RESUMO
The use of porous polymer monoliths functionalized with silver nanoparticles is introduced in this work for high-sensitivity surface-enhanced Raman scattering (SERS) detection. Preparation of the SERS detection elements is a simple process comprising the synthesis of a discrete polymer monolith section within a silica capillary, followed by physically trapping silver nanoparticle aggregates within the monolith matrix. A SERS detection limit of 220 fmol for Rhodamine 6G is demonstrated, with excellent signal stability over a 24 h period. The capability of the SERS-active monolith for label-free detection of biomolecules was demonstrated by measurements of bradykinin and cytochrome c. The SERS-active monoliths can be readily integrated into miniaturized micrototal-analysis systems for online and label-free detection for a variety of biosensing, bioanalytical, and biomedical applications.
Assuntos
Nanopartículas Metálicas/química , Polímeros/análise , Polímeros/química , Análise Espectral Raman/métodos , Bradicinina/análise , Bradicinina/química , Citocromos c/análise , Citocromos c/química , Polimerização/efeitos da radiação , Porosidade , Dióxido de Silício/química , Prata/química , Propriedades de Superfície , Raios UltravioletaRESUMO
A two-dimensional microfluidic system is presented for intact protein separations combining isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) employing in situ photopolymerized polyacrylamide (PAAm) gels. The PAAm gels are used for multiple functions. In addition to serving as a highly-resolving separation medium for gel electrophoresis, discrete polyacrylamide gel plugs are used to enable the efficient isolation of different on-chip media including anolyte, catholyte, and sample/ampholyte solutions for IEF. The gel plugs are demonstrated as on-chip reagent containers, holding defined quantities of SDS for on-chip SDS-protein complexation, and enabling the use of a discontinuous buffer system for sample band sharpening during SDS-PAGE. The 2-D chip also employs several unique design features including an angled isoelectric focusing channel to minimize sample tailing, and backbiasing channels designed to achieve uniform interdimensional sample transfer. Separation results using E. coli cell lysate are presented using a 10-channel chip with and without the discontinuous buffer system, with resolving power more than doubled in the former case. Further improvements in separation resolution are demonstrated using a 20-channel chip design.
Assuntos
Eletroforese em Microchip/instrumentação , Eletroforese em Gel de Poliacrilamida/instrumentação , Focalização Isoelétrica/instrumentação , Proteínas/análise , Resinas Acrílicas/química , Soluções Tampão , Eletroforese em Gel Bidimensional/instrumentação , Desenho de Equipamento , Escherichia coli , Reprodutibilidade dos TestesRESUMO
A facile method enabling the integration of elastomeric valves into rigid thermoplastic microfluidic chips is described. The valves employ discrete plugs of elastomeric polydimethylsiloxane (PDMS) integrated into the thermoplastic substrate and actuated using a threaded stainless steel needle. The fabrication process takes advantage of poly(ethylene glycol) (PEG) as a sacrificial molding material to isolate the PDMS regions from the thermoplastic flow channels, while yielding smooth contact surfaces with the PDMS valve seats. The valves introduce minimal dead volumes, and provide a simple mechanical means to achieve reproducible proportional valving within thermoplastic microfluidic systems. Burst pressure tests reveal that the valves can withstand pressures above 12 MPa over repeated open/close cycles without leakage, and above 24 MPa during a single use, making the technology well suited for applications such as high performance liquid chromatography. Proportional valve operation is demonstrated using a multi-valve chemical gradient generator fabricated in cyclic olefin polymer.
