新型仿生納米材料的制備及其在生物檢測中的應(yīng)用研究
發(fā)布時間：2018-11-12 11:59
【摘要】：新型納米材料(金屬納米簇、石墨烯等)由于其獨特的性質(zhì)受到研究者的青睞,并廣泛地用于生物醫(yī)學(xué)檢測�；诩{米材料的分析方法簡便、快捷、易操作、靈敏,在化學(xué)分析、生物傳感、疾病檢測、環(huán)境監(jiān)測等領(lǐng)域具有潛在的應(yīng)用前景。隨著相關(guān)學(xué)科的迅速發(fā)展以及相關(guān)新技術(shù)的引入,光電(如熒光和電化學(xué))檢測的靈敏度、準(zhǔn)確度和選擇性日益提高,應(yīng)用范圍遍及眾多領(lǐng)域,已經(jīng)發(fā)展成為一種十分重要且有效的化學(xué)分析手段。本論文設(shè)計和制備了熒光金銀納米簇以及相關(guān)的光電化學(xué)傳感器,通過光電手段對人類健康和安全密切相關(guān)的小分子和金屬離子進(jìn)行識別檢測。另外,鑒于金納米簇優(yōu)良的熒光特性和生物相容性,作為一類新型的小尺寸的熒光探針應(yīng)用于細(xì)胞活體成像以及腫瘤治療。具體工作開展如下：首先,創(chuàng)新性地以胞嘧啶核苷為模板原位快速合成熒光增強、水溶性的金銀復(fù)合納米簇,并對其紫外-可見吸收光譜、熒光光譜、粒徑形貌、熒光壽命、熒光量子產(chǎn)率以及金元素和銀元素的價態(tài)和含量進(jìn)行了表征。研究結(jié)果表明,該金銀復(fù)合納米簇在370 nm激發(fā)下具有強烈的黃色熒光(560nm發(fā)射),粒徑大約為1.50nm,量子產(chǎn)率約9%,平均熒光壽命6.07μs,并且具有良好的熒光穩(wěn)定性、分散性以及水溶性,由此奠定了其在生物分析中的潛在應(yīng)用價值。在此基礎(chǔ)上,我們對銀離子快速顯著增強金納米簇?zé)晒獾臋C(jī)理進(jìn)行了探討,并把這種快速原位合成熒光增強的金銀復(fù)合納米簇的方法用于血清中疾病相關(guān)分子的檢測。相關(guān)研究結(jié)果進(jìn)一步顯示了金銀納米簇在生物分析和診斷中應(yīng)用的潛在可能性。在上述研究工作的基礎(chǔ)上,本論文利用金納米簇優(yōu)良的熒光特性發(fā)展了一種非標(biāo)記的、雙功能的快速高靈敏選擇性檢測銀離子和汞離子的新方法。研究結(jié)果表明,銀離子能夠快速顯著地增強金納米簇的熒光并形成具有強烈黃色熒光的金銀復(fù)合納米簇,而汞離子可以迅速猝滅增強后的金銀納米簇的熒光。據(jù)此,我們設(shè)計了雙功能熒光探針并用于實際樣品湖水中檢測銀離子和汞離子,檢測限分別達(dá)到10 nM和30 nM。進(jìn)一步地,本論文探討和闡明了相關(guān)熒光探針的傳感機(jī)理：銀離子增強金納米簇?zé)晒庵饕墙饘僭鰪姛晒?MEF)效應(yīng)即被還原后的銀單質(zhì)與Au NCs作用增強熒光；汞離子對金銀復(fù)合納米簇?zé)晒獾拟鐧C(jī)理是由于汞離子和銀離子之間高親和力的嗜金屬作用Hg2+-Ag~+。這種非標(biāo)記的、雙功能的熒光開關(guān)探針制備簡單、快速,在環(huán)境污染和健康監(jiān)測方面具有實際可行的應(yīng)用價值�；铙w成像需要熒光金屬納米簇具有長的激發(fā)和發(fā)射波長,以避免生物體自身熒光的干擾。因此,本論文利用一氧化碳還原的方法成功制備了谷胱甘肽穩(wěn)定的金納米簇,該納米簇易溶于水、熒光穩(wěn)定且紅光發(fā)射。相關(guān)形貌和光譜表征顯示金納米簇具有強烈的熒光(量子產(chǎn)率7.6%),水溶液中激發(fā)波長520 nm,發(fā)射波長640 nm,平均粒徑約為2nm�；谠摕晒饨鸺{米簇構(gòu)建了熒光生物傳感器。通過利用Fe3+引發(fā)的聚集熒光猝滅效應(yīng),實現(xiàn)了Fe3+的選擇性識別檢測,檢測限達(dá)0.3 μM,并成功實現(xiàn)對湖水、自來水中鐵離子的檢測。進(jìn)一步地,將該金納米簇用于細(xì)胞和活體熒光成像,不僅成功實現(xiàn)了活體腫瘤部位的快速近紅外(710nm)熒光成像,而且通過簡單自組裝的方法構(gòu)建了水溶性卟啉衍生物功能化的金納米簇復(fù)合體系,同時實現(xiàn)了腫瘤近紅外成像和光熱治療,有效地抑制了腫瘤的生長。光、電信號在生物醫(yī)學(xué)檢測中的地位不分伯仲。因此,本論文工作還研究和探討了基于石墨烯和鉑納米顆粒等光電材料自組裝修飾電極的生物電化學(xué)傳感器及其在生物醫(yī)學(xué)檢測中的應(yīng)用。論文構(gòu)建了一個基于石墨烯-鉑(RGO-Pt)納米復(fù)合物的過氧化氫(H_2O_2)傳感器成功用于檢測細(xì)胞釋放的H_2O_2。首先,通過物理吸附和電沉積的方法把石墨烯和鉑納米顆粒修飾到玻碳電極上,形成納米材料修飾的電化學(xué)傳感器。該傳感器對過氧化氫具有優(yōu)異的電催化還原作用,能夠快速增強響應(yīng)電流以及明顯降低H_2O_2的還原過電位,具有較低的檢測限(0.2μM)、較寬的線性范圍(0.5μM-3.475 mM)和較高的檢測靈敏度(459士3mA M-1 cm-2)。更重要的是,該過氧化氫傳感器能夠成功地用于高靈敏檢測細(xì)胞刺激下釋放的過氧化氫。綜上所述,本論文設(shè)計制備和組裝了新型納米材料,基于其優(yōu)異的性能,以熒光分析法和電化學(xué)法為研究手段,進(jìn)一步將新型納米材料用于生物醫(yī)學(xué)檢測。通過被測定物質(zhì)引起的金屬納米簇?zé)晒鈴姸然蛘唠娀瘜W(xué)傳感器響應(yīng)電流變化而實現(xiàn)定量分析測定,建立了基于光電信號的高靈敏檢測分析傳感平臺。金屬納米簇在生物體系的應(yīng)用優(yōu)勢成為研究生物大分子的構(gòu)型、生物分子之間的相互作用機(jī)制的有力工具,為細(xì)胞和活體標(biāo)記示蹤研究提供了新的思路和途徑,使得腫瘤等疾病治療更加精準(zhǔn)靶向。
[Abstract]:The new type of nano-material (metal nanoclusters, graphene, etc.) is widely used for biomedical detection because of its unique properties. The method is simple, rapid, easy to operate and sensitive based on the nano material, and has potential application prospect in the fields of chemical analysis, biological sensing, disease detection, environmental monitoring and the like. With the rapid development of the related subjects and the introduction of related new technologies, the sensitivity, accuracy and selectivity of photoelectricity (such as fluorescence and electrochemical) detection are increasing, and the application range is in many fields, and has been developed into a very important and effective chemical analysis method. In this paper, the fluorescent gold and silver nanoclusters and the related photoelectric chemical sensors are designed and prepared, and the small molecules and metal ions which are closely related to human health and safety