节点文献
蛋白质—金属纳米粒子体系荧光增强效应及其分析应用
The Fluoresence Enhancement Effect of Protein-Metal Nanoparticle and the Analytical Application
【作者】 高希宝；
【导师】 杨景和；
【作者基本信息】 山东大学 ， 分析化学， 2007， 博士
【摘要】 近年来，随着纳米科技的兴起，金属纳米粒子以其独特的光学和电学性质、良好的稳定性、小尺寸和表面效应以及独特的生物亲和性，使其在医药、卫生分析以及生化免疫等领域显示了潜在的价值，引起广大科技工作者的兴趣。金属纳米粒子独特的表面效应是其具有优良性能以及与其他材料复合时表现出来的独特性能的关键。金属纳米微粒的粒径、形状以及排列情况与其紫外一可见吸收光谱、表面增强拉曼散射（SERS）光谱、共振散射光谱以及荧光光谱之间有强烈的依赖关系。金属纳米颗粒与荧光分子直接结合或经修饰后连接，可以改变荧光体系的紫外-可见吸收光谱、增强表面拉曼散射光谱和共振散射光谱，对荧光光谱的影响随金属纳米颗粒的种类以及荧光分子的种类不同可产生猝灭作用也可产生增强作用。本论文以分析化学、生物化学以及材料化学为研究背景，结合纳米科学技术手段，并利用荧光光谱、吸收光谱、光散射光谱、园二色谱、透射电子显微镜和高分辨透射电子显微镜、荧光寿命以及Zeta电位等测定技术，研究了各种蛋白质对各种金属纳米荧光体系的荧光增强作用，探讨了蛋白质与金属纳米粒子结合及其荧光增强作用的机理，建立了利用金属纳米粒子作为荧光探针来测定微量蛋白质的分析方法。论文的第一章阐述了金属纳米颗粒的制备方法、金属纳米颗粒的应用、研究进展以及发展趋势。共引用文献191篇。论文的第二章研究了蛋白质对金纳米颗粒近红外荧光的增强效应及其分析应用。利用液相还原法制备了不同大小的金纳米颗粒。吸收光谱研究指出，大颗粒胶体金只在250nm处有吸收，随胶体金粒径减小至21nm，在525nm处出现新的吸收峰，且其强度随纳米颗粒的减小而增强，并伴有吸收峰的兰移。研究发现，15nm的金纳米颗粒能够发射近红外荧光，其激发和发射峰分别为538nm和811.2nm。同时还发现，蛋白质能够明显增强金纳米近红外荧光强度，并研究了影响荧光增强效应的各种因素。实验指出，在最佳实验条件下（即15nm的纳米金颗粒，pH7.0时），蛋白质浓度在一定范围内与体系的荧光强度呈线性关系，P450、BSA、HRP、和HSA的线性范围分别是2.3×10-7-1.0×10-5mol／L、2.0×10-7-1.5×10-5mol／L、1.5×10-7-1.5×10-5mol／L和1.5×10-7-1.5×10-5mol／L，它们的检出限分别达到2.4×10-8mol／L、2.2×10-8mol／L、2.0×10-8mol／L和2.0×10-8mol／L，可见该方法具有较高的灵敏度。该方法已用于实际样品的分析，其结果令人满意。本文以BSA蛋白质金纳米荧光体系为代表，利用Zeta电位、荧光寿命、TEM电镜、吸收光谱、共振散射光谱、园二色谱以及荧光光谱等技术，研究了体系中蛋白质与金纳米颗粒间的相互作用和蛋白质对金纳米颗粒近红外荧光的增强机理。研究表明，金纳米颗粒表面荷负电，能与蛋白质结合。认为胶体金以蛋白质为模板能够在其表面发生聚集，金纳米体系按一定的规律定向排列，纳米粒子聚集后间距减小，导致表面等离子体传播特性的改变以及局域表面等离子体模式和表面等离子体模式的相互作用，这种相互作用受到外围环境的介电特性的影响。同时，金属粒子所产生的等离子体可以增强其表面周围环境的电场，这种增强的电场可以和周围环境发生相互作用，其表现为如吸收光谱兰移等光谱特性的改变。这可能是使整个金标记蛋白体系荧光强度增强的部分原因。而蛋白质为金纳米颗粒提供的疏水性环境是荧光体系荧光增强的另一原因。论文的第三章研究了铕纳米颗粒的制备、光谱性质和蛋白质对其荧光的增强效应及其分析应用。利用单宁酸做还原剂，首次将金属铕从它的硝酸盐中还原出来，制成金属铕纳米颗粒。以硫辛酸做修饰剂，修饰铕纳米颗粒，并与蛋白质结合。比较修饰、结合蛋白质前后纳米颗粒的光学性质的变化。研究指出，通过还原剂的不同用量可以控制生成的铕纳米颗粒的大小，随着还原剂单宁酸用量的逐渐减少，生成的铕纳米颗粒直径不断增大。各种粒径的铕纳米颗粒都在275nm处有最大吸收，且随着铕纳米颗粒直径的增大，吸收峰的位置没有变化而吸收峰的强度逐渐增强。研究发现，铕纳米颗粒能够发射紫外荧光，其激发和发射峰分别在275nm和380nm左右；随着纳米颗粒的增大，其荧光强度逐渐增强而发射峰逐渐兰移。铕纳米颗粒这种荧光性质的尺寸效应明显不同于贵金属纳米的荧光尺寸效应（随着纳米颗粒的增大，荧光峰红移）。铕纳米颗粒经硫辛酸修饰后荧光强度有所降低，但与蛋白质结合后荧光强度明显增强，且发射峰位置兰移。同时，研究了影响体系荧光强度的各种影响因素。在最佳条件下，即20nm铕颗粒经硫辛酸修饰后，在pH6.0的磷酸盐溶液中和SDBS存在下，体系荧光强度的增加与蛋白质浓度在一定范围内呈线性关系，BSA、HRP、P450、OMP以及NSE的线性范围分别为6.0×10<sup>-8-1.2×10-5g／ml、2.0×10-8-1.5×10-5g／ml、6.0×10-8-1.4×10-5g／ml、2.0×10-8-1.8×10-5g／ml和3.0×10-8-1.2×10-5g／ml。它们的检出限分别为3.2×10-8g／ml、1.0×10-8g／ml、2.9×10-8g／ml、9.8×10-9g／ml和1.2×10-8g／ml。可见该方法有较高的灵敏度和较宽的线性范围。该方法已用于实际样品分析，结果令人满意。该文还研究了蛋白质与铕纳米颗粒间的相互作用，并探讨了蛋白质对铕纳米荧光增强的机理。研究表明，铕纳米颗粒通过硫辛酸与蛋白质结合，并发生了能量转移，即BSA将吸收的能量通过分子间能量转移的形式转移给铕纳米颗粒，从而使铕纳米颗粒的特征荧光强度增强。SDBS的存在使体系的荧光强度进一步增强，一方面是由于SDBS也将吸收的能量传递给Eu-lipoic acid-BSA体系，使得Eu-lipoic acid-BSA体系荧光增强；另一方面，SDBS和蛋白质为体系所提供的疏水环境能够减少络合物和水分子之间的碰撞，从而减少因碰撞而导致体系的能量损失。因此，体系的荧光量子产率提高，体系的荧光强度明显增强。论文的第四章研究了铽纳米颗粒的制备、光谱性质和蛋白质对其荧光的增强效应及其分析应用。利用单宁酸做还原剂，首次将金属铽从它的硝酸盐中还原出来，制成金属铽纳米颗粒。以巯基丙酸做修饰剂，修饰铽纳米颗粒，并与蛋白质结合。比较修饰、结合蛋白质前后纳米颗粒的光学性质的变化。研究表明，通过还原剂的不同用量可以控制生成的铽纳米颗粒的大小，随着还原剂单宁酸用量的逐渐增加，生成的铽纳米颗粒直径不断减小。各种粒径的铽纳米颗粒都在275nm处有最大吸收，且随着铽纳米颗粒直径的增大，吸收峰的位置没有明显变化而吸收峰的强度逐渐增强。研究指出，铽纳米颗粒能够发射紫外荧光，其激发和发射峰分别位于256nm和388nm左右；且随着纳米颗粒变小，荧光强度逐渐变弱，但发射峰位置没有变化。