RESUMO
Many fundamental questions of astrophysics, biochemistry, and geology rely on the ability to accurately and precisely measure the mass and abundance of isotopes. Taken a step further, the capacity to perform such measurements on intact molecules provides insights into processes in diverse biological systems. Described here is the coupling of a combined atomic and molecular (CAM) ionization source, the liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma, with a commercially available ThermoScientific Fusion Lumos mass spectrometer. Demonstrated for the first time is the ionization and isotopically resolved fingerprinting of a long-postulated, but never mass-spectrometrically observed, bi-metallic complex Hg:Se-cysteine. Such a complex has been implicated as having a role in observations of Hg detoxification by selenoproteins/amino acids. Demonstrated as well is the ability to mass spectrometrically-resolve the geochronologically important isobaric 87Sr and 87Rb species (Δm ~ 0.3 mDa, mass resolution m/Δm ≈ 1,700,000). The mass difference in this case reflects the beta-decay of the 87Rb to the stable Sr isotope. These two demonstrations highlight what may be a significant change in bioinorganic and atomic mass spectrometry, with impact expected across a broad spectrum of the physical, biological, and geological sciences. Graphical Abstract "".
Assuntos
Espectrometria de Massas/instrumentação , Animais , Pressão Atmosférica , Monitoramento Ambiental/instrumentação , Desenho de Equipamento , Análise de Injeção de Fluxo/instrumentação , Humanos , Isótopos/análise , Mercúrio/análise , Selenocisteína/análise , Selenoproteínas/químicaRESUMO
This work presents a multisyringe flow injection analysis (MSFIA) system for the automatic spectrophotometric determination of total iron in wine. The reaction is based on the complexation of Fe(II) with 2-(5-bromo-2-pyridylazo)-5-(diethylamino)-phenol (Br-PADAP). Ascorbic acid was used as reducing reagent for Fe(III) to Fe(II) and, in this way, to determine the total iron content in wine. The absorbance of the Fe(II)-(Br-PADAP)2 complex was measured at 748â¯nm. The proposed method provided a working rage from 0.36 to 5â¯mgâ¯L-1 of Fe(II), with a detection limit of 0.11â¯mgâ¯L-1 of Fe(II), a relative standard deviation of 0.42% (3â¯mgâ¯L-1 of Fe(II), nâ¯=â¯10), and a 46â¯h-1 injection throughput. The system is very simple, rapid and selective, and has been successfully applied to determine total iron in red, rosé and white wine without any need for sample pre-treatment steps. The results agree well with ICP-AES, which used as a reference method.
Assuntos
Análise de Injeção de Fluxo/instrumentação , Análise de Alimentos/instrumentação , Ferro/análise , Seringas , Vinho/análise , Ácido Ascórbico/química , Automação , Compostos Azo/química , Ferro/química , Limite de DetecçãoRESUMO
A simple, reliable flow injection analysis (FIA)-tandem mass spectrometric (MS/MS) method was developed for the determination of gemini surfactants, designated as 16-3-16, 16(Py)-S-2-S-(Py)16 and 16-7N(GK)-16, as gene delivery agents in cellular matrix. 16-3-16 is a conventional gemini surfactant bearing two quaternary amines, linked by a 3-carbon spacer region, 16(Py)-S-2-S-(Py)16 contains two pyridinium head groups, while 16-7N(GK)-16 bears a glycine-lysine di-peptide in the space region. The method was fully validated according to USFDA guidelines. It is the first time that FIA-MS/MS method was developed for the quantification of gemini surfactants, belonging to different structural families. The method was superior to existing liquid chromatographic (LC)-MS/MS methods in terms of sensitivity and time of analysis. Positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode were used on a triple quadrupole-linear ion trap (4000 QTRAP®) instrument. Deuterated internal standards were used to correct for matrix effects and variations in ionization within the ESI source. Isotope dilution standard curves were established in cellular matrix, with a linear range of 10 nM-1000 nM for 16-3-16 and 16(Py)-S-2-S-(Py)16, and 20 nM-2000 nM for 16-7N(GK)-16. The precision, accuracy, recovery and stability were all within the acceptable ranges as per the USFDA guidelines. The method was successfully applied for the quantification of target gemini surfactants in the nuclear fraction of PAM 212 keratinocyte cells treated with nanoparticles, which varied significantly and may explain differences in the observed efficiency and/or toxicity of these gemini surfactants in gene delivery.
