Design and fabrication of uniquely shaped thiol-ene microfibers using a two-stage hydrodynamic focusing design.

Microfluidic systems have advantages that are just starting to be realized for materials fabrication. In addition to the more common use for fabrication of particles, hydrodynamic focusing has been used to fabricate continuous polymer fibers. We have previously described such a microfluidics system which has the ability to generate fibers with controlled cross-sectional shapes locked in place by in situ photopolymerization. The previous fiber fabrication studies produced relatively simple round or ribbon shapes, demonstrated the use of a variety of polymers, and described the interaction between sheath-core flow-rate ratios used to control the fiber diameter and the impact on possible shapes. These papers documented the fact that no matter what the intended shape, higher flow-rate ratios produced rounder fibers, even in the absence of interfacial tension between the core and sheath fluids. This work describes how to fabricate the next generation of fibers predesigned to have a much more complex geometry, as exemplified by the "double anchor" shape. Critical to production of the pre-specified fibers with complex features was independent control over both the shape and the size of the fabricated microfibers using a two-stage hydrodynamic focusing system. Design and optimization of the channels was performed using finite element simulations and confocal imaging to characterize each of the two stages theoretically and experimentally. The resulting device design was then used to generate thiol-ene fibers with a unique double anchor shape. Finally, proof-of-principle functional experiments demonstrated the ability of the fibers to transport fluids and to interlock laterally.

Catch and release: integrated system for multiplexed detection of bacteria.

An integrated system with automated immunomagnetic separation and processing of fluidic samples was demonstrated for multiplexed optical detection of bacterial targets. Mixtures of target-specific magnetic bead sets were processed in the NRL MagTrap with the aid of rotating magnet arrays that entrapped and moved the beads within the channel during reagent processing. Processing was performed in buffer and human serum matrixes with 10-fold dilutions in the range of 10(2)-10(6) cells/mL of target bacteria. Reversal of magnets' rotation post-processing released the beads back into the flow and moved them into the microflow cytometer for optical interrogation. Identification of the beads and the detection of PE fluorescence were performed simultaneously for multiplexed detection. Multiplexing was performed with specifically targeted bead sets to detect E. coli 0157.H7, Salmonella Common Structural Antigen, Listeria sp., and Shigella sp., dose-response curves were obtained, and limits of detection were calculated for each target in the buffer and clinical matrix. Additional tests demonstrated the potential for using the MagTrap to concentrate target from larger volumes of sample prior to the addition of assay reagents.

Spinning magnetic trap for automated microfluidic assay systems.

While sophisticated analyses have been performed using lab-on-chip devices, in most cases the sample preparation is still performed off chip. The global need for easy-to-use, disposable testing devices necessitates that sample processing is automated and that transport complexity between the processing and analytical components is minimal. We describe a complete sample manipulation unit for performing automated target capture, efficient mixing with reagents, and controlled target release in a microfluidic channel, using an array of spinning magnets. The "MagTrap" device consists of 6 pairs of magnets in a rotating wheel, situated immediately beneath the microchannel. Rotation of the wheel in the direction opposite to the continuous flow entraps and concentrates the bead-target complexes and separates them from the original sample matrix. As the wheel rotates and the active pair of magnets moves away from the microchannel, the beads are released and briefly flow downstream before being trapped and pulled upstream by the next pair of magnets. This dynamic and continuous movement of the beads ensures that the full surface area of each bead is exposed to reagents and prevents aggregation. The release of the target-bead complexes for further analysis is facilitated by reversing the rotational direction of the wheel to sweep the beads downstream. Sample processing with the MagTrap was demonstrated for the detection of E. coli in a range of concentrations (1 × 10(3), 1 × 10(4) and 1 × 10(6) cells ml(-1)). Results show that sample processing with the MagTrap outperformed the standard manual protocols, improving the detection capability while simultaneously reducing the processing time.

UV polymerization of hydrodynamically shaped fibers.

Most natural and man-made fibers have circular cross-sections; thus the properties of materials composed of non-circular fibers are largely unexplored. We demonstrate the technology for fabricating fibers with predetermined cross-sectional shape. Passive hydrodynamic focusing and UV polymerization of a shaped acrylate stream produced metre-long fibers for structural and mechanical characterization.

