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At least 523 records · Page 29Linked to original sources

PubMed2002

Plastic biochannel hybridization devices: a new concept for microfluidic DNA arrays.

Conventional DNA hybridization assay kinetics depends solely on the diffusion of target to surface-bound probes, causing long hybridization times. In this study, we examined the possibilities of accelerating the hybridization process by using microfluidic channels ("biochannels") made of polycarbonate, optionally with an integrated pump. We produced two different devices to study these effects: first, hybridization kinetics was investigated by using an eSensor electrochemical DNA detection platform allowing kinetic measurements in homogenous solution. We fabricated an integrated cartridge for the chip comprising the channel network and a micropump for the oscillation of the hybridization mixture to further overcome diffusion limitations. As a model assay, we used an assay for the detection of single-nucleotide polymorphisms in the HFE-H gene. Second, based on the biochannel approach, we constructed a plastic microfluidic chip with a network of channels for optical detection of fluorescent-labeled targets. An assay for the simultaneous detection of four pathogenic bacteria surrogate strains from multiple samples was developed for this device. We observed high initial hybridization velocities and a fast attainment of equilibrium for the biochannel with integrated pump. Experimental results were compared with predictions generated by computer simulations.

Computer Simulation↗

PubMed2002

Sample pre-concentration by isotachophoresis in microfluidic devices.

We have designed microfluidic devices with the aim of coupling isotachophoresis (ITP) with zone electrophoresis (ZE) as a method to increase the concentration limit of detection in microfluidic devices. We used plastic multi-channel chips, designed with long sample injection channel segments, to increase the sample loading. The chip was designed to allow stacking of the sample into a narrow band by discontinuous ITP buffers and subsequent separation in the ZE mode. In the ITP-ZE mode, with a 2-cm long sample injection plug, sensitivity was increased by 400-fold over chip ZE and we found that the separation performance after the ITP stacking was comparable to that of regular chip ZE. We report sub-picomolar limits of detection of fluorescently labeled ACLARA eTag reporter molecules electrokinetically injected from cell lysate sample matrixes containing moderate salt concentrations. We evaluated sample injections from buffers with varied ionic strengths and found that efficient stacking and separations were obtained in both low and high conductivity buffers, including physiological buffer with at least 140 mM salt. We applied ITP-ZE to the analysis of a cell surface protease (ADAM 17) which used live intact cells in physiological buffers with detection limits below 10 cells/assay.

Buffers↗

PubMed2003

Lattice Boltzmann model of microfluidics in the presence of external forces.

We present here a lattice Boltzmann model in the presence of external force fields to describe electrokinetic microfluidic phenomena and consider pressure as the only external force to drive liquid flow. Our results from a 9-bit square lattice Boltzmann model are in good agreement with recent experimental data in a pressure-driven microchannel flow that could not be fully described by electrokinetic theory. The differences between the predicted and the experimental Reynolds numbers from pressure gradients are well within 5%. Results suggest that the lattice Boltzmann model described here is an effective computational tool for predicting the more complex microfluidic systems that might be problematic using conventional methods.

Journal Article↗

PubMed2003

Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis.

Modern DNA sequencing 'factories' have revolutionized biology by completing the human genome sequence, but in the race to completion we are left with inefficient, cumbersome, and costly macroscale processes and supporting facilities. During the same period, microfabricated DNA sequencing, sample processing and analysis devices have advanced rapidly toward the goal of a 'sequencing lab-on-a-chip'. Integrated microfluidic processing dramatically reduces analysis time and reagent consumption, and eliminates costly and unreliable macroscale robotics and laboratory apparatus. A microfabricated device for high-throughput DNA sequencing that couples clone isolation, template amplification, Sanger extension, purification, and electrophoretic analysis in a single microfluidic circuit is now attainable.

Base Sequence↗

PubMedDate not supplied

Isolation of motile spermatozoa from semen samples using microfluidics.

