ABSTRACT
Attention deficit hyperactivity disorder (ADHD) is a neurological condition frequently identified in early childhood and frequently co-occurs with other neuropsychological disorders, particularly autism. Viloxazine hydrochloride, a non-stimulant medication, has recently gained approval for treating attention-deficit hyperactivity disorder. This paper describes the first spectrofluorimetric method for precisely measuring the content of viloxazine in pharmaceutical capsules and rat plasma. This method employed NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) as a fluorescent probe, which transformed viloxazine in an alkaline environment into a remarkably sensitive fluorescent adduct. Upon excitation at 476 nm, this adduct becomes detectable at a wavelength of 536 nm. The method was validated using ICH criteria, revealing acceptable linearity across a concentration range of 200-2000 ng/ml and high sensitivity with LOD and LOQ values of 46.774 ng/ml and 141.741 ng/ml, respectively. This method was adeptly applied in a pharmacokinetic study of viloxazine in rat plasma following a single oral dose (10 mg/kg), yielding a mean peak plasma concentration (Cmax) of 1721 ng/ml, achieved within 1.5 h. Furthermore, the environmental impact of the technique was assessed using two greenness assessment tools, revealing a notable level of eco-friendliness and sustainability.
Subject(s)
Fluorescent Dyes , Spectrometry, Fluorescence , Viloxazine , Animals , Rats , Fluorescent Dyes/chemistry , Viloxazine/chemistry , Viloxazine/pharmacokinetics , Viloxazine/blood , Male , Molecular Structure , 4-Chloro-7-nitrobenzofurazan/chemistry , Administration, OralABSTRACT
High fructose intake is an important cause of metabolic disease. Due to the increasing prevalence of metabolic diseases worldwide, the development of an accurate and efficient tool for monitoring fructose in food is urgently needed to control the intake of fructose. Herein, a new fluorescent probe NBD-PQ-B with 7-nitrobenz-2-oxa-1, 3-diazole (NBD) as the fluorophore, piperazine (PQ) as the bridging group and phenylboronic acid (B) as the recognition receptor, was synthesized to detect fructose. The fluorescence of NBD-PQ-B increased linearly at 550 nm at an excitation wavelength of 497 nm with increasing fructose concentration from 0.1 to 20 mM. The limit of detection (LOD) of fructose was 40 µM. The pKa values of NBD-PQ-B and its fructose complexes were 4.1 and 10.0, respectively. In addition, NBD-PQ-B bound to fructose in a few seconds. The present technique was applied to determine the fructose content in beverages, honey, and watermelon with satisfactory results. Finally, the system could not only be applied in an aqueous solution with a spectrophotometer, but also be fabricated as a NBD-PQ-B/polyvinyl oxide (PEO) film by electrospinning for on-site food analysis simply with the assistance of a smartphone.
Subject(s)
Fluorescent Dyes , Food Analysis , Fructose , Spectrometry, Fluorescence , Fructose/analysis , Fluorescent Dyes/chemistry , Spectrometry, Fluorescence/methods , Food Analysis/methods , Limit of Detection , Honey/analysis , Beverages/analysis , 4-Chloro-7-nitrobenzofurazan/chemistryABSTRACT
Influenza viruses represent a major threat to human health and are responsible for seasonal epidemics, along with pandemics. Currently, few therapeutic options are available, with most drugs being at risk of the insurgence of resistant strains. Hence, novel approaches targeting less explored pathways are urgently needed. In this work, we assayed a library of nitrobenzoxadiazole derivatives against the influenza virus A/Puerto Rico/8/34 H1N1 (PR8) strain. We identified three promising 4-thioether substituted nitrobenzoxadiazoles (12, 17, and 25) that were able to inhibit viral replication at low micromolar concentrations in two different infected cell lines using a haemagglutination assay. We further assessed these molecules using an In-Cell Western assay, which confirmed their potency in the low micromolar range. Among the three molecules, 12 and 25 displayed the most favourable profile of activity and selectivity and were selected as hit compounds for future optimisation studies.
