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1.
ACS Appl Mater Interfaces ; 11(18): 16822-16829, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-30977357

RESUMO

The construction of ratiometric fluorescence assay has displayed fantastic advantages in improving semi-quantitative visualization capability by presenting successive color changes. Herein, long-wavelength emission nitrogen-doped carbon dots (N-CDs) were developed for intrinsic ratiometric detection of silver ions (Ag+) and glutathione (GSH), accompanied by visualization fluorescence variation of orange and green. The label-free N-CDs were favorably obtained through one-step hydrothermal synthesis and displayed single long-wavelength emission at 618 nm under the excitation wavelength of 478 nm. Interestingly, a ratio rising peak emerges at 532 nm and the emission at 618 nm decreases with the introduction of Ag+, which exhibits ratiometric fluorescence emission characteristics ( I618nm/ I532nm) in the range of 0-140 µM with significant fluorescence varying from orange to green. Furthermore, the fluorescence of CDs@Ag(I) can be effectively ratiometric recovered by virtue of a specific reaction of GSH with Ag+, which is accompanied by the fluorescence of the solution returning from green to orange. In addition, the N-CDs hold excellent biocompatibility which can be implemented as the visualization biosensing platform for intracellular determination of Ag+ and GSH, demonstrating that proposed N-CDs have tremendous potential in biological systems.


Assuntos
Técnicas Biossensoriais , Carbono/química , Glutationa/isolamento & purificação , Prata/isolamento & purificação , Materiais Biocompatíveis/química , Glutationa/química , Nitrogênio/química , Imagem Óptica , Pontos Quânticos/química , Prata/química
2.
Biosens Bioelectron ; 133: 154-159, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30927679

RESUMO

In this work, an ultrasensitive electrochemiluminescence (ECL) biosensor was constructed using poly-L-lysine (PLL) as a novel co-reactant of luminol and poly(luminol/aniline) nanorods loaded reduced graphene oxide (PLA@rGO) as nanoprobe, which enable highly sensitivity detection of glutathione (GSH). To the best of our knowledge, it is the first time that PLL was used for the co-reactant of luminol. Notably, about a 5-fold enhancement was obtained compared with the individual PLA@rGO using GCE. Due to the remarkable quenching effect between the excited state of PLL and the reduced form of GSH in the ECL system of luminol/PLL, the ECL sensing platform exhibited wide linear ranges of 1.0 × 10-9-1.0 × 10-4 M and 1.0 × 10-4-1.0 × 10-2 M and a low detection limit of 7.7 × 10-10 M. Simultaneously, the biosensor was also successfully applied to detect GSH in human serum sample with high recoveries. Hence, this work would open a new platform for the wide application of PLL in immunoassay and various sensors.


Assuntos
Técnicas Biossensoriais , Glutationa/isolamento & purificação , Grafite/química , Polilisina/química , Compostos de Anilina/química , Glutationa/química , Ouro/química , Humanos , Luminol/química , Nanopartículas Metálicas/química
3.
Sensors (Basel) ; 19(2)2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30634480

RESUMO

A novel turn-on fluorescence assay was developed for the rapid detection of glutathione (GSH) based on the inner-filter effect (IFE) and redox reaction. Molybdenum disulfide quantum dots (MoS2 QDs), which have stable fluorescent properties, were synthesized with hydrothermal method. Manganese dioxide nanosheets (MnO2 NSs) were prepared by exfoliating the bulk δ-MnO2 material in bovine serum albumin (BSA) aqueous solution. The morphology structures of the prepared nanoparticles were characterized by transmission electron microscope (TEM). Studies have shown that the fluorescence of MoS2 QDs could be quenched in the presence of MnO2 NSs as a result of the IFE, and is recovered after the addition of GSH to dissolve the MnO2 NSs. The fluorescence intensity showed a good linear relationship with the GSH concentration in the range 20⁻2500 µM, the limit of detection was 1.0 µM. The detection method was applied to the analysis of GSH in human serum samples. This simple, rapid, and cost-effective method has great potential in analyzing GSH and in disease diagnosis.