以巯基丙酸做修饰剂，修饰铽纳米颗粒，可与蛋白质结合，且结合后体系的荧光强度明显增强，并伴随发射峰位置兰移。同时，研究了影响体系荧光强度的各种影响因素。在最佳实验条件下(即20nm铽纳米颗粒，pH6.8的磷酸盐缓冲溶液，CTAB浓度为5.0×10-5mol／L时)，体系荧光强度的增加与蛋白质浓度在一定范围内呈线性关系，BSA、HRP、P450、OMP以及NSE的线性范围分别为8.0×10-8-1.0×10-5g／ml、3.0×10-8-8.0×10-5g／ml、5.0×10-8-1.2×10-5g／ml、3.0×10-8-9.0×10-5g／ml和4.0×10-8-1.1×10-5g／ml。它们的检出限分别为3.4×10-8g／ml、2.1×10-8g／ml、1.9×10-8g／ml、8.9×10-9g／ml和1.1×10-8g／ml。可见该方法有较高的灵敏度和较宽的线性范围。该方法已用于实际样品分析，结果令人满意。本文还以BSA蛋白质铽纳米荧光体系为代表，研究了蛋白质与铽纳米颗粒间的相互作用，并探讨了蛋白质增强铽纳米荧光荧光强度的机理。研究认为，铽纳米颗粒通过巯基丙酸与蛋白质结合，并发生了能量转移，即BSA将吸收的能量通过分子间能量转移的形式传递给铽，从而使铽的特征荧光强度增强。CTAB的加入使体系的荧光强度增强，一方面是由于CTAB也能将能量传递给铽纳米荧光体系，使得Tb-mercaptopropionic acid-BSA体系荧光增强；另一方面，CTAB和蛋白质为体系所提供的疏水环境能够减少络合物和水分子之间的碰撞，从而减少因碰撞而导致体系的能量损失。因此，体系的荧光量子产率提高和荧光强度明显增强。论文的第五章研究了金属纳米粒子与蛋白质间的荧光增强作用。将前期工作中新发现的金纳米颗粒近红外荧光特性以及新纳米材料金属铕纳米颗粒的荧光性质与金属纳米粒子表面荧光增强效应结合起来，研究金属纳米颗粒与蛋白质间的荧光增强效应，为建立样品用量少而灵敏的蛋白质荧光分析方法奠定了基础。本文将金属（金、铕）纳米颗粒及其标记物固定在石英玻璃表面上，不仅研究了金属纳米岛膜表面对蛋白质荧光的增强作用，而且研究了蛋白质对金属纳米岛膜荧光的增强作用。研究表明，P450能够明显增强金纳米岛膜的紫外荧光（λex=230nm，λem=400nm）和近红外荧光（λex=538nm，λem=811.2nm）；而胶原蛋白隔离的金纳米岛膜对BSA的荧光有大的增强作用。与金纳米岛膜相比，铕纳米岛膜表面的增强作用较差。由于固体支架的准确固定、石英片的材质和厚度的均匀成度等因素都对金属纳米岛膜定量分析的准确度产生影响，所以，该方法还存在实验操作中的具体问题，有待于进一步研究。机理研究表明，首先，蛋白质与金纳米颗粒结合后可以提供较强的疏水微环境，从而使体系荧光强度增强。另外，在金岛膜的作用下，BSA中酪氨酸残基的荧光得到了明显的增强，并且已经超过了色氨酸残基的同步荧光强度。这是因为低量子产率的酪氨酸残基分子接近金岛膜纳米粒子后，荧光猝灭碰撞明显减少，并且辐射速率比猝灭速率更快，从而导致了低量子产率荧光基团的荧光强度得到显著增强。本论文的主要特点和创新点1．研究发现，15nm左右的金纳米颗粒能够发射近红外荧光，其激发和发射峰分别为538nm和811.2nm；随着纳米颗粒的减小，其荧光强度逐渐增强，且荧光峰位置不变；而蛋白质能够明显增强该近红外荧光强度。实验表明，在最佳实验条件下，体系的荧光强度的增加值与蛋白质浓度在一定范围内呈线性关系。据此以金纳米颗粒为荧光探针建立了蛋白质近红外荧光测定新方法。利用Zeta电位、TEM、吸收光谱、荧光光谱、共振散射光谱、圆二色谱和芘探针等技术研究了体系中蛋白质与金纳米颗粒间的相互作用，并提出了蛋白质对金纳米颗粒近红外荧光的增强机理。2．利用单宁酸做还原剂，首次制备了单一金属铕纳米颗粒，并研究了其光谱性质。研究发现，铕纳米颗粒能够发射紫外荧光，其激发和发射峰分别为275nm和380nm；随着纳米颗粒的增大，其荧光强度逐渐增强，而荧光峰逐渐兰移。可见，铕纳米颗粒的这种荧光性质的尺寸效应明显不同于贵金属纳米颗粒的荧光尺寸效应。研究指出，在SDBS存在下，蛋白质能够明显增强经硫辛酸修饰的铕纳米颗粒的荧光，且发射峰位置兰移。在最佳实验条件下，体系荧光强度的增加与蛋白质浓度在一定范围内呈线性关系。并以此建立了以铕纳米颗粒作为荧光探针的蛋白质新的测定方法。3．利用单宁酸做还原剂，首次制备了单一金属铽纳米颗粒，并研究了其光谱性质。研究发现，铽纳米颗粒能够发射紫外荧光，其激发和发射峰分别为256nm和388.2nm；铽纳米颗粒越小，荧光强度越弱，但其荧光峰位置几乎没有变化。研究指出，在CTAB存在下，蛋白质能够明显增强巯基丙酸修饰的铽纳米颗粒的荧光强度，且发射峰位置兰移。在最佳实验条件下，体系荧光强度的增加与蛋白质浓度在一定范围内呈线性关系。并以此建立了蛋白质定新的荧光光度法。4．本文利用Zeta电位、吸收光谱、共振光散射光谱、圆二色光谱和同步荧光光谱技术研究了蛋白质分别与铕、铽纳米颗粒间的相互作用和蛋白质对纳米颗粒荧光的增强机理。研究认为，铕和铽纳米颗粒分别通过硫辛酸和巯基丙酸与蛋白质相结合并发生了能量转移，即蛋白质吸收的能量通过分子间能量转移给铕和铽纳米颗粒，从而引起铕和铽纳米颗粒荧光的增强。表面活性剂SDBS和CTAB的加入能分别使铕和铽纳米颗粒体系的荧光强度增强，一方面是由于表面活性剂能分别参与两个体系的能量转移，另一方面表面活性剂和蛋白质能提供疏水环境，使体系的荧光量子产率提高，导致两体系的荧光明显增强。5．利用金属（金、铕）纳米岛膜表面不仅研究了它对蛋白质荧光的增强作用，而且也研究了蛋白质对金属纳米岛膜荧光的增强作用。研究表明，金纳米岛膜的增强作用优于铕纳米岛膜的增强作用。P450能明显增强金纳米岛膜的紫外荧光和近红外荧光；而用胶原蛋白作为隔离层的金纳米岛膜对BSA荧光有较大的增强作用。上述研究不仅扩展了金属纳米颗粒的范围，而且丰富了金属纳米颗粒的光谱性质，因而对纳米材料和纳米生物技术的发展具有重要的意义。
