Peter B. Howell

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.

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.

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.

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.

A combinatorial approach to microfluidic mixing

A new computational approach to the modeling and design of efficient microfluidic mixers is demonstrated. The mixers created provide far more rapid mixing than previous designs. A set of mixer components is created and mapped using a traditional Navier–Stokes fluid solver. The maps are used to quickly model the effect each component has on the lateral distribution of species in the channel. For a mixer of a given length, all the possible combinations of components can be evaluated, and the best mixer for a given metric can be found. Although the mixers presented in this study are short (length-to-width ratios below 8), they show degrees of mixing comparable to much longer mixers found in the literature.

Automated processing integrated with a microflow cytometer for pathogen detection in clinical matrices.

A spinning magnetic trap (MagTrap) for automated sample processing was integrated with a microflow cytometer capable of simultaneously detecting multiple targets to provide an automated sample-to-answer diagnosis in 40 min. After target capture on fluorescently coded magnetic microspheres, the magnetic trap automatically concentrated the fluorescently coded microspheres, separated the captured target from the sample matrix, and exposed the bound target sequentially to biotinylated tracer molecules and streptavidin-labeled phycoerythrin. The concentrated microspheres were then hydrodynamically focused in a microflow cytometer capable of 4-color analysis (two wavelengths for microsphere identification, one for light scatter to discriminate single microspheres and one for phycoerythrin bound to the target). A three-fold decrease in sample preparation time and an improved detection limit, independent of target preconcentration, was demonstrated for detection of Escherichia coli 0157:H7 using the MagTrap as compared to manual processing. Simultaneous analysis of positive and negative controls, along with the assay reagents specific for the target, was used to obtain dose-response curves, demonstrating the potential for quantification of pathogen load in buffer and serum.

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.

Hydrodynamically directed multiscale assembly of shaped polymer fibers

A long-sought goal of material science is the development of fabrication processes by which synthetic materials can be made to mimic the multiscale organization many natural materials utilize to achieve unique functional and material properties. Here we demonstrate how the microfluidic fabrication of polymer fibers can take advantage of hydrodynamic forces to simultaneously direct assembly at the molecular and micron scales. The microfluidic device generates long fibers by initiating polymerization of a continuously flowing fluid via UV irradiation within the microfluidic channel. Prior to polymerization, hydrodynamic shear forces direct molecular scale assembly and a combination of hydrodynamic focusing and advection driven by grooves in the channel walls manipulate the cross-sectional shape of the pre-polymer stream. Polymerization subsequently locks in both molecular scale alignment and micron-scale fiber shape. This simple method for generating structures with multiscale organization could be useful for fabricating materials with multifunctionality or enhanced mechanical properties.

Designing Microfluidic Components for Enhanced Surface Delivery Using a Genetic Algorithm Search

*† ‡ § The Toolbox approach to the automated design of microfluidic components is extended to include a genetic algorithm search of candidate designs. Performance metrics for characterizing surface delivery are described, and the software is applied to choose sequences of grooves to add to a rectangular microchannel in order to optimize surface delivery for pressure-driven flow. Initial searches using five groove shapes identify designs that perform much better than standard mixers found in the literature. These initial searches produced two sets of competing designs, and each set was dominated by a different subset of the allowable groove shapes. Additional targeted searches that limited the groove choices to each of these two subsets produced additional significant improvements in the designs.