Assuntos
Técnicas Biossensoriais/métodos , Glutationa/isolamento & purificação , Nanopartículas Metálicas/química , Pontos Quânticos/química , Dissulfetos/química , Corantes Fluorescentes/química , Glutationa/sangue , Ouro/química , Humanos , Limite de Detecção , Compostos de Manganês/química , Molibdênio/química , Óxidos/química , Telúrio/química
4.
Biosens Bioelectron ; 124-125: 89-95, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30343161

RESUMO

An electroanalysis strategy has been developed for probing glutathione (GSH) separately in hela and yeast cells based on the displacement reaction route using melamine-copper (MA-Cu) nanocomposites. Herein, MA-Cu nanocomposites were initially synthesized by the controlled supermolecular self-assembly process showing various morphological structures depending on the MA-to-Cu ratios used. It was discovered that the electrodes modified with rod-like MA-Cu nanocomposites could achieve the stable electrochemical output of solid-state CuCl at a low potential, which might circumvent the possible interference from co-existing electroactive substances in complicated backgrounds like cells. More importantly, the yielded CuCl signals would decrease selectively induced by GSH through the specific Cu-GSH interaction that would trigger the displacement of CuCl into non-electroactive complex. The MA-Cu nanorods-modified electrodes can allow for the detection of GSH with the concentrations linearly ranging from 0.010 to 300.0 µM. Subsequently, the feasibility of the developed electroanalysis strategy was demonstrated for the evaluation of GSH separately in the extractions of hela and yeast cells, promising the wide applications in the clinical and food analysis fields.


Assuntos
Técnicas Biossensoriais , Glutationa/isolamento & purificação , Nanocompostos/química , Cobre/química , Eletrodos , Glutationa/química , Células HeLa , Humanos , Limite de Detecção , Triazinas/química , Leveduras/química
5.
Mikrochim Acta ; 185(7): 321, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884923

RESUMO

Polystyrene nanofibers were coated with copper nanoparticles (CuNPs) by a combination of electrospinning and in-situ reduction of Cu(II) using sodium borohydride as the reductant. The CuNPs on the nanofibers were characterized by energy dispersive spectrometry, scanning electron microscopy and transmission electron microscopy. A cartridge was packed with the nanofibers which then were activated with methanol and water. Glutathione (GSH) is found to quantitatively adsorbed by the packed cartridge at pH 3.0, and then can be desorbed with aqueous 2-mercaptoethanol and detected, after derivatization with ortho-phthalaldehyde, via high performance liquid chromatography with fluorometric detection. Under optimized conditions, the method has a 1.1 ng·mL-1 detection limit and a response that is linear in the 10-1000 ng·mL-1 GSH concentration range. The recoveries of GSH from artificial urine spiked at three levels (80, 400 and 800 ng·mL-1) are in the range of 94.6-98.6% with relative standard deviations (RSD) of <4.5% (n = 5). The method was applied to assessing the differences in urinary GSH between high-risk infants and healthy infants. The results show that the levels of GSH of normal infants are significantly higher than those of high-risk infants (P < 0.05). Graphical abstract Schematic of the preparation of CuNP-assembled nanofibers and the mechanism of extracting glutathione (GSH). GSH can be extracted by this material based on a strong interaction between the sorbent and GSH. This is attributed to the formation of Cu-S bonds between Cu and -SH.


Assuntos
Cobre/química , Glutationa/análise , Glutationa/isolamento & purificação , Nanopartículas Metálicas/química , Nanofibras/química , Poliestirenos/química , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão , Formaldeído/química , Glutationa/química , Espectrometria de Fluorescência , Água/química
6.
Sci Rep ; 8(1): 7243, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29740145

RESUMO

The influences of various factors, including the symbiosis established with the roots of specific tree species, on the production of volatiles in the fruiting bodies of Tuber magnatum have not been investigated yet. Volatiles in T. magnatum fruiting bodies were quantitatively and qualitatively determined by both PTR-MS and GC-MS in order to compare the accuracy of the two methods. An electronic nose was also used to characterize truffle samples. The influence of environmental changes on the antioxidant capabilities of fruiting bodies was also determined. Statistically significant differences were found between fruiting bodies with different origins. The relationship between the quality of white truffle fruiting bodies and their specific host plant is described along with an analysis of metabolites other than VOCs that have ecological roles. Our results indicate that the geographical origin (Italy and Istria) of the fruiting bodies is correlated with the quantity and quality of volatiles and various antioxidant metabolites. This is the first report characterizing antioxidant compounds other than VOCs in white truffles. The correlation between geographical origin and antioxidant contents suggests that these compounds may be useful for certifying the geographical origin of truffles.