【Abstract】 In the recent years, along with the rise of nano science and technology, metal naoparticles are widely used and studied by spacious researchers in medicine, sanitary analysis and bio-chemical immunity, and in which showed potential application value, because of the particular optical and electrical properties, high stability, small dimension and superficial effects and its unique bio-affinities. The unique superficial effects of metal nanoparticles are the key properties to exhibit excellent and particular capabilities when composite with other materials. The UV-Visible spectra, surface-enhanced Raman scattering （SERS）, Resonance Light Scattering（RLS） and fluorescence spectra of metal nanoparticles have strong relations with the size, shape and particle arrangement of metal nanoparticles, and the direct combination or connected by modification with fluorescent molecules can also influence these spectra of fluorescent system. The influences on fluorescence spectra of metal nanoparticles may bring about fluorescent quenching or fluorescent enhancement, depend on the categories of metal nanoparticles and fluorescent molecules.Based on the researches of analytical chemistry, bio-chemistry and material chemistry, combined with the nano-science and technology, and using the techniques of fluorescence, absorption, light scattering and Circular Dichroism （CD） spectro-scopies, Transmission Electron Microscope（TEM）, High-resolution Transmission Electron Microscope, and Zeta potential measurement, we studied the fluorescent enhancement effects of protein-metal nanoparticle systems, discussed the interaction and the fluorescence enhancement mechanism, and established the new fluorescent methods for the determination of trace proteins with metal nanoparticles as the probe.In the first part of the thesis, we summarized the research progresses of metal nano-science, the methods of preparation and the usage of metal nanoparticles., predicted the trends and prospects of metal nano-science in the future. 191 references were cited.In the second part of this thesis, the near infrared fluorescence of AuNPs and its enhancement by proteins were studied. We used reduction method in solution toproduce different size gold nanoparticles. The studies of their absorption spectroscopyshowed that big gold nanoparticles have absorption peaks only at 250nm, along withthe decrease of particle size to 21 nm, a new absorption peak emerged at 525nm, andits intensity was increased with the decrease of particle size, absorption peak shiftedto shorter wavelength. It was found that 15nm gold nanonanoparticle has a nearinfrared fluorescent emission, its excition and emission peaks were 538nm and811.2nm, respectively. Result also showed that the infrared fluorescent intensities ofgold nanoparticles were greatly enhanced by proteins, and the factors which caninfluence the fluorescence enhancement were also studied. Experimental resultshowed that, under the optimized conditions, that is 15nm gold nanoparticles, andpH7.0 phosphoric buffer solution, there was the linear relationship between thefluorescent intensities of this nano-system and the concentrations of proteins in adefinite range of concentrations, the linear ranges of P450, BSA, HRP, and HSA were2.3×10-7-1.0×10-5mol/L, 2.0×10-7-1.5×10-5mol/L, 1.5×10-7-1.5×10-5mol/L, and1.5×10-7-1.5×10-5mol/L, respectively, and