Assuntos
Análise de Injeção de Fluxo/métodos , Técnicas de Transferência de Genes , Tensoativos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Linhagem Celular Tumoral , Núcleo Celular/química , Análise de Injeção de Fluxo/instrumentação , Queratinócitos , Camundongos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
This study reports the preparation and characterization of nickel/lead hydroxide nanoparticles used to construct electrochemical sensors, which were investigated for amperometric quantification of N-acetylcysteine (NAC). The newly synthesised material presents good uniformity, with the lead (II) ions homogenously incorporated into the alpha nickel hydroxide crystal structure, confirmed by X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy analyses. Films of nanoparticles (3â¯nm in size) were prepared on conductive fluorine-doped tin oxide-coated glass slides and used connected to a specially built batch injection analysis (BIA) cell with a capacity of only 4â¯mL and the electrode positioned in the bottom. To attain optimal analytical performance, the main parameters for BIA measurements (volume injected, different velocities of injection and best distance of the pipette from the electrode) were evaluated, as was the working potential, to determine the optimal conditions. Linear responses were obtained for the concentration range from 20 to 220 µmol L-1, and the limits of detection (3σ/slope) and quantification (10σ/slope) were calculated as 0.23 µmol L-1 and 0.70 µmol L-1, respectively. The new NAC sensor does not exhibit a memory effect and has enormous potential utility in the quantitative determination of N-acetylcysteine in drugs. The results of the analysis of NAC obtained using BIA presented good concordance with those obtained by chromatography. The analytical frequency attained using BIA (120 analysis h-1) compares very favourably with the one obtained using chromatography (6 analysis h-1).
Assuntos
Acetilcisteína/análise , Técnicas Eletroquímicas , Análise de Injeção de Fluxo , Hidróxidos/química , Nanoestruturas/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Análise de Injeção de Fluxo/instrumentaçãoRESUMO
Chemiluminescence (CL) of copper nanoclusters (CuNCs) induced by cerium (IV) (Ce(IV)) or potassium permanganate (KMnO4 ) in acidic medium was observed. The potential application of CuNCs CL in analytical chemistry was also demonstrated using trihexyphenidyl hydrochloride (THP) as an example based on its enhancing CL intensity for the CuNCs-Ce(IV)/KMnO4 systems. The excited state of the CuNCs acted as a luminophore in the CuNCs-Ce(IV) system, while CuNCs played the role of reductant in the CuNCs-KMnO4 system. The increased CL intensity for Ce(IV)-CuNCs system was proportional to the THP concentrations in the range of 0.1 to 10.0 µM. The detection limit was 49.0 nM and the relative standard deviation was 2.2% for 2.0 µM THP (n = 11). The proposed method was applied to detect THP in pharmaceutical formulations and human plasma samples.
