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1.
Anal Chem ; 93(3): 1749-1756, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33351590

RESUMO

Significant efforts have been made to develop robust and reliable methods for simultaneous biothiols determination in different matrices, but there still exist the problems such as easy oxidation, tedious derivatization, and difficulty in discrimination, which brings unsatisfactory results in their accuracy and fast quantification in biological samples. To overcome these problems, a simultaneous biothiols detection method combining a "selenium signature" chemical probe and paper spray mass spectrometry (PS-MS) was proposed. In the strategy, the modified-paper substrate is used to enhance the analytical performance. Chemical probe Ebselen-NH2 that has a specific response to biothiols was designed and covalently fixed on the surface of an oxidized paper substrate. By the identification of derivatized product with distinctive selenium isotope distribution and employment of the optimized PS-MS method, qualitative and quantitative analysis of five biothiols including glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), N-acetylcysteine (Nac), and homocysteine (Hcy) were realized. Biothiols in plasma and cell lysates were measured with satisfactory results. The established method not only provides a novel protocol for simultaneous determination of biothiols, but also is helpful for understanding the biological and clinical roles played by these bioactive small molecules.


Assuntos
Acetilcisteína/análise , Cisteína/análise , Dipeptídeos/análise , Corantes Fluorescentes/química , Glutationa/análise , Papel , Selênio/química , Técnicas Biossensoriais , Cisteína/análogos & derivados , Humanos , Espectrometria de Massas
2.
J Chromatogr A ; 1637: 461806, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33360435

RESUMO

The development of plant-based protein polymers to employ in biofilm production represents the promising intersection between material science and sustainability, and allows to obtain biodegradable materials that also possess excellent physicochemical properties. A possible candidate for protein biopolymer production is phaseolin, a storage protein highly abundant in P Vulgaris beans. We previously showed that transformed tobacco chloroplasts could be employed to express a mutated phaseolin carrying a signal peptide (directing it into the thylakoids) also enriched of a cysteine residue added to its C-terminal region. This modification allows for the formation of inter-chain disulfide bonds, as we previously demonstrated, and should promote polymerization. To verify the effect of the peptide modification and to quantify polymer formation, we employed hollow-fiber flow field-flow fractionation coupled to UV and multi-angle laser scattering detection (HF5-UV-MALS): HF5 allows for the selective size-based separation of phaseolin species, whereas MALS calculates molar mass and conformation state of each population. With the use of two different HF5 separation methods we first observed the native state of P.Vulgaris phaseolin, mainly assembled into trimers, and compared it to mutated phaseolin (P*) which instead resulted highly aggregated. Then we further characterized P* using a second separation method, discriminating between two and distinct high-molecular weight (HMW) species, one averaging 0.8 × 106 Da and the second reaching the tens of million Da. Insight on the conformation of these HMW species was offered from their conformation plots, which confirmed the positive impact of the Cys modification on polymerization.


Assuntos
Biopolímeros/química , Cisteína/análise , Fabaceae/química , Miniaturização , Proteínas de Plantas/química , Tabaco/genética , Fracionamento por Campo e Fluxo/métodos , Luz , Peso Molecular , Transcriptoma
3.
Food Chem ; 337: 128008, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32920267

RESUMO

This study investigated the role of furfuryl alcohol (FFA) in the formation of furfurylthiol (FFT), the most important odorant in roasted coffee, using in-bean and spiking experiments. Green beans were spiked with FFA, and after roasting FFT was quantified by stable isotope dilution analysis. The FFT level in the roasted beans increased dose-dependently with addition of FFA. Additionally, beans were spiked with isotopically labelled d2-FFA which generated isotopically labelled d2-FFT after roasting. However, no labelled furfural was observed. The results unambiguously show that FFA serves as a precursor of FFT in coffee. On the other hand, the data indicate that furfural stems not from oxidation of FFA and plays no major role as precursor for FFT formation during coffee roasting. The suggested formation pathway leads from FFA to the furfuryl cation, then protein-bound S-furfuryl-l-cysteine and by subsequent elimination to FFT.