Assuntos
Antioxidantes/isolamento & purificação , Carpóforos/química , Saccharomycetales/química , Compostos Orgânicos Voláteis/isolamento & purificação , Antioxidantes/classificação , Antioxidantes/metabolismo , Ácido Ascórbico/isolamento & purificação , Ácido Ascórbico/metabolismo , Betulaceae/microbiologia , Betulaceae/fisiologia , Nariz Eletrônico , Carpóforos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Geografia , Glutationa/isolamento & purificação , Glutationa/metabolismo , Itália , Metaboloma , Análise Multivariada , Populus/microbiologia , Populus/fisiologia , Quercus/microbiologia , Quercus/fisiologia , Saccharomycetales/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Simbiose/fisiologia , Compostos Orgânicos Voláteis/classificação , Compostos Orgânicos Voláteis/metabolismo
7.
Redox Biol ; 16: 359-380, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29627744

RESUMO

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.


Assuntos
Dissulfetos/isolamento & purificação , Metaboloma , Estresse Oxidativo , Compostos de Sulfidrila/isolamento & purificação , Cromatografia Líquida , Dissulfetos/sangue , Dissulfetos/urina , Glutationa/sangue , Glutationa/isolamento & purificação , Glutationa/urina , Humanos , Espectrometria de Massas , Oxirredução , Compostos de Sulfidrila/sangue , Compostos de Sulfidrila/urina
8.
Biosens Bioelectron ; 112: 93-99, 2018 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-29702388

RESUMO

Herein, a visible light photoelectrochemical (PEC) platform based on polyaniline (PANI) and nanoMoS2 composites as optoelectronic material for glutathione detection without any auxiliary of biomolecules or labeled materials was developed. Firstly, the nanoMoS2 was prepared via a simple ultrasound exfoliation method. The PANI was synthesized by chemical oxidative polymerization method. Then composite of PANI and nanoMoS2 was used to modify gold electrode. It was found that the composite membrane showed excellent PEC properties. And glutathione enhanced the PEC signal greatly. Based on this finding a method for glutathione detection was fabricated. Under the optimum conditions, the linear response of glutathione concentrations ranged from 1.0 × 10-10 to 1.0 × 10-4 mol L-1 was obtained with a detection limit of 3.1 × 10-11 mol L-1. The relative standard deviation was 2.9% at 2.0 × 10-9 M (n = 10). This method showed high sensitivity and simpleness which opened up a new promising signal-on PEC platform for future bioassay.


Assuntos
Técnicas Biossensoriais , Glutationa/isolamento & purificação , Neoplasias/diagnóstico , Compostos de Anilina/química , Técnicas Eletroquímicas , Glutationa/química , Ouro/química , Humanos , Limite de Detecção , Nanocompostos/química , Titânio/química
9.
Molecules ; 23(2)2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29470399

RESUMO

A selective and ratiometric turn-on fluorescent probe was designed and synthesized by using a novel dicyanoisophorone-based derivative and acrylate moiety. The probe displayed high stability and good selectivity to cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). It also exhibited rapid response to Cys within 180 s. Most importantly, the fluorescence intensity ratio at 590 and 525 nm (I590/I525) was linearly dependent on the Cys concentration in the range from 0 to 40 µM and the detection limit calculated to be 0.48 µM. This probe was also applied for bioimaging of intracellular Cys in living HeLa cells with low cytotoxicity.


