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At least 685 records · Page 38Linked to original sources

PubMed2006

Microfluidic cartridges preloaded with nanoliter plugs of reagents: an alternative to 96-well plates for screening.

In traditional screening with 96-well plates, microliters of substrates are consumed for each reaction. Further miniaturization is limited by the special equipment and techniques required to dispense nanoliter volumes of fluid. Plug-based microfluidics confines reagents in nanoliter plugs (droplets surrounded by fluorinated carrier fluid), and uses simple pumps to control the flow of plugs. By using cartridges pre-loaded with nanoliter plugs of reagents, only two pumps and a merging junction are needed to set up a screen. Screening with preloaded cartridges uses only nanoliters of substrate per reaction, and requires no microfabrication. The low cost and simplicity of this method has the potential of replacing 96-well and other multi-well plates, and has been applied to enzymatic assays, protein crystallization and optimization of organic reactions.

Chemical Precipitation↗

PubMed2005

Continuous on-line concentration based on dynamic pH junction for trimethoprim and sulfamethoxazole by microfluidic capillary electrophoresis combined with flow injection analysis system.

A novel, rapid and continuous on-line concentration approach based on dynamic pH junction for the analysis of trimethoprim (TMP) and sulfamethoxazole (SMZ) by microfluidic capillary electrophoresis (CE) combined with flow injection analysis is developed in this paper. Stacking is due to decreases in the velocity of analytes when migrating from the low-pH sample zone (sample was dissolved in 50 mM HCl) to a relatively high-pH buffer (30 mM phosphate buffer, pH 8.5) filled in the capillary. This results in 2.9-4.7-fold improvement in concentration sensitivity relative to conventional capillary electrophoresis methods. The separation could be achieved within 2 min and sample throughput rate can reach up to 38 h(-1).

Electrophoresis, Capillary↗

PubMed2006

Glucose microfluidic biosensors based on immobilizing glucose oxidase in poly(dimethylsiloxane) electrophoretic microchips.

Here we reported a novel microfluidic biosensor with an on-column immobilized enzyme microreactor. The fabrication approach of this biosensor is simple and the enzyme microreactors with controlled sizes can be placed at any desired position on the microchip. Taking glucose oxidase (GOx) as an example, electroosmotic flow (EOF) as a driving force and amperometry as a detection method, the performance of biosensors were modulated by changing the length of enzyme reactor from 0.5 cm to 3 cm, and the linear ranges were changed from 0-8.0 mM to 0-30.0 mM with the detection limits from 42 microM to 6.5 microM. The enzyme reactor remained its 65% activity after 23 days storage. It also showed good anti-interference ability and was used to quantify glucose in human serum samples.

Biosensing Techniques↗

PubMed2005

Analysis of mitochondrial DNA in microfluidic systems.

Abnormalities in mitochondrial function play a major role in many human diseases. It is often of critical importance to ascertain what proportion of the mitochondria within a cell, or cells, bear a given mutation (the mitochondrial "demographics"). In this work, a rapid, novel, on-chip procedure was used, in which a restriction enzyme was employed to excise a mitochondrial DNA (mtDNA) sequence from plasmid DNA that acted as a prototypical mitochondrial genome. The DNA was then denatured, reassembled to form duplexes, fluorescently labelled and analysed. This method was able to differentiate between a homogeneous population and a heterogeneous population. Using a microfluidic chip, the method could be performed in about 45 min, even without robotics or multiplexed operation, whereas conventional methods of analysis require days to perform. This method may ultimately form the basis for a means of characterizing the mitochondrial demographics of a single cell.

DNA, Mitochondrial↗

PubMed2006

Simple approaches to close the open structure of microfluidic chips and connecting them to the macro-world.

Microchip electrophoresis has a great potential to improve the speed and throughput of chemical and biochemical analyses. Conventional electrophoresis microchip fabrication methods comprise the main steps of channel formation, cover plate binding and access hole construction. While the fabrication of appropriate cover plates and their bonding process are quite essential to the creation of closed microfluidic networks, connection means of microchips to the macro-world is one of the most important parts of microchip fabrication. In this paper the most commonly used approaches are discussed for cover plate connector fabrication in conjunction with high and low temperature glue-less binding processes. The microchannels in the glass substrate were fabricated by sawing and powder blasting under regular laboratory settings, i.e., not necessitating the use of a clean-room environment, making in this way broader availability for electrophoresis microchip technology.

