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At least 469 records · Page 26Linked to original sources
PubMed2003

Effects of liquid conductivity differences on multi-component sample injection, pumping and stacking in microfluidic chips.

As an increasing number of processes are being integrated into Lab-on-a-chip devices, there is an increasing need for flexible and accurate sample manipulation techniques for effective transport and separation. Conductivity differences between running buffer and analyte samples can arise as a product of on-chip processing, or by design. The two situations studied here are sample pumping (where bulk transport is increased and separation of charged analytes is delayed using a relatively high conductivity sample), and sample stacking (where bulk transport is decreased and separation of charged analytes is expedited using a relatively low conductivity sample). A recently developed dynamic loading method for on-chip sample injection in a straight-cross channel configuration is applied here to both pumping and stacking cases. A key characteristic of the dynamic loading method is the ability to inject samples of high concentration density and uniformity of any length. By employing the conductivity differences alone, the effectiveness of either sample transport or sample separation are shown to improve over the uniform conductivity case. Then it is demonstrated that increasing the sample length, through dynamic loading, greatly increases the effectiveness of sample pumping, evidenced in an eight-fold increase in peak height as well as a decrease in total sample length at a downstream detector. Dynamic loading in the sample stacking case was shown to also increase peak intensity height (three-fold) in rapid separations. These results demonstrate that the dynamic loading technique, used in conjunction with strategic conductivity differences, significantly extends the capabilities of microfluidic chips.

Journal Article↗
PubMed2004

A new two-chip concept for continuous measurements on PMMA-microchips.

A new concept for continuous measurements on microchips is presented. A PMMA (polymethylmethacrylate) based capillary electrophoresis chip with integrated conductivity detection is combined with a second chip, which undertakes the task of fluid handling and electrical connections. The combination of electrokinetic and hydrodynamic flows allows long-term continuous stable analyses with good reproducibilities of migration time and peak heights of analytes. The two-chip system is characterized in terms of stability and reproducibility of separation and detection of small ions. Relative standard deviations of <1% and 3% respectively for retention times and peak heights during long-term measurements can be achieved. The new system combines simple handling and automated analysis without the need for refilling, cleaning or removal of the separation chip after one or several measurements.

Journal Article↗
PubMed2004

Planar chip device for PCR and hybridization with surface acoustic wave pump.

We have developed a microfluidic device operating at a planar surface instead of a closed channel network. The fluid is transported in single droplets using surface acoustic waves (SAW) on a piezoelectric LiNbO(3) substrate. The surface of the piezo is chemically structured to induce high contact angles of the droplets or enclose areas where the liquid can wet the substrate. Combining the SAW technique with thin film resistance heaters, a biological analysis chip with integrated DNA amplification by PCR and hybridization was designed. To prevent evaporation of the PCR reagents at high temperatures the sample is enclosed in droplets of mineral oil. On this chip the SAW resolves dried primers, shifts the oil capped liquid between the two heaters and mixes during hybridization. The chip is able to perform a highly sensitive, fast and specific PCR with a volume as low as 200 nl. During the temperature cycles an online monitoring of the DNA concentration is feasible with an optical unit, providing a sensitivity of 0.1 ng. After PCR the product is moved to the second heater for the hybridization on a spotted DNA array. With our chip we were able to detect a single nucleotide polymorphism (SNP) responsible for the Leiden Factor V syndrome from human blood.

Acoustics↗
PubMed2005

A resistive-pulse sensor chip for multianalyte immunoassays.

MultiAnalyte immunoassays are often required to diagnose a pathologic condition. Here, we show how resistive-pulse sensing and multiple artificial pores can be integrated together on a single chip to detect different antigens rapidly and simultaneously. We use multiple pores on a single chip to detect the size change of latex colloids upon specific antigen-antibody binding on the colloid surface. As a proof-of-principle, we demonstrate our ability to detect simultaneously human G-CSF and GM-CSF antigens on a single chip. Our novel technique is a scalable technology that can lead to the sensing of at least N2 antigens simultaneously with an N N array of pores on a single chip.

Antigen-Antibody Reactions↗
PubMed2005

The autofluorescence of plastic materials and chips measured under laser irradiation.

