Timothy J. Shepodd

Reversed-phase electrochromatography of amino acids and peptides using porous polymer monoliths

Efficient and rapid separation of minute levels of amino acids and bioactive peptides is of significant importance in the emerging field of proteomics as well as in the clinical and pharmaceutical arena. We have developed novel UV-initiated acrylate-based porous polymer monoliths as stationary phases for capillary- and chip-electrochromatography of cationic, anionic, and neutral amino acids and peptides, followed by absorbance or laser-induced fluorescence detection. The rigid monoliths are cast-to-shape and are tunable for charge and hydrophobicity. For separations at low pH, monoliths containing quaternary amine moieties were used to achieve high electroosmotic flow, and for high pH separations monoliths with acidic sulfonic acid groups were employed. Efficient and reproducible separations of phenylthiohydantoin-labeled amino acids, native peptides, and amino acids and peptides labeled with naphthalene-2,3-dicarboxaldehyde (NDA) were achieved using both negatively- and positively-charged polymer monoliths in capillaries. Separation efficiencies in the range of 65,000-371,000 plates/m were obtained with capillary electrochromatography. Buffer composition and the degree of column hydrophobicity were studied systematically to optimize separations. The monoliths were also cast in the microchannels of glass chips and electrochromatographic separation followed by laser-induced fluorescence detection of three NDA-labeled bioactive peptides was obtained.

Voltage-addressable on/off microvalves for high-pressure microchip separations

We present a microchip-based, voltage-addressable on/off valve architecture that is fundamentally consistent with the pressures and solvents employed for high-pressure liquid chromatography. Laser photopatterning of polymer monoliths inside glass microchannels is used to fabricate mobile fluid control elements, which are opened and closed by electrokinetic pressures. The glass substrates and crosslinked polymer monoliths operate in water-acetonitrile mixtures and have been shown to hold off pressures as high as 350 bar (5000 p.s.i.). Open/closed flow ratios of 10(4) to 10(6) have been demonstrated over the pressure range 1.5-70 bar (20-1000 p.s.i.), and the pressure-leak relationship shows the potential for valving control of flow through packed or monolithic chromatography columns. We expect that this valve platform will enable multiplexing of multiple chromatographic separations on single microchips.

Microfluidic routing of aqueous and organic flows at high pressures: fabrication and characterization of integrated polymer microvalve elements

This paper presents the first systematic engineering study of the impact of chemical formulation and surface functionalization on the performace of free-standing microfluidic polymer elements used for high-pressure fluid control in glass microsystems. System design, chemical wet-etch processes, and laser-induced polymerization techniques are described, and parametric studies illustrate the effects of polymer formulation, glass surface modification, and geometric constraints on system performance parameters. In particular, this study shows that highly crosslinked and fluorinated polymers can overcome deficiencies in previously-reported microvalve architectures, particularly limited solvent compatibility. Substrate surface modification is shown effective in reducing the friction of the polymer-glass interface and thereby facilitating valve actuation. A microchip one-way valve constructed using this architecture shows a 2 x 10(8) ratio of forward and backward flow rates at 7 MPa. This valve architecture is integrated on chip with minimal dead volumes (70 pl), and should be applicable to systems (including chromatography and chemical synthesis devices) requiring high pressures and solvents of varying polarity.

Mobile Flow Control Elements for High-Pressure Micro-Analytical Systems Fabricated Using in-Situ Polymerization

A method for rapidly fabricating a family of robust fluid control elements in microfluidic channels is presented. The polymer devices are lithographically defined in situ in glass microfluidic channels in a few seconds on a benchtop. The devices are capable of controlling fluid flow in microchannels at pressures exceeding 5000 psi (340 bar) and can be actuated in milliseconds. In this work we demonstrate chip-based devices, including a piston, check-valve, and a 10 nanoliter pipette.

Microfluidic Gene Arrays for Rapid Genomic Profiling

Genomic analysis tools have recently become an indispensable tool for the evaluation of gene expression in a variety of experiment protocols. Two of the main drawbacks to this technology are the labor and time intensive process for sample preparation and the relatively long times required for target/probe hybridization. In order to overcome these two technological barriers we have developed a microfluidic chip to perform on chip sample purification and labeling, integrated with a high density genearray. Sample purification was performed using a porous polymer monolithic material functionalized with an oligo dT nucleotide sequence for the isolation of high purity mRNA. These purified mRNA’s can then rapidly labeled using a covalent fluorescent molecule which forms a selective covalent bond at the N7 position of guanine residues. These labeled mRNA’s can then released from the polymer monolith to allow for direct hybridization with oligonucletide probes deposited in microfluidic channel. To allow for rapid target/probe hybridization high density microarray were printed in microchannels. The channels can accommodate array densities as high as 4000 probes. When oligonucleotide deposition is complete, these channels are sealed using a polymer film which forms a pressure tight seal to allow sample reagent flow to the arrayed probes. This process will allow for real time target to probe hybridization monitoring using a top mounted CCD fiber bundle combination. Using this process we have been able to perform a multi-step sample preparation to labeled target/probe hybridization in less than 30 minutes. These results demonstrate the capability to perform rapid genomic screening on a high density microfluidic microarray of oligonucleotides.

