新型仿生纳米材料的制备及其在生物检测中的应用研究
发布时间：2018-11-12 11:59
【摘要】：新型纳米材料(金属纳米簇、石墨烯等)由于其独特的性质受到研究者的青睐,并广泛地用于生物医学检测。基于纳米材料的分析方法简便、快捷、易操作、灵敏,在化学分析、生物传感、疾病检测、环境监测等领域具有潜在的应用前景。随着相关学科的迅速发展以及相关新技术的引入,光电(如荧光和电化学)检测的灵敏度、准确度和选择性日益提高,应用范围遍及众多领域,已经发展成为一种十分重要且有效的化学分析手段。本论文设计和制备了荧光金银纳米簇以及相关的光电化学传感器,通过光电手段对人类健康和安全密切相关的小分子和金属离子进行识别检测。另外,鉴于金纳米簇优良的荧光特性和生物相容性,作为一类新型的小尺寸的荧光探针应用于细胞活体成像以及肿瘤治疗。具体工作开展如下：首先,创新性地以胞嘧啶核苷为模板原位快速合成荧光增强、水溶性的金银复合纳米簇,并对其紫外-可见吸收光谱、荧光光谱、粒径形貌、荧光寿命、荧光量子产率以及金元素和银元素的价态和含量进行了表征。研究结果表明,该金银复合纳米簇在370 nm激发下具有强烈的黄色荧光(560nm发射),粒径大约为1.50nm,量子产率约9%,平均荧光寿命6.07μs,并且具有良好的荧光稳定性、分散性以及水溶性,由此奠定了其在生物分析中的潜在应用价值。在此基础上,我们对银离子快速显著增强金纳米簇荧光的机理进行了探讨,并把这种快速原位合成荧光增强的金银复合纳米簇的方法用于血清中疾病相关分子的检测。相关研究结果进一步显示了金银纳米簇在生物分析和诊断中应用的潜在可能性。在上述研究工作的基础上,本论文利用金纳米簇优良的荧光特性发展了一种非标记的、双功能的快速高灵敏选择性检测银离子和汞离子的新方法。研究结果表明,银离子能够快速显著地增强金纳米簇的荧光并形成具有强烈黄色荧光的金银复合纳米簇,而汞离子可以迅速猝灭增强后的金银纳米簇的荧光。据此,我们设计了双功能荧光探针并用于实际样品湖水中检测银离子和汞离子,检测限分别达到10 nM和30 nM。进一步地,本论文探讨和阐明了相关荧光探针的传感机理：银离子增强金纳米簇荧光主要是金属增强荧光(MEF)效应即被还原后的银单质与Au NCs作用增强荧光；汞离子对金银复合纳米簇荧光的猝灭机理是由于汞离子和银离子之间高亲和力的嗜金属作用Hg2+-Ag~+。这种非标记的、双功能的荧光开关探针制备简单、快速,在环境污染和健康监测方面具有实际可行的应用价值。活体成像需要荧光金属纳米簇具有长的激发和发射波长,以避免生物体自身荧光的干扰。因此,本论文利用一氧化碳还原的方法成功制备了谷胱甘肽稳定的金纳米簇,该纳米簇易溶于水、荧光稳定且红光发射。相关形貌和光谱表征显示金纳米簇具有强烈的荧光(量子产率7.6%),水溶液中激发波长520 nm,发射波长640 nm,平均粒径约为2nm。基于该荧光金纳米簇构建了荧光生物传感器。通过利用Fe3+引发的聚集荧光猝灭效应,实现了Fe3+的选择性识别检测,检测限达0.3 μM,并成功实现对湖水、自来水中铁离子的检测。进一步地,将该金纳米簇用于细胞和活体荧光成像,不仅成功实现了活体肿瘤部位的快速近红外(710nm)荧光成像,而且通过简单自组装的方法构建了水溶性卟啉衍生物功能化的金纳米簇复合体系,同时实现了肿瘤近红外成像和光热治疗,有效地抑制了肿瘤的生长。光、电信号在生物医学检测中的地位不分伯仲。因此,本论文工作还研究和探讨了基于石墨烯和铂纳米颗粒等光电材料自组装修饰电极的生物电化学传感器及其在生物医学检测中的应用。论文构建了一个基于石墨烯-铂(RGO-Pt)纳米复合物的过氧化氢(H_2O_2)传感器成功用于检测细胞释放的H_2O_2。首先,通过物理吸附和电沉积的方法把石墨烯和铂纳米颗粒修饰到玻碳电极上,形成纳米材料修饰的电化学传感器。该传感器对过氧化氢具有优异的电催化还原作用,能够快速增强响应电流以及明显降低H_2O_2的还原过电位,具有较低的检测限(0.2μM)、较宽的线性范围(0.5μM-3.475 mM)和较高的检测灵敏度(459士3mA M-1 cm-2)。更重要的是,该过氧化氢传感器能够成功地用于高灵敏检测细胞刺激下释放的过氧化氢。综上所述,本论文设计制备和组装了新型纳米材料,基于其优异的性能,以荧光分析法和电化学法为研究手段,进一步将新型纳米材料用于生物医学检测。通过被测定物质引起的金属纳米簇荧光强度或者电化学传感器响应电流变化而实现定量分析测定,建立了基于光电信号的高灵敏检测分析传感平台。金属纳米簇在生物体系的应用优势成为研究生物大分子的构型、生物分子之间的相互作用机制的有力工具,为细胞和活体标记示踪研究提供了新的思路和途径,使得肿瘤等疾病治疗更加精准靶向。
[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.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318;TB383.1
，
本文编号：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.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318;TB383.1
，
本文编号：2327046
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