Construction And Applications Of High-reliability Optical Imaging Probes

Optical imaging technology is a basic tool for detecting various analytes and monitoring biological events,with high temporal and spatial resolution and imaging sensitivity.In the past few decades,a large number of optical imaging probes based on organic small molecule fluorescent dyes,fluorescent proteins and nanomaterials have been constructed.By detecting and imaging various targets under physiological conditions,such as ions,small biological molecules,proteins and nucleic acids,optical imaging molecular probes can not only realize early diagnosis of disease and real-time monitoring of disease progression,but also provide a quantitative index reference for the design,development and efficacy evaluation of new drugs.Optical imaging technology is a visual imaging technology that uses the luminescence intensity of a luminescent probe as a detection signal.It mainly includes fluorescence imaging technology that requires real-time light excitation and bioluminescence imaging technology that does not require real-time light excitation.Traditional fluorescent imaging probes require real-time light excitation,which results in a strong tissue self-luminescece background,which reduces the sensitivity and specificity of in vivo imaging.However,traditional bioluminescence imaging technology requires enzymes and substrates to detect luminescence signals,and the signals are often affected by the microenvironment where the enzyme is located and the distribution of the substrate in the organism.These defects of the current optical imaging probes will reduce the reliability in analysis and detection,and limit their applications in biomedical imaging.Therefore,how to design high-specificity and high-sensitivity optical imaging molecular probes,and achieve high-reliability biosensing and bioimaging,is the focus of this thesis.In order to solve the current challenges faced by activated optical imaging molecular probes in biosensing and bioimaging applications,this thesis designs and constructs a series of highly reliable optical imaging probes and applies them to the detection and imaging of enzymes and reactive oxygen species in living cells,tissues and mice models.The details are as follows:1)In Chapter 2,in order to track the mitophagy process under hypoxic conditions in real time,we developed a nitroreductase and p H-activated near-infrared fluorescent probe(NIR-HMA)for mitophagy visualization.NIR-HMA can monitor the p H changes of mitochondria in a ratiometric manner after activation of the over-expressed nitroreductase under cell hypoxia,so as to visualize the process of mitophagy induced by hypoxia.Using NIR-HMA,we can not only track the formation of autophagosomes and autolysosomes,but also distinguish the degree of hypoxia-induced mitophagy in different cells.Using the cyclic hypoxia-reoxygenation model,we applied NIR-HMA to study the role of mitophagy in hypoxic environment.It was observed that the fluorescence ratio decreased after reoxygenation,and the level of mitophagy increased further after hypoxia,which indicated that mitophagy may be the self-protection process of cells adapting to hypoxia.This work provides an attractive method for real-time visualization of relevant physiological processes in hypoxic environments.2)In Chapter 3,in order to solve the problem that traditional enzyme-activated probes are less accurate in bioimaging and disease diagnosis,we developed a leucine aminopeptidase(LAP)and monoamine oxidase(MAO)activated near-infrared fluorescent probe(NML)is used for specific detection and precise imaging of liver diseases.NML is composed of a near-infrared fluorophore and a tandem substrate chain of two enzymes through a self-eliminating linker.Only when the LAP and MAO are present at the same time,can the fluorophore be activated to emit fluorescence,which greatly improves the specificity of its detection.In the serum test,NML can effectively distinguish carbon tetrachloride-induced liver cirrhosis and paracetamol-induced liver injury.Importantly,in clinical serum sample testing,NML also demonstrated the ability to specifically detect.Therefore,this smart molecular probe shows great potential in clinical liver disease diagnosis.3)In Chapter 4,in order to solve the problem of fluorescence imaging technology being easily interfered by biological background,we built an afterglow nanoprobe platform that does not require real-time light excitation,and designed the first afterglow imaging probe(RAN)for quantitative detection and imaging of nitric oxide(NO).By introducing a ratiometric detection strategy based on afterglow resonance energy transfer(ARET),RAN can effectively avoid the problem of the decay of the afterglow intensity over time of traditional afterglow probes,greatly improving the detection reliability of the probe.In addition,RAN has excellent chemical stability and light stability,convenient synthesis,general post-modification strategies and ideal nano-scale size.Therefore,the probe shows great application potential in specificity and reliability detection.4)In Chapter 5,based on the advantages of RAN in quantitative detection and low-background bioimaging,we apply it to the detection and afterglow imaging of NO in the macrophage polarization model.The results showed that both the drug-induced RAW264.7 macrophage polarization process had high levels of NO production.Since real-time light excitation is not required,afterglow imaging has higher imaging signal-to-background ratio and sensitivity than fluorescence imaging.In addition,we also imaged the NO produced in drug-induced macrophage immunotherapy to achieve real-time evaluation of the effect of immunotherapy,which is of great significance for the screening of immunotherapy drugs.