Assuntos
Cisteína/isolamento & purificação , Corantes Fluorescentes/química , Isocianatos/química , Imagem Molecular , Acrilatos/química , Cisteína/química , Glutationa/química , Glutationa/isolamento & purificação , Homocisteína/química , Homocisteína/isolamento & purificação , Humanos
10.
Stem Cell Reports ; 10(2): 600-614, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29307581

RESUMO

The core functions of stem cells (SCs) are critically regulated by their cellular redox status. Glutathione is the most abundant non-protein thiol functioning as an antioxidant and a redox regulator. However, an investigation into the relationship between glutathione-mediated redox capacity and SC activities is hindered by lack of probe. Here, we demonstrate that cyanoacrylamide-based coumarin derivatives are ratiometric probes suitable for the real-time monitoring of glutathione levels in living SCs. These probes revealed that glutathione levels are heterogeneous among subcellular organelles and among individual cells and show dynamic changes and heterogeneity in repopulating SCs depending on oxidative stress or culture conditions. Importantly, a subpopulation of SCs with high glutathione levels exhibited increased stemness and migration activities in vitro and showed improved therapeutic efficiency in treating asthma. Our results indicate that high glutathione levels are required for maintaining SC functions, and monitoring glutathione dynamics and heterogeneity can advance our understanding of the cellular responses to oxidative stress.


Assuntos
Antioxidantes/metabolismo , Glutationa/metabolismo , Mitocôndrias/metabolismo , Células-Tronco/metabolismo , Citosol/metabolismo , Glutationa/isolamento & purificação , Proteínas de Fluorescência Verde/genética , Humanos , Oxirredução , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio
11.
Biosens Bioelectron ; 99: 251-258, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-28772228

RESUMO

In this work, photoelectrochemical (PEC) sensors based on carbon dots (CDs) were developed for ultrasensitive detection of glutathione (GSH) without additional catalysts. In this PEC sensing system, CDs exhibited both photoelectric and catalytic properties. Silver nanoparticles (AgNPs), graphene oxide (GO), and mesoporous silica (MS) were introduced in order to enhance the sensing properties of CDs for GSH. Among the different hybrid nanocomposites, CDs@MS based PEC sensors exhibited the best sensing properties: the sensitivity and limit of detection (LOD) for GSH were found to be 57.6nAµM- 1 and 6.2nM (S/N = 3), respectively, in the linear range 0.02-4µM. In addition, the developed PEC sensors showed a high selectivity for GSH even with interferences of other biological thiols and amino acids. The PEC sensor was successfully applied for GSH detection in human serum and probing of myocardial infarction (MI) conditions by estimating the amount of GSH in the myocardial cells of mice, which had been treated with different ischemia/ischemia-reperfusion times. These results indicated that the CDs based hybrid nanocomposites are promising candidates for the development of PEC biosensors with enhanced sensing performances.


Assuntos
Técnicas Biossensoriais , Glutationa/isolamento & purificação , Infarto do Miocárdio/diagnóstico , Processos Fotoquímicos , Compostos de Cádmio/química , Carbono/química , Glutationa/química , Grafite/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Nanocompostos/química , Pontos Quânticos/química , Prata
12.
Artigo em Inglês | MEDLINE | ID: mdl-29100759

RESUMO

A simple "one-pot" derivatization and liquid-liquid extraction (LLE) procedure was developed for GC-MS analysis of reduced glutathione (GSH) analysis in erythrocytes. The metabolite was extracted by 5% (w/v) TCA, the supernatant treated with ECF and ethanol-pyridine media, the derivative separated and detected by gas chromatography-mass spectrometry using a short non-polar capillary GC column at a high column-head pressure. Total analysis time was 11min. The process was optimized by a Design of Experiment. The method was validated showing a good linearity over the 25.4-813.4µM concentration range, providing satisfactory results in terms of intra-day and inter-day precision as well as an optimal accuracy. The new method was evaluated in a pilot study involving patients with severe protein malnutrition. Comparison of this group with a group of healthy subjects revealed significantly lower GSH concentrations in erythrocytes in the former, thus proving that the described GC-MS method could be employed for fast and simple GSH analysis in clinical studies.


