RESUMO
An extensive study of capillary flow of fluids with various viscosities in straight and periodically constricted microchannels with different surface wettability is presented. Capillary filling speed in hydrophilic, less hydrophilic, and hydrophobic microchannels were experimentally monitored and compared with the Washburn theoretical model. For all liquids, a linear relationship was found between the square of propagation distance and time, which is expected for Newtonian fluids. Experimental results indicated slower velocity compared to the theoretical prediction due to simplifications of the Washburn model. Capillary filling speed of fluids into long-fluororinated chain silane modified channels confirmed the expected lyophobic nature of the coating (i.e., not favorable for either hydrophilic or hydrophobic liquids). Presence of the precursor film ahead of the three-phase contact line in the microscopic level was demonstrated. White light and fluorescent images confirmed the presence of precursor film and capillary evaporation at the interface. Evaporation enhanced the deviation between experimental and theoretical results due to continuous wettability alteration of penetrating fluid.
RESUMO
Nanostructured nickel (Ni) and nickel oxide (NiO) electrodes were fabricated on Ni foils using the glancing angle deposition (GLAD) technique. Cyclic voltammetry and amperometry showed the electrodes enable non-enzymatic electrochemical determination of glucose in strongly alkaline media. Under optimized conditions of NaOH concentration and working potential (~ 0.50 V vs. Ag/AgCl), the GLAD electrodes performed far better than bare Ni foil electrodes, with the GLAD NiO electrode showing an outstanding sensitivity (4400 µA mM-1 cm-2), superior detection limit (7 nM), and wide dynamic range (0.5 µM-9 mM), with desirable selectivity and reproducibility. Based on their performance at a low concentration, the GLAD NiO electrodes were also used to quantify glucose in artificial urine and sweat samples which have significantly lower glucose levels than blood. The GLAD NiO electrodes showed negligible response to the common interferents in glucose measurement (uric acid, dopamine, serotonin, and ascorbic acid), and they were not poisoned by high amounts of sodium chloride. Graphical abstract The figures depict (A) SEM image of vertical post-GLAD NiO electrodes used for non-enzymatic electrochemical glucose monitoring, and (B) calibration plots of the three different electrodes.
Assuntos
Técnicas Biossensoriais , Glicemia/análise , Técnicas Eletroquímicas , Nanoestruturas/química , Níquel/química , Eletrodos , Humanos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Two kinds of electrochemical impedimetric biosensors for the detection of E. coli O157:H7 are described and compared. They were fabricated using self-assembled layers of thiolated protein G (PrG-thiol) on (i) planar gold electrodes and (ii) gold nanoparticles (Au NPs) modified gold electrodes. The fabrications of the biosensors were characterized using cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy and atomic force microscopy techniques. The modification of the planar gold electrode by Au NPs via self-assembled monolayer of 1,6-hexadithiol as a linker molecule increased the electrochemically active surface area by about 2.2 times. The concentration of PrG-thiol and its incubation time, as well as the concentration of IgG were optimized. The Au NP-based biosensor exhibited a limit of detection of 48 colony forming unit (cfu mL-1) which is 3 times lower than that of the planar gold electrode biosensor (140 cfu mL-1). It also showed a wider dynamic range (up to 107 cfu mL-1) and sensitivity. The improved analytical performance of the Au NP-modified biosensor is ascribed to the synergistic effect between the Au NPs and the PrG-thiol scaffold. The biosensor exhibited high selectivity for E. coli O157:H7 over other bacteria such as Staphylococcus aureus and Salmonella typhimurium. Graphical abstract Schematic representations of sensor fabrication using Au NP-modified electrode (HKEC = heat- killed E. coli O157:H7).
