New Organic Fluorescence Probes And Graphitic Carbon Nitride Nanosheets Nanomaterials Based Emerging Fluorescence Biosensor Technology

Compared with the traditional analysis method,Fluorescence biosensor detection technology,as a new biological analysis technology,provides higher sensitivity,faster response,better selectivity,lower cost and the advantages of real-time,in situ,online and continuous monitoring in complex systems,which attracts more and more attention in the field of biochemical analysis and medical diagnosis.The organic fluorescence probe can be widely used in the detection of biological macromolecules（nucleic acid,protein,etc.）,inorganic small molecules（oxidative free radicals,metal cations,an ions,etc.）,biological sulfhydryl small molecules（cysteine,glutathione）and so on because of its low preparation cost,simple operation,high sensitivity,strong selectivity,low biotoxicity and high fluorescence quantum yield.Therefore,the development of a new type of organic fluorescence probe will promote the development of a new method of fluorescence biosensor.Graphite carbon nitride（g-C₃N₄）nanosheets,as a new type of two-dimensional nanomaterials,can be used as fluorescent imaging tracers an d nanocarriers in cells with the advantages of good water solubility,high fluorescence quantum yield,large specific surface area and low toxicity.In addition,the nanosheets,with the generation of singlet oxygen under light conditions,can be used as photosensitive reagents for photodynamic therapy of tumor cells.These properties will make g-C₃N₄ nanosheets have a very high application value in the development of highly sensitive fluorescent biosensor methods and disease diagnosis and treatment.Combined with the main problems in the field of biomedical research,we have developed some new fluorescent biosensor methods for biomarker detection,cell imaging and disease diagnosis and treatment by using organic fluorescence probes and g-C₃N₄nanosheets.The mian contents are described as follows:In chapter 2,We have developed a novel turn-on mitochondrion-targeting fluorescence probe via reduction induced intramolecular charge transfer for fast mitochondrial methionine sulfoxide reductases（Msrs）imaging i n living cells.We design four fluorescent probes for Msrs by conjugating methyl phenyl sufoxide,a well-known Msrs substrate,with different small electron with-drawing cationic moieties.Fluorescence signal is switched on upon Msrs-mediated reduction of the electron withdrawing sulfoxide to an electron dona ting sulfide,generating fluorophores of distinct donor-acceptor structure and large Stokes shift.Among them,MSP1 is demonstrated to exhibit high sensitivity,fast response and high specificity toward Msr A in vitro.Meanwhile,the hydrophobic methylpyrid ium endows the probe with excellent mitochondrial targeting ability.Furthermore,MSP1 is capable of detecting and imaging mitochondrial Msrs in living cells.Moreover,MSP1 is successfully introduced to detect reduced Msrs activity in a cellular model for Parkinson’s disease.To our knowledge,this is the first mitochondrial-targeting fluorescent probe that enables detection and imaging of mitochondrial Msrs in living cells.In chapter 3,Based on the intramolecular charge transfer effect（ICT）,we have designed a near infrared fluorescent dye DCX-OH with long Stokes shift by combining benzo cyanopyranone（electron-withdrawing group）with hydroxyl occupied electron group（electro n-donating group）,and then we use2-diphenylphosphonic acid to quench the fluorescence of DCX-OH by forming an ester bond with DCX-OH to obtain the probe DCX-TPP for the detection and cell imaging of hyponitric acid（HNO）.The probe in the detection of HN O has the advantages of long Stokes shift（160 nm）,high sensitivity,high response speed and like.Further more,our probe has a good imaging ability to detect the changes of HNO content produced by exogenous HNO and endophytic HNO,which provides an effective detection tool for further revealing the physiological function of HNO.In chapter 4,we have developed a novel fluorescent graphitic carbon nitride nanosheet（g-C₃N₄）based biosensor for highly sensitive,label-free detection of alkaline phosphatase（ALP）.The biosensor relies on the high affinity interaction between PPi（natural substrate of ALP）and Cu²⁺,which prevents coordination between the g-C₃N₄ nanosheet and Cu²⁺.In the presence of ALP,PPi is catalyzed to become transformed into phosphate（Pi）with a much weaker interaction with Cu²⁺,which was choosed to coordinate with the g-C₃N₄nanosheet and led to the quenched fluorescence of g-C₃N₄nanosheet via photoinduced electron transfer（PET）.The proposed method is rapid,simple and implemented in a homogeneous format.Compared to existing methods for ALP assay typically involving the use of synthetic substrates,the developed approach utilizes a natural substrate of ALP in biological systems,which may afford higher catalytic efficiency toward t his substrate and thus provide much better sensitivity for ALP detection.This novel biosensor is demonstrated to enable quantitative analysis of ALP in a wide range from 0.1 to1000 UL^-1 with a low detection limit of 0.08 UL^-1,which is among the most sensitive assays for ALP.It is expected that the developed method may provide a low-cost,convenient,rapid and highly sensitive platform for ALP-based clinical diagnostics and biomedical applications.In chapter 5,we show a novel phototherapeutic nanoplatform based on g-C₃N₄nanosheets by intracellular messenger RNA triggered catalytic hairpin assembly（CHA）for fluorescence imaging guided photodynamic therapy（PDT）.The integrated CHA for fluorescence signal amplification enables highly sensitive and specific imaging of intracellular m RNA,while g-C₃N₄ nanosheets can be as favorable nanocarriers,fluorescence tracer agent for imaging and effective photosensitizers for PDT.Our nanoplatfo rm provides not only a sensitive and specific intracellular m RNA imaging method but aso an imaging guided highly efficient PDT for cancer cells.The developed nanoplatform provides a clue that the g-C₃N₄ nanosheets as biocompatible 2D layered layered nanom aterials hold the promising application for cancer diagnosis and therapy.