Subject(s)
4-Chloro-7-nitrobenzofurazan/pharmacology , Antiviral Agents/pharmacology , Influenza A Virus, H1N1 Subtype/drug effects , 4-Chloro-7-nitrobenzofurazan/chemical synthesis , 4-Chloro-7-nitrobenzofurazan/chemistry , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cell Survival/drug effects , Cells, Cultured , Dogs , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity RelationshipABSTRACT
We report a non-antibody GLUT1 inhibitor probe NBDQ that is 30 times more sensitive than the traditional GLUT1 transportable tracer for cancer cell imaging and Warburg effect-based tumor detection. NBDQ reveals significant advantages in terms of tumor selectivity, fluorescence stability and in vivo biocompatibility in xenograft tumor imaging, including triple-negative breast cancer.
Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Biomarkers, Tumor/analysis , Deoxyglucose/analogs & derivatives , Fluorescent Dyes/chemistry , Glucose Transporter Type 1/antagonists & inhibitors , Triple Negative Breast Neoplasms/diagnostic imaging , 4-Chloro-7-nitrobenzofurazan/chemistry , Animals , Biocompatible Materials/chemistry , Cell Line, Tumor , Cell Membrane Permeability , Deoxyglucose/chemistry , Glucose Transporter Type 1/genetics , Humans , Mice , Multimodal Imaging , Neoplasms, Experimental , Optical Imaging , Positron-Emission TomographyABSTRACT
Melanoma cells have high glycolytic capacity. Glucose uptake is a key rate-limiting step in glucose utilization. Here we describe a simple protocol for measuring direct glucose uptake in living melanoma cells by flow cytometry.
Subject(s)
Flow Cytometry/methods , Glucose/metabolism , Melanoma/metabolism , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/chemistry , Biological Transport , Cell Culture Techniques/methods , Cell Line, Tumor , Deoxyglucose/analogs & derivatives , Deoxyglucose/chemistry , Fluorescence , Fluorescent Dyes/chemistry , HumansABSTRACT
The 7-nitrobenzo[c][1,2,5]oxadiazole (NBD) derivative NBDHEX (compound 1) and its analogue MC3181 (compound 2) have been found to be potent inhibitors of tumor cell growth in vitro and therapeutically active and safe in mice bearing human melanoma xenografts. To enhance the aqueous solubility of these compounds, we synthesized the hemisuccinate of 1 (compound 3) and the phosphate monoesters of 1 and 2 (compound 4 and 5, respectively). These novel NBD derivatives displayed a solubility in the conventional phosphate-buffered saline up to 150-fold higher than that of 1, and up to 4-fold higher than that of 2. Notably, solubility of phosphates 4 and 5 in a potassium phosphate buffer at pH 7.4, was up to 500-fold higher than that of 1, and ~10-fold higher than that of 2. Compounds 3-5 retained high cytotoxicity towards cultured human melanoma and osteosarcoma cells and were cleaved in vitro by both human and murine hydrolases, thus releasing the corresponding parent compound (i.e., 1 or 2). Interestingly, esters 3-5 displayed high inhibitory activity towards the glutathione transferase (GST) isoform GSTP1-1 and showed a reactivity towards reduced glutathione comparable to that of the respective parent compound. Finally, both 4 and 5 were safe and effective when administered intravenously or orally as an aqueous solution to mice xenografted with A375 human melanoma tumors. Collectively, these results and the previously observed synergistic interaction between 1 and 2 and various approved anticancer drugs, suggest the possible utility of phosphates 4 and 5 as single agents and in combination regimens in cancers with unmet medical need, including melanoma.
