| Luminescence is a natural phenomenon of electromagnetic radiation emission(in the form of ultraviolet,visible,or infrared light),which can be described as a form of energy release when an exciting-state molecule relaxes to its ground state.The essence of luminescence is the process through which many different forms of excitation energy is converted into light and release the latter by certain substances.According to the sources of excitation energy,luminescence can be divided into many types,such as thermoluminescence,electroluminescence,photoluminescence(fluorescence and phosphorescence),bioluminescence and chemiluminescence.Bioluminescence,as the name suggests,refers to certain compounds produce and release photons with specific enzymes in living organisms;while chemiluminescence refers to some(exothermic)reactions release energy in the form of light when the products in the excited state return to the ground state.Therefore,both bioluminescence and chemiluminescence are the process of transformation from chemical energy to light energy.Bioluminescence can be considered as a spectacular type chemiluminescence where bioluminescent enzymes are required.Luminescence has been known well and applied in daily life for quite a long time.For examples,luminol has been used for the blood detection in forensic science.Besides,we can find oxalate esters in "light stick devices" and acridinium esters in immunoassay clinically.Moreover,various luminescent substances are used in environmental analysis,food&drug control and biomedicine research,including immol/Lunoassay or non-immol/Lunoassay,diagnosis as well as biosensor design.Luminescence imaging,including bioluminescence imaging(BLI)and chemiluminescence imaging(CLI),has been emerged based on the concept of luminescence recent years.Compared with imaging technology,this novel noninvasive approach is more sensitive,convenient and faster,having a promising application potential and commercial prospect in celluo and in vivo.Intensively studied,D-luciferin(aminoluciferin)-firefly luciferase system has been chosen in the maj ority of BLI system.In the presence of ATP,Mg2+and O2,D-luciferin(aminoluciferin)can be oxidized by firefly luciferase by which visible light is released.Using "cage" strategy,6'-hydroxy(amino)group of D-luciferin(aminoluciferin)can be modified to develop luminescent probes for the detection of certain bioactive molecules or emzymes.Reactive oxygen species(ROS)are required for triggering chemiluminescence of luminol,oxalate esters and acridinium esters.Considering that chemiluminescence of 1,2-dioxetanes can be triggered in aqueous system under mild conditions without ROS,this moiety is suitable for biological applications.The "cage" strategy can also be used in chemiluminescent probe design.When 3-phenol moiety is protected by certain groups,chemiluminescent probes for some analytes or enzymes will be obtained.Based on probe development experience and medical chemistry knowledge,we designed several luminescent probes in this dissertation.The topics mainly include three parts:firstly,we modified amonoluciferin with azo moiety to develop a bioluminescent probe for hypoxia detection;secondly,we obtained palladium chemiluminescence probe by connecting the butyne group to 1,2-dioxetane;thirdly,based on 1,2-dioxetane,we designed and synthesized probes for the biothiol chemiluminescent assay;fourthly,based on 1,2-dioxetane,chemiluminescent probe for the biothiol chemiluminescent assay was developed and evaluated;fifthly,we commenced the study on hypochlorite chemiluminescent probe,also based on 1,2-dioxetane.Part ? Study on hypoxia bioluminescent probeDefined as lower-than-normal oxygen conditions,hypixa is a pathogenic characteristic of solid tumors owing to absent or abnormal vasculature in the tumor microenvironment(TME).As an important factor of TME,hypoxia has a pivotal role in tumor progression,angiogenesis,metastasis,invasion and resistance to immune system and therapy.It is known that hypoxia emerges when the diameter of solid tumor increases to approximate 350 ?m in the body.As a result,hypoxia imaging is essential in diagnosis of cancer in early stage.Different kinds of imaging system have been explored and developed for hypoxia detection,including positron emission tomography(PET)and magnetic resonance imaging(MRI),phosphorescence imaging and fluorescent imaging,etc.In order to establish the approach for hypoxia detection in vivo,we developed a BLI system based on luciferin-firefly luciferase.In the current exploration,we managed to introduce an azo group into firefly luciferin to develop a hypoxia bioluminescent probe HBL using the “caging” strategy,based on the previous works in our group.As azo group is sensitive to hypoxia,HBL can be reduced to aminoluciferin by CYP450 reductase which is more active under low oxygen pressure.After aminoluciferin is recognized by luciferase,bioluminescence will be observed with certain devices.The results manifested that probe HBL is able to monitor hypoxia visually via CYP450 reductase with a high sensitivityand fine resolution in living cells and animals.Moreover,the experimental data establish a clear correlation between dynamic hypoxia degrees and tumors with different size.Part ? Study on palladium chemiluminescent probe As an important transition metal catalyst,palladium is extensively used in many areas including electronics,petroleum industry,automobile industry and fine chemicals engineering.However,palladium is hard to be biodegraded and easy to be enriched through the food chain so that brings harm to the environment as well as people's health.Therefore,methods for detection of palladium in environmental and biological samples effectively,accurately and quickly are desirable.There are many traditional methods for palladium detection by which accurate detection can be achieved rapidly and sensitively.However,these methods require complicated sample-pretreatment procedures,well-controlled experimental conditions,expensive facilities and highly-trained individuals.Using “cage” strategy,herein we managed to introduce butynyl group to 1,2-dioxetane moiety generating the palladium chemiluminescent probe(PCL).It was supposed that electronically