Assuntos
Fenômenos Mecânicos , Técnicas Analíticas Microfluídicas/instrumentação , Polímeros/química , Pressão , Temperatura , Alcenos/química , Dimetilpolisiloxanos/química , Gases/química , Microtecnologia , Permeabilidade , Polietilenoglicóis/química , Solventes/química , Propriedades de SuperfícieRESUMO
Polymer microfluidic chips employing in situ photopolymerized polymethacrylate monoliths for high-performance liquid chromatography separations of peptides is described. The integrated chip design employs a 15 cm long separation column containing a reversed-phase polymethacrylate monolith as a stationary phase, with its front end seamlessly coupled to a 5 mm long methacrylate monolith which functions as a solid-phase extraction (SPE) element for sample cleanup and enrichment, serving to increase both detection sensitivity and separation performance. In addition to sample concentration and separation, solvent splitting is also performed on-chip, allowing the use of a conventional LC pump for the generation of on-chip nanoflow solvent gradients. The integrated platform takes advantage of solvent bonding and a novel high-pressure needle interface which together enable the polymer chips to withstand internal pressures above 20 MPa (approximately 2900 psi) for efficient pressure-driven HPLC separations. Gradient reversed-phase separation of fluorescein-labeled model peptides and BSA tryptic digest are demonstrated using the microchip HPLC system. Online removal of free fluorescein and enrichment of labeled proteins are simultaneously achieved using the on-chip SPE column, resulting in a 150-fold improvement in sensitivity and a 10-fold reduction in peak width in the following microchip gradient LC separation.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Peptídeos/isolamento & purificação , Ácidos Polimetacrílicos/química , Extração em Fase Sólida/instrumentação , Animais , Bovinos , Fluoresceína-5-Isotiocianato/química , Injeções , Peptídeos/química , Peptídeos/metabolismo , Pressão , Reprodutibilidade dos Testes , Coloração e Rotulagem , Tripsina/metabolismoRESUMO
In an effort to develop new tools for diagnosing influenza in resource-limited settings, we fabricated a polycarbonate (PC)-polydimethylsiloxane (PDMS) hybrid microchip using a simple epoxy silica sol-gel coating/bonding method and employed it in sensitive detection of influenza virus with Europium nanoparticles (EuNPs). The incorporation of sol-gel material in device fabrication provided functionalized channel surfaces ready for covalent immobilization of primary antibodies and a strong bonding between PDMS substrates and PC supports without increasing background fluorescence. In microchip EuNP immunoassay (µENIA) of inactivated influenza viruses, replacing native PDMS microchips with hybrid microchips allowed the achievement of a 6-fold increase in signal-to-background ratio, a 12-fold and a 6-fold decreases in limit-of-detection (LOD) in influenza A and B tests respectively. Using influenza A samples with known titers, the LOD of influenza µENIA on hybrid microchips was determined to be ~10(4) TCID50 titer/mL and 10(3)-10(4) EID50 titer/mL. A comparison test indicated that the sensitivity of influenza µENIA enhanced using the hybrid microchips even surpassed that of a commercial laboratory influenza ELISA test. In addition to the sensitivity improvement, assay variation was clearly reduced when hybrid microchips instead of native PDMS microchips were used in the µENIA tests. Finally, infectious reference viruses and nasopharyngeal swab patient specimens were successfully tested using µENIA on hybrid microchip platforms, demonstrating the potential of this unique microchip nanoparticle assay in clinical diagnosis of influenza. Meanwhile, the tests showed the necessity of using nucleic acid confirmatory tests to clarify ambiguous test results obtained from prototype or developed point-of-care testing devices for influenza diagnosis.
Assuntos
Dimetilpolisiloxanos/química , Imunoensaio/instrumentação , Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , Dispositivos Lab-On-A-Chip , Nanopartículas Metálicas/química , Resinas Epóxi/química , Desenho de Equipamento , Análise de Falha de Equipamento , Európio/química , Humanos , Vírus da Influenza A/imunologia , Influenza Humana/diagnóstico , Influenza Humana/imunologia , Nanopartículas Metálicas/ultraestrutura , Transição de Fase , Cimento de Policarboxilato/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Dióxido de Silício/químicaRESUMO
High-sensitivity and rapid flow-through immunosensors based on photopolymerized surface-reactive polymer monoliths are investigated. The porous monoliths were synthesized within silica capillaries from glycidyl methacrylate and ethoxylated trimethylolpropane triacrylate precursors, providing a tortuous pore structure with high surface area for the immobilization of antibodies or other biosensing ligands. The unique morphology of the monolith ensures efficient mass transport and interactions between solvated analyte molecules and covalently immobilize antibodies anchored to the monolith surface, resulting in rapid immunorecognition. The efficacy of this approach is demonstrated through a direct immunoassay model using anti-IgG as a monolith-bound capture antibody and fluorescein-labeled IgG as an antigen. In situ antigen measurements exhibited a linear response over a concentration range between 0.1 and 50 ng/mL with 5 min assay times, while controllable injection of 1 µL volumes of antigen through the monolith elements yielded a mass detection limit of 100 pg ((â¼700amol). These results suggest that porous monolith supports represent a flexible and promising material for the fabrication of rapid and sensitive immunosensors suitable for integration into capillary or microfluidic devices.
