基于纳米粒子的荧光传感新体系研究
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摘要
纳米粒子是指在三维尺度上至少有一维处于纳米尺度范围(1～100nm)的物质,具有独特的化学性质和物理性质,呈现出常规材料不具备的优越性,是当前科技领域的研究热点。纳米粒子在生物技术、环境检测、纳米光学、纳米电子学、医药、陶瓷、磁性材料和防护材料等诸多领域有极好的应用潜力。
本文研究了纳米粒子(纳米金、碳点)在检测生物小分子如氨基酸、蛋白质和金属离子中的作用。纳米金(gold nanoparticles,AuNPs)是一种重要的纳米材料,具有独特的电学、光学性质及良好的生物亲和性,是荧光团的超强猝灭剂。碳点(Carbon Dots,CDs)是继量子点之后的新型碳质纳米材料,因其生物相容性好、毒性低的特点,特别适合生物体系分析。
本文以纳米金和碳点为研究对象,主要研究内容如下：
(1)本文基于荧光内滤效应,建立了一种使用纳米金高灵敏测定生物氨基酸的方法。半胱氨酸在许多生化过程中起着至关重要的作用,造血功能不足、白细胞减少及牛皮癣等疾病都和体内半胱氨酸的缺乏有关,因此半胱氨酸的高灵敏检测具有非常重要的意义。荧光素的荧光光谱与纳米金的吸收光谱部分重叠,以纳米金做为吸收体,荧光素做为发光体,构造了荧光传感体系。纳米金可以猝灭荧光素的荧光,而半胱氨酸的巯基能与纳米金形成Au-S键导致纳米金聚集及其吸光度减弱,从而使荧光素由于内滤光效应而减弱的荧光恢复。在选定的实验条件下,荧光素的荧光强度的增加值与半胱氨酸的浓度在5.0×10-8～1.0×10-6mol／L范围内呈良好的线性关系,检测限为1×10-8mol／L。
(2)阿尔茨海默病(alzheimer's disease, AD),又称老年痴呆症,是一种中枢神经退行性疾病,严重影响着老年人的身体健康和生活质量。β-淀粉样蛋白(βAmyloid,AP)是AD的病理学特征。因此Aβ的高灵敏检测对AD的早期诊断具有十分重要的意义。利用Aβ的不同聚集态对胰蛋白酶的不同作用,建立了一种监测Aβ1-42聚集过程的方法。分散态的Aβ可以使trypsin的活性保持,而纤维化的Ap (fAβ)与trypsin形成trypsin/fAβ配合物,抑制trypsin的活性。通过纳米金和Arg6体系来检测trypsin的活性,从而监测到Ap的聚集状态。
(3)利用荧光碳点与硫黄素T(Thioflavin T,ThT)之间的内滤光效应测定Cu2+。细胞内的Cu2+在生理学和病理学方面起非常重要的作用,体内Cu2+浓度的变化会导致一些严重的神经变性疾病。利用荧光CDs的荧光谱与ThT的激发和发射谱存在较大重叠的性质,构建了CDs/ThT内滤光体系。ThT会吸收CDs发出的荧光从而使ThT的荧光增加,而Cu2+是一种猝灭剂可以使CDs/ThT的荧光猝灭,并且ThT的荧光强度与Cu2+的浓度呈良好的线性关系。从而确立了一种测定Cu2+的新方法,且在Cu2+测定方面有很好的应用前景。
Nanoparticles, defined as a substance with the scale between1-100nm at least in one-dimensional of three-dimensional space, with a unique chemical and physical properties,showing the advantages that the conventional materials doesn't have, is the hot point of the current scientific and technological fields.Nanoparticles has excellent potential application in biotechnology, environmental testing, nanochemistry, nanoelectronics, medicine ceramics,magnetic materials,protective materials, and many other fields.
In this thesis,the role of Nanoparticles (gold nanoparticles, carbon dots) in the detection of small biological molecules such as amino acids, proteins and metal ions is researched.Gold nanoparticles,an important nanomaterials with unique electrical, optical properties and good biocompatibility, is the super quencher of the fluorophore. Carbon dots.the new carbonaceous nanoparticles following quantum dots,is particularly suitable for biological systems analysis because of its good biocompatibility and low toxicity.
