RESUMEN
S-nitrosothiols (RSNOs) are very important biomolecules that play crucial roles in many physiological and physiopathological processes. They act as NO-donors and are candidates for future medicines. Their identification and quantitation are therefore important for biomedical applications. One, two or more RSNOs can then be combined to design a drug and therefore, the quantification of each is important to establish an acceptable quality control process. Till date, miniaturized devices have been used to detect RSNOs based on their total quantitation without a preceding separation step. This study reports on an original and integrated microdevice allowing for the successive electrokinetic separation of low molecular weight RSNOs, their decomposition under metal catalysis, and their quantitation by amperometric detection of the produced nitrite in the end-channel arrangement, leading to their quantitation in a single run. For this purpose, a commercial SU-8/Pyrex microfluidic system was coupled to a portable and wireless potentiostat. Different operating and running parameters were optimized to achieve the best analytical data, allowing for an LOD equal to 20 µM. The simultaneous separation of S-nitrosoglutathione and S-nitrosocysteine was successfully obtained within 75 s. The proposed methodology using SU-8/Pyrex microfluidic devices opens new possibilities to investigate future drug candidates for NO-donors.
Asunto(s)
Cisteína/análogos & derivados , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , S-Nitrosoglutatión/análisis , S-Nitrosotioles/análisis , Catálisis , Cobre/química , Cisteína/análisis , Cisteína/síntesis química , Cisteína/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Límite de Detección , Técnicas Analíticas Microfluídicas/instrumentación , S-Nitrosoglutatión/síntesis química , S-Nitrosoglutatión/química , S-Nitrosotioles/síntesis química , S-Nitrosotioles/químicaRESUMEN
This paper describes the use of ionic solutions as sensing electrodes for capacitively coupled contactless conductivity detection on electrophoresis microchips. Initially, two channels were engraved in a PMMA holder by using a CO2 laser system and sealed with a thin adhesive membrane. PDMS electrophoresis chips were fabricated by soft lithography and reversibly sealed against the polymer membrane. Different ionic solutions were investigated as metalless electrodes. The electrode channels were filled with KCl solutions prepared in conductivity values from approximately 10 to 40 S/m. The best analytical response was achieved using the KCl solution with 21.9 S/m conductivity (2 mol/L). Besides KCl, we also tested NaCl and LiCl solutions for actuating as detection electrodes. Taking into account the same electrolyte concentration (2 mol/L), the best response was recorded with KCl solution due to its higher ionic conductivity. The optimum operating frequency (400 kHz) and the best sensing electrode (2 mol/L KCl) were used to monitor electrophoretic separations of a mixture containing K(+) , Na(+) , and Li(+) . The use of liquid solutions as sensing electrodes for capacitively coupled contactless conductivity detection measurements has revealed great performance to monitor separations on chip-based devices, avoiding complicated fabrication schemes to include metal deposition and encapsulation of electrodes. The LOD values were estimated to be 28, 40, and 58 µmol/L for K(+) , Na(+) , and Li(+) , respectively, what is comparable to that of conventional metal electrodes. When compared to the use metal electrodes, the proposed approach offers advantages regarding the easiness of fabrication, simplicity, and lower cost per device.
Asunto(s)
Electroforesis por Microchip/instrumentación , Conductividad Eléctrica , Electrodos , Electroforesis por Microchip/métodos , Diseño de Equipo , Líquidos Iónicos/química , Metales Alcalinos/química , Metales Alcalinos/aislamiento & purificación , Cloruro de Potasio/químicaRESUMEN
S-Nitrosoglutathione (GSNO) is a very important biomolecule that has crucial functions in many physiological and physiopathological processes. GSNO acts as NO donor and is a candidate for future medicines. This work describes, for the first time, the separation and the detection of GSNO and its decomposition products using capillary electrophoresis coupled to mass spectrometry (CE-MS). The separation was performed in slightly alkaline medium (pH 8.5) under positive-ionization MS detection. The identification of three byproducts of GSNO was formally performed for the first time: oxidized glutathione (GSSG), glutathione sulfinic acid (GSO2H), and glutathione sulfonic acid (GSO3H). GSO2H and GSO3H are known to have important biological activity, including inhibition of the glutathione transferase family of enzymes which are responsible for the elimination of many mutagenic, carcinogenic, and pharmacologically active molecules. We observed, after the ageing of GSNO in the solid state, that the proportion of both GSSG and GSO3H increases whereas that of GSO2H decreases. These results enabled us to propose an oxidation scheme explaining the formation of such products.
