RESUMO
BACKGROUND: Lipids are regulators of insulitis and ß-cell death in type 1 diabetes development, but the underlying mechanisms are poorly understood. Here, we investigated how the islet lipid composition and downstream signaling regulate ß-cell death. METHODS: We performed lipidomics using three models of insulitis: human islets and EndoC-ßH1 ß cells treated with the pro-inflammatory cytokines interlukine-1ß and interferon-γ, and islets from pre-diabetic non-obese mice. We also performed mass spectrometry and fluorescence imaging to determine the localization of lipids and enzyme in islets. RNAi, apoptotic assay, and qPCR were performed to determine the role of a specific factor in lipid-mediated cytokine signaling. RESULTS: Across all three models, lipidomic analyses showed a consistent increase of lysophosphatidylcholine species and phosphatidylcholines with polyunsaturated fatty acids and a reduction of triacylglycerol species. Imaging assays showed that phosphatidylcholines with polyunsaturated fatty acids and their hydrolyzing enzyme phospholipase PLA2G6 are enriched in islets. In downstream signaling, omega-3 fatty acids reduce cytokine-induced ß-cell death by improving the expression of ADP-ribosylhydrolase ARH3. The mechanism involves omega-3 fatty acid-mediated reduction of the histone methylation polycomb complex PRC2 component Suz12, upregulating the expression of Arh3, which in turn decreases cell apoptosis. CONCLUSIONS: Our data provide insights into the change of lipidomics landscape in ß cells during insulitis and identify a protective mechanism by omega-3 fatty acids. Video Abstract.
Assuntos
Ácidos Graxos Ômega-3 , Ilhotas Pancreáticas , N-Glicosil Hidrolases , Camundongos , Animais , Humanos , Ilhotas Pancreáticas/metabolismo , Morte Celular , Citocinas/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Insaturados , Fosfatidilcolinas/metabolismoRESUMO
Ambient mass spectrometry imaging (MSI) is a powerful technique that allows for the simultaneous mapping of hundreds of molecules in biological samples under atmospheric conditions, requiring minimal sample preparation. We have developed nanospray desorption electrospray ionization (nano-DESI), a liquid extraction-based ambient ionization technique, which has proven to be sensitive and capable of achieving high spatial resolution. We have previously described an integrated microfluidic probe, which simplifies the nano-DESI setup, but is quite difficult to fabricate. Herein, we introduce a facile and scalable strategy for fabricating microfluidic devices for nano-DESI MSI applications. Our approach involves the use of selective laser-assisted etching (SLE) of fused silica to create a monolithic microfluidic probe (SLE-MFP). Unlike the traditional photolithography-based fabrication, SLE eliminates the need for the wafer bonding process and allows for automated, scalable fabrication of the probe. The chamfered design of the sampling port and ESI emitter significantly reduces the amount of polishing required to fine-tune the probe thereby streamlining and simplifying the fabrication process. We have also examined the performance of a V-shaped probe, in which only the sampling port is fabricated using SLE technology. The V-shaped design of the probe is easy to fabricate and provides an opportunity to independently optimize the size and shape of the electrospray emitter. We have evaluated the performance of SLE-MFP by imaging mouse tissue sections. Our results demonstrate that SLE technology enables the fabrication of robust monolithic microfluidic probes for MSI experiments. This development expands the capabilities of nano-DESI MSI and makes the technique more accessible to the broader scientific community.
Assuntos
Microfluídica , Espectrometria de Massas por Ionização por Electrospray , Camundongos , Animais , Espectrometria de Massas por Ionização por Electrospray/métodos , Nanotecnologia/métodos , TecnologiaRESUMO
Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that enables molecular imaging of biological samples with high spatial resolution. We have recently developed an integrated microfluidic probe (iMFP) for nano-DESI mass spectrometry imaging (MSI) that significantly enhances the robustness of the technique. In this study, we designed a new probe that enables imaging of biological samples with high spatial resolution. The new probe design features smaller primary and spray channels and an entirely new configuration of the sampling port that enables robust imaging of tissues with a spatial resolution of 8-10 µm. We demonstrate the spatial resolution, sensitivity, durability, and throughput of the iMFP by imaging mouse uterine and brain tissue sections. The robustness of the high-resolution iMFP allowed us to perform first imaging experiments with both high spatial resolution and high throughput, which is particularly advantageous for high-resolution imaging of large tissue sections of interest to most MSI applications. Overall, the new probe design opens opportunities for mapping of biomolecules in biological samples with high throughput and cellular resolution, which is important for understanding biological systems.