A microfluidic device was designed with two parallel laminar flow channels where non-motile spermatozoa and debris would flow along their initial streamlines and exit one outlet, whereas motile spermatozoa had an opportunity to swim into a parallel stream and exit a separate outlet. Motile sperm samples were prepared with density gradient separation (n = 5). Sperm motility was assessed the following day after exposing aliquots to polydimethylsiloxane (PDMS) used to construct the device. There was no difference in sperm motility when compared with unexposed aliquots (P > 0.05). Unprocessed semen samples (n = 10) were placed in wider channels and sperm motility and strict morphology were assessed from sorted outlets. Sperm motility increased from 44 +/- 4.5% to 98 +/- 0.4% (P < 0.05) and morphology increased from 10 +/- 1.05% to 22 +/- 3.3% (P < 0.05) following processing. Finally, density gradient prepared samples (n = 6) containing 5 x 10(6) motile spermatozoa/ml and 50 x 10(6) round immature germ cells/ml were sorted and assessed in a similar fashion. The ratio of motile spermatozoa to round immature germ cells in the wide inlet (1:10) was significantly improved in the thin outlet (33:1) (P < 0.05). This microfluidic device provides a novel method for isolating motile, morphologically normal spermatozoa from semen samples without centrifugation. This technology may prove useful in isolating motile spermatozoa from oligozoospermic samples, even with high amounts of non-motile gamete and/or non-gamete cell contamination. A movie sequence showing streaming and sorting of spermatozoa may be purchased for viewing on the internet at www.rbmonline.com/Article/847 (free to web subscribers).

Cell Separation↗

PubMed2001

Velocity measurement of particles flowing in a microfluidic chip using Shah convolution Fourier transform detection.

A noninvasive radiative technique, based on Shah convolution Fourier transform detection, for velocity measurement of particles in fluid flows in a microfluidic chip, is presented. It boasts a simpler instrumental setup and optical alignment than existing measurement methods and a wide dynamic range of velocities measurable. A glass-PDMS microchip with a layer of patterned Cr to provide multiple detection windows which are 40 microns wide and 70 microns apart is employed. The velocities of fluorescent microspheres, which were electrokinetically driven in the channel of the microfluidic chip, were determined. The effects of increasing the number of detection windows and sampling period were investigated. This technique could have wide applications, ranging from the determination of the velocity of particles in pressure-driven flow to the measurement of electrophoretic mobilities of single biological cells.

Algorithms↗

PubMed2001

A polymeric microfluidic chip for CE/MS determination of small molecules.

A polymeric microfluidic chip made of Zeonor 1020 was fabricated using conventional embossing techniques to perform capillary electrophoresis for selected ion monitoring and selected reaction monitoring mass spectrometric detection of small molecules. A silicon master was microfabricated using photolithographic and dry etching processes. The microfluidic channel was embossed in the plastic from a silicon master. The embossed chip was thermally bonded with a Zeonor 1020 cover to form an enclosed channel. This channel (60-microm width, 20-microm depth, 2.0- and 3.5-cm length) provided capillary electrophoresis (CE) separation of polar small molecules without surface treatment of the polymer. A microsprayer coupled via a microliquid junction provided direct electrospray mass spectrometric detection of CE-separated components. An electric field of 0.5-2 kV/cm applied between the microsprayer and a separation buffer reservoir produced a separation of carnitine, acylcarnitine, and butylcarnitine with separation efficiencies ranging from 1,650 to 18,000 plates. Injection quantities of 0.2 nmol of these compounds produced a separation of the targeted polar small molecules without surface treatment of the polymer-abundant ion current signals and baseline separation of these compounds in less than 10 s. These results suggest the feasibility of polymeric chip-based devices for ion spray CE/MS applications.

Acetylcarnitine↗

PubMed2001

Electrokinetically driven microfluidic chips with surface-modified chambers for heterogeneous immunoassays.