Rapid Separation of Peptides and Amino Acids in Glass Microchips by Reversed-Phase Electrochromatography

We have developed microfabricated glass chips for reversed-phase separation followed by laser-induced fluorescence detection of peptides and amino acids. Hydrophobic acrylate-based porous polymer monoliths were cast in the channels by photopolymerization. Use of UV-light for initiation of polymerization allows for selective patterning of stationary phase in the channels for optimal design of injection and detection regions. Charged functionalities such as either sulfonic acids or quaternary amines were incorporated during polymerization for generation of electroosmotic flow. These monoliths can be cast in situ in less than 10 minutes, are very reproducible with respect to separation characteristics, and allow easy manipulation of separation parameters such as charge, hydrophobicity, and pore size. Moreover, the solvent used to cast the polymer is chosen so as to allow electroosmotic flow to flush the channels thereby obviating any need for application of high pressures for conditioning. Rapid (50 seconds) and efficient (up to 600,000 plates/m) separations of peptides and amino acids were achieved in the microchip.

Savannah River Site/K Area Complex getter life extension report.

The K Area Complex (KAC) at the Savannah River Site (SRS) has been utilizing HiTop hydrogen getter material in 9975 Shipping Containers to prevent the development of flammable environments during storage of moisture-containing plutonium oxides. Previous testing and subsequent reports have been performed and produced by Sandia National Laboratories (SNL) to demonstrate the suitability and longevity of the getter during storage at bounding thermal conditions. To date, results have shown that after 18 months of continuous storage at 70 C, the getter is able to both recombine gaseous hydrogen and oxygen into water when oxygen is available, and irreversibly getter (i.e. scavenge) hydrogen from the vapor space when oxygen is not available, both under a CO{sub 2} environment. [Refs. 1-5] Both of these reactions are catalytically enhanced and thermodynamically favorable. The purpose of this paper is to establish the justification that maintaining the current efforts of biannual testing is no longer necessary due to the robust performance of the getter material, the very unlikely potential that the recombination reaction will fail during storage conditions in KAC, and the insignificant aging effects that have been seen in the testing to date.

Biological detection and tagging using tailorable, reactive, highly fluorescent chemosensors.

This program was focused on the development of a fluorogenic chemosensor family that could tuned for reaction with electrophilic (e.g. chemical species, toxins) and nucleophilic (e.g. proteins and other biological molecules) species. Our chemosensor approach utilized the fluorescent properties of well-known berberine-type alkaloids. In situ chemosensor reaction with a target species transformed two out-of-plane, weakly conjugated, short-wavelength chromophores into one rigid, planar, conjugated, chromophore with strong long wavelength fluorescence (530-560 nm,) and large Stokes shift (100-180 nm). The chemosensor was activated with an isourea group which allowed for reaction with carboxylic acid moieties found in amino acids.

Performance testing of aged hydrogen getters against criteria for interim safe storage of plutonium bearing materials.

Hydrogen getters were tested for use in storage of plutonium-bearing materials in accordance with DOEs Criteria for Interim Safe Storage of Plutonium Bearing Materials. The hydrogen getter HITOP was aged for 3 months at 70 C and tested under both recombination and hydrogenation conditions at 20 and 70 C; partially saturated and irradiated aged getter samples were also tested. The recombination reaction was found to be very fast and well above the required rate of 45 std. cc H2h. The gettering reaction, which is planned as the backup reaction in this deployment, is slower and may not meet the requirements alone. Pressure drop measurements and {sup 1}H NMR analyses support these conclusions. Although the experimental conditions do not exactly replicate the deployment conditions, the results of our conservative experiments are clear: the aged getter shows sufficient reactivity to maintain hydrogen concentrations below the flammability limit, between the minimum and maximum deployment temperatures, for three months. The flammability risk is further reduced by the removal of oxygen through the recombination reaction. Neither radiation exposure nor thermal aging sufficiently degrades the getter to be a concern. Future testing to evaluate performance for longer aging periods is in progress.

Development of porous polymer monoliths for reverse-phase chromatography of proteins.

The polymers developed in this project are intended for use as a stationary phase in reverse-phase chromatography of proteins, where the mobile phase is a solution of acetonitrile and a phosphate buffer, 6.6 pH. A full library of pore sizes have been developed ranging from 0.41{micro}m to 4.09 {micro}m; these pore sizes can be determined by the solvent ratio of tetrahydrofuran:methoxyethanol during polymerization. A column that can separate proteins in an isocratic mode would be a vast improvement from the common method of separating proteins through gradient chromatography using multiple solvents. In the stationary phase, the main monomers have hydrophobic tails, lauryl acrylate and steryl acrylