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Biomedical subjects

J M Ramsey

Publications and source records attributed to J M Ramsey.

At least 19 recordsLinked to original sources
PubMed2001

Flow cytometry of Escherichia coli on microfluidic devices.

Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip). The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence. The E. coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz. The observed labeling efficiencies for the dyes and antibody were greater than 94%.

Cell Membrane Permeability↗
PubMed2001

Electroosmotically induced hydraulic pumping with integrated electrodes on microfluidic devices.

Electroosmotic manipulation of fluids was demonstrated using thin metal electrodes integrated within microfluidic channels at the substrate and cover plate interface. Devices were fabricated by photolithographically patterning electrodes on glass cover plates that were then bonded to polymeric substrates into which the channels were cast. Polymeric substrates were used to provide a permeable membrane for the transport and removal of gaseous electrolysis products generated at the electrodes. Electroosmotic flow between interdigitated electrodes was demonstrated and provided electric field-free pumping of fluids in sections of the channel outside of the electrode pairs. The resultant pumping velocities were shown to be dependent on the applied voltage, not on the applied field strength, and independent of the length of the electroosmotically pumped region.

Electrochemistry↗
PubMed2001

Two-dimensional electrochromatography/capillary electrophoresis on a microchip.

A two-dimensional separation system on a microfabricated device was demonstrated using open-channel electrochromatography as the first dimension and capillary electrophoresis as the second dimension. The first dimension was operated under isocratic conditions, and the effluent from the first dimension was repetitively injected into the second dimension every few seconds. A 25-cm separation channel with spiral geometry for open-channel electrochromatography was chemically modified with octadecylsilane and coupled to a 1.2-cm straight separation channel for capillary electrophoresis. Fluorescently labeled products from tryptic digests of beta-casein were analyzed in 13 min with this system.

Caseins↗
PubMed2001

On-chip proteolytic digestion and analysis using "wrong-way-round" electrospray time-of-flight mass spectrometry.

Rapid protein digestion and analysis using a hybrid microchip nanoelectrospray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupled to a disposable nanospray emitter. Reactions between substrate and enzyme (trypsin), mixed off-chip and then immediately loaded into a sample reservoir on the device, are monitored in real time following the onset of electrospray. Protein cleavage products are determined at the optimum pH for generating tryptic fragments, directly from the digestion buffer using "wrong-way-round" electrospray, i.e., monitoring (MH)+ ions from basic solutions. Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomole or even attomole quantities of analyte/spectrum using peptide mass fingerprinting, loading 0.1-2 pmol/microL of sample on the chip. The sequence coverage for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique.

Amino Acid Sequence↗
PubMed2001

Electrophoretic injection bias in a microchip valving scheme.

The pinched injection strategy, implemented on microfabricated fluidic devices (microchips), was investigated for an electrophoretic injection bias. Both the sample loading and dispensing steps were found to contribute to the injection bias whereby neutral species were injected preferentially to anionic species. In the sample loading step, neutral species filled a larger volume in the cross intersection than anionic species. Similarly, in the dispensing step, a larger volume of neutral analyte was injected than anionic analyte. Up to a 27% difference in injected volumes was observed. Fluorescently labeled amino acids were used as model analytes.

Aspartic Acid↗
PubMed2001

The ITS-2 of the nuclear rDNA as a molecular marker for populations, species, and phylogenetic relationships in Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease.

The nucleotide sequences of the rDNA second internal transcribed spacer (ITS-2) of 31 populations of 12 and 3 species of the two main Triatominae tribes Triatomini and Rhodniini, including the most important Chagas disease vectors, were obtained. Sequence comparisons and parsimony, distance, and maximum-likelihood analyses indicate that ITS-2 is a useful marker for resolving supraspecific, specific, subspecific, and even sometimes population-level relationships in Triatominae. Results were markedly different between species of Triatomini and Rhodniini, suggesting polyphyly. Phylogenetic trees support an old divergence between South American and North-Central American Triatomini and query the validity of some genera (Dipetalogaster, Psammolestes). The very low sequence variation between species of the phyllosoma complex suggests that subspecific ranking would be more appropriate. Triatoma dimidiata proves to be a clearly differentiated species, with several populations evidencing a clinal variation along a north-south axis and a population from Yucatan showing differences consistent with specific status.

Animals↗
PubMed2000

Space charge effects on resolution in a miniature ion mobility spectrometer.

Miniaturization of ion mobility spectrometry (IMS) is expected to have many advantages, as well as difficulties, in the separation of chemical species at atmospheric pressure. We report the results of studies of a miniature ion mobility spectrometer that has a drift channel 1.7 mm in diameter, the smallest cross section reported to date. The miniature cell contains a homogeneous drift field and is operated at atmospheric pressure. The miniature IMS has been characterized by measuring both negative and positive ion spectra using a frequency-quadrupled Nd: YAG laser on samples of NO, O2, and methyl iodide; a useful resolution (> 10) was achieved with an operating voltage of 500 V. Peak broadening due to Coulomb repulsion was determined to have a major effect on the resolution of the miniature device.

Journal Article↗
PubMed2000

Microchip devices for high-efficiency separations.

We have fabricated a 25-cm-long spiral-shaped separation channel on a glass microchip with a footprint of only 5 cm x 5 cm. Electrophoretic separation efficiencies for dichlorofluoroscein (DCF) on this chip exceeded 1,000,000 theoretical plates and were achieved in under 46 s at a detection point 22.2 cm from the injection cross. The number of theoretical plates increased linearly with the applied voltage, and at a separation field strength of 1,170 V/cm, the rate of plate generation was approximately 21,000 plates/s. The large radii of curvature of the turns minimized the analyte dispersion introduced by the channel geometry as evidenced by the fact that the effective diffusion coefficient of DCF was within a few percent of that measured on a microchip with a straight separation channel over a wide range of electric field strengths. A micellar electrokinetic chromatography separation of 19 tetramethylrhodamine-labeled amino acids was accomplished in 165 s with an average plate number of 280,000. The minimum resolution between adjacent peaks for this separation was 1.2.

