RESUMO
Biological thiols importantly regulate the intracellular redox activity and metabolic level, but many of the developed probes for biothiols are facing difficulty in effectively distinguishing GSH from Cys/Hcy due to the similarity in mechanism. In this work, despite the previous pattern of "Logic Gate", we reported the concept of "Fluorescence Fusion" for the first time to achieve only one excitation-emission process. The exploited the probe, MZ-NBD, could quickly measure GSH in 10 min with a large Stokes shift (130 nm). Though the reacting mechanism was similar, only GSH could cause the "Fluorescence Fusion" with only one strong fluorescence response while Cys/Hcy caused two peaks. Adjusting the excitation wavelength could hardly split the fused peak into two. Though image recognition by artificial intelligence could easily distinguish the patterns of peaks, here we used the signal-treating method to realize the high selectivity towards GSH. Moreover, MZ-NBD could be utilized for rapid detection of GSH in living MCF-7 cells, which was more suitable for GSH than using the "Logic Gate" strategy. More than introducing a novel probe with the new concept, this work was meaningful as the linker of traditional reaction-based fluorescent probes and potential image recognition by artificial intelligence, thus led to various future researches in inter-disciplines.
Assuntos
Cisteína , Glutationa , Inteligência Artificial , Fluorescência , Corantes Fluorescentes , Glutationa/isolamento & purificação , Homocisteína , Humanos , Células MCF-7RESUMO
OBJECTIVE: In this study, we aimed to maximize glutathione (GSH) production by a metabolically engineered Yarrowia lipolytica strain using a small-scale optimization approach. RESULTS: A three levels four factorial Box-Behnken Design was used to assess the effect of pH, inulin extract, yeast extract and ammonium sulfate concentrations on cell growth and to generate a mathematical model which predict optimal conditions to maximize biomass production and thus GSH titer. The obtained results revealed that only yeast and inulin extract concentrations significantly affect biomass production. Based on the generated model, a medium composed of 10 g/L of yeast extract and 10 g/L of inulin extract from Jerusalem artichoke was used to conduct batch cultures in 2 L bioreactor. After 48 h of culture, the biomass and the glutathione titer increased by 55% (5.8 gDCW/L) and 61% (1011.4 mg/L), respectively, as compared to non-optimized conditions. CONCLUSION: From the obtained results, it could be observed that the model established from small scale culture (i.e. 2 mL) is able to predict performance at larger scale (i.e. 2 L bioreactor, two orders of magnitude scale-up). Moreover, the results highlight the ability of the optimized process to ensure high titer of glutathione using a low-cost carbon source.
Assuntos
Reatores Biológicos , Glutationa/biossíntese , Engenharia Metabólica , Yarrowia/genética , Sulfato de Amônio/farmacologia , Técnicas de Cultura Celular por Lotes , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Fermentação , Glutationa/isolamento & purificação , Inulina/farmacologia , Modelos Teóricos , Leveduras/químicaRESUMO
Systemic Lupus Erythematosus is an autoimmune disease with symptoms pervasive to all organ systems. It affects more females as compared to males (in the ratio 9:1). Oxidative stress plays a major role in the pathogenesis of SLE and other autoimmune diseases. In order to understand the relationship between cell specific oxidative stress and the severity of SLE, this research study involving the estimation of intracellular ROS accumulation in T and NK cell was conducted on SLE patients of North Indian Population. At the same time, to estimate anti-oxidant defense, Keap1 and Nrf2 levels were estimated in these cell types. The relationship between the expression of Killer immunoglobulin receptors i.e., KIR2DL4 & KIR3DL1 and oxidative stress was also evaluated as these receptors are imperative for the function and self-tolerance of NK cells.Oxidative stress was raised along with Keap1 and Nrf2 in T and NK cell subsets in SLE patients. The expression of KIR2DL4 was raised and that of KIR3DL1 was reduced in the NK cells of patients. The intensity of change in expression and its significance varied among the subsets. Nrf2 expression was raised in these species against oxidative stress as the antioxidant defense mechanism pertaining to Keap1-Nrf2 pathway, but the adequacy of response needs to be understood in further studies. The expression of KIR2DL4 and KIR3DL1 varied among the patient and healthy controls and the expression of the latter was found to have a significant positive relationship with plasma Glutathione(reduced) concentration.
