基于半导体量子点和石墨烯量子点的功能性荧光纳米生物传感器的构建及在生物医学分析中的应用

发布时间：2018-05-25 22:48

  本文选题：CdTe量子点 + 石墨烯量子点　； 参考：《吉林大学》2017年博士论文

【摘要】：纳米技术是纳米科学领域中迅速发展且分支最广的一项科学技术。近年来纳米技术和光电技术的蓬勃发展,使利用不同纳米材料构建的纳米传感器的开发进入了新的阶段。其中,半导体量子点(QDs)自从出现起就广受关注。他们荧光强度强,量子产率高,具有尺寸依赖的可调谐发射波长,而且发射峰窄而对称,这些优良的性质使他们成为研究生命科学的重要工具。而作为新一类荧光碳基材料的石墨烯量子点(GQDs)也在近几年来取得广泛关注。GQDs这种新兴的荧光纳米材料具有出色的生物相容性,独特的光学性质以及容易被修饰的材料表面和边缘。作为生物传感器中最具代表性的两种荧光纳米材料,他们已经被用于多种领域,如水质监测,体液检测,临床诊断,药物传输,基因诊断,生物成像等。另外,通过对他们进行表面功能化,在其表面连接各种各样的修饰物,就可以利用修饰物与目标分子的亲和作用实现特异性检测。对QDs或GQDs功能化能够丰富纳米材料在生物传感器中的应用,以此达到检测尽可能多的分析物的目的。本论文分别以功能化的CdTeQDs和GQDs为荧光纳米探针,构建了一系列荧光纳米生物传感器,并分别研究了其在生命小分子,酶活性等生物医学分析领域的应用,具体内容如下:第一部分,我们简要介绍了当前荧光纳米生物传感器中所使用的最具有代表性的两种荧光纳米材料——半导体量子点和石墨烯量子点进行简要介绍,主要包括它们的基本特性、制备方法,发光机理,表面功能化及其在生物医学分析领域中构建荧光纳米生物传感器的应用。第二部分,我们基于L-半胱氨酸包覆的CdTeQDs构建了一个简单灵敏的荧光探针用于在人类体液中检测腺苷5'-三磷酸(ATP)。首先我们在水相中合成了一系列具有不同尺寸的L-半胱氨酸包覆的CdTeQDs(L-cys-CdTeQDs)。通过研究Zn~(2+)对不同尺寸的L-cys-CdTeQDs的荧光调制作用,我们发现Zn~(2+)可以与QDs表面的L-半胱氨酸有效配位,猝灭比表面积更大的小尺寸的L-cys-CdTeQDs的荧光;另一方面,在ATP存在的情况下,ATP的磷酸基团通过Zn-O-P键对Zn~(2+)具有很高的亲和作用,Zn~(2+)通过金属-配体配位作用优先与ATP结合,导致Zn~(2+)对L-cys-CdTeQDs的调制作用被抑制,由此恢复L-cys-CdTeQDs的荧光。QDs的荧光强度与ATP浓度在5-50μmol L~(-1)范围内成正比,ATP的检测限为2.07μmol L~(-1)。相比于其它生物体内重要的磷酸盐,该传感体系显示对ATP良好的选择性,并成功应用于人血清样品中ATP的测定。第三部分,我们设计了一种简单、方便、高敏感性的荧光“off-on”体系,利用N-乙酰基半胱氨酸包覆的CdTeQDs(NAC-CdTeQDs)对胰蛋白酶进行检测。我们通过回流加热法在水溶液中合成了不同尺寸的NAC-CdTeQDs,通过引入牛血红蛋白(Hb)形成QDs/Hb复合体系。Hb通过静电吸引和表面配位的协同作用附着在QDs表面,拉近了两者之间的距离,使电子从NAC-CdTeQDs向Hb转移,从而导致Hb对QDs的荧光猝灭作用。Hb在胰蛋白酶的作用下会水解成小的多肽类,并且释放出不活跃的血红素分子,从而减弱了Hb对QDs荧光强度的影响,QDs的荧光恢复。我们利用Hb和血红素分子对QDs荧光的影响不同来检测胰蛋白酶的活性。QDs荧光强度的恢复与胰蛋白酶浓度的对数成比例,线性范围是0.2~40 ng m L~(-1),检出限是0.144 ng m L~(-1)。我们利用胰蛋白酶的抑制剂模型来证实该系统的可行性。大豆胰蛋白酶抑制剂的IC50值是3.06μg m L~(-1)。我们建立了一种荧光实时检测胰蛋白酶及其抑制剂的方法,相比其他的生物酶检测体系,该方法表现出高选择性和灵敏性,用于人体尿液样本中的胰蛋白酶检测时取得满意的结果。第四部分,我们利用多巴胺功能化的CdTeQDs(QDs-DA)作为荧光探针,用于在生物体液中检测L-组氨酸。首先,CdTe与多巴胺共价连接形成一种表面具有邻苯二酚结构的荧光传感器。由于Ni~(2+)和QDs-DA的邻苯二酚结构之间的强配位相互作用,QDs-DA的荧光强度可以被Ni~(2+)猝灭。因为Ni~(2+)与L-组氨酸的高亲和力,在L-组氨酸存在下,Ni~(2+)优选与L-组氨酸结合,使QDs-DA的荧光强度恢复。恢复的QDs-DA的荧光强度与L-组氨酸的浓度在1.0×10-6~1.0×10-4 mol L~(-1)范围内成比例,检测限为5.0×10-7 mol L~(-1)。所建立的方法在其他常见氨基酸存在的情况下对L-组氨酸的选择性良好,用于人血清样品中L-组氨酸的测定,结果令人满意。第五部分,我们通过简单的自上而下水热法制备出了具有黄绿色荧光的GQDs,并通过Cr(VI)和抗坏血酸的氧化还原反应调制GQDs的荧光强度,连续检测了抗坏血酸(AA)和酸性磷酸酶(ACP)。AA与Cr2O72-可以发生氧化还原反应生成Cr3+,基于Cr3+在GQDs上的静电吸附以及Cr3+与GQDs表面上的-COOH和-OH基团之间的强螯合作用,GQDs可以与Cr3+之间发生电子转移作用导致荧光信号猝灭,猝灭程度与AA的浓度成比例。这一体系进一步用于酸性磷酸酶(ACP)的选择性检测。磷酸酶底物抗坏血酸磷酸酯钠(AAP)可以通过ACP水解得到AA。然后,AA通过与Cr2O72-氧化还原反应得到Cr3+,导致GQDs的荧光猝灭,通过体系荧光信号的变化间接检测ACP的浓度。由此我们构建了基于Cr(VI)的氧化还原调制的GQDs荧光纳米传感器,用于顺序检测AA和ACP。该方法显示出对AA和ACP的高度选择性和抗干扰能力,并在实际样品测定中获得了令人满意的结果。第六部分,我们利用rGQDs和生物聚合物之间的自组装和解组装作用构建了一种无标记的碱性磷酸酶(ALP)生物传感器。我们先用Na BH4化学还原了发黄绿色荧光的GQDs,得到了发蓝光的还原型石墨烯量子点(rGQDs),且荧光强度得到明显提高。接着,利用壳聚糖(CS)与rGQDs静电作用形成复合体系,通过结构变化改变rGQDs的荧光强度。当将ALP的酶解底物(NaPO_3)_6引入到自组装复合体系时,因为其更强的电负性,可以作为rGQDs/CS复合体系的解组装试剂将rGQDs从复合体系中竞争下来,“turn-on”体系荧光;最后将ALP引入到解组装体系中,ALP可以水解(NaPO_3)_6,使CS重新与rGQDs组合在一起,恢复自组装体系并“turn-off”复合体系的荧光。猝灭的荧光强度与ALP的活性存在一定的比例关系,因此我们可以构建一个由(NaPO_3)_6调制的,基于rGQDs/CS复合体系的“turn-on-off”ALP荧光生物传感器。该传感器检测范围宽,对ALP有高选择性,且在实际样品的检测中也得到了良好的应用。上述的几种荧光传感器的构建对于推进生化分析和医学诊断研究,以及进一步探索基于量子点的纳米材料生物传感应用具有重要意义。