Assuntos
Microfluídica , Espectrometria de Massas por Ionização por Electrospray , Camundongos , Animais , Espectrometria de Massas por Ionização por Electrospray/métodos , Encéfalo/diagnóstico por imagemRESUMO
Nanospray desorption electrospray mass spectrometry imaging (nano-DESI MSI) enables quantitative mapping of hundreds of molecules in biological samples with minimal sample pretreatment. We have recently developed an integrated microfluidic probe (iMFP) for nano-DESI MSI. Herein, we describe an improved design of the iMFP for the high-throughput imaging of tissue sections. We increased the dimensions of the primary and spray channels and optimized the spray voltage and solvent flow rate to obtain a stable operation of the iMFP at both low and high scan rates. We observe that the sensitivity, molecular coverage, and spatial resolution obtained using the iMFP do not change to a significant extent as the scan rate increases. Using a scan rate of 0.4 mm/s, we obtained high-quality images of mouse uterine tissue sections (scan area: 3.2 mm × 2.3 mm) in only 9.5 min and of mouse brain tissue (scan area: 7.0 mm × 5.4 mm) in 21.7 min, which corresponds to a 10-15-fold improvement in the experimental throughput. We have also developed a quantitative metric for evaluating the quality of ion images obtained at different scan rates. Using this metric, we demonstrate that the quality of nano-DESI MSI data does not degrade substantially with an increase in the scan rate. The ability to image biological tissues with high throughput using iMFP-based nano-DESI MSI will substantially speed up tissue mapping efforts.
Assuntos
Microfluídica , Espectrometria de Massas por Ionização por Electrospray , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Diagnóstico por Imagem , Camundongos , Solventes/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Ambient ionization based on liquid extraction is widely used in mass spectrometry imaging (MSI) of molecules in biological samples. The development of nanospray desorption electrospray ionization (nano-DESI) has enabled the robust imaging of tissue sections with high spatial resolution. However, the fabrication of the nano-DESI probe is challenging, which limits its dissemination to the broader scientific community. Herein, we describe the design and performance of an integrated microfluidic probe (iMFP) for nano-DESI MSI. The glass iMFP, fabricated using photolithography, wet etching, and polishing, shows comparable performance to the capillary-based nano-DESI MSI in terms of stability and sensitivity; a spatial resolution of better than 25â µm was obtained in these first proof-of-principle experiments. The iMFP is easy to operate and align in front of a mass spectrometer, which will facilitate broader use of liquid-extraction-based MSI in biological research, drug discovery, and clinical studies.
Assuntos
Técnicas Analíticas Microfluídicas , Útero/citologia , Animais , Feminino , Camundongos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Clinical application of direct sampling electrospray ionization mass spectrometry (ESI-MS) remains limited due to problems associated with very "dirty" sample matrices. Herein we report on a microfluidic platform that allows direct mass spectrometric analysis of serum samples of microliter sizes. The platform integrates in-line paper adsorption-based sample clean-up and voltage assisted liquid desorption ESI-MS/MS (VAL DESI-MS/MS) to detect multiple targeted compounds of clinical interest. Adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) were selected as model analytes. Simultaneous quantification of these compounds in human serum samples was demonstrated. For all the three compounds, linear calibration curves were obtained in a concentration range from 0.20 to 20.0 µmol/L with r2 values ≥ 0.996. Limits of detection were 0.019, 0.015, and 0.011 µmol/L for AMP, ADP, and ATP, respectively. Recovery was found in the range from 96.5% to 103.5% at spiking concentrations of 0.25 and 2.50 µmol/L. The results indicate that the proposed microfluidic mass spectrometric platform is robust and effective. It may have a potential in clinical analysis.