Assuntos
Cobre/química , Medições Luminescentes/métodos , Nanoestruturas/química , Triexifenidil/análise , Calibragem , Cério/química , Cisteína/química , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodos , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Oxirredução , Permanganato de Potássio/química , Espectrofotometria Ultravioleta/métodos , Espectroscopia de Infravermelho com Transformada de Fourier , Comprimidos/análise , Triexifenidil/sangueRESUMO
The determination of iron and copper exploiting a microsequential injection lab-on-valve system with online spectrophotometric detection is described. A new, environmental friendly 3-hydroxy-4-pyridinone chelator, functionalized with a polyethylene glycol chain (MRB12) to improve water solubility, was used for iron determination. For copper determination, 1-(2-pyridylazo)-2-naphthol (PAN) was used. Different parameters affecting the formation of the complexes were studied, namely sample volume, reagent concentration, and buffer composition and concentration. The optimized conditions, 150µL of sample volume and 250mgL-1 of MRB12 for iron determination, and 200µL of sample and 120mgL-1 of PAN for copper determination, enabled an LOD of 15 and 18µgL-1 for iron and copper, respectively. The robustness of the developed procedure was assessed by the calculation of the relative standard deviation (RSD), 5% for iron determination and 2% for copper determination. The accuracy of the method was assessed comparing the results with two certified samples (RD<7.5%) and calculating recovery percentages with five river water samples (average<107%).
Assuntos
Cobre/análise , Análise de Injeção de Fluxo/instrumentação , Água Doce/análise , Ferro/análise , Rios/química , Espectrofotometria/métodos , Análise de Injeção de Fluxo/métodos , Naftóis/química , Polietilenoglicóis/química , Piridonas/químicaRESUMO
Quantitative metabolomics has become much more important in clinical research in recent years. Individual differences in matrix effects (MEs) and the injection order effect are two major factors that reduce the quantification accuracy in liquid chromatography-electrospray ionization-mass spectrometry-based (LC-ESI-MS) metabolomics studies. This study proposed a postcolumn infused-internal standard (PCI-IS) combined with a matrix normalization factor (MNF) strategy to improve the analytical accuracy of quantitative metabolomics. The PCI-IS combined with the MNF method was applied for a targeted metabolomics study of amino acids (AAs). D8-Phenylalanine was used as the PCI-IS, and it was postcolumn-infused into the ESI interface for calibration purposes. The MNF was used to bridge the AA response in a standard solution with the plasma samples. The MEs caused signal changes that were corrected by dividing the AA signal intensities by the PCI-IS intensities after adjustment with the MNF. After the method validation, we evaluated the method applicability for breast cancer research using 100 plasma samples. The quantification results revealed that the 11 tested AAs exhibit an accuracy between 88.2 and 110.7%. The principal component analysis score plot revealed that the injection order effect can be successfully removed, and most of the within-group variation of the tested AAs decreased after the PCI-IS correction. Finally, targeted metabolomics studies on the AAs showed that tryptophan was expressed more in malignant patients than in the benign group. We anticipate that a similar approach can be applied to other endogenous metabolites to facilitate quantitative metabolomics studies.
Assuntos
Neoplasias da Mama/sangue , Cromatografia Líquida/normas , Metabolômica/normas , Espectrometria de Massas por Ionização por Electrospray/normas , Neoplasias da Mama/patologia , Calibragem , Cromatografia Líquida/métodos , Feminino , Análise de Injeção de Fluxo/instrumentação , Humanos , Metabolômica/métodos , Variações Dependentes do Observador , Fenilalanina/sangue , Análise de Componente Principal , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Triptofano/sangueRESUMO
Metabolomics based on direct mass spectrometry (MS) analysis, either by direct infusion or flow injection of crude sample extracts, shows a great potential for metabolic fingerprinting because of its high-throughput screening capability, wide metabolite coverage and reduced time of analysis. Considering that numerous metabolic pathways are significantly perturbed during the initiation and progression of diseases, these metabolomic tools can be used to get a deeper understanding about disease pathogenesis and discover potential biomarkers for early diagnosis. In this work, we describe the most common metabolomic platforms used in biomedical research, with special focus on strategies based on direct MS analysis. Then, a comprehensive review on the application of direct MS fingerprinting in clinical issues is provided.