Asunto(s)
Electroforesis Capilar , Disulfuro de Glutatión/análisis , Espectrometría de Masas , S-Nitrosoglutatión/análisis , Ácidos Sulfínicos/análisis , Ácidos Sulfónicos/análisis , Electroforesis Capilar/métodos , Disulfuro de Glutatión/aislamiento & purificación , Espectrometría de Masas/métodos , Oxidación-Reducción , S-Nitrosoglutatión/aislamiento & purificación , Ácidos Sulfínicos/aislamiento & purificación , Ácidos Sulfónicos/aislamiento & purificaciónRESUMEN
A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a 2-fold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.
Asunto(s)
Microfluídica/instrumentación , Óxido Nítrico/biosíntesis , Análisis de la Célula Individual , Linfocitos T/metabolismo , Humanos , Células Jurkat , Estándares de ReferenciaRESUMEN
Advances in the instrumentation and materials for photopolymerization 3D printing aided the use of this powerful technique in the fabrication of microfluidic devices. The costs of printers and supplies have been reduced to the point where this technique becomes attractive for prototyping microfluidic systems with good resolution. With all the development of multi-material 3D printers, most of the microfluidic devices prepared by photopolymerization 3D printing are based on a single substrate material. We developed a digital light processing multi-material 3D printer where two or more resins can be used to prepare complex objects and functional microfluidic devices. The printer is based on a vat inclination system and embedded peristaltic pumps that allow the injection and removal of resins and cleaning step between material changes. Although we have built the whole system, the modification can be incorporated into commercially available printers. Using a high-resolution projector, microfluidic channels as narrow as 43 µm were obtained. We demonstrate the printing of multi-material objects containing flexible, rigid, water-soluble, fluorescent, phosphorescent, and conductive (containing PEDOT or copper nanoparticles) resins. An example of a microfluidic chip containing electrodes for electrochemical detection is also presented.
RESUMEN
The fabrication of microfluidic devices through fused deposition modeling (FDM) 3D printing has faced several challenges, mainly regarding obtaining microchannels with suitable transparency and sizes. Thus, the use of this printing system to fabricate microdevices for analytical and bioanalytical applications is commonly limited when compared to other printing technologies. However, for the first time, this work shows a systematic study to improve the potential of FDM 3D printers for the fabrication of transparent microfluidic devices. Several parameters and printing characteristics were addressed in both theoretical and experimental ways. It was found that the geometry of the printer nozzle plays a significant role in the thermal radiation effect that limits the 3D printing resolution. This drawback was minimized by adapting an airbrush tip (0.2 mm orifice diameter) to a conventional printer nozzle. The influence of the height and width of the extruded layer on the resolution and transparency in 3D-printed microfluidic devices was also addressed. Following the adjustments proposed, microchannels were obtained with an average width of around 70 µm ± 11 µm and approximately 80% visible light transmission (for 640 µm thickness). Therefore, the reproducibility and resolution of FDM 3D printing could be improved, and this achievement can expand the capability of this printing technology for the development of microfluidic devices, particularly for analytical applications.
Asunto(s)
Dispositivos Laboratorio en un Chip , Impresión Tridimensional , Sistemas de Liberación de Medicamentos , Reproducibilidad de los ResultadosRESUMEN
Microfluidic devices based on textile threads have interesting advantages when compared to systems made with traditional materials, such as polymers and inorganic substrates (especially silicon and glass). One of these significant advantages is the device fabrication process, made more cheap and simple, with little or no microfabrication apparatus. This review describes the fundamentals, applications, challenges, and prospects of microfluidic devices fabricated with textile threads. A wide range of applications is discussed, integrated with several analysis methods, such as electrochemical, colorimetric, electrophoretic, chromatographic, and fluorescence. Additionally, the integration of these devices with different substrates (e.g., 3D printed components or fabrics), other devices (e.g., smartphones), and microelectronics is described. These combinations have allowed the construction of fully portable devices and consequently the development of point-of-care and wearable analytical systems.
Asunto(s)
Dispositivos Laboratorio en un Chip , Textiles , Colorimetría , Sistemas de Atención de Punto , Teléfono InteligenteRESUMEN
A simple, fast, and inexpensive masking technology without any photolithographic step to produce glass microchannels is proposed in this work. This innovative process is based on the use of toner layers as mask for wet chemical etching. The layouts were projected in graphic software and printed on wax paper using a laser printer. The toner layer was thermally transferred from the paper to cleaned glass surfaces (microscope slides) at 130 degrees C for 2 min. After thermal transference, the glass channel was etched using 25% (v/v) hydrofluoric acid (HF) solution. The toner mask was then removed by cotton soaked in acetonitrile. The etching rate was approximately 7.1 +/- 0.6 microm min(-1). This process is economically more attractive than conventional methods because it does not require any sophisticated instrumentation and it can be implemented in any chemical/biochemical laboratory. The glass channel was thermally bonded against a flat glass cover and its analytical feasibility was investigated using capacitively coupled contactless conductivity detection (C(4)D) and laser-induced fluorescence (LIF) detection.