Assuntos
Anticorpos/química , Técnicas Biossensoriais/instrumentação , Análise de Injeção de Fluxo/instrumentação , Imunoensaio/instrumentação , Polímeros/química , Espectrometria de Fluorescência/instrumentação , Anticorpos/imunologia , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
Fabrication of microfluidic systems from polymeric materials is attractive because of simplicity and low cost. Unfortunately, the surfaces of many polymeric materials can adsorb biological samples. Therefore, it is necessary to modify their surfaces before these polymeric materials can be used for separation and analysis. Oftentimes it is difficult to modify polymeric surfaces because of their resistance to chemical reaction. Recently, we introduced a surface-reactive acrylic polymer, poly(glycidyl methacrylate-co-methyl methacrylate) (PGMAMMA), which can be modified easily and is suitable for fabrication of microfluidic devices. Epoxy groups on the surface can be activated by air plasma treatment, hydrolysis, or aminolysis. In this work, the resulting hydroxyl or amino groups were reacted with 2-bromoisobutylryl bromide to introduce an initiator for surface-initiated atom-transfer radical polymerization (SI-ATRP). Polyethylene glycol (PEG) layers grown on the surface using this method were uniform, hydrophilic, stable, and resistant to protein adsorption. Contact angle measurement and X-ray photoelectron spectroscopy (XPS) were used to characterize activated polymer surfaces, initiator-bound surfaces, and PEG-grafted surfaces. We obtained excellent capillary electrophoresis (CE) separations of proteins and peptides with the PEG-modified microchips. A separation efficiency of 4.4 x 10(4) plates for a 3.5 cm long separation channel was obtained.
Assuntos
Compostos de Epóxi/química , Metacrilatos/química , Procedimentos Analíticos em Microchip/métodos , Técnicas Analíticas Microfluídicas/métodos , Polimetil Metacrilato/química , Aminas/química , Eletroforese Capilar , Radicais Livres/química , Hidrocarbonetos Bromados/química , Hidrólise , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Polietilenoglicóis/química , Análise Espectral , Propriedades de Superfície , Fatores de TempoRESUMO
In-channel atom transfer radical polymerization (ATRP) was used to graft a PEG layer on the surface of microchannels formed in poly(glycidyl methacrylate)-co-(methyl methacrylate) (PGMAMMA) microfluidic devices. The patterned and cover plates were first anchored with ATRP initiator and then thermally bonded together, followed by pumping a solution containing monomer, catalyst, and ligand into the channel to perform ATRP. A PEG-functionalized layer was grafted on the microchannel wall, which resists protein adsorption. X-ray photoelectron spectroscopy (XPS) was used to investigate the initiator-bound surface, and EOF was measured to evaluate the PEG-grafted PGMAMMA microchannel. Fast, efficient, and reproducible separations of amino acids, peptides, and proteins were obtained using the resultant microdevices. Separation efficiencies were higher than 1.0x10(4) plates for a 3.5 cm separation microchannel. Compared with microdevices modified using a previously reported ATRP technique, these in-channel modified microdevices demonstrated better long-term stability.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Aminoácidos/isolamento & purificação , Caseínas/metabolismo , Eletroforese Capilar/métodos , Humanos , Lactoglobulinas/isolamento & purificação , Metilmetacrilatos/química , Peptídeos/isolamento & purificação , Polimetil Metacrilato/química , Hidrolisados de Proteína/isolamento & purificação , Proteínas/isolamento & purificação , Albumina Sérica/isolamento & purificaçãoRESUMO
In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteínas de Escherichia coli/isolamento & purificação , Focalização Isoelétrica/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Resinas Acrílicas/química , Técnicas Analíticas Microfluídicas/métodosRESUMO
A poly(ethylene glycol)-functionalized acrylic copolymer was developed for fabrication of microfluidic devices that are resistant to protein and peptide adsorption. Planar microcapillary electrophoresis (microCE) devices were fabricated from this copolymer with the typical cross pattern to facilitate sample introduction. In contrast to most methods used to fabricate polymeric microchips, the photopolymerization-based method used with the copolymer reported in this work was of the soft lithography type, and both patterning and bonding could be completed within 10 min. In a finished microdevice, the cover plate and patterned substrate were bonded together through strong covalent bonds. Additionally, because of the resistance of the copolymer to adsorption, fabricated microfluidic devices could be used without surface modification to separate proteins and peptides. Separations of fluorescein isothiocyanate-labeled protein and peptide samples were accomplished using these new polymeric microCE microchips. Separation efficiencies as high as 4.7 x 10(4) plates were obtained in less than 40 s with a 3.5-cm separation channel, yielding peptide and protein peaks that were symmetrical.