Journal Article↗
PubMed2000

A microfabricated fluidic device for performing two-dimensional liquid-phase separations.

A microfabricated fluidic device that combines micellar electrokinetic chromatography and high-speed open-channel electrophoresis on a single structure for the rapid automated two-dimensional analysis of peptides has been devised and demonstrated. The microchip operates by rapidly sampling and analyzing effluent in the second dimension from the first dimension. Second-dimension analyses are performed and completed every few seconds, with total analysis times of less than 10 min for tryptic peptides. The peak capacity of the two-dimensional separations has been estimated to be in the 500-1000 range. The orthogonality of the separation techniques, an important factor for maximizing peak capacity or resolution elements, was verified by examining each technique independently for peptide separations. The two-dimensional separation strategy was found to greatly increase the resolving power over that obtained for either dimension alone.

Chromatography↗
PubMed2000

Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling.

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.

Electrophoresis, Capillary↗
PubMed2000

Novel microfabricated device for electrokinetically induced pressure flow and electrospray ionization mass spectrometry.

A novel microchip device for electrospray ionization has been fabricated and interfaced to a time-of-flight mass spectrometer. Fluid is electrokinetically transported through the chip to a fine fused-silica capillary inserted directly into a channel at the edge of the device. Electrospray is established at the tip of the capillary, which assures a stable, efficient spray. The electric potential necessary for electrospray generation and the voltage drop for electroosmotic pumping are supplied through an electrically permeable glass membrane contacting the fluidic channel holding the capillary. The membrane is fabricated on the microchip using standard photolithographic and wet chemical etching techniques. Performance relative to other microchip electrospray sources has been evaluated and the device tested for potential use as a platform for on-line electrophoretic detection. Sensitivity was found to be approximately three orders of magnitude better than spraying from the flat edge of the chip. The effect of the capillary on electroosmotic flow was examined both experimentally and theoretically.

Pressure↗
PubMed2000

Integrated microchip-device for the digestion, separation and postcolumn labeling of proteins and peptides.

A microchip device was demonstrated that integrated enzymatic reactions, electrophoretic separation of the reactants from the products and post-separation labeling of proteins and peptides prior to detection. A tryptic digestion of oxidized insulin B-chain was performed in 15 min under stopped flow conditions in a heated channel, and the separation was completed in 1 min. Localized thermal control of the reaction channel was achieved using a resistive heating element. The separated reaction products were then labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and detected by laser-induced fluorescence. A second reaction at elevated temperatures was also demonstrated for the on-chip reduction of disulfide bridges using insulin as a model protein. This device represents one of the highest levels, to date, of monolithic integration of chemical processes on a microchip.

Hydrolysis↗
PubMed2000

Integrated system for rapid PCR-based DNA analysis in microfluidic devices.

An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (approximately 1 min/ cycle) and efficient DNA amplification on-chip followed by electrophoretic sizing and detection on the same chip. An on-chip DNA concentration technique has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration injection scheme enables detection of PCR products after performing as few as 10 thermal cycles, with a total analysis time of less than 20 min. The starting template copy number was less than 15 per injection volume.

DNA↗
PubMed2000

Laser desorption/in situ chemical ionization aerosol mass spectrometry for monitoring tributyl phosphate on the surface of environmental particles.

The possibility of using real-time aerosol mass spectrometry (RTAMS) for the detection of surface-adsorbed tributyl phosphate (TBP) as an alkali metal adduct has been investigated. Environmental particles contain variable amounts of easily ionizable alkali metals. During laser desorption of surface-adsorbed TBP molecules, Na+ and K+ ions are generated by the interaction of the laser radiation with the particle's material. The alkali metal ions serve as in situ chemical ionization reagents of the neutral analyte molecules. The effect of laser fluence on the signal intensities of the potassium ion and cationized TBP was also studied. The best performance of the instrument was observed with laser fluences that produce high abundances of K+ but low abundances of ions from the particle's bulk material. The relatively low laser fluence, necessary to produce potassium ions, prevents the excessive fragmentation of the analyte. The instrument is capable of real-time monitoring of submonolayer coverage of TBP on the surface of micron-sized particles.

Organophosphates↗
PubMed2000

Characterization of cellular optoporation with distance.

We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation of adherent cells, three spatial zones were present which were distinguished by the viability of the cells and the loading efficiency (or number of extracellular molecules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (> 60 microns under these conditions), nearly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a substrate for protein kinase C and the cellular contents then analyzed by capillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for future work, cells were optoporated and loaded with a fluorophore in the enclosed channels of microfluidic devices.

Amino Acid Sequence↗
PubMed2000

Effects of the electric field distribution on microchip valving performance.

Valving characteristics on microfluidic devices were controlled through manipulation of the electric field strengths during both the sample loading and dispensing steps. Three sample loading profiles for the constant volume valve (pinched injection) in conjunction with four dispensing schemes were investigated to study valving performance. The sample confinement profiles for the sample loading step consisted of a weakly pinched sample, a medium pinched sample, and a strongly pinched sample. Four dispensing schemes varied the electric field strengths in the sample and sample waste channels relative to the analysis channel to control the volume of the sample dispensed from the valve. The axial extent of the sample plug decreased as the electric field strengths in the sample and sample waste channels were raised relative to the analysis channel. In addition, a trade-off existed between sample plug length and sensitivity.

Animals↗