are identified and detected by means of photoelectric means. In addition, in view of the excellent fluorescence characteristics and biocompatibility of the gold nanoclusters, a novel small-size fluorescent probe is used for cell in-vivo imaging and tumor treatment. The method comprises the following steps of: firstly, carrying out in-situ rapid synthesis of the fluorescence-enhanced and water-soluble gold-silver composite nano-cluster by using the cytoskeleton as a template, and carrying out ultraviolet-visible absorption spectrum, fluorescence spectrum, particle size appearance and fluorescence lifetime of the gold-silver composite nano-cluster; The fluorescence quantum yield and the valence state and content of the gold and silver elements were characterized. The results show that the gold and silver composite nanoclusters have strong yellow fluorescence (560nm emission) under the excitation of 370 nm, the particle size is about 1. 50nm, the quantum yield is about 9%, the average fluorescence lifetime is 6.07. m thereby laying a potential application value in the biological analysis. On this basis, we discussed the mechanism of the rapid and significant enhancement of the fluorescence of the gold nanoclusters, and the method of the rapid in-situ synthesis of the fluorescence-enhanced gold and silver complex nanoclusters was used to detect the disease-related molecules in the serum. The results of the study further show the potential for the application of gold and silver nanoclusters in biological analysis and diagnosis. On the basis of the above research work, a new method for rapidly and highly sensitive selective detection of silver ions and mercury ions with a non-labeled and double function was developed by using the excellent fluorescence characteristics of the gold nanoclusters. The results show that the silver ion can enhance the fluorescence of the gold nanoclusters rapidly and form the gold and silver composite nanoclusters with strong yellow fluorescence, and the mercury ions can rapidly and rapidly destroy the fluorescence of the enhanced gold and silver nano-clusters. As a result, we designed a dual-function fluorescent probe and used to detect silver ions and mercury ions in the actual sample lake, with a detection limit of 10 nM and 30 nM, respectively. Further, this paper discusses and expounds the sensing mechanism of the relevant fluorescent probe: the silver ion-enhanced gold nano-cluster fluorescence is mainly the metal-enhanced fluorescence (MEF) effect, that is, the silver simple substance after being reduced and the effect of the Au NCs enhance the fluorescence; The effect of mercury ion on the fluorescence of gold and silver composite nanoclusters is due to the high affinity of Hg 2 +-Ag ~ + between the mercury ions and the silver ions. The non-labeled and double-function fluorescent switch probe has the advantages of simple and rapid preparation, and practical application value in the aspects of environmental pollution and health monitoring. In-vivo imaging requires that the fluorescent metal nanoclusters have a long excitation and emission wavelength to avoid the interference of the organism's own fluorescence. In this paper, a method of carbon monoxide reduction was used to successfully prepare the glutathione-stabilized gold nanoclusters, which were soluble in water, with stable fluorescence and red light emission. The related morphology and spectral characterization show that the gold nanoclusters have a strong fluorescence (70.6% quantum yield), the excitation wavelength in the aqueous solution is 520 nm, the