the detection limits were 2.44×10-8mol/L,2.21×10-8mol/L, 1.99×10-8mol/L and 2.01×10-8mol/L, respectively. So this methodwas sensitive. This method was used for the analysis of some specific samples and theresults were satisfactory. We take BSA as example to discuss the mechanism of theinteraction and the fluorescence enhancement of gold nanoparticles by proteins usingthe techniques of Zeta measurement, fluorescent life times, Transmission ElectronMicroscope（TEM）, fluorescence spectroscopy, absorption spectroscopy, lightscattering, and Circular Dichroism spectroscopy（CD）. Result showed that goldnanoparticles can directly connect to proteins, aggregated orderly around the proteinaccording to the particle size, shorten the distance between gold nanoparticles,changed the transmitting properties of surface plasma and resulted in the interactionsbetween the modes of local surface plasma and the surface plasma. These interactionswere influenced by the dielectric properties of peripheral environment. At the sametime, the plasma produced by metal nanoparticle can increase the electronic field ofits peripheral environment, and the increased electronic field can interact with the peripheral environment. This may be the part reasons that make the fluorescence intensity of nanogold labeled proteins increase. Additionally the hydrophobic environment provided by proteins can also lead to the increase of infrared fluorescent intensity of gold nano-system.In the third part of the thesis, the preparation method and the optical properties of alone metal europium nanoparticles and the fluorescence enhancement effects of the system were studied. Use tannic acid as reducer, for the first time, to reduce europium ions to europium nanoparticles, which were modified with lipoic acid in order to connect with proteins. Results showed that different amount of reducers can produce different size of nanoparticles, the lesser tannic acid used, the larger the size of europium nanoparticles. All sizes of europium nanoparticles have absorption peaks at 275nm, but the fluorescence intensity enhances along with the increase in particle size. Result showed that 20nm europium nanoparticles have UV-emission peaks at 380nm with the excitation of 275nm, which were increased and blue shifted along with the increase of particle size. This size effect of europium nanoparticle was different to that of noble metal nanoparticle obviously （the emission peak was red shifted along with the increase of size）. The fluorescence intensity was decreased by the modification of lipoic acid but greatly increased by the proteins with a blue shift of the emission peak. The factors which could influence the fluorescent intensities of the nano-system were also studied at the same time. Under the optimized conditions, that is, lipoic acid modified 20nm europium nanoparticles, in the presence of SDBS and pH6.0 phosphoric buffer solution, the fluorescent intensities of this nano-system