Assuntos
Coffea/química , Furanos/química , Odorantes/análise , Compostos de Sulfidrila/química , Café , Cisteína/análise , Temperatura Alta
4.
Food Chem ; 338: 128011, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32950006

RESUMO

This study is aimed to comparatively investigate the phytochemical profiles, focusing on the nutritional and phytochemical properties of common garlic (Allium sativum L.; CG) and elephant garlic (EG) (Allium ampeloprasum var. holmense) collected from the Val di Chiana area (Tuscany, Italy). The results showed a lower amount of fibers, demonstrating a higher digestibility of the bulb, and sulfur-containing compounds in EG rather than in CG. Untargeted metabolomic profiling followed by supervised and unsupervised statistics allowed understanding the differences in phytochemical composition among the two bulbs, both as raw bulbs, processed following the in vitro gastrointestinal digestion process. Typical sulfur-containing compounds, such as alliin and N-gamma-glutamyl-S-allyl cysteine, could notably be detected in lower amounts in EG. EG maintains a distinct phytochemical signature during in vitro gastrointestinal digestion. Our findings support the distinct sensorial attributes of the bulbs.


Assuntos
Allium/química , Allium/metabolismo , Compostos Fitoquímicos/análise , Antioxidantes/análise , Cisteína/análogos & derivados , Cisteína/análise , Digestão , Alho/química , Alho/metabolismo , Itália , Metabolômica , Fenóis/análise , Raízes de Plantas/química , Metabolismo Secundário , Especificidade da Espécie , Compostos de Enxofre/análise
5.
Anal Chim Acta ; 1113: 43-51, 2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32340668

RESUMO

This study fabricated a dual-emission probe consisting of monolayer MoS2 quantum dots (M - MoS2 QDs) and L-cystine-linked boron-dipyrromethene (L-Cys-BODIPY) molecules for ratiometric sensing of biothiols, thiol product-related enzyme reactions, and ratiometric imaging of glutathione (GSH)-related reactions in HeLa cells. The formation of L-Cys-BODIPY-adsorbed M - MoS2 QDs (named as BODIPY-M-MoS2 QDs) was demonstrated by comparing them with M - MoS2 QDs using transmission electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. The BODIPY-M-MoS2 QDs exhibited dual-emission bands, excellent biocompatibility, and good resistance to photobleaching. It was found that the adsorbed L-Cys-BODIPY molecules rarely quenched the fluorescence of M - MoS2 QDs, and meanwhile, they were self-quenched by π-π stacking between each BODIPY backbones. The presence of biothiols induced the reduction of weakly fluorescent L-Cys-BODIPY to strongly fluorescent of L-cysteine-conjugated BODIPY. Since having a much higher molar absorption coefficient than L-Cys-BODIPY, the liberated L-cysteine-conjugated BODIPY behaved as an effective inner filter to absorb the excitation light and subsequently quenched the fluorescence of M - MoS2 QDs. The appearance of L-cysteine-conjugated BODIPY could barely affected to the fluorescence lifetime of M - MoS2 QDs, confirming the inner filter effect of L-cysteine-conjugated BODIPY onto the fluorescence of M - MoS2 QDs. The present probe not only provided a linear ratiometric response to 1-10 mM GSH, 1-10 µM cysteine, and 1-10 µM of homocysteine but also remarkably showed the ratiometric detection of thiol products from the reactions of 1-900 units L-1 S-adenosylhomocysteine (SAH) hydrolase and SAH as well as 1-850 units L-1 GSH reductase and disulfide GSH. Additionally, the present probe was well-suited for ratiometric imaging of intracellular GSH levels in non-treated and drug-treated HeLa cells.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , Pontos Quânticos/química , Compostos de Boro/química , Cistina/química , Dissulfetos/química , Células HeLa , Humanos , Limite de Detecção , Molibdênio/química , Espectrometria de Fluorescência/métodos
6.
Anal Chim Acta ; 1105: 139-146, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32138912

