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At least 307 records · Page 17Linked to original sources
PubMed2004

Fabrication of poly(methyl methacrylate) microfluidic chips by atmospheric molding.

A greatly simplified method for fabricating poly(methyl methacrylate) (PMMA) separation microchips is introduced. The new protocol relies on UV-initiated polymerization of the monomer solution in an open mold under ambient pressure. Silicon microstructures are transferred to the polymer substrate by molding a methyl methacrylate solution in a sandwich (silicon master/Teflon spacer/glass plate) mold. The chips are subsequently assembled by thermal sealing of the channel and cover plates. The new fabrication method obviates the need for specialized replication equipment and reduces the complexity of prototyping and manufacturing. Variables of the fabrication process were assessed and optimized. The new method compares favorably with common fabrication techniques, yielding high-quality devices with well-defined channel and injection-cross structures, and highly smoothed surfaces. Nearly 100 PMMA chips were replicated using a single silicon master, with high chip-to-chip reproducibility (relative standard deviations of 1.5 and 4.7% for the widths and depths of the replicated channels, respectively). The relatively high EOF value of the new chips (2.12 x 10(-4) cm(2) x V(-1) x s(-1)) indicates that the UV polymerization process increases the surface charge and hence enhances the fluidic transport. The attractive performance of the new CE microchips has been demonstrated in connection with end-column amperometric and contactless-conductivity detection schemes. While the new approach is demonstrated in connection with PMMA microchips, it could be applied to other materials that undergo light-initiated polymerization. The new approach brings significant simplification of the process of fabricating PMMA devices and should lead to a widespread low-cost production of high-quality separation microchips.

Journal Article↗
PubMed2005

Microfluidic selection and retention of a single cardiac myocyte, on-chip dye loading, cell contraction by chemical stimulation, and quantitative fluorescent analysis of intracellular calcium.

A microfluidic method to study the contraction of a single cardiac myocyte (heart muscle cell) has been developed. This method integrates various single-cell operations as well as on-chip dye loading, and quantitative analysis of intracellular calcium concentration, [Ca2+]i. After the channel enlargement by on-chip etching to accommodate large-sized cardiac myocytes, a single cell is selected and retained at a V-shaped cell retention structure within the microchip. Owing to the fragile property of the cardiac myocytes that could easily be damaged by centrifugation, the calcium-sensitive fluorescent dye was loaded in the cell by on-chip dye loading. This on-chip method minimized the damage to the cells from the use of a centrifuge in the conventional method and provided a way of cellular analysis of fragile cells. Subsequently, quantitative analysis of [Ca2+]i of a single cardiac myocyte by fluorescence measurement was achieved for the first time in a microfluidic chip, thanks to the intracellular calcium stimulant of ionomycin. The resting [Ca2+]i of the cardiomyocyte determined was consistent with the literature value. From the spontaneous contraction study, it was found that fluorescence intensity cannot represent the [Ca2+]i variation accurately, which implied the importance of the quantitative analysis of [Ca2+]i.

Animals↗
PubMed2005

Multiple and simultaneous detection of specific bacteria in enriched bacterial communities using a DNA microarray chip with randomly generated genomic DNA probes.

A DNA microarray chip for detecting the presence of specific bacterial strains was developed using random genomic probes derived from genomic DNA, i.e., without any sequence information. Thirteen bacteria from different genuses were selected as targets. For the fabrication of the random genomic probes, genomic DNA from pure cultures of each bacterium was fractionated using several pairs of restriction endonucleases. After size fractionation of the genomic DNA fragments, random genomic libraries for each bacterium were constructed. From the library, specific probes were amplified by PCR and the probes were affixed to a slide glass to fabricate the DNA microarray chip. The results from tests with pure and mixed cultures of the bacteria used in the fabrication of the chips showed specific responses and only a small portion of cross-hybridization. This DNA microarray chip was also tested to detect the presence of specific bacteria in mixed populations. In these tests, it was demonstrated that this system provided a fast and specific response to the presence of bacterial species in mixed samples, even in activated sludge samples. This indicates that any DNA microarray chip for the detection of specific bacteria can be fabricated using the same protocols as presented in this study without requiring any genus level sequence information from pure isolates.

Bacterial Typing Techniques↗
PubMed2005

Hand-held microanalytical instrument for chip-based electrophoretic separations of proteins.

The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).

Electrophoresis, Microchip↗
PubMed2004

Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing.