Dynamic reversibility of hydrodynamic focusing for recycling sheath fluid.

The phenomenon of "unmixing" has been demonstrated in microfluidic mixers, but here we manipulate laminar flow streams back to their original positions in order to extend the operational utility of an analytical device where no mixing is desired. Using grooves in the channel wall, we passively focus a sample stream with two sheath streams to center it in a microchannel for optical analysis. Even though the sample stream is completely surrounded by sheath fluid, reversing the orientation of the grooves in the channel walls returns the sample stream to its original position with respect to the sheath streams. We demonstrate the separation of the sample stream from the contiguous sheath streams and the recycling of the sheath fluid using the reversibility of laminar flow. Polystyrene microspheres and fluorescent dye were used to quantify the performance of the unsheathing process. We found that the maximum numbers of microspheres and all of the fluorescent dye were recaptured at sheath recycling levels <92%. The use of this sheathing technique has previously been demonstrated in a sensitive microflow cytometer; the unsheathing capability now provides the opportunity to recover particles from the sensor with minimal dilution or to recycle the sheath fluid for long-term unattended operation.

Fluorescence-based sensing of 2,4,6-trinitrotoluene (TNT) using a multi-channeled poly(methyl methacrylate) (PMMA) microimmunosensor.

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1-10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT.

A simple sheath-flow microfluidic device for micro/nanomanufacturing: fabrication of hydrodynamically shaped polymer fibers.

A simple sheath flow microfluidic device is used to fabricate polymer micro/nanofibers that have precisely controlled shapes and sizes. Poly(methylmethacrylate) (PMMA) was used as the model polymer for these experiments. The sheath-flow device uses straight diagonal and chevron-shaped grooves integrated in the top and bottom walls of the flow channel to move sheath fluid completely around the polymer stream. Portions of the sheath stream are deflected in such a way as to define the cross-sectional shape of the polymer core. The flow-rate ratio between the sheath and core solution determines the fiber diameter. Round PMMA fibers with a diameter as small as 300 nm and flattened fibers with a submicron thickness are demonstrated.

Multi-wavelength microflow cytometer using groove-generated sheath flow.

A microflow cytometer was developed that ensheathed the sample (core) fluid on all sides and interrogated each particle in the sample stream at four different wavelengths. Sheathing was achieved by first sandwiching the core fluid with the sheath fluid laterally via fluid focusing. Chevron-shaped groove features fabricated in the top and bottom of the channel directed sheath fluid from the sides to the top and bottom of the channel, completely surrounding the sample stream. Optical fibers inserted into guide channels provided excitation light from diode lasers at 532 and 635 nm and collected the emission wavelengths. Two emission collection fibers were connected to PMTs through a multimode fiber splitter and optical filters for detection at 635 nm (scatter), 665 nm and 700 nm (microsphere identification) and 565 nm (phycoerythrin tracer). The cytometer was capable of discriminating microspheres with different amounts of the fluorophores used for coding and detecting the presence of a phycoerythrin antibody complex on the surface of the microspheres. Assays for Escherichia coli were compared with a commercial Luminex flow cytometer.

Two simple and rugged designs for creating microfluidic sheath flow.

A simple design capable of 2-dimensional hydrodynamic focusing is proposed and successfully demonstrated. In the past, most microfluidic sheath flow systems have often only confined the sample solution on the sides, leaving the top and bottom of the sample stream in contact with the floor and ceiling of the channel. While relatively simple to build, these designs increase the risk of adsorption of sample components to the top and bottom of the channel. A few designs have been successful in completely sheathing the sample stream, but these typically require multiple sheath inputs and several alignment steps. In the designs presented here, full sheathing is accomplished using as few as one sheath input, which eliminates the need to carefully balance the flow of two or more sheath inlets. The design is easily manufactured using current microfabrication techniques. Furthermore, the sample and sheath fluid can be subsequently separated for recapture of the sample fluid or re-use of the sheath fluid. Designs were demonstrated in poly(dimethylsiloxane) (PDMS) using soft lithography and poly(methyl methacrylate) (PMMA) using micromilling and laser ablation.