[Abstract]:Nanotechnology is a rapid development and widely branched science and technology in the field of nanoscience. In recent years, the development of nanotechnology and photoelectric technology has made the development of nano sensors developed with different nanomaterials into a new stage. Among them, semiconductor quantum dots (QDs) have attracted much attention since they appeared. Their fluorescence intensity Strong, high quantum yield, with a size dependent tunable emission wavelength and a narrow and symmetrical emission peak, these excellent properties make them an important tool for the study of life science. As a new class of fluorescent carbon based materials, graphene quantum dots (GQDs) have also gained wide attention in recent years to pay attention to the new fluorescent nanomaterials, such as.GQDs It has excellent biocompatibility, unique optical properties and surface and edge of easily modified materials. As the most representative two kinds of fluorescent nanomaterials in biosensors, they have been used in many fields, such as water quality monitoring, body fluid detection, clinical diagnosis, drug transmission, gene diagnosis, biological imaging and so on. They carry out surface functionalization, connect a variety of modifiers on their surface, and make use of the affinity between the modifier and the target molecules to achieve specific detection. The functionalization of QDs or GQDs can enrich the application of nanomaterials in biosensors, so as to achieve the purpose of detecting as many analytical objects as possible. A series of fluorescent nanoscale biosensors are constructed by the energetic CdTeQDs and GQDs as fluorescent nanoprobes, and their applications in biomedical analysis fields such as small molecules of life, enzyme activity and other fields are studied. The first part is a brief introduction to the most representative of the current fluorescent nano biosensors. Two kinds of fluorescent nanomaterials - semiconductor quantum dots and graphene quantum dots are briefly introduced, including their basic properties, preparation methods, luminescence mechanism, surface functionalization and the application of fluorescent nano biosensors in biomedical analysis. The second part, based on the L- cysteine coated CdTeQDs A simple and sensitive fluorescent probe was constructed to detect adenosine 5'- three phosphoric acid (ATP) in human body fluid. First, we synthesized a series of L- cysteine coated CdTeQDs (L-cys-CdTeQDs) with different sizes in the aqueous phase. By studying the fluorescence modulation of Zn~ (2+) for different sizes of L-cys-CdTeQDs, we found Zn~ (2+). With the effective coordination of L- cysteine on the surface of QDs, the fluorescence of a small size L-cys-CdTeQDs larger than the surface area is quenched; on the other hand, in the presence of ATP, the phosphoric acid group of ATP has a high affinity to Zn~ (2+) through the Zn-O-P bond, and Zn~ (2+) combines the metal ligand coordination with ATP, leading to Zn~. The modulation effect of QDs was suppressed, thus the fluorescence intensity of the fluorescence.QDs of L-cys-CdTeQDs was restored to a positive ratio with the concentration of ATP in the range of 5-50 Mu mol L~ (-1), and the detection limit of ATP was 2.07 Mu mol L~ (-1). Compared to the important phosphate in other organisms, the sensing system showed good selectivity for ATP, and was successfully applied to human serum samples. In the third part, we designed a simple, convenient, Gao Min sensitive fluorescence "off-on" system, using N- acetyl cysteine coated CdTeQDs (NAC-CdTeQDs) to detect trypsin. We synthesized different sizes of NAC-CdTeQDs in aqueous solution by reflux heating, and formed Hb by introducing bovine hemoglobin (Hb). The QDs/Hb composite system.Hb adheres to the surface of QDs by the synergistic effect of electrostatic attraction and surface coordination, drawing the distance between the two and transferring electrons from NAC-CdTeQDs to Hb, resulting in the fluorescence quenching effect of Hb on QDs, and.Hb will hydrolyze into small peptides under the action of trypsin, and release the inactive heme molecules. The effect of Hb on the fluorescence intensity of QDs and the fluorescence recovery of QDs were reduced. We used Hb and heme molecules to detect the difference in the fluorescence of QDs. The fluorescence intensity of trypsin was detected in the logarithm of the logarithm of the trypsin concentration, the linear range was 0.2~40 ng m L~ (-1), and the detection limit was 0.144 ng. The white enzyme inhibitor model is used to confirm the feasibility of the system. The IC50 value of the soybean trypsin inhibitor is 3.06 g m L~ (-1). We have established a method of real-time fluorescence detection of trypsin and its inhibitors. Compared with other biological enzyme detection systems, the method shows high selectivity and sensitivity, which is used in human urine samples. The fourth part, we use dopamine functionalized CdTeQDs (QDs-DA) as a fluorescence probe to detect L- histidine in biological fluids. First, CdTe and dopamine covalent together to form a surface with catechol structure fluorescence sensor. Due to Ni~ (2+) and QDs-DA catechol. The strong ligand interaction between the structures, the fluorescence intensity of QDs-DA can be quenched by Ni~ (2+). Because of the high affinity of Ni~ (2+) with L- histidine, Ni~ (2+) combines with L- histidine in the presence of L- histidine. The fluorescence intensity of QDs-DA is restored. The fluorescence intensity of the resumed QDs-DA and the concentration of histidine is 1 * * 10-4. The range is proportional to the detection limit of 5 x 10-7 mol L~ (-1). The method established is good for the L- histidine in the presence of other common amino acids. The results are satisfactory for the determination of L- histidine in human serum samples. The fifth part, we have prepared a yellowish green fluorescence