In this paper, Gold nanoparticles and carbon dots are studied, the main contents are as follows:
(1)This article establish a highly sensitive method for the determination of biological amino acids based on the fluorescence inner filter effect of gold nanoparticles.Cysteine plays a vital role in many biochemical processes.Lack of cysteine in vivo will lead to the disease of Hematopoietic insufficiency,leukopenia, psoriasis and so on,hence highly sensitive detection of cysteine is very important.The fluorescence spectrum of fluorescein and the absorption spectrum of gold nanoparticles can be partially overlapped.Constructed a fluorescent sensing system with gold nanoparticles as the absorbent and fluorescein as the fluorophore. Gold nanoparticles could quench the fluorescence of fluorescein,while the sulfhydryl of cysteine formate the Au-S key with gold nanoparticles,thus cause the aggregation of gold nanoparticle with its reduced absorbance,finally fluorescence of fluorescein recovered.Under the selected experimental conditions,the enhancemant value of the fluorescence intensity of fluorescein showed a good linear relationship with the concentration of cystein in the range of5.0×l0-8-1.0×l0-6mol/L,and the detection limit is1×10-8mol/L.
(2)Alzheimer's disease(AD),is a degenerative disease of the central nervous,which seriously affect the health and quality of life of the elderly.β-amyloid (Aβ) is the pathological feature of AD,therefore highly sensitive detection of Aβ has a very important meaning for the early diagnosis of AD. The different aggregation state of Aβ have different effect for trypsin, thus could be used to monitor the aggregation process of Aβ. Soluble Aβ can maintain the activity of trypsin,while fiber Aβ (fAβ) can form the trypsin/fAβ complex with trypsin and inhibit its activity.Thus aggregation state of Aβ can be monitored by detecting the activity of trypsin in the gold nanoparticles and Arg6system.
(3)Build the inner filter effect system between fluorescent carbon dots(CDs) and thioflavine T (ThT) for the determination of Cu2+.The Cu2+in cells play an important role in the physiology and pathology,and the change of the concentration of Cu2+in vivo will cause some serious neurodegenerative diseases.There is a big overlap between the fluorescence spectrum of CDs and excitation and emission spectrum of ThT, which could be used to build a CDs/ThT inner filter system for the detection of Cu2+.ThT will absorb the fluorescence emitted by CDs and the fluorescence intensity of ThT increase, and that Cu2+is a quencher can quench the fluorescence of CDs/ThT system,and there is a good linear relationship between fluorescence intensity of ThT and the concentration of Cu2+.Thus a new method for the detection of Cu2+was established and has good application prospects in the detection of Cu2+.
本文研究了纳米粒子(纳米金、碳点)在检测生物小分子如氨基酸、蛋白质和金属离子中的作用。纳米金(gold nanoparticles,AuNPs)是一种重要的纳米材料,具有独特的电学、光学性质及良好的生物亲和性,是荧光团的超强猝灭剂。碳点(Carbon Dots,CDs)是继量子点之后的新型碳质纳米材料,因其生物相容性好、毒性低的特点,特别适合生物体系分析。
本文以纳米金和碳点为研究对象,主要研究内容如下：
(1)本文基于荧光内滤效应,建立了一种使用纳米金高灵敏测定生物氨基酸的方法。半胱氨酸在许多生化过程中起着至关重要的作用,造血功能不足、白细胞减少及牛皮癣等疾病都和体内半胱氨酸的缺乏有关,因此半胱氨酸的高灵敏检测具有非常重要的意义。荧光素的荧光光谱与纳米金的吸收光谱部分重叠,以纳米金做为吸收体,荧光素做为发光体,构造了荧光传感体系。纳米金可以猝灭荧光素的荧光,而半胱氨酸的巯基能与纳米金形成Au-S键导致纳米金聚集及其吸光度减弱,从而使荧光素由于内滤光效应而减弱的荧光恢复。在选定的实验条件下,荧光素的荧光强度的增加值与半胱氨酸的浓度在5.0×10-8～1.0×10-6mol／L范围内呈良好的线性关系,检测限为1×10-8mol／L。
(2)阿尔茨海默病(alzheimer's disease, AD),又称老年痴呆症,是一种中枢神经退行性疾病,严重影响着老年人的身体健康和生活质量。β-淀粉样蛋白(βAmyloid,AP)是AD的病理学特征。因此Aβ的高灵敏检测对AD的早期诊断具有十分重要的意义。利用Aβ的不同聚集态对胰蛋白酶的不同作用,建立了一种监测Aβ1-42聚集过程的方法。分散态的Aβ可以使trypsin的活性保持,而纤维化的Ap (fAβ)与trypsin形成trypsin/fAβ配合物,抑制trypsin的活性。通过纳米金和Arg6体系来检测trypsin的活性,从而监测到Ap的聚集状态。
(3)利用荧光碳点与硫黄素T(Thioflavin T,ThT)之间的内滤光效应测定Cu2+。细胞内的Cu2+在生理学和病理学方面起非常重要的作用,体内Cu2+浓度的变化会导致一些严重的神经变性疾病。利用荧光CDs的荧光谱与ThT的激发和发射谱存在较大重叠的性质,构建了CDs/ThT内滤光体系。ThT会吸收CDs发出的荧光从而使ThT的荧光增加,而Cu2+是一种猝灭剂可以使CDs/ThT的荧光猝灭,并且ThT的荧光强度与Cu2+的浓度呈良好的线性关系。从而确立了一种测定Cu2+的新方法,且在Cu2+测定方面有很好的应用前景。
