Boron-chelating fluorescent probe (BOPB) in the red region combined with CE-LIF for the detection of NO in mice liver.

Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8-(3',4'-Diamino phenyl)-3,5-(2-hydroxyphenyl)-dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE-LIF detection. Therefore, BOPB was used in CE-LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB-T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H3 BO3 -NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10(-9) -5.0 × 10(-7) M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization-based CE-LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.

One step physically adsorbed coating of silica capillary with excellent stability for the separation of basic proteins by capillary zone electrophoresis.

The coating of capillary inner surface is considered to be an effective approach to suppress the adsorption of proteins on capillary inner surface in CE. However, most of coating materials reported are water-soluble, which may dissolve in BGE during the procedure of electrophoresis. In this study, a novel strategy for selection of physically coating materials has been illustrated to get coating layer with excellent stability using materials having poor solubility in commonly used solvents. Taking natural chitin as example (not hydrolyzed water soluble chitosan), a simple one step coating method using chitin solution in hexafluoroisopropanol was adopted within only 21 min with good coating reproducibility (RSDs of EOF for within-batch coated capillaries of 1.55% and between-batch coated capillaries of 2.31%), and a separation of four basic proteins on a chitin coated capillary was performed to evaluate the coating efficacy. Using chitin coating, the adsorption of proteins on capillary inner surface was successfully suppressed with reversed and stable EOF, and four basic proteins including lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A were baseline separated within 16 min with satisfied separation efficiency using 20 mM pH 2.0 H3PO4-Na2HPO4 as back ground electrolyte and 20 kV as separation voltage. What is more important, the chitin coating layer could be stable for more than two months during this study, which demonstrates that chitin is an ideal material for preparing semi-permanent coating on bare fused silica capillary inner wall and has hopeful potential in routine separation of proteins with CE.

The natural degradation of benzophenone at low concentration in aquatic environments.

The natural degradation caused by sun irradiation and microbes in aquatic environments is of major significance in the elimination process of benzophenone (BP). In this study, the fate of BP in surface water at a low concentration of 10 µg/L was investigated, including both photodegradation and microbial degradation. The result showed that the photodegradation rate of BP was affected by several parameters such as the initial concentration, continuous input, and the presence of the analogue, ions and small molecules. Meanwhile, the rate of microbial degradation of BP was mainly influenced by the kind and amount of microbes in the environmental water.

Capillary electrophoresis strategy to monitor the released and remaining nitric oxide from the same single cell using a specially designed water-soluble fluorescent probe.

Gasotransmitters including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have attracted more and more attention in the past decades due to their unique signaling and functions. However, as a fundamental issue in the investigations of gasotransmitters, the cell membrane permeability and release behavior of them is controversial in reports because of the lack of an efficient approach to determine gasotransmitters released out of and remaining in the same cells simultaneously. To solve such problem, taking NO as representative, a robust and facile strategy has been reported based on a completely water-soluble fluorescent probe and a commercially available capillary electrophoresis system. A specially designed boron-dipyrromethene (BODIPY)-based fluorescent probe with two sulfonate groups, disodium 2,6-disulfonate-1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl) difluoroboradiaza-s-indance (TMDSDAB), has been developed. As a turn-on fluorescent probe, TMDSDAB can react with NO promptly in aqueous media, and 540-fold enhancement of fluorescence is obtained. Using TMDSDAB, the trapping and quantification of NO released out of and remaining in the same single cell was achieved by capillary electrophoresis with laser-induced fluorescence detection. The limit of detection is 0.5 nM for NO. The proposed method has been applied to estimate the release behavior of single macrophages, and the results indicated that the cell membrane should be a barrier to NO diffusion.

A new BODIPY-based long-wavelength fluorescent probe for chromatographic analysis of low-molecular-weight thiols.

A new long-wavelength fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene (DMDSPAB-I), was designed and synthesized for thiol labeling in high-performance liquid chromatography (HPLC). The excitation and emission wavelengths of DMDSPAB-I are 620 and 630 nm, respectively, with a high fluorescence quantum yield of 0.557, which is advantageous in preventing interference of intrinsic fluorescence from complex biological matrices and enabling high sensitivity HPLC. Based on DMDSPAB-I, a reversed-phase HPLC method was developed for measuring low-molecular-weight thiols including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. After the specific reaction of DMDSPAB-I with thiols, baseline separation of all six stable derivatives was achieved through isocratic elution on a C18 column within 25 min, with the limits of detection (signal-to-noise ratio = 3) from 0.24 nmol L(-1) for glutathione to 0.72 nmol L(-1) for penicillamine. The proposed method was validated in part by measuring thiols in blood samples from mice, with recoveries of 95.3-104.3%.