Assuntos
Difosfato de Adenosina/sangue , Monofosfato de Adenosina/sangue , Trifosfato de Adenosina/sangue , Técnicas Eletroquímicas , Dispositivos Lab-On-A-Chip , Papel , Adsorção , Humanos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A method based on microfluidic voltage-assisted liquid desorption electrospray ionization-tandem mass spectrometry (VAL-DESI-MS/MS) has been developed for fast quantification of free amino acids in food. Food extracts were transferred to the microfluidic platform and analyzed by liquid desorption ESI-MS/MS. Deuterated aspartic acid (i.e., 2,2,3-d3-Asp) was used as internal standard for analysis. The method had linear calibration curves with r2 values > 0.998. Limits of detection were at the level of sub µM for the amino acids tested, i.e., glutamic acid (Glu), arginine (Arg), tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). To validate the proposed method in food analysis, extracts of Cordyceps fungi were analyzed. Amino acid contents were found in the range from 0.63 mg/g (Tyr in Cordyceps sinensis) to 4.44 mg/g (Glu in Cordyceps militaris). Assay repeatability (RSD) was ≤ 5.2% for all the five amino acids measured in all the samples analyzed. Recovery was found in the range from 95.8 to 105.1% at two spiking concentrations of 0.250 mg/g and 1.00 mg/g. These results prove that the proposed microfluidic VAL-DESI-MS/MS method offers a quick and convenient means of quantifying free amino acids with accuracy and repeatability. Therefore, it may have potential in food analysis for nutritional and quality assessment purposes. Graphical abstract.
Assuntos
Aminoácidos/análise , Análise de Alimentos/métodos , Microfluídica , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
This work describes a novel method for quantification of miRNAs based on multistage signal amplification (MSA) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The multistage signal amplification involves hybridization enrichment of miRNA targets with a DNA probe-magnetic bead conjugate, target recycling amplification with a duplex-specific nuclease, and acid hydrolysis of the reporter molecules producing free nucleobases. Nucleobases thus generated are quantified by LC-ESI-MS/MS with specificity and repeatability. Taking miR-21 as the model target, biological samples such as serum and cell cultures were analyzed by using the present protocol. The analytical results indicate that facile and cost-effective quantifications of miRNA targets can be achieved by using the popular LC-ESI-MS/MS technique, and very importantly, without an isolation of total RNAs from the sample prior to the quantitative assay. The assay for miR-21 detection had a linear calibration curve in the range from 0.2 pM to 0.25 nM with a limit of detection of 60 fM. Analysis of MCF-7 cells treated with toremifene (a potent inhibitor of breast cancer cell growth) revealed that the content of miRNA-21 decreased by ca. 50%, and the decrease was dose-dependent.
Assuntos
Cromatografia Líquida/métodos , MicroRNAs/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Sondas de DNA/química , Sondas de DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desoxirribonucleases/química , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Células MCF-7 , MicroRNAs/genética , Hibridização de Ácido NucleicoRESUMO
MicroRNAs (miRNAs) are associated with physiological and pathological processes. They are recognized as biomarkers for diseases diagnosis and treatment evaluation. Herein we propose a simple and cost-effective HPLC method for quantitative assay of target miRNAs with femtomolar sensitivity, single-base discrimination selectivity and low background. The assay is based on an innovative signal-on strategy. In this strategy, polyadenylation of poly(A) polymerase extends an all 'A' sequence at the end of target miRNA, and the substantially increased number of adenine bases are labeled with 2-Chloroacetaldehyde (CAA) to open a signal-on mode and realize a signal amplification. The linearly amplified fluorescence signal is separated from other inference signals and quantified by high performance liquid chromatography with fluorescence detection (HPLC-FD). Combining with affinity magnetic solid phase extraction (MSPE), the method is well suited for analysis of complex biological samples such as serum and cell lysate with nearly zero background fluorescence. Taking miRNA-21 as the model analyte, this absolute quantification method has a limit of detection of 200 fM and a linear calibration curve (R2 = 0.999) in the range from 2.00 pM to 1.00 nM. Using locked nucleic acid (LNA) modified probes rather than ssDNA probes, the assay selectivity is improved. Moreover, analysis of bovine serum and cell lysate samples by using the method is demonstrated. Intracellular content of miRNA-21 is found to be 0.0150 amol/cell in MCF-7 cells with an assay repeatability of 4.0% (RSD, n = 3). The present HPLC quantification of miRNA offers an accurate, reliable, and cost-effective means for quantitative assay of miRNAs occurring in biological samples. Also importantly, it eliminates the need for total RNA isolation for the analysis. It may be useful for more effective diagnosis of diseases and therapeutic evaluation.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , DNA de Cadeia Simples/genética , MicroRNAs/análise , Animais , Bioensaio , Calibragem , Bovinos , Técnicas de Cultura de Células , Humanos , Limite de Detecção , Células MCF-7 , MicroRNAs/genética , Técnicas de Amplificação de Ácido NucleicoRESUMO
