It is difficult to mix solutions in microchannels. Under typical operating conditions, flows in these channels are laminar-the spontaneous fluctuations of velocity that tend to homogenize fluids in turbulent flows are absent, and molecular diffusion across the channels is slow. We present a passive method for mixing streams of steady pressure-driven flows in microchannels at low Reynolds number. Using this method, the length of the channel required for mixing grows only logarithmically with the Péclet number, and hydrodynamic dispersion along the channel is reduced relative to that in a simple, smooth channel. This method uses bas-relief structures on the floor of the channel that are easily fabricated with commonly used methods of planar lithography.
This paper describes a simple, versatile method of generating gradients in composition in solution or on surfaces using microfluidic systems. This method is based on controlled diffusive mixing of species in solutions that are flowing laminarly, at low Reynolds number, inside a network of microchannels. We demonstrate the use of this procedure to generate (1) gradients in the compositions of solutions, measured directly by colorimetric assays and (2) gradients in topography of the surfaces produced by generating concentration gradients of etching reagents, and then using these gradients to etch profiles into the substrate. The lateral dimensions of the gradients examined here, which went from 350 to 900 μm, are determined by the width of the microchannels. Gradients of different size, resolution, and shape have been generated using this method. The shape of the gradients can be changed continuously (dynamic gradients) by varying the relative flow velocities of the input streams of fluids. The method is experimentally simple and highly adaptable, and requires no special equipment except for an elastomeric relief structure that can be readily prepared by rapid prototyping. This technique provides a new platform with which to study phenomena that depend on gradients in concentration, especially dynamic phenomena in cell biology (chemotaxis and haptotaxis) and surface chemistry (nucleation and growth of crystals, etching, and Marangoni effects).
Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero ("cliff" gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant ("hill" gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.
This paper describes the generation of gradients having complex shapes in solution using microfluidic networks. Flowing multiple streams of fluid each carrying different concentrations of substances laminarly and side-by-side generated step concentration gradients perpendicular to the direction of the flow. Appropriately designed networks of microchannels for controlled diffusive mixing of substances generated a range of shapes for the gradients, including linear, parabolic, and periodic. The lateral dimensions of the gradients ranged from 900 to 2200 μm. This paper also demonstrates the generation of overlapping gradients composed of different species. Since solutions in the microfluidic network exist as steady states and are continuously renewed, the gradients established in the capillaries are spatially and temporally constant and can be maintained easily for periods of hours. Using laminar flow to generate gradients should be useful in both biological and nonbiological research.
Little is known about the influence of substrate-bound gradients on neuronal development, since it has been difficult to fabricate gradients over the distances typically required for biological studies (a few hundred micrometers). This article demonstrates a generally applicable technique for the fabrication of substratebound gradients of proteins with complex shapes, using laminar flows in microchannels. Gradients that range from pure laminin to pure BSA were formed in solution by using a network of microchannels, and these proteins were allowed to adsorb onto a homogeneous layer of poly-L-lysine. Rat hippocampal neurons were cultivated on these substrate-bound gradients. Analysis of optical images of these neurons showed that axon specification is oriented in the direction of increasing surface density of laminin. Linear gradients in laminin adsorbed from a gradient in solution having a slope of ١[laminin] > about 0.06 g (ml⅐m) ؊1 (defined by dividing the change of concentration of laminin in solution over the distance of the gradient) orient axon specification, whereas those with ١[laminin] < about 0.06 g (ml⅐m) ؊1 have no effect. microfluidics ͉ neuronal polarity ͉ hippocampal G radients of chemoattractant and chemorepellent substances play central roles in controlling the development of the brain (1). Although the influence of gradients of soluble substances on neuronal behavior has been studied extensively and has been used to unravel the molecular and cellular mechanisms of axon guidance (2), much less is known about gradients of substrate-bound substances (3). Generating consistent substrate-bound gradients over the distances required for biological studies (a few hundred micrometers) has been difficult, and this difficulty hindered the investigation of the role of immobilized gradients in neuronal development (e.g., establishment of cellular polarity and axon guidance) and cell migration. This article demonstrates a generally applicable technique for the fabrication of substrate-bound gradients that uses laminar flow of fluids in microchannels; we have described the microfluidic systems used to form these gradients (4). By generating gradients in the concentration of proteins in solution and allowing them to adsorb on a surface, we fabricated substratebound gradients that range from pure laminin to pure BSA immobilized on a uniform layer of poly-L-lysine (PLL) over distances of a few hundred micrometers. We demonstrate that axon specification of rat hippocampal neurons cultivated on these gradients is oriented in the direction of increasing concentration of laminin. We demonstrate that gradients of extracellular molecules adsorbed on the surface are capable of orienting the polarity of cultured hippocampal neurons.Hippocampal neurons in culture develop their characteristic structural and functional polarity in a stereotyped sequence of developmental events (5). Cells first attach to the substrate and then develop processes. When new processes start to form, they cannot be distinguished as either axons o...
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