Assuntos
Doença de Alzheimer/metabolismo , Diabetes Mellitus/metabolismo , Espectrometria de Massas/métodos , Metabolômica/métodos , Neoplasias/metabolismo , Obesidade/metabolismo , Animais , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodos , Humanos , Espectrometria de Massas/instrumentação , Redes e Vias Metabólicas , Metaboloma , Metabolômica/instrumentaçãoRESUMO
Pharmaceutical development is greatly hindered by the poor predictive power of existing in vitro models for drug efficacy and toxicity testing. In this work, we present a new and multilayer organs-on-a-chip device that allows for the assessment of drug metabolism, and its resultant drug efficacy and cytotoxicity in different organ-specific cells simultaneously. Four cell lines representing the liver, tumor (breast cancer and lung cancer), and normal tissue (gastric cells) were cultured in the compartmentalized micro-chambers of the multilayer microdevice. We adopted the prodrug capecitabine (CAP) as a model drug. The intermediate metabolites 5'-deoxy-5-fluorocytidine (DFUR) of CAP that were metabolized from liver and its active metabolite 5-fluorouracil (5-FU) from the targeted cancer cells and normal tissue cells were identified using mass spectrometry. CAP exhibited strong cytoxicity on breast cancer and lung cancer cells, but not in normal gastric cells. Moreover, the drug-induced cytotoxicity on cells varied in various target tissues, suggesting the metabolism-dependent drug efficacy in different tissues as exisits in vivo. This in vitro model can not only allow for characterizing the dynamic metabolism of anti-cancer drugs in different tissues simultaneously, but also facilitate the assessment of drug bioactivity on various target tissues in a simple way, indicating the utility of this organs-on-chip for applications in pharmacodynamics/pharmacokinetics studies, drug efficacy and toxicity testing.
Assuntos
Capecitabina/farmacocinética , Capecitabina/toxicidade , Dispositivos Lab-On-A-Chip , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Técnicas de Cultura de Órgãos/instrumentação , Testes de Toxicidade/instrumentação , Células A549 , Órgãos Bioartificiais , Capecitabina/administração & dosagem , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodos , Células Hep G2 , Humanos , Análise do Fluxo Metabólico/instrumentação , Análise do Fluxo Metabólico/métodos , Neoplasias Experimentais/patologia , Técnicas de Cultura de Órgãos/métodos , Análise Serial de Tecidos/instrumentação , Testes de Toxicidade/métodos , Vísceras/efeitos dos fármacos , Vísceras/metabolismo , Vísceras/patologiaRESUMO
Sex steroids are involved in many physiological and pathological processes. The determination of sex steroids is essential to understand the mechanisms of human health and diseases. Derivatization techniques could specifically enhance the sensitivities for sex steroids with a given functional group. However, no derivatization reagents are available for profiling multifunctional sex steroids, including phenolic estrogens, ketolic androgens, and ketolic progestogens, in a single analytical run. In the present study, a novel method involving ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) was developed for profiling both ketolic and phenolic sex steroids in human serum using an automated injection program combined with diverter valve switch and step analysis (AIDSA). The human serum, prepared through liquid-liquid extraction and subsequently derivatized using Girard P offline, was automatically injected twice under the automated injection program. For the first injection, Girard P-derivatized ketolic sex steroids were loaded onto the column, and subsequently, the second injection and online derivatization of the same sample using dansyl chloride were performed in the injector needle for 15 min. The dansyl-labeled phenolic sex steroids were then loaded onto the column. The diverter valve worked in coordination with the injection program to import the derivatized sex steroids and remove excess derivatization reagents. The two types of derivatives were individually analyzed in a step-by-step manner. In addition, online dansyl derivatization and Girard P derivative analyses were simultaneously implemented to shorten the total analysis time. This method was well validated and applied to determine the sex steroid levels in human serum.