Subject(s)
4-Chloro-7-nitrobenzofurazan/metabolism , Antineoplastic Agents/metabolism , Glutathione S-Transferase pi/antagonists & inhibitors , Glutathione S-Transferase pi/metabolism , Neoplasms/metabolism , Water/metabolism , 4-Chloro-7-nitrobenzofurazan/chemistry , 4-Chloro-7-nitrobenzofurazan/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Esters/chemistry , Esters/metabolism , Female , Glutathione Transferase/antagonists & inhibitors , Glutathione Transferase/metabolism , Humans , Male , Mice , Mice, Nude , Neoplasms/drug therapy , Solubility , Water/chemistry , Xenograft Model Antitumor Assays/methodsABSTRACT
During an effort to find insulin mimetic compounds, the leaves of Gymnema inodorum were shown to have a stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation on a 70% ethanol extract of G. inodorum was applied to yield two new (1 and 2) and two known (8 and 9) oleanane triterpenoids with a methyl anthranilate moiety together with five further new oleanane triterpenoids (3-7). The chemical structures of all isolates were determined based on their spectroscopic data, including IR, UV, NMR, and mass spectrometric analysis. The isolated compounds (1-9) were determined for their stimulatory activities on glucose uptake in differentiated 3T3-L1 adipocyte cells using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe. Three compounds (3, 5, and 9) showed stimulatory effects on the uptake of 2-NBDG in 3T3-L1 adipocyte cells. Chemicals with a methyl anthranilate moiety have been considered as crucial contributors of flavor odor in foods, and quantitative analysis showed the content of compound 8 to be 0.90 ± 0.01 mg/g of the total extract. These results suggest that the leaves of G. inodorum have the potential to be used as an antidiabetic functional food or tea.
Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Deoxyglucose/analogs & derivatives , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Oleanolic Acid/analogs & derivatives , Triterpenes/pharmacology , 3T3-L1 Cells , 4-Chloro-7-nitrobenzofurazan/chemistry , 4-Chloro-7-nitrobenzofurazan/pharmacology , Animals , Biological Transport/drug effects , Cell Differentiation/drug effects , Deoxyglucose/chemistry , Deoxyglucose/pharmacology , Glucose/analysis , Gymnema , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/isolation & purification , Insulin/chemistry , Insulin/metabolism , Mice , Molecular Structure , Oleanolic Acid/chemistry , Oleanolic Acid/isolation & purification , Oleanolic Acid/pharmacology , Plant Leaves , Triterpenes/chemistry , Triterpenes/isolation & purificationABSTRACT
A nitrobenzoxadiazole-based fluoroprobe (NBD-Bu) is designed to probe cellular metabolic activity in cancer and normal cells. NBD-Bu shows a significant fluorescence enhancement upon selective binding to the transport protein serum albumin in PBS buffer at ambient conditions. Encouraged by this finding, the site- specificity of NBD-Bu has been explored through a competitive displacement assay in the presence of site-specific markers such as warfarin and ibuprofen. Notably, even at micromolar concentrations, the probe possesses the ability to displace the site marker drug ibuprofen, efficiently. Subsequently, high-resolution fluorescence imaging results consolidated the potential of NBD-Bu for detection of abnormal cellular metabolic activity.
Subject(s)
4-Chloro-7-nitrobenzofurazan/chemistry , Serum Albumin/chemistry , Animals , CHO Cells , Cell Line , Cricetulus , HeLa Cells , Humans , Magnetic Resonance Spectroscopy , Microscopy, Confocal , Serum Albumin, Bovine/chemistry , Serum Albumin, Human/chemistryABSTRACT
Physiological processes rely on initial recognition events between cellular components and other molecules or modalities. Biomolecules can have multiple sites or mode of interaction with other molecular entities, so that a resolution of the individual binding events in terms of spatial localization as well as association and dissociation kinetics is required for a meaningful description. Here we describe a trichromatic fluorescent binding- and displacement assay for simultaneous monitoring of three individual binding sites in the important transporter and binding protein human serum albumin. Independent investigations of binding events by X-ray crystallography and time-resolved dynamics measurements (switchSENSE technology) confirm the validity of the assay, the localization of binding sites and furthermore reveal conformational changes associated with ligand binding. The described assay system allows for the detailed characterization of albumin-binding drugs and is therefore well-suited for prediction of drug-drug and drug-food interactions. Moreover, conformational changes, usually associated with binding events, can also be analyzed.
Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Boron Compounds/chemistry , Ibuprofen/chemistry , Lauric Acids/chemistry , Serum Albumin, Human/chemistry , Warfarin/chemistry , 4-Chloro-7-nitrobenzofurazan/chemistry , Binding Sites , Crystallography, X-Ray , Fluorescence , Humans , Molecular Dynamics Simulation , Molecular StructureABSTRACT
The demanding metabolic needs of cancer cells are met by aerobic glycolysis. While whole-body PET imaging methods exist for evaluating this metabolic response, these are not ideal for local, more detailed regions such as mucosal surfaces. Fluorescence imaging of glucose analogs with similarities to radiolabeled deoxyglucose used in PET, namely, fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), offers such an alternative, particularly as this glucose analog may be delivered by local topical delivery. In this chapter, methods for in vivo epithelial imaging in a preclinical hamster model for oral cancer and oral epithelial dysplasia are described. Outlined are methods for preparation and in vivo delivery of 2-NBDG by topical application to the oral mucosa followed by fluorescence imaging to compare fluorescence responses between neoplasia and control mucosa or to monitor changes in fluorescence signal with time in both groups.
Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Carcinoma, Squamous Cell/metabolism , Deoxyglucose/analogs & derivatives , Fluorescent Dyes/chemistry , Glucose/metabolism , Intravital Microscopy/methods , Mouth Neoplasms/metabolism , Neoplasms, Experimental/metabolism , 4-Chloro-7-nitrobenzofurazan/administration & dosage , 4-Chloro-7-nitrobenzofurazan/chemistry , Administration, Topical , Animals , Carcinoma, Squamous Cell/pathology , Deoxyglucose/administration & dosage , Deoxyglucose/chemistry , Mesocricetus , Mouth Neoplasms/pathology , Neoplasms, Experimental/pathologyABSTRACT
Direct determination of linagliptin (LIN) using fluorimetry has been limited because LIN releases a very weak fluorescence signal. Accordingly, it should be derivatized first with a fluorogenic reagent to enhance its fluorescence and consequentially the sensitivity required for its bioanalysis. This is the first description of a spectrofluorimetric method for LIN quantification in human plasma. The suggested method exploits the nucleophilic nature of its amino group to react with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer at pH 8.5 to yield a strong fluorescent product with excitation and emission wavelengths of 459 and 529 nm, respectively. Experimental variables concerning the conditions of reaction and fluorescence intensity were carefully investigated and optimized. The linearity range was from 1.0-100 ng ml-1 with a lower detection limit and a lower quantification limit of 0.60 ng ml-1 and 1.82 ng ml-1 , respectively. Validation of the suggested method has been accomplished and the application to LIN analysis in commercial tablets as well as in human plasma resulted in a mean per cent recovery of 100.12 ± 1.57 and 99.65 ± 1.22, respectively. The developed method was proven to be a promising, simple and fast alternative bioanalytical method for LIN quantification in clinical and bioequivalence research.
Subject(s)
4-Chloro-7-nitrobenzofurazan/chemistry , Body Fluids/chemistry , Linagliptin/blood , Humans , Linagliptin/chemistry , Molecular Structure , Spectrometry, FluorescenceABSTRACT
Cerebral glycogen is principally localized in astrocytes rather than in neurons. Glycogen metabolism has been implicated in higher brain functions, including learning and memory, yet the distribution patterns of glycogen in different types of astrocytes have not been fully described. Here, we applied a method based on the incorporation of 2-NBDG, a D-glucose fluorescent derivative that can trace glycogen, to investigate glycogen's distribution in the brain. We identified two types of astrocytes, namely, 2-NBDGI (glycogen-deficient) and 2-NBDGII (glycogen-rich) cells. Whole-cell patch-clamp and fluorescence-activated cell sorting (FACS) were used to separate 2-NBDGII astrocytes from 2-NBDGI astrocytes. The expression levels of glycogen metabolic enzymes were analyzed in 2-NBDGI and 2-NBDGII astrocytes. We found unique glycogen metabolic patterns between 2-NBDGI and 2-NBDGII astrocytes. We also observed that 2-NBDGII astrocytes were mainly identified as fibrous astrocytes but not protoplasmic astrocytes. Our data reveal cell type-dependent glycogen distribution and metabolism patterns, suggesting diverse functions of these different astrocytes.