Electrophoresis, Microchip↗

PubMed2006

Application of microfluidic chip with integrated optics for electrophoretic separations of proteins.

This paper describes the fabrication, the characterization and the applications of a capillary electrophoresis microchip. This hybrid device (glass/PDMS) features channels and optical waveguides integrated in one common substrate. It can be used for electrophoretic separation and fluorimetric detection of molecules. The microfluidic performance of the device is demonstrated by capillary zone and gel electrophoresis of proteins.

Carbocyanines↗

PubMed2006

A capillary-based microfluidic instrument suitable for immunoaffinity chromatography.

The analysis of biological samples to produce clinical or research data often requires measurement of analytes from complex biological matrices and limited volumes. Miniaturized analytical systems capable of minimal sample consumption and reduced analysis times have been employed to meet this need. The small footprint of this technology offers the potential for portability and patient point-of-care testing. A prototype microfluidic system has been developed and is presented for potential rapid assessment of clinical samples. The system has been designed for immunoaffinity chromatography as a means of separating analytes of interest from biological matrices. The instrument is capable of sub-microliter sample injection and detection of labeled antigens by long wavelength laser-induced fluorescence (LIF). The laboratory-constructed device is assembled from an array of components including two syringe pumps, a nano-gradient mixing chip, a micro-injector, a diode laser, and a separation capillary column made from a polymer/silica (PEEKsil) tube. An in-house program written with LabVIEW software controls the syringe pumps to perform step gradient elution and collects the LIF signal as a chromatogram. Initial columns were packed with silica beads to evaluate the system. Optimization of the device has been achieved by measuring flow accuracy with respect to column length and particle size. Syringe size and pressure effects have also been used to characterize the capability of the pumps. Based on test results, a 200-microm x 25-mm column packed with 1-microm silica beads was chosen for use with a 500-microL syringe. The system was tested for mixer proportioning by pumping different compositions of buffer and fluorescent dye solutions in a stepwise fashion. A linear response was achieved for increasing concentrations of fluorescent dye by online mixing (R2=0.9998). The effectiveness of an acidic gradient was confirmed by monitoring pH post-column and measuring premixed solutions offline. Finally, assessment of detectability was achieved by injecting fluorescent dye solutions and measuring the signal from the LIF detector. The limit of detection for the system with these solutions was 10.0 pM or 10.0 amol on-column. As proof-of-principle, immunoaffinity chromatography was demonstrated with immobilized rabbit anti-goat IgG and a fluorescent dye-goat IgG conjugate as a model antigen.

Animals↗

PubMed2006

A microfluidic fluorous solid-phase extraction chip for purification of amino acids.

An electrokinetically-driven microfluidic chip was developed to realize beads-based solid-phase extraction (SPE) of amino acids. This chip uses a two-level (deep/shallow) poly(dimethylsiloxane) (PDMS) microchannel network to confine the fluorous reversed-phase silica beads within the SPE chamber. The mixture of fluorous tagged and non-tagged amino acids was carried into the fluorous solid-phase extraction (F-SPE) chamber by electrokinetic pumping and was successfully separated and extracted. By adding a reference material to the sample, the extraction efficiency of the eluted fluorous-tagged amino acid was calculated using the detection results from mass spectrometry (MS). The F-SPE microchips showed good reproducibility and efficiency, yielding an average extraction efficiency of 55% with a RSD of 10.6% under the typical experimental conditions.

Amino Acids↗

PubMed2005

Using nanoliter plugs in microfluidics to facilitate and understand protein crystallization.

Protein crystallization is important for determining protein structures by X-ray diffraction. Nanoliter-sized plugs--aqueous droplets surrounded by a fluorinated carrier fluid--have been applied to the screening of protein crystallization conditions. Preformed arrays of plugs in capillary cartridges enable sparse matrix screening. Crystals grown in plugs inside a microcapillary may be analyzed by in situ X-ray diffraction. Screening using plugs, which are easily formed in PDMS microfluidic channels, is simple and economical, and minimizes consumption of the protein. This approach also has the potential to improve our understanding of the fundamentals of protein crystallization, such as the effect of mixing on the nucleation of crystals.