RESUMO

An effective ratiometric fluorescent probe based on silicon particles/gold nanoclusters (SiNPs/AuNCs) nanohybrid has been fabricated and applied to be a "on-off-on" switch sensing platform for detection of Hg2+ and cysteine. In this elaborated sensing platform, the SiNPs just acted as internal reference signal, providing a build-in correction for background interferences and environmental effects, to which the AuNCs as a signal report unit for Hg2+ response was covalently grafted by amidation reaction. The fluorescence intensity of SiNPs/AuNCs could be effectively quenched upon adding Hg2+, accompanied with an easily distinguishable fluorescent color change. The ratiometric fluorescence signal (F649/F511) of the established nanoprobe was linearly proportional to the concentration of Hg2+ ranging from 0.02 to 24 µM with a low detection limit of 5.6 nM, which is below the guideline value of Hg2+ in drinking water set by the World Health Organization. Interestingly, upon addition of cysteine, the Hg2+-quenched fluorescence intensity was recovered gradually. Furthermore, the approach developed has also been utilized for Hg2+ detection in real complex biological samples with satisfactory results. More importantly, benefiting from the good water-solubility and excellent biocompatibility, this nanoprobe can monitor the intracellular Hg2+ and cysteine in living cells, indicating its potential applications in advanced biosensing and bioimaging.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Mercúrio/análise , Nanopartículas/química , Imagem Óptica , Animais , Água Potável/química , Ouro/química , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Tamanho da Partícula , Silício/química , Espectrometria de Fluorescência , Propriedades de Superfície
7.
Mikrochim Acta ; 187(4): 229, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170465

RESUMO

Black phosphorus quantum dots (BP QDs) with small size are synthesized using an easy to operate thermal method. It was found that BP QDs possess oxidase-mimicking activity. They can catalyze the oxidation of the substrate 3,3',5,5'-tetramethylbenzidine to produce a blue-colored product even in the absence of hydrogen peroxide. Active oxygen species are proved to be involved in the reaction through the experiments of radical scavenging and electron spin resonance. Biothiols including reduced glutathione and cysteine inactivate the oxidase-mimicking activity of BP QDs, concomitant to the fading of the blue solution. This provides the  base for a colorimetric method for the determination of glutathione and cysteine. The decreased absorbance at 652 nm displays linear response to the concentrations of glutathione ranging from 0.1 to 5.0 µmol L-1, and cysteine from 0.1 to 10.0 µmol L-1. The detection limits are 0.02 µmol L-1 and 0.03 µmol L-1 for glutathione and cysteine, respectively. Successive determinations of 1.0 µmol L-1 glutathione and 5.0 µmol L-1 cysteine solution give relative standard deviations of 0.8% and 1.7% (n = 11), respectively. As a preliminary application, the practicability of the method was evaluated by the determination of glutathione in pharmaceutical preparations. This work not only discovers a useful oxidase mimics but also sets up a reliable platform based on BP QDs in colorimetric detection. Graphical abstract Schematic representation of colorimetric determination for biothiols through inactivating oxidase mimetic-like catalytic activity of black phosphorus quantum dots (BP QDs) on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with dissolved oxygen to produce its blue oxidized product (oxTMB).


Assuntos
Técnicas Biossensoriais , Colorimetria , Cisteína/análise , Glutationa/análise , Oxirredutases/química , Fósforo/química , Pontos Quânticos/química , Cisteína/metabolismo , Glutationa/metabolismo , Estrutura Molecular , Oxirredutases/metabolismo , Fósforo/metabolismo
8.
Org Biomol Chem ; 18(7): 1487-1492, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32026925

RESUMO

As an important biothiol in living cells, cysteine is closely related to oxidative damage in living organisms. Sulfite from cysteine metabolism in living cells plays a crucial role in maintaining homeostasis in an organism, and the unbalance of sulfite in vivo would lead to multiple diseases. Thus the development of a new fluorescent probe for cysteine metabolism is needed urgently in mitochondria which are the main place of cysteine metabolism. Herein we construct a novel targeting mitochondria fluorescent probe CP-K based on the FRET mechanism to visualize sulfite in living MCF-7 cells. Probe CP-K displays a large Stokes shift of 150 nm, a low detection limit (26.3 nM) and "naked eye" detection after the addition of HSO3-. Importantly, it is appropriate for imaging the endogenous sulfite from cysteine metabolism in living cells.


