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PubMedDate not supplied

Microfluidics device for single cell gene expression analysis in Saccharomyces cerevisiae.

We have measured single-cell gene expression over time using a microfluidics-based flow cell which physically traps individual yeast using microm-sized structures (yeast jails). Our goal was to determine variability of gene expression within a cell over time, as well as variability between individual cells. In our flow cell system, yeast jails are fabricated out of PDMS and gene expression is visualized using fluorescently-tagged proteins of interest. Previously, single-cell yeast work has been done using micromanipulation on agar, or FACS. In the present device agar is eliminated, resulting in a superior optical system. The flow of media through the flow cell washes daughter cells away, eliminating the need for micromanipulation. Unlike FACS, the described device can track individual yeast over a time course of many hours. The flow cells are compatible with the needs of quantitative fluorescence microscopy, and allow simultaneous measurements to be done on a large number of individual yeast. We used these flow cells to determine the expression of HSP104-GFPand RAS2-YFP, genes known to affect yeast life span. The results demonstrate inter-cell variation in expression of both genes that could not have been detected without this single-cell analysis.

Bacterial Proteins↗

PubMed2001

Analysis of recombinant adenoviruses using an integrated microfluidic chip-based system.

The use of recombinant virus for gene therapy requires rigorous quality control methods to ensure that the viral vector preparations are functional and safe. A viral identity test is performed in which the viral payload, or transgene, is PCR amplified, followed by digestion with restriction enzymes that yields a characteristic "fingerprint." These DNA fragments are typically analyzed by agarose gel electrophoresis. The ethidium bromide-stained gels are photographed or scanned and the results are sufficient for a qualitative or semiquantitative identity confirmation of the viral product. We have investigated the use of an integrated microfluidic chip-based system as a new tool in the quality control testing of a recombinant, adenoviral, gene therapy product. The chip-based method was found to be very sensitive, requiring 100-fold less sample and only one-third the time compared to the agarose gel method. The automated data analysis sizes and quantitates the DNA fragments, thus yielding a more thorough, reproducible, sensitive, and rapid analysis.

Adenoviruses, Human↗

PubMed2005

Feature characterization of microfabricated microfluidic chips by PDMS replication and CCD imaging.

This paper presents a new approach for the metrological characterization of microfabricated features on microfluidic chips, based on a combination of poly(dimethylsiloxane) (PDMS) replication and charge-coupled device (CCD) imaging. A PDMS replicate was cast from the original chip sample, and a 2-mm thick sample slice was cut from the replica at the cross-section to be studied. The digital image of the revealed structural profile was captured by a CCD camera under a microscope, and the image was processed using specially-developed algorithms for CCD image calibration and edge detection. Depth and width measurements obtained using the method agreed well with those gained using a stylus profiler and universal measuring microscope, with a deviation of below 0.9 mum, while profile distortions of deeper structures using stylus profilers were avoided. The method is reliable, non-destructive, and cheap and simple to implement in any analytical laboratory.

Journal Article↗

PubMed2003

Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes.

Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A(2) on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.

Enzymes↗

PubMed2001

Theoretical analysis of molecular diffusion in pressure-driven laminar flow in microfluidic channels.

The T-sensor is a microfluidic analytical device that operates at low Reynolds numbers to ensure entirely laminar flow. Diffusion of molecules between streams flowing side by side may be observed directly. The pressure-driven velocity profile in the duct-shaped device influences diffusive transport in ways that affect the use of the T-sensor to measure molecular properties. The primary effect is a position-dependent variation in the extent of diffusion that occurs due to the distribution of residence time among different fluid laminae. A more detailed characterization reveals that resultant secondary concentration gradients yield variations in the scaling behavior between diffusive displacement and elapsed time in different regions of the channel. In this study, the time-dependent evolution of analyte distribution has been quantified using a combination of one- and two-dimensional models. The results include an accurate portrayal of the shape of the interdiffusion region in a representative T-sensor assay, calculation of the diffusive scaling law across the width of the channel, and quantification of artifacts that occur when making diffusion coefficient measurements in the T-sensor.

Biophysical Phenomena↗

PubMed2002

Microfluidic technologies in clinical diagnostics.