This article presents the first example of a microfluidic chip for heterogeneous bioassays using a locally immobilized biospecific layer and operated electrokinetically. The reaction chamber has picoliter dimensions and is integrated into a network of microchannels etched in glass. The high affinity of protein A (PA) for rabbit immunoglobulin G (rIgG) was exploited for chip testing, with PA being immobilized on microchannel walls and fluorescently labeled (Cy5) rIgG serving as sample. It was possible to operate the chip in an immunoaffinity chromatographic manner, using electrokinetically pumped solutions. Concentration of antibody from dilute solution onto the solid phase was demonstrated, with signal gains of approximately 30 possible. A dose-response curve for Cy5-rIgG was obtained for concentrations down to 50 nM, for an incubation time of 200 s. The flexibility of chip layout was demonstrated for competitive immunoassay of rIgG, using both a combined sample/tracer incubation and sequential addition of these solutions. With assay times generally below 5 min for this unoptimized device, the microfluidic approach described shows great potential for many high-throughput screening applications.

Carbocyanines↗

PubMed2001

Adaptation of a surface plasmon resonance biosensor with microfluidics for use with small sample volumes and long contact times.

The efficient delivery of sample to surface-immobilized sites is a key element in biosensing. For a surface plasmon resonance (SPR) biosensor, this has been addressed by constant flow through a microfluidic system with a sample injection loop (Sjölander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345). The present study describes an alternative mode of sample delivery without constant unidirectional flow. It was implemented on a commercial Biacore X SPR biosensor equipped with a microfluidic cartridge, but with the fluidic handling performed by an externally computer-controlled syringe pump. We demonstrate that sample volumes as low as 2 microL can be reproducibly positioned to cover the sensor surfaces, manipulated in a serial fashion, efficiently mixed by applying an oscillatory flow pattern, and fully recovered. Compared to the traditional continuous unidirectional flow configuration, we found very similar kinetic responses at high analyte concentrations and slightly slower responses at low concentrations, most likely due to depletion of analyte from the small sample volumes due to surface binding. With the antibody-antigen systems tested, binding parameters were obtained that are generally within 10% of those from conventional experiments. In the new configuration, biosensor experiments can be conducted without the usual constraints in the surface contact time that are correlated with sample volume and mass transport rate. This can translate to improved detection limits for slow reactions and can facilitate kinetic and thermodynamic binding studies.

Biosensing Techniques↗

PubMed2001

Development of a fully integrated analysis system for ions based on ion-selective optodes and centrifugal microfluidics.

A fully integrated, miniaturized analysis system for ions based on a centrifugal microfluidics platform and ion-selective optode membranes is described. The microfluidic architecture is composed of channels, five solution reservoirs, a measuring chamber, and a waste reservoir manufactured onto a disk-shaped substrate of poly(methyl methacrylate). Ion-selective optode membranes, composed of plasticized poly(vinyl chloride) impregnated with an ionophore, a proton chromoionophore, and a lipophilic anionic additive, were cast, with a spin-on device, onto a support layer and then immobilized on the disk. Fluid propulsion is achieved by the centrifugal force that results from spinning the disk, while a system of valves is built onto the disk to control flow. These valves operate based on fluid properties and fluid/substrate interactions and are controlled by the angular frequency of rotation. With this system, we have been able to deliver calibrant solutions, washing buffers, or "test" solutions to the measuring chamber where the optode membrane is located. An analysis system based on a potassium-selective optode has been characterized. Results indicate that optodes immobilized on the platform demonstrate theoretical responses in an absorbance mode of measurement. Samples of unknown concentration can be quantified to within 3% error by fitting the response function for a given optode membrane using an acid (for measuring the signal for a fully protonated chromoionophore), a base (for fully deprotonated chromoionophore), and two standard solutions. Further, the ability to measure ion concentrations by employing one standard solution in conjunction with acid and base and with two standards alone were studied to delineate whether the current architecture could be simplified. Finally, the efficacy of incorporating washing steps into the calibration protocol was investigated.

Calibration↗

PubMed2001

Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye.

