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At least 847 records · Page 47Linked to original sources

PubMed2005

Simultaneous multiple immunoassays in a compact disc-shaped microfluidic device based on centrifugal force.

BACKGROUND: We explored the potential of a microfluidic device based on centrifugal force as an immunoassay platform by examining the imprecision of assays carried out with 200 nL of sample. METHODS: Biotinylated antibodies against alpha-fetoprotein (AFP), interleukin-6 (IL-6), and carcinoembryonic antigen [(CEA); 0.1 g/L each in 15 mmol/L phosphate-buffered saline (PBS) containing 0.1 mL/L Tween 20] were attached to a microcolumn packed with streptavidin-coated particles. A 200-nL sample was then allowed to pass through the microcolumn for 240 s, followed by Alexa 647-labeled detection antibody (7.5 mg/L in 15 mmol/L PBS containing 10 g/L bovine serum albumin). The flow rate was controlled by altering the rotational speed. Up to 104 sandwich type immunoassays were completed within 50 min. RESULTS: For AFP, IL-6, and CEA the detection limits were, respectively, 0.15, 1.25, and 1.31 pmol/L. Inter- and intraassay imprecisions (CVs) were <10% and <20%, respectively, for analyte concentrations >5 pmol/L. The CEA antibody had the lowest affinity according to fluorescence image analysis of the microcolumn region. The result was confirmed in a comparative study using BIAcore 3000. CONCLUSIONS: Day-to-day (total) imprecision (CV) of immunoassays on the compact disc-shaped device are <20%. Analysis of fluorescence images allows rapid ranking of antibodies according to their affinities.

Animals↗

PubMed2006

Fabrication of polydimethylsiloxane microfluidics using SU-8 molds.

We detail the widely prevalent technique of polydimethylsiloxane (PDMS) molding using SU-8 for creating microfluidic chambers and channels. Although other techniques such as injection molding are more apt for mass manufacturing and cost-effective, PDMS molding is used almost exclusively for rapid prototyping in corporate and research environments because of its simplicity and fast turnaround time.

Dimethylpolysiloxanes↗

PubMed2006

Laser ablation as a fabrication technique for microfluidic devices.

The use of microfluidic devices is making rapid inroads in the modern laboratory. Traditionally, devices have been manufactured in silica owing to its well-understood surface chemistry and micromachining techniques that are ubiquitous in the microelectronics industry. Recently researchers have begun to utilize devices fabricated from polymer substrates as an alternative to glass. Reasons include total cost and the ability to tailor physical and chemical properties. However, traditional microfabrication techniques of molds that are used to fabricate polymer devices by imprinting or injection molding are subject to similar limitations as those associated with silica. In a research and development environment, it is important that researchers have access to fabrication techniques that are rapid and easily implemented with a variety of polymer substrates. Laser ablation has been proven to be such a technique, and this chapter describes the fabrication of a simple injection tee utilizing this technique. This chapter also examines the practical details involved in laser ablation from the perspective of a scientist who desires to utilize a turn-key system.

Lasers↗

PubMed2005

Measuring single-bond rupture forces using high electric fields in microfluidic channels and DNA oligomers as force tags.

The disruption force of specific biotin-streptavidin bonds was determined using DNA oligomers as force tags. Forces were generated by an electric field acting on a biotinylated fluorescently labeled DNA oligomer. DNA oligomers were immobilized via biotin-streptavidin bonds on the walls of microfluidic channels. Channel layout and fluid-based deposition process were designed to enable well-defined localized deposition of the oligomers in a narrow gap of the microchannel. Electric fields of up to 400 V/cm were applied and electric field induced desorption of DNA oligomers was observed. At T approximately 30 degrees C, field-induced desorption of both a 12 mer as well as a 48 mer yielded a streptavidin-biotin disruption force of 75 fN. Streptavidin-functionalized surfaces remained intact and could be reloaded with biotinylated oligomers. At approximately 20 degrees C, however, no field-induced unbinding of the oligomers was observed at electric field strength of up to 400 V/cm, indicating a significant temperature dependence of the bond strength.

Biophysics↗

PubMed2005

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.

Agrin↗

PubMed2006

DNA mutation detection and analysis using miniaturized microfluidic systems.

