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Biomedical subjects

J Michael Ramsey

Publications and source records attributed to J Michael Ramsey.

14 recordsLinked to original sources
PubMed2005

Microchip separations in reduced-gravity and hypergravity environments.

Microfabricated fluidics technology, e.g., lab-on-a-chip devices, offers many attractive features for performing chemistry and biochemistry on space-based platforms. We have constructed a portable, battery-operated microfluidic platform that was tested under reduced gravity and hypergravity conditions that would be experienced in space flight and launch. This device consisted of a microchip, microchip holder, two 0-8-kV high-voltage power supplies, a high-voltage switch, a solid-state diode-pumped green laser, an optical train, a channel photomultiplier, and an inertial mass measurement unit all under the control of a laptop computer and powered by 10 D-cell alkaline batteries. The unit was tested on NASA's reduced gravity research aircraft at gravity levels that are relevant to NASA's intended use of bioreporter-based microchips for environmental monitoring of space and planetary environments on manned and unmanned spacecraft. Over the course of two flights, 834 fast electrophoretic separations of four amino acids were performed under a variety of gravitational environments including zero-g, Martian-g, lunar-g, and approximately 1.8-g. All separations were performed in less than 12 s and automatically analyzed. After correction with an internal migration standard, the migration time reproducibilities were all <1% relative standard deviation.

Journal Article↗
PubMed2005

Stacking due to ionic transport number mismatch during sample sweeping on microchips.

Sample stacking can occur in isoconductive buffer systems as a result of ion transport mismatches that cause changes in buffer conductivity during electrophoresis. Fluorescence imaging was used to examine this effect in the sweeping of hydrophobic dyes with sodium dodecyl sulfate (SDS) on microchips. Imaging revealed the occurrence of a stacking effect in a sodium borate buffer system in which the sample buffer and SDS-containing run buffer had the same initial conductivity. Injected sample plugs were first swept by SDS micelles and the swept band was then stacked at the trailing end of the sample zone. This effect is due to changes in conductivity at both the front and back interfaces of the injected sample plug and can be modeled by moving boundary equations. Maximum signal enhancements of 86-, 160- and 560-fold were obtained for Rhodamine 560, Rhodamine B and Rhodamine 6G, respectively, by the combination of sweeping and stacking within a 1 cm section of microchannel. Based on sample sweeping/stacking and manipulation of the electric field polarity, a method of trapping and concentrating analyte from multiple injections was also demonstrated.

Buffers↗
PubMed2005

Static and dynamic acute cytotoxicity assays on microfluidic devices.

Static and dynamic acute toxicity assays of cells were performed on microfluidic devices where materials were hydraulically transported. Static assays were performed by incubating cells with an agent in a microchip reservoir and optically interrogating the cells after hydrodynamic focusing at a cross intersection. Dynamic assays were performed on a microchip with a 25-cm-long spiral channel where the cells were mixed with an agent and optically monitored 0.1, 12, and 22 cm from the point of mixing. The incubation time was determined by the time needed for cells to transit from the mixing location to the point of detection. Cell viability was determined using the ratio of fluorescence signals from membrane permeant (calcein) and membrane impermeant (propidium iodide) stains. The model system used in this study was the viability of Jurkat cells in the presence of the agent Triton X-100). An average LC50 value of 138 microM for Triton X-100 was obtained for an incubation period of 7-12 min using the static assay. LC50 values obtained with the dynamic assay for 25- and 47-s incubation times were 290 and 250 microM Triton X-100, respectively. Higher LC50 values for the dynamic assay were expected due to the shorter incubation times.

Cell Survival↗
PubMed2005

Preconcentration of proteins on microfluidic devices using porous silica membranes.

Fluorescently labeled proteins were electrophoretically concentrated on microfabricated devices prior to separation and laser-induced fluorescence detection on the same device. The proteins were concentrated using a porous silica membrane between adjacent microchannels that allowed the passage of buffer ions but excluded larger migrating molecules. Concentrated analytes were then injected into the separation column for analysis. Two basic microchip designs were tested that allowed sample concentration either directly in the sample injector loop or within the microchannel leading from the sample reservoir to the injector. Signal enhancements of approximately 600-fold were achieved by on-chip preconcentration followed by SDS-CGE separation. Preconcentration for CE analysis in both coated and uncoated open channels was also demonstrated. Fluorescently labeled ovalbumin could be detected at initial concentrations as low as 100 fM by using a combination of field-amplified injection and preconcentration at a membrane prior to CE in coated channels.

Electrophoresis, Capillary↗
PubMed2004

Strategy for repetitive pinched injections on a microfluidic device.

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

Electrochemistry↗
PubMed2004

High-pressure ion trap mass spectrometry.

The effects of buffer gas pressure on ion trap stability, mass resolution/calibration, and choice of mass scanning are described. Pressure effects were treated phenomenologically by adding a drag term to the ion equations of motion. The resulting collisional damping enlarges the mass-dependent stability region but reduces the region in which mass-selective resonance ejection can be performed. The pressure effects can be reduced by increasing the frequency of the alternating quadrupole field.

Journal Article↗
PubMed2004

Genetic analysis of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) and arbitrarily primed PCR: gel analysis compared with microchip gel electrophoresis.

