| H2O2 as a member of reactive oxygen species(ROS)plays important role from toxin to signaling agent.There are many photoelectrochemical or sensing devices have been developed for studying the ROS expanded in breadth and depth.Although optical sensing technologies have been designed attractive modalities for visualizing the generation of H2O2 and for exploring the role of this potentially harmful molecule in both pathology and physiology,the strategies have huge challenges in fast,real time,sensitive,and in situ understanding the generation of H2O2.The main reasons are as follows:first,most biological oxygen is used in aerobic respiration and it takes up only 1%to 2%oxygen to produce reactive oxygen species,so the H2O2 in organism have a lower concernation;second,the life span of H2O2 is shorter from produce to decomposition under physiological conditions and it is difficult to direct quantitative and qualitative;third,quantifying H2O2 in cytokines or growth factors stimulated pathways are very important,but the current imaging methods are mostly limited to monochromatic response causing quantitative difficulty.Although the borate ester based probes have been able to monitor the existence of endogenous H2O2,it is hardly to detect H2O2 in many physiological pathways for its inherent lower concentration.This thesis took the above-mentioned problems as research objectives,and several nanoprobes were successfully designed for H2O2 detection.First,borate ester,namely,H2O2 recognition site,functionalized hemicyenine probes were synthesized.By optimizing the para-position substituent group of recognition site,the response time of the selected probe to H2O2 was shortened,which realized the rapid detection of H2O2.Second,after screening,we got the excellent performance fluorophore,BMC3,as both target-activated trigger and the dual-emission fluorescence modulator to TPGQD42O.Upon two-photon excitation,ratio-dependent probe,TPGQD420-BMC3,displays a green-to-blue resolved emission band in quantifying H2O2 from mentioned pathways.Third,we synthesized signal amplification based micelle probe,namely,PMPC-Bpe@amylose,and applied to sensitive recognize of H2O2 related diseases in biopsy.Finally,the prepared cascade signal amplification based micelle probe,namely,PMPC-Bpe-BHQ2@SQ,was used for sensitive imaging ultra low concentration of H2O2 in cellular pathway stimulated by growth factors.This thesis mainly studies are as follows:1)Borate Ester Probe for H2O2 Rapid Detection Based on Regulation of Substituent Group.Existed probes containing borate ester recognition site in the response time of H2O2 have gone far beyond the life span of H2O2 in both pathology and physiology.Under biological detection,the probes react slowly with H2O2 and it can’t realize real-time quantitative assay.Therefore,design an optical probes that quick respond to H2O2 is very necessary.To this end,we designed and synthesized of 6 kinds of hemicyenine based probe that had different substituents in para-position of recognition site.With the help of dynamic scanning,we studied the influence on different substituents of BMCs to the response speed of H2O2.We found that when different substituents in para-position of recognition site registrated from electron-withdrawing groups to strong electron-withdrawing groups,the speed from two hours cut down to fifteen minutes.Through optimization substituents,we finally got the fastest probe in response speed to H2O2.2)Target-activated Modulation of Dual-color and Two-photon Fluorescence of Graphene Quantum Dots for in Vivo Imaging of Hydrogen Peroxide.The development of nanoprobes suitable for two-photon microscopy techniques is highly desirable for mapping biological species in living systems.However,at the current stage,the nanoprobes are restricted to single-color fluorescence changes,making it unsuitable for quantitative detection.To circumvent this problem,we designed a H2O2-activated modulation dual-emission and two-photon graphene quantum dot(TPGQD420)for imaging of H2O2.For specific recognition of H2O2 and lighting the fluorescence of TPGQD420,a boronate ester-functionalized merocyanine(BMC)fluorophore was used as both target-activated trigger and the dual-emission fluorescence modulator.Upon two-photon excitation at 740 nm,TPGQD420-BMC3 displays a green-to-blue resolved emission band in response to H2O2 with an emission shift of 110 nm,and the H2O2 can be determined from 0.2 to 40μM with a detection limit of 0.05μM.Moreover,the fluorescence response of the TPGQD420-BMC3 toward H2O2 is rapid and extremely specific.The feasibility of the proposed method is demonstrated by two-photon ratiometrically mapping the production