Identification of genetic sequence variations occurring on a population-wide scale is key to unraveling the complex interactions that are the underlying cause of many medical disorders and diseases. A critical need exists, however, for advanced technology to enable DNA mutation analysis to be performed with significantly higher throughput and at significantly lower cost than is currently attainable. Microfluidic systems offer an attractive platform to address these needs by combining the ability to perform rapid analysis with a simplified device format that can be inexpensively mass-produced. This paper will review recent progress toward developing these next-generation systems and discuss challenges associated with adapting these technologies for routine laboratory use.

DNA Mutational Analysis↗

PubMed2005

Macromolecular crystallization with microfluidic free-interface diffusion.

Fluidigm Corp. released the Topaz 1.96 and 4.96 crystallization chips in the fall of 2004. Topaz 1.96 and 4.96 are the latest evolution of Fluidigm's microfluidics crystallization technologies that enable ultra-low-volume rapid screening for macromolecular crystallization. Topaz 1.96 and 4.96 are similar to each other but represent a major redesign of the Topaz system and have substantially improved ease of automation and ease of use, improved efficiency and even further reduced the amount of material needed. With the release of the new Topaz system, Fluidigm continues to set the standard in low-volume crystallization screening, which is having an increasing impact in the field of structural genomics and more generally in structural biology. It is likely that further optimization and increased utility of the Topaz crystallization system will emerge. It is also probable that further innovation and the emergence of competing technologies will be seen.

Crystallization↗

PubMed2006

High-throughput multi-antigen microfluidic fluorescence immunoassays.

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.

Antigens↗

PubMed2003

Intracellular HSP72 detection in HL60 cells using a flow cytometry system based on microfluidic analysis.

HSP72 is an important marker for various environmental stresses and diseases, and many researchers need to detect HSP72 levels in various cells. We have therefore developed an assay to monitor intracellular heat-shock protein 72 expression on a microfluidic Lab-on-a-chip platform. We established this method to detect HSP72 intracellularly by antibody staining with DNA counterstaining. The Lab-on-a-chip technology is simple and efficient when performing flow cytometric assays. By permeabilizing the cells for the delivery of antibodies, we were able to show HSP72 expression after 30 min heat-shock at 44 degrees C and then at various post-incubation times at 37 degrees C. We compared our method to a conventional flow cytometer and an enzyme immunoassay technique.

Biological Assay↗

PubMed2003

Identifying Mycobacterium species and strain typing using a microfluidic labchip instrument.

We developed schemes for rapid identification of Mycobacterium species and strain typing using a microfluidic labchip instrument. A 439-bp region of the gene that codes for the 65-kDa heat shock protein (hsp65), which has sequence polymorphisms specific for most mycobacterial species, was examined using PCR-restriction analysis (PRA). We performed PRA in duplicate, using 2 strains each of 12 species, and observed that fragment sizes (bp) determined automatically by the instrument were consistently smaller than the correct sizes for each of the species as determined by sequence analysis (mean variance, < 7 bp). Mycobacterium tuberculosis isolates were typed with the labchip instrument using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, which determines the number of copies of repeated units at 12 loci in the genome based on product size after PCR amplification. Seven strains with one to six repeat copies at each locus were examined. Sizes were smaller by a mean of 13.47 bp compared with correct sizes predicted by sequence analysis, but could be used to correctly identify all strains types. Isolates of Mycobacterium chelonae and Mycobacterium abscessus were typed using randomly amplified polymorphic DNA (RAPD) electrophoresis, and patterns obtained using the labchip instrument were compared with multilocus enzyme electrophoresis (MEE) types. Patterns were distinct and reproducible for all strains except those with closely related MEE types. The labchip instrument is a versatile alternative for sizing mycobacterial DNA fragments.

DNA, Bacterial↗

PubMed2005

Validation of oligonucleotide microarray data using microfluidic low-density arrays: a new statistical method to normalize real-time RT-PCR data.

