本文選題：雙發(fā)射比率熒光探針 + CdTe量子點��； 參考：《河北大學》2017年碩士論文

【摘要】：近年來,納米材料由于其獨特的性質(zhì)得到了廣大研究者的青睞,以納米材料為基礎構(gòu)建的各種生物傳感平臺已廣泛應用于生物化學研究領域。本文分別利用量子點和金納米粒子這兩種納米材料構(gòu)建了檢測總蛋白和腎素濃度的熒光傳感平臺,得到初步滿意的結(jié)果。具體如下:本文設計了一種新型的雙發(fā)射比率熒光探針用于總蛋白含量的檢測。首先通過靜電層層自組裝技術(shù)將發(fā)射綠色熒光的CdTe量子點組裝到發(fā)射紅色熒光的聚苯乙烯基質(zhì)染料熒光微球表面,得到一種新型的復合微球探針,該探針在單波長紫外光激發(fā)下,呈現(xiàn)出區(qū)分明顯的兩個發(fā)射峰。由于外層綠色熒光強度明顯大于內(nèi)層紅色熒光的強度,此時探針整體呈現(xiàn)綠色熒光。Cu2+能夠選擇性的猝滅探針外層量子點的熒光,對內(nèi)層微球中的染料幾乎無影響,因此,當Cu2+存在的情況下,由于外層綠色熒光的猝滅,探針整體呈現(xiàn)紅色熒光。在堿性條件下,蛋白質(zhì)具有將Cu2+還原為Cu+的特性,因此在蛋白質(zhì)存在時,由于Cu2+被還原為Cu+,對探針外層不再猝滅,這時探針恢復為綠色熒光。蛋白質(zhì)的含量與探針的熒光光譜圖和熒光顏色直接相關(guān),依據(jù)此原理進行總蛋白的測定。結(jié)果表明,當總蛋白濃度在0.05 mg/mL-4.0 mg/m L范圍內(nèi)時,與探針的比率(外層量子點的熒光強度與內(nèi)層熒光微球的熒光強度的比值)呈良好的線性關(guān)系。探針的熒光顏色從紅色逐步變?yōu)辄S色直到綠色,實現(xiàn)了可視化分析。利用該方法檢測實際樣品脫脂牛奶中總蛋白含量時,與經(jīng)典BCA法測得的數(shù)據(jù)基本一致,由此可證明該方法的可靠性和實用性。由于該方法在利用熒光光譜圖對總蛋白含量定量的同時,還可以通過探針顏色的變化進行半定量,因此,可進一步推廣做成比色卡,以達到更加便捷檢測總蛋白濃度的目的。除此之外,本文還將金納米粒子(gold nanoparticles,AuNPs)和熒光素標記的腎素底物結(jié)合作為熒光共振能量轉(zhuǎn)移傳感平臺,建立了腎素濃度的檢測方法。首先設計了一條特殊的多肽,能夠被腎素特異性的水解,在該多肽的C端用5(6)-異硫氰酸熒光素(fluorescein isothiocyanate,FITC)修飾,N端用生物素(biotin)標記。然后將鏈霉親合素(streptavidin,STV)標記的AuNPs與biotin標記的底物特異性結(jié)合,得到AuNP/多肽/FITC的結(jié)構(gòu),此時,由于FITC與AuNPs距離較近,產(chǎn)生能量共振轉(zhuǎn)移,FITC的熒光被AuNPs猝滅。當有腎素存在,該多肽被腎素特異性水解,帶有FITC的肽鏈殘基從AuNPs表面游離出來,此時AuNPs與FITC之間的能量共振轉(zhuǎn)移消失,FITC熒光恢復。體系中FITC的熒光強度與腎素的濃度有關(guān),并且腎素濃度在0.01?g/mL-0.2?g/mL范圍內(nèi)時,與體系的熒光強度成正比。此外,該方法還可用于腎素抑制劑的篩選,在臨床診斷和藥物開發(fā)方面具有一定的應用潛力。
[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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.3

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相關(guān)博士學位論文 前1條

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本文編號：1916157