Assuntos
Cisteína/análise , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Mitocôndrias/química , Cisteína/metabolismo , Humanos , Células MCF-7 , Mitocôndrias/metabolismo , Estrutura Molecular , Imagem Óptica
9.
Nat Commun ; 11(1): 793, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034152

RESUMO

Fluorescence-based technologies have revolutionized in vivo monitoring of biomolecules. However, significant technical hurdles in both probe chemistry and complex cellular environments have limited the accuracy of quantifying these biomolecules. Herein, we report a generalizable engineering strategy for dual-emission anti-Kasha-active fluorophores, which combine an integrated fluorescein with chromene (IFC) building block with donor-π-acceptor structural modification. These fluorophores exhibit an invariant near-infrared Kasha emission from the S1 state, while their anti-Kasha emission from the S2 state at around 520 nm can be finely regulated via a spirolactone open/closed switch. We introduce bio-recognition moieties to IFC structures, and demonstrate ratiometric quantification of cysteine and glutathione in living cells and animals, using the ratio (S2/S1) with the S1 emission as a reliable internal reference signal. This de novo strategy of tuning anti-Kasha-active properties expands the in vivo ratiometric quantification toolbox for highly accurate analysis in both basic life science research and clinical applications.


Assuntos
Bioquímica/métodos , Corantes Fluorescentes/química , Imagem Molecular/métodos , Células A549 , Animais , Benzopiranos/química , Cisteína/análise , Feminino , Fluoresceína/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Glutationa/análise , Células Hep G2 , Humanos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Piranos/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Espironolactona/química
10.
Anal Chim Acta ; 1104: 125-131, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32106943

RESUMO

We report on the synthesis of manganese oxide doped CDs (MnOx-CDs) by a hydrothermal strategy using manganese (III) acetylacetonate (Mn(III) (C5H7O2)3) as the only raw materials. The MnOx-CDs exhibit water solubility, favorable biocompatibility, low cytotoxicity, and show blue fluorescence with excitation/emission maxima at 326/442 nm with a quantum yield of 11.3%, allowing efficient cellular imaging. The MnOx-CDs have a reversible temperature-sensitive fluorescent property in vitro within 10-60 °C, which can also be used as a sensitive thermometer in living cells. By a scratch assay, the MnOx-CDs can restrain the migration of HepG2 cancer cells, which make the MnOx-CDs be attractive candidates for liver cancer adjuvant treatment. Besides, the fluorescence of the MnOx-CDs is quenched in the presence of Fe3+ due to the formation of a nonfluorescent MnOx-CDs-Fe3+ complex between oxygen-containing groups on the surface of MnOx-CDs and Fe3+, and the quenched fluorescence of MnOx-CDs can be turn-on by dissociation of MnOx-CDs-Fe3+ complexes by biothiols including L-cysteine, homocysteine and glutathione. Therefore, the Fe3+ and biothiols can be sequentially detected with high reliability and accuracy via exploiting the on-off-on nanosensor at room temperature, respectively. Further application to detection biothiols in human serum indicated that the probe was practicality and feasibility in medical field.


Assuntos
Técnicas Biossensoriais/métodos , Carbono/química , Compostos de Manganês/química , Imagem Óptica/métodos , Óxidos/química , Pontos Quânticos/química , Compostos de Sulfidrila/análise , Técnicas Biossensoriais/instrumentação , Cisteína/análise , Corantes Fluorescentes/química , Células Hep G2 , Temperatura Alta , Humanos , Imagem Óptica/instrumentação , Espectrometria de Fluorescência
11.
Chem Commun (Camb) ; 56(20): 3065-3068, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32049076

RESUMO

Herein, a novel stacked hexagonal prism, Ag@Ni-MOF-1, was designed and developed as an integrated SERS platform not only for successfully catalyzing the in situ synthesis of DA-quinone under physiological pH, but also for establishing an approach for specific determination of Cys, an important species in the brain related to Alzheimer's disease (AD).