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/métodos , Escherichia coli O157/isolamento & purificação , Técnicas Biossensoriais/métodos , Espectroscopia Dielétrica , Técnicas Eletroquímicas/instrumentação , Eletrodos , Proteínas de Escherichia coli , Ouro , Nanopartículas MetálicasRESUMO
The separation behavior of 6.5-66 kDa proteins in silica particle array-based sieves formed by colloidal self-assembly in microchips is reported across a pore size range of 22-103 nm. The protein separation and resolution improves markedly with decreasing pore size. The variation of electrophoretic mobility with molecular weight of SDS-protein complexes and with particle size was evaluated using the Ogston sieving equation for a random pore gel structure, and using the modified Giddings equation developed by Wirth for uniform pore structures. The Wirth/Giddings equation provides the best fit for estimation of molecular weight of proteins, and demonstrates that even though experimental evidence shows there is some dispersion in measured pore sizes, these structures can best be described as having a uniform pore size.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Nanoestruturas/química , Análise Serial de Proteínas/métodos , Proteínas/isolamento & purificação , Tamanho da Partícula , Porosidade , Proteínas/químicaRESUMO
We report on a facile method to stabilize colloidal self-assembled (CSA) nanoparticles packed in microchannels for high speed size-based separation of proteins. Silica nanoparticles, self-assembled in a network of microfluidic channels, were stabilized with a methacrylate polymer prepared in situ through photopolymerization. The entrapment conditions were investigated to minimize the effect of the polymer matrix on the structure of the packing and the separation properties of the CSA beds. SEM shows that the methacrylate matrix links the nanoparticles at specific sphere-sphere contact points, improving the stability of the CSA structure at high electric fields (up to at least 1800 V/cm), allowing fast and efficient separation. The %RSD of the protein migration times varied between 0.3 and 0.5% (n = 4, in 1 day) and <0.83% over a period of 7 days (n = 28 runs) in a single device, at high field strength. The overall %RSD of protein migration times from chip-to-chip across a single fabrication run was 4.3% (n = 3) and between fabrication runs was 11% (n = 35), with 87% fabrication yield, demonstrating reproducible packing and entrapment behavior. The optimized entrapped CSA beds demonstrated better separation performance (plate height, H â¼ 200 nm) than similarly prepared on-chip CSA beds without the polymer entrapment. Polymer-entrapped CSA beds also exhibited superior protein resolving power: the minimum resolvable molecular weight difference of proteins in the polymer-entrapped CSA bed is 0.6 kDa versus â¼9 kDa for the native silica CSA bed (i.e. without polymer entrapment).
Assuntos
Coloides/química , Nanopartículas/química , Proteínas/isolamento & purificação , Técnicas Analíticas Microfluídicas , Tamanho da Partícula , Polímeros/química , Proteínas/análise , Proteínas/química , Reprodutibilidade dos Testes , Dióxido de Silício/químicaRESUMO
This paper demonstrates the integration of complementary split-ring resonators (CSSRs) with digital microfluidics (DMF) sample manipulation for passive, on-chip radio-frequency (RF) sensing. Integration is accomplished by having the DMF and the RF-sensing components share the same ground plane: by designing the RF-resonant openings directly into the ground plane of a DMF device, both droplet motion and sensing are achieved, adding a new on-board detection mode for use in DMF. The system was modelled to determine basic features and to balance various factors that need to be optimized to maintain both functionalities (DMF-enabled droplet movement and RF detection) on the same chip. Simulated and experimental results show good agreement. Using a portable measurement setup, the integrated CSSR sensor was used to effectively identify a series of DMF-generated drops of ethanol-water mixtures of different compositions by measuring the resonant frequency of the CSSR. In addition, we show that a binary solvent system (ethanol/water mixtures) results in consistent changes in the measured spectrum in response to changes in concentration, indicating that the sensor can distinguish not only between pure solvents from each other, but also between mixtures of varied compositions. We anticipate that this system can be refined further to enable additional applications and detection modes for DMF systems and other portable sensing platforms alike. This proof-of-principle study demonstrates that the integrated DMF-CSSR sensor provides a new platform for monitoring and characterization of liquids with high sensitivity and low consumption of materials, and opens the way for new and exciting applications of RF sensing in microfluidics.