Assuntos
Androgênios/sangue , Automação , Estrogênios/sangue , Análise de Injeção de Fluxo , Extração Líquido-Líquido , Progestinas/sangue , Cromatografia Líquida de Alta Pressão/instrumentação , Análise de Injeção de Fluxo/instrumentação , Humanos , Extração Líquido-Líquido/instrumentação , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
Overcoming transport barriers to delivery of therapeutic agents in tumors remains a major challenge. Focused ultrasound (FUS), in combination with modern nanomedicine drug formulations, offers the ability to maximize drug transport to tumor tissue while minimizing toxicity to normal tissue. This potential remains unfulfilled due to the limitations of current approaches in accurately assessing and quantifying how FUS modulates drug transport in solid tumors. A novel acoustofluidic platform is developed by integrating a physiologically relevant 3D microfluidic device and a FUS system with a closed-loop controller to study drug transport and assess the response of cancer cells to chemotherapy in real time using live cell microscopy. FUS-induced heating triggers local release of the chemotherapeutic agent doxorubicin from a liposomal carrier and results in higher cellular drug uptake in the FUS focal region. This differential drug uptake induces locally confined DNA damage and glioblastoma cell death in the 3D environment. The capabilities of acoustofluidics for accurate control of drug release and monitoring of localized cell response are demonstrated in a 3D in vitro tumor mode. This has important implications for developing novel strategies to deliver therapeutic agents directly to the tumor tissue while sparing healthy tissue.
Assuntos
Antineoplásicos/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Monitoramento de Medicamentos/instrumentação , Análise de Injeção de Fluxo/instrumentação , Dispositivos Lab-On-A-Chip , Neoplasias Experimentais/tratamento farmacológico , Sonicação/instrumentação , Acústica/instrumentação , Técnicas de Cultura Celular por Lotes/instrumentação , Linhagem Celular Tumoral , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Sistemas Microeletromecânicos/instrumentação , Neoplasias Experimentais/patologia , Impressão Tridimensional/instrumentaçãoRESUMO
We present a microfluidic chip that generates linear concentration gradients of multiple solutes that are orthogonally-aligned to each other. The kinetics of gradient formation was characterized using a fluorescent tracer matching the molecular weight of small inhibitory drugs. Live-cell signalling and motility experiments were conducted to demonstrate the potential uses and advantages of the device. A431 epidermoid carcinoma cells, where EGF induces apoptosis in a concentration-dependent manner, were simultaneously exposed to gradients of MEK inhibitor and EGF receptor (EGFR) inhibitor. By monitoring live caspase activation in the entire chip, we were able to quickly assess the combinatorial interaction between MEK and EGFR pathways, which otherwise would require costly and time consuming titration experiments. We also characterized the motility and morphology of MDA-MB-231 breast cancer cells exposed to orthogonal gradients of EGF and EGFR inhibitor. The microfluidic chip not only permitted the quantitative analysis of a population of cells exposed to drug combinations, but also enabled the morphological characterization of individual cells. In summary, our microfluidic device, capable of establishing concentration gradients of multiple compounds over a group of cells, facilitates and accelerates in vitro cell biology experiments, such as those required for cell-based drug combination assays.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Bioensaio/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Análise de Injeção de Fluxo/instrumentação , Dispositivos Lab-On-A-Chip , Neoplasias Experimentais/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Combinação de Medicamentos , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Neoplasias Experimentais/patologia , Neoplasias Experimentais/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resultado do TratamentoRESUMO
A simple and sensitive flow injection chemiluminescence (CL) method was developed for determination of nalidixic acid by application of CdS quantum dots (QDs) in KMnO4-morin CL system in acidic medium. Optical and structural features of L-cysteine capped CdS quantum dots which were synthesized via hydrothermal approach were investigated using X-ray diffraction (XRD), scanning electron microscopy (SEM), photoluminescence (PL), and ultraviolet-visible (UV-Vis) spectroscopy. Moreover, the potential mechanism of the proposed CL method was described using the results of the kinetic curves of CL systems, the spectra of CL, PL and UV-Vis analyses. The CL intensity of the KMnO4-morin-CdS QDs system was considerably increased in the presence of nalidixic acid. Under the optimum condition, the enhanced CL intensity was linearly proportional to the concentration of nalidixic acid in the range of 0.0013 to 21.0 mg L(-1), with a detection limit of (3σ) 0.003 mg L(-1). Also, the proposed CL method was utilized for determination of nalidixic acid in environmental water samples, and commercial pharmaceutical formulation to approve its applicability. Furthermore, corona discharge ionization ion mobility spectrometry (CD-IMS) method was utilized for determination of nalidixic acid and the results of real sample analysis by two proposed methods were compared. Comparison the analytical features of these methods represented that the proposed CL method is preferable to CD-IMS method for determination of nalidixic acid due to its high sensitivity and precision.