BACKGROUND: Laboratory instrumentation and analytical devices are becoming smaller, simpler, and smarter. This trend to miniaturization extends to fluid handling and fluid analysis. However, fluid behavior undergoes significant changes as geometric scale decreases. The laminar flow behavior of fluids in microfluidic devices must be accommodated in the design and development of clinical and bio-clinical miniaturized systems. CONCLUSION: The scale of chemical and clinical analysis systems will continue to decrease. The capability to manufacture smaller fluidic devices and to quantitatively monitor smaller volumes of liquids bring this process of miniaturization into the domain of laminar flow. New and enabling technologies are being developed using the unique diffusion-based characteristics of the laminar flow domain for sample preparation and analysis. These new analytical systems will have a significant impact on the future of clinical diagnostics.

Biomechanical Phenomena↗

PubMed2002

Bioanalysis in microfluidic devices.

Microfabricated bioanalytical devices (also referred to as laboratory-on-a-chip or micro-TAS) offer highly efficient platforms for simultaneous analysis of a large number of biologically important molecules, possessing great potential for genome, proteome and metabolome studies. Development and implementation of microfluidic-based bioanalytical tools involves both established and evolving technologies, including microlithography, micromachining, micro-electromechanical systems technology and nanotechnology. This article provides an overview of the latest developments in the key device subject areas and the basic interdisciplinary technologies. Important aspects of DNA and protein analysis, interfacing issues and system integration are all thoroughly discussed, along with applications for this novel "synergized" technology in high-throughput separations of biologically important molecules. This review also gives a better understanding of how to utilize these technologies as well as to provide appropriate technical solutions to problems perceived as being more fundamental.

Chemistry Techniques, Analytical↗

PubMed2002

Capillary electrophoresis-electrochemistry microfluidic system for the determination of organic peroxides.

A microfluidic analytical system for the separation and detection of organic peroxides, based on a microchip capillary electrophoresis device with an integrated amperometric detector, was developed. The new microsystem relies on the reductive detection of both organic acid peroxides and hydroperoxides at -700 mV (vs. Ag wire/AgCl). Factors influencing the separation and detection processes were examined and optimized. The integrated microsystem offers rapid measurements (within 130 s) of these organic-peroxide compounds, down to micromolar levels. A highly stable response for repetitive injections (RSD 0.35-3.12%; n = 12) reflects the negligible electrode passivation. Such a "lab-on-a-chip" device should be attractive for on-site analysis of organic peroxides, as desired for environmental screening and industrial monitoring.

Electrochemistry↗

PubMed2002

Characterizing electroosmotic flow in microfluidic devices.

The current-monitoring method was used to measure the electroosmotic flow (EOF) in borosilicate glass capillaries and zeonor plastic microfluidic devices. The surface of the zeonor devices must be oxidized to support EOF and this treatment shows signs of aging within 6 days. Oxidized zeonor devices showed the same response to changes in applied field, pH, and ionic concentration as the capillaries. The effects of several common dynamic surfactant coatings on the walls were also studied (0.1%, v/v solutions of POP-6, POP4, Pluronics L81, and NP-40). These generally significantly suppressed the EOF but required several days to stabilize.

Buffers↗

PubMed1999

Measurement of electroosmotic flow in plastic imprinted microfluid devices and the effect of protein adsorption on flow rate.

Several commercially available plastic materials were used as substrates in the fabrication of microfluid channels for biochemical analysis. Protocols for fabrication using the wire-imprinting method are reported for polystyrene, polymethylmethacrylate and a copolyester material. Channel sealing was accomplished by low-temperature bonding of a substrate of similar material; therefore, each channel was composed of a single material on all sides. The electroosmotic flow in 25-microm imprinted channels was evaluated for each substrate material. The copolyester material exhibited the highest electroosmotic flow mobility of 4.3 x 10(-4) cm2 V(-1) s(-1) which is similar to that previously reported for fused-silica capillaries. Polystyrene exhibited the lowest electroosmotic flow mobility of 1.8 x 10(-4) cm2 V(-1) s(-1). Plots of linear velocity versus applied electric field strength were linear from 100 V cm(-1) to 500 V cm(-1) indicating that heat dissipation is effective for all substrates in this range. Electroosmotic flow was reevaluated in the plastic channels following incubation in antibody solution to access the non-specific binding characteristics of a common biochemical reagent onto the substrate materials. All materials tested showed a high degree of non-specific adsorption of IgG as indicated by a decrease in the electroosmotic flow mobility in post-incubation testing.