Assuntos
Células Matadoras Naturais/metabolismo , Lúpus Eritematoso Sistêmico/genética , Estresse Oxidativo , Receptores KIR2DL4/metabolismo , Receptores KIR3DL1/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Glutationa/isolamento & purificação , Humanos , Índia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Serina Endopeptidases/metabolismo , Linfócitos T/metabolismoRESUMO
In this work, a simple device for extraction glutathione (GSH) in biological fluids using a hybrid monolithic material within a polypropylene syringe is developed. For this purpose, glycidyl methacrylate-based monolith was firstly prepared within this housing material, and the polymer was modified with different ligands (ammonia, cysteamine and cystamine). The resulting materials (containing amine or thiol groups, respectively) were then functionalized with gold nanoparticles (AuNPs). The hybrid material that gave the largest AuNPs coverage was selected as solid-phase (SPE) sorbent and several variables affecting the extraction recovery of this compound were investigated. Under optimal conditions, GSH was quantitatively retained at pH 6.0, and then it was desorbed with aqueous dithiothreitol solution and determined, after derivatization with o-phthaldialdehyde, via reversed-phase LC with fluorometric detection. The limit of detection was ca. 1.5 ng mL-1, and the reproducibility between extraction units was below 8% (expressed as relative standard deviation), which demonstrates the robustness of the method. The developed material was also applied for the extraction of GSH in saliva and urine samples yielding recoveries ranging from 86 to 105%.
Assuntos
Glutationa/isolamento & purificação , Ouro/química , Nanopartículas Metálicas/química , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão/métodos , Compostos de Epóxi/química , Glutationa/análise , Glutationa/urina , Humanos , Limite de Detecção , Metacrilatos/química , Reprodutibilidade dos Testes , Saliva/química , SeringasRESUMO
In the last decades, enzyme mimics have been regarded as strong substitutes to natural enzymes. The construction of biosensors based on these enzyme mimics with competitive catalytic activity and substrate specificity has attracted a lot of research interest. Herein, for the first time, we investigated the capability of nanoscale FeS2 to serve as enzyme mimics. Then, a facile and effective biosensor is fabricated based on its intrinsic peroxidase-like catalytic activity. In the presence of H2O2, FeS2 nanoparticles (NPs) possess high peroxidase-like activity to 3,3',5,5'-tetramethylbenzidine (TMB) oxidation, which can be ascribed to the generation of hydroxyl radicals (·OH) from the H2O2 decomposition catalyzed by FeS2 NPs. As for TMB, the resulting Michaelis-Menten constant (Km) value of FeS2 NPs is found to be about 12 times lower than that of natural horseradish peroxidase (HRP), highlighting the superiority of FeS2 NPs. Based on these intriguing observations, a reliable colorimetric method is then developed for detection of H2O2 and glutathione (GSH) by a simple mix-and-detect strategy. The detection limits of H2O2 and GSH are as low as 0.91 µM and 0.15 µM (3σ/slope), respectively. Moreover, FeS2 NPs can also catalyse the photoluminescence (PL) substrate terephthalic acid (TA) under the assistance of H2O2. This work remarkably extends the utilization of FeS2 NPs in the construction of colorimetric and PL biosensors in the fields of biosensing, environmental monitoring, and medical diagnosis.
