| Biological coenzyme NADH plays an important role in cellular energy metabolism.Due to its intrinsic fluorescence properties,many NADH-based metabolic indicators,such as mean fluorescence lifetime of NADH,optical redox ratio and NADH free/bound ratio,have been established and widely applied in cancer detection and cellular metabolism researches.The fluorescence dynamics of NADH is significant for the correct understanding and application of these metabolic indicators.Although nsresolved fluorescence dynamics of NADH has been extensively studied,modern ultrafast fluorescence spectroscopy techniques have almost never been applied in this field.The ultrafast fluorescence dynamics of NADH has been studied for the first time in this thesis,by using femtosecond fluorescence upconversion spectrophotofluorometer combined with a variety of steady-state and time-resolved spectral apparatus.Besides,we have also investigated the ultrafast fluorescence dynamics of two NADH model molecules,and the interaction between NADH and two dehydrogenase proteins.This thesis shows the first systematic and comprehensive study of ultrafast fluorescence dynamics in NADH field.The main research results are as follows:1.The fs-resolved fluorescence dynamics of NADH was studied,which revealed the co-existence of quasi-static self-quenching(QSSQ)and biological water solvation in free NADH.The potential effects of QSSQ component in fluorescence imaging studies of NADH and the method to reduce those effects have been discussed.Furthermore,by constructing time-resolved emission spectrum(TRES)and hydration correlation function (),we found that the solvation dynamics could be described in two different time scales: 1.4 ps for bulk water and 27 ps for biological water.2.Since the QSSQ and the biological water solvation process occur at the same time scales,which is not conducive to the in-depth research of QSSQ component,the ultrafast fluorescence dynamics of NADH in the weak hydrogen bond environment has been further studied.By reducing the hydrogen bond strength,we succeeded in inhibiting the contribution from biological water solvation process,and realized the separated observation of QSSQ component in NADH.Besides,our results also provided more direct experimental evidence for the existence of QSSQ phenomenon.3.NADH model molecules have greatly promoted the studies on the origin of the nanosecond lifetime components.In this thesis,two NADH model molecules,1-benzyl-1,4-dihydroniacinamide(BNAH)and reduced nicotinamide mononucleotide(NMNH),were chosen and their femtosecond fluorescence dynamics were studied.By comparing the difference of ultrafast dynamics between the two model molecules and NADH,we preliminarily revealed the molecular origins of QSSQ phenomenon in free NADH.4.NADH free/bound ratio has been widely applied in real-time monitoring of many metabolism-related diseases.The free/bound ratio is generally considered to represent the concentration ratio of free and protein-bound NADH in cells.In this thesis,two dehydrogenase proteins,malate dehydrogenase(MDH)and lactate dehydrogenase(LDH),were selected as the typical binding sites of NADH,and the ultrafast fluorescence dynamics of protein-bound NADH was studied.It was found that the dehydrogenase binding sites significantly abolished the QSSQ component in NADH.These results suggest that NADH free/bound ratio resolved from conventional FLIM techniques significantly underestimates the concentration of free NADH,which may mislead the interpretation of many energy metabolism phenomena.Therefore,it is necessary to carry out the correction in practical application,and the correction method is also proposed in this thesis.In summary,we have systematically studied the ultrafast fluorescence dynamics of free and protein-bound NADH,and elucidated their differences in QSSQ composition and its influence on metabolic imaging research.This work can fill the gap of NADH femtosecond resolved fluorescence research,and promote its applications in biomedical fields. |
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