Nanoparticles, defined as a substance with the scale between1-100nm at least in one-dimensional of three-dimensional space, with a unique chemical and physical properties,showing the advantages that the conventional materials doesn't have, is the hot point of the current scientific and technological fields.Nanoparticles has excellent potential application in biotechnology, environmental testing, nanochemistry, nanoelectronics, medicine ceramics,magnetic materials,protective materials, and many other fields.
In this thesis,the role of Nanoparticles (gold nanoparticles, carbon dots) in the detection of small biological molecules such as amino acids, proteins and metal ions is researched.Gold nanoparticles,an important nanomaterials with unique electrical, optical properties and good biocompatibility, is the super quencher of the fluorophore. Carbon dots.the new carbonaceous nanoparticles following quantum dots,is particularly suitable for biological systems analysis because of its good biocompatibility and low toxicity.
In this paper, Gold nanoparticles and carbon dots are studied, the main contents are as follows:
(1)This article establish a highly sensitive method for the determination of biological amino acids based on the fluorescence inner filter effect of gold nanoparticles.Cysteine plays a vital role in many biochemical processes.Lack of cysteine in vivo will lead to the disease of Hematopoietic insufficiency,leukopenia, psoriasis and so on,hence highly sensitive detection of cysteine is very important.The fluorescence spectrum of fluorescein and the absorption spectrum of gold nanoparticles can be partially overlapped.Constructed a fluorescent sensing system with gold nanoparticles as the absorbent and fluorescein as the fluorophore. Gold nanoparticles could quench the fluorescence of fluorescein,while the sulfhydryl of cysteine formate the Au-S key with gold nanoparticles,thus cause the aggregation of gold nanoparticle with its reduced absorbance,finally fluorescence of fluorescein recovered.Under the selected experimental conditions,the enhancemant value of the fluorescence intensity of fluorescein showed a good linear relationship with the concentration of cystein in the range of5.0×l0-8-1.0×l0-6mol/L,and the detection limit is1×10-8mol/L.
(2)Alzheimer's disease(AD),is a degenerative disease of the central nervous,which seriously affect the health and quality of life of the elderly.β-amyloid (Aβ) is the pathological feature of AD,therefore highly sensitive detection of Aβ has a very important meaning for the early diagnosis of AD. The different aggregation state of Aβ have different effect for trypsin, thus could be used to monitor the aggregation process of Aβ. Soluble Aβ can maintain the activity of trypsin,while fiber Aβ (fAβ) can form the trypsin/fAβ complex with trypsin and inhibit its activity.Thus aggregation state of Aβ can be monitored by detecting the activity of trypsin in the gold nanoparticles and Arg6system.
(3)Build the inner filter effect system between fluorescent carbon dots(CDs) and thioflavine T (ThT) for the determination of Cu2+.The Cu2+in cells play an important role in the physiology and pathology,and the change of the concentration of Cu2+in vivo will cause some serious neurodegenerative diseases.There is a big overlap between the fluorescence spectrum of CDs and excitation and emission spectrum of ThT, which could be used to build a CDs/ThT inner filter system for the detection of Cu2+.ThT will absorb the fluorescence emitted by CDs and the fluorescence intensity of ThT increase, and that Cu2+is a quencher can quench the fluorescence of CDs/ThT system,and there is a good linear relationship between fluorescence intensity of ThT and the concentration of Cu2+.Thus a new method for the detection of Cu2+was established and has good application prospects in the detection of Cu2+.
引文
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