Profiling studies using microarrays to measure messenger RNA (mRNA) expression frequently identify long lists of differentially expressed genes. Differential expression is often validated using real-time reverse transcription PCR (RT-PCR) assays. In conventional real-time RT-PCR assays, expression is normalized to a control, or housekeeping gene. However, no single housekeeping gene can be used for all studies. We used TaqMan Low-Density Arrays, a medium-throughput method for real-time RT-PCR using microfluidics to simultaneously assay the expression of 96 genes in nine samples of chronic lymphocytic leukemia (CLL). We developed a novel statistical method, based on linear mixed-effects models, to analyze the data. This method automatically identifies the genes whose expression does not vary significantly over the samples, allowing them to be used to normalize the remaining genes. We compared the normalized real-time RT-PCR values with results obtained from Affymetrix Hu133A GeneChip oligonucleotide microarrays. We found that real-time RT-PCR using TaqMan Low-Density Arrays yielded reproducible measurements over seven orders of magnitude. Our model identified numerous genes that were expressed at nearly constant levels, including the housekeeping genes PGK1, GAPD, GUSB, TFRC, and 18S rRNA. After normalizing to the geometric mean of the unvarying genes, the correlation between real-time RT-PCR and microarrays was high for genes that were moderately expressed and varied across samples.

Algorithms↗

PubMed2006

Shear-dependent aggregation characteristics of red blood cells in a pressure-driven microfluidic channel.

Though the aggregation of red blood cells (RBCs) is a major determinant of blood viscosity, there have not been any available techniques to measure the effect of RBC aggregation on blood viscosity over a range of shear rates. The microfluidic shearing technique with vibration has been applied to an aggregometer for measuring the dynamic aggregation characteristic of RBCs. In measuring backscattered light intensity I(t) and pressure p(t) over time, both aggregation and the stress-shear rate information can be determined simultaneously. The feasibility and accuracy of the new aggregation measurement technique has been demonstrated to correlate with blood viscosity for normal and heated blood. We found that RBC aggregability showed shear-dependent behavior, which can be correlated directly with shear-thinning blood viscosity. The present measurements of the dynamic aggregation characteristic over shear rate enable the interpretation of the shear-rate dependent blood viscosity, which is greatly affected by RBC aggregation.

Adult↗

PubMed2006

Geometrical focusing of cells in a microfluidic device: an approach to separate blood plasma.

It is well known that when a suspension of cells flows in small vessels (arterioles or venules), there exists a cell-free layer of a few microns adjacent to the vascular walls. Using an in vitro model, we show experimentally that for a fixed flow rate a geometrical constriction in the flow can artificially enhance the cell-free layer. Also, we show that rapid variation of the geometry coupled to the deformability of the cells can dramatically modify their spatial distribution in the channel. The effects of the constriction geometry, flow rate, suspending fluid viscosity, cell concentration, and cell deformability are studied and the results are interpreted in terms of a model of the hydrodynamic drift of an ellipsoidal cell in a shear flow. We propose a microfluidic application of this focusing effect for separation of the red blood cells from the suspending plasma.

Blood Flow Velocity↗

PubMed2000

A microfluidic system for high-speed reproducible DNA sizing and quantitation.

Microfabrication technology was used to develop a system consisting of disposable glass chips containing etched channels, reagents including polymer matrix and size standards, computer-controlled instrumentation for performing electrophoretic separations and fluorescence detection of double-stranded DNA, and software for automated data analysis. System performance was validated for separation and quantitation reproducibility using samples varying in amount and size of DNA fragments, buffer composition, and salt concentrations. Several applications of the microfluidic system for DNA analysis have been demonstrated, such as of polymerase chain reaction (PCR) products, sizing of plasmid digests, and detection of point mutations by restriction fragment length polymorphism (RFLP) mapping.

Animals↗

PubMed2000

Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface.