Assuntos
Benzoquinonas/síntese química , Materiais Biomiméticos/química , Dopamina/síntese química , Níquel/química , Compostos Organometálicos/química , Prata/química , Benzoquinonas/química , Catálise , Cisteína/análise , Dopamina/química , Humanos , Concentração de Íons de Hidrogênio , Compostos Organometálicos/síntese química , Análise Espectral Raman
12.
Analyst ; 145(4): 1179-1183, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-31898708

RESUMO

Biological nanopore technology has the advantages of high selectivity and high reproducibility for characterizing single biomolecules. However, it is challenging to achieve protein sequencing owing to the heterogeneous charge distributions of the protein and the small structural difference from each amino acid. Here, we took the inherent electrochemically confined sensing interface of the aerolysin nanopore to enhance its interaction with single amino acids. The results showed that single cysteine molecules, a highly reactive amino acid in aging and neurodegenerative diseases, could be captured and monitored by an aerolysin nanopore as it produced distinctive current blockages with a prolonged statistical duration of 0.11 ± 0.02 ms at +120 mV. This is the first report of the detection of a single amino acid molecule by a biological nanopore directly without any modification and labelling. This study facilitates the direct detection of single amino acids by regulating the characteristic interaction between the single amino acids and the designed sensing interface of aerolysin nanopores.


Assuntos
Toxinas Bacterianas/química , Técnicas Biossensoriais/métodos , Cisteína/análise , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/química , Toxinas Bacterianas/metabolismo , Cisteína/química , Limite de Detecção , Bicamadas Lipídicas/química , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Conformação Proteica
13.
Anal Chim Acta ; 1097: 238-244, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910965

RESUMO

Hydrogen sulfide (H2S), cysteine (Cys) and homocysteine (Hcy) play critical roles in human pathologies and there are close interconnections between them in their generation and metabolism in living systems. To elucidate their complex interplay networks, single-molecule fluorescent probes enabling simultaneous detection of H2S and Cys/Hcy from distinct emission channels have been becoming indispensable tools. In this report, we have rationally developed a novel fluorescent probe, NC, for H2S and Cys/Hcy by integrating an amino 7-nitro-1, 2, 3-benzoxadiazole (NBD) moiety and an azide group into the coumarin platform. NC exhibited good selectivity and sensitivity for the discriminatory detection of H2S and Cys/Hcy, and its capability for imaging of intracellular H2S and Cys/Hcy was proved.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Homocisteína/análise , Sulfeto de Hidrogênio/análise , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Estrutura Molecular , Imagem Óptica , Células Tumorais Cultivadas
14.
Anal Chim Acta ; 1097: 245-253, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910966

RESUMO

In this work, we based on an iminocoumarin borate complex to design and synthesize a red-emitting fluorescent probe, THQ-BF2-O-NBD, to discriminatively detect Cys/Hcy and GSH with a large Stokes shift (100 nm). This probe responded to GSH to display red fluorescence with λmax = 630 nm. In contrast, two well-separated emission bands with λmax = 565 and 630 nm were observed in the solution of THQ-BF2-O-NBD with Cys/Hcy. The probe was capable to distinguish Cys/Hcy from GSH in real biological systems.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , Animais , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Cinética , Estrutura Molecular , Espectrometria de Fluorescência , Células Tumorais Cultivadas , Peixe-Zebra
15.
Parasitol Int ; 76: 102062, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31978597