of endogenous H2O2 in living cells as well as in deep-tissues of murine mode at 0-600μm.To the best of our knowledge,this is the first paradigm to rationally design a dual-emission and two-photon nanoprobe via fluorescence modulation of GQDs with switchable molecules,which will extend new possibility to design powerful molecular tools for in vivo bioimaging applications.3)Visual Biopsy by Hydrogen Peroxide-induced Signal Amplification.Visual biopsy has attracted special interest by surgeons due to its simplicity and practicality;however,the limited sensitivity of the technology makes it difficult to achieve an early diagnosis.To circumvent this problem,herein,we designed a visual signal amplification strategy for establishing a marker-recognizable biopsy that enables to early cancer diagnosis.In our proposed approach,H2O2 was selected as a underlying marker for its compact relationship in cancer progression.For selectively recognition of H2O2 in the process of visual biopsy,a benzylbenzeneboronic acid pinacol ester-decorated copolymer,namely,PMPC-Bpe,was synthesized,affording the final formation of the H2O2-responsive micelles,in which amylose was trapped.The presence of H2O2 activates the boronate ester recognition site and induces it releasing abundant indicator amylose leading to signal amplification.The indicator came across the solution of KI/I2 added to the sample and the formative amylose-KI/I2 complex has a distinct blue color at 574 nm for visual amplification detection.The feasibility of the proposed method is demonstrated by visualizing the H2O2 content of cancer at different stages and three kinds of actual cancerous samples.As far as we know,this is the first paradigm to rationally design a signaling amplification-based molecular recognizable biopsy for visual and sensitive disease identification,which will extend new possibilities for marker-recognization and signal amplification based biopsy in disease progressing.4)In Situ Cascade Signal Amplification:Ultrasensitive Detection of Hydrogen Peroxide Generation in Living Cells.The designing of cascade assays strategies for sensing ultralow analyte concentrations is of crucial importance.However,the strategies are not fit for vivo detection in recent years for needing to add assistant tools,such as enzyme or special signal reporter.To overcome the shortcomings we constructed,for the first time,an intracellular cascade signal amplification strategy by using biological matrix to amplify the fluorescence signal.As a proof-of-concept,micelle based nanoprobe,PMPC-Bpe-BHQ2@SQ,was designed by decorating BHQ2,a quenching agent that can quench fluorescence of wrappage and enhance sensitivity of the nanoprobe,and benzylbenzeneboronic acid pinacol ester,H2O2 recognition site,with alkynyl functionalized poly(carbonate)s backbone via“click"-reaction,in which SQ-dye,a squarylium dye that can greatly enhance the fluorescence quantum yields by intracellular biological matrix,was trapped.The presence of H2O2 activates the borate ester recognition site and induces it become the hydrophilic end destabilized the micelles,thus releasing the SQ-dye generating primary fluorescence enhancement.Moreover,the released SQ-dye can bind with biological proteins,leading to secondary fluorescent enhancement,realizing in situ quadratic fluorescence signal amplification in living systems.The feasibility of the proposed method is demonstrated as in situ for understanding the mechanism of H2O2 generation both growth factors stimulated pathway in cells and stimulated by testosterone in vivo.As far as we know,this is the first paradigm to rationally design in situ cascade signal amplification for sensitive detection of H2O2 generation in stimulated pathways,which will extend new possibilities for realizing cascade signal amplification in vivo.Above-mentioned rational designed probes have realized the detection of H2O2 in a fast,real time,and sensitive way.However,the understanding of the importance of cysteine oxidation and of the roles it plays in redox biology lack powerful chemical toolbox for elucidation of oxidative modifications in biological settings.We need to expand our search toward novel nucleophiles that show enhanced reactivity and selectivity toward sulfenic acid.Moreover,the emergence of protein sulfinylation as a cell signaling mechanism motivates the development of new approaches that can identify protein targets that are susceptible to sulfinylation.Numerous biological pathways and systems have abilities to produce H2O2.Therefore,we should integrate this enigmatic ROS in the processes of protein function regulation in future. |
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