Assuntos
Antibacterianos/análise , Compostos de Cádmio/química , Flavonoides/química , Medições Luminescentes/métodos , Ácido Nalidíxico/análise , Pontos Quânticos/química , Sulfetos/química , Poluentes Químicos da Água/análise , Cisteína/química , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Desenho de Equipamento , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodos , Água Subterrânea/análise , Limite de Detecção , Medições Luminescentes/instrumentação , Permanganato de Potássio/química , Pontos Quânticos/ultraestruturaRESUMO
A novel flow-programming setup based on the sequential injection principle is herein proposed for on-line monitoring of temporal events in cell permeation studies. The permeation unit consists of a Franz cell with its basolateral compartment mixed under mechanical agitation and thermostated at 37 °C. The apical compartment is replaced by commercially available Transwell inserts with a precultivated cell monolayer. The transport of drug substances across epithelial cells genetically modified with the P-glycoprotein membrane transporter (MDCKII-MDR1) is monitored on-line using rhodamine 123 as a fluorescent marker. The permeation kinetics of the marker is obtained in a fully automated mode by sampling minute volumes of solution from the basolateral compartment in short intervals (10 min) up to 4 h. The effect of a P-glycoprotein transporter inhibitor, verapamil as a model drug, on the efficiency of the marker transport across the cell monolayer is thoroughly investigated. The analytical features of the proposed flow method for cell permeation studies in real time are critically compared against conventional batch-wise procedures and microfluidic devices.
Assuntos
Automação/métodos , Células Epiteliais/metabolismo , Análise de Injeção de Fluxo/métodos , Verapamil/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Células Epiteliais/química , Análise de Injeção de Fluxo/instrumentação , Humanos , Cinética , Rodamina 123/química , Rodamina 123/metabolismo , Verapamil/químicaRESUMO
A novel design of reusable microfluidic platform that generates a stationary nanoliter droplet array (SNDA) for cell incubation and analysis, equipped with a complementary array of individually addressable electrodes for each microwell is studied. Various solute concentration gradients were generated between the wells where dielectrophoresis (DEP) was used to characterize the effect of the gradients on the cell's response. The feasibility of generating concentration gradients and observation of DEP responses was demonstrated using a gradient of salts in combination with microparticles and viable cells. L1210 Lymphoma cells were used as the model cells in these experiments. Lymphoma cells' cross-over frequency (COF) decreased with increasing stress conditions. Specifically, a linear decrease in the cell COF was measured as a function of solution tonicity and blebbistatin dose. Lymphoma cells were incubated under a gradient of the chemotherapeutic agent doxorubicin (DOX), which led to saturation in the cell-COF response at 30 nM DOX, demonstrating the potential of the platform in screening of label-free drugs.