RESUMO

A platyhelminth, Spirometra erinaceieuropaei, belonging to the class Cestoda, causes human sparganosis, and infection with its larva results in subtle inflammation in the body of its host. We previously reported the purification of a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF) from the excretory/secretory products of S. erinaceieuropaei plerocercoids that may be involved in immuno-modification. We determined the sequence of P-ISF from the N-terminal and the internal 10 amino acids of P-ISF using degenerate PCR and 5'- and 3'-RACE methods. The putative gene encoding P-ISF was 1443 bp long and the gene contained 10 exons and 9 introns in a genomic DNA of size 5205 bp. P-ISF consists of 480 amino acids including the N-terminal signal peptide sequence, and has two unknown domains,-cestoda cysteine-rich domains (CCDs) and a fibronectin type III domain between the two CCDs. All cysteine residues were conserved in the two CCDs, which shared 62% amino acid identities. Homologous analysis revealed that the CCDs were homologous with an unknown protein of Diphyllobothrium latum. To produce specific antibodies, we expressed recombinant P-ISF (rP-ISF) using wheat germ protein synthetic system. P-ISF was localized in the sub-cutaneous tissues and the parenchymal tissues of plerocercoids. Transcription of P-ISF was detected only in plerocercoid stage, but not in adult stage. Western blotting also showed a band in plerocercoide stage but not in adult. The rP-ISF did not suppress nitrite production in RAW 264.7 cells stimulated with LPS, and this might be due to lack of carbohydrate chains in the recombinant protein.


Assuntos
Glicoproteínas/genética , Proteínas de Helminto/genética , Spirometra/genética , Animais , Clonagem Molecular , Cisteína/análise , Cisteína/genética , Feminino , Fibronectinas/genética , Genoma Helmíntico , Camundongos , Camundongos Endogâmicos C57BL , Sinais Direcionadores de Proteínas , Células RAW 264.7 , Proteínas Recombinantes/genética , Organismos Livres de Patógenos Específicos
16.
Talanta ; 210: 120612, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987202

RESUMO

A new strategy endowing probe with large Stokes shift, high quantum yield and red emission was developed by incorporating tetrahydroquinoxaline unit and five-membered pyrrolidine ring on fluorophore. As demonstrated, a novel probe was rationally designed and synthesized for sensitive monitor of cellular cysteine (Cys) and endogenous aminoacylase with large Stokes shift and high quantum yield.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Imagem Óptica , Pirrolidinas/química , Teoria Quântica , Quinoxalinas/química , Animais , Células Cultivadas , Corantes Fluorescentes/síntese química , Camundongos , Estrutura Molecular , Pirrolidinas/síntese química , Quinoxalinas/síntese química , Células RAW 264.7
17.
J Pharm Biomed Anal ; 180: 113066, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31891875

RESUMO

Cysteine is a sulfur-containing amino acid which plays an outstanding role in many biological pathways in mammals. The analysis and quantification of native cysteine remains a critical issue due to its highly reactive thiol group evolving to the disulfide cystine derivative through oxidation reaction. Aimed at improving the derivative stability, cysteine was labelled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), which reacts with both amino and thiol groups. The derivatization was optimized and the chemical identity of the reaction product was assessed via high-resolution mass spectrometry. The NBD-cysteine derivative resulted stable for 10 days. This derivative was enantioresolved (α and RS equal to 1.25 and 2.70, respectively) thanks to a (R,R)-Whelk-O1 phase with the following chromatographic setting: eluent, MeOH/water-90/10 (v/v) with 15 mM ammonium formate (pwsH 6.0); column temperature, 35 °C; flow rate, 1.0 mL/min. The developed method was validated following the ICH guidelines and applied for the quality control of a L-cysteine containing dietary supplement.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cisteína/análise , Cisteína/normas , Suplementos Nutricionais/análise , Suplementos Nutricionais/normas , Espectrometria de Massas/métodos , Cápsulas , Cisteína/química , Estabilidade de Medicamentos , Limite de Detecção , Reprodutibilidade dos Testes , Estereoisomerismo
18.
Talanta ; 209: 120580, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892055

RESUMO

In this study, a mitochondria-specific fluorescent probe for efficient ratiometric detection of Cys was designed and investigated. Probe 1 is composed of a xanthylene skeleton and a benzyl group containing an acryloyl moiety. The probe showed excellent water solubility, good selectivity and sensitivity toward Cys over other analytes, and afforded an extremely low detection limit of 33.7 nM. The possible detection mechanism was ascertained by HRMS analysis. Moreover, probe 1 had excellent mitochondrial-targeting ability (the Pearson's correlation coefficient was 0.96), and was capable of monitoring endogenous Cys in living HeLa cells by dual channel ratiometric bioimaging, demonstrating its significant potential in biological applications.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Mitocôndrias/química , Xantenos/química , Células HeLa , Humanos , Limite de Detecção , Microscopia Confocal/métodos , Imagem Óptica/métodos
19.
Spectrochim Acta A Mol Biomol Spectrosc ; 230: 118030, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-31951867

