Studies On New Low-background And Highly Sensitive Fluorescence Detection Methods For Nitric Oxide Based On Near-infrared BODIPY Dyes

During the metabolism of organism, a variety of active free radicals are continuously produced, such as hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide anion (O2·-), hydroxyl radical (·OH), nitric oxide (NO), peroxynitrite (ONOO-) and so on. Free radicals at low concentrations are important regulators from normal physiological conditions to pathological states of life, while the overproduced free radicals induce oxidative damages to the organism. Since the function of free radicals in biological system closely depends on their concentrations, it is of great importance to develop rapid, sensitive and accurate methods for the determination of free radicals in complex biological matrices. It has been reported that small-molecule fluorescent probes can efficiently trap free radicals and achieve high sensitivity, high selectivity and real-time detection of free radicals in complex samples.The paper has made a comprehensive review on the research development of small-molecule fluorescent probes for the free radicals. With NO as the study object, two novel near-infrared (NIR,>600nm) fluorescence probes,8-(3,4-diaminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)naphtho[b,g]-s-indacene (DANPBO-H) and8-(3,4-diaminophenyl)-1,7-dimcthyl-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)n aphtho[b,g]-s-indacene (DANPBO-M) have been designed and synthesized in order to red-shift the excitation and emission wavelengths to NIR region, and improve the cell retention of NO fluorescence probes and corresponding triazoles. Combined with high performance liquid chromatography and fluorescence detection (HPLC-FD), fluorescence microscope and fluorescence spectrophotometer, the DANPBOs were used to determinate NO in cells, blood, animal and plant tissues. The proposed methods are simple, fast and demonstrate low background interferences and high sensitivity and selectivity, which could realize the monitoring of slight changes of NO in biological samples under different environments.The research mainly includes the following aspects:(1) With the1,2,3,4-tetrahydronaphalene rings rigidly fused to the pyrrole fragments of BODIPY chromophor, two NIR fluorescence probes,8-(3,4-diaminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)naphtho[b,g]-s-indacene (DANPBO-H) and8-(3,4-diaminophenyl)-1,7-dimethyl-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)naphtho [b,g]-s-indacene (DANPBO-M), for NO detection have been designed, synthesized and characterized. The fluorescence properties and cell retention of DANPBOs and their NO derivatives (DANPBO-Ts) are studied. The fluorescence quantum yields of DANPBO-H and DANPBO-M are0.0004and0.001, respectively. After the reaction with NO, the fluorescence quantum yields increase by400-fold and550-fold, respectively. The excitation and emission wavelengths of DANPBO-H-T are621nm and631nm, and those of DANPBO-M-T are609nm and619nm, respectively. The fluorescence of NO derivatives is stable for at least24h under the radiation of xenon lamp, and is rarely influenced by pH and solvents. The primary research indicates that DANPBOs and DANPBO-Ts can be retained well in cells for at least3.5h without any leakage.(2) With the detection wavelengths of DANPBO-H and its derivative in NIR region, the background interference from biological molecules can be obviously reduced. Using DANPBO-H as a pre-column derivatizing reagent for NO, a new HPLC-FD method has been established to detect NO in various biological samples. The derivatization reaction was completed at35?. for20min and the product was eluted in3.5min using MeOH/THF/50mM pH7.0H3Cit-NaOH buffer (81/7/12, v/v/v) as mobile phase and a Cs column as stationary phase. The linear calibration range of the proposed method is6.0×10-9to2.00×10-7M and the detection limit (S/N=3) reaches to5.5×10-9M (S/N=3). The chromatograms of rice, mice tissues and cell biological samples matrixes were studied at NIR detection wavelengths (621/631nm), and visible detection wavelengths (496/505nm) under the same mobile phase. In contrast to the serious background interference detected at496/505nm, there was almost no background interference from the biological matrices under the excitation and emission wavelengths of621nm and631nm. The proposed method has been validated by the determination of NO in organs and blood of mice, rice pangen and radicle, the human vascular endothelial (ECV-304) cells, and the mouse macrophage (RAW264.7) cells. The spiked recoveries range from95to108%with RSDs below2.3%. Compared to the previous fluorescent probes for NO in HPLC, DANPBO-H exhibits excellent resistance to the background interference from various complex biological matrices, which results in a flat and clean baseline in sample analysis favorable for the separation and detection.(3) Based on the excellent cell retention of DANPBOs and DANPBO-Ts, DANPBOs were used to image NO in ECV-304cells and RAW264.7cells. The results indicated that DANPBOs and its solvent (acetone) used for NO imaging in the studies are almost nontoxic to cells. DANPBOs can avoid the autofluorescence interference from biological matrices, and DANPBOs and DANPBO-Ts can be well retained in cells for at least3.5h. This fact suggests that the superior intracellular retention of DANPBOs and DANPBO-Ts attributes to their good lipophilicity, which avoids the introducing of ester groups to the probes for improving the cell retention of probes. On this basis, DANPBO-H was used to image NO released by RAW264.7cells, and the imaging conditions is as follows:2.5?g/mL LPS stimulation for16h, and3.0×10-6M DANPBO-H incubated for35min. This method not only improves the sensitivity and the quality of the fluorescence imaging, but also can be adapted to monitor process and long-term tracking of the cell activity.(4) NIR DANPBOs can minimize background interference such as absorption and autofluorescence from biological matrices, reduce the scattered light and improve tissues penetration depth and achieve noninvasive imaging. DANPBOs can sensitively capture NO in injured liver and emit bright red fluorescence while there is no obvious fluorescence from normal liver. The results show that DANPBOs have great application potential for in vivo imaging, and the method also provides an effective visual way to study the relationship between the disease and NO.(5) Combined with stereo fluorescence microscope, DANPBO-H has been used to detect the NO in rice radical. Endogenous NO in rice radicle was imaged by using this method, and the results confirmed that NO in rice radical could be produced by nitrogen oxide synthase pathway and nitric acid, nitrite reductase pathway. Furthermore, the production of NO in rice radicals was investigated under various abiotic stress conditions, such as salt, drought and high and low temperature. The results demonstrated that the amount of NO in rice radicle obviously increased under those abiotic stress conditions in addition to high temperature stress condition. The proposed method is fast, simple, intuitive, non-destructive detection of NO in plant tissue, and can be used to study the sources of NO and monitor the slight changes of NO in plants under different conditions.(6) Combined with fluorescence stereo microscope, DANPBO-H was used to study the effect of the plant hormones, such as alpha-naphthalene acetic acid, indole acetic acid, indole butyric acid, gibberellin, abscisic acid, salicylic acid, on the production of NO in rice radicle. The results show that the amount of NO produced in rice radicle has obvious change after treatment with those plant hormones. The fluorescence tissue imaging method can be fast, real-time, sensitive and intuitive to reflect those changes, and is a powerful tool to study the relationships of plant hormones and NO.(7) Using DANPBO-H to derivative NO, a convenient, fast, direct, sensitive fluorescence spectrophotometer method for the determination of NO in biological samples was developed. The linear calibration range is4.0×10-8to5.0×10-7M and the detection limit (S/N=3) is2.3×10-9M. The proposed method has been validated by the determination of NO in gardenia, onions and mung bean sprouts. The spiked recoveries range from95.65to104.76%with RSDs below2.21%. This method can be used to directly detect NO in many biological samples, and has significant values in the analysis and monitoring of NO in conventional analysis.