Assuntos
Separação Celular/instrumentação , Quimiotaxia/fisiologia , Eletroforese/instrumentação , Análise de Injeção de Fluxo/instrumentação , Dispositivos Lab-On-A-Chip , Análise Serial de Tecidos/instrumentação , Quimiotaxia/efeitos dos fármacos , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Miniaturização , Nanotecnologia/instrumentaçãoRESUMO
Quantifying cell-to-cell variability in drug response dynamics is important when evaluating therapeutic efficacy. For example, optimizing latency reversing agents (LRAs) for use in a clinical "activate-and-kill" strategy to purge the latent HIV reservoir in patients requires minimizing heterogeneous viral activation dynamics. To evaluate how heterogeneity in latent HIV activation varies across a range of LRAs, we tracked drug-induced response dynamics in single cells via live-cell imaging using a latent HIV-GFP reporter virus in a clonal Jurkat T cell line. To enable these studies in suspension cells, we designed a simple method to capture an array of single Jurkat T cells using a passive-flow microfluidic device. Our device, which does not require external pumps or tubing, can trap hundreds of cells within minutes with a high retention rate over 12 hours of imaging. Using this device, we quantified heterogeneity in viral activation stimulated by transcription factor (TF) activators and histone deacetylase (HDAC) inhibitors. Generally, TF activators resulted in both faster onset of viral activation and faster rates of production, while HDAC inhibitors resulted in more uniform onset times, but more heterogeneous rates of production. Finally, we demonstrated that while onset time of viral gene expression and rate of viral production together predict total HIV activation, rate and onset time were not correlated within the same individual cell, suggesting that these features are regulated independently. Overall, our results reveal drug-specific patterns of noisy HIV activation dynamics not previously identified in static single-cell assays, which may require consideration for the most effective activate-and-kill regime.
Assuntos
Separação Celular/instrumentação , HIV/fisiologia , HIV/ultraestrutura , Inibidores de Histona Desacetilases/administração & dosagem , Dispositivos Lab-On-A-Chip , Ativação Viral/fisiologia , Bioensaio/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/instrumentação , HIV/efeitos dos fármacos , Humanos , Células Jurkat , Microscopia de Fluorescência/instrumentação , Análise Serial de Tecidos/instrumentação , Ativação Viral/efeitos dos fármacos , Latência ViralRESUMO
The electrodynamic ion funnel facilitates efficient focusing and transfer of charged particles in the higher-pressure regions (e.g., ion source interfaces) of mass spectrometers, thus providing increased sensitivity. An "off-axis" ion funnel design has been developed to reduce the source contamination and interferences from, e.g. ESI droplet residue and other poorly focused neutral or charged particles with very high mass-to-charge ratios. In this study, a dual ion funnel interface consisting of an orthogonal higher pressure electrodynamic ion funnel (HPIF) and an ion funnel trap combined with a triple quadrupole mass spectrometer was developed and characterized. An orthogonal ion injection inlet and a repeller plate electrode was used to direct ions to an ion funnel HPIF at a pressure of 9-10 Torr. Key factors for the HPIF performance characterized included the effects of RF amplitude, the DC gradient, and operating pressure. Compared to the triple quadrupole standard interface more than 4-fold improvement in the limit of detection for the direct quantitative MS analysis of low abundance peptides was observed. The sensitivity enhancement in liquid chromatography selected reaction monitoring (LC-SRM) analyses of low-abundance peptides spiked into a highly complex mixture was also compared with that obtained using both a commercial S-lens interface and an in-line dual-ion funnel interface.
Assuntos
Análise de Injeção de Fluxo , Peptídeos/análise , Cromatografia Líquida de Alta Pressão/instrumentação , Análise de Injeção de Fluxo/instrumentação , Espectrometria de Massas/instrumentação , PressãoRESUMO
Cancer, developmental biology and tissue injury present multiple examples where groups of cells residing in close proximity communicate via paracrine factors. It is nearly impossible to dissect such cellular interactions in vivo and is quite challenging in vitro. The goal of this study is to utilize a reconfigurable microfluidic device in order to study paracrine signal exchange between groups of primary hepatocytes in vitro. Previously, we demonstrated that hepatocytes residing on protein spots containing collagen and hepatocyte growth factor (HGF) spots expressed epithelial (hepatic) phenotypes and also rescued them in neighboring hepatocytes on collagen spots that did not receive direct HGF stimulus. Herein, we designed a microfluidic device with parallel fluidic channels separated by retractable (reconfigurable) walls and employed this device to investigate interactions between groups of HGF-stimulated and unstimulated hepatocytes. Using a novel reconfigurable microfluidic device, we demonstrate that cultivation of HGF-containing protein spots upregulates the production of endogenous HGF in hepatocytes and that these HGF molecules diffuse over, causing phenotype enhancement in the recipient cells. We also show that selective treatment of the recipient hepatocytes with a c-met inhibitor (SU11274) diminishes the rescue effect, as gauged by the down-regulation of albumin and HGF expression. Our study is one of the first to demonstrate paracrine signaling via HGF in primary hepatocytes. More broadly, tools and methods described here may be used to study paracrine signaling in other types of cells and will have relevance for various fields of biomedical research from cancer to immunology.