The good, the bad, and the tiny: a review of microflow cytometry.

Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities-such as automated sample staining-are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers.

Fabrication of polymer microfluidic systems by hot embossing and laser ablation.

Fabrication of microfluidic channels in common commercially available thermoplastic materials can be easily accomplished using hot embossing or ultraviolet (UV) laser ablation. Hot embossing involves replication of a microfluidic network in a polymer substrate from a stamp (or template) fabricated in silicon or metal. UV laser ablation is performed by either exposing the polymer substrate through a mask or by using a laser direct-write process. The resulting polymer microfluidic channels are most often sealed with another polymer piece using thermal bonding or solvent bonding to complete the fabrication procedure. Unlike their silicon and glass counterparts, polymer microfluidic systems can be fabricated by these methods in less than 1 h, making the materials attractive for both research prototyping and commercialization.

Toolbox for the design of optimized microfluidic components.

A computational "toolbox" for the a priori design of optimized microfluidic components is presented. These components consist of a microchannel under low-Reynolds number, pressure-driven flow, with an arrangement of grooves cut into the top and bottom to generate a tailored cross-channel flow. An advection map for each feature (i.e., groove of a particular shape and orientation) predicts the lateral transport of fluid within the channel due to that feature. We show that applying these maps in sequence generates an excellent representation of the outflow distribution for complex designs that combine these basic features. The effect of the complex three-dimensional flow field can therefore be predicted without solving the governing flow equations through the composite geometry, and the resulting distribution of fluids in the channel is used to evaluate how well a component performs a specified task. The generation and use of advection maps is described, and the toolbox is applied to determine optimal combinations of features for specified mixer sizes and mixing metrics.

A microfluidic mixer with grooves placed on the top and bottom of the channel.

A new microfluidic mixer is presented consisting of a rectangular channel with grooves placed in the top and bottom. This not only increases the driving force behind the lateral flow, but allows for the formation of advection patterns that cannot be created with structures on the bottom alone. Chevrons, pointing in opposite directions on the top and bottom, are used to create a pair of vortices positioned side by side. Stripes running the width of the channel generate a pair of vertically stacked vortices. Computational fluid dynamics (CFD) simulations are used to model the behavior of the systems and provide velocity maps at cross-sections within the mixer. Experiments demonstrate the mixing that results when two segregated species enter the mixer side-by-side and pass through two cycles of the mixer (i.e., two alternating sets of four stripes and four chevrons).

Design and evaluation of a Dean vortex-based micromixer.

A mixer, based on the Dean vortex, is fabricated and tested in an on-chip format. When fluid is directed around a curve under pressure driven flow, the high velocity streams in the center of the channel experience a greater centripetal force and so are deflected outward. This creates a pair of counter-rotating vortices moving fluid toward the inner wall at the top and bottom of the channel and toward the outer wall in the center. For the geometries studied, the vortices were first seen at Reynolds numbers between 1 and 10 and became stronger as the flow velocity is increased. Vortex formation was monitored in channels with depth/width ratios of 0.5, 1.0, and 2.0. The lowest aspect ratio strongly suppressed vortex formation. Increasing the aspect ratio above 1 appeared to provide improved mixing. This design has the advantages of easy fabrication and low surface area.

Micellar affinity gradient focusing: a new method for electrokinetic focusing.

This report describes a new method for the concentration and separation of neutral and/or hydrophobic analytes based on a combination of the analytes' electrophoretic mobility, and affinity for partitioning into a micellar phase. Micellar affinity gradient focusing (MAGF) works by creating a gradient in the micellar retention factor. An electric field is applied along the channel to cause the (negatively charged) micelles to move from the region of high retention to the region of low retention, and the mobile phase is forced to move from the region of low retention to the region of high retention. Consequently, the analyte moves into the gradient region from both directions where it is concentrated at a point where its total velocity is zero. Different analytes, which interact differently with the micelles, will have zero total velocity at different points along the gradient, and will thereby be simultaneously concentrated and separated.