emission wavelength is 640 nm, and the average particle size is about 2nm. a fluorescence biosensor is built on the basis of the fluorescent gold nanoclusters. The detection of Fe ~ (3 +) in water and tap water was successfully realized by using the effect of the aggregation and fluorescence induced by Fe3 +. The detection limit of Fe ~ (3 +) was detected. The detection limit of Fe ~ (3 +) was 0.3. m Further, the gold nanoclusters are used for cell and in-vivo fluorescence imaging, not only the rapid near-infrared (710nm) fluorescence imaging of the in-vivo tumor site is successfully realized, at the same time, the near-infrared imaging and photothermal treatment of the tumor are realized, and the growth of the tumor is effectively inhibited. The position of the optical and electrical signals in the biomedical detection is not the same. Therefore, this paper also studies and discusses the bio-electrochemical sensor based on the self-assembly modified electrode of the photoelectric material such as the graphene and the platinum nano-particles and the application of the biological electrochemical sensor in the biomedical detection. In this paper, a hydrogen peroxide (H _ 2O _ 2) sensor based on the nano-composite of graphene-platinum (RGO-Pt) was successfully used to detect the H _ 2O _ 2 released by the cells. firstly, the graphene and the platinum nano-particles are modified on the glass-carbon electrode by a method of physical adsorption and electro-deposition to form a nano-material-modified electrochemical sensor. The sensor has excellent electrocatalytic reduction effect on hydrogen peroxide, can rapidly enhance the response current and obviously reduce the reduction potential of the H _ 2O _ 2, has a lower detection limit (0.2. mu.M), a wide linear range (0.5. mu.M-3.475 mM) and a higher detection sensitivity (459 + 3mA M-1 cm-2). More importantly, the hydrogen peroxide sensor can be used successfully for highly sensitive detection of hydrogen peroxide released under cell stimulation. To sum up, a new type of nano-material is designed and assembled in this paper. Based on its excellent performance, the new type of nano-material is used for biomedical detection by using fluorescence analysis method and electrochemical method as the research means. and a high-sensitivity detection and analysis sensing platform based on a photoelectric signal is established by carrying out quantitative analysis and measurement on the fluorescence intensity of the metal nano cluster or the change of the response current of the electrochemical sensor caused by the measured substance. The application of the metal nanoclusters in the biological system has become a powerful tool for studying the configuration of the biological macromolecule and the interaction mechanism between the biological molecules, and provides a new thought and a way for the cell and the in-vivo marker tracing research, so that the treatment of the disease such as the tumor is more accurate and targeted.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R318;TB383.1
，
本文編號：2327046