Animals↗

PubMed2005

Rare cancer cell analyzer for whole blood applications: automated nucleic acid purification in a microfluidic disposable card.

One current challenge facing point-of-care cancer detection is that existing methods make it difficult, time consuming and too costly to (1) collect relevant cell types directly from a patient sample, such as blood and (2) rapidly assay those cell types to determine the presence or absence of a particular type of cancer. We present a proof of principle method for an integrated, sample-to-result, point-of-care detection device that employs microfluidics technology, accepted assays, and a silica membrane for total RNA purification on a disposable, credit card sized laboratory-on-card ('lab card") device in which results are obtained in minutes. Both yield and quality of on-card purified total RNA, as determined by both LightCycler and standard reverse transcriptase amplification of G6PDH and BCR-ABL transcripts, were found to be better than or equal to accepted standard purification methods.

Fusion Proteins, bcr-abl↗

PubMed2003

Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection.

Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235 microm depth and 25 microm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H2O2. All immunosensors were capable of detecting atrazine at the sub-microg l(-1) level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 microg l(-1), and IC50 values of 0.096 and 0.130 microg l(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCl buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87-102 and 88-124% at atrazine fortification levels of 0.5-3 and 80-240 microg l(-1), respectively.

Adsorption↗

PubMed2003

High-efficiency, two-dimensional separations of protein digests on microfluidic devices.

High-efficiency, two-dimensional separations of tryptic digests were achieved using glass microfluidic devices. Following micellar electrokinetic chromatography (MEKC) separations in a 19.6-cm-long serpentine channel, the peptides were rapidly sampled into a 1.3-cm-long second-dimension channel, where they were separated by capillary electrophoresis (CE). The turns in the serpentine channel were asymmetrically tapered to minimize geometrical contributions to band broadening and to provide ample channel length for high-efficiency chromatographic separations. Analysis of rhodamine B injections routinely produced plate numbers of 230000 and 40000 in the first (MEKC) and second (CE) dimensions, respectively, corresponding to plate heights of 0.9 and 0.3 microm. The electric field strengths were 200 V/cm for MEKC and 2400 V/cm for CE. In analysis times less than 15 min, two-dimensional separation of bovine serum albumin tryptic digest produced a peak capacity of 4200 (110 in the first dimension and 38 in the second dimension). The system was used to identify a peptide from a tryptic digest of ovalbumin using standard addition and to distinguish between tryptic digests of human and bovine hemoglobin.

Animals↗

PubMed2003

Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices.

This paper describes the compatibility of poly(dimethylsiloxane) (PDMS) with organic solvents; this compatibility is important in considering the potential of PDMS-based microfluidic devices in a number of applications, including that of microreactors for organic reactions. We considered three aspects of compatibility: the swelling of PDMS in a solvent, the partitioning of solutes between a solvent and PDMS, and the dissolution of PDMS oligomers in a solvent. Of these three parameters that determine the compatibility of PDMS with a solvent, the swelling of PDMS had the greatest influence. Experimental measurements of swelling were correlated with the solubility parameter, delta (cal(1/2) cm(-3/2)), which is based on the cohesive energy densities, c (cal/cm(3)), of the materials. Solvents that swelled PDMS the least included water, nitromethane, dimethyl sulfoxide, ethylene glycol, perfluorotributylamine, perfluorodecalin, acetonitrile, and propylene carbonate; solvents that swelled PDMS the most were diisopropylamine, triethylamine, pentane, and xylenes. Highly swelling solvents were useful for extracting contaminants from bulk PDMS and for changing the surface properties of PDMS. The feasibility of performing organic reactions in PDMS was demonstrated by performing a Diels-Alder reaction in a microchannel.

Dimethylpolysiloxanes↗

PubMed2004

A packaging technique for polymer microfluidic platforms.

A new technique, resin-gas injection, has been developed for bonding and surface modification of polymer microfluidic devices. This method can easily bond biochips with complex flow patterns. A cascade micromixer and a multichannel DNA sequencing chip were demonstrated experimentally. By adding surface modification agents, the interfacial free energy of the substrate with water can be controlled. Local modification of the channel surface can also be achieved through sequential resin-gas injection in conjunction with a masking technique. For application, this technique is used to form a layer of dry monolithic stationary hydrogel on the walls of a microchannel, serving as a sieving material for electrophoresis separation of DNA fragments. The reagent loading and the electrophoresis separation efficiency of this technique were compared experimentally with the conventional linear polymer solution method used in the microchannel-based DNA sequencing process. It is found that our method has the advantages of more user-friendly operation, easier and faster sample loading, but slightly less separation efficiency.

