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At least 451 records · Page 25Linked to original sources
PubMed2004

Electrotransfection of mammalian cells using microchannel-type electroporation chip.

Transfection of DNA molecules into mammalian cells with electric pulsations, which is so-called electroporation, is a powerful and widely used method that can be directly applied to gene therapy. However, very little is known about the basic mechanisms of DNA transfer and cell response to the electric pulse. We developed a microelectroporation chip with poly(dimethylsiloxane) (PDMS) to investigate the mechanism of electroporation as a first step of DNA transfer and to introduce the benefits of miniaturization into the genetic manipulation. The microelectroporation chip has a microchannel with a height of 20 microm and a length of 2 cm. Owing to the transparency of PDMS, we could in situ observe the uptake process of propidium iodide (PI) into SK-OV-3 cells, which shows promise in visualization of gene delivery in living cells. We also noticed the geometric effect on the degree of electroporation in microchannels with diverse channel width. This experimental result shows that the geometry can be another parameter to be considered for the electroporation when it is performed in microchannels with an exponential decaying pulse generator. Cell culturing is possible within the microelectroporation chip, and we also successfully transfected SK-OV-3 cells with enhanced green fluorescent protein genes, which demonstrates the feasibility of the microelectroporation chip in genetic manipulation.

Animals↗
PubMed2004

On-chip nanoliter-volume multiplex TaqMan polymerase chain reaction from a single copy based on counting fluorescence released microchambers.

A novel method for multiplex TaqMan PCR in nanoliter volumes on a highly integrated silicon microchamber array is described. Three different gene targets, related to beta-actin, sex-determining region Y (SRY), and Rhesus D (RhD) were amplified and detected simultaneously on the same chip by using three different types of human genomic DNA as the templates. The lack of cross-contamination and carryover was shown using alternate dispensing of mineral oil-coated microchambers containing template and those without template. To confirm the specificity of our system to beta-actin, SRY, and RhD genes, we employed the larger volume PCR samples to a commercial real-time PCR system, SmartCycler. The samples were cycled with the same sustaining temperatures as with the microchamber array. Instead of the conventional method of DNA quantification, counting the number of the fluorescence released microchambers in consequence to TaqMan PCR was employed to our chip. This simple method of observing the end point signal had provided a dynamic quantitative range. Stochastic amplification of 0.4 copies/reaction chamber was achieved. The microfabricated PCR chip demonstrated a rapid and highly sensitive response for simultaneous multiple-target detection, which is a promising step toward the development of a fully integrated device for the "lab-on-a-chip" DNA analysis.

Actins↗
PubMed2006

UV embossed polymeric chip for protein separation and identification based on capillary isoelectric focusing and MALDI-TOF-MS.

This paper demonstrates a ultraviolet (UV)-embossed polymeric chip for protein separation by capillary isoelectric focusing (CIEF) and identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The polymeric chip was replicated by a UV-embossing technique using a soft rubber mold. Five diverse widely investigated families of UV-cured formulations were examined for MALDI ionization efficiency of bovine serum albumin (BSA) samples spotted on the polymer surfaces. The signal and signal-to-noise ratio from the polyester formulation were each 12 times those obtained with PMMA (a commonly used polymer material for MALDI) at picomole sample concentration. A polyester chip was successfully used to carry out CIEF to separate proteins, followed by MALDI-TOF-MS identification. Issues related to the successful chip fabrication and protein separation and identification are discussed.

Electrophoresis, Capillary↗
PubMed2006

Improved sensitivity and physical properties of sol-gel protein chips using large-scale material screening and selection.

Protein chips are a powerful emerging technology with extensive biomedical applications. However, the development of optimal, economical surface materials capable of maintaining the activity of embedded proteins is a challenge. Here, we introduce a new optimized, low-cost, sol-gel biomaterial for use in protein chips with femtogram-level sensitivity. A novel protein chip material with significantly improved physical properties and sensitivity was produced using unique screening and selection methods. Using this platform, the sensitive, specific detection of the interactions between an HIV antigen and its antibody and between a cyclin-kinase protein pair was observed. This study is the first to demonstrate the detection of protein-protein interactions on sol-gel microarrays and describes an important improvement in the physical properties of sol-gel-derived protein chip materials for biomedical research.