Assuntos
Eritrócitos/química , Ésteres do Ácido Fórmico/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glutationa/sangue , Glutationa/isolamento & purificação , Extração Líquido-Líquido/métodos , Glutationa/química , Humanos , Limite de Detecção , Modelos Lineares , Projetos Piloto , Reprodutibilidade dos Testes
13.
Anal Biochem ; 539: 39-44, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-28993139

RESUMO

Glutamine, glutamate and glutathione are key modulators of excessive oxidative stress in tumor cells. In this study, we developed a rapid and accurate HILIC-MS/MS method to simultaneously determine concentrations of cellular glutamine, glutamate and glutathione. A bared silica HILIC column was employed to analyze these polar metabolites. The LC-MS parameters were optimized to achieve high sensitivity and selectivity. The analysis can be completed within 4 min under optimal conditions. The method was validated in terms of accuracy, precision, and linearity. Intra-day (n = 9) precision was within 2.68-6.24% among QCs. Inter-day precision (n = 3) was below 12.4%. The method accuracy was evaluated by the recovery test, and the accuracy for three analytes were between 91.6 and 110%. The developed method was applied to study antioxidant function of GLS2 in non-small cell lung cancer cells. Changes in concentrations of glutamine, glutamate and glutathione revealed that the overexpression of GLS2 could effectively decrease oxidative stress. In summary, this study developed a rapid HILIC-MS/MS method for quantification of GLS2-related metabolites that could facilitate elucidation of the role of GLS2 in tumor development.


Assuntos
Cromatografia Líquida de Alta Pressão , Ácido Glutâmico/análise , Glutaminase/metabolismo , Glutamina/análise , Glutationa/análise , Espectrometria de Massas em Tandem , Linhagem Celular Tumoral , Ácido Glutâmico/isolamento & purificação , Glutamina/isolamento & purificação , Glutationa/isolamento & purificação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção
14.
Nutr. hosp ; 34(1): 59-64, ene.-feb. 2017. tab
Artigo em Inglês | IBECS | ID: ibc-161142

RESUMO

Introduction: Breast milk contains molecules needed for the development of children; the integrity and function of these molecules is affected by the presence of pro-oxidants. Protein carbonyls are mainly produced as a result of the interaction of metals with reactive oxygen species (ROS), which may initiate a chain reaction that promotes molecular oxidation. Objective: This study aimed to determine the association between the concentration of protein carbonyls with the concentration of trace elements (lead [Pb], cadmium [Cd] and selenium [Se]), superoxide radical (O2 •-) production, and glutathione (GSH) content, as well with the activity of the main antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx], glutathione reductase [GR] and glutathione S-transferase [GST]) in breast milk. Methods: In this study 108 transitional milk samples (7-10 days) were analyzed. Antioxidant enzyme activities, O2 •- production, protein carbonyl and GSH concentrations were analyzed by spectrophotometry. Trace element concentration was quantified by atomic absorption spectrophotometry. Generalized linear modelling was used to assess the relationship between protein carbonyls concentration with oxidative stress indicators and trace elements concentration. Results: Cd and Pb were detected in 21.3 and 55.6% of breast milk samples, respectively. The median concentration of Cd was 0.01 μg L-1 (0.01-3.52 μg L-1) and Pb concentration was 2.61 μg L-1 (0.08-195.20 μg L-1). According to the best-fi t model, the main factors contributing to protein carbonyl concentrations were the activity of GPx, GR, and concentration of GSH, Se, Pb and Cd. Conclusions: According to the generalized linear model, the activity of GPx and GR, could help explain protein oxidation induced by Pb and Cd in breast milk (AU)