Simultaneous determination of primary and secondary phenethylamines in biological samples by high-performance liquid chromatographic method with fluorescence detection.

Phenylalanine is an essential amino acid and its metabolites relate to various physiological and immune functions of living organisms. To monitor the alteration of concentration of primary and secondary phenethylamines including N-methyltyramine, octopamine, tyramine, tyrosine and phenylalanine in the metabolic pathway of phenylalanine, a sensitive and selective reversed-phase high-performance liquid chromatographic method has been developed in this study. The identification and quantification of phenethylamines were performed by fluorescent detection after pre-column derivatization with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene, an excellent fluorescent probe which could react with both primary and secondary amino groups simultaneously. The derivatization was carried out at 25 °C for 25 min, and the separation was performed on a C18 column within 20 min. The linear ranges were from 2.0 to 100 nM for phenylalanine and tyramine to 5.0 to 250 for tyrosine and octopamine, with the detection limits of 0.1 nM for octopamine, tyramine, tyrosine and phenylalanine and 0.2 nM for N-methyltyramine (signal-to-noise ratio=3), which allowed for the sure determination of phenethylamines at trace levels in the real samples without complex pretreatment or enrichment during multitudinous samples analysis. The proposed method has been validated by the analysis of the five target compounds in biological samples with spiked recoveries of 96.4-104.4% and the relative standard deviation of 1.0 and 4.4%.

Dual labeling for simultaneous determination of nitric oxide, glutathione and cysteine in macrophage RAW264.7 cells by microchip electrophoresis with fluorescence detection.

A simple, rapid and efficient method based on microchip electrophoresis coupled with fluorescence detection (MCE-FLD) was developed for simultaneous determination of nitric oxide (NO), glutathione (GSH) and cysteine (Cys) using dual labeling strategy. Two highly reactive fluorogenic probes, 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene (DAMBO) and 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), were used for labeling NO and thiols, respectively, under physiological conditions. The rapid separation and sensitive detection of the derivatives were achieved on a glass microchip within 70s in a running buffer of 20mM H3Cit-Na2HPO4 solution (pH 7.4) containing 15% (v/v) acetonitrile at a separation voltage of 2400V. The limits of detection (S/N=3) for NO, GSH and Cys were 7.0, 3.0 and 2.0nM, respectively. The proposed method was validated by measuring intracellular levels of NO and biothiols in macrophage RAW264.7 cells.

Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near-infrared laser-induced fluorescence detection using a new BODIPY-based probe as labeling reagent.

A CZE with near-infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ-glutamylcysteine, cysteinylglycine, and N-acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N-acetylcysteine to 0.31 nmol/L for γ-glutamylcysteine, which are better than or comparable to those reported with other derivatization-based CE-LIF methods. As the first trial of NIR CE-LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5-104.3%.

Subacute effect of decabromodiphenyl ethane on hepatotoxicity and hepatic enzyme activity in rats.

Information regarding decabromodiphenyl ethane (DBDPE) effects on hepatotoxicity and metabolism is limited. In the present study, Wistar rats were given oral DBDPE at different doses. DBDPE induced oxidative stress, elevated blood glucose levels, increased CYP2B2 mRNA, CYP2B1/2 protein, 7-pentoxyresorufin O-depentylase (PROD) activity, and induced CYP3A2 mRNA, CYP3A2 protein, and luciferin benzylether debenzylase (LBD) activity. UDPGT activity increased with its increasing exposure levels, suggesting that oral DBDPE exposure induces drug-metabolizing enzymes in rats via the CAR/PXR signaling pathway. The induction of CYPs and co-regulated enzymes of phase II biotransformation may affect the homeostasis of endogenous substrates, including thyroid hormones, which may, in turn, alter glucose metabolism.

Highly sensitive low-background fluorescent probes for imaging of nitric oxide in cells and tissues.

