發(fā)布時間：2018-05-25 22:48

  本文選題：CdTe量子點 + 石墨烯量子點　； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：納米技術(shù)是納米科學(xué)領(lǐng)域中迅速發(fā)展且分支最廣的一項科學(xué)技術(shù)。近年來納米技術(shù)和光電技術(shù)的蓬勃發(fā)展,使利用不同納米材料構(gòu)建的納米傳感器的開發(fā)進入了新的階段。其中,半導(dǎo)體量子點(QDs)自從出現(xiàn)起就廣受關(guān)注。他們熒光強度強,量子產(chǎn)率高,具有尺寸依賴的可調(diào)諧發(fā)射波長,而且發(fā)射峰窄而對稱,這些優(yōu)良的性質(zhì)使他們成為研究生命科學(xué)的重要工具。而作為新一類熒光碳基材料的石墨烯量子點(GQDs)也在近幾年來取得廣泛關(guān)注。GQDs這種新興的熒光納米材料具有出色的生物相容性,獨特的光學(xué)性質(zhì)以及容易被修飾的材料表面和邊緣。作為生物傳感器中最具代表性的兩種熒光納米材料,他們已經(jīng)被用于多種領(lǐng)域,如水質(zhì)監(jiān)測,體液檢測,臨床診斷,藥物傳輸,基因診斷,生物成像等。另外,通過對他們進行表面功能化,在其表面連接各種各樣的修飾物,就可以利用修飾物與目標(biāo)分子的親和作用實現(xiàn)特異性檢測。對QDs或GQDs功能化能夠豐富納米材料在生物傳感器中的應(yīng)用,以此達到檢測盡可能多的分析物的目的。本論文分別以功能化的CdTeQDs和GQDs為熒光納米探針,構(gòu)建了一系列熒光納米生物傳感器,并分別研究了其在生命小分子,酶活性等生物醫(yī)學(xué)分析領(lǐng)域的應(yīng)用,具體內(nèi)容如下:第一部分,我們簡要介紹了當(dāng)前熒光納米生物傳感器中所使用的最具有代表性的兩種熒光納米材料——半導(dǎo)體量子點和石墨烯量子點進行簡要介紹,主要包括它們的基本特性、制備方法,發(fā)光機理,表面功能化及其在生物醫(yī)學(xué)分析領(lǐng)域中構(gòu)建熒光納米生物傳感器的應(yīng)用。第二部分,我們基于L-半胱氨酸包覆的CdTeQDs構(gòu)建了一個簡單靈敏的熒光探針用于在人類體液中檢測腺苷5'-三磷酸(ATP)。首先我們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)。通過研究Zn~(2+)對不同尺寸的L-cys-CdTeQDs的熒光調(diào)制作用,我們發(fā)現(xiàn)Zn~(2+)可以與QDs表面的L-半胱氨酸有效配位,猝滅比表面積更大的小尺寸的L-cys-CdTeQDs的熒光;另一方面,在ATP存在的情況下,ATP的磷酸基團通過Zn-O-P鍵對Zn~(2+)具有很高的親和作用,Zn~(2+)通過金屬-配體配位作用優(yōu)先與ATP結(jié)合,導(dǎo)致Zn~(2+)對L-cys-CdTeQDs的調(diào)制作用被抑制,由此恢復(fù)L-cys-CdTeQDs的熒光。QDs的熒光強度與ATP濃度在5-50μmol L~(-1)范圍內(nèi)成正比,ATP的檢測限為2.07μmol L~(-1)。相比于其它生物體內(nèi)重要的磷酸鹽,該傳感體系顯示對ATP良好的選擇性,并成功應(yīng)用于人血清樣品中ATP的測定。第三部分,我們設(shè)計了一種簡單、方便、高敏感性的熒光“off-on”體系,利用N-乙酰基半胱氨酸包覆的CdTeQDs(NAC-CdTeQDs)對胰蛋白酶進行檢測。我們通過回流加熱法在水溶液中合成了不同尺寸的NAC-CdTeQDs,通過引入牛血紅蛋白(Hb)形成QDs/Hb復(fù)合體系。Hb通過靜電吸引和表面配位的協(xié)同作用附著在QDs表面,拉近了兩者之間的距離,使電子從NAC-CdTeQDs向Hb轉(zhuǎn)移,從而導(dǎo)致Hb對QDs的熒光猝滅作用。Hb在胰蛋白酶的作用下會水解成小的多肽類,并且釋放出不活躍的血紅素分子,從而減弱了Hb對QDs熒光強度的影響,QDs的熒光恢復(fù)。我們利用Hb和血紅素分子對QDs熒光的影響不同來檢測胰蛋白酶的活性。