A new method for on-chip sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins is reported. Miniaturization of SDS-PAGE has attracted significant attention because it offers rapid analysis times, excellent resolution, high throughput, and the potential for integration and automation, as compared to conventional counterparts. The presented on-chip SDS-PAGE technique employed photolithographically patterned, cross-linked gels fabricated in situ in <20 min. The effects of sieving gel composition on the migration properties of fluorescently labeled protein standards (ranging in molecular weight from 14.2 to 66 kDa) were quantified, as was the ability of the gels to function as a sieving matrix for biologically relevant species. Ferguson analysis was employed to calculate retardation coefficients and free solution mobilities. In conjunction with fluorescence imaging, the on-chip SDS-PAGE separation mechanism was evaluated in terms of separation performance indexes, as well as limiting behaviors (i.e., free solution mobility, exclusion characteristics). The photolithographically fabricated gels employed for on-chip SDS-PAGE allowed rapid (<30 s) separations of proteins in short separation lengths (4 mm) with efficiencies as high as 4.41 x 10(5) plates/m. The on-chip SDS-PAGE separations were approximately 100 times faster than conventional slab gel SDS-PAGE (60 min) and occurred in a fraction of the separation length required by slab gels. The migration behavior of protein standards correlated well with molecular weight and allowed molecular weight determination for interleukin-2, fibroblast growth factor, insulin-like growth factor, and tetanus toxin C-fragment.

Cross-Linking Reagents↗
PubMed2000

A micromachined chip-based electrospray source for mass spectrometry.

A micromachining process is described for fabricating a mass spectrometry electrospray source on a silicon chip. The process utilizes polymer (parylene) layers to form a system of chambers, filters, channels, and hollow needle structures (electrospray emitters) that extend more than a millimeter beyond the edge of the silicon substrate. The use of photoresist as the sacrificial layer facilitates the creation of long channels. Access to the channel structures on the chip is through a port etched through the silicon substrate that also serves as a sample reservoir. A reusable chip holder consisting of two plastic plates and an elastomer gasket provides the means to mount the chip in front of the mass spectrometer inlet and make electrical and gas connections. The electrospray emitters have tapered tips with 5 microns x 10 microns rectangular openings. The shape of the tip can be varied depending on the shape of the mask used to protect the parylene structures during the final plasma etch. The parylene emitters are physically robust and require only a high electric field to achieve stable electrospray operation over a period of a few hours. Direct comparisons with conventional glass or fused silica emitters indicated very similar performance with respect to signal strength and stability, spectral quality, and endurance. The automated MS/MS analysis of a mixture of tryptic peptides was no more difficult and yielded nearly identical results as the analysis of the same sample using a conventional nanospray device. This work demonstrates that an efficient electrospray interface to mass spectrometry can be integrated with other on-chip structures and mass-produced using a batch process.

Mass Spectrometry↗
PubMed2005

Regulation of cytochrome P450 2E1 by heat shock protein 90-dependent stabilization and CHIP-dependent proteasomal degradation.

Alcohol-inducible cytochrome P450 2E1 (CYP2E1) has the most rapid turnover of any member of this large family of membrane-bound oxygenases, and its degradation rate is altered profoundly by various substrates, such as ethanol and CCl(4). CYP2E1 is degraded by the ubiquitin-proteasome pathway, and because the hsp90/hsp70-based chaperone machinery is often involved in maintaining the balance between protein integrity and degradation by this pathway, we have asked whether CYP2E1 is regulated by the chaperone machinery. We show here that treatment of transformed human skin fibroblasts stably expressing CYP2E1 with the hsp90 inhibitor radicicol results in CYP2E1 degradation that is inhibited by the proteasome inhibitor lactacystin. Immunoadsorption of hsp90 from cytosol of HEK cells expressing the truncated CYP2E1(Delta3-29) yields coadsorption of CYP2E1(Delta3-29). Cotransfection of HEK cells with both the truncated CYP2E1 and the hsp70-dependent E3 ubiquitin ligase CHIP results in CYP2E1(Delta3-29) degradation, and CYP2E1(Delta3-29) co-immunoadsorbs with myc-CHIP from cytosol of cotransfected cells. Purified, bacterially expressed CYP2E1(Delta3-29) is ubiquitylated in a CHIP-dependent manner when it is incubated with a purified system containing the E1 ubiquitin activating enzyme, E2, and CHIP. CYP2E1 is the first P450 shown to be an hsp90 "client" protein that can be ubiquitylated by the hsp70-dependent E3 ubiquitin ligase CHIP. Our observations lead to a general model of how substrates, such as ethanol, can regulate the interaction of CYP2E1 with the chaperones hsp90 and hsp70 to profoundly alter enzyme turnover.

Acetylcysteine↗
PubMed2006

Structure and interactions of the helical and U-box domains of CHIP, the C terminus of HSP70 interacting protein.