A technique is described for the measurement of fluid temperatures in microfluidic systems based on temperature-dependent fluorescence. The technique is easy to implement with a standard fluorescence microscope and CCD camera. In addition, the method can be used to measure fluid temperatures with micrometer spatial resolution and millisecond time resolution. The efficacy of the method is demonstrated by measuring temperature distributions resulting from Joule heating in a variety of microfluidic circuits that are electrokinetically pumped. With the equipment used for these measurements, fluid temperatures ranging from room temperature to 90 degrees C were measured with a precision ranging from 0.03 to 3.5 degrees C-dependent on the amount of signal averaging done. The spatial and temporal resolutions achieved were 1 microm and 33 ms, respectively.

Journal Article↗

PubMed2001

Pressure-driven laminar flow in tangential microchannels: an elastomeric microfluidic switch.

This paper describes laminar fluid flow through a three-dimensional elastomeric microstructure formed by two microfluidic channels, fabricated in layers that contact one another face-to-face (typically at a 90 degree angle), with the fluid flows in tangential contact. There are two ways to control fluid flow through these tangentially connected microchannels. First, the flow profiles through the crossings are sensitive to the aspect ratio of the channels; the flow can be controlled by applying external pressure and changing this aspect ratio. Second, the flow direction of an individual laminar stream in multiphase laminar flow depends on the lateral position of the stream within the channel; this position can be controlled by injecting additional streams of fluid into the channel. We describe two microfluidic switches based on these two ways for controlling fluid flow through tangential microchannels and present theoretical arguments that explain the observed dependence of the flow profiles on the aspect ratio of the channels.

Journal Article↗

PubMed2001

Microfluidic arrays of fluid-fluid diffusional contacts as detection elements and combinatorial tools.

This paper describes microfluidic systems that can be used to investigate multiple chemical or biochemical interactions in a parallel format. These three-dimensional systems are generated by crossing two sets of microfluidic channels, fabricated in two different layers, at right angles. Solutions of the reagents are placed in the channels; in different modes of operation, these solutions can be either flowing or stationary-the latter is important when one set of channels is filled with viscous gels with immobilized reagents. At every crossing, the channels are separated either by a single membrane or by a composite separator comprising a membrane, a microwell, and a second membrane. These components allow diffusive mass transport and minimize convective transport through the crossing. Polycarbonate membranes with 0.1-1-microm vertical pores were used to fabricate the devices. Each crossing of parallel channels serves as an element in which chemical or biochemical interactions can take place; interactions can be detected by monitoring changes in fluorescence and absorbance. These all-organic systems are straightforward to fabricate and to operate and may find applications as portable microanalytical systems and as tools in combinatorial research.

Journal Article↗

PubMed2001

Monolithic porous polymer for on-chip solid-phase extraction and preconcentration prepared by photoinitiated in situ polymerization within a microfluidic device.

Monolithic porous polymers have been prepared by photoinitiated polymerization within the channels of a microfluidic device and used for on-chip solid-phase extraction and preconcentration. The preparation of the monolithic material with hydrophobic and ionizable surface chemistries is easily achieved by copolymerization of butyl methacrylate with ethylene dimethacrylate, or 2-hydroxyethyl methacrylate and [2-(methacryloyloxy)ethyl]trimethylammonium chloride with ethylene dimethacrylate, respectively. The porous properties, and consequently the flow resistance, of the monolithic device are controlled by the use of a mixture of hexane and methanol as a porogenic mixture. This mixture was designed to meet the specific requirements for pore formation within macroporous monoliths useful in the microfluidic formats. The low flow resistance enables high flow rates of up to 10 microL/min, which corresponds to a linear flow velocity of 50 mm/s and far exceeds the flow velocities typical of the common analytical microchips. The function of the monolithic concentration device was first demonstrated using very dilute solutions of Coumarin 519. The performance in a more realistic application was then demonstrated with the enrichment of a hydrophobic tetrapeptide and also of green fluorescent protein for which an increase in concentration by a factor as high as 10(3) was achieved.

Biotechnology↗

PubMed2001

SPR imaging measurements of 1-D and 2-D DNA microarrays created from microfluidic channels on gold thin films.

