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Biomedical subjects

Paul W Bohn

Publications and source records attributed to Paul W Bohn.

23 records · Page 2Linked to original sources
PubMed2004

Surface adsorption and transfer of organomercaptans to colloidal gold and direct identification by matrix assisted laser desorption/ionization mass spectrometry.

The adsorption characteristics of two different organomercaptan adsorbates, 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA) and the peptide Cys-Lys-Trp-Ala-Lys-Trp-Ala-Trp (CKWAKWAK), on colloidal Au were studied, and the conjugates produced were characterized by UV-vis spectroscopy, transmission electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Fluorescence difference measurements of free thiols in solution were used to assemble surface adsorption isotherms on Au colloid revealing surface coverages of 1.0 x 10(14) molecules cm(-2) for SAMSA and 3.1 x 10(14) molecules cm(-2) for CKWAKWAK. The free energies of adsorption were calculated to be -48.4 kJ/mol for SAMSA and -49.2 kJ/mol for CKWAKWAK. UV-visible absorption spectroscopy and transmission electron microscopy reveal that the thiol/colloid conjugates flocculate under conditions where the net charge per colloid is small or neutral and that flocculated colloids can be resuspended by a change in pH to more basic for the acidic SAMSA/Au conjugates or to more acidic for the basic CKWAKWAK/Au conjugates. The reversible flocculation allows the conjugates to be readily separated from free adsorbate in solution and thereby prepared for further characterization. CKWAKWAK/colloid conjugates were analyzed by MALDI-MS, and the mass spectra show (M + H)(+), (M + Na)(+), and (M + K)(+) ions attributable to the peptide. The manipulations studied here constitute a powerful complement to microfluidic-based separation and analysis methods. Conjugating mass-limited analytes to Au colloids makes it possible to sequester and transfer small quantities of analytes with high efficiency.

Adsorption↗
PubMed2004

Quantitative submonolayer spatial mapping of Arg-Gly-Asp-containing peptide organomercaptan gradients on gold with matrix-assisted laser desorption/ionization mass spectrometry.

Peptides containing the tripeptide sequence Arg-Gly-Asp (RGD) have the ability to bind to members of the integrin superfamily of cell-surface receptors and direct cellular adhesion and haptotaxis. The goal of this work is the development of a rapid and effective method for the quantitative submonolayer spatial composition mapping of surfaces displaying molecular assemblies of RGD-containing organomercaptan peptides on a Au surface using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS). Quantitation of the RGD peptide is achieved by determining the peak intensity of the protonated molecular ion, (M + H)+, relative to the (M + H)+ peak for an internal standard, which is similar chemically but with glutamic acid (E) substituted for aspartic acid (D). Using optimized sample preparation procedures, a bilinear calibration was obtained between the quantitative peak intensity ratio and the mole fraction of the RGD-containing peptide. Quantitative compositions were determined with relative standard deviations of <10%, even in the presence of 10x spot-to-spot variations in the absolute signal intensities, by using this internal standard approach. This MALDI-MS quantitative analysis method was employed to probe variable-width two-component counterpropagating electrochemically generated gradients of the two peptides, prepared by coupling in-plane electrochemical potential gradients with the electrosorption reactions of organothiols to vary the composition laterally. The measured lateral composition profiles match the quasi-linear potential gradient model and yield profiles that overlap to a high degree of fidelity in potential space. Thus, MALDI-MS spatial composition mapping should become a powerful tool for the preparation of designed surfaces facilitating the study of cellular adhesion and motility and cell-cell interactions.

Gold↗
PubMed2003

Nanocapillary array interconnects for gated analyte injections and electrophoretic separations in multilayer microfluidic architectures.

An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyses. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyses.

Arginine↗
PubMed2003

Gateable nanofluidic interconnects for multilayered microfluidic separation systems.

The extension of microfluidic devices to include three-dimensional fluidic networks allows complex fluidic and chemical manipulations but requires innovative methods to interface fluidic layers. Externally controllable interconnects, employing nuclear track-etched polycarbonate membranes containing nanometer-diameter capillaries, are described that produce hybrid three-dimensional fluidic architectures. Controllable nanofluidic transfer is achieved by controlling applied bias, polarity, and density of the immobile nanopore surface charge and the impedance of the nanocapillary array relative to the microfluidic channels. Analyte transport between vertically separated microchannels has three stable transfer levels, corresponding to zero, reverse, and forward bias. The transfer can even depend on the properties of the analyte being transferred such as the molecular size, illustrating the flexible character of the analyte transfer. In a specific analysis implementation, nanochannel array gating is applied to capillary electrophoresis separations, allowing selected separated components to be isolated for further manipulation, thereby opening the way for preparative separations at attomole analyte mass levels.

Electrophoresis, Capillary↗