The heat-shock proteins Hsp70 and Hsp90 play a crucial role in regulating protein quality control both by refolding and by preventing the aggregation of misfolded proteins. It has recently been shown that Hsp70 and Hsp90 act not only in protein refolding but also cooperate with the C terminus of Hsp70 interacting protein (CHIP), a multidomain ubiquitin ligase, to mediate the degradation of unfolded proteins. We present the crystal structure of the helical linker domain and U-box domain of zebrafish CHIP (DrCHIP-HU). The structure of DrCHIP-HU shows a symmetric homodimer. The conformation of the helical linker domains and the relative positions of the helical and U-box domains differ substantially in DrCHIP-HU from those in a recently published structure of an asymmetric dimer of mammalian (mouse) CHIP. We used an in vitro ubiquitination assay to identify residues, located on two long loops and a central alpha helix of the CHIP U-box domain, that are important for interacting with the ubiquitin-conjugating enzyme UbcH5b. In addition, we used NMR spectroscopy to define a complementary interaction surface located on the N-terminal alpha helix and the L4 and L7 loops of UbcH5b. Our results provide insights into conformational variability in the domain arrangement of CHIP and into U-box-mediated recruitment of UbcH5b for the ubiquitination of Hsp70 and Hsp90 substrates.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Carotenoid content, physicochemical, and sensory qualities of deep-fried carrot chips as affected by dehydration/rehydration, antioxidant, and fermentation.

Carrot slices were subjected to one of the following experiments prior to deep-frying: (A) dehydration/rehydration, (B) soaking in different antioxidants, and (C) fermentation with/without blanching. There were no significant differences (P > or = 0.05) in carotenoid contents among carrot chips treated with/without dehydration. Soaking in sodium metabisulfite resulted in the highest carotenoid content and lightness (L), redness (a), and yellowness (b) values among the antioxidant treatments. Fermentation without blanching significantly decreased (P < 0.05) carotenoid content, vitamin A activity, and fat content. Dehydration and fermentation with blanching significantly increased (P < 0.05) the lightness (L), redness (a), and yellowness (b) values of the chips. Dehydration/rehydration, but not antioxidant and fermentation, significantly decreased (P < 0.05) the water activity of the chips. The textural values of carrot chips prepared using sodium metabisulfite, without dehydration and without fermentation, were the lowest among other treatments which suggests the crispiest. Carrot chips prepared using sodium metabisulfite, without dehydration and without fermentation, had the highest carotenoid content and retention, and the highest overall acceptability score.

Antioxidants↗
PubMed2002

Changes in compositional parameters of tubers of potato (Solanum tuberosum) during low-temperature storage and their relationship to chip processing quality.

A four-year study of a number of compositional parameters of potato tubers during low-temperature storage was conducted to examine the compositional differences between cold-tolerant (low sugar-accumulating) and cold-sensitive (high sugar-accumulating) tubers in relation to potato chip processing quality. Compositional parameters analyzed included sucrose, reducing sugars, nitrogen, protein, ascorbic acid, and dry matter content. Pearson correlation analysis of the data illustrated that chip color was most closely correlated with reducing sugar concentration. Multiple regression analysis revealed that the relative contribution of each parameter to chip color varied greatly among the cultivars and selections evaluated and from season to season. This analysis demonstrates that the quantitative relationships between the measured compositional parameters and chip color were not sufficient to provide a general predictive index of chip color quality for tubers processed directly from low-temperature storage.

Ascorbic Acid↗
PubMed2000

Physicochemical, microbiological and sensory changes in stored plantain chips.

Plantain chips were produced from unripe plantain fruits. The chips were packed in polyethylene bags and stored at 30 +/- 2 degrees C for 1, 2 and 3 months, respectively. The storage stability of the chips was evaluated by analyzing periodically for changes in the physicochemical, microbiological and sensory characteristics. Protein and ash contents of the chips did not change (p > 0.05) over time. Fat decreased slightly (p > 0.05) over time. Vitamin C decreased and was negatively correlated with storage time (r = -95; p < 0.05). Peroxide value and microbial count increased with storage (p < 0.05). Sensory scores for chips did not change (p > 0.05) at 2 months storage, but, thereafter, decreased (p < 0.05).

Fats↗
PubMed2003

Chip ligating human genomic DNA serves as storage material and template for polymerase chain reaction.

A chip was developed to store DNA for medical research. The optional restriction site fixed on the chip can randomly ligate with whole human genomic DNA treated by the corresponding restriction enzyme. PCR can then use the chip as template DNA. Moreover, a chip fixing two restriction sites (e.g. EcoRI and HindIII) showed the amplification by PCR for any location of genomic DNA. Repetitive PCRs have confirmed that a DNA chip can be stored by at -4 degrees C for 2 years.

DNA↗
PubMed2004

Development and evaluation of a protein microarray chip for diagnosis of hepatitis C virus.

A protein chip diagnostic kit was developed for the diagnosis of hepatitis C virus (HCV) based on the protein chip technique and the immuno-concentration method. This kit was designed for low-density protein chips and also for the availability of multiple sample screening. Applicability of the chip was evaluated using 96 blood specimens and the results were compared to results of an anti-HCV enzyme immunoassay (EIA) test. With further development, the technology associated with the development of this chip could be applied to the simultaneous detection of multiple protein-protein, protein-ligand interactions.