RESUMO
In this work, we report the direct electrochemical oxidation of fentanyl using commercial screen-printed carbon electrodes (SPCEs) modified with carboxyl-functionalized carbon nanofibers (fCNFs). CNFs have surface chemistry and reactivity similar to carbon nanotubes (CNTs), yet they are easier to produce and are of a lower cost than CNTs. By monitoring the current produced during the electrochemical oxidation of fentanyl, variables such as fCNF loading, fentanyl accumulation time, electrolyte pH, and differential pulse voltammetry parameters were optimized. Under an optimized set of conditions, the fCNF/SPCEs responded linearly to fentanyl in the concentration range of 0.125-10 µM, with a limit of detection of 75 nM. The fCNF/SPCEs also demonstrated excellent selectivity against common cutting agents found in illicit drugs (e.g., glucose, sucrose, caffeine, acetaminophen, and theophylline) and interferents found in biological samples (e.g., ascorbic acid, NaCl, urea, creatinine, and uric acid). The performance of the sensor was also successfully tested using fentanyl spiked into an artificial urine sample. The straightforward electrode assembly process, low cost, ease of use, and rapid response make the fCNF/SPCEs prime candidates for the detection of fentanyl in both physiological samples and street drugs.
RESUMO
A microfluidic device that performs "in space" sample fractionation, collection, and preconcentration for proteomics is described. Effluents from a 2.75 mm long fractionation channel, focused via sheath flow, were sequentially delivered into an array of 36-collection channels containing monolithic polymer beds for SPE. Optimum conditions for the device design, and simultaneous photolytic fabrication of 36 monolithic columns in the 36 channels, as well as for their proper performance in electrokinetic sample fractionation and collection are described. A hydrophobic butyl methacrylate-based monolithic porous polymer was copolymerized with an ionizable monomer, acryloamido-methyl-propane sulfonate, to form a polymer monolith for SPE that also sustains cathodic electroosmotic flow. The SPE bed was made deep enough to greatly reduce the linear flow rate within the bed, in order to compensate for the lower electroosmotic mobility of the cationically charged SPE bed relative to the glass walled device. Under these conditions, electrokinetic fractionation of a protein sample resulted in tightly focused sample zones delivered into each of the 36-channel polymer beds with no observed crosscontamination. Monolithic columns showed reproducible performance with preconcentration factor of 30 for 2 min loading time. The ability to fractionate, collect, and preconcentrate samples on a microfluidic platform will be especially useful for automated or continuous operation of these devices in proteomics research.
Assuntos
Eletroforese Capilar/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Extração em Fase Sólida/instrumentação , Eletroforese Capilar/métodos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Metacrilatos/química , Polímeros/química , Proteômica/instrumentação , Proteômica/métodos , Reprodutibilidade dos Testes , Albumina Sérica/química , Extração em Fase Sólida/métodosRESUMO
A molecularly imprinted sol-gel is reported for selective and sensitive electrochemical determination of the drug naloxone (NLX). The sensor was developed by combining molecular imprinting and sol-gel techniques and electrochemically grafting the sol solution onto a functionalized multiwall carbon nanotube modified indium-tin oxide (ITO) electrode. The sol-gel layer was obtained from acid catalyzed hydrolysis and condensation of a solution composed of triethoxyphenylsilane (TEPS) and tetraethoxysilane (TES). The fabrication, structure and properties of the sensing material were characterized via scanning electron microscopy, spectroscopy and electrochemical techniques. Parameters affecting the sensor's performance were evaluated and optimized. A sensor fabricated under the optimized conditions responded linearly between 0.0 µM and 12 µM NLX, with a detection limit of 0.02 µM. The sensor also showed good run-to-run repeatability and batch-to-batch performance reproducibility with relative standard deviations (RSD) of 2.5-7.8% (n = 3) and 9.2% (n = 4), respectively. The developed sensor displayed excellent selectivity towards NLX compared to structurally similar compounds (codeine, fentanyl, naltrexone and noroxymorphone), and was successfully used to measure NLX in synthetic urine samples yielding recoveries greater than 88%.
