT对ATP的传感性质。研究结果表明:Job's-plot结果显示探针Cou-PT与ATP能形成1:1复合物,结合常数为Kapp=3.12×104 M-1,CD光谱和DLS结果说明ATP的加入使聚噻吩主链构象发生了从非平面到平面构象的转变并且发生聚集。Cou-PT在加入ATP前后荧光光谱呈比率变化,同时吸收光谱红移伴随溶液颜色变化,所以该探针也可用于裸眼检测ATP。此外,该探针对ATP表现出优异的选择性,ADP,AMP或各种其他阴离子的加入没有引起荧光及紫外光谱明显的变化。Cou-PT还被用于在生理pH条件下实时监测ATPase催化的ATP水解反应,并初步实现了对活细胞的荧光成像。
[Abstract]:Adenosine triphosphate (ATP) is an important biological small molecule in organism, which plays a key role in many life processes. Abnormal concentration of ATP often leads to many diseases, so it is simple, rapid and sensitive. Selective detection methods are of great significance not only for the basic research of life process, but also for clinical diagnosis, drug analysis and so on. Fluorescence technique has been widely used in the detection of bioactive substances because of its advantages of simple operation, high sensitivity, good selectivity and easy in situ imaging. The fluorescence signal of ATP fluorescence probe reported at present is usually based on the change of single wavelength emission intensity. This method is easy to be affected by the external environment and has low signal-to-noise ratio (SNR). However, the ratio fluorescence probe, which combines the substrate with the probe, exhibits excitation or emission shift, and two fluorescence emission intensities change with the change of the substrate. The detection was performed using the ratio of fluorescence intensity at these two different wavelengths. This method can improve the dynamic response range of detection, establish internal standard and greatly weaken the interference of other factors. It is a convenient and effective way to improve the sensitivity of fluorescent probe. In recent years, fluorescent probes based on conjugated polymers have attracted wide attention due to their good photoelectric properties and signal amplification effects. Polythiophene derivatives are sensitive to external stimuli due to their conformation and aggregation morphology. The fluorescence spectra of polythiophene derivatives will change significantly under the inducement of the analyte, and the color of the solution will change obviously. Therefore, as a fluorescence and colorimetric double-mode detection probe materials are sought after by many researchers. In this paper, a ratio ATP fluorescence probe (Cou-PT) was designed and synthesized. Coumarin fluorescence group was introduced into the side chain of polythiophene (PT). Coumarin fluorescence group was used as energy donor for FRET and conjugated polythiophene as FRET energy receptor. The detection principle is as follows: the unbonded Cou-PT adopts the random coil conformation. Once ATP is added, Cou-PT forms a complex with ATP. As a result, the main chain of polythiophene changed from random cluster conformation to plane conformation and further aggregation between chains occurred. The fluorescence quenching of the system and the red shift of UV spectrum were observed. In order to regulate the FRET efficiency of coumarin fluorescence group to the main chain of polythiophene, the Cou-PT absorption spectrum and the emission spectrum of coumarin fluorescence group were overlapped before and after the addition of ATP, and the two mechanisms of fluorescence quenching induced by coupling aggregation of coumarin fluorescence group to polythiophene were also studied. The ratio detection of ATP is realized. In this paper, the sensing properties of ratio probe Cou-PT to ATP were systematically studied by means of fluorescence spectrum, UV absorption spectrum, circular dichroism spectrum and dynamic light scattering spectroscopy. The results of Job's-plot show that Cou-PT and ATP can form 1:1 complex, and the binding constant is Kapp=3.12 脳 104M-1. The CD spectra and DLS results show that the main conformation of polythiophene changes from non-plane to plane conformation and aggregates with the addition of ATP, and the fluorescence spectrum of Cou-PT changes proportionally before and after the addition of ATP. At the same time, the red shift of absorption spectrum is accompanied by the color change of solution, so the probe can also be used for naked eye detection of ATP.. In addition, the probe showed excellent selectivity to ATP, and the addition of ADP,AMP or various other anions did not cause obvious changes in fluorescence and UV spectra. Cou-PT was also used to monitor the hydrolysis of ATP catalyzed by ATPase in real-time under physiological pH conditions. The fluorescence imaging of living cells was preliminarily realized.
【学位授予单位】：天津理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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