Quantitative analysis of chemotaxis towards toluene by Pseudomonas putida in a convection-free microfluidic device.

Chemotaxis has been shown to be beneficial for the migration of soil-inhabiting bacteria towards industrial chemical pollutants, which they degrade. Many studies have demonstrated the importance of this microbial property under various circumstances; however, few quantitative analyses have been undertaken to measure the two essential parameters that characterize the chemotaxis of bioremediation bacteria: the chemotactic sensitivity coefficient χ(0) and the chemotactic receptor constant K(c). The main challenge to determine these parameters is that χ(0) and K(c) are coupled together in non-linear mathematical models used to evaluate them. In this study we developed a method to accurately measure these parameters for Pseudomonas putida in the presence of toluene, an important pollutant in groundwater contamination. Our approach uses a multilayer microfluidic device to expose bacteria to a convection-free linear chemical gradient of toluene that is stable over time. The bacterial distribution within the gradient is measured in terms of fluorescence intensity, and is then used to fit the parameters Kc and χ(0) with mathematical models. Critically, bacterial distributions under chemical gradients at two different concentrations were used to solve for both parameters independently. To validate the approach, the chemotaxis parameters of Escherichia coli strains towards α-methylaspartate were experimentally derived and were found to be consistent with published results from related work.

Controlled microfluidic interfaces.

The microfabrication technologies of the semiconductor industry have made it possible to integrate increasingly complex electronic and mechanical functions, providing us with ever smaller, cheaper and smarter sensors and devices. These technologies have also spawned microfluidics systems for containing and controlling fluid at the micrometre scale, where the increasing importance of viscosity and surface tension profoundly affects fluid behaviour. It is this confluence of available microscale engineering and scale-dependence of fluid behaviour that has revolutionized our ability to precisely control fluid/fluid interfaces for use in fields ranging from materials processing and analytical chemistry to biology and medicine.

2D separations on a 1D chip: gradient elution moving boundary electrophoresis-chiral capillary zone electrophoresis.

A new method is described for two-dimensional (2D) separations using a microfluidic chip normally employed for single dimension electrophoresis. The method employs a combination of gradient elution moving boundary electrophoresis (GEMBE) and chiral capillary zone electrophoresis (CZE). The simplicity of the first dimension GEMBE method enables its implementation in the injection channel of a conventional electrophoresis chip, simplifying the design and operation of the device. The method was used for high resolution 2D chiral separations of a mixture of amino acids considered as possible signatures of extant or extinct life for solar system exploration. The enantiomers of aspartic acid, glutamic acid, serine, alanine, and valine were all resolved as well as glycine (achiral) and several unidentified impurities, giving an estimated peak capacity of 35 for the region between valine and glycine. The results highlight the need for high peak capacity separations for chiral amino acid analysis if accurate enantiomeric ratios are to be determined.

Magnetic connectors for microfluidic applications.

We present a new type of microfluidic connector that employs a ring magnet on one side of the microfluidic chip and a disc magnet on the other side to produce a sealed connection between external tubing and inlets or outlets of microfluidic devices. The connectors are low-cost, simple to use and assemble, and reusable. We used numerical (finite element) simulations in order to optimize their geometry. Configurations that achieve interfacial forces in the range of 2 N to 15 N are discussed. Several types of gasket materials were explored. Finally, we demonstrate an application of these connectors in a microfluidic device used to generate liposomes.

A vacuum manifold for rapid world-to-chip connectivity of complex PDMS microdevices.

The lack of simple interfaces for microfluidic devices with a large number of inlets significantly limits production and utilization of these devices. In this article, we describe the fabrication of a reusable manifold that provides rapid world-to-chip connectivity. A vacuum network milled into a rigid manifold holds microdevices and prevents leakage of fluids injected into the device from ports in the manifold. A number of different manifold designs were explored, and all performed similarly, yielding an average of 100 kPa (15 psi) fluid holding pressure. The wide applicability of this manifold concept is demonstrated by interfacing with a 51-inlet microfluidic chip containing 144 chambers and hundreds of embedded pneumatic valves. Due to the speed of connectivity, the manifolds are ideal for rapid prototyping and are well suited to serve as "universal" interfaces.

The microfluidic palette: a diffusive gradient generator with spatio-temporal control.

This paper describes a new microfluidic device called the "microfluidic palette", capable of generating multiple spatial chemical gradients simultaneously inside a microfluidic chamber. The unique aspect of this work is that chemical gradients are generated by diffusion, without convection, and can either be held constant over long periods, or modified dynamically. We characterized a representative device with a 1.5 mm circular chamber where diffusion takes place, and three access-ports for the delivery and removal of solutes. The gradient stabilizes in approximately 15 min for small molecules and can be maintained constant indefinitely. We demonstrate overlapping gradients with different spatial location and a controlled rotation of a diffusive gradient around its centre. Finally, we demonstrate the applicability of this tool to study the chemotactic response of the bacteria P. aeruginosa to glucose.

Using pattern homogenization of binary grayscale masks to fabricate microfluidic structures with 3D topography.

Because fluids at the microscale form three dimensional interfaces and are subject to three dimensional forces, the ability to create microstructures with modulated topography over large areas could greatly improve control over microfluidic phenomena (e.g., capillarity and mass transport) and enable exciting novel microfluidic applications. Here, we report a method for the fabrication of three-dimensional relief microstructures, based on the emergence of smooth features when a photopolymer is exposed to UV light through a transparency mask with binary motifs. We show that homogeneous features emerge under certain critical conditions that are also common to other, apparently unrelated, phenomena such as the emergence of macroscopic continuum properties of composite materials and the rates of ligand binding to cell membrane receptors. This fabrication method is simple and inexpensive, and yet it allows for the fabrication of microstructures over large areas (centimetres) with topographic modulation of features with characteristic dimensions smaller than 100 micrometres.