Assuntos
Técnicas Biossensoriais , Glutationa/isolamento & purificação , Peróxido de Hidrogênio/isolamento & purificação , Benzidinas/química , Materiais Biomiméticos/química , Glucose/química , Glutationa/química , Humanos , Peróxido de Hidrogênio/química , Nanopartículas/química , Oxirredução , Peroxidase/químicaRESUMO
Fundamental understandings and precise control of nanoparticle growth in the complex biological environment are crucial to broadening their potential applications in tissue imaging. Herein, we report that glutathione (GSH), a widely used capping ligand for precise control of the size of gold nanoparticle (AuNP) down to single-atom level in test tubes, can also be used to direct the selective growth of the AuNPs in the mitochondria of renal tubule cells as well as hippocampus cells in the tissues. Precise control of this growth process can lead to the formation of both ultrasmall AuNPs with near-infrared luminescence and large plasmonic AuNPs. The observed selective growth of the AuNPs is likely due to unique GSH storage function of the mitochondria. Using a different ligand, ß-glucose thiol, we also found that the brush border of the intestine for glucose absorption became the major site for the growth of luminescent AuNPs. These findings suggest that selective growth of AuNPs in the biological tissues can indeed be directed with specific ligands, opening up a new avenue to tissue labeling and future development of artificial bionano hybrid systems.
Assuntos
Glutationa/isolamento & purificação , Ouro/farmacologia , Nanopartículas Metálicas/química , Glucose/química , Glutationa/química , Ouro/química , Hipocampo/efeitos dos fármacos , Humanos , Túbulos Renais/efeitos dos fármacos , Ligantes , Luminescência , Mitocôndrias/efeitos dos fármacos , Compostos de Sulfidrila/química , Engenharia Tecidual/métodosRESUMO
A hollow-fiber liquid-phase microextraction (HF-LPME) method was established for purification and enrichment of glutathione (GSH) in human saliva followed by a miniaturized capillary electrophoresis with amperometric detection system (mini-CE-AD). Based on regulating isoelectric point and increasing salt effect to modify donor phase, HF-LPME could provide high enrichment efficiency for GSH up to 471 times, and the extract was directly injected for mini-CE-AD analysis. The salt-effect enhanced HF-LPME/mini-CE-AD method has been successfully applied to saliva analysis, and acceptable LOD (0.46 ng/mL, S/N = 3) and recoveries (92.7-101.3%) could be obtained in saliva matrix. The sample pretreatment of this developed method was simple and required no derivatization, providing a potential alternative for non-invasive fluid analysis using portable instrument.
Assuntos
Eletroforese Capilar/métodos , Glutationa , Microextração em Fase Líquida/métodos , Saliva/química , Glutationa/análise , Glutationa/isolamento & purificação , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Cloreto de Sódio/químicaRESUMO
Metallofullerenes have attracted considerable attention as potential novel noninvasive high-relaxivity magnetic resonance contrast agents. However, the applications of metallofullerenes as stimuli-responsive biosensors to monitor biological processes are still scarce. Herein, manganese-fullerenes core-shell nanocomposites are prepared via a facile one-pot approach to achieve GSH-activatable magnetic resonance/fluorescence bimodal imaging functions. The nanocomposites initially have a FRET-induced quenched fluorescence, and water-resisting stimulated low T1-MRI contrast. Upon exposure to GSH, collapse of the outer MnO2 shell led to reconstruction of the nanoprobes and subsequently resulted in multicolor fluorescence recovery and longitudinal (T1) relaxivity enhancement (r1 value up to 29.8 mM-1 s-1 at 0.5 T based on Mn ion). Our work demonstrates feasibility of using fullerenes to fabricate activatable probes for molecular imaging of GSH, which may promote the development of new fullerene-based stimuli-responsive multimodal probes for the detection and regulation of particular biological processes in vivo.
Assuntos
Técnicas Biossensoriais , Fulerenos/química , Glutationa/isolamento & purificação , Imagem Molecular/métodos , Animais , Meios de Contraste/química , Meios de Contraste/farmacologia , Fluorescência , Glutationa/química , Humanos , Íons/química , Imageamento por Ressonância Magnética , Manganês/química , Compostos de Manganês/química , Metais/química , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Nanocompostos/químicaRESUMO
Developing near-infrared (NIR) persistent luminescence (PersL) bioprobes has attracted increasing attention due to their ultra-low autofluorescence and deep tissue penetration. Accurate imaging of glutathione (GSH) in vivo is of great significance to provide a real-time visualization of pathologies. Herein, manipulating metal ions as the switch of NIR afterglow was demonstrated to construct NIR PersL nanoprobes for the first time, which was highly efficient and much simpler than the luminescence resonance energy transfer (LRET) principle. As a proof-of-concept application, an activatable NIR PersL nanoprobe (Probe-1) built on core-shell structure was rationally fabricated for detection of GSH via employing Cu2+ as the quencher. In light of unique NIR PersL feature and flexible functionality, Probe-1 exhibited greatly enhanced analytical performances for GSH detection, and particularly imaging of tumor in living mouse. This approach without limits of spectral overlap from the LRET principle provided a new way to develop NIR PersL bioprobes for autofluorescence-free detection.