Asunto(s)
Electroforesis por Microchip/instrumentación , Vidrio , Microquímica/instrumentación , Microfluídica/instrumentación , Acetonitrilos/química , Diseño de Equipo , Gossypium/metabolismo , Calor , Ácido Fluorhídrico/química , Materiales Manufacturados , Microquímica/métodos , Técnicas Analíticas Microfluídicas , Miniaturización , Plásticos , Cuarzo/química , Silicio/químicaRESUMEN
This paper presents the use of elastomeric polyurethane (PU), derived from castor oil (CO) biosource, as a new material for fabrication of microfluidic devices by rapid prototyping. Including the irreversible sealing step, PU microchips were fabricated in less than 1h by casting PU resin directly on the positive high-relief molds fabricated by standard photolithography and nickel electrodeposition. Physical characterization of microchannels was performed by scanning electron microscopy (SEM) and profilometry. Polymer surface was characterized using contact angle measurements and the results showed that the hydrophilicity of the PU surface increases after oxygen plasma treatment. The polymer surface demonstrated the capability of generating an electroosmotic flow (EOF) of 2.6 x 10(-4)cm(2)V(-1)s(-1) at pH 7 in the cathode direction, which was characterized by current monitoring method at different pH values. The compatibility of PU with a wide range of solvents and electrolytes was tested by determining its degree of swelling over a 24h period of contact. The performance of microfluidic systems fabricated using this new material was evaluated by fabricating miniaturized capillary electrophoresis systems. Epinephrine and l-DOPA, as model analytes, were separated in aqueous solutions and detected with end-channel amperometric detection.
Asunto(s)
Electroforesis por Microchip/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Poliuretanos/química , Electroforesis Capilar/métodos , Epinefrina/análisis , Epinefrina/química , Levodopa/análisis , Levodopa/química , Microscopía Electrónica de Rastreo , Reproducibilidad de los ResultadosRESUMEN
Methacryloxypropyl-modified poly(dimethylsiloxane) rubbers were obtained from poly(dimethylsiloxane), PDMS, and methacryloxypropyltrimethoxysilane, MPTMS, by polycondensation reactions. The modified rubbers, prepared with 20 and 30% (v/v) of MPTMS, were used as substrates for microchannel fabrication by the CO(2) laser ablation technique. Raman imaging spectroscopy was used for the surface characterization, showing the homogeneity of the rubbery material, with uniform distribution of the crosslinking centers. Under the experimental conditions used, damage to the rubber from the CO(2) laser radiation used for the channel engraving was not observed. Correlation maps of the surface were obtained in order to spatially evaluate the modification inside and outside the channels. The correlations between the methacryloxypropyl-modified poly(dimethylsiloxane) rubbers and MPTMS (spectral range of 1800-1550 cm(-1)) and PDMS (spectral range of 820-670 cm(-1)) precursors were higher than 0.95 and 0.99, respectively. In addition, Raman imaging spectroscopy allows monitoring the topography of the fabricated microchannel.
Asunto(s)
Dimetilpolisiloxanos/química , Microtecnología/métodos , Espectrometría Raman , Compuestos de Organosilicio , Goma/química , Silanos/químicaRESUMEN
Capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C(4)D) was used for rapid, accurate and simultaneous determination of free fluoride and monofluorophosphate (MFP) in six different toothpaste samples. A buffer solution containing 15 mmol L(-1) histine, 25 mmol L(-1) lactic acid, and 2.5 mmol L(-1) tetradecyltrimethylammonium bromide (TTAB) was used as background electrolyte (BGE). A complete separation of the analytes and the internal standard (tartrate) could be attained in less than 2.5 min. The limits of detection (LOD) and quantification (LOQ) were, respectively, 0.17 and 0.57 mg L(-1) for free fluoride and 0.70 and 2.33 mg L(-1) for MFP. Recoveries ranging from 85 to 107% were obtained for samples spiked with standard solutions of free fluoride or MFP. The CE-C(4)D method was compared to an ion-selective electrode (ISE) method and the results were in good agreement. More importantly, the CE-C(4)D method demonstrates the advantage of being able to determine MFP without a prior hydrolysis step.