[Abstract]:The new type of nano-material (metal nanoclusters, graphene, etc.) is widely used for biomedical detection because of its unique properties. The method is simple, rapid, easy to operate and sensitive based on the nano material, and has potential application prospect in the fields of chemical analysis, biological sensing, disease detection, environmental monitoring and the like. With the rapid development of the related subjects and the introduction of related new technologies, the sensitivity, accuracy and selectivity of photoelectricity (such as fluorescence and electrochemical) detection are increasing, and the application range is in many fields, and has been developed into a very important and effective chemical analysis method. In this paper, the fluorescent gold and silver nanoclusters and the related photoelectric chemical sensors are designed and prepared, and the small molecules and metal ions which are closely related to human health and safety are identified and detected by means of photoelectric means. In addition, in view of the excellent fluorescence characteristics and biocompatibility of the gold nanoclusters, a novel small-size fluorescent probe is used for cell in-vivo imaging and tumor treatment. The method comprises the following steps of: firstly, carrying out in-situ rapid synthesis of the fluorescence-enhanced and water-soluble gold-silver composite nano-cluster by using the cytoskeleton as a template, and carrying out ultraviolet-visible absorption spectrum, fluorescence spectrum, particle size appearance and fluorescence lifetime of the gold-silver composite nano-cluster; The fluorescence quantum yield and the valence state and content of the gold and silver elements were characterized. The results show that the gold and silver composite nanoclusters have strong yellow fluorescence (560nm emission) under the excitation of 370 nm, the particle size is about 1. 50nm, the quantum yield is about 9%, the average fluorescence lifetime is 6.07. m thereby laying a potential application value in the biological analysis. On this basis, we discussed the mechanism of the rapid and significant enhancement of the fluorescence of the gold nanoclusters, and the method of the rapid in-situ synthesis of the fluorescence-enhanced gold and silver complex nanoclusters was used to detect the disease-related molecules in the serum. The results of the study further show the potential for the application of gold and silver nanoclusters in biological analysis and diagnosis. On the basis of the above research work, a new method for rapidly and highly sensitive selective detection of silver ions and mercury ions with a non-labeled and double function was developed by using the excellent fluorescence characteristics of the gold nanoclusters. The results show that the silver ion can enhance the fluorescence of the gold nanoclusters rapidly and form the gold and silver composite nanoclusters with strong yellow fluorescence, and the mercury ions can rapidly and rapidly destroy the fluorescence of the enhanced gold and silver nano-clusters. As a result, we designed a dual-function fluorescent probe and used to detect silver ions and mercury ions in the actual sample lake, with a detection limit of 10 nM and 30 nM, respectively. Further, this paper discusses and expounds the sensing mechanism of the relevant fluorescent probe: the silver ion-enhanced gold nano-cluster fluorescence is mainly the metal-enhanced fluorescence (MEF) effect, that is, the silver simple substance after being reduced and the effect of the Au NCs enhance the fluorescence; The effect of mercury ion on the fluorescence of gold and silver composite nanoclusters is due to the high affinity of Hg 2 +-Ag ~ + between the mercury ions and the silver ions. The non-labeled and double-function fluorescent switch probe has the advantages of simple and rapid preparation, and practical application value in the aspects of environmental pollution and health monitoring. In-vivo imaging requires that the fluorescent metal nanoclusters have a long excitation and emission wavelength to avoid