A microfluidic device is described in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector and a protein digestion bed on a monolithic substrate. A large channel, 800 microm wide, 150 microm deep and 15 mm long, was created to act as a reactor bed for trypsin immobilized on 40-60 microm diameter beads. Separation was performed in channels etched 10 microm deep, 30 microm wide and about 45 mm long, feeding into a capillary, attached to the chip with a low dead volume coupling, that was 30 mm in length, with a 50 microm i.d. and 180 microm o.d. Sample was pumped through the reactor bed at flow rates between 0.5 and 60 microL/min. The application of this device for rapid digestion, separation and identification of proteins is demonstrated for melittin, cytochrome c and bovine serum albumin (BSA). The rate and efficiency of digestion was related to the flow rate of the substrate solution through the reactor bed. A flow rate of 1 or 0.5 microL/min was found adequate for complete consumption of cytochrome c or BSA, corresponding to a digestion time of 3-6 min at room temperature. Coverage of the amino acid sequence ranged from 92% for cytochrome c to 71% for BSA, with some missed cleavages observed. Melittin was consumed within 5 s. In contrast, a similar extent of digestion of melittin in a cuvet took 10-15 min. The kinetic limitations associated with the rapid digestion of low picomole levels of substrate were minimized using an integrated digestion bed with hydrodynamic flow to provide an increased ratio of trypsin to sample. This chip design thus provides a convenient platform for automated sample processing in proteomics applications.

Animals↗

PubMed2001

Design of an interface to allow microfluidic electrophoresis chips to drink from the fire hose of the external environment.

An interface design is presented that facilitates automated sample introduction into an electrokinetic microchip, without perturbing the liquids within the microfluidic device. The design utilizes an interface flow channel with a volume flow resistance that is 0.54-4.1 x 10(6) times lower than the volume flow resistance of the electrokinetic fluid manifold used for mixing, reaction, separation, and analysis. A channel, 300 microm deep, 1 mm wide and 15-20 mm long, was etched in glass substrates to create the sample introduction channel (SIC) for a manifold of electrokinetic flow channels in the range of 10-13 microm depth and 36-275 microm width. Volume flow rates of up to 1 mL/min were pumped through the SIC without perturbing the solutions within the electrokinetic channel manifold. Calculations support this observation, suggesting a leakage flow to electroosmotic flow ratio of 0.1:1% in the electrokinetic channels, arising from 66-700 microL/min pressure-driven flow rates in the SIC. Peak heights for capillary electrophoresis separations in the electrokinetic flow manifold showed no dependence on whether the SIC pump was on or off. On-chip mixing, reaction and separation of anti-ovalbumin and ovalbumin could be performed with good quantitative results, independent of the SIC pump operation. Reproducibility of injection performance, estimated from peak height variations, ranged from 1.5-4%, depending upon the device design and the sample composition.

Animals↗

PubMed2001

Continuous concentration of bacteria in a microfluidic flow cell using electrokinetic techniques.

A novel method for the concentration of bacterial solutions is presented that implements electrokinetic techniques, zone electrophoresis (ZE) and isoelectric focusing (IEF), in a microfluidic device. The method requires low power (< 3e-5 W) and can be performed continuously on a flowing stream. The device consists of two palladium electrodes held in a flow cell constructed from layers of polymeric film held together by a pressure-sensitive adhesive. Both ZE and IEF are performed with carrier-free solutions in devices in which the electrodes are in intimate contact with the sample fluid. IEF experiments were performed using natural pH gradients; no carrier ampholyte solution was required. Experiments performed in buffer alone resulted in significant electroosmotic flow. Pretreatment of the sample chamber with bleach followed by a concentrated solution of cationic detergent effectively suppressed electroosmotic flow.

Bacteria↗

PubMed2001

Integrated self-calibration via electrokinetic solvent proportioning for microfluidic immunoassays.

On-board generation of a set of calibration standards was demonstrated within a microfluidic device designed to perform immunoassay. Electrokinetic flow was used to proportionally mix the antibody (Ab) to bovine serum albumin (BSA) and a diluting buffer, to provide varying Ab concentrations for downstream mixing with fluorescently labeled BSA (BSA*). Mixing ratios were determined from electrical impedance modeling of the fluidic network using P-SPICE software, and peak heights for the labeled species were analyzed relative to the concentration calculated from the model. For dilution and separation of fluorescently labeled amino acids, a linear calibration curve was obtained for mixing ratios of 0.118 to 7.46. A linear calibration curve was obtained for the immunoassay calibration using dilution ratios between 0.197 and 5.077. Deviations were observed at larger extremes, possibly due to leakage effects at intersections. Peak height reproducibility was +/- 3% for the immunoassay, using diluted monoclonal Ab in mouse ascites fluid as the analyte. Recovery for on-chip calibration was 92 +/- 6% versus calibrants prepared off-chip, indicating a small bias.

Amino Acids↗