Microfluidic channels fabricated from poly(dimethylsiloxane) (PDMS) are employed in surface plasmon resonance imaging experiments for the detection of DNA and RNA adsorption onto chemically modified gold surfaces. The PDMS microchannels are used to (i) fabricate "1-D" single-stranded DNA (ssDNA) line arrays that are used in SPR imaging experiments of oligonucleotide hybridization adsorption and (ii) create "2-D" DNA hybridization arrays in which a second set of PDMS microchannels are placed perpendicular to a 1-D line array in order to deliver target oligonucleotide solutions. In the 1-D line array experiments, the total sample volume is 500 microL; in the 2-D DNA array experiments, this volume is reduced to 1 microL. As a demonstration of the utility of these microfluidic arrays, a 2-D DNA array is used to detect a 20-fmol sample of in vitro transcribed RNA from the uidA gene of a transgenic Arabidopsis thaliana plant. It is also shown that this array fabrication method can be used for fluorescence measurements on chemically modified gold surfaces.

Gold↗

PubMed2002

Integrated microfluidic electrophoresis system for analysis of genetic materials using signal amplification methods.

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), was performed within a microfluidic chip. The presence of DNA from methicillin-resistant Staphylococcus aureus was determined by signal amplification of a specific DNA sequence. The microfluidic device consisted of four channels intersecting to mix the sample and reagents within 55 s, as they were directed toward the reactor coil by electrokinetic pumping. The 160-nL CPT reactor occupied approximately 220 mm2. Gel-free capillary electrophoresis separation of the biotin- and fluorescein-labeled probe from the probe fragments was performed on-chip following the on-chip reaction. An off-chip CPT reaction, with on-chip separation gave a detection limit of 2 fM (0.03 amol) target DNA and an amplification factor of 85,000. Calibration curves, linear at <5% probe fragmentation, obeyed a power law relationship with an argument of 0.5 [target] at higher target DNA concentrations for both on-chip and off-chip CPT reaction and analysis. An amplification factor of 42,000 at 250 fM target (25,000 target molecules) was observed on-chip, but the reaction was approximately 4 times less sensitive than off-chip under the conditions used. Relative SD values for on-chip CPT were 0.8% for the peak migration times, 9% for the area of intact probe peak, and 8% for the fragment/probe peak area ratio.

DNA↗

PubMed2002

Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.

A solid-object printer was used to produce masters for the fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS). The printer provides an alternative to photolithography for applications where features of > 250 microm are needed. Solid-object printing is capable of delivering objects that have dimensions as large as 250 x 190 x 200 mm (x, y, z) with feature sizes that can range from 10 cm to 250 microm. The user designs a device in 3-D in a CAD program, and the CAD file is used by the printer to fabricate a master directly without the need for a mask. The printer can produce complex structures, including multilevel features, in one unattended printing. The masters are robust and inexpensive and can be fabricated rapidly. Once a master was obtained, a PDMS replica was fabricated by molding against it and used to fabricate a microfluidic device. The capabilities of this method are demonstrated by fabricating devices that contain multilevel and tall features, devices that cover a large area (approximately 150 cm2), and devices that contain nonintersecting, crossing channels.

Animals↗

PubMed2002

Biomolecular recognition on well-characterized beads packed in microfluidic channels.

We describe a new approach for the analysis of biomolecular recognition in microfluidic channels. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer (FRET). We establish a model system that detects the FLAG epitope. The assay can potentially detect subfemtomole quantities of antibody with a high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter-to-submicroliter volumes of analyte fluid. Kinetic and equilibrium constants for the reaction of this receptor-ligand pair are obtained through modeling of kinetic responses of the affinity microcolumn and are consistent with those obtained by flow cytometry. Because of the correlation between kinetic and equilibrium data obtained for the microcolumns, quantitative analysis can be done prior to the steady-state end point of the recognition reaction. This method has the promise of combining the utility of affinity chromatography with the advantage of direct, quantitative, and real-time analysis and the cost-effectiveness of microanalytical devices. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes and molecular assembly and microsystem-based multianalyte determinations.

Antibodies, Monoclonal↗