RESUMO
We describe a microfluidic device in which integrated tryptic digestion, SPE, CE separation and electrospray ionization for MS are performed. The chip comprised of 10 × 30 µm channels for CE, and two serially connected 150 µm deep, 800 µm wide channels packed with 40 to 60 µm diameter beads, loaded with either immobilized trypsin, reversed-phase packing or both. On-chip digestion of cytochrome c using the trypsin bed showed complete consumption of the protein in 3 min, in contrast to the 2 h required for conventional solution phase tryptic digestion. SPE of 0.25 µg/mL solutions of the peptides leu-enkephalin, angiotensin II and LHRH gave concentration enhancements in the range of 4.4-12, for a ten times nominal volume ratio. A 100 nM cytochrome c sample concentrated 13.3 times on-chip gave a sequence coverage of 85.6%, with recovery values ranging from 41.2 to 106%. The same sample run without SPE showed only five fragment peaks and a sequence coverage of 41.3%. When both on-chip digestion and SPE (13.3 volume ratio concentration enhancement) were performed on 200 nM cytochrome c samples, a sequence coverage of 76.0% and recovery values of 21-105% were observed. Performing on-chip digestion alone on the same sample gave only one significant fragment peak. The above digestion/peptide concentration step was compared to on-chip protein concentration by SPE followed by on-chip digestion with solution phase trypsin. Both procedures gave similar recovery results; however, much larger trypsin autodigestion interference in the latter approach was apparent.
Assuntos
Eletroforese em Microchip/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microextração em Fase Sólida/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Tripsina/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Fatores de TempoRESUMO
We present three generations of microchip-based "in-space" sample fractionators and collectors for use in proteomics. The basic chip design consisted of a single channel for CE separation of analytes that then intersects a fractionation zone feed into multiple high aspect ratio microchannels for fractionation of separated components. Achievements of each generation are discussed in relation to important design criteria. CE-separated samples were electrokinetically driven to multiple collection channels in sequence without cross-contamination under the protection of sheath streams. A 36-channel fractionator demonstrated the efficacy of a high-throughput fractionator with no observed cross-contamination. A mixture of IgG and BSA was used to test the efficiency of the fractionator and collector. CE of the fractionated samples was performed on the same device to verify their purity. Our demonstration proved to be efficient and reproducible in obtaining non-contaminated samples over 15 sample injections. Experimental results were found to be in close agreement with PSpice simulation in terms of flow behavior, contamination control and device performance. The design presented here has a great potential to be integrated in proteomic platforms.
Assuntos
Eletroforese Capilar/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Proteômica/instrumentação , Animais , Bovinos , Fracionamento Químico/instrumentação , Desenho de Equipamento , Imunoglobulina G/isolamento & purificação , Miniaturização/instrumentação , Soroalbumina Bovina/isolamento & purificaçãoRESUMO
Glancing angle deposition (GLAD) was used to fabricate nanostructured silicon (Si) thin films with highly controlled morphology for use in laser desorption/ionization mass spectrometry (DIOS-MS). Peptides, drugs and metabolites in the mass range of 150-2500 Da were readily analyzed. The best performance was obtained with 500 nm thick films deposited at a deposition angle of 85 degrees . Low background mass spectra and attomole detection limits were observed with DIOS-MS for various peptides. Films used after three months of dry storage in ambient conditions produced mass spectra with negligible low-mass noise following a 15 min UV-ozone treatment. The performance of the Si GLAD films was as good as or better than that reported for electrochemically etched porous silicon and related materials, and was superior to matrix-assisted laser desorption/ionization (MALDI)-MS for analysis of mixtures of small molecules between 150-2500 Da in terms of background chemical noise, detection limits and spot-to-spot reproducibility. The spot-to-spot reproducibility of signal intensities (100 shots/spectrum) from 21 different Si GLAD film targets was +/-13% relative standard deviation (RSD). The single shot-to-shot reproducibility of signals on a single target was +/-19% RSD (n = 7), with no indication of sweet spots or mute spots.
