基于新型荧光传感器检测总蛋白和肾素浓度

发布时间：2018-05-20 20:40

本文选题：双发射比率荧光探针 + CdTe量子点　；参考：《河北大学》2017年硕士论文

【摘要】：近年来,纳米材料由于其独特的性质得到了广大研究者的青睐,以纳米材料为基础构建的各种生物传感平台已广泛应用于生物化学研究领域。本文分别利用量子点和金纳米粒子这两种纳米材料构建了检测总蛋白和肾素浓度的荧光传感平台,得到初步满意的结果。具体如下:本文设计了一种新型的双发射比率荧光探针用于总蛋白含量的检测。首先通过静电层层自组装技术将发射绿色荧光的CdTe量子点组装到发射红色荧光的聚苯乙烯基质染料荧光微球表面,得到一种新型的复合微球探针,该探针在单波长紫外光激发下,呈现出区分明显的两个发射峰。由于外层绿色荧光强度明显大于内层红色荧光的强度,此时探针整体呈现绿色荧光。Cu2+能够选择性的猝灭探针外层量子点的荧光,对内层微球中的染料几乎无影响,因此,当Cu2+存在的情况下,由于外层绿色荧光的猝灭,探针整体呈现红色荧光。在碱性条件下,蛋白质具有将Cu2+还原为Cu+的特性,因此在蛋白质存在时,由于Cu2+被还原为Cu+,对探针外层不再猝灭,这时探针恢复为绿色荧光。蛋白质的含量与探针的荧光光谱图和荧光颜色直接相关,依据此原理进行总蛋白的测定。结果表明,当总蛋白浓度在0.05 mg/mL-4.0 mg/m L范围内时,与探针的比率(外层量子点的荧光强度与内层荧光微球的荧光强度的比值)呈良好的线性关系。探针的荧光颜色从红色逐步变为黄色直到绿色,实现了可视化分析。利用该方法检测实际样品脱脂牛奶中总蛋白含量时,与经典BCA法测得的数据基本一致,由此可证明该方法的可靠性和实用性。由于该方法在利用荧光光谱图对总蛋白含量定量的同时,还可以通过探针颜色的变化进行半定量,因此,可进一步推广做成比色卡,以达到更加便捷检测总蛋白浓度的目的。除此之外,本文还将金纳米粒子(gold nanoparticles,AuNPs)和荧光素标记的肾素底物结合作为荧光共振能量转移传感平台,建立了肾素浓度的检测方法。首先设计了一条特殊的多肽,能够被肾素特异性的水解,在该多肽的C端用5(6)-异硫氰酸荧光素(fluorescein isothiocyanate,FITC)修饰,N端用生物素(biotin)标记。然后将链霉亲合素(streptavidin,STV)标记的AuNPs与biotin标记的底物特异性结合,得到AuNP/多肽/FITC的结构,此时,由于FITC与AuNPs距离较近,产生能量共振转移,FITC的荧光被AuNPs猝灭。当有肾素存在,该多肽被肾素特异性水解,带有FITC的肽链残基从AuNPs表面游离出来,此时AuNPs与FITC之间的能量共振转移消失,FITC荧光恢复。体系中FITC的荧光强度与肾素的浓度有关,并且肾素浓度在0.01?g/mL-0.2?g/mL范围内时,与体系的荧光强度成正比。此外,该方法还可用于肾素抑制剂的筛选,在临床诊断和药物开发方面具有一定的应用潜力。
[Abstract]:In recent years, nanomaterials have been favored by many researchers because of their unique properties. Various biosensor platforms based on nanomaterials have been widely used in the field of biochemistry research. In this paper, a fluorescence sensing platform for the determination of total protein and renin concentration was constructed using quantum dots and gold nanoparticles, respectively, and the results were satisfactory. The main results are as follows: a novel double emission ratio fluorescence probe was designed for the detection of total protein content. Firstly, the CdTe quantum dots emitting green fluorescence were assembled onto the surface of the red fluorescent polystyrene matrix dye fluorescent microspheres by electrostatic layer self-assembly technique, and a novel composite microsphere probe was obtained. The probe exhibits two distinct emission peaks under single wavelength UV excitation. Because the green fluorescence intensity of the outer layer is obviously higher than that of the inner layer red fluorescence, the whole probe presents the green fluorescence. Cu2 can selectively quench the fluorescence of the probe's outer quantum dot, and has little effect on the dye in the inner layer microsphere. In the presence of Cu2, the whole probe showed red fluorescence due to the quenching of green fluorescence in the outer layer. In alkaline condition, the protein has the property of reducing Cu2 to Cu. Therefore, in the presence of protein, Cu2 is reduced to Cu, and the outer layer of the probe is no longer quenched, so the probe returns to green fluorescence. The content of protein is directly related to the fluorescence spectrum and color of the probe, and the total protein is determined according to this principle. The results showed that when the total protein concentration was in the range of 0. 05 mg/mL-4.0 mg/m / L, there was a good linear relationship between the ratio of the fluorescence intensity of the outer quantum dot and the fluorescence intensity of the inner fluorescent microsphere. The fluorescence color of the probe gradually changed from red to yellow to green, realizing visual analysis. When the total protein content in skim milk is detected by this method, it is consistent with the data obtained by classical BCA method, which can prove the reliability and practicability of this method. Since the method can be used to quantify the total protein content by fluorescence spectroscopy, it can also be semi-quantified by the change of the probe color, so it can be further extended to make a colorimetric card, so that the detection of total protein concentration can be more convenient. In addition, gold nanoparticles gold nanoparticles-AuNPs) and fluorescein labeled renin substrates were combined as a fluorescence resonance energy transfer sensing platform to establish a method for the determination of renin concentration. Firstly, a special peptide was designed, which could be hydrolyzed by renin specifically. The C-terminal of the peptide was labeled with a N-terminal modified with fluorescein isothiocyanate and labeled with biotin. Then the AuNPs labeled by streptavidinine was specifically bound to the substrate labeled with biotin, and the structure of AuNP/ polypeptide / FITC was obtained. At this time, due to the proximity between FITC and AuNPs, the fluorescence of FITC produced energy resonance transfer was quenched by AuNPs. In the presence of renin, the peptide was hydrolyzed specifically by renin, and the residue of peptide chain with FITC was dissociated from the surface of AuNPs. The energy resonance transfer between AuNPs and FITC disappeared and the fluorescence of FITC recovered. The fluorescence intensity of FITC in the system is related to the concentration of renin, and when the concentration of renin is in the range of 0.01?g/mL-0.2?g/mL, it is directly proportional to the fluorescence intensity of the system. In addition, the method can be used to screen renin inhibitors, and has potential in clinical diagnosis and drug development.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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