Plastic materials have the potential to substitute for glass substrates used in microfluidic and microTAS systems adding flexibility in materials' choices. Optical quality plastic materials with a low autofluorescence are crucial for optimal detection by fluorescence and laser induced fluorescence techniques. This paper summarizes a series of optical investigations on commercially available plastic chip materials (PMMA, COC, PC, PDMS) and chips made from those materials. Intrinsic optical constants of plastic materials-refractive index for bulk materials-determined by spectroscopic ellipsometry and transmission spectroscopy in the visible range are presented. The laser-induced autofluorescence of materials and chips was assessed at four laser wavelengths, namely, 403, 488, 532 and 633 nm. Considerable bleaching of the autofluorescence was observed under continuous laser illumination. Overall, the longer wavelength laser excitation sources yielded less autofluorescence. PDMS exhibited the least autofluorescence and was comparable to BoroFloat glass. In all cases, chips exhibited slightly higher autofluorescence than the raw plastic materials from which they had been made.

Equipment Failure Analysis↗
PubMed2006

Design and fabrication of a multilayered polymer microfluidic chip with nanofluidic interconnects via adhesive contact printing.

The design and fabrication of a multilayered polymer micro-nanofluidic chip is described that consists of poly(methylmethacrylate) (PMMA) layers that contain microfluidic channels separated in the vertical direction by polycarbonate (PC) membranes that incorporate an array of nanometre diameter cylindrical pores. The materials are optically transparent to allow inspection of the fluids within the channels in the near UV and visible spectrum. The design architecture enables nanofluidic interconnections to be placed in the vertical direction between microfluidic channels. Such an architecture allows microchannel separations within the chip, as well as allowing unique operations that utilize nanocapillary interconnects: the separation of analytes based on molecular size, channel isolation, enhanced mixing, and sample concentration. Device fabrication is made possible by a transfer process of labile membranes and the development of a contact printing method for a thermally curable epoxy based adhesive. This adhesive is shown to have bond strengths that prevent leakage and delamination and channel rupture tests exceed 6 atm (0.6 MPa) under applied pressure. Channels 100 microm in width and 20 microm in depth are contact printed without the adhesive entering the microchannel. The chip is characterized in terms of resistivity measurements along the microfluidic channels, electroosmotic flow (EOF) measurements at different pH values and laser-induced-fluorescence (LIF) detection of green-fluorescent protein (GFP) plugs injected across the nanocapillary membrane and into a microfluidic channel. The results indicate that the mixed polymer micro-nanofluidic multilayer chip has electrical characteristics needed for use in microanalytical systems.

Journal Article↗
PubMed2006

Initial study of two-phase laminar flow extraction chip for sample preparation for gas chromatography.

A sample preparation method for gas chromatography using a two-phase, laminar flow extraction PDMS/glass chip has been developed. A stable two-phase laminar interface was obtained by surface modification, and the organic extraction phase and the aqueous sample phase were separated effectively when the two-phase laminar flows exit the chip. Experiments were conducted on the chip to extract ephedrine from aqueous solution. Good reproducibility was obtained over the entire range of ephedrine concentration using the extraction chips (CV range 2.7%-4.5%). Effects of salt and solvent on extraction efficiency were studied.

Chromatography, Gas↗
PubMed2006

An integrated digital microfluidic chip for multiplexed proteomic sample preparation and analysis by MALDI-MS.

To realize multiplexed sample preparation on a digital microfluidic chip for high-throughput Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), several fluidic functions need to be integrated. These include the generation of multiple droplets from a reservoir and parallel in-line sample purification. In this paper, we develop two critical new functions in handling protein solutions and standard proteomic reagents with electrowetting-on-dielectric (EWOD) actuation, leading to an integrated chip for multiplexed sample preparation for MALDI-MS. The first is a voltage sequence designed to generate a series of droplets from each of the three reservoirs--proteomic sample, rinsing fluid, and MALDI reagents. It is the first time that proteomic reagents have been dispensed using EWOD in an air (as opposed to oil) environment. The second is a box-in-box electrode pattern developed to allow droplet passing over dried sample spots, making the process of in-line sample purification robust for parallel processing. As a result, parallel processing of multiple sample droplets is demonstrated on the integrated EWOD-MALDI-MS chip, an important step towards high-throughput MALDI-MS. The MS results, collected directly from the integrated devices, are of good quality, suggesting that the tedious process of sample preparation can be automated on-chip for MALDI-MS applications as well as other high-throughput proteomics applications.

Angiotensin II↗
PubMed2006

A capillary-PDMS hybrid chip for separations-based sensing of neurotransmitters in vivo.

