Microfluidics for ultrasmall-volume biological analysis.
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Biochemistry↗
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Results for “Microfluidics”
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Homeland security and bioterrorism applications: detection of bioweapon pathogens by microfluidic-based electrophoretic DNA analysis.
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Bacteria↗
Precise patterning in microfluidic devices.
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Comment↗
[Relation between the axonal membrane microfluidity and excitability].
The olfactory nerve of the garfish, the rabbit vagus nerve and the sciatic nerve of the frog labelled with pyrene or triethylammonium-butyl-pyrene show during the action potential a transient decrease in the Ie/Im ratio which suggest a small transient decrease in nerve membrane fluidity.
Animals↗
Computer-controlled microcirculatory support system for endothelial cell culture and shearing.
Endothelial cells (ECs) lining the inner lumen of blood vessels are continuously subjected to hemodynamic shear stress, which is known to modify EC morphology and biological activity. This paper describes a self-contained microcirculatory EC culture system that efficiently studies such effects of shear stress on EC alignment and elongation in vitro. The culture system is composed of elastomeric microfluidic cell shearing chambers interfaced with computer-controlled movement of piezoelectric pins on a refreshable Braille display. The flow rate is varied by design of channels that allow for movement of different volumes of fluid per variable-speed pump stroke. The integrated microfluidic valving and pumping system allowed primary EC seeding and differential shearing in multiple compartments to be performed on a single chip. The microfluidic flows caused ECs to align and elongate significantly in the direction of flow according to their exposed levels of shear stress. This microfluidic system overcomes the small flow rates and the inefficiencies of previously described microfluidic and macroscopic systems respectively to conveniently perform parallel studies of EC response to shear stress.
Cell Culture Techniques↗
Numerical simulation of polymerization in interdigital multilamination micromixers.
Free radical polymerization in microfluidic devices modeled with the help of numerical simulations is discussed. The simulation method used allows the simultaneous solvation of partial differential equations resulting from the hydrodynamics, thermal and mass transfer (convection, diffusion and chemical reaction). Three microfluidic devices are modeled, two interdigital multilamination micromixers respectively with a large and short focusing section, and a simple T-junction followed by a microtube reactor together considered as a bilamination micromixer with a large focusing section. The simulations show that in spite of the heat released by the polymerization reaction, the thermal transfer in such microfluidic devices is high enough to ensure isothermal conditions. Moreover, for low radial Peclet number, microfluidic devices with a large focusing section can achieve better control over the polymerization than a laboratory scale reactor as the polydispersity index obtained is very close to the theoretical limiting value. As the characteristic dimension of the microfluidic device increases, i.e. for high radial Peclet number, the reactive medium cannot be fully homogenized by the diffusion transport before leaving the system resulting in a high polydispersity index and a loss in the control of the polymerization.
Biological Transport↗
Microautosamplers for discrete sample injection and dispensation.
Microfluidic systems show considerable potential for use in the continuous reaction and analysis of biosamples for various applications, such as drug screening and chemical synthesis. Typically, microfluidic chips are externally connected with large-scale autosamplers to inject specific volumes of discrete samples in the continuous monitoring and analysis of multiple samples. This paper presents a novel microelectromechanical system (MEMS)-based autosampler capable of performing the discrete injection and dispensation of variable-volume samples. This microdevice can be integrated with other microfluidic devices to facilitate the continuous monitoring and analysis of multiple biosamples. By means of electroosmotic focusing and switching controlled by the direct application of electric sources on specific fluid reservoirs, a precise sample volume can be injected into the specified outlet port. Fluorescence dye images verify the performance of the developed device. An injection-and-washing scheme is developed to prevent cross-contamination during the continuous injection of different samples. This approach renders feasible the injection of several discrete samples using a single microchip. Compared to its large-scale counterparts, the developed microautosampler is compact in size, has low fabrication costs, is straightforward to control, and most importantly, is readily integrated with other microfluidic devices (e.g., microcapillary electrophoresis chips) to form a microfluidic system capable of the continuous monitoring and analysis of bioreactions. The proposed microautosampler could be promising towards realizing the micrototal analysis system (mu-TAS) concept.
Electrophoresis, Microchip↗
Lab-on-a-chip sample preparation using laminar fluid diffusion interfaces--computational fluid dynamics model results and fluidic verification experiments.