was linear with the concentrations of proteins in a definite range of protein concentrations, the linear ranges of BSA, HRP, P450, OMP and NSE were 6.0×10-8-1.2×10-5g/ml 2.0×10-8-1.5×10-5g/ml 6.0×10-8-1.4×10-5g/ml, 2.0×10-8-1.8×10-5g/ml and 3.0×10-8-1.2×10-5g/ml, respectively, and their detection limits were 3.2×10-8g/ml, 1.0×10-8g/ml, 2.9×10-8g/ml, 9.8×10-9g/ml and 1.2×10-8g/ml respectively. Therefore, this method was sensitive with a broader linear range. We have used this method to some specific samples and the results are satisfactory. We have also discussed the interaction of proteins with nanoparticles and the mechanism of the fluorescence enhancement of europium nanoparticle system by trace proteins. It was considered that europium nanoparticle conjugated with proteins through lipoic acid modifier, and there was an energy transfer between them, that was, the energy absorbed by the protein was transferred to europium nanoparticles through intermolecular energy transfer, which maked the fluorescence intensity of europium nanoparticle increase. The existence of SDBS could also enhance the fluorescence intensity of this system , firstly because SDBS also transferred absorbed energy to Eu-lipoic acid-protein system to make the fluorescence intensity of europium nanoparticle increase, another reason was that both SDBS and protein could provide the hydrophobic environment for europium nanoparticles, which could decrease the opportunities of collision between water molecules and the conjugates, resulting in the decrease of the energy loss due to the collisions. So the fluorescent quantum yield increased and the fluorescent intensities enhanced.In the fourth part of the thesis, the preparation method and the optical properties of alone metal terbium nanoparticles and the fluorescence enhancement effect of the system were studied. Use tannic acid as reducer, for the first time, to reduce terbium ions to terbium nanoparticles, which was modified with mercaptopropionic acid in order to connect with proteins. Results showed that different amount of reducers can produce different size of nanoparticles, the more tannic acid used, the lesser the size of terbium nanoparticles. All sizes of terbium nanoparticles have absorption peaks at 275nm, but the fluorescence intensity enhances along with the increase in particle size. It is found that terbium nanoparticle has UV-emission peak at 388.2nm by the excitation of 256nm , and the fluorescence intensity was increased without emission peak changes along with the increase of particle size. Terbium nanopartlce could conjugate with proteins by modifying its surface with mercaptopropionic acid, and make the fluorescence intensity of the conjugate enhanced obviously accompanied by a blue shift of emission peak. The factors which could influence the fluorescent intensities of the nano-system were also studied at the same time. Under the optimized conditions, that is, mercaptopropionic acid