QDs熒光強度的恢復(fù)與胰蛋白酶濃度的對數(shù)成比例,線性范圍是0.2~40 ng m L~(-1),檢出限是0.144 ng m L~(-1)。我們利用胰蛋白酶的抑制劑模型來證實該系統(tǒng)的可行性。大豆胰蛋白酶抑制劑的IC50值是3.06μg m L~(-1)。我們建立了一種熒光實時檢測胰蛋白酶及其抑制劑的方法,相比其他的生物酶檢測體系,該方法表現(xiàn)出高選擇性和靈敏性,用于人體尿液樣本中的胰蛋白酶檢測時取得滿意的結(jié)果。第四部分,我們利用多巴胺功能化的CdTeQDs(QDs-DA)作為熒光探針,用于在生物體液中檢測L-組氨酸。首先,CdTe與多巴胺共價連接形成一種表面具有鄰苯二酚結(jié)構(gòu)的熒光傳感器。由于Ni~(2+)和QDs-DA的鄰苯二酚結(jié)構(gòu)之間的強配位相互作用,QDs-DA的熒光強度可以被Ni~(2+)猝滅。因為Ni~(2+)與L-組氨酸的高親和力,在L-組氨酸存在下,Ni~(2+)優(yōu)選與L-組氨酸結(jié)合,使QDs-DA的熒光強度恢復(fù)�；謴�(fù)的QDs-DA的熒光強度與L-組氨酸的濃度在1.0×10-6~1.0×10-4 mol L~(-1)范圍內(nèi)成比例,檢測限為5.0×10-7 mol L~(-1)。所建立的方法在其他常見氨基酸存在的情況下對L-組氨酸的選擇性良好,用于人血清樣品中L-組氨酸的測定,結(jié)果令人滿意。第五部分,我們通過簡單的自上而下水熱法制備出了具有黃綠色熒光的GQDs,并通過Cr(VI)和抗壞血酸的氧化還原反應(yīng)調(diào)制GQDs的熒光強度,連續(xù)檢測了抗壞血酸(AA)和酸性磷酸酶(ACP)。AA與Cr2O72-可以發(fā)生氧化還原反應(yīng)生成Cr3+,基于Cr3+在GQDs上的靜電吸附以及Cr3+與GQDs表面上的-COOH和-OH基團之間的強螯合作用,GQDs可以與Cr3+之間發(fā)生電子轉(zhuǎn)移作用導(dǎo)致熒光信號猝滅,猝滅程度與AA的濃度成比例。這一體系進一步用于酸性磷酸酶(ACP)的選擇性檢測。磷酸酶底物抗壞血酸磷酸酯鈉(AAP)可以通過ACP水解得到AA。然后,AA通過與Cr2O72-氧化還原反應(yīng)得到Cr3+,導(dǎo)致GQDs的熒光猝滅,通過體系熒光信號的變化間接檢測ACP的濃度。由此我們構(gòu)建了基于Cr(VI)的氧化還原調(diào)制的GQDs熒光納米傳感器,用于順序檢測AA和ACP。該方法顯示出對AA和ACP的高度選擇性和抗干擾能力,并在實際樣品測定中獲得了令人滿意的結(jié)果。第六部分,我們利用rGQDs和生物聚合物之間的自組裝和解組裝作用構(gòu)建了一種無標(biāo)記的堿性磷酸酶(ALP)生物傳感器。我們先用Na BH4化學(xué)還原了發(fā)黃綠色熒光的GQDs,得到了發(fā)藍光的還原型石墨烯量子點(rGQDs),且熒光強度得到明顯提高。接著,利用殼聚糖(CS)與rGQDs靜電作用形成復(fù)合體系,通過結(jié)構(gòu)變化改變rGQDs的熒光強度。當(dāng)將ALP的酶解底物(NaPO_3)_6引入到自組裝復(fù)合體系時,因為其更強的電負性,可以作為rGQDs/CS復(fù)合體系的解組裝試劑將rGQDs從復(fù)合體系中競爭下來,“turn-on”體系熒光;最后將ALP引入到解組裝體系中,ALP可以水解(NaPO_3)_6,使CS重新與rGQDs組合在一起,恢復(fù)自組裝體系并“turn-off”復(fù)合體系的熒光。猝滅的熒光強度與ALP的活性存在一定的比例關(guān)系,因此我們可以構(gòu)建一個由(NaPO_3)_6調(diào)制的,基于rGQDs/CS復(fù)合體系的“turn-on-off”ALP熒光生物傳感器。該傳感器檢測范圍寬,對ALP有高選擇性,且在實際樣品的檢測中也得到了良好的應(yīng)用。上述的幾種熒光傳感器的構(gòu)建對于推進生化分析和醫(yī)學(xué)診斷研究,以及進一步探索基于量子點的納米材料生物傳感應(yīng)用具有重要意義。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：O657.3
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本文編號：1934942