Microfluidic structures for the generation of laminar fluid diffusion interfaces (LFDIs) for sample preparation and analysis are discussed. Experimental data and the results of fluid modeling are shown. LFDIs are generated when two or more streams flow in parallel in a single microfluidic structure without any mixing of the fluids other than by diffusion of particles across the diffusion interface. It has been shown that such structures can be used for diffusion-based separation and detection applications. The method has been applied to DNA desalting, the extraction of small proteins from whole blood samples, and the detection of various constituents in whole blood, among other examples. In this paper the design and manufacture of self-contained microfluidic cartridges for the extraction of small molecules from a mixture of small and large molecules by diffusion is demonstrated. The cards are operated without any external instrumentation, and use hydrostatic pressure as the driving force. The performance of the cartridges is illustrated by separating fluorescein from a mixture of fluorescein and dextran of molecular weight 2 x 10(6). In a single pass, 98.6% of dextran was retained in the product whereas 43.1% of fluorescein was removed. The method is adjustable for different separation requirements, and computational fluid dynamics (CFD) models are shown that demonstrate the tuning of various microfluidic parameters to optimize separation performance. Other applications of LFDIs for establishment of stable concentration gradients, and the exposure of chemical constituents or biological particles to these concentration gradients are shown qualitatively. Microfluidic chips have been designed for high-throughput screening applications that enable the uniform and controlled exposure of cells to lysing agents, thus enabling the differentiation of cells by their sensitivity to specific agents in an on-chip cytometer coupled directly to the lysing structure.
Chemistry Techniques, Analytical↗
Micrometer-sized supported lipid bilayer arrays for bacterial toxin binding studies through total internal reflection fluorescence microscopy.
In this article, we present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy. The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated distearoylphosphatidylcholine:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside G(T1b) or G(M1). The ganglioside-populated SLB arrays were then exposed to either cholera toxin B subunit or tetanus toxin C fragment. Binding was assayed on planar substrates by total internal reflection fluorescence microscopy down to 100 pM concentration for cholera toxin subunit B and 10 nM for tetanus toxin fragment C. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is influenced by the microenvironment of the SLB and the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions.
Bacterial Toxins↗
Electroosmotic guiding of sample flows in a laminar flow chamber.
The so-called address-flow principle is described: a valveless, electroosmotically driven technology used for controlling the stream profile in a laminar flow chamber. The method is explained, and a theoretical description and experimental verification are presented. Adjustment of the flow of two electroosmotically controlled guiding streams, running parallel to a central sample stream, can be used for positioning the sample stream in the dimension perpendicular to the flow direction. The results presented show that address-flow microfluidics allow easy and accurate control of sample stream position and width. The electroosmotic flow (EOF)-controlled guiding of microfluidic flows described in this paper, is a new unit operation that might aid in separation and collection in microfluidic devices. One possible application of address-flow microfluidics is guiding of capillary electrophoresis-separated components over a multisensor array, in order to perform affinity assays.
Animals↗
High-efficiency electrokinetic micromixing through symmetric sequential injection and expansion.
Rapid electric field switching is an established microfluidic mixing strategy for electrokinetic flows. Many such microfluidic mixers are variations on the T- or Y-form channel geometry. In these configurations, rapid switching of the electric field can greatly improve initial mixing over that achieved with static-field mixing. Due to a fundamental lack of symmetry, however, these strategies produce lingering cross-channel concentration gradients which delay complete mixing of the fluid stream. In this paper, a field switching microfluidic mixing strategy which utilizes a symmetric sequential injection geometry with an expansion chamber to achieve high efficiency microfluidic mixing is demonstrated experimentally. A three-inlet injector sequentially interlaces two dissimilar incoming solutions. Downstream of the injector, the sequence enters an expansion chamber resulting in a dramatic (two orders of magnitude) decrease in Peclet number and rapid axial diffusive mixing. The outlet concentration may be accurately varied over the full spectrum by tuning the duty cycle of the field switching waveform. The chips are designed with input from a previous numerical study, manufactured in poly(dimethylsiloxane) using soft-lithography based microfabrication, and tested using fluorescence microscopy. In the context of on-chip chemical processing for analytical operations, the demonstrated mixing strategy has several features: high mixing efficiency (99%), compact axial length (2.3 mm), steady outflow velocity, and readily variable outlet concentration (0.15 < c* < 0.95).