Quantitative assay of microRNAs (miRNAs) with mass spectrometric detection currently suffers from two major disadvantages, i.e., being insufficient in sensitivity and requiring an extraction or chromatographic separation prior to MS detection. In this work, we developed a facile and sensitive assay of targeted miRNAs based on the combination of cyclic enzymatic amplification (CEA) with microfluidic voltage-assisted liquid desorption electrospray ionization tandem mass spectrometry (VAL-DESI-MS/MS). The single-stranded DNA (ssDNA) probe was designed to have a sequence complementary to the miRNA target with an extension of a two-base nucleotide fragment (i.e., CpC) at the 3'-position as MS signal reporter, thus being easy to prepare and high in stability. In the proposed CEA-VAL-DESI-MS/MS assay, an ssDNA probe was added to a sample solution, forming a DNA-miRNA hybrid. Duplex-specific nuclease (DSN) was then added to cleave specifically the DNA probe in the heteroduplex strands. As the hybridization-cleavage cycle repeated itself for many rounds, a large quantity of CpC molecules was produced that was quantified by VAL-DESI-MS/MS with accuracy and specificity. miRNA-21 was tested as the model target. The assay had a linear calibration equation in the range from 2.5 pM to 1.0 nM with a limit of detection of 0.25 pM. Determination of miRNA-21 in cellular samples was demonstrated. miRNA-21 was found to be 95.3 ± 13.95 amol ( n = 3) in 100 mouse peritoneal macrophages with a recovery of 94.2 ± 2.6% ( n = 3). Interestingly, analysis of exosomes secreted from these cells revealed that exposure of the cells to chemical stimuli caused a 3-fold increase in exosomal level of miRNA-21. The results suggest that the proposed assay may provide an accurate and cost-effective means for quantification of targeted miRNAs in biomedical samples.
Assuntos
MicroRNAs/análise , Microfluídica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Calibragem , Sondas de DNA , DNA de Cadeia Simples/química , Limite de Detecção , Macrófagos Peritoneais/química , Camundongos , Reprodutibilidade dos TestesRESUMO
Signal suppression by sample matrix in direct electrospray ionization-mass spectrometric (ESI-MS) analysis hampers its clinical and biomedical applications. We report herein the development of a microfluidic voltage-assisted liquid desorption electrospray ionization (VAL-DESI) source to overcome this limitation. Liquid DESI is achieved for the first time in a microfluidic format. Direct analysis of urine, serum, and cell lysate samples by using the proposed microfluidic VAL-DESI-MS/MS method to detect chemical compounds of biomedical interest, including nucleosides, monoamines, amino acids, and peptides is demonstrated. Analyzing a set of urine samples spiked with dihydroxyphenylalanine (DOPA) showed that the assay had a linear calibration curve with r2 value of 0.997 and a limit of detection of 0.055 µM DOPA. The method was applied to simultaneous quantification of nucleosides, that is, cytidine, adenosine, uridine, thymidine, and guanosine in cell lysates using 8-bromoadenosine as internal standard. Adenosine was found most abundant at 26.5 ± 0.57 nmol/106 cells, while thymidine was least at 3.1 ± 0.31 nmol/106 cells. Interestingly, the ratio of adenosine to deoxyadenosine varied significantly from human red blood cells (1.07 ± 0.06) to cancerous cells, including lymphoblast TK6 (0.52 ± 0.02), skin melanoma C32 (0.82 ± 0.04), and promyelocytic leukemia NB4 cells (0.38 ± 0.06). These results suggest that the VAL-DESI-MS/MS technique has a good potential in direct analysis of biofluids. Further, because of the simplicity in its design and operation, the proposed microfluidic liquid DESI source can be fabricated as a disposable device for point-of-care measurements.
Assuntos
Cafeína/sangue , Cafeína/urina , Técnicas Analíticas Microfluídicas , Espectrometria de Massas por Ionização por Electrospray , Cafeína/química , Eritrócitos/química , Humanos , Estrutura Molecular , Células Tumorais CultivadasRESUMO
Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-µL flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (n = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with r2 value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (n = 5). The detection limit was estimated to be 2.9 × 107 exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h.