Antibodies↗
PubMed2000

A dc microplasma on a chip employed as an optical emission detector for gas chromatography

A micromachined plasma chip is coupled to a conventional gas chromatograph to investigate its performance as an optical emission detector. The device employs a 180-nL plasma chamber in which an atmospheric pressure dc glow discharge is generated in helium. Applied power is 9 mW (770 V, 12 microA) and helium flow rate 320 nL s(-1). A number of carbon-containing compounds are detected in the column effluent by recording the emission at 519 nm. For hexane, the detector has a linear dynamic range of over two decades and a minimum detectability of 10(-12) g s(-1) (800 ppb). The detector signal shows a marked peak broadening and tailing when compared with the signal of a flame ionization detector. This is mainly attributed to dead volumes and chromatographic processes introduced by the connecting tubing and the chip glass channels. The device was operated for more than 24 h without a significant change in performance. Operation is stable and instrumental requirements are simple. Future use of the detector chip in conventional gas chromatography or as an integrated detector in on-chip gas chromatography is discussed.

Journal Article↗
PubMed2005

Building addressable libraries: the use of electrochemistry for spatially isolating a heck reaction on a chip.

Pd(0) was generated at preselected sites on an electrochemically addressable chip and then utilized to effect a Heck reaction. The Pd(0) was confined to the preselected electrodes with the use of allylmethyl carbonate. Unlike most mediated electrochemical reactions, the electrolysis in this case was not used to convert a stoichiometric process into a catalytic one by recycling the metal. Instead, the unique environment of the chip was used to interfere with a catalytic process to make it stoichiometric. This was done to gain spatial control over the reaction. The development of a strategy for conducting Pd(0)-catalyzed reactions on the chips should greatly expand the synthetic chemistry available for building chip-based libraries.

Combinatorial Chemistry Techniques↗
PubMed2004

Building addressable libraries: the use of electrochemistry for generating reactive Pd(II) reagents at preselected sites on a chip.

A Pd(II) reagent has been generated at preselected sites on an electrochemically addressable chip. The reagent was used to effect the Wacker oxidation of an olefin substrate bound to the chip near the electrode. The use of ethyl vinyl ether in the solution above the chip effectively kept the Pd(II) reagent generated at the preselected electrode from migrating to neighboring electrodes and initiating Wacker oxidations at unwanted sites on the chip.

2,4-Dinitrophenol↗
PubMed2006

Building addressable libraries: spatially isolated, chip-based reductive amination reactions.

A Pd(II) reagent has been generated at preselected sites on an electrochemically addressable chip and used to effect the oxidation of the neighboring alcohols on the polymer coating the chip's surface. The resulting carbonyls were then used to accomplish site-selective reductive amination reactions on the chips. The work demonstrates that the confinement strategy developed for spatially isolated Wacker oxidations to specific sites on the chips is general and can be used for other Pd(II)-based reactions.

Alcohols↗
PubMed2002

Flavor and texture of banana chips dried by combinations of hot air, vacuum, and microwave processing.

The behavior of 16 volatile compounds of banana during a combination of air-drying (AD) and vacuum microwave-drying (VMD) of banana chips was characterized. Samples were AD to remove 60, 70, 80, or 90% of moisture (wet basis) and then subjected to VMD to achieve a final moisture content of 3% (dry basis). Banana slices were also dehydrated using only AD, VMD, and freeze-drying (FD) for comparison. Samples that underwent more VMD had significantly lower levels of volatile compounds, which is attributed to the decreased formation of an impermeable solute layer on the surface of the chips. High values for water solubility and relative volatility of compounds correlated with losses during VMD; however, additional factors appear to influence the behavior of compounds during VMD processing. The optimal process of 90%AD/10%VMD yielded crisper banana chips with significantly higher volatile levels and sensory ratings than AD chips.

Desiccation↗
PubMed1998

Phase I trial of edatrexate plus carboplatin in advanced solid tumors: amelioration of dose-limiting mucositis by ice chip cryotherapy.