Introducción: la leche materna contiene las moléculas necesarias para el desarrollo de los niños; la integridad y función de estas moléculas se afecta por la presencia de prooxidantes. Los carbonilos proteicos se producen principalmente como resultado de la interacción de metales con especies reactivas de oxígeno (ERO), los cuales pueden iniciar una reacción en cadena que promueve la oxidación molecular. Objetivo: este estudio tiene como objetivo determinar la asociación entre la concentración de carbonilos proteicos con la concentración de elementos traza (plomo [Pb], cadmio [Cd] y selenio [Se]), producción de radical superóxido (O2 •-), y contenido de glutatión (GSH), así como con la actividad de las principales enzimas (superóxido dismutasa [SOD], catalasa [CAT], glutatión peroxidasa [GPx], glutatión reductasa [GR] y glutatión S-transferasa [GST]) en leche materna. Métodos: en este estudio se analizaron 108 muestras de leche de transición (7-10 días). La actividad de las enzimas antioxidantes, producción de O2 •-, concentración de carbonilos proteicos y GSH se analizaron por espectrofotometría. La concentración de elementos traza se cuantificó por espectrometría de absorción atómica. Se utilizó un modelo lineal generalizado para evaluar la relación entre la concentración de carbonilos proteicos con los indicadores de estrés oxidativo y las concentraciones de elementos traza. Resultados: Cd y Pb fueron detectados en 21,3 y 55,6% de las muestras de leche materna, respectivamente. La mediana de la concentración de Cd fue 0,01 μg l-1 (0,01-3,52 μg l-1) y para la concentración de Pb fue 2,61 μg l-1 (0,08-195,20 μg l-1). De acuerdo con el modelo de mejor ajuste, los principales factores de afectan la concentración de carbonilos proteicos, son la actividad de GPx y GR, y las concentraciones de GSH, Se, Pb y Cd. Conclusiones: de acuerdo con el modelo lineal generalizado, la actividad de GPx y GR podría ayudar a explicar la oxidación proteica, inducida por Pb y Cd en leche materna (AU)


Assuntos
Humanos , Lactente , Estresse Oxidativo/fisiologia , Leite Humano/fisiologia , Proteínas do Leite/biossíntese , Marcadores Inorgânicos/análise , Antioxidantes/análise , Oligoelementos/isolamento & purificação , Superóxidos/análise , Glutationa/isolamento & purificação , Leite Humano/enzimologia
15.
Biosens Bioelectron ; 90: 403-409, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27825881

RESUMO

Glutathione (GSH) and cysteine (Cys) play different roles in biological systems, thus the discrimination between them is of great importance. Herein we report a multi-emissive fluorescent probe for the selective detection of GSH and Cys. The probe was composed of covalently linked BODIPY and coumarin fluorophores. The BODIPY fluorophore was designed to react with GSH and Cys and generate different products with distinct photophysical properties, and the coumarin fluorophore acted as an internal standard. The probe exhibited green emission in aqueous solution. Upon addition of Cys, it yielded nitrogen-substituted BODIPY with weak fluorescence and free coumarin with blue emission. In the presence of glutathione, it generated mono- and di-sulfur substituted BODIPY and coumarin, resulting in various emission colors at different concentrations of GSH. Interestingly, the solution exhibited white fluorescence at GSH concentration of 0.4mM. The probe was capable of detecting and imaging GSH and Cys in living HeLa cells, indicating its significant potential in biological applications.


Assuntos
Técnicas Biossensoriais , Cisteína/isolamento & purificação , Corantes Fluorescentes/química , Glutationa/isolamento & purificação , Compostos de Boro/química , Cumarínicos/química , Cisteína/química , Glutationa/química , Células HeLa , Humanos
16.
Biosens Bioelectron ; 89(Pt 2): 919-926, 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-27818045

RESUMO

Hydrogen sulfide is a critical biological messenger, but few biologically compatible methods are available for its detection in vivo. Here, we describe the design and synthesis of a novel azide-functionalized near-infrared probe, NIR-Az, for a hydrogen sulfide assay in which a self-immolative linker is incorporated between the azide moiety and phenolic dihydroxanthene fluorophore from a cyanine dye. A large "turn-on" near-infrared fluorescence signal results from the reduction of the azide group of the fluorogenic moiety to an amine, in which the self-immolative linker also enhances the accessibility of NIR-Az to hydrogen sulfide. NIR-Az can select hydrogen sulfide from among 16 analytes, including cysteine, glutathione, and homocysteine. By exploiting the superior properties of NIR-Az, such as its good biocompatibility and rapid cell internalization, we successfully demonstrated its usefulness in monitoring both the concentration- and time-dependent variations of hydrogen sulfide in living cells and animals (detection limit less than 0.26µM), thereby providing a powerful approach for probing hydrogen sulfide chemistry in biological systems.