Small-molecule fluorescent probes in combination with fluorescent microscopy can be a powerful tool to provide real-time detection and high spatiotemporal resolution of transient molecules in cells and bodies. For the design of fluorescent probes for transient molecule imaging, high detection sensitivity is crucial. In this report, two new fluorescent probes, 8-(3,4-diaminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro)naphtho[b,g]-s-indacene (DANPBO-H) and 8-(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), have been developed for nitric oxide (NO) imaging. The detection sensitivity has been efficiently improved by use of these probes through increasing NO detection signals and decreasing background fluorescence. Fluorescence in the far-red region is enhanced by 400- and 550-fold after reaction with NO is achieved and remains stable for at least 24 h under the irradiation of xenon lamp. Excitation and emission wavelengths longer than 600 nm and excellent intracellular retention of these probes and their NO products create dark background inside and outside cells and tissues. What is more, the excellent intracellular retention of these compounds is obtained by their strong lipophilicity, which is a novel design concept diametrically opposite to the traditional approaches. The high sensitivity and dark background make DANPBO-H and DANPBO-M competitive for NO imaging in cells and tissues. The lipophilicity-based intracellular retention mechanism as a design strategy has great potential in the development of fluorescent probes for bioimaging.

Development of a potential method based on microchip electrophoresis with fluorescence detection for the sensitive determination of intracellular thiols in RAW264.7 cells.

This paper, for the first time, reported the development of a simple, rapid, and reliable method for the separation and sensitive determination of four thiol compounds including homocysteine, cysteine, glutathione, and N-acetylcysteine based on glass MCE with fluorescence detection using a highly reactive fluorogenic probe, 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), as the labeling reagent. TMPAB-o-M reacted selectively with thiols to produce highly fluorescent derivatives and the highest derivatization efficiency was achieved within 6 min in physiological conditions. After the optimization of separation conditions, a baseline separation of the four thiol compounds was achieved with the detection limits ranging from 2 nM for glutathione to 4 nM for cysteine (S/N = 3) and RSDs (n = 5) in the range of 3.2-3.8%. The proposed method was significantly sensitive compared to those using electrochemical or even LIF detection in MCE-based setup reported previously, and applied to the determination of intracellular thiols in macrophage RAW264.7 cells.

N-methyl-2-pyrrolidonium methyl sulfonate acidic ionic liquid as a new dynamic coating for separation of basic proteins by capillary electrophoresis.

A simple and economical CE method has been developed for the analysis of four model basic proteins by employing N-methyl-2-pyrrolidonium methyl sulfonate ionic liquid (IL) as the dynamic coating material based on the interaction of both between electrostatic attraction and hydrogen bond, and between the organic cations of IL and the inner surface of bare fused-silica capillary. The N-methyl-2-pyrrolidonium-based IL modified capillary not only generated a stable suppressed electroosmotic flow, but also effectively eliminated the wall adsorption of proteins. Several important parameters such as the IL concentration, pH values, and concentrations of the background electrolyte were optimized to improve the separation of basic proteins. Consequently, under the optimum separation conditions, a satisfied separation of basic proteins including lysozyme, cytochrome c, ribonuclease A, and α-chymotrypsinogen A with theoretical plates ranging from 2.09 × 10(5) to 4.48 × 10(5) plates/m had been accomplished within 15 min. The proposed method first illustrated the effect of hydrogen bond between coating material and inner capillary surface on the coating, which should be a new strategy to design and select more effective coating materials to form more stable coatings in CE.

Novel B,O-chelated fluorescent probe for nitric oxide imaging in Raw 264.7 macrophages and onion tissues.

A novel fluorescent probe based on B,O-chelated dipyrromethene chromophore in far-visible and near-infrared spectral region (600-900 nm), boron chelated 8-(3,4-diaminophenyl)-3,5-bis(2-hydroxyphenyl)-4-bora-3a,4a-diaza-s-indancene (BOPB), has been first developed for nitric oxide (NO) imaging. BOPB, a turn-on fluorescent probe, can react with NO rapidly under physiological condition. The reaction product of BOPB with NO, BOPB-T, emits bright red fluorescence at 643 nm when excited at 622 nm. Meanwhile, BOPB-T displays high fluorescent quantum yield of 0.21 and good photostability. The selectivity for NO over other reactive oxygen/nitrogen species and ascorbic acid has been investigated and BOPB has good specificity for the detection of NO. MTT assay shows that the toxicity of BOPB (below 10 µM) to living cells can be neglected. Based on these investigations, BOPB has been used for NO imaging in Raw 264.7 cells and onion tissues. Meanwhile, mechanical injury to onion tissues results in a brighter fluorescence around the wound, which indicates that more NO has been produced in plant tissues in response to external stimuli. Our studies illustrate that BOPB has advantages of high sensitivity, low background interference and little photo damage on fluorescence imaging of NO.

