Origin Of Metal-enhanced Fluorescent In Solution Phase And Application Of Fluorescence Protein In Gene Regulation

In order to meet the technical requirements of the rapid development in biologyfiled,also accompanied by the gradually developed of the fluorescent material andmicroscopy,fluorescence technology has become a very important technique in manyareas of biological research.In recent years more and more researchers focued on thein vivo detection,such as labeling some specific tissues in the cells to study theirstructure,or labeling label a protein by a fluorescence protein to measure itsexpression level.Due to the higher authenticity and credibility of in situ detecion invivo than in vitro,more and more people began to pay attention to this area.The mostimportant technique of in situ detection was how to label the targeting molecules.Two kinds of lables are widely used now.One was that using the fluorscencemoecules specifically binding to the target,and studying the target molecules bydetecting the fluorescence labels.The other form of label was fluorescence protein.The protein labeled by a fluorescence protein(FP)was extremely specific,and theexpression level of target gene was reported and detected by the FP.Here wediscussed these two forms of fluorescence labelsFor the first form,the current key issue was still looking for a promisingcandidate fluorescence label,which had better optical properties than dye and lowertoxicity than Quantum Dot.Due to the excellent characteristics of the rare earthcomplexes,many researchers considered it as a very promising fluorescence materialfor in vivo detection.Because all the moelecules inside the cells were situated insolution environment,we need to find a rare earth complexe which had goodfluorescence properties.In order to do that,we synthesized Eu complexes,and studiedits optical characteristic in solution phase.We also tried to enhance the fluorescenceintensity of the complexes in solution by Ag nanoparticles,and discussed the originsof metal enhanced fluorescence in solution.Luminescence enhancement and quenching of Eu(TTA)₃·2H₂O complex in DMF(N,N-dimethylformamide)solution containing silver nanoparticles were observed, which depended on the concentrations of both europium complex and silvernanoparticles.Their origins were discussed based on absorption spectra,excitationand emission spectra,and luminescent decay dynamics.The results indicated thatwhen the concentration of Eu complex was high enough,strong interaction amongcomplex molecules occurred.The presence of Ag nanoparticles definitely decreasedthe interaction among complex molecules.In the solution containing Ag nanoparticlesthe electronic-dipole transition rate of5D0-⁷F₂increased due to enhanced local fieldsurrounding Eu³⁺ions,while the nonradiative transition rate from5D0decreasedowing to decreased resonant energy transfer among europium complex molecules.These two factors led to the luminescence enhancement of europium complex.As tothe luminescent quenching,was attributed to absorption competition between Agnanoparticles and europium complex at excited wavelength.We also studied the wavelength dependence of Ag nanoparticles enhanced Eucomplexes fluorescence in solution.We selected three different wavelengthes,269nm,341nm and375nm as the excitation light,and explored how the excitation andemission intensity vary with increasing of Ag nanoparticles concentration.When wedicussed the absorption spectra,excitation and emission spectra,we found that Agnanoparticles could not only increase the electronic-dipole transition rate of Eu³⁺,butalso increase the excitation intensity and excitation efficiency.We also found that boththe enhancement factor of excitation and emission light intensity were very differentwhen we used different excitation wavelength.The results also showed that thepresence of Ag nanoparticles also reduced the efficiency of energy transfer betweenEu³⁺and organic ligands(TTA).Finally,we concluded that Ag nanoparticles not onlyincreased the radiative transition,decreased nonradiative transition,but also enhancedthe local field effect of Ag nanoparticles,increased excitation efficiency of thecomplex solution,reduced internal energy transfer efficiency of Eu complexemolecule.All these factors worked together,finally enhanced the fluorescence of Eucomplex in solution.For the fluorescence protein,since it also was a protein,so it did not have atoxic effect on cell.Meanwhile,the genetically encoded FPs were extremely specific when it was used to label a protein.These advantages made it became the mostsuitable fluorescence label in the in vivo detection since the GFP was discovered.Asfurther development of GFP and finding of its variants with different fluorescencebehaviour,the fluorescence protein has been used by more and more researchers indifferent biolodical field.We constructed a tetR self-repressor gene regulation circuit,and used Venus asthe reporter of transcription factor TetR.Here,Venus is a YFP variant whichmaturated very fast and had higher fluorescence signal than many other kindfluorescence proteins.In order to make sure the copy number of the self-repressorgene regulation circuit is single-copy,we integrated it on the chromosome of E.coli.We induced the cells under different concentration of aTc to change the biochemicalreaction parameters of the regutated circuit,such as the binding and unbinding ratesbetween TetR and oprator tetO.We explored the expression level distribution of TetRin different biochemical environment by monitoring fluorescence of Venus.Ourresults showed that when the binding/unbinding were much faster than thesynthesis/degradation of protein,the concentration distribution of TetR in all the cellsexhibited one peak under the data from flow cytometry.But when we tuned thecondition of inducing to make the the binding/unbinding were even slower than thesynthesis/degradation,we found that there were two peaks happened,which meantthat there were two population cells with different expression level of regulatorprotein even when the same kind of cells grew in same environment.This result fittedto the theoretical prediction which was also got in our group very well.It must be asignificant breakthrough in the research filed of cell differentiation.