RESUMO
Performing localized chemical events on surfaces is critical for numerous applications. We earlier invented the microfluidic probe (MFP), which circumvented the need to process samples in closed microchannels by hydrodynamically confining liquids that performed chemistries on surfaces (Juncker et al. Nat. Mater. 2005, 4, 622-628). Here we present a new and versatile probe, the vertical MFP (vMFP), which operates in the scanning mode while overcoming earlier challenges that limited the practical implementation of the MFP technology. The key component of the vMFP is the head, a microfluidic device (â¼1 cm(2) in area) consisting of glass and Si and having microfluidic features fabricated in-plane in the Si layer. The base configuration of the head has two micrometer-size channels that inject/aspirate liquids and terminate at the apex which is â¼1 mm(2). In scanning mode, the head is oriented vertically with the apex parallel to the surface with typical spacing of 1-30 µm. Such length scales and using flow rates from nanoliters/second to microliters/second allow chemical events to be performed on surfaces with tens of picoliter quantities of reagents. Before scanning, the head is clipped on a holder for leak-free, low dead volume interface assembly, providing a simple world-to-chip interface. Surfaces are scanned by mounting the holder on a computer-controlled stage having â¼0.1 µm resolution in positioning. We present detailed steps to fabricate vMFP heads having channels with dimensions from 1 µm × 1 µm to 50 µm × 50 µm for liquid localization over areas of 10-10,000 µm(2). Additionally, advanced design strategies are described to achieve high yield in fabrication and to support a broad range of applications. These include particulate filters, redundant aperture architectures, inclined flow-paths that service apertures, and multiple channels to enable symmetric flow confinement. We also present a method to characterize flow confinement and estimate the distance between the head and the surface by monitoring the evolution of a solution of fluorescently labeled antibody on an activated glass surface. This flow characterization reveals regimes of operation suitable for different surface topographies. We further integrate the dispensing of immersion liquid to the vMFP head for processing surfaces for extended periods of time (â¼60 min). The versatility of the vMFP is exemplified by patterning fluorescently labeled proteins, inactivation of cells using sodium hypochlorite, and staining living NIH fibroblasts with Cellomics. These applications are enabled by the compact design of the head, which provides easy access to the surface, simplifies alignment, and enables processing surfaces having dimensions from the micrometer to the centimeter scale and with large topographical variations. We therefore believe that ease-of-operation, reconfigurability, and conservative use of chemicals by the vMFP will lead to its widespread use by microtechnologists and the chemical and biomedical communities.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Animais , Desenho de Equipamento , Hidrodinâmica , Imersão , Camundongos , Células NIH 3T3 , Propriedades de SuperfícieRESUMO
Microfluidic networks (microFNs) were used to pattern biomolecules with high resolution on a variety of substrates (gold, glass, or polystyrene). Elastomeric microFNs localized chemical reactions between the biomolecules and the surface, requiring only microliters of reagent to cover square millimeter-sized areas. The networks were designed to ensure stability and filling of the microFN and allowed a homogeneous distribution and robust attachment of material to the substrate along the conduits in the microFN. Immunoglobulins patterned on substrates by means of microFNs remained strictly confined to areas enclosed by the network with submicron resolution and were viable for subsequent use in assays. The approach is simple and general enough to suggest a practical way to incorporate biological material on technological substrates.
Assuntos
Dimetilpolisiloxanos , Vidro , Ouro , Imunoglobulina G , Poliestirenos , Silicones , Adesividade , Animais , Fenômenos Químicos , Físico-Química , Galinhas , Ensaio de Imunoadsorção Enzimática , Borracha , Propriedades de SuperfícieRESUMO
Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. This method, called affinity-contact printing (alphaCP), uses a structured elastomer derivatized with ligands against the target molecules. After the target molecules have been captured, they are printed from the elastomer onto a variety of surfaces. The ligand remains on the stamp for reuse. In contrast with conventional affinity chromatography, here dissociation and release of captured molecules to the substrate are achieved mechanically. We demonstrate this technique by extracting the cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from tissue homogenates and cell culture lysates and patterning affinity-purified NgCAM on polystyrene to stimulate the attachment of neuronal cells and guide axon outgrowth.