Highly sensitive determination of nitric oxide in biologic samples by a near-infrared BODIPY-based fluorescent probe coupled with high-performance liquid chromatography.

Nitric oxide (NO) acts as an important regulator and mediator in numerous processes of biological systems. In this work, the analytical potential of a novel near-infrared (NIR, >600 nm) BODIPY-based fluorescent probe for NO, 8-(3,4-diaminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro) naphtho[b, g]s-indacene (DANPBO-H) has been evaluated in high performance liquid chromatography (HPLC). In 25 mM pH 6.50 borate buffer, DANPBO-H reacted with NO to give the corresponding triazole, DANPBO-H-T, at 35 °C for 20 min. DANPBO-H-T was eluted using a mobile phase of methanol/tetrahydrofuran/50mM pH 7.00 H3Cit-NaOH buffer (81:7:12, v/v/v) in 4 min on a C8 column and detected with fluorescence detection at excitation and emission wavelengths of 621 and 631 nm, respectively. The limit of detection (LOD) (signal-to-noise=3) reached to 5.50×10(-10) M. Excellent selectivity was observed against other reactive oxygen/nitrogen species. Various representative biological matrixes including the whole blood and organs of mice, the pangen and radical of rice, human vascular endothelial (ECV-304) cells and mouse macrophage (RAW 264.7) cells were used to verify the feasibility and resistance to interfering effects from complex biological sample matrixes of the developed DANPBO-H-based HPLC method. Compared to the existing derivatization-based HPLC methods for NO, the proposed method eliminates interfering effects from complex biological sample matrixes efficiently owing to the fluorescence detection in the NIR region, and is more advantageous and robust for the sensitive and selective determination of NO in complex biological samples.

Determination of neurotransmitter amino acids in mouse central nervous system by CE-LIF.

An MEKC method with LIF detection has been developed for the determination of seven neurotransmitter amino acids (NAAs) using 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-S-indacene as the labeling reagent. After derivatization at room temperature for 30 min, the seven target NAAs including glycine, alanine, γ-aminobutyric acid, taurine, glutamine, glutamic acid, and aspartic acid were separated in running buffer, which consisted of 70 mM pH 4.00 H3 PO4 /Na3 PO4 buffer, 5.5 mM cetyltrimethyl ammonium bromide and 20% v/v acetonitrile within 17 min. The LODs were 2 ~ 14 × 10(-10) M without interference from other coexisting amino acids. The proposed method has been applied to the analysis of NAAs in the central nervous systems of healthy mice and those with Alzheimer's disease with recoveries of 92-104%.

"Off-on" red-emitting fluorescent probes with large Stokes shifts for nitric oxide imaging in living cells.

Fluorescent probes with larger Stokes shifts in the far-visible and near-infrared spectral region (600-900 nm) are more superior for cellular imaging and biological analysis due to avoiding light scattering interference, reducing autofluorescence from biological sample and encouraging deeper tissue penetration in vivo imaging. In this work, two bis-methoxyphenyl-BODIPY fluorescent probes for the detection of nitric oxide (NO) have been firstly synthesized. Under physiological conditions, these probes can react with NO to form the corresponding triazoles with 250- and 70-fold turn-on fluorescence emitting at 590 and 620 nm, respectively. Moreover, the triazole forms of these probes have large Stokes shifts of 38 nm, in contrast to 10 nm of existing BODIPY probes for NO. Excellent selectivity has been observed against other reactive oxygen/nitrogen species, ascorbic acid and biological matrix. After the evaluation of MTT assay, new fluorescent probes have been successfully applied to fluorescence imaging of NO released from RAW 264.7 macrophages by co-stimulation of lipopolysaccharide and interferon-γ. The experimental results indicate that our fluorescent probes can be powerful candidates for fluorescence imaging of NO due to the low background interference and high detection sensitivity.

Determination of fatty acids in bio-samples based on the pre-column fluorescence derivatization with 1,3,5,7-tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene by high performance liquid chromatography.