PURPOSE: Edatrexate (10-Edam) is a methotrexate analog with improved intracellular transport, polyglutamation, and antitumor activity compared to the parent compound. Edatrexate shows schedule-dependent synergism with platinum compounds in preclinical studies. We performed a phase I trial to determine toxicities and establish the maximum tolerated dose (MTD) of edatrexate in combination with carboplatin. Based on the short initial plasma half-life of edatrexate, prophylactic ice chip cryotherapy was used to reduce the severity of mucositis. PATIENTS AND METHODS: Forty-six chemotherapy-naive patients with advanced solid tumors were treated. Edatrexate was given weekly for 5 doses (50% on day 8), and then every other week, followed by carboplatin at a fixed dose of 350 mg/m2 on day 1 and then every 4 weeks for 8 cycles. Edatrexate dose was increased at increments of 10 mg/m2/dose level beginning at 60 mg/m2/week (range 60-120 mg/m2). RESULTS: All patients were assessable for toxicity and response analysis. The median number of cycles administered per patients was 4. This combination chemotherapy regimen was well tolerated. Using ice chip cryotherapy, no grade IV mucositis was observed. Grade III mucositis occurred in only 7/46 pts and was not dose-related. Protocol-defined dose-limiting toxicity occurred at a edatrexate dose level of 120 mg/m2, yielding an MTD of 110 mg/m2. Responding tumor types included non-small cell and small lung cancer, head and neck cancer, and bladder cancer. CONCLUSIONS: 1 ) This phase I study demonstrated the safety and tolerability of this edatrexate and carboplatin combination. 2) Dose-limiting mucositis did not occur allowing escalation of edatrexate dose above levels previously achieved with this edatrexate dose schedule. This was most likely a result of prophylactic ice chip cryotherapy. 3) An edatrexate dose of 110 mg/m2 with ice chip cryotherapy is recommended for Phase II trials of this combination.

Adult↗
PubMed2003

Evaluation of antioxidant activity of methanolic extract from peanut hulls in fried potato chips.

Antioxidant activity of methanolic extract from peanut hulls (MEPH) was evaluated in fried potato chips during storage at 25 degrees C and 45 degrees C. Free fatty acids (FFA) and peroxide values (POV) were used as criteria to assess MEPH as an antioxidant. After six months storage at 45 degrees C potato chips treated with 1200 and 1600 ppm of MEPH showed lower values of FFA (0.083, 0.080%) and POV (30.0, 29.0 mEq/kg) than the control samples (FFA 0.377%, and POV 180 mEq/kg). Potato chips treated with BHA and BHT showed POVs of 29.0 and 25.0 mEq/kg whereas FFA values were 0.0086 and 0.074%, respectively, after six months storage at 45 degrees C. These results illustrate that MEPH, at various concentrations, exhibited very strong antioxidant activity which was almost equal to synthetic antioxidants (BHA & BHT). Subjective evaluation studies also showed that potato chips treated with 1200-1600 ppm MEPH after six months storage at 45 degrees C, were organoleptically acceptable.

Antioxidants↗
PubMed2004

Microfluidic ELISA: on-chip fluorescence imaging.

Fluorescent reactions of a heterogeneous sandwich enzyme-linked immunoassay (ELISA) in an all-PDMS [poly (dimethylsiloxane)] microfluidic device were detected using a cooled charge coupled device (CCD) camera interfaced with an epifluorescence microscope. The study represents preliminary efforts to integrate biochemical reactions and detection on-chip using the "hybrid" detection approach. In initial experiments, the PDMS chip microsensor was successfully used to quantify a model analyte (sheep IgM) with sensitivity down to 17nM. Thus, we demonstrate here the extension of this hybrid integrated technique to on-chip imaging and quantification of light emission from a biochemical immunoassay in PDMS chip.

Animals↗
PubMed2004

Dead volume free micro-machined electro-spray chip for mass spectrometry.

