生物荧光探针的分子动力学研究与应用
发布时间：2018-09-11 09:32
【摘要】：在探究生命领域时,荧光技术被认为是最为有效的手段之一。时间分辨荧光光谱相比于稳态荧光光谱不易受荧光分子浓度、激发波长和功率等因素的影响,近年来备受研究者们的关注。在研究不发光的生物分子和细胞时,研究者们需要借助荧光探针,其中使用最广的主要包括有机小分子荧光探针、氨基酸多肽荧光探针、荧光蛋白探针和基于新型纳米发光材料的探针等。在本文中,我们将利用时间分辨荧光光谱来研究不同类型探针分子的荧光动力学,并将它们用于生物应用研究。氨苯乙烯类的荧光分子是典型的有机小分子探针。我们合成了 o-DASPMI和p-DASPMI,并分别用稳态荧光光谱、皮秒级的时间分辨荧光光谱和飞秒级时间分辨荧光光谱技术来表征它们的荧光性质。o-DASPMI和p-DASPMI斯托克斯位移超过150nm,荧光寿命分别为6.6ps和12.4ps.超短的荧光寿命使得DASPMI的荧光可以用来测量TCSPC或者FLIM设备的仪器响应函数(IRF)。在本文中,我们利用DASPMI荧光测得的IRF分别拟合了罗丹明B(RhB)、酰磺罗丹明B(SRB)和SRB与RNA适配子SRB2m复合体的荧光寿命,拟合的效果与散射光作为IRF 一致。实验结果证明DASPMI的荧光可以用作TCSPC或者FLIM设备的IRF检测,并有效的解决了探测器颜色效应等问题。在缓冲液中,游离的p-DASPMI的荧光寿命为12.4ps,与BSA结合后出现两个更长的荧光寿命,分别是0.9 ns和2.6 ns.通过荧光寿命的比重和DAS光谱,我们将0.9 ns和2.6 ns分别对应到BSA的ⅢA区和ⅡA区。通过比较荧光寿命变化、寿命比重变化和参数(α2τ2+α3τ3)/α1τ1与参数α3τ3/α2τ2的变化,我们研究了 pH和Cu2+对蛋白质结构的影响。结果表明借助时间分辨荧光光谱,p-DASPMI即能更灵敏的定量检测蛋白质浓度,也能同时对游离态的探针和不同绑定位点处的探针进行监控,实现了对蛋白质多个位点的结构研究。pH对细胞内的生理过程起到至关重要的作用。借助pH探针实时观察生理环境的pH波动是非常有意义的。我们合成了多种基于Trp-X的肽链,研究了其荧光性质随pH的变化,并尝试将其接在多肽上,用于测量多肽周围环境的pH值。我们首先研究Trp-Trp双肽和它的三种衍生物(NATrp2Me、NBTrp2和Trp2Me)在不同pH下的荧光光谱,发现裸露的氨基是Trp-X对pH响应的关键。然后我们研究Trp-X在多肽中的性质。在多肽Trp-X-Ala-Ser中,Trp-X仍然保持着pH的响应。相比于 Trp-Trp-Ala-Ser(WWAS),Trp-Ala-Ala-Ser(WAAS)和 Trp-Glu-Ala-Ser(WEAS)的荧光量子产率更高、荧光寿命更长,对pH的响应更敏感。干扰实验中,金属,阳离子虽然干扰了探针稳态荧光强度,但对时间分辨荧光光谱几乎没有影响。研究结果表明,Trp-X双肽的平均荧光寿命可以超灵敏、高选择性的标定多肽周围的pH环境。roUnaG-BR作为一种新型的氧化还原探针,不仅克服GFP类荧光蛋白在厌氧环境下表达不佳的问题,还实现了可随时"点亮"荧光的功能。但是roUnaG-BR是一种单通道激发和发射的荧光探针,饱和氧化态和饱和还原态之间的平均荧光寿命只有0.3ns的变化,很难采用稳态荧光强度或者平均荧光寿命来标定细胞内的氧化还原状态。本文采用时间分辨荧光光谱研究roUnaG-BR,并通过对比UnaG-BR的荧光和结构性质,发现在还原态的体系中,2.2ns荧光寿命对应的分子构型占主导地位;而在氧化态的体系中,0.2ns荧光寿命对应的分子构型占主导地位。采用参数α3/α1和α3τ3/α1τ1却可以获得10倍和8.1倍的动态变化范围,这比稳态荧光的7倍还要大。结果表明,借助时间分辨荧光技术,单激发通道和单发射通道的roUnaG-BR可以定量标定细胞内的氧化还原状态,并可获得比稳态荧光强度和平均荧光寿命更高的灵敏度。
[Abstract]:Time-resolved fluorescence spectroscopy is considered to be one of the most effective means to explore the field of life. Compared with steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy is not easily affected by fluorescence molecular concentration, excitation wavelength and power. Recently, it has attracted much attention of researchers. The most widely used fluorescent probes include organic small molecule fluorescent probes, amino acid polypeptide fluorescent probes, fluorescent protein probes and probes based on novel nanoluminescent materials. O-DASPMI and p-DASPMI were synthesized and characterized by steady-state fluorescence spectroscopy, picosecond time-resolved fluorescence spectroscopy and femtosecond time-resolved fluorescence spectroscopy respectively. The fluorescence lifetime of DASPMI is 6.6 PS and 12.4 PS at 150 nm, respectively. The fluorescence lifetime of DASPMI can be used to measure the instrumental response function (IRF) of TCSPC or FLIM equipment. In this paper, the IRF measured by DASPMI fluorescence is used to fit the fluorescence lifetime of Rhodamine B (RhB), Sulfonyl Rhodamine B (SRB) and SRB-SRB-SRB2m complexes, respectively. The experimental results show that the fluorescence of DASPMI can be used as the IRF detection of TCSPC or FLIM equipment, and the color effect of detector can be effectively solved. In the buffer solution, the fluorescence lifetime of the free p-DASPMI is 12.4 ps, and two longer fluorescence lifetimes appear after binding with BSA, respectively, are 0.9 PS. NS and 2.6 ns. The ratios of fluorescence lifetime and DAS spectra of 0.9 ns and 2.6 ns correspond to the regions III A and II A of BS A, respectively. The effects of pH and Cu 2+ on the structure of protein were studied by comparing the changes of fluorescence lifetime, specific gravity of lifetime and the parameters (alpha 2_2+alpha 3 3)/alpha 1 and the parameters alpha 3_3/alpha 2. Time-resolved fluorescence spectroscopy, p-DASPMI can detect protein concentration more sensitively and quantitatively, and also can monitor the free probe and probe at different binding sites simultaneously. The structure of multiple protein sites can be studied. pH plays an important role in the physiological process of cells. We have synthesized a variety of Trp-X-based peptide chains and studied their fluorescence properties as a function of pH. We have attempted to graft them onto polypeptides to measure the pH of the environment around the peptides. We first studied the fluorescence spectra of Trp-Trp dipeptide and its three derivatives (NATrp2Me, NBTrp2 and Trp2Me) at different pHs. It was found that the exposed amino group was the key to the pH response of Trp-X. Then we studied the properties of Trp-X in polypeptides. In polypeptide Trp-X-Ala-Ser, Trp-X still maintained pH response. Compared with Trp-Trp-Ala-Ser (WWAS), Trp-Ala-Ala-Ser (WAAS) and Trp-Glu-Ala-Ser (WEAS), Trp-Ala-Ser had higher fluorescence quantum yield, longer fluorescence lifetime and more sensitive response to pH. The results show that the average fluorescence lifetime of Trp-X dipeptide can be highly sensitive and selective to calibrate the pH environment around the peptide. As a new redox probe, roUnaG-BR can not only overcome G but also overcome G. However, roUnaG-BR is a single-channel fluorescent probe. The average fluorescence lifetime between saturated oxidized and reduced states is only 0.3 ns. It is difficult to use steady-state fluorescence intensity or average fluorescence lifetime. In this paper, time-resolved fluorescence spectroscopy was used to study the fluorescence and structural properties of roUnaG-BR. It was found that the molecular configurations corresponding to the fluorescence lifetime of 2.2ns were dominant in the reduced system, and the molecular configurations corresponding to the fluorescence lifetime of 0.2ns were dominant in the oxidized system. The results show that roUnaG-BR with single excitation channel and single emission channel can quantitatively calibrate the redox state of cells and obtain stronger fluorescence than that with steady state fluorescence. The sensitivity of degree and average fluorescence lifetime is even higher.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3
本文编号：2236308
[Abstract]:Time-resolved fluorescence spectroscopy is considered to be one of the most effective means to explore the field of life. Compared with steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy is not easily affected by fluorescence molecular concentration, excitation wavelength and power. Recently, it has attracted much attention of researchers. The most widely used fluorescent probes include organic small molecule fluorescent probes, amino acid polypeptide fluorescent probes, fluorescent protein probes and probes based on novel nanoluminescent materials. O-DASPMI and p-DASPMI were synthesized and characterized by steady-state fluorescence spectroscopy, picosecond time-resolved fluorescence spectroscopy and femtosecond time-resolved fluorescence spectroscopy respectively. The fluorescence lifetime of DASPMI is 6.6 PS and 12.4 PS at 150 nm, respectively. The fluorescence lifetime of DASPMI can be used to measure the instrumental response