modified 20nm terbium nanoparticles, in the presence of CTAB and pH6.8 phosphoric buffer solution, the fluorescent intensities of this nano-system was linear with the concentrations of proteins in a definite range of protein concentrations, the linear ranges of BSA, HRP, P450, OMP and NSE were 8.0×10-8-1.0×10-5g/rnl 3.0×10-8-8.0×10-5g/ml 5.0×10-8-1.2×10-5g/ml, 3.0×10-8- 9.0×10-5g/ml and 4.0×10-8-1.1×10-5g/ml respectively, and the detection limits were 3.4×10-8g/ml, 2.1×10-8g/ml, 1.9×10-8g/ml. 8.9×10-9g/ml and 1.1×10-8g/ml, respectively. So this method was sensitive with a broader linear range. This method has be used for the analysis of some specific samples and the results are satisfactory. We take BSA as example to discuss the interaction of proteins with nanoparticles and the mechanism of the fluorescent enhancement of terbium nanoparticle system by proteins. It was considered that terbium nanoparticle conjugated with proteins through mercaptopropionic acid modifier and there was an energy transfer between them , that was, the energy absorbed by BSA was transferred to terbium through intermolecular energy transfer, which maked the fluorescence intensity of terbium nanoparticle increase. While CTAB could enhance the fluorescence intensity of this system, firstly because CTAB could also transfer absorbed energy to Tb-mercaptopropionic acid-protein system to make the fluorescence intensity of terbium nanoparticle increase, another reason was that both CTAB and BSA could provide the hydrophobic environment for terbium nanoparticles, which decreased the opportunities of collision between water molecules and the conjugates, resulting in the decrease of the energy loss due to the collisions. So the fluorescent quantum yield increased and the fluorescent intensities enhanced.In the fifth part of this thesis, the fluorescence enhancement between metal nanoparticle island film and the proteins were studied. We integrate our new research works such as infrared fluorescence of AuNGs and fluorescence properties of rare earth metal nanoparticles, with the fluorescence surface enhancement effect, to study the fluorescence enhancement between nanoparticle and proteins, in order to build up the sensitive new method of microanalysis. The metal nanoparticles （Au, Eu） and the proteins are fixed on the surface of quartz slides, we study not only the fluorescence enhancement of the proteins brought by nanop-metal island film surface, but also the fluorescence enhancement of metal nanoparticle island film by proteins. Result showed that the UV-fluorescent （λex=230nm,λem=400nm） and infrared fluorescent （λex=538nm,λem=811.2nm） intensities of AuNP island film were greatly increased by p450, while the fluorescent intensity of BSA was greatly increased by the AuNP island film （λex=280nm,λem=350nm）. Contrary to the nano-gold island film, the fluorescence enhancement of nano-europium island film was very weak. Because that the factors, such as fixing of solid