Design and fabrication themes of a dead volume free electro-spray chip for mass spectrometry based on mechanical micro-machining approach is discussed in this article. To implement the dead volume free concept, a cantilever needle is embedded inside the micro-channel of the spray-chip such that there is no connecting joint between the sample wall and the spray-tip. To ensure that the proposed spray-chip is able to produce efficient Taylor cone, embedded needles with different cone angles are fabricated to produce an efficient experiment. Experiments to distinguish amphetamine by a LCQ type mass spectrometry are conducted. Experimental results show that the proposed dead volume free electro-spray chip is able to reach the desired goal. It is also found from the experiments that the ringlike cross-section of the needle embedded micro-channel is critical to the stability of the Taylor cone at the spray-tip. To ensure a stable Taylor cone can be produced at the spray-tip, an embedded needle with a smaller diameter is desired.

Amphetamine↗
PubMed2004

Negative dielectrophoretic force assisted construction of ordered neuronal networks on cell positioning bioelectronic chips.

Developing new methods and technologies in order to pattern neurons into regular networks is of utmost scientific interest in the field of neurological research. An efficient method here is developed for trapping neurons and constructing ordered neuronal networks on bioelectronic chips by using arrayed negative dielectrophoretic (DEP) forces. A special bioelectronic chip with well defined positioning electrode arrays was designed and fabricated on silicon substrate. When a high frequency AC signal was applied, the cell positioning bioelectronic chip (CPBC) is able to provide a well-defined non-uniform electric field, and thus generate negative DEP forces. The parameters, such as size of positioning electrode, conductivity of working solution, amplitude and frequency of power signal and cell concentration, were investigated to optimize the performance of the CPBC. When the neuron suspension was added onto the energized bioelectronic chip, the neurons were immediately trapped and quickly formed the predetermined pattern. Neurons may adhere and then be cultured directly on the CPBC, and show good neuron viability and neurite development. The formation of the ordered neuronal networks after two-week culture demonstrates that negative dielectrophoretic force assisted construction of ordered neuronal networks is effective, and it could be used to assist in monitoring functional activities of neuronal networks.

Animals↗
PubMed2001

The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation.

The folding of both wild-type and mutant forms of the cystic-fibrosis transmembrane-conductance regulator (CFTR), a plasma-membrane chloride-ion channel, is inefficient. Most nascent CFTR is retained in the endoplasmic reticulum and degraded by the ubiquitin proteasome pathway. Aberrant folding and defective trafficking of CFTRDeltaF508 is the principal cause of cystic fibrosis, but how the endoplasmic-reticulum quality-control system targets CFTR for degradation remains unknown. CHIP is a cytosolic U-box protein that interacts with Hsc70 through a set of tetratricorepeat motifs. The U-box represents a modified form of the ring-finger motif that is found in ubiquitin ligases and that defines the E4 family of polyubiquitination factors. Here we show that CHIP functions with Hsc70 to sense the folded state of CFTR and targets aberrant forms for proteasomal degradation by promoting their ubiquitination. The U-box appeared essential for this process because overexpresion of CHIPDeltaU-box inhibited the action of endogenous CHIP and blocked CFTR ubiquitination and degradation. CHIP is a co-chaperone that converts Hsc70 from a protein-folding machine into a degradation factor that functions in endoplasmic-reticulum quality control.

Animals↗
PubMed2000

Analysis of yeast protein kinases using protein chips.

We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.

Adenosine Triphosphate↗
PubMed2002

Peptide chips for the quantitative evaluation of protein kinase activity.

Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. Despite the promise of these chips, their development for quantitative assays has been limited by several factors, including a lack of well-defined surface chemistries to immobilize peptides, the heterogeneous presentation of immobilized ligands, and nonspecific adsorption of protein to the substrate. This paper describes a peptide chip that overcomes these limitations, and demonstrates its utility in activity assays of the nonreceptor tyrosine kinase c-Src. The chip was prepared by the Diels-Alder-mediated immobilization of the kinase substrate AcIYGEFKKKC-NH(2) on a self-assembled monolayer of alkanethiolates on gold. Phosphorylation of the immobilized peptides was characterized by surface plasmon resonance, fluorescence, and phosphorimaging. Three inhibitors of the enzyme were quantitatively evaluated in an array format on a single, homogeneous substrate.

Amino Acid Sequence↗
PubMed1998

Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips.

Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

Biosensing Techniques↗