the interference of the organism's own fluorescence. In this paper, a method of carbon monoxide reduction was used to successfully prepare the glutathione-stabilized gold nanoclusters, which were soluble in water, with stable fluorescence and red light emission. The related morphology and spectral characterization show that the gold nanoclusters have a strong fluorescence (70.6% quantum yield), the excitation wavelength in the aqueous solution is 520 nm, the emission wavelength is 640 nm, and the average particle size is about 2nm. a fluorescence biosensor is built on the basis of the fluorescent gold nanoclusters. The detection of Fe ~ (3 +) in water and tap water was successfully realized by using the effect of the aggregation and fluorescence induced by Fe3 +. The detection limit of Fe ~ (3 +) was detected. The detection limit of Fe ~ (3 +) was 0.3. m Further, the gold nanoclusters are used for cell and in-vivo fluorescence imaging, not only the rapid near-infrared (710nm) fluorescence imaging of the in-vivo tumor site is successfully realized, at the same time, the near-infrared imaging and photothermal treatment of the tumor are realized, and the growth of the tumor is effectively inhibited. The position of the optical and electrical signals in the biomedical detection is not the same. Therefore, this paper also studies and discusses the bio-electrochemical sensor based on the self-assembly modified electrode of the photoelectric material such as the graphene and the platinum nano-particles and the application of the biological electrochemical sensor in the biomedical detection. In this paper, a hydrogen peroxide (H _ 2O _ 2) sensor based on the nano-composite of graphene-platinum (RGO-Pt) was successfully used to detect the H _ 2O _ 2 released by the cells. firstly, the graphene and the platinum nano-particles are modified on the glass-carbon electrode by a method of physical adsorption and electro-deposition to form a nano-material-modified electrochemical sensor. The sensor has excellent electrocatalytic reduction effect on hydrogen peroxide, can rapidly enhance the response current and obviously reduce the reduction potential of the H _ 2O _ 2, has a lower detection limit (0.2. mu.M), a wide linear range (0.5. mu.M-3.475 mM) and a higher detection sensitivity (459 + 3mA M-1 cm-2). More importantly, the hydrogen peroxide sensor can be used successfully for highly sensitive detection of hydrogen peroxide released under cell stimulation. To sum up, a new type of nano-material is designed and assembled in this paper. Based on its excellent performance, the new type of nano-material is used for biomedical detection by using fluorescence analysis method and electrochemical method as the research means. and a high-sensitivity detection and analysis sensing platform based on a photoelectric signal is established by carrying out quantitative analysis and measurement on the fluorescence intensity of the metal nano cluster or the change of the response current of the electrochemical sensor caused by the measured substance. The application of the metal nanoclusters in the biological system has become a powerful tool for studying the configuration of the biological macromolecule and the interaction mechanism between the biological molecules, and provides a new thought and a way for the cell and the in-vivo marker tracing research, so that the treatment of the disease such as the tumor is more accurate and targeted.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R318;TB383.1
，
本文編號：2327046
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