The Fluoresence Enhancement Effect Of Protein-Metal Nanoparticle And The Analytical Application

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.