| ObjectiveThis research aims to study the effect of olfactory marker protein(OMP)on the fluorescence enhancement of europium and gold nanoparticles,to develop a sensitive method for the quantitative determination of proteins,and to study the interacting mechanism between europium,gold nanoparticles and proteins.Methods1.Interactions and applications between europium nanoparticles and proteinsThe Eu(NO3)3 solution was reduced by tannic acid under vigorous stirring at 30℃water bath for 30 min and cooled to ambient temperature.Europium nanoparticles were obtained after the solution was centrifuged,scrubbed and metered volume. Nanoeuropium-OMP conjugate was synthesized by the similar method.Lipoic acid was added while the solution was stirred and OMP was added to the solution at 37℃water bath for 8 hours.The fluorophotometer was used to observe the effect of OMP on the fluorescence enhancement of europium nanoparticles and relationship between fluorescence changes and the concentration of OMP.The resonance light scattering, absorption spectra,transmission electron microscope and circular dichroism spectra techniques were also used to study the interacting mechanism between europium nanoparticles and proteins.2.Interactions and applications between gold nanoparticles and proteinsThe AuCl3 solution was reduced by trisodium citrate under vigorous stirring at boiling for 15 min and cooled to ambient temperature.Gold nanoparticles were obtained after the solution was centrifuged,scrubbed and metered volume.Nanogold-OMP conjugate was synthesized by the similar method.Lipoic acid was added while the solution was stirred and OMP was added to the solution at 37℃water bath for 8 hours.The fluorophotometer was used to observe the effect of OMP on the near-infrared fluorescence enhancement of gold nanoparticles and relationship between fluorescence changes and the concentration of OMP. The resonance light scattering,absorption spectra,transmission electron microscope and circular dichroism spectra techniques were also used to study the interacting mechanism between gold nanoparticles and proteins.Results1.Interactions and applications between europium nanoparticles and proteinsUnder the excitation of 271 nm,the fluorescence intensity at 379 nm of europium nanoparticles can be greatly enhanced by the addition of OMP.Under the optimum conditions,there is a linear relationship between the enhancement of fluorescence intensity and the concentration of proteins in the range of 2.0×10-8to 8.0×10-6g/mL for OMP, 2.0×10-8to 9.0×10-6g/mL for HRP,6.0×10-8to 1.2×10-5g/mL for P450,6.0×10-8to 1.0×10-5g/mL for BSA and 3.0×10-8to 1.2×10-5g/mL for NSE,and their limits of detection are 9.8×10-9g/mL,1.0×10-8g/mL,2.9×10-8g/mL,3.2×10-8g/mL and 1.2×10-8g/mL.This method can be used to determine the concentration of OMP in simulated sample and the result is satisfactory.The study of mechanism indicates that the interaction of europium nanoparticles,lipoic acid and OMP makes the construction of OMP change to form Eu-lipoic acid-OMP conjugate.Under the excitation of 271 nm,an energy transfer process may occur in this system,that is,tryptophane and tyrosine residue of OMP transmit the absorbed energy to europium nanoparticles through lipoic acid and notably enhance the fluorescence intensity.2.Interactions and applications between gold nanoparticles and proteinsExperiment indicates that the near-infrared fluorescence intensity at 811 nm of gold nanoparticles can be significantly enhanced by the presence of OMP under the excitation of 538 nm.Based on this,a sensitive method for the quantitative determination of proteins was established.Under the optimum conditions,there is a linear relationship between the enhancement of fluorescence intensity and the concentration of proteins in the range of 2.0×10-8to 9.0×10-6g/mL for OMP,3.0×10-8to 8.0×10-6g/mL for HRP,8.0×10-8to 1.2×10-5g/mL for P450,5.0×10-8to 1.0×10-5g/mL for BSA and 5.0×10-8to 8.0×10-6g/mL for NSE,and their limits of detection are 6.2×10-9g/mL,8.4×10-9g/mL,5.5×10-9g/mL, 6.0×10-9g/mL and 9.7×10-9g/mL.This method can be used to determine the concentration of OMP in simulated sample and the result is satisfactory.The mechanism research shows that lipoic acid connects gold nanoparticles and OMP by hydrosulfuryl to form Au-lipoic acid-OMP compound.This conjugate has crystal lattice construction and causes the change in transmission property of surface plasma,thus notably enhances the near-infrared fluorescence intensity of gold nanoparticles.Conclusions1.OMP can greatly enhance the fluorescence intensity of europium nanoparticles as well as the near-infrared fluorescence intensity of gold nanoparticles.2.The effect of OMP on the fluorescence enhancement of europium nanoparticles and gold nanoparticles can be used to determine the concentration of proteins and the result is satisfactory.3.The energy transfer among europium nanoparticles,lipoic acid and OMP may enhance the fluorescence intensity of europium nanoparticles,while the fluorescence enhancement of gold nanoparticles by OMP is caused by the change in transmission property of surface plasma.The main features and creative points of this thesis are as follows:1.Make use of good luminescent property of lanthanide and high sensitivity of nano-metal material in biomolecule detection to prepare europium nanoparticles and establish a novel method for protein determination.2.Study the effect of OMP on the near-infrared fluorescence enhancement of gold nanoparticles based on past research to raise sensitivity of protein determination.3.Use many research techniques,such as fluorescence,resonance light scattering, absorption spectra,transmission electron microscope to investigate the interacting mechanism of these systems.
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