Assuntos
Técnicas Biossensoriais , Sulfeto de Hidrogênio/isolamento & purificação , Espectrometria de Fluorescência , Animais , Azidas/química , Cisteína/química , Cisteína/isolamento & purificação , Fluorescência , Corantes Fluorescentes/química , Glutationa/química , Glutationa/isolamento & purificação , Sulfeto de Hidrogênio/química
17.
Biosens Bioelectron ; 90: 117-124, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27886598

RESUMO

A fluorescent probe (1) for distinguishing amongst biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), is developed based on different cascade reactions. The key design feature of fluorescent probe 1 is the integration of two potential reaction groups for the thiol and amino groups of biothiols in one molecule. By reacting with the halogen atom and α, ß-unsaturated malonitrile in probe 1, Cys, Hcy and GSH can generate a total of three main products with distinct photophysical properties. Probe 1 shows a strong fluorescence turn-on response to Cys with blue-green emission by using an excitation wavelength of 390nm. At an excitation wavelength of 500nm, probe 1 responds to GSH over Cys and Hcy and emits strong orange fluorescence. The discrimination of biothiols can be demonstrated by cell imaging experiments, indicating that probe 1 can be a useful tool for the selective imaging of Cys and GSH in living cells.


Assuntos
Técnicas Biossensoriais , Cisteína/isolamento & purificação , Glutationa/isolamento & purificação , Homocisteína/isolamento & purificação , Cumarínicos/química , Corantes Fluorescentes/química , Humanos , Nitrilos/química
18.
Biosens Bioelectron ; 90: 69-74, 2017 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27886603

RESUMO

Nanozymes are increasingly used as components in assays and diagnostics. Here, we describe a rapid and highly sensitive colorimetric assay for the detection and quantification of glutathione (GSH) employing MnO2 nanosheets as an artificial oxidase. In the assay pale yellow 3,3´,5,5´-tetramethylbenzidine (TMB) is oxidized to a blue product (oxTMB) under catalyzing of MnO2 nanosheets with a significant change in absorption at 650nm. GSH selectively inhibits this reaction with a detection limit of 300nM. The high specificity of inhibition by GSH allows this system to be used to determine the GSH concentrations in human serum samples. The MnO2 nanosheet-based assay is simple, rapid, sensitive and selective for the quantification of GSH and surpasses detection methods based on other MnO2 nanomaterials.


Assuntos
Técnicas Biossensoriais , Glutationa/isolamento & purificação , Nanopartículas Metálicas/química , Oxirredutases/química , Colorimetria , Glutationa/química , Humanos , Limite de Detecção , Compostos de Manganês/química , Óxidos/química
19.
Free Radic Biol Med ; 97: 85-94, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27212018

RESUMO

Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0ng/mL for GSH and 1.0ng/mL for GSSG with lower limits of detection at 0.5ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases.


Assuntos
Cromatografia Líquida/métodos , Dissulfeto de Glutationa/isolamento & purificação , Glutationa/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Animais , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células MCF-7 , Camundongos , Oxirredução , Estresse Oxidativo
20.
Sci Rep ; 6: 24730, 2016 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-27126222

RESUMO

Integrating droplet-based microfluidics with mass spectrometry is essential to high-throughput and multiple analysis of single cells. Nevertheless, matrix effects such as the interference of culture medium and intracellular components influence the sensitivity and the accuracy of results in single-cell analysis. To resolve this problem, we developed a method that integrated droplet-based microextraction with single-cell mass spectrometry. Specific extraction solvent was used to selectively obtain intracellular components of interest and remove interference of other components. Using this method, UDP-Glc-NAc, GSH, GSSG, AMP, ADP and ATP were successfully detected in single MCF-7 cells. We also applied the method to study the change of unicellular metabolites in the biological process of dysfunctional oxidative phosphorylation. The method could not only realize matrix-free, selective and sensitive detection of metabolites in single cells, but also have the capability for reliable and high-throughput single-cell analysis.


Assuntos
Gotículas Lipídicas/química , Microextração em Fase Líquida/métodos , Análise de Célula Única/métodos , Espectrometria de Massas por Ionização por Electrospray , Monofosfato de Adenosina/análise , Monofosfato de Adenosina/isolamento & purificação , Glucosamina/análogos & derivados , Glucosamina/análise , Glucosamina/isolamento & purificação , Glutationa/análise , Glutationa/isolamento & purificação , Humanos , Células MCF-7 , Microfluídica , Solventes/química
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