DNA↗

PubMed2004

Surface modification of the channels of poly(dimethylsiloxane) microfluidic chips with polyacrylamide for fast electrophoretic separations of proteins.

The electrophoresis of proteins was investigated using poly(dimethylsiloxane) (PDMS) microfluidic chips whose surfaces were modified with polyacrylamide through atom-transfer radical polymerization. PDMS microchips were made using a glass replica to mold channels 10 microm high and 30 microm wide, with a T-intersection. The surface modification of the channels involved surface oxidation, followed by the formation of a self-assembled monolayer of benzyl chloride initiators, and then atom-transfer radical polymerization to grow a thin layer of covalently bonded polyacrylamide. The channels filled spontaneously with aqueous buffer due to the hydrophilicity of the coating. The resistance to protein adsorption was studied by open-channel electrophoresis for bovine serum albumin labeled with fluorophor. A plate height of 30 microm, corresponding to an efficiency of 33 000 plates/m, was obtained for field strengths from 18 to 889 V/cm. The lack of dependence of plate height on field strength indicates that there is no detectable contribution to broadening from adsorption. A 2- to 3-fold larger plate height was obtained for electrophoresis in a 50-cm polyacrylamide-coated silica capillary, and the shape of the electropherogram indicated the efficiency is limited by a distribution of species. The commercial capillary exhibited both reversible and irreversible adsorption of protein, whereas the PDMS microchip exhibited neither. A separation of lysozyme and cytochrome c in 35 s was demonstrated for the PDMS microchip.

Acrylic Resins↗

PubMed2004

Potentiometric titrations in a poly(dimethylsiloxane)-based microfluidic device.

This paper describes a microfluidic device, fabricated in poly(dimethylsiloxane), that is used for potentiometric titrations. This system generates step gradients of redox potentials in a series of microchannels. These potentials are probed by microelectrodes that are integrated into the chip; the measured potentials were used to produce a titration curve from which the end point of a reaction was measured.

Dimethylpolysiloxanes↗

PubMed2004

Strategy for repetitive pinched injections on a microfluidic device.

A microfluidic valve was fabricated with a cross intersection and two tee intersections in close proximity and evaluated for repetitive pinched injections. Electrokinetic forces were used to mobilize the sample and control diffusive transport at a cross intersection to produce sample plugs of short axial extent in an analysis channel similar to the standard pinched valve. The addition of a tee intersection in the sample channel maintained the sample close to the injection valve under "pullback"conditions allowing more rapid loading into the cross intersection. A second tee intersection allowed unidirectional transport in the analysis channel enabling loading of subsequent injections during an analysis. The two tee intersections were each located 80 microm from the cross intersection. Injection frequencies of 1, 2.5, 5, and 10 Hz were tested with a duty cycle of 0.5 for sample loading and dispensing. With 1 kV applied to the microchip during dispensing, the relative standard deviation of the peak areas for 15 injections was 1.6%. The peak width (4sigma) for the repetitive injections increased from 71 to 96 microm compared to a standard pinched injection due to the presence of the tee intersection in the analysis channel.

Electrochemistry↗

PubMed2005

Addressable electric fields for size-fractioned sample extraction in microfluidic devices.

Fraction collection following electrophoresis is of major importance for a variety of biological analyses. These assays typically need to identify specific fractions in the separated sample for further processing and require extraction of one or a group of fragments. In this paper, we have developed and characterized a technique to generate addressable electric fields for improved extraction during electrophoresis in microfluidic devices. The addressable electric field is achieved by applying a low bias voltage (1-2 V) to microelectrode pairs within the electrophoresis microchannel. Theoretical analysis shows the purity of the extracted sample can be improved as much as 30% over extraction without the shaped electric fields, and nearly 100% predicted yield can be achieved. We also describe the theoretical design of shaped electric fields by characterizing the optimal electrode geometry, field strength, channel configuration, and electrophoretic migration behavior needed for efficient band extraction.

DNA↗