scaffold, the quality of quartz slide, and the equality of the thickness, can influence the fluorescence enhancement between protein and metal nano-island film, so in this method, there are experimental problems need to further study. The mechanism study showed that, at first, the strong hydrophobic environment provided by the proteins increased the intensity of the nano-sysytem, another reason was that the fluorescence intensity of the tyrosine residues with lower quantum yield in the proteins was greatly enhanced when it adjacent to the surface of nano-gold island film, even greater than that of tryptophan residue, which is attributed to that when the tyrosine residue adjacent to the surface of nano-gold island film, the fluorescence quenching from the collision with water molecules decreased, and the rate of emission is greater than the rate of quenching, so as to increase the fluorescence intensity of lower quantum yield fluorescent group greatly.The characteristics and innovations of the thesis are as follows:1. It is found that 15nm gold nanoparticle has a near infrared fluorescence emission of 811.2nm with the excitation peak of 538nm, the fluorescence intensity was increased along with the decrease of particle size without the shift of the emission peak, and the infrared fluorescence intensity of this system was greatly enhanced by proteins. Experimental results showed that under the optimized conditions, the fluorescent intensities of this nano-system was linear with the concentrations of proteins in a definite range of protein concentrations. According to this, a new infrared fluorescence method for the determination of proteins was established by using nano-gold as fluorescence probe.We studied the interaction between nanogold and proteins by Zeta potential measurement, TEM and fluorescence, absorption, light scattering, Circular Dichroism （CD） spectroscopies, and put forward the mechanism of infrared fluorescence enhancement of nano-gold particles by the proteins.2. Using tannic acid as reducer, for the first time, to reduce europium ions to metal europium nanoparticles, and the optical properties were studied. Study found that europium nanoparticles could emit UV fluorescence with the emission peaks of 380nm and the excitation peaks of 275nm, its intensity was increased with a concomitant blue shift of the emission peak along with the increase of particle size. This size effect of europium nanoparticle was different to that of noble metal nanoparticle obviously. The investigation showed that at the existence of SDBS, proteins can greatly enhance the fluorescence intensity of europium nanopartlce modified by lipoic acid with a blue shift of emission peak. Under the optimized conditions, the fluorescent intensities of this nano-system was linear with the concentrations of proteins in a definite range of protein concentrations. Therefore, a new analytical method for the determination of proteins was established by