Assuntos
Comunicação Autócrina/fisiologia , Separação Celular/instrumentação , Análise de Injeção de Fluxo/instrumentação , Hepatócitos/fisiologia , Dispositivos Lab-On-A-Chip , Comunicação Parácrina/fisiologia , Animais , Anistreplase , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Feminino , Hepatócitos/citologia , Ratos , Ratos Endogâmicos LewRESUMO
L-Cysteine capped CdS quantum dots (QDs) were synthesized through a facile hydrothermal method and characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), photoluminescence (PL) and UV-Vis spectroscopy. The light emitted from KMnO4-L-cysteine capped CdS QDs reaction in acidic medium was applied as a simple and sensitive chemiluminescence (CL) system for determination of dexamethasone. The CL intensity of KMnO4-L-cysteine capped CdS QDs CL system was remarkably enhanced in the presence of dexamethasone. Under optimum experimental conditions, the enhanced CL intensity was related to dexamethasone concentration in the range of 0.004-25.0 mg L(-1), with the detection limit (3σ) of 0.0013 mg L(-1). The analytical applicability of the proposed CL system was assessed by determining dexamethasone in spiked environmental water samples and pharmaceutical formulation. The analytical performances of proposed flow-injection CL method for the determination of dexamethasone were compared with those obtained by corona discharge ionization ion mobility spectrometry (CD-IMS) method. The proposed CL system exhibits a higher sensitivity and precision than the CD-IMS method for the determination of dexamethasone.
Assuntos
Anti-Inflamatórios/análise , Compostos de Cádmio/química , Dexametasona/análise , Medições Luminescentes/instrumentação , Pontos Quânticos/química , Sulfetos/química , Poluentes Químicos da Água/análise , Cisteína/química , Monitoramento Ambiental/instrumentação , Desenho de Equipamento , Análise de Injeção de Fluxo/instrumentação , Limite de Detecção , Luminescência , Água/análiseRESUMO
Tissue intermittent hypoxia (IH) occurs in obstructive sleep apnea, sickle cell anemia, physical exercise and other conditions. Poor gas solubility and slow diffusion through culture media hampers mimicking IH-induced transitions of O(2) in vitro. We aimed to develop a system enabling exposure of cultured cells to IH and to validate such exposure by real-time O(2) measurements and cellular responses. Standard 24-well culture plates and plates with bottoms made from a gas permeable film were placed in a heated cabinet. Desired cycling of O(2) levels was induced using programmable solenoids to purge mixtures of 95% N(2) + 5% CO(2) or 95% O(2) + 5% CO(2). Dissolved oxygen, gas pressure, temperature, and water evaporation were measured during cycling. IH-induced cellular effects were evaluated by hypoxia inducible factor (HIF) and NF-κB luciferase reporters in HEK296 cells and by insulin secretion in rat insulinoma cells. Oxygen cycling in the cabinet was translated into identical changes of O(2) at the well bottom in gas permeable, but not in standard cultureware. Twenty-four hours of IH exposure increased HIF (112%), NF-κB (111%) and insulin secretion (44%). Described system enables reproducible and prolonged IH exposure in cultured cells while controlling for important environmental factors.