RESUMO
Microchip-based bead-packed columns for electrochromatography are described for several types of stationary phases. Chromatography columns 2 mm in length were used for the separation of proteins and peptides by size- and ion-exchange modes of separation, respectively. In size-exclusion electrochromatograpgy, FITC-IgG and FITC-insulin were baseline resolved in less than 10 s, with efficiencies of up to 139,000 plates/m for FITC-insulin. In strong cation-exchange electrochromatography, a mixture of three fluorescently labeled peptides was baseline resolved in less than 40 s, with efficiencies up to 400,000 plates/m. The RSD for the analytes retention times were<3% in both size-exclusion and ion-exchange modes of separations. The use of a 1-mm-long reverse-phase column for the semiquantitative evaluation of pharmaceutical formulations in drug solubility tests illustrates the use of this microfluidic chip-based electrochromatographic approach to drug development.
Assuntos
Eletrocromatografia Capilar/instrumentação , Microfluídica/instrumentação , Resinas de Troca de Cátion , Preparações Farmacêuticas/químicaRESUMO
We report on the development of a regenerable sensitive immunosensor based on electrochemical impedance spectroscopy for the detection of type 5 adenovirus. The multi-layered immunosensor fabrication involved successive modification steps on gold electrodes: (i) modification with self-assembled layer of 1,6-hexanedithiol to which gold nanoparticles were attached via the distal thiol groups, (ii) formation of self-assembled monolayer of 11-mercaptoundecanoic acid onto the gold nanoparticles, (iii) covalent immobilization of monoclonal anti-adenovirus 5 antibody, with EDC/NHS coupling reaction on the nanoparticles, completing the immunosensor. The immunosensor displayed a very good detection limit of 30 virus particles/ml and a wide linear dynamic range of 10(5). An electrochemical reductive desorption technique was employed to completely desorb the components of the immunosensor surface, then re-assemble the sensing layer and reuse the sensor. On a single electrode, the multi-layered immunosensor could be assembled and disassembled at least 30 times with 87% of the original signal intact. The changes of electrode behavior after each assembly and desorption processes were investigated by cyclic voltammetry, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy techniques.
Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/isolamento & purificação , Técnicas Biossensoriais , Adenoviridae/genética , Espectroscopia Dielétrica , Técnicas Eletroquímicas , Ouro/química , Humanos , Nanopartículas Metálicas/químicaRESUMO
We report on the development of an electrochemical reductive desorption protocol for repeated regeneration of gold electrodes modified with multi-layers of self-assembled surfaces for use in electrochemical sensing. The gold electrodes were first modified with 1,6-hexanedithiol to which gold nanoparticles were attached in a subsequent modification step. Attachment of thiolated single-stranded nucleic acid oligomers to the gold nanoparticles completed the electrochemical sensor. The changes of electrode behavior after each assembly and desorption processes were investigated by cyclic voltammetry, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy techniques. The self-assembled sensor showed a wide dynamic range (0.1-100 nM), a low detection limit (20 pM) and high reproducibility (4.4% RSD) for complementary nucleic acid target molecules, along with reusability. On a single gold electrode, the complete sensor-target structure could be assembled and disassembled at least four times with 90% of its original signal intact.