Adsorption↗

PubMed2003

Visualization and numerical modelling of microfluidic on-chip injection processes.

Sample injection processes accomplished using a microfluidic-cross chip are investigated experimentally and numerically. Fluorescent dye is employed to differentiate the sample solution from the pure buffer. Different sample geometries are achieved using different applied electric fields and dyes with different electrophoretic mobilities. Of particular interest here are concentration-dense samples with large axial extent (extending beyond the intersection). The ability to load and subsequently dispense these large axial extent samples is predicted numerically and verified experimentally by direct visualization. Containing more mass, larger samples exhibited lower concentration gradients, making them less sensitive to diffusion and well-suited to transport once dispensed. In the loading process, however, larger samples were found to be more sensitive to pressure effects than more focused samples. This was investigated by imaging sample geometries under various applied fields in the presence of a constant pressure gradient. Laplace pressure originating from differential meniscus curvatures in the reservoirs was found to be the most significant source of such pressure disturbances in these geometries.

Journal Article↗

PubMed2003

A dynamic loading method for controlling on-chip microfluidic sample injection.

A new technique for controlling discrete sample injection in straight-cross microfluidic chips is presented here. This technique involves a three-part process with a dynamic loading step in between the steady-state loading step and the dispensing step. During the intermediate step, sample is pumped into the intersection and into the three connecting channels. The key features of this technique are the ability to dynamically control the sample size and the ability to inject well-defined samples at the original sample concentration. Injections of these samples with lengths varying from 2 channel widths (100 microm) to 20 channel widths (millimeter-sized) are demonstrated. The sample concentration profiles obtained are compared with those of focused and less-focused pinched-valve injections. In applications such as high-speed capillary zone electrophoresis, this technique can provide an increase in signal with a small increase in sample length. This technique is especially applicable to many large-sample applications in which the offset twin-T microchip has been previously employed.

Journal Article↗

PubMed2001

Electroosmotic properties of microfluidic channels composed of poly(dimethylsiloxane).

Microfluidic devices fabricated from polymers exhibit great potential in biological analyses. Poly(dimethylsiloxane) (PDMS) has shown promise as a substrate for rapid prototyping of devices. Despite this, disagreement exists in the literature as to the ability of PDMS to support electroosmotic (EO) flow and the stability of that flow over time. We demonstrate that in low ionic strength solutions near neutral in pH. oxidized PDMS had a four-fold greater EO mobility (mu(eo)) compared to native PDMS. The greater mu(eo) was maintained irrespective of whether glass or PDMS was used as a support forming one side of the channel. This enhanced mu(eo) was preserved as long as the channels were filled with an aqueous solution. Upon exposure of the channels to air, the mobility decreased by a factor of two with a half-life of 9 h. The EO properties of the air-exposed, oxidized PDMS were regenerated by exposure to strong base. High ionic strength, neutral in pH buffers compatible with living eukaryotic cells diminished the EO flow in the oxidized PDMS devices to a much greater extent than in the native PDMS devices. For analyses utilizing intact and living cells, oxidation of PDMS may not be an effective strategy to substantially increase the mu(eo).

Dimethylpolysiloxanes↗

PubMed2002

PDMS device for patterned application of microfluids to neuronal cells arranged by microcontact printing.

A microfluidic device in polydimethylsiloxane (PDMS) consisting of an eight lines micro-injection array integrated in a base flow channel has been realized. The device is assembled from multiple PDMS parts, which have been moulded using notably micromachined masters in SU-8 photoresist. In contact with a planar substrate, up to eight independent laminar flow lines with cross-sections of 100 x 200 microm(2) can be generated. Dedicated for the application of pharmaceutical compounds to electrogenic cells in vitro, this device was tested with a neuronal cell line, Mz1-cells. These were cultured on lines of laminin deposited onto polystyrene substrates by microcontact printing. We were able to inject into this culture multiple lines of coloured PBS in parallel to the orientation of cellular growth. No mixing between the individual flow lines did occur.

Animals↗

PubMed2003

Selective detection of L-glutamate using a microfluidic device integrated with an enzyme-modified pre-reactor and an electrochemical detector.

