Synthesis Of New Fluorescent Probe And In Vivo Fluorescent Imaging For Nitroreductase

Nitroreductase（NTR）is a kind of endogenous flavoproteinase with reducing effect,and can reduce nitroaromatic compounds to the corresponding amines in the presence of nicotinamide adenine dinucleotide（NADH）or nicotinamide adenine dinucleotide phosphate（NADPH）,which serve as an electron donor.NTR can be found in the bacteria species and biological tissue,and is usually overexpressed in hypoxic tumor cells such as HeLa cells,A549 cells,MCF-7 cells and V79 cells.So far,a lot of the fluorescent probes have been synthesized and analytical methods for evaluating the activity of nitroreductase have been developed using the fluorescent probes.However,the most of nitroreductase fluorescent probes exbit problems in detecting time,exciatation and emission wavelength,such as long detection time,short excitation and emission wavelengths,Therefore,it is necessary to employ a new nitroreductase fluorescent probe with advantages including specific selectivity,high sensitivity,and short detecting time,long excitation and emission wavelengths（650-900nm）,which can be used to the biological imaging and quantitative detection of nitroreductase.Cyanine laser dyes have been widely used in the synthesis of nitroreductase fluorescent probes due to their special chemical structure,high extinction coefficient as well as good photophysical and photochemical properties.The purpose of this thesis is to develop a new near-infare fluorescent probe for detecting nitroreductase based on principles of molecular recognition and molecular fluorescence sensing.A new near-infrared nitroreductase fluorescent probe was developed based on and 4-nitrobenzyl bromide,using new indocyanine green（IR820）as the precursor compound of the fluorophore,using 4-nitrobenzyl bromide as reconginition and quenching group,using nitroreductase as detection target,A novel methond of"signal-on" fluorescene probe analysis for evaluating nitroreductase activity was established using the new fluorescene probe.At the same time,the regularity of the cyanine fluorescent probes for detecting nitroreductase was discussed,and the feasibility study of the application of the fluorescent probe in biological imaging was carried out.The research results of this paper provide a new method for the maging detection of nitroreductase in metastatic tumor tissues and important reference information for the synthesis of near-infrared nitroreductase fluorescent probes and bioimaging analysis,which promote related research in the field of life analytical chemistry.This work was finally supported by National Natural Science Foundation of China,"Development of smart sensors based on mobile phone with blood glucose meter and its application in quantitative assessment of DNA peroxidation damage"（No.21729501,）.This master’s degree thesis is composed of three parts,which are introduction,research report,research conclusion and outlook.There are four chapters in total.The first chapter is the general introduction,the second and third chapters are the experiment report,and the last chapter is the experiment conclusion and outlook of this paper.Chapter one is the general introduction,which briefly summarizes the basic principles and characteristics of fluorescence analysis,and the biological functions of nitroreductase and its analysis methods;the classification and application of common fluorescent probes and the design of nitroreductase fluorescent probes Synthesis strategy research and research progress;Finally,the research purpose and research content of this thesis are introduced.The classification and application of the common fluorescent probes have been described,Meanwhile the principle and synthesis strategy,research progress of nitroreductase fluorescent probes were described in detail as far as possible.Finally,the research purpose and research content of this thesis were presented.The second chapter presents the development of synthesis method of one new nearinfare fluorescent probe.A new near-infare fluorophore compound was synthesized by using the cyanine-based laser dye new indocyanine green（IR820）and resorcinol as raw materials;a new fluorescent probe was synthesized by introducing 4-nitrobenzyl bromide to the fluopohore compound as recognition group and fluorescence quenching group.Both them was characterized by ESI-MS,UV-Vis absorption spectrum and fluorescene spectra,the experimental results showed that the actual molecular weights of the two new compounds were consistent with their theoretical calculations,both new fluorophore and probe have two distinct absorption peaks,for fluorophore λ1=623 nm,λ2=696 nm,λmax=623 nm,ε=2.31×104 L/mol·cm-1,λem=720 nm;for probe λ1=585 nm,λ2=699 nm,λmax=585 nm,ε=2.31×104 L/mol·cm-1,and no fluorescene was observed in visible and near-infare range.In the third chapter,a fluorescene methond for the detection of nitroreductase was developed,which was based on that the synthesized probe has no fluorescence signal in the range of 690～800 nm in phosphate buffer in the absence of NADH and nitroreductase,while the synthesized probe produces strong fluorescence signal at 720nm in the presence of NADH and nitroreductase.The measurement conditions including concentration of NADH,pH,reaction time and temperature were optimized to be 0.10 M PBS（pH=7.40）-150 μM NADH at 37℃ for 12 min.In the optimized condition,the fluorescene signal was in propotion to the concentration of nitroreductase in the range from 0.002 to 1.0μg/mL,and the detection of limit was achieved to be 1 ng/mL nitroreductase.Importantly,the synthesized probe has been successfully applied to the fluorescence imaging of nitroreductase in tumor cells and living zebrafish.The fourth chapter is the conclusion and outlook.This chapter summarizes the work of this thesis and discusses the shorfalls,and looks forward to the further work.