Assuntos
Biotecnologia/métodos , Técnicas de Cultura de Células/métodos , Animais , Axônios/metabolismo , Células COS , Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Células Cultivadas , Embrião de Galinha , Relação Dose-Resposta a Droga , Imunoglobulina G/metabolismo , Cinética , Microscopia de Fluorescência , Modelos Biológicos , Neurônios/metabolismo , Poliestirenos/metabolismo , Ligação Proteica , Proteínas/análise , Proteínas/isolamento & purificação , Fatores de Tempo , TransfecçãoRESUMO
Interaction between tumor cells and the microenvironment is key in initiation, progression, and invasiveness of cancer. In particular, mesenchymal stem cells (MSCs) are recruited to the sites of developing tumors, thus promoting metastasis formation. Although it is well known that MSCs migrate and integrate in the tumor microenvironment (TME), their fate and function inside the tumor is still not clear. In this study, we analyzed the role played by MSCs in breast cancer oncogenesis. Data indicate that interaction of breast cancer cells with MSCs results in an increased proliferation and metabolic activity of breast cancer cells, partially due to MSC-derived microvesicles that are shed in the TME. Moreover, we addressed the question of whether we could modulate such interaction by acting on P2X-mediated intercellular communication. By inhibiting P2X-mediated purinergic signaling, we succeeded in reducing both the cancerogenic as well as the metastatic potential of breast cancer cells co-cultured with MSCs, in 2D as well as in 3D in vitro models. Data obtained demonstrate for the first time that the trophic effect of MSCs on breast cancer cell growth is exerted via ionotropic purinergic signaling, thus suggesting the inhibition of the purinergic signaling system as a potential target for therapeutic intervention.
Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Neoplásicas/citologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Técnicas de Cocultura , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores Purinérgicos P2X/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologiaRESUMO
The microfluidic probe (MFP) is a non-contact, scanning microfluidic technology for local (bio)chemical processing of surfaces based on hydrodynamically confining nanoliter volumes of liquids over tens of micrometers. We present here a compact MFP (cMFP) that can be used on a standard inverted microscope and assist in the local processing of tissue sections and biological specimens. The cMFP has a footprint of 175 × 100 × 140 mm(3) and can scan an area of 45 × 45 mm(2) on a surface with an accuracy of ±15 µm. The cMFP is compatible with standard surfaces used in life science laboratories such as microscope slides and Petri dishes. For ease of use, we developed self-aligned mounted MFP heads with standardized "chip-to-world" and "chip-to-platform" interfaces. Switching the processing liquid in the flow confinement is performed within 90 s using a selector valve with a dead-volume of approximately 5 µl. We further implemented height-compensation that allows a cMFP head to follow non-planar surfaces common in tissue and cellular ensembles. This was shown by patterning different macroscopic copper-coated topographies with height differences up to 750 µm. To illustrate the applicability to tissue processing, 5 µm thick M000921 BRAF V600E+ melanoma cell blocks were stained with hematoxylin to create contours, lines, spots, gradients of the chemicals, and multiple spots over larger areas. The local staining was performed in an interactive manner using a joystick and a scripting module. The compactness, user-friendliness, and functionality of the cMFP will enable it to be adapted as a standard tool in research, development and diagnostic laboratories, particularly for the interaction with tissues and cells.
Assuntos
Dispositivos Lab-On-A-Chip , Melanoma/patologia , Manejo de Espécimes , Humanos , Dispositivos Lab-On-A-Chip/instrumentação , Dispositivos Lab-On-A-Chip/métodos , Microscopia/instrumentação , Microscopia/métodos , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Coloração e Rotulagem/instrumentação , Coloração e Rotulagem/métodosRESUMO
Immunoassays are widely used for medical diagnostics and constitute the principal method of detecting pathogenic agents and thus of diagnosing many diseases. These assays, which are most often performed in well plates, would be greatly improved by a practical method to pattern a series of antigens on a flat surface and to localize their binding to many analytes. But no obvious method exists to expose a planar surface successively to a series of antigens and analytes. Here, we present miniaturized mosaic immunoassays based on patterning lines of antigens onto a surface by means of a microfluidic network (muFN). Solutions to be analyzed are delivered by the channels of a second muFN across the pattern of antigens. Specific binding of the target antibodies with their immobilized antigens on the surface results in a mosaic of binding events that can readily be visualized in one screening using fluorescence. It is thus possible to screen solutions for antibodies in a combinatorial fashion with great economy of reagents and at a high degree of miniaturization. Such mosaic-format immunoassays are compatible with the sensitivity and reliability required for immunodiagnostic methods.