A chip fabricated by multilayer soft lithography of poly(dimethylsiloxane) was created for separations-based sensing of neurotransmitters in vivo. The chip incorporated a pneumatically actuated peristaltic pump and valving system to combine low-flow push-pull perfusion sampling, on-line derivatization, and flow-gated injection onto an embedded fused-silica capillary for high speed separation of amine neurotransmitters from the brain of living animals. Six 160 microm wide by 10 microm high control channels, actuated with an overlapping 60 degrees pulse sequence, simultaneously drove sample and buffers through fluidic channels of the same dimensions. Tunable sampling flow rates of 40 to 130 nL min(-1) and separation buffer flow rates of 380 to 850 nL min(-1) were achieved with actuation frequencies between 3 and 10 Hz. On-line sampling of amine neurotransmitters with separation efficiencies in excess of 250,000 plates, detection limits of approximately 40 nM, and relative standard deviations of 4% for glutamate and aspartate were achieved in vitro. Electropherograms with resolution of gamma-aminobutyric acid, glutamine, taurine, serine, glycine, o-phosphorylethanolamine, glutamate, and aspartate could be collected every 30 s for over 4 h in vivo. It was also shown that pharmacological agents could be delivered and subsequent changes in neurotransmitter profile could be measured when delivering either 70 mM K+ artificial cerebrospinal fluid or 200 microM l-trans-pyrrolidine-2,4-dicarboxilic acid with the chip. These results demonstrate the ability of this chip to sample and monitor chemicals in the complex environment of the central nervous system with high selectivity and sensitivity over extended periods.

Animals↗
PubMed2002

A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos.

AGAMOUS-like-15 (AGL15) is a member of the MADS-domain family of DNA-binding regulatory factors that accumulates preferentially in tissue developing in an embryonic mode. To better understand how AGL15 functions, we developed a chromatin immunoprecipitation (ChIP) approach to isolate genes regulated directly by AGL15. ChIP allows purification of in vivo protein-DNA complexes. The co-purified DNA is recovered and used to isolate the putatively regulated gene. Several tests must be performed to show that the putative downstream target gene is truly regulated by the DNA-binding protein. The DNA-binding regulatory protein must interact with cis regulatory elements. The downstream gene expression pattern should respond to the level of the trans-acting regulatory factor. The cis element should be able to confer regulation in response to the trans-acting factor. We describe, in this report, our ChIP protocol, and discuss in detail, tests to confirm regulation by AGL15 for two targets identified by ChIP. These targets are referred to as Downstream Target of AGL15 (DTA1 and DTA2). Expression of DTA1, which encodes a protein with high similarity to GA-2 oxidase-like proteins, is induced by AGL15. DTA2 encodes a novel protein and expression of this target is repressed by AGL15.

Arabidopsis↗
PubMed2002

Evaluation of Affymetrix Gene Chip sensitivity in rat hippocampal tissue using SAGE analysis. Serial Analysis of Gene Expression.

DNA microarrays are a powerful tool for monitoring thousands of transcript levels simultaneously. However, the use of DNA microarrays in studying the central nervous system faces several challenges. These include the detection of low-abundance transcripts in highly complex tissue as well as estimating relatively low-magnitude changes in transcript levels in response to experimental manipulation. Many transcripts important to brain function have low expression levels or are expressed in relatively few cells, making them difficult to detect in the complex background of brain tissue. The aim of the present study is to evaluate the sensitivity of Gene Chip detection of transcripts in brain by using results from serial analysis of gene expression (SAGE) studies. The results of this comparison indicate that Affymetrix Gene Chips, like SAGE, only reliably detect medium- to high-abundance transcripts and that detection of low-abundance transcripts, many of which have great relevance to biological function in brain, is inconsistent. Specifically, we estimate that Gene Chips reliably detect no more than 30% of the hippocampal transcriptome when using a gross hippocampal dissection as the source tissue. This report provides the first broad evaluation of Affymetrix Gene Chip sensitivity relevant to studying the brain.

Animals↗
PubMedDate not supplied

Arthroscopic removal of dorsoproximal chip fractures of the proximal phalanx in standing horses.