A microfluidic device integrated with a nanoliter volume enzyme pre-reactor and an enzyme-modified electrode was developed for the highly selective continuous measurement of glutamate (Glu). The device consists mainly of two glass plates. One plate incorporates an electrochemical cell that consists of working electrode (WE), reference electrode (RE) and counter electrode (CE). The WE is modified with a bilayer film of Os-polyvinylpyrridine-based mediator containing horseradish peroxidase (Os-gel-HRP). The WE was operated at -50 mV versus Ag. The other plate has a thin layer flow channel integrated with a pre-reactor. The reactor has a number of micropillars (20 microm in diameter, 20 microm high and separated from each other by a 20 microm gap) modified with ascorbate oxidase (AAOx) to eliminate L-ascorbic acid (AA). The enzymatic oxidation of AA is superior to that obtained with our previously reported pre-electrolysis type micro-reactor since electrochemically reversible transmitters such as catecholamines do not provide a cathodic current at the WE. In addition, the high operation potential of the pre-reactor causes unknown electroactive species, which also cause interference at the detection electrode. As a result, we were able to detect 1 microM Glu continuously at a low flow rate even when AA concentration was 100 microM.

Ascorbic Acid↗

PubMed2001

Microfluidic components for protein characterization.

The use of microfluidic components to create an analytical toolbox for the very rapidly growing field of proteomics is described. This toolbox provides novel generic analytical solutions that are highly adaptable for analysis of various biomolecules, ranging from high to low abundant. The components are fabricated using silicon micromachining and consist of a microchip immobilised enzyme reactor (microIMER), a piezoelectric microdispenser and high-density nanovial target plates. This microtechnology based platform interfaces matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF-MS) to a wide range of upstream sample handling and/or analytical techniques. Examples of applications such as rapid on-line digestion (12 s) and sample preparation of proteins, interfacing to capillary liquid chromatography (100 attomol sensitivity), and in-vial chemistry on femtomol amounts of sample are presented.

Bioreactors↗

PubMed2000

Plastic microfluidic devices modified with polyelectrolyte multilayers

Control of the polymer surface chemistry is a crucial aspect of development of plastic microfluidic devices. When commercially available plastic substrates are used to fabricate microchannels, differences in the EOF mobility from plastic to plastic can be very high. Therefore, we have used polyelectrolyte multilayers (PEMs) to alter the surface of microchannels fabricated in plastics. Optimal modification of the microchannel surfaces was obtained by coating the channels with alternating layers of poly(allylamine hydrochloride) and poly(styrene sulfonate). Polystyrene (PS) and poly(ethylene terephthalate) glycol (PETG) were chosen as substrate materials because of the significant differences in the polymer chemistries and in the EOF of channels fabricated in these two plastic materials. The efficacy of the surface modification has been evaluated using XPS and by measuring the EOF mobility. When microchannels prepared in both PS and PETG are modified with PEMs, they demonstrate very similar electroosmotic mobilities. The PEMs are easily fabricated and provide a means for controlling the flow direction and the electroosmotic mobility in the channels. The PEM-coated microchannels have excellent wettability, allowing facile filling of the channels. In addition, the PEMs produce reproducible results and are robust enough to withstand long-term storage.

Journal Article↗

PubMed2001

A picoliter-volume mixer for microfluidic analytical systems.

Mixing confluent liquid streams is an important, but difficult operation in microfluidic systems. This paper reports the construction and characterization of a 100-pL mixer for liquids transported by electroosmotic flow. Mixing was achieved in a microfabricated device with multiple intersecting channels of varying lengths and a bimodal width distribution. All channels running parallel to the direction of flow were 5 microm in width whereas larger 27-microm-width channels ran back and forth through the parallel channel network at a 45 degrees angle. The channel network composing the mixer was approximately 10 microm deep. It was observed that little mixing of the confluent solvent streams occurred in the 100-microm-wide, 300-microm-long mixer inlet channel where mixing would be achieved almost exclusively by diffusion. In contrast, after passage through the channel network in the approximately 200-microm-length static mixer bed, mixing was complete as determined by confocal microscopy and CCD detection. Theoretical simulations were also performed in an attempt to describe the extent of mixing in microfabricated systems.

Algorithms↗