Assuntos
DNA/análise , Técnicas Eletroquímicas/instrumentação , Ouro/química , Nanopartículas/química , Compostos de Sulfidrila/química , Sequência de Bases , Técnicas Biossensoriais/instrumentação , Sondas de DNA/química , Espectroscopia Dielétrica , Impedância Elétrica , Eletrodos , Desenho de Equipamento , Limite de Detecção , Espectroscopia Fotoeletrônica , Reprodutibilidade dos TestesRESUMO
Both 6 and 8-channel integrated microfluidic sample pretreatment devices capable of performing "in space" sample fractionation, collection, preconcentration and elution of captured analytes via sheath flow assisted electrokinetic pumping are described. Coatings and monolithic polymer beds were developed for the glass devices to provide cationic surface charge and anodal electroosmotic flow for delivery to an electrospray emitter tip. A mixed cationic ([2-(methacryloyloxy)ethyl] trimethylammonium chloride) (META) and hydrophobic butyl methacrylate-based monolithic porous polymer, photopolymerized in the 6- or 8-fractionation channels, was used to capture and preconcentrate samples. A 0.45 wt% META loaded bed generated comparable anodic electroosmotic flow to the cationic polymer PolyE-323 coated channel segments in the device. The balanced electroosmotic flow allowed stable electrokinetic sheath flow to prevent cross contamination of separated protein fractions, while reducing protein/peptide adsorption on the channel walls. Sequential elution of analytes trapped in the SPE beds revealed that the monolithic columns could be efficiently used to provide sheath flow during elution of analytes, as demonstrated for neutral carboxy SNARF (residual signal, 0.08% RSD, n = 40) and charged fluorescein (residual signal, 2.5% n = 40). Elution from monolithic columns showed reproducible performance with peak area reproducibility of ~8% (n = 6 columns) in a single sequential elution and the run-to-run reproducibility was 2.4-6.7% RSD (n = 4) for elution from the same bed. The demonstrated ability of this device design and operation to elute from multiple fractionation beds into a single exit channel for sample analysis by fluorescence or electrospray mass spectrometry is a crucial component of an integrated fractionation and assay system for proteomics.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Proteômica , Benzopiranos/química , Eletro-Osmose , Fluoresceína/química , Corantes Fluorescentes/química , Técnicas Analíticas Microfluídicas/instrumentação , Peptídeos/análise , Peptídeos/isolamento & purificação , Polímeros/química , Porosidade , Proteínas/análise , Proteínas/isolamento & purificação , Extração em Fase Sólida , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We report a variety of procedures for fabricating confinement-induced polymer coatings, used to eliminate non-specific protein adsorption and to control electroosmotic flow for microchip capillary electrophoresis. The coating strategy generates relatively thick polymer wall coatings (100-700 nm) and can easily be tuned by adjusting the monomer concentration. 2-hydroxyethyl methacrylate (HEMA) polymer coating, photopatterned in microfluidic channels, effectively reduced protein non-specific adsorption and rendered high efficiency (N/m=â¼3 × 106) for protein separation. The coating strategy provides rapid and effective means to create robust wall coatings, with the ability to photograft various surface chemistries onto the coating. [2-(methacryloyloxy) ethyl] trimethylammonium chloride grafted HEMA coated channels showed high durability and reproducibility for generating EOF (RSD=2.6%, n=64) over a period of 15 days. Sulfobetaine methacrylate grafted HEMA coated channels allowed separation of BSA digest, 15 peaks resolved in 25s, with an average N/m of 4 × 105.