RESUMO

In this study, a new 4,5-quinolimide-based fluorescent sensor BNC was synthesized and characterized. BNC showed single selectivity for Cu2+via the "turn-off" fluorescence among various common metal ions. After forming a 1:1 stoichiometric complex with Cu2+, the detection limit (LOD) of BNC for Cu2+ was measured to be 0.44 µM. Subsequently, the in situ generated BNC-Cu2+ complex had been used for sensing Cys with the LOD of 1.5 µM through the displacement strategy, resulting in the revivable emission of BNC. According to the "off-on-off" fluorescence cycle of BNC generated by the alternate addition of Cu2+ and Cys, a reversible memorized device with "read-write-read-erase" behavior was constructed at the molecular level. Furthermore, the recoveries of Cu2+ in lake water with BNC were in the range of 95.0-105%. And sequential fluorescence imagings of BNC for Cu2+ and Cys were successfully applied in living yeast cells.


Assuntos
Técnicas Biossensoriais/métodos , Cobre/análise , Cisteína/análise , Corantes Fluorescentes/química , Quinolinas/química , Saccharomyces cerevisiae/metabolismo , Poluentes Químicos da Água/análise , Limite de Detecção , Imagem Óptica , Espectrometria de Fluorescência
20.
Mater Sci Eng C Mater Biol Appl ; 108: 110369, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923961

RESUMO

Bionanocomposite materials were developed from the assembly of polymer-coated copper-cystine high-aspect ratio structures (CuHARS) and cellulose fibers. The coating of the metal-organic materials with polyallylamine hydrochloride (PAH) allows their covalent linkage to TEMPO-oxidized cellulose by means of EDC/NHS. The resulting materials can be processed as films or macroporous foams by solvent casting and lyophilization, respectively. The films show good mechanical behavior with Young's moduli around 1.5 GPa as well as resistance in water, while the obtained foams show an open network of interconnected macropores with average diameters around 130 µm, depending on the concentration of the initial suspension, and compression modulus values around 450 kPa, similar to other reported freeze-dried nanocellulose-based aerogels. Based on these characteristics, the cellulose/PAH-CuHARS composites are promising for potential biomedical applications as implants or wound dressing materials. They have proved to be effective in the decomposition of low molecular weight S-nitrosothiols (RSNOs), similar to those existing in blood, releasing nitric oxide (NO). This effect is attributed to the presence of copper in the crystalline structure of the CuHARS building unit, which can be gradually released in the presence of redox species like ascorbic acid, typically found in blood. The resulting biomaterials can offer the interesting properties associated with NO, like antimicrobial activity as preliminary tests showed here with Escherichia coli and Staphylococcus epidermidis. In the presence of physiological concentration of RSNOs the amount of generated NO (around 360 nM) is not enough to show bactericidal effect on the studied bacteria, but it could provide other properties inherent to NO even at low concentration in the nM range like anti-inflammatory and anti-thrombotic effects. The cytotoxic effect recorded of the films on rat brain endothelial cells (BMVECs) is least significant and proves them to be friendly enough for further biological studies.


Assuntos
Materiais Biocompatíveis/química , Celulose/química , Cobre/química , Cistina/química , Óxido Nítrico/análise , Animais , Encéfalo/irrigação sanguínea , Catálise , Sobrevivência Celular/efeitos dos fármacos , Celulose/ultraestrutura , Contagem de Colônia Microbiana , Cisteína/análogos & derivados , Cisteína/análise , Módulo de Elasticidade , Células Endoteliais/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Cinética , Microvasos/citologia , Poliaminas/química , Porosidade , Ratos , S-Nitrosotióis/análise , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimento
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