Electromagnetic Fields↗
Measuring single-cell susceptibility to antibiotics within monoclonal bacterial populations.
The emergence of new resistant bacterial strains is a worldwide challenge. A resistant bacterial population can emerge from a single cell that acquires resistance or persistence. Hence, new ways of tackling the mechanism of antibiotic response, such as single cell studies are required. It is necessary to see what happens at the single cell level, in order to understand what happens at the population level. To date, linking the heterogeneity of single-cell susceptibility to the population-scale response to antibiotics remains challenging due to the trade-offs between the resolution and the field of view. Here we present a platform that measures the ability of individual E. coli cells to form small colonies at different ciprofloxacin concentrations, by using anchored microfluidic drops and an image and data analysis pipelines. The microfluidic results are benchmarked against classical microbiology measurements of antibiotic susceptibility, showing an agreement between the pooled microfluidic chip and replated bulk measurements. Further, the experimental likelihood of a single cell to form a colony is used to provide a probabilistic antibiotic susceptibility curve. In addition to the probabilistic viewpoint, the microfluidic format enables the characterization of morphological features over time for a large number of individual cells. This pipeline can be used to compare the response of different bacterial strains to antibiotics with different action mechanisms.
Anti-Bacterial Agents↗
Microfluidics/CMOS orthogonal capabilities for cell biology.
The study of individual cells and cellular networks can greatly benefit from the capabilities of microfabricated devices for the stimulation and the recording of electrical cellular events. In this contribution, we describe the development of a device, which combines capabilities for both electrical and pharmacological cell stimulation, and the subsequent recording of electrical cellular activity. The device combines the unique advantages of integrated circuitry (CMOS technology) for signal processing and microfluidics for drug delivery. Both techniques are ideally suited to study electrogenic mammalian cells, because feature sizes are of the same order as the cell diameter, approximately 50 microm. Despite these attractive features, we observe a size mismatch between microfluidic devices, with bulky fluidic connections to the outside world, and highly miniaturized CMOS chips. To overcome this problem, we developed a microfluidic flow cell that accommodates a small CMOS chip. We simulated the performances of a flow cell based on a 3-D microfluidic system, and then fabricated the device to experimentally verify the nutrient delivery and localized drug delivery performance. The flow-cell has a constant nutrient flow, and six drug inlets that can individually deliver a drug to the cells. The experimental analysis of the nutrient and drug flow mass transfer properties in the flowcell are in good agreement with our simulations. For an experimental proof-of-principle, we successfully delivered, in a spatially resolved manner, a 'drug' to a culture of HL-1 cardiac myocytes.
Cell Culture Techniques↗
Manufacturing monodisperse chitosan microparticles containing ampicillin using a microchannel chip.
The purpose of this study was using a developed microfluidic chip to prepare size-controlled monodisperse chitosan microparticles encapsulating ampicillin. Our strategy is that a chitosan aqueous solution (the disperse phase) is fed into the microfluidic chip equipped with a cross-junction microchannel, and is sheared by the viscous oil flows (the continuous phase) to form monodisperse semi-product, chitosan emulsions. These fine emulsions are then gelled into stability upon gelation by injection of copper sulfate solution at the terminal microchannel of the microfluidic chip, and finally the uniform chitosan microparticles are formed in an efficient manner. The proposed chip is fabricated by a CO(2) laser machine on a conventional poly methyl methacrylate (PMMA) substrate. This microfluidic chip has four inlet ports, one cross-channel and one outlet port. We have demonstrated that one can control the size of chitosan microparticles from 100 to 800 microm in diameter (with a variation less than 5%) by altering the relative sheath/sample flow rate ratio. Experimental data showed that when given a steady continuous phase (oil flow), the emulsion size increases with the increase in average velocity of the dispersed phase flow (sample flow). In addition, the release of the model drug (ampicillin) from these microspheres is proved to be once-daily for clinical application. We also revealed that appropriate particle sizes for different release patterns are predictable, enabling better applications of chitosan as a drug carrier.