Assuntos
Carbocianinas/análise , Cromatografia em Gel/métodos , Vesículas Extracelulares/química , Corantes Fluorescentes/análise , Linhagem Celular , Fluorescência , Humanos , Lipossomos/química , Tamanho da PartículaRESUMO
Capillary electrophoresis-based single cell analysis has become an essential approach in researches at the cellular level. However, automation of single cell analysis has been a challenge due to the difficulty to control the number of cells injected and the irreproducibility associated with cell aggregation. Herein we report the development of a new microfluidic platform deploying the double nano-electrode cell lysis technique for automated analysis of single cells with mass spectrometric detection. The proposed microfluidic chip features integration of a cell-sized high voltage zone for quick single cell lysis, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Built upon this platform, a microchip electrophoresis-mass spectrometric method (MCE-MS) has been developed for automated single cell analysis. In the method, cell introduction, cell lysis, and MCE-MS separation are computer controlled and integrated as a cycle into consecutive assays. Analysis of large numbers of individual PC-12 neuronal cells (both intact and exposed to 25mM KCl) was carried out to determine intracellular levels of dopamine (DA) and glutamic acid (Glu). It was found that DA content in PC-12 cells was higher than Glu content, and both varied from cell to cell. The ratio of intracellular DA to Glu was 4.20±0.8 (n=150). Interestingly, the ratio drastically decreased to 0.38±0.20 (n=150) after the cells are exposed to 25mM KCl for 8min, suggesting the cells released DA promptly and heavily while they released Glu at a much slower pace in response to KCl-induced depolarization. These results indicate that the proposed MCE-MS analytical platform may have a great potential in researches at the cellular level.
Assuntos
Eletroforese em Microchip/instrumentação , Espectrometria de Massas/instrumentação , Análise de Célula Única/instrumentação , Animais , Automação/instrumentação , Automação/métodos , Dopamina/análise , Dopamina/metabolismo , Eletrodos , Eletroforese em Microchip/métodos , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Dispositivos Lab-On-A-Chip , Espectrometria de Massas/métodos , Células PC12 , Cloreto de Potássio/farmacologia , Ratos , Análise de Célula Única/métodosRESUMO
Chemical stimulus-induced neurotransmitter release from neuronal cells is well-documented. However, the dynamic changes in neurochemical release remain to be fully explored. In this work, a three-layered microfluidic chip was fabricated and evaluated for studying the dynamics of neurotransmitter release from PC-12 cells. The chip features integration of a nanoliter sized chamber for cell perfusion, pneumatic pressure valves for fluidic control, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Deploying this platform, a microchip electrophoresis-mass spectrometric method (MCE-MS) was developed to simultaneously quantify important neurotransmitters, including dopamine (DA), serotonin (5-HT), aspartic acid (Asp), and glutamic acid (Glu) without need for labeling or enrichment. Monitoring neurotransmitter release from PC-12 cells exposed to KCl (or alcohol) revealed that all four neurotransmitters investigated were released. Two release patterns were observed, one for the two monoamine neurotransmitters (i.e., DA and 5-HT) and another for the two amino acid neurotransmitters. Release dynamics for the two monoamine neurotransmitters was significantly different. The cells released DA most quickly and heavily in response to the stimulation. After exposure to the chemical stimulus for 4 min, the DA level in the perfusate from the cells was 86% lower than that at the beginning. Very interestingly, the cells started to release 5-HT in large quantities when they stopped releasing DA. These results suggest that DA and 5-HT are packaged into different vesicle pools and they are mobilized differently in response to chemical stimuli. The microfluidic platform proposed is proven useful for monitoring cellular release in biological studies.