Subject(s)
Astrocytes/metabolism , Glycogen/metabolism , Single-Cell Analysis/methods , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/chemistry , Animals , Astrocytes/chemistry , Cells, Cultured , Cerebral Cortex/metabolism , Deoxyglucose/analogs & derivatives , Deoxyglucose/chemistry , Glucose , Glycogen/analysis , Glycogen/deficiency , Mice , Mice, Inbred C57BL , Neurons/metabolismABSTRACT
A sensitive dual mode chemosensor NS-1 comprising Nitrobenzoxadiazole and salicylhydrazide conjugate has been synthesized via single step reaction. The probe NS-1 is characterized by analytical techniques such as multi nuclear NMR techniques and Mass spectrometry. The probe is showing a strong change in color from yellow to red on treatment of Cd(II) ions, interestingly its shows bright "Switch-ON" fluorescence state upon binding of Cd2+ ions in buffer solution whereas other cations did not showed any color change as well as fluorescent change. Interestingly the probe NS-1 did not results in the any color and fluorescence change with the adding together of Zn(II) ions, hence the probe is able to differentiate between Cd(II) ions from Zn(II). Further the color change and turn-on fluorescence can be rationalized by the interruption of internal charge transfer upon binding of Cd(II) ions with NS-1. The Internal charge transfer disturbance led to fluorescence change of the receptor NS-1 upon addition of Cd2+ has been further supported by TD-DFT calculations. The limit of detection was found to be 6.31 nano molar. The association constant was found to be 7.97*104 M-1 using Benesi-Hildebrand plot method. MTT assay suggesting that the probe NS-1 is least toxic to cells and it will be widely applicable to image Cd(II) ions in living cells via fluorescence imaging.
Subject(s)
Cadmium/analysis , Microscopy, Fluorescence , 4-Chloro-7-nitrobenzofurazan/chemistry , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Ions/chemistry , Limit of Detection , Spectrometry, Fluorescence/methodsABSTRACT
The direct determination of alogliptin benzoate (ALO) using fluorescence has not yet been accomplished because ALO cannot fluoresce naturally. Accordingly, it should be derivatized first with a fluorogenic reagent to enhance the sensitivity required for its bioanalysis. This method is the first spectrofluorimetric assay for ALO quantification exploiting the nucleophilic nature of its amino group to react with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer at pH 8.5 to produce a strong fluorescent compound that is excited at and emits at wavelengths 470 and 527 nm, respectively. Experimental variables concerning the conditions of reaction and fluorogenic intensity were carefully investigated and optimized. Linearity was from 1-250 ng ml-1 with a lower detection limit of 0.29 ng ml-1 and a lower quantification limit of 0.88 ng ml-1 . Validation of the current study was accomplished with mean per cent recovery of 100.62 ± 1.59 in tablets and 99.86 ± 0.82 in human plasma. Furthermore, the current method has been utilized in the bioanalysis of ALO in real rat plasma after oral administration with a simple specimen preparation. The developed method has proven to be a promising alternative method for ALO analysis in bioequivalence studies.
Subject(s)
4-Chloro-7-nitrobenzofurazan/chemistry , Benzoates/blood , Fluorescent Dyes/chemistry , Piperidines/blood , Spectrometry, Fluorescence , Uracil/analogs & derivatives , Animals , Benzoates/chemistry , Benzoates/pharmacokinetics , Humans , Male , Molecular Structure , Piperidines/chemistry , Piperidines/pharmacokinetics , Quantum Theory , Rats , Rats, Wistar , Spectrometry, Fluorescence/instrumentation , Uracil/blood , Uracil/chemistry , Uracil/pharmacokineticsABSTRACT
The antitumor agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (1) is a potent inhibitor of GSTP1-1, a glutathione S-transferase capable of inhibiting apoptosis by binding to JNK1 and TRAF2. We recently demonstrated that, unlike its parent compound, the benzoyl ester of 1 (compound 3) exhibits negligible reactivity towards GSH, and has a different mode of interaction with GSTP1-1. Unfortunately, 3 is susceptible to rapid metabolic hydrolysis. In an effort to improve the metabolic stability of 3, its ester group has been replaced by an amide, leading to N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexyl)benzamide (4). Unlike 3, compound 4 was stable to human liver microsomal carboxylesterases, but retained the ability to disrupt the interaction between GSTP1-1 and TRAF2 regardless of GSH levels. Moreover, 4 exhibited both a higher stability in the presence of GSH and a greater cytotoxicity towards cultured A375 melanoma cells, in comparison with 1 and its analog 2. These findings suggest that 4 deserves further preclinical testing.