Enzyme-Linked Immunosorbent Assay↗
PubMed2001

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins.

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.

Animals↗
PubMed2001

Smart single-chip gas sensor microsystem.

Research activity in chemical gas sensing is currently directed towards the search for highly selective (bio)chemical layer materials, and to the design of arrays consisting of different partially selective sensors that permit subsequent pattern recognition and multi-component analysis. Simultaneous use of various transduction platforms has been demonstrated, and the rapid development of integrated-circuit technology has facilitated the fabrication of planar chemical sensors and sensors based on three-dimensional microelectromechanical systems. Complementary metal-oxide silicon processes have previously been used to develop gas sensors based on metal oxides and acoustic-wave-based sensor devices. Here we combine several of these developments to fabricate a smart single-chip chemical microsensor system that incorporates three different transducers (mass-sensitive, capacitive and calorimetric), all of which rely on sensitive polymeric layers to detect airborne volatile organic compounds. Full integration of the microelectronic and micromechanical components on one chip permits control and monitoring of the sensor functions, and enables on-chip signal amplification and conditioning that notably improves the overall sensor performance. The circuitry also includes analog-to-digital converters, and an on-chip interface to transmit the data to off-chip recording units. We expect that our approach will provide a basis for the further development and optimization of gas microsystems.

Journal Article↗
PubMed1986

Evaluation of the biochemical effects of CHIP in normal and tumour-bearing C3H mice.

The biochemical effects of CHIP have been studied in C3H mice with and without transplanted mammary tumour. The maximum tolerated dose of CHIP was first determined by lethality and intestinal crypt assays to be 40 mg kg-1 and this dose was used to assay the time course of gastric distension and the pattern of drug distribution. A high level of CHIP uptake was found in liver as well as kidney. For this reason, tests for both kidney and liver damage were undertaken up to 60 days post-treatment using a dose of 10 mg kg-1 Neoplatin for comparison. Despite the high level of platinum drug uptake in liver, there was no biochemical evidence of hepatocellular or cholestatic damage. From the renal point of view, there was the expected rise in serum urea after Neoplatin but not after CHIP and there was also a rise in urinary NAG after Neoplatin in tumour bearing mice. There was, however, evidence of suppression of protein levels including enzymes, following treatment with both drugs. Tumour-bearing mice respond differently from normal mice following treatment with platinum drugs. The study confirms that CHIP is less toxic than Neoplatin.

Acetylglucosaminidase↗
PubMed2005

Development of anangiogenesis-focused cDNA chip and validation of its functionality.

DNA chip has been used as a powerful tool to study the genetic reprogramming of cells and its link to cellular phenotype such as angiogenesis. To evaluate the angiogenesis related genetic reprogramming more efficiently, we here developed an angiogenesis-focused cDNA chip containing 153 angiogenesis related genes arrayed in duplicate on a slide glass. In order to validate the functionality of the angiogenesis-focused cDNA chip, we examined gene expression profiles in HT1080 cells treated with either fetal bovine serum, a well known pro-angiogenic factor, or trichostatin A, a known angiogenesis inhibitor, using the cDNA chip. All duplicate data from the analysis are well matched with each other and gene expression profiles are well consistent with previously reported data. These results demonstrate that the angiogenesis-focused cDNA chip developed here can be a useful tool towards angiogenesis- related researches.

Angiogenesis Inducing Agents↗
PubMed2006

CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70.

Exposure of cells to various stresses often leads to the induction of a group of proteins called heat shock proteins (HSPs, molecular chaperones). Hsp70 is one of the most highly inducible molecular chaperones, but its expression must be maintained at low levels under physiological conditions to permit constitutive cellular activities to proceed. Heat shock transcription factor 1 (HSF1) is the transcriptional regulator of HSP gene expression, but it remains poorly understood how newly synthesized HSPs return to basal levels when HSF1 activity is attenuated. CHIP (carboxy terminus of Hsp70-binding protein), a dual-function co-chaperone/ubiquitin ligase, targets a broad range of chaperone substrates for proteasomal degradation. Here we show that CHIP not only enhances Hsp70 induction during acute stress but also mediates its turnover during the stress recovery process. Central to this dual-phase regulation is its substrate dependence: CHIP preferentially ubiquitinates chaperone-bound substrates, whereas degradation of Hsp70 by CHIP-dependent targeting to the ubiquitin-proteasome system occurs when misfolded substrates have been depleted. The sequential catalysis of the CHIP-associated chaperone adaptor and its bound substrate provides an elegant mechanism for maintaining homeostasis by tuning chaperone levels appropriately to reflect the status of protein folding within the cytoplasm.

Animals↗