Research On Reversible Bioluminescence Probe For Redox Status Based On Inner Filter Effect

The balance of the redox state in the organism is an important factor for the normal physiological activities of the body and the stable operation of the physiological metabolic process in the aerobic environment.Once the redox status balance is imbalance,various diseases such as tumors,atherosclerosis,neurodegenerative diseases,and diabetes will be caused.Therefore,it is of great scientific significance to develop high-efficiency analytical methods suitable for real-time monitoring of the dynamic changes of redox-active species in organisms.Compared with traditional photoluminescence technology,bioluminescence does not require external excitation to generate optical signals,avoids a series of problems such as background signal interference and sample damage,and exhibits the advantages of high sensitivity and high signal-to-noise ratio in biological imaging.However,the current bioluminescent probes mostly rely on the substrate cage design strategy,which has the defect of irreversible analysis,and cannot distinguish between the transient physiological changes of redox active species and the pathological oxidative stress.In response to this problem,this thesis uses the principle of the inner filter effect to convert the absorption signal change into the bioluminescence signal change.To modulate the bioluminescent signal generated by the substrate-enzyme reaction rather than the bioluminescent reaction itself,then constructed a redox reversible molecular recognition unit on the absorber that overlaps with the bioluminescence spectrum,so as to realize the reversible cycle monitoring of redox active substances,and apply it to the dynamic imaging analysis of redox in vivo.The specific research contents are as follows:1.Research on reversible H2O2 bioluminescence probes.A H2O2 reversible bioluminescence absorber IFC was constructed,and its absorption spectrum overlapped with the bioluminescence emission spectrum of the substrate D-Luc,which could produce an inner filter effect and turn off the bioluminescence.After reacting with H2O2,the absorber IFC was oxidized,the structure changes,the inner filter effect disappears,and the bioluminescence recovers.When the light absorber IFC is reduced,the internal filtering bioluminescence can be performed again.The bioluminescent probe composed of the absorber IFC and the substrate D-Luc has a good reversible response to H2O2,and has been successfully used for reversible monitoring of intracellular H2O2.2.Study on bioluminescent probes of H2O2/SO2 redox cycle.A class of redox reversible responsive absorbers(IFA-1,IFA-2,IFA-3)were constructed,and it was found that the absorber IF A-1 had the largest overlap between the absorption spectrum and the bioluminescence emission spectrum of the substrate D-Luc,and the inner filter effect is the best,which can filter the bioluminescent signal generated by the enzymatic reaction.SO2 derivatives can undergo nucleophilic reaction with the light absorber IFA-1 to destroy its conjugated system,eliminate the inner filter effect,and restore the bioluminescence signal.When the oxide H2O2 is added,the conjugated structure of the light absorber IFA-1 can be restored,and the bioluminescence can be weakened again.Through bioluminescence imaging in vitro and in cells,it is known that the light absorber IFA-1 has excellent reversible characteristics,and it has been successfully applied to the dynamic imaging analysis of H2O2/SO2 redox in vivo.