Subject(s)
4-Chloro-7-nitrobenzofurazan/pharmacology , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Glutathione S-Transferase pi/antagonists & inhibitors , 4-Chloro-7-nitrobenzofurazan/chemical synthesis , 4-Chloro-7-nitrobenzofurazan/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Benzamides/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Glutathione S-Transferase pi/metabolism , Humans , Hydrolysis , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Asymmetrical lipid nanoparticles are interesting nanocarriers for charged molecules, like nucleic acids. They promise control over inner and outer charge. High charge density on the inside is favourable for efficient condensation and charge neutralisation of highly charged biopharmaceuticals, while a neutral or slightly negative outer layer promotes biocompatibility. The main goal of this work was the development and characterisation of asymmetric liposomes, prepared using water-in-oil (w/o) nanoemulsions of phospholipids (PLs) and squalene in a centrifugal field. This method enables the control over the lipid composition of each monolayer. Liposomes were prepared by passing PL w/o nanoemulsions through an oil-water interface previously saturated with PLs. We used N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE) or N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3- phosphocholine (NBD-PC) as a fluorescent marker for either the inner or outer lipid layer and plasmid DNA (pDNA) as nucleic acid payload. The final liposomes had sizes below 200 nm and polydispersity indexes of 0.3 and had a bilayer asymmetry of 70%, thus shielding the charge of positive PLs in the inner bilayer leaflet. Final formulations were examined using negative staining transmission electron microscopy (TEM). Plasmid encapsulation efficiency of the method was 10-15%. Our results indicate that the w/o nanoemulsion-centrifugation method allows the successful production of liposomes with tailored features for encapsulation of nucleic acid therapeutics.
Subject(s)
Emulsions/chemistry , Liposomes/chemistry , Nanoparticles/chemistry , Nucleic Acids/chemistry , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/chemistry , Fluorescent Dyes , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Phospholipids/chemistry , Squalene/chemistryABSTRACT
A long-wavelength fluorescent probe NR-CY was developed for simultaneous identification of cysteine/glutathione and sulphide by combining the derivative of Nile red with 7-nitrobenzofurazan. The response of NR-CY to thiols is regulated by intramolecular charge transfer and photoinduced electron transfer mechanisms. For sulphide at 560â¯nm, cysteine at 475â¯nm and glutathione at 425â¯nm, different absorbance increases can be observed. NR-CY can detect cysteine at fluorescence emission 543â¯nm and distinguish sulphide from other analytes by kinetic experiments at 636â¯nm. The probe showed a rapid response to these thiols (cysteine was 90â¯s and sulphide was 30â¯s). In addition, NR-CY has been successfully applied to live MCF-7 cell imaging.
Subject(s)
4-Chloro-7-nitrobenzofurazan/chemistry , Cysteine/analysis , Fluorescent Dyes/chemistry , Glutathione/analysis , Homocysteine/analysis , Oxazines/chemistry , Cysteine/chemistry , Glutathione/chemistry , Homocysteine/chemistry , Humans , MCF-7 CellsABSTRACT
Deoxyribonucleic acid (DNA) has been used as a material for a variety of applications, including surface functionalization for cell biological or in vitro reconstitution studies. Use of DNA-based surface functionalization eliminates limitations of multiplexing posed by traditionally used methods in applications requiring spatially segregated surface functionalization. Recently, we have reported a stochastic, membrane fusion-based strategy to fabricate multicomponent membrane array substrates displaying spatially segregated protein ligands using biotin-streptavidin and Ni-NTA-polyhistidine interactions. Here, we report the delivery of DNA oligonucleotide-conjugated lipid molecules to membrane corrals, allowing spatially segregated membrane corral functionalization in a membrane microarray. Incubation of microbeads coated with the supported membrane resulted in an exchange of lipid contents with planar membrane corrals present on a micropatterned substrate. Increases in the system temperature and membrane corral size resulted in alterations in the rate constant of lipid exchange, which are in agreement with our previously developed analytical model and further confirm that lipid exchange is a diffusion-based process that takes place after the formation of a long "fusion-stalk" between the two membranes. We take advantage of the physical dimensions of the fusion-stalk with a large aspect ratio to deliver DNA oligonucleotide-conjugated lipid molecules to membrane corrals. We believe that the ability to functionalize membrane corrals with DNA oligonucleotides significantly increases the utility of the stochastic fusion-mediated lipid delivery strategy in the functionalization of biomolecules such as DNA or DNA-conjugated protein ligands.