Ampicillin↗
Fully integrated miniature device for automated gene expression DNA microarray processing.
A DNA microarray with 12,000 features was integrated with a microfluidic cartridge to automate the fluidic handling steps required to carry out a gene expression study of the human leukemia cell line (K562). The fully integrated microfluidic device consists of microfluidic pumps/mixers, fluid channels, reagent chambers, and a DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated into the cartridge to provide pumping of liquid solutions. The device was completely self-contained: no external pressure sources, fluid storage, mechanical pumps, mixers, or valves were necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Fluidic experiments were performed to study the on-chip washing efficiency and uniformity. A single-color transcriptional analysis of K562 cells with a series of calibration controls (spiked-in controls) to characterize this new platform with regard to sensitivity, specificity, and dynamic range was performed. The device detected sample RNAs with a concentration as low as 0.375 pM. Experiment also showed that the performance of the integrated microfluidic device is comparable with the conventional hybridization chambers with manual operations, indicating that the on-chip fluidic handling (washing and reaction) is highly efficient and can be automated with no loss of performance. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps in genomic analysis.
Bacteriophage lambda↗
Early mammalian embryo development depends on cumulus removal technique.
Cumulus removal (CR) at the zygote stage is necessary for most mammalian in vitro production (IVP). Present techniques use high fluidic stresses (vortexing) or mechanical stress with enzymatic treatment (pipetting) to remove cumulus. Herein a recently developed microfluidic device for cumulus removal from zygotes is compared with traditional vortexing. Microfluidic CR (microFCR) increased development on day 2 (20 +/- 4% to 35 +/- 6%, p < 0.01) and blastocyst formation at day 8 (33 +/- 1% to 57 +/- 5%, p < 0.01) when compared to vortex CR. Vortexing effects on embryo development were studied; 15, 30 and 120 s vortex doses. Development at day 2 was inversely proportional to duration of vortexing. An in situ transcription assay was used to assess biochemical activity of zygotes after cumulus removal. There was a spike of RNA transcription of vortexed zygotes at 2 h post CR not seen in the microfluidic treatment. These results suggest the potential for microfluidic methods to enhance production efficiencies while providing insight into basic developmental mechanisms.
Animals↗
Performing microchannel temperature cycling reactions using reciprocating reagent shuttling along a radial temperature gradient.
This study develops a novel temperature cycling strategy for executing temperature cycling reactions in laser-etched poly(methylmethacrylate) (PMMA) microfluidic chips. The developed microfluidic chip is circular in shape and is clamped in contact with a circular ITO heater chip of an equivalent diameter. Both chips are fabricated using an economic and versatile laser scribing process. Using this arrangement, a self-sustained radial temperature gradient is generated within the microfluidic chip without the need to thermally isolate the different temperature zones. This study demonstrates the temperature cycling capabilities of the reported microfluidic device by a polymerase chain reaction (PCR) process using ribulose 1,5-bisphosphate carboxylase large subunit (rbcL) gene as a template. The temperature ramping rate of the sample inside the microchannel is determined from the spectral change of a thermochromic liquid crystal (TLC) solution pumped into the channel. The present results confirm that a rapid thermal cycling effect is achieved despite the low thermal conductivity of the PMMA substrate. Using IR thermometry, it is found that the radial temperature gradient of the chip is approximately 2 degrees C mm(-1). The simple system presented in this study has considerable potential for miniaturizing complex integrated reactions requiring different cycling parameters.
Equipment Design↗
Behaviour and design considerations for continuous flow closed-open-closed liquid microchannels.
This paper introduces a method of combining open and closed microchannels in a single component in a novel way which couples the benefits of both open and closed microfluidic systems and introduces interesting on-chip microfluidic behaviour. Fluid behaviour in such a component, based on continuous pressure driven flow and surface tension, is discussed in terms of cross sectional flow behaviour, robustness, flow-pressure performance, and its application to microfluidic interfacing. The closed-open-closed microchannel possesses the versatility of upstream and downstream closed microfluidics along with open fluidic direct access. The device has the advantage of eliminating gas bubbles present upstream when these enter the open channel section. The unique behaviour of this device opens the door to applications including direct liquid sample interfacing without the need for additional and bulky sample tubing.
Equipment Design↗