Steady flow generation in microcirculatory systems.

In this paper we explore the mechanical generation of steady-non pulsatile-flow in microfluidic systems. The rationale of the paper is inspired in the example of cardiovascular systems where at the microscale (i.e. capillaries) the flow is steady rather than pulsatile to optimize performance. We present a solution to the generation of steady flow in engineered microfluidic systems either in open or closed loop configurations via the use of disc pumps. The disc pump consists of a flat rotating disc and utilizes both viscous drag and centrifugal force to achieve pumping. Experiments using single loop and double loop microfluidic systems are presented to characterize the disc pump. Continuous flow generated by the disc pumps can be used to separate particles based on size using recirculating loops and for extraction of small particles without disturbing the concentration of bigger particles. The potential impact of this technology includes sample separation and extraction techniques into portable microfluidic labs-on-a-chip, and long term culture systems for cells in suspension.

Capillary inserts in microcirculatory systems.

Microfluidic loops (i.e. closed fluid paths) pose specific practical challenges such as priming, introducing analytes or reagents in a controlled way and sampling products. In this technical note we address these three issues using a removable part of the microchannel that we call a 'capillary insert'.

Magnetically-driven biomimetic micro pumping using vortices.

Planar micropumps utilizing vortices shed by an oscillating ferromagnetic bar are presented. The movement of the bar is induced by magnetic coupling with an external spinning magnet. Thus, energy transfer is achieved without physical contact or need of any on-chip power source. To create vortices inside the chip, the Reynolds number is locally increased with the oscillation of the bar. The utilization of the vortices as a tool for efficient transfer of energy is inspired by the behaviour of swimming animals and flying insects in nature. The pumps operate in two different scales (milli-scale and micro-scale) in order to take advantage of both. Experiments are presented characterizing the pumps and their flow patterns. The range of operation of the pumps is from 3 microl min(-1) to 600 microl min(-1), though smaller flow rates are also possible.

Pneumatic valves in folded 2D and 3D fluidic devices made from plastic films and tapes.

We present a rapid prototyping technique that expands elastomeric valving capabilities to devices made from thin materials such as plastic films and tapes. The time required from conception to full fabrication of functional devices is within a few hours. A key characteristic of this technology is that devices are thin (typically less than 0.5 mm in thickness), which allows for the fabrication of devices with many layers. This feature also permits folding of devices into 3D structures having fully functional valves. We illustrate this concept with the fabrication of a 25 mm-per-side cube whose walls contain microfluidic channels and valves. Control of liquid delivery through the faces of the cube is demonstrated with a chemotaxis experiment of C. elegans migrating within the enclosed volume of the cube as stimuli are delivered through the walls of the cube to the interior faces.

A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array.

A novel microscale device has been developed to enable the one-step continuous flow assembly of monodisperse nanoscale liposomes using three-dimensional microfluidic hydrodynamic focusing (3D-MHF) in a concentric capillary array. The 3D-MHF flow technique displays patent advantages over conventional methods for nanoscale liposome manufacture (i.e., bulk-scale alcohol injection and/or sonication) through the on-demand synthesis of consistently uniform liposomes without the need for post-processing strategies. Liposomes produced by the 3D-MHF device are of tunable size, have a factor of two improvement in polydispersity, and a production rate that is four orders of magnitude higher than previous MHF methods, which can be attributed to entirely radially symmetric diffusion of alcohol-solvated lipid into an aqueous flow stream. Moreover, the 3D-MHF platform is simple to construct from low-cost, commercially-available components, which obviates the need for advanced microfabrication strategies necessitated by previous MHF nanoparticle synthesis platforms.

A robust diffusion-based gradient generator for dynamic cell assays.

This manuscript describes a new method to generate purely diffusive chemical gradients that can be modified in time. The device is simple in its design and easy to use, which makes it amenable to study biological processes that involve static or dynamic chemical gradients such as chemotaxis. We describe the theory underlying the convection-free gradient generator, illustrate the design to implement the theory, and present a protocol to align multiple layers of double sided tape and laminates to fabricate the device. Using this device, a population of mammalian cells was exposed to different concentrations of a toxin within a concentration gradient in a 48 h experiment. Cells were probed dynamically by cycling the gradient on and off, and cell response was monitored using time-lapse fluorescence microscopy. The experiment and results illustrate the type of applications involving dynamic cell behavior that can be targeted with this type of gradient generator.

Measurement and validation of cell-based assays with microfluidics at the National Institute of Standards and Technology.

The National Institute of Standards and Technology (NIST) is the National Metrology Institute for the USA. Our mission is to advance measurement science, standards and technology in ways that enhance economic security and improve quality of life in the USA. Due to the increased need for technologies that advance biological research and the many new and exciting innovations in microfluidics, our projects are aimed at engineering well-controlled microenvironments for quantitative measurements of cell behavior in microfluidic systems. Cell-based microfluidics at NIST is a highly multidisciplinary activity and is greatly influenced by NIST programs in biochemical sciences, materials science, engineering and information technology. Although there are many microfluidic-related activities ongoing at NIST, we will focus on projects related to cell-based measurements in this article.