function (IRF) of TCSPC or FLIM equipment. In this paper, the IRF measured by DASPMI fluorescence is used to fit the fluorescence lifetime of Rhodamine B (RhB), Sulfonyl Rhodamine B (SRB) and SRB-SRB-SRB2m complexes, respectively. The experimental results show that the fluorescence of DASPMI can be used as the IRF detection of TCSPC or FLIM equipment, and the color effect of detector can be effectively solved. In the buffer solution, the fluorescence lifetime of the free p-DASPMI is 12.4 ps, and two longer fluorescence lifetimes appear after binding with BSA, respectively, are 0.9 PS. NS and 2.6 ns. The ratios of fluorescence lifetime and DAS spectra of 0.9 ns and 2.6 ns correspond to the regions III A and II A of BS A, respectively. The effects of pH and Cu 2+ on the structure of protein were studied by comparing the changes of fluorescence lifetime, specific gravity of lifetime and the parameters (alpha 2_2+alpha 3 3)/alpha 1 and the parameters alpha 3_3/alpha 2. Time-resolved fluorescence spectroscopy, p-DASPMI can detect protein concentration more sensitively and quantitatively, and also can monitor the free probe and probe at different binding sites simultaneously. The structure of multiple protein sites can be studied. pH plays an important role in the physiological process of cells. We have synthesized a variety of Trp-X-based peptide chains and studied their fluorescence properties as a function of pH. We have attempted to graft them onto polypeptides to measure the pH of the environment around the peptides. We first studied the fluorescence spectra of Trp-Trp dipeptide and its three derivatives (NATrp2Me, NBTrp2 and Trp2Me) at different pHs. It was found that the exposed amino group was the key to the pH response of Trp-X. Then we studied the properties of Trp-X in polypeptides. In polypeptide Trp-X-Ala-Ser, Trp-X still maintained pH response. Compared with Trp-Trp-Ala-Ser (WWAS), Trp-Ala-Ala-Ser (WAAS) and Trp-Glu-Ala-Ser (WEAS), Trp-Ala-Ser had higher fluorescence quantum yield, longer fluorescence lifetime and more sensitive response to pH. The results show that the average fluorescence lifetime of Trp-X dipeptide can be highly sensitive and selective to calibrate the pH environment around the peptide. As a new redox probe, roUnaG-BR can not only overcome G but also overcome G. However, roUnaG-BR is a single-channel fluorescent probe. The average fluorescence lifetime between saturated oxidized and reduced states is only 0.3 ns. It is difficult to use steady-state fluorescence intensity or average fluorescence lifetime. In this paper, time-resolved fluorescence spectroscopy was used to study the fluorescence and structural properties of roUnaG-BR. It was found that the molecular configurations corresponding to the fluorescence lifetime of 2.2ns were dominant in the reduced system, and the molecular configurations corresponding to the fluorescence lifetime of 0.2ns were dominant in the oxidized system. The results show that roUnaG-BR with single excitation channel and single emission channel can quantitatively calibrate the redox state of cells and obtain stronger fluorescence than that with steady state fluorescence. The sensitivity of degree and average fluorescence lifetime is even higher.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3
【参考文献】
相关博士学位论文 前2条
1 贾梦辉;基于超快荧光光谱的蛋白表面水合化动力学研究[D];华东师范大学;2016年
2 袁玉峰;新型银纳米材料与生物辅酶的精密光谱特性研究[D];华东师范大学;2016年
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