Assuntos
Eletroforese em Microchip/métodos , Técnicas Analíticas Microfluídicas , Peptídeos/análise , Polietilenoglicóis/química , Polímeros/química , Proteínas/análise , Acrilatos/química , Adsorção , Polímeros/síntese química , Reprodutibilidade dos TestesRESUMO
Because of their surface heterogeneity, proteins readily adsorb on polymeric substrates via various interactions, which adversely affects the performance of polymeric microfluidic devices in electrophoresis-based protein/peptide analysis. Therefore, it is necessary to use surface modification techniques such as dynamic coating or more complicated permanent surface modification, which has broader application and better performance, to render the polymeric microchannels protein-resistant. This manuscript is a review of the surface chemistry of microfluidic devices used for electrophoretic separations of proteins and peptides. The structural complexity of proteins as it relates to adsorption is described, followed by a review of the mechanisms and structural characteristics of protein-resistant surfaces. Permanent surface modification techniques used in grafting protein-resistant materials onto the surfaces of electrophoresis microchannels fabricated from polymer substrates are summarized and successful examples are presented.
Assuntos
Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Técnicas Analíticas Microfluídicas , Peptídeos/análise , Proteínas/análise , Polímeros/química , Propriedades de SuperfícieRESUMO
A novel approach to integrating a buffer ion-permeable membrane in a poly(glycidyl methacrylate-co-methyl methacrylate) micro electric field gradient focusing (muEFGF) device is described. A weir structure on which the membrane was positioned was fabricated between the separation channel and field gradient-generating channel. Before formation of the membrane, the surface of the polymeric microdevice was treated for covalent bonding of the membrane. Following surface modification, a prepolymer solution containing poly(ethylene glycol) acrylate/methacrylate and Tris-HCl buffer was loaded into the microdevice. Low-pressure nitrogen gas was then purged through the separation and field gradient-generating channels to remove the prepolymer solution from these channels. Residual prepolymer solution was retained on the weir structure due to surface tension. Finally, the premembrane was cured in place on the weir using UV radiation. Using a muEFGF device, green fluorescent protein (GFP) was concentrated 4000-fold. Separation of GFP and R-phycoerythrin, and selective elution of GFP from a protein mixture containing GFP, FITC-labeled casein, and FITC-labeled hemoglobin were also demonstrated. It was found that the membrane conductivity and presence of carboxylic acid impurities in the membrane strongly affected the behavior of the muEFGF device.
Assuntos
Eletricidade , Membranas Artificiais , Micro-Ondas , PolímerosRESUMO
A surface-reactive acrylic polymer, poly(glycidyl methacrylate-co-methyl methacrylate) (PGMAMMA), was synthesized and evaluated for suitability as a substrate for fabrication of microfluidic devices for chemical analysis. This polymer has good thermal and optical properties and is mechanically robust for cutting and hot embossing. A key advantage of this polymeric material is that the surface can be easily modified to control inertness and electroosmotic flow using a variety of chemical procedures. In this work, the procedures for aminolysis, photografting of linear polyacrylamide, and atom-transfer radical polymerization on microchannel surfaces in PGMAMMA substrates were developed, and the performance of resultant microfluidic electrophoresis devices was demonstrated for the separation of amino acids, peptides, and proteins. Separation efficiencies as high as 4.6 x 10(4) plates for a 3.5-cm-long separation channel were obtained. The results indicate that PGMAMMA is an excellent substrate for microfabricated fluidic devices, and a broad range of applications should be possible.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Polímeros/química , Aminas/química , Aminoácidos/química , Etilenodiaminas/química , Fluoresceína-5-Isotiocianato , Concentração de Íons de Hidrogênio , Metilmetacrilatos/química , Nitrogênio/química , Peptídeos/química , Proteínas/química , Reprodutibilidade dos Testes , Solventes , TemperaturaRESUMO
Polymeric materials have emerged as appealing alternatives to conventional inorganic substrates for the fabrication of microscale analytical systems; however, native polymeric surfaces typically require covalent modification to ensure optimum biocompatibility. 2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA microcapillary electrophoresis (muCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins on microchannel surfaces. Experiments using fluorescein isothiocyanate-conjugated bovine serum albumin indicated that both column efficiency and migration time reproducibility were 1 order of magnitude better with derivatized compared to untreated PMMA muCE chips. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA muCE chips. All analyses were completed in less than 60 s, and separation efficiencies as high as 5.2 x10(4) plates for a 3.5-cm-long separation channel were obtained. These results demonstrate the general applicability of surface-grafted PMMA microdevices for a broad range of protein analyses.