Cellular Nitric Oxide Analysis Based On New Amphiphilic Fluorescent Probes

Cells are the basic structural and functional units of living organisms.Major organelles found in eukaryotic cells include plasma membrane,mitochondria,lysosomes,nucleus and so on,which are capable of performing their own functions independently and working synergistically to maintain normal functions of cells.Each organelle needs specific chemical species and the appropriate microenvironment to work.As an important intra-and inter-cellular signaling molecule,nitric oxide(NO)is produced within the cell and acts intracellularly and extracellularly to participate in a variety of physiological and pathological processes.Therefore,it is of great significance to develop efficient analytical methods for NO with subcellular resolution or at single-cell levels,which is helpful for the studies of the functional mechanism of NO in living systems.Analytical methods based on small molecule fluorescent probes are powerful tools to monitor NO in biological samples with advantages of high selectivity,sensitivity and spatiotemporal resolution.Compared to the rapid development of fluorescent probes and analytical methods for intracecullar NO,the fluorescence analysis of NO released out of cells,especially for single cells,is extremely limited.This dissertation reviews the analytical approaches based on small molecule fluorescent probes for NO and recent development of fluorescent probes with subcellular resolution.Aimed at the detection of NO released out of living cells,an amphiphilic fluorescent probe,disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene(DSDMHDAB),has been designed and synthesized.With a hydrophobic C16 alkyl chain acting as the membrane anchor and a hydrophilic fluorescence sensor head being kept outside the cell,DSDMHDAB could trap NO released out of cells more efficiently and be used for fluorescence imaging.Using confocal laser scanning microscope(CLSM)and capillary electrophoresis laser-induced fluorescence(CE-LIF)detection,various effective,sensitive and practical methods for visualization and single-cell quantitative analysis of NO released out of living cells as well as both intra-and extracellular NO have been established using DSDMHDAB and other lipophilic fluorescent probes for NO.The major research work in this dissertation is described as follows:(1)With the introduction of a hydrophobic alkyl chain and hydrophilic sulfonates to the BODIPY fluorophore,a novel amphiphilic fluorescent probe,disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene(DSDMHDAB),has been designed,synthesized and characterized.The critical micellar concentration(CMC)of DSDMHDAB was estimated to be 1.0 mM,and 0.01%Triton X-100 was added to the bulk solution to avoid the accumulation of DSDMHDAB on the surface of bulk solution when DSDMHDAB concentration is lower than 1.0 mM.The fluorescence quantum yield of DSDMHDAB is 0.003 and that of its NO derivative,DSDMHDAB-T,is 0.183.The excitation and emission wavelengths of DSDMHDAB-T are 515 nm and 529 nm,respectively.The fluorescence intensity of DSDMHDAB and DSDMHDAB-T has slight change with the variation of pH from 2.0 to 8.0 or under the laser irradiation for 12 h,which indicates that DSDMHDAB is suitable for monitoring NO in living systems.(2)Taking advantage of the amphiphilicity,DSDMHDAB has been successfully used to monitor the extracellular release of NO from living cells in combination with confocal laser scanning microscope(CLSM).After incubation with cells for 2 min at room temperature,DSDMHDAB can accumulate on the outer surface of the plasma membrane uniformly and respond to NO released out of cells rapidly and sensitively with low cytotoxicity.RAW 264.7 cells were pre-incubated with 0.5 ?g/mL LPS and 0.01 ?g/mL IFN-y for 12 h to stimulate the production of NO,whereas ECV-304 cells were incubated with 10.0 ?M JS-K(a GST-activated NO generator)for 30 min to simulate the release of NO.Upon following incubation with 5.0 ?M DSDMHDAB for 2 min,the diffusion of NO across the plasma membrane has been trapped rapidly and observed directly with the excitation at 488 nm.Moreover,NO released from RAW 264.7 cells and HeLa cells induced by actinomycin D has been imaged.The proposed method is intuitive,fast and sensitive,which opens a new way for studying the intercellular signal transduction of NO in various physiological and pathological processes.(3)A new method for simultaneous visualization of intra-and extracellular NO based on dual-color fluorescence imaging has been established by using two fluorescent probes with the same NO trapper.The green-emitting amphiphilic fluorescent probe,DSDMHDAB,was used for extracellular NO and the red-emitting lipophilic one,4,4-difluoro-8-(3,4-diaminophenyl)-3,5-bis(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-i ndacene(p-MOPB),was for intracellular NO.RAW 264.7 cells pretreated with 500 ng/mL LPS and 500 U/mL IFN-y for 12 h were incubated with 5.0 ?M DSDMHDAB and 5.0 ?M p-MOPB for 20 min,and then subjected to dual-color fluorescence imaging with the simultaneous excitation by lasers of 488 nm and 543 nm.In addition,cell lysates were analyzed by high performance liquid chromatography and fluorescence detection(HPLC-FLD)to evaluate the relative content of intra-and extracellular NO.The proposed visualization method can reflect the real-time distribution and relative content of intra-and extracellular NO rapidly and directly,and provide a new approach for the research on the homeostasis and release behavior of NO.(4)Using DSDMHDAB as a labeling reagent,a capillary electrophoresis laser-induced fluorescence(CE-LIF)detection method has been developed for the determination of NO released out of single cells,and applied to the analysis of RAW 264.7 cells.Using 20 mM pH 7.4 H3BO3-Na2B4O7 solution containing 10 mM SDS and 20%(v/v)acetonitrile as a running buffer,the separation of DSDMHDAB-T was carried out within 4 min.The linear calibration range is 37-3700 amol with a detection limit(S/N = 3)of 11.6 amol.The proposed method with the integration of single-cell sampling,lysis,separation and detection is fast,sensitive and effective for the determination of NO released from single cells.(5)Simultaneous determination of intra-and extracellular NO of single RAW 264.7 cells based on a dual labeling strategy has been accomplished using offline/online derivatization capillary electrophoresis laser-induced fluorescence(CE-LIF)detection.The amphiphilic fluorescent probe DSDMHDAB and the lipophilic fluorescent.probe 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene(DAMBO)with the similar excitation and emission wavelengths and the same NO trapper were adopted to label extra-and intracellular NO,respectively.In a running buffer of 40 mM pH 7.4 Tris-H3BO3 and HEPES mixed solutions containing 15 mM SDS and 20%(v/v)acetonitrile,a baseline separation of the two NO derivatives derived from offline derivatization was achieved within 4 min without obvious interference.Detection limits(S/N = 3)of intra-and extracellular NO were 3.0 amol and 10.7 amol,respectively.Furthermore,in order to avoid the interference among cells and make the results more accurate,single-cell sampling,derivatization,lysis,separation and detection have been integrated in the capillary and online analysis of single cells has been performed to determine intra-and extracellular NO.Detection limits(S/N = 3)were 2.4 amol and 8.1 amol for intra-and extracellular NO,respectively.The proposed method based on mild and fast derivatization as well as the commercial-available instrument has advantages of high efficiency,sensitivity,accuracy,practicability and versatility,which provides a special and practical technical platform for the studies of the relative content and functional mechanisms of intra-and extracellular NO at the single-cell level.