Molecular Kinetics Of Fluorescent Biosensors And Its Applications

Fluorescent measurement is one of the best methods for biological researches.Time-resolved fluorescence has been caught more and more attentions in recent years because of its resistance towards changes of molecular concentration,excitation wavelength,excitation power,etc.In order to label nonluminous biological molecules or cells,various fluorescent biosensors have been developed,mainly including organic molecules,amino acids or polypeptides,fluorescent proteins,and semiconductor quantum dots.In this dissertation,we study excited state dynamics of fluorescent probes by time-resolved fluorescence technique and apply them to biological detection.Styryl molecules are classical types of organic dyes.In this dissertation,two pyridinium styryl dyes,o-DASPMI and p-DASPMI,were synthesized and characterized by steady-state fluorescence spectra as well as picosecond and femtosecond time-resolved fluorescence spectra.Both dyes exhibited large Stokes shifts over 150 nm,and their fluorescence lifetimes were 6.6 ps and 12.4 ps,respectively.Because of their short fluorescence lifetimes,DASPMIs were used to measure Instrument Response Function(IRF)employed in time-correlated single-photon counting(TCSPC).The fluorescence transients of these DASPMIs were used as IRFs for iterative deconvolution fitting of the time resolved fluorescence decay profiles of Rhodamine B(RhB),sulforhodamine B(SRB),and the SRB-SRB2m RNA aptamer complex.The quality of fittings by DASPMIs method was consistently equivalent to that of fittings by scattering light method.These results indicated that DASPMI-derived IRFs could be widely applied to time-resolved spectroscopy and fluorescence lifetime imaging microscopy(FLIM).p-DASPMI showed a short lifetime of 12.4 ps in pH buffers.When it complexed with BSA,two longer lifetimes of 0.9 ns and 2.6 ns were observed.The amplitudes of lifetimes and decay associated spectra(DAS)showed that p-DASPMI molecules bound to subdomains ⅢA and ⅡA of BSA.Comparing the changes of lifetimes,amplitudes,(α2τ2 + α3τ3)/α1τ1 and α3τ3/α2τ2,we studied the conformational variants of BSA induced by pH and Cu2+.All results indicated that p-DASPMI could be used to monitor multi locations of proteins simultaneously,when it interacted with other molecules.pH plays a vital role in various cellular activities.Thus,real-time observation of the local pH through a pH indicator is very important for studying many physiological processes.In this dissertation,Trp-X peptides were synthesized to detect pH values.We firstly studied pH response of Trp-Trp dipeptide and its derivatives(NATrp2Me,NBTrp2 and Trp2Me)by steady-state and time-resolved fluorescence spectra.We discovered that the exposed amino is pivotal for pH-dependence of Trp-X.Moreover,an artificially synthesized tetrapeptide(Trp-Trp-Ala-Ser,WWAS)confirmed pH sensitivity of N-terminal Trp-Trp residues.pH values could be quantitatively determined from fluorescence intensities and lifetimes of N-terminal Trp-Trp residue.In Trp-X peptides,both of the terminal amino group and the second amino acid had great influences on fluorescence of the indole ring in tryptophan.Comparing with WWAS,two new tetrapeptides,Trp-Ala-Ala-Ser(WAAS)and Trp-Glu-Ala-Ser(WEAS),had higher fluorescence quantum yields,longer fluorescence lifetimes,and more sensitive pH response.In interference experiments,metal ions did not disturbed time-resolved fluorescence at all.As novel genetically encoded pH indicators,N-terminal Trp-X could be fused to proteins for monitoring environmental pH values in studies of polypeptides or proteins.roUnaG-BR,as the first redox probe based on UnaG,can not only turn on fluorescence by bilirubin,but also work well in the absence of oxygen.Unlike GFP,roUnaG-BR has one excitation channel and emission channel,which makes it difficult to label redox states quantitatively based on steady-state fluorescence intensity.By time-resolved fluorescence technique,we found that fluorescence lifetime of the most molecules in reduced state was 2.2 ns,while in oxidation state it was 0.2 ns.Labeling redox state with parameters α3/α1 and α3τ3/α1τ1 obtained a 10-folds and 8.1-folds increase,which is higher than the 7-folds increase of steady-state fluorescence intensity.Live cell experiments proved this method was valid in quantitatively detecting redox state in vivo with a higher sensitivity than average lifetime method.