OBJECTIVE: To report a technique for, and outcome after, arthroscopic removal of dorsoproximal chip fractures of a proximal phalanx in standing horses. STUDY DESIGN: Retrospective study. ANIMALS OR SAMPLE POPULATION: A total of 104 horses, 1 to 13 years of age, with a dorsoproximal chip fracture of a proximal phalanx. METHODS: Horses were restrained in standing stocks and sedated with intravenous xylazine, detomidine, or a combination of both drugs. Local analgesia was achieved with 2% mepivacaine administered intra-articularly and by subcutaneous infiltration in a crescent-shaped block dorsal to the fetlock. Sterile drapes were placed on the surgical field, and impervious drapes were used on the hoof and floor. Arthroscopic portals were created in the dorsal pouch of the metacarpophalangeal or metatarsophalangeal joints to remove chip fractures. Statistical analysis was performed on race records for all Thoroughbred racehorses and compared with previously published studies. Operative and hospitalization times were compared with those of the general hospital population, and risks associated with general anesthesia were examined. RESULTS: No major operative or postoperative complications occurred. Ninety-one percent of racehorses raced after surgery with 78% returning to race at the same or higher level. CONCLUSION: Standing arthroscopic surgery can be performed successfully to remove dorsoproximal chip fractures of the proximal phalanx. CLINICAL RELEVANCE: Standing arthroscopic surgery is a valid alternative treatment for experienced surgeons to avoid the expense and potential risks associated with general anesthesia.

Anesthesia, Local↗
PubMed2004

Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry.

DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to site-specifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence.

Alkynes↗
PubMed2005

The distribution of response spectra in the lateral geniculate nucleus compared with reflectance spectra of Munsell color chips.

This paper compares the spectral response curves of cells in the lateral geniculate nucleus (LGN) with the reflectance spectra of a large sample of Munsell color chips. By examining the color chips with methods used by neural response researchers and the LGN cells with methods used by psychophysical color researchers, we obtain insights that may be useful for advancing knowledge in both fields. For LGN cells, the prevailing view is that they tend to be clustered into distinct types or along discernible lines or planes when data obtained from selected light stimuli are represented in a three-dimensional space derived from cone contributions. In contrast, the Munsell color chips are viewed as rather evenly distributed in a three-dimensional perceptual space based on the psychophysical judgment of surface colors. We demonstrate that, when the Munsell chips are viewed in the space typically applied to LGN cells, the distribution appears similar to that of the cells and vice versa. We show why this result occurs and suggest that it has implications for studies in both fields.

Animals↗
PubMed2005

Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach.

Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. We have developed a chromatin immunoprecipitation (ChIP)-chip strategy for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. We report the use of ChEST in Drosophila to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. Our approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome.

Animals↗
PubMed2001

Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip.

A hybrid circuit of a semiconductor chip and synaptically connected neurons was implemented and characterized. Individual nerve cells from the snail Lymnaea stagnalis were immobilized on a silicon chip by microscopic picket fences of polyimide. The cells formed a network with electrical synapses after outgrowth in brain conditioned medium. Pairs of neurons were electronically interfaced for noninvasive stimulation and recording. Voltage pulses were applied to a capacitive stimulator on the chip to excite the attached neuron. Signals were transmitted in the neuronal net and elicited an action potential in a second neuron. The postsynaptic excitation modulated the current of a transistor on the chip. The implementation of the silicon-neuron-neuron-silicon circuit constitutes a proof-of-principle experiment for the development of neuroelectronic systems to be used in studies on neuronal signal processing, neurocomputation, and neuroprosthetics.

Animals↗
PubMed2003

Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips.

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30-40 min. Up to several hundred SNPs can be assayed on a 36-mm2 chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.

Biosensing Techniques↗
PubMed2003

Dimerization of the human E3 ligase CHIP via a coiled-coil domain is essential for its activity.

The Hsp70-interacting E3-ubiquitin ligase CHIP has been implicated in the decision as to whether a target protein enters the refolding or the degradation pathway. To further characterize the activity of CHIP we purified untagged Homo sapiens and Drosophila melanogaster CHIP (hCHIP, dCHIP). In contrast to other E3-ubiquitin ligases, both hCHIP and dCHIP proteins formed homodimers at physiological concentrations. We identified a predicted coiled-coil region in a mixed charge segment of the hCHIP and dCHIP sequence and found it to be necessary and sufficient for dimer formation. A mutant of hCHIP lacking this segment (hCHIPdelta-(128-229)) was incapable of dimer formation, but the segment by itself (hCHIP-(128-229)) readily dimerized. Furthermore, we demonstrated that dimerization is a prerequisite for activity of hCHIP in the reconstituted ubiquitination assay. Control of dimerization may thus provide a mechanism for regulation of CHIP activity.

Animals↗