by a simple top-down hydrothermal method. GQDs and Cr (VI) and ascorbic acid redox reaction modulate the fluorescence intensity of GQDs, and the oxidation redox reaction of ascorbic acid (AA) and acid phosphatase (ACP).AA is continuously detected to produce Cr3+, based on the electrostatic adsorption of Cr3+ in GQDs and the strong chelation between the Cr3 + on the surface of the Cr3 + and the Cr2O72-. The electron transfer between GQDs and Cr3+ leads to the quenching of the fluorescence signal, and the degree of quenching is proportional to the concentration of AA. This system is further used for the selective detection of acid phosphatase (ACP). The phosphatase substrate, ascorbate phosphate sodium (AAP) can be hydrolyzed to AA. by ACP, and AA is obtained by the redox reaction of Cr2O72- to Cr. 3+, which leads to the fluorescence quenching of GQDs, detects the concentration of ACP indirectly through the changes in the fluorescence signal of the system. Thus, we construct a GQDs fluorescent nano sensor based on Cr (VI) redox modulation, which is used for sequential detection of AA and ACP., which shows the high selectivity and anti-interference ability to AA and ACP, and has been obtained in the actual sample determination. In the sixth part, we constructed an unlabeled alkaline phosphatase (ALP) biosensor using the self-assembly and assembly of rGQDs and biopolymers. We first used Na BH4 to chemically restore the yellow green fluorescence GQDs, and obtained the blue light of the prototype graphene quantum dots (rGQDs) and the fluorescence intensity. Then, the composite system of chitosan (CS) and rGQDs was used to form a composite system, and the fluorescence intensity of rGQDs was changed by structural change. When the enzyme substrate (NaPO_3) _6 of ALP was introduced into the self assembled composite system, because of its stronger electronegativity, it could be used as a solution assembly reagent for the rGQDs/CS composite system to take rGQDs from the composite system. In the competition, "turn-on" system fluorescence; finally, the ALP is introduced into the solution assembly system, and ALP can hydrolyze (NaPO_3) _6, so that CS is recombined with rGQDs to restore the fluorescence of the self-assembly system and the "turn-off" composite system. The fluorescence intensity of the quenching is proportional to the activity of ALP, so we can build a one The "turn-on-off" ALP fluorescent biosensor based on (NaPO_3) _6, based on the rGQDs/CS composite system, has a wide range of detection range, high selectivity for ALP and good application in the detection of actual samples. The construction of these fluorescent sensors is for the advancement of biochemical and medical diagnosis research, and the following. It is of great significance to explore the application of nanoscale materials based on quantum dots for biosensing.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3
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本文编号：1934942