using europium nanoparticles as the fluorescence probe.3. Using tannic acid as reducer, for the first time, to reduce terbium ions to metal terbium nanoparticles, and the optical peoperties were studied. Study found that terbium nanoparticles have UV-emission peak of 388.2nm with the excitation peak of 256nm, its intensity was increased along with the increase of particle size. The study showed that at the existence of CTAB, proteins can enhance the fluorescence intensity of terbium nanopartlce modified by mercaptopropionic acid. Under the optimized conditions, the fluorescent intensities of this nano-system was linear with the concentrations of proteins in a definite range of protein concentrations. Therefore, a new analytical method for the determination of proteins was established by using terbium nanoparticles as the fluorescence probe.4. We discussed the interaction between europium, terbium nanoparticles and proteins and the fluorescence enhancement of europium, terbium nanoparticle systems by proteins, respectively, using the techniques of Zeta potential measurement and fluorescence, absorption, light scattering and Circular Dichroism （CD） spectroscopies. Study concluded that europium and terbium nanoparticles conjugated with proteins through lipoic acid and mercaptopropionic acid, respectively, and the energy transfers were occurred, that was, the energy absorbed by BSA was transferred to both europium and terbium, respectively, through intermolecular energy transfer, resulting in the increase of fluorescence intensities of both europium and terbium nanoparticles enhanced. In addition, SDBS and CTAB can also enhance the fluorescence intensities of both europium and terbium nanoparticle systems, respectively, firstly because SDBS and CTAB can transfer the absorbed energy to Eu-lipoic acid-protein and Tb-mercaptopropionic acid-protein systems to make the fluorescence intensities of europium and terbium nanoparticles increase, another reason is that the hydrophobic environment provided by SDBS-protein and CTAB-protein can increase the fluorescent quantum yield of two systems and make their fluorescent intensities enhance.5. Using the nano-metal （Au, Eu） island films, we studied not only the fluorescence enhancement of the proteins brought by nano-metal island film, but also the fluorescence enhancement of nano-metal island film by the proteins. Results showed that the effect of surface enhanced fluorescence of nano-gold island film was better than that of nano-europium island film. P450 can enhance both the intensities of UV-fluorescence and infrared fluorescences of nano-gold island film, while the nano-gold island film with the collagen can increase the fluorescence intensity of BSA.The studies mentioned above not only broaden the ranges of metal nanoparticles, but also enriched optical properties of metal nanoparticles; as a result, these studies are very important to the development of nano-materials and nano-biotechnique.