Assuntos
Eletroforese em Microchip/instrumentação , Fragmentos de Peptídeos/isolamento & purificação , Polímeros/química , Animais , Bovinos , Eletro-Osmose , Eletroforese em Microchip/métodos , Metacrilatos/química , Fragmentos de Peptídeos/análise , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificaçãoRESUMO
We evaluate the compatibility and performance of polymer monolith solid phase extraction beds that incorporate cationic charge, with a polycationic surface coating, PolyE-323, fabricated within microfluidic glass chips. The PolyE-323 is used to reduce protein and peptide adsorption on capillary walls during electrophoresis, and to create anodal flow for electrokinetically driven nano-electrospray ionization mass spectrometry. A hydrophobic butyl methacrylate-based monolithic porous polymer was copolymerized with an ionizable monomer, [2-(methacryloyloxy)ethyl] trimethylammonium chloride to form a polymer monolith for solid phase extraction that also sustains anodal electroosmotic flow. Exposure of the PolyE-323 coating to the monolith forming mixture affected the performance of the chip by a minor amount; electrokinetic migration times increased by â¼5%, and plate numbers were reduced by an average of 5% for proteins and peptides. 1-mm long on-chip monolithic solid phase extraction columns showed reproducible, linear calibration curves (R(2)=0.9978) between 0.1 and 5 nM BODIPY at fixed preconcentration times, with a capacity of 2.4 pmol or 0.92 mmol/L of monolithic column for cytochrome c. Solution phase on-bed trypsin digestion was conducted by capturing model protein samples onto the monolithic polymer bed. Complete digestion of the proteins was recorded for a 30 min stop flow digestion, with high sequence coverage (88% for cytochrome c and 56% for BSA) and minimal trypsin autodigestion product. The polycationic coating and the polymer monolith materials proved to be compatible with each other, providing a high quality solid phase extraction bed and a robust coating to reduce protein adsorption and generate anodal flow, which is advantageous for electrospray.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Fragmentos de Peptídeos/análise , Extração em Fase Sólida/instrumentação , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Tripsina/metabolismo , Eletro-Osmose , Microscopia Eletrônica de Varredura , Fragmentos de Peptídeos/metabolismo , Polimerização , Ácidos Polimetacrílicos/química , Proteínas/isolamento & purificação , Proteínas/metabolismoRESUMO
The integration of porous structures into microchannels is known to enable unique and useful separations both in electrophoresis and chromatography. Etched pillars and other nanostructures have received considerable interest in recent years as a platform for creating microchannels with pores tailored to specific applications. We present a versatile method for integration of three-dimensionally sculptured nano- and micro-structures into PDMS microchannels. Glancing angle deposition was used to fabricate nanostructures that were subsequently embedded in PDMS microchannels using a sacrificial resist process. With this technique, an assortment of structures made from a wide selection of materials can be integrated in PDMS microchannels; some examples of this versatility, including chiral and chevron nanostructures, are demonstrated. We also present a working device made using this process, separating 6/10/20 kbp and 10/48 kbp DNA mixtures in a DNA fractionator containing GLAD-deposited SiO(2) vertical posts as the separating medium. The separation mechanism was verified to resemble that found in prior fractionation devices, using total internal reflection fluorescence microscopy. GLAD fabrication enables insertion of three-dimensional structures into microchannels that cannot be fabricated with any existing techniques, and this versatility in structural design could facilitate new developments in on-chip separations.
RESUMO
Integration of a packed column onto a microchip for performance of capillary electrochromatography (CEC) is described. The quartz device incorporated a cross-injector, and a double weir trapping design for formation of 1, 2 and 5 mm long CEC columns. Three fluorescent dyes were baseline-resolved with plate numbers of 330,(330,000 plates/m; height equivalent to a theoretical plate, H = 3.0 microm) for BODIPY 493/503, 360 (360,000 plates/m; H = 2.8 microm) for rhodamine 123 and 244 (244,000 plates/m; H = 4.1 microm) for acridine orange (AO) with 500 V applied on a 1 mm long column. The 2 mm column yielded approximately 1.8 times more theoretical plates than did the 1 mm column, when operated at the same flow rate. Van Deemter plots were obtained for the three column lengths, showing increased plate height for the 5 mm length. A 2 mm column gave peak height and area relative standard deviation (RSD) values of 2.5 and 3.3%, respectively, as averages for the three dyes (n = 15). The RSD for the dye retention times was 1% (n = 6) over one day, and 3% (n = 30) over five days. Indirect fluorescence detection of thiourea and of amino acids was possible using a neutral indicator dye (BODIPY 493/503), with a detection limit of 10 microM for amino acids.