Assuntos
Técnicas Biossensoriais , Glutationa/isolamento & purificação , Neoplasias/diagnóstico por imagem , Animais , Cobre/química , Modelos Animais de Doenças , Transferência Ressonante de Energia de Fluorescência , Glutationa/genética , Humanos , Raios Infravermelhos , Luminescência , Camundongos , Nanopartículas/química , Neoplasias/genéticaRESUMO
Radical mediated oxidation of polyunsaturated fatty acids (PUFA) is known to generate a series of polyoxygenated cyclic products (PUFA-On, n ≥ 3). Here, we describe the characterization of glutathione (GSH) conjugates bound to polyoxygenated docosahexaenoic (DHA-On, n = 3-9), arachidonic (ARA-On, n = 3-7), α-linolenic (ALA-O3), and linoleic (LA-O3) acid species. Similar conjugates were also characterized for N-acetylcysteine (NAC) and Cu,Zn-superoxide dismutase (SOD1). Extensive LC-MS/MS characterization using a synthetic α-linolenic hydroxy-endoperoxide (ALA-O3) derivative revealed at least two types of mechanisms leading to thiol adduction: a mechanism involving the nucleophilic attack by thiolate anion on 1,2-dioxolane to form a sulfenate ester-bonded conjugate and a mechanism involving cleavage of the dioxolane to form a α,ß-unsaturated carbonyl followed by the Michael addition reaction. Finally, we detected a GSH conjugate with hydroxy-endoperoxide derived from linoleic acid (LA-O3) in mice liver. In summary, our study reveals the formation of a series of thiol conjugates that are bound to highly oxygenated PUFA species. GSH conjugates described in our study may potentially play relevant roles in redox and inflammatory processes, especially under high oxygen tension conditions.
Assuntos
Ácidos Graxos Insaturados/química , Glutationa/química , Animais , Cromatografia Líquida , Ácidos Graxos Insaturados/metabolismo , Glutationa/isolamento & purificação , Glutationa/metabolismo , Fígado/química , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Oxirredução , Peróxidos/química , Peróxidos/metabolismo , Espectrometria de Massas em TandemRESUMO
In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C4 D-LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 15 mM 2-amino-2-hydroxymethyl-propane-1,3-diol, and 2 mM 18-crown-6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double-opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C4 D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.
Assuntos
Eletroforese Capilar/métodos , Espectrometria de Fluorescência/métodos , Testes Respiratórios/métodos , Ácidos Carboxílicos/análise , Ácidos Carboxílicos/isolamento & purificação , Condutividade Elétrica , Desenho de Equipamento , Glutationa/análise , Glutationa/isolamento & purificação , Humanos , Íons/análise , Íons/isolamento & purificação , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Lágrimas/químicaRESUMO
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/químicaRESUMO
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 1.0â¯×â¯10-9-1.0â¯×â¯10-4 M and 1.0â¯×â¯10-4-1.0â¯×â¯10-2 M and a low detection limit of 7.7â¯×â¯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/químicaRESUMO
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 20â»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/químicaRESUMO
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⯵M. 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/químicaRESUMO
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/químicaRESUMO
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/metabolismoRESUMO
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 <â¯10â¯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/urinaRESUMO
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 1.0â¯×â¯10-10 to 1.0â¯×â¯10-4 molâ¯L-1 was obtained with a detection limit of 3.1â¯×â¯10-11 mol L-1. The relative standard deviation was 2.9% at 2.0â¯×â¯10-9 M (nâ¯=â¯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/químicaRESUMO
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.