Subject(s)
DNA/chemistry , Lipid Bilayers/chemistry , Oligodeoxyribonucleotides/chemistry , Streptavidin/chemistry , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/chemistry , Biotin/chemistry , Diffusion , Membrane Microdomains , Microspheres , Particle Size , Phosphatidylcholines/chemistryABSTRACT
Nowadays, the safety of cosmetics is a widespread concern. Amines are common cosmetic additives. Some of them such as amino acids are beneficial. Another kind of amines, however, ε-aminocaproic acid (EACA) is prohibited to add into cosmetics for its adverse reactions. In this study, a simple, rapid, sensitive and eco-friendly one-step ultrasonic-assisted extraction and derivatization (UAE-D) method was developed for determination of EACA and amino acids in cosmetics by coupling with high-performance liquid chromatography (HPLC). By using this sample preparation method, extraction and derivatization of EACA and amino acids were finished in one step in ultrasound field. During this procedure, 4-fluoro-7-nitrobenzofurazan (NBD-F)was applied as derivatization reagent. The extraction conditions including the amount of NBD-F, extraction and derivatization temperature, the ultrasonic vibration time and pH value of the aqueous phase were evaluated. Meanwhile, the extraction mechanism was investigated. Under optimized conditions, the method detection limits were 0.086-0.15⯵g/L, and method quantitation limits were 0.29-0.47⯵g/L with RSDs less than 3.7% (nâ¯=â¯3). The recoveries of EACA and amino acids obtained from cosmetic samples were in range from 76.9% to 122.3%. Amino acids were found in all selected samples and quantified in range from 1.9⯱â¯0.9 to 677.2⯱â¯17.9⯵g/kg. And EACA was found and quantified with the contents of 1284.3⯱â¯22.1⯵g/kg in a toner sample. This UAE-D-HPLC method shortened and simplified the sample pretreatment as well as enhanced the sensitivity of analytical method. In our record, only 10â¯min was needed for the total sample preparation process. And the method detection limits were two orders of magnitude less than literature reports. Furthermore, we reduced the consumption of solvent and minimized the usage of organic solvents, which made our method moving towards green analytical chemistry. In brief, our UAE-D-HPLC method is a simple, rapid, sensitive and eco-friendly analytical method for the determination of EACA and amino acids in cosmetics.
Subject(s)
Amino Acids/analysis , Aminocaproic Acid/analysis , Chromatography, High Pressure Liquid , Cosmetics/chemistry , Solid Phase Extraction/methods , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/chemistry , Amino Acids/isolation & purification , Aminocaproic Acid/isolation & purification , Hydrogen-Ion Concentration , Limit of Detection , Solvents/chemistry , Sonication , TemperatureABSTRACT
Gymnema sylvestre (Retz.) R.Br. ex Sm. (Asclepiadaceae) is a well-known Ayurvedic anti-sweet plant for the treatment of type 2 diabetes mellitus. Although it was previously proposed that G. sylvestre exhibits chemical variation based on geography, most research on G. sylvestre has used material originating from India. Morphological and anatomical descriptions, ITS1-5.8S-ITS2 DNA sequencing, and acid hydrolysis analyses showed that G. sylvestre samples from Vietnam are distinguishable from those of Indian origin and thus suggest a dissimilarity among G. sylvestre samples with different geographic distributions. An LC-MS-guided strategy targeting 3ß-glucuronide oleane-triterpenes in the Vietnamese G. sylvestre variety led to the isolation of four known compounds and nine previously undescribed compounds, named gymnemosides ND1-ND9. None of the isolated compounds were reported in the Indian sample, further supporting the geo-diversity of G. sylvestre. Three compounds, gymnemosides ND7-9, exerted significant stimulatory effects on the uptake of 2-NBDG in 3T3-L1 adipocyte cells and thus have potential as lead molecules for anti-diabetes agents.