excited /w-oxybenzoate anion released once butynyl moiety is cleaved via metal-catalyzed reaction.Subsequnently,the excited intermediate undergoes an electron transfer process to return to the ground state,according to Cl EEL(Chemically Initiated Electron Exchange Luminescence)mechanism.Finally,the extra energy released in the form of light.The results suggested that PCL is capable of monitoring palladium ion visually in vitro,in cellulo and in vivo.Although the instability of carbonate structure in PCL may drive down the LODs in the basic system or in complex biological samples,this experience can still help us expand the palladium imaging toolkit and applications of chemiluminescence technology.Part ? Study on biothiol chemiluminescent probes It is known to us that hydrosulfuryl groups often perform as active sites in biological big molecules.Meanwhile,Small molecular biothiols,such as cysteine(Cys),homocysteine(Hey)and glutathione(GSH),are significant to regulate the redox homeostasis and metabolism in the body,it has been reported that abnormity of biological thiol levels has extremely close relationship with various cardiovascular and neurodegenerative diseases.Therefore,detection and the quantification of biothiols is desirable for both academic research and clinical applications.Many conventional techniques are capable for detection of biothiols qualitatively and quantitatively.As a result of high sensitivity,high temporal and spatial resolution,and noninvasiveness,luminescent probes provide us another approach for the determination of biological thiol species.Considering the nucleophilicity of hydronsulfuryl group,alkenyl or chloroalkyl moiety was chosen as the reaction site of Michael addition.According to this strategy,we designed and synthesized two biothiol chemiluminescent probes,TCL-1 and TCL-2.based on 1,2-dioxetane scaffold.The recognition group will be cleaved by biothiols to generate the high-energetic intermediate which would emit photons through CIEEL mechanism.According to the results of related experiments,we discovered the instability of TCL-2 in aqueous environment made it improper for bioimaging assays.Fortunately, TCL-1 is capable to monitor biothiol such as cysteine in in aqueous solution visually in vitro,in cellulo and in vivo.It is also able to determine biothiols in complex biological samples.As a result,the exploration will expand biothiol visualization toolkit and applications of chemiluminescence technology.PART ? Study on hydrogen selenide chemiluminescent probeAs an indispensable microelement in human and animal bodies,selenium performs as redox active sites in selenoproteins.These selenoproteins(and selenoenzymes)play significant roles in immune regulation,body growth,lipid oxidation,antiphlogosis,detoxification and cancer prevention and therapy.Sodium selenite is commonly used as an inorganic selenium supplement clinically.It has been found that this affordable molecule can be potentially applied in cancer treatment.When reduced by GSH and thioredoxin system in vivo,selenite is converted into endogenic H2Se that has certain physiological and pathological activities.Therefore,a reliable and rapid approach for H2Se detection is required with the purpose of further exploration on its physiological functions and anticancer mechanism.In view of the stronger nucleophilicity of H2Se compared with H2S,we managed to connected 1,2-dithiane to 1,2-dioxetane in order to develop a chemiluminescent probe named SeCL for H2Se detection.Fortunately,the results of preliminary activity tests were just the same as we expected.With high sensitivity and selectivity,SeCL is capable for H2Se imaging in living cells.We hope the novel chemiluminescent probe can be applied for imaging H2Se in vivo to understand its physiological functions and anticancer mechanism clearly.PART ? Study on hypochlorite chemiluminescent probeHypochlorous acid,a potent oxidant against bacteria and fungi in mammalian oganisms,plays vital roles in health protection.Nevertheless,over-expressed hypochlorite may lead to destruction of cellular structure,injury of normal tissue and a variety of disease such as cardiovascular disease,neurodegenerative disorders,acute coronary syndrome,atherosclerosis,chronic infectious arthritis,lung and kidney injury,and even cancer.Therefore,an imaging method for hypochlorite detection in vivo is significant and necessary for diagnosis of certain diseases and clarification of immune response process.To date,the technology limits the full comprehension of hypochlorite in physiological and pathological progresses.A series of electrochemical analysis and optical imaging analysis methods for hypochlorite detection have been established.While optical imaging is preferable for its low cost,high selectivity and sensitivity as well as fine resolution.Based on the addition mechanism of hypochlorite towards thio esters,we chose dimethyl aminothioformate moiety as recognition group to modify 1,2-dioxetane luminophore.Finally,the hypochlorite chemiluminescent probe HCCL was designed and synthesized.The probe was found to be valid for hypochlorite detection in vitro according to the results of preliminary imaging assays.We expect a good capability of our probe for hypochlorite imaging in celluo and in vivo in follow-up experiments.Thus,a convenient,rapid and reliable platform would be established for understanding physiological and pathological activities of hypochlorite intensivelyIn summary,five kinds of small molecular luminescence probe introduced in this dissertation can realized luminescence imaging of hypoxia,palladium ion,biothiols,hydrogen selenide and hypochlorite respectively.These approaches can be used in vitro,in celluo and in vivo.Despite good performance of these probes in related experiments,there is still a long way to go for clinical application.To get high luminous quantum yield and longer emission wavelength for deep tissue imaging,further modification of the luminophores is necessary.Besides,better recognition groups should be designed according to the knowledge of chemical biology.Overall,BLI and CLI methods can be applied in drug development,life process research and clinical detection in the future. |
PDF Full Text Request