1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene (TMBB-EDAN) has been designed and synthesized as a highly fluorescent labeling reagent for carboxylic acids. By using TMBB-EDAN, a sensitive and rapid method based on high performance liquid chromatography-fluorescence detection for the determination of twelve fatty acids (FAs) in bio-samples has been developed. Under optimized conditions, these FAs were tagged with TMBB-EDAN in the presence of 1-ethyl-3-(3-dimethyla-minopropyl) carbodiamide at 20°C for 30min and then the baseline separation was achieved on a C18 column with a linear gradient elution in 26min. With fluorescence detection at λex/λem=490nm/510nm, the linear ranges of FAs were from 3.0 to 300nM and the detection limits with a signal-to-noise ratio of 3 were in the 0.2-0.4nM range. The proposed method offers advantages of milder derivatization condition and much better sensitivity for the determination of FAs, when compared to the reported fluorescence derivatization-based methods.

Determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene.

Altered levels of thiols in biological fluids are considered to be an important indicator for several diseases. In this article, 1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene is proposed as a fluorescent derivatization reagent for the determination of thiols including glutathione, cysteine, N-acetylcysteine, and homocysteine by HPLC. Under the optimized derivatization and separation conditions, a baseline separation of all the four derivatives has been achieved using isocratic elution on an RP C(8) column within 26 min. With fluorescence detection at 505 and 525 nm for the excitation and emission, respectively, the LODs (S/N = 3) are from 0.2 nM (glutathione) to 0.8 nM (cysteine). The feasibility of this method in real samples has been evaluated by the determination of thiols in human plasma from the healthy persons and hypertensive patients with recoveries of 92-105.3%.

Impact of traffic emissions on local air quality and the potential toxicity of traffic-related particulates in Beijing, China.

OBJECTIVE: Air-borne particulates from different sources could have different physicochemical properties and inflammatory potentials. This study aims to characterize the chemical compositions and the toxicity of ambient particulate matter (PM) associated with traffic emissions. METHODS: The concentrations of trace elements, organic carbon (OC), elemental carbon (EC) and polycyclic aromatic hydrocarbons (PAHs) in PM2.5 and PM10 were measured in samples collected at sites in Beijing, China. Their toxic effects on the pulmonary system of rats were investigated. Biochemical parameters (LDH, T-AOC, TP) and inflammatory cytokine(IL-6, IL-1, TNF-a) levels were measured in the lungs of rats exposed to traffic-related PM. Oxidative damage was observed. PM samples were taken from a near road site and an off road site in summer time in 2006. RESULTS: The concentrations of the USEPA priority pollutant PAHs in both PM10 and PM2.5 were higher (299.658 and 348.412) at the near road site than those (237.728 and 268.472) at the off road site. The similar trend was observed for the concentrations of trace elements in PM. Compared to coarse particles (PM10), fine particles (PM2.5) have a greater adsorption capacity to enrich toxic elements than inhalable particles. Decrease in antioxidant capacity and an increase in the amount of lipid peroxidation products in rat lung tissues was observed. CONCLUSION: The findings of the present study suggest that the differing inflammatory responses of PM collected from the two road sites might have been mediated by the differing physicochemical characteristics.

Determination of thiols by capillary micellar electrokinetic chromatography with laser induced fluorescence detection using 1,3,5,7-tetramethyl-8-phenyl-(4-iodoacetamido) difluoroboradiaza-s-indacene as labeling reagent.

A sensitive and effective micellar electrokinetic capillary chromatography with laser-induced fluorescence detection approach was described for the determination of low molecular-mass thiols using 1,3,5,7-tetramethyl-8-phenyl-(4-iodoacetamido) difluoroboradiaza-s-indacene as the labeling reagent. After precolumn derivatization, baseline separation of six thiol compounds including cysteine, glutathione, N-acetylcysteine, homocysteine, 6-mercaptopurine, and penicillamine were achieved within 18 min. The optimal running buffer was composed of mixtures involving 25 mM sodium dodecyl sulfate, 25% (v/v) acetonitrile and 15 mM sodium phosphate buffer, pH 7.5. The detection limits (S/N = 3) were found as low as 40 pM under argon ion laser-induced fluorescence detector (λ(ex)/λ(em) = 488/520 nm), which were much better than the reported approaches. The accuracy and specificity of this assay for real samples were assured by a standard addition method. The proposed method has been applied to the analysis of thiols both in human plasma and plum flower samples with recoveries of 92.0-109.4%.