OBJECTIVES: To assess the applicability of a newly emerging microchip gel electrophoresis for rapid strain differentiation among clinical isolates of Pseudomonas aeruginosa, and to compare this technique with the traditional gel method for DNA separation. METHODS: One hundred clinical strains of P. aeruginosa obtained from a hospital in northwestern Ohio were tested for reactivity to 3 serotype-specific monoclonal antibodies by enzyme-linked immunosorbent assay. Twelve strains (4 from each serogroup) were selected for DNA analysis by polymerase chain reaction (PCR)-based, single primer DNA fingerprinting methods with 3 different primers: 1 enterobacterial repetitive intergenic consensus PCR and 2 arbitrarily primed PCRs. The PCR products were analyzed by agarose slab gel and microchip gel electrophoresis. RESULTS: Of the 100 clinical isolates tested, 39% (4%, 14%, and 21%) were found to be serotypes 0:3, 0:6, and 0:11, respectively. Twelve strains were chosen for DNA analysis by PCR. The PCR products were analyzed by agarose slab gel electrophoresis and on microchips to determine interspecies diversity. Both methods demonstrated that different serotypes exhibited different electrophoretic patterns. Two strains (clinical strains 6 and 7, serotype 0:6) showed identical patterns, indicating a high degree of relatedness. CONCLUSION: In all cases, there was concordance between the electrophoretic patterns detected by the two methods. The capability of conducting both PCR and microchip gel electrophoresis offers an opportunity for an automated and rapid method for genetic analysis and differentiation among strains of P. aeruginosa and other microorganisms.

Bacterial Typing Techniques↗
PubMed2003

Microfluidic devices for the high-throughput chemical analysis of cells.

A microfluidic device is reported that integrated cell handling, rapid cell lysis, and electrophoretic separation and detection of fluorescent cytosolic dyes. The device function was demonstrated using Jurkat cells that were loaded with the fluorogenic dyes - carboxyfluorescein diacetate, Oregon green carboxylic acid diacetate, or Calcein AM. The loaded cells were hydrodynamically transported from the cell-containing reservoir to a region on the microfluidic device where they were focused and then rapidly lysed using an electric field. Complete lysis was accomplished in <33 ms. The hydrolyzed, fluorescent dyes in the cell lysate were automatically injected into a separation channel on the device and detected 3 mm downstream of the injection point. The total separation time was approximately 2.2 s with absolute migration time reproducibilities of <1% and efficiencies ranging from 2300 to 4000 theoretical plates. Results from 139 cells are reported. A small fraction of these cells, approximately 9%, were found to enzymatically hydrolyze the loaded dyes in a manner significantly different from the majority of the cells. Cell analysis rates of 7-12 cells/min were demonstrated and are >100 times faster than those reported using standard bench-scale capillary electrophoresis.

Algorithms↗
PubMed2003

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

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

Animals↗
PubMed2003

Analysis of volatile organic compounds in air with a micro ion trap mass analyzer.

Analysis of several volatile organic compounds in air has been demonstrated with a micro ion trap mass analyzer equipped with a semipermeable membrane sampling inlet. MS/MS of selected compounds was also shown to be feasible with the miniature ion trap and could be used to improve sensitivity by reducing background noise.

Journal Article↗
PubMed2003

Sample filtration, concentration, and separation integrated on microfluidic devices.

Microfabricated devices integrating sample filtration, solid-phase extraction, and chromatographic separation with solvent programming were demonstrated. Filtering of the sample was accomplished at the sample inlet with an array of seven channels each 1 microm deep and 18 microm wide. Sample concentration and separation were performed on channels 5 microm deep and 25 microm wide coated with a C18 phase, and elution was achieved under isocratic, step, or linear gradient conditions. For the solid-phase extraction, signal enhancement factors of 400 over a standard injection of 1.0 s were observed for a 320-s injection. Four polycyclic aromatic compounds were resolved by open channel electrochromatography in under 50 s. Chip operation was unaffected by the presence of the 5-microm silica particles at the filter entrance.

Journal Article↗
PubMed2002

Double resonance ejection in a micro ion trap mass spectrometer.

Ion ejection from a cylindrical micro ion trap by resonance excitation of the secular motion is observed to be strongly dependent on the frequency of the secular motion at resonance. Both the intensity of the ion signal and the mass resolution of the resulting mass spectrum are increased when the ion secular frequency is approximately that of a nonlinear resonance of the trap. The resonances are attributed to electrical as well as geometrical considerations.

Journal Article↗
PubMed2002

Genomes to Life "Center for Molecular and Cellular Systems": a research program for identification and characterization of protein complexes.

Goal 1 of Department of Energy's Genomes to Life (GTL) program seeks to identify and characterize the complete set of protein complexes within a cell. Goal 1 forms the foundation necessary to accomplish the other objectives of the GTL program, which focus on gene regulatory networks and molecular level characterization of interactions in microbial communities. Together this information would allow cells and their components to be understood in sufficient detail to predict, test and understand the responses of a biological system to its environment. The Center for Molecular and Cellular Systems has been established to identify and characterize protein complexes using high through-put analytical technologies.A dynamic research program is being developed that supports the goals of the Center by focusing on the development new capabilities for sample preparation and complex separations, molecular level identification of the protein complexes by mass spectrometry, characterization of the complexes in living cells by imaging techniques, and bioinformatics and computational tools for the collection and interpretation of data and formation of databases and tools to allow the data to be shared by the biological community.

Animals↗