Assuntos
Eletroforese , Microfluídica , Neurotransmissores/análise , Espectrometria de Massas por Ionização por Electrospray , Animais , Ácido Aspártico/análise , Dopamina/análise , Ácido Glutâmico/análise , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Nanoestruturas/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Cloreto de Potássio/farmacologia , Ratos , Serotonina/análiseRESUMO
Previous work has established that D-serine (D-Ser) plays important roles in certain neurological processes. Study on its uptake/storage and release by neuronal cells is highly significant for elucidating relevant mechanisms. In this work, PC-12 cells were incubated with racemic Ser (100 µM each enantiomer). After incubation, both intra- and extracellular levels of D-Ser and L-Ser were quantified by chiral microchip electrophoresis with mass spectrometric detection. It was found the cells preferably took up D-Ser over L-Ser. After 120 min incubation, D-Ser percentage ([D-Ser]/([D-Ser] + [L-Ser]) in the culture media changed from 50% to 9% while inside the cells it increased from 13% to 67%. Small neutral amino acids such as threonine impaired D-Ser uptake. Ser release was studied by using PC-12 cells preloaded with D-Ser. KCl, Glu, and Gly evoked Ser release. Interestingly, while depolarization by KCl evoked release of Ser as a D-Ser/L-Ser mixture of 1:1 ratio, the stereoisomeric composition of Ser released due to Glu exposure varied with the exposure time, ranging from 73% D-Ser (i.e., [D-Ser] > [L-Ser]) at 2 min to 44% (i.e., [D-Ser] < [L-Ser]) at 14 min, clearly indicating a stereochemical preference for D-Ser in Ser release from neuronal cells evoked by Glu-receptor activation.
Assuntos
Neurônios/metabolismo , Serina/metabolismo , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Eletroforese em Microchip/métodos , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Espectrometria de Massas/métodos , Células PC12 , Cloreto de Potássio/metabolismo , Ratos , EstereoisomerismoRESUMO
A microchip electrophoresis-mass spectrometric (MCE-MS) method was developed for fast chiral analysis. The proposed MCE-MS platform deployed a glass/PDMS hybrid microchip with an easy-to-fabricate monolithic nanoelectrospray emitter. Enantiomeric MCE separation was achieved by means of the partial filling technique. A novel chip design with an arm channel connecting to the middle of the MCE separation channel for delivering the chiral selector was tested and proven valid. Enantiomeric separation of3.4-dihydroxyphenylalanine (DOPA), glutamic acid (Glu), and serine (Ser), the selected test compounds,were achieved within 130 s with resolution values (R(s)) of 2.4, 1.1, and 1.0, respectively. The proposed chiral MCE-MS assay was sensitive and had detection limits of 43 nM for l-DOPA and 47 nM for d-DOPA.The analytical platform was well suited for studies of stereochemical preference in living cells because it integrated cell culture, sample injection, chiral separation, and MS detection into a single platform.Metabolism of DOPA in human SH-SY5Y neuronal cells was studied as a model system. On-chip incubation of SH-SY5Y cells with racemic DOPA was carried out, and the incubation solution was injected and in-line assayed at time intervals. It was found that l-DOPA concentration decreased gradually as incubation time increased while the concentration of coexisting d-DOPA remained constant. The results firmly indicated that SH-SY5Y cells metabolized l-DOPA effectively while left d-DOPA intact.
Assuntos
Di-Hidroxifenilalanina/química , Eletroforese em Microchip/métodos , Ácido Glutâmico/química , Espectrometria de Massas/métodos , Serina/química , Linhagem Celular , Células/química , Células/metabolismo , Di-Hidroxifenilalanina/metabolismo , Humanos , EstereoisomerismoRESUMO
A fast microchip electrophoresis-nano-electrospray ionization-mass spectrometric method (MCE-nanoESI-MS) was developed for analysis of amino acids in biological samples. A glass/poly(dimethylsiloxane) hybrid microchip with a monolithic nanoESI emitter was used in the platform. The proposed MCE-nanoESI-MS analytical method showed high separation efficiency for amino acids. Baseline separation of an amino acid mixture containing Lys, Arg, Val, Tyr, and Glu was completed within 120 s with theoretical plate numbers of >7,500. The method was applied to study cellular release of excitatory amino acids (i.e., aspartic acid (Asp) and glutamic acid (Glu)) under chemical stimulations. Linear calibration curves were obtained for both Asp and Glu in a concentration range from 1.00 to 150.0 µM. Limits of detection were found to be 0.37 µM for Asp and 0.33 µM for Glu (S/N = 3). Assay repeatability (relative standard deviation, n = 6) was 4.2 and 4.5%, for Asp and Glu at 5.0 µM, respectively. In the study of cellular release, PC-12 nerve cells were incubated with alcohol at various concentrations for 1 h. Both extra- and intracellular levels of Asp and Glu were measured by the proposed method. The results clearly indicated that ethanol promoted the release of both Asp and Glu from the cells.
Assuntos
Aminoácidos/análise , Eletroforese em Microchip/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Aminoácidos/metabolismo , Animais , Eletroforese em Microchip/economia , Desenho de Equipamento , Limite de Detecção , Células PC12 , Ratos , Espectrometria de Massas por Ionização por Electrospray/economia , Fatores de TempoRESUMO
Integration of a pressure-driven make-up flow (MUF) into a microchip electrophoresis (MCE) platform in order to facilitate its coupling with electrospray ionization-mass spectrometric detection (ESI-MS) is described. In the glass/PDMS hybrid microchip, a MUF channel was made to intersect with the MCE separation channel at an angle of 45°. The MUF was generated by a syringe pump. Microscopic image results from simulation studies showed that the pressure-driven MUF and the potential-driven electroosmotic flow in the MCE separation channel could be run separately without interfering with each other and mixed well at the joint point by adjusting either the MUF flow rate or the potential applied for MCE separation. The MUF had several desirable functions, including making the start of electrospray easy and cleaning the nanoESI emitter continuously when not spraying. High separation efficiency was achieved with the proposed MCE-nanoESI-MS system in separating an amino acid mixture containing glutamine, serine, threonine, phenylalanine, and glutamic acid. All of them were baseline separated from each other within 3 min. Plate numbers of >10,000 (on a 2.5 cm MCE separation channel) were obtained. The analytical platform also showed a linear response for quantification of DOPA with a detection limit (S/N=3) of 0.10 µM. In addition, on-line derivatization of MCE elutes in order to enhance MS detection sensitivity was easily carried out by adding the tagging reagent into the MUF. These results indicated that the present system might have a good potential in MCE-MS applications.
Assuntos
Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , Aminoácidos/análise , Aminoácidos/isolamento & purificação , Limite de Detecção , Nanotecnologia/instrumentação , Pressão , Reprodutibilidade dos TestesRESUMO
This paper describes a novel detection system based on small-angle optical deflection from the collinear configuration of a microfluidic chip. In this system, the incident light beam was focused on the microchannel through the edge of a lens, resulting in a small deflection angle that deviated 20° from the collinear configuration. The emitted fluorescence was collected through the center of the same lens and delivered to a photomultiplier tube in the vertical direction; the reflection light of the chip plate was kept away from the detector. In contrast to traditional confocal and nonconfocal laser-induced fluorescence detection systems, background levels resulting from scattered excitation light, reflection and refraction from the microchip was significantly eliminated. Significant enhancement of the signal-to-noise ratio was obtained by shaping a laser beam that combined an attenuator with a spectral filter to optimize laser power and the dimensions of the laser beam. FITC and FITC-labeled amino acid were used as model analytes to demonstrate the performance sensitivity, separation efficiency, and reproducibility of this detection system by using a hybrid polydimethylsiloxane/glass microfluidic device. The limit of detection of FITC was estimated to be 2 pM (0.55 zmol) (S/N = 3). Furthermore, the single cell analysis for the determination of intracellular glutathione in a single 3T3 mouse fibroblast cell was demonstrated. The results suggest that the proposed optical arrangements will be promising for development of sensitive, low-cost microfluidic systems.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Aminoácidos/análise , Aminoácidos/química , Animais , Desenho de Equipamento , Fluoresceína-5-Isotiocianato/análise , Fluoresceína-5-Isotiocianato/química , Glutationa/análise , Glutationa/química , Espaço Intracelular/química , Camundongos , Células NIH 3T3 , Óptica e Fotônica/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodosRESUMO
A facile capillary electrophoresis (CE) method was developed for the screening of monoamine oxidase B (MAO-B) inhibitors in natural extracts. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds, and detected by their UV absorbance at 280 nm. Conditions for the separation of substrates, products and enzyme were optimized. The optimal buffer composition was 50 mM N-2-hydroxyethyl-piperazine-N'-2-ethane sulphonic acid (HEPES) solution containing 10 mM SDS (pH=7.4). Under the optimal condition, the baseline separation of substrates, products and enzyme was achieved within 2 min. The present method was used to determine MAO-B kinetic constants, K(i), K(m) and IC50 based on quantitative of the substrate peak area compared with the reference electropherogram obtained from without the inhibitor. A validation study shows good reproducibility for both migration time (RSD=1.8%) and peak area (RSD=3.9%). Finally, the screening of 16 natural extracts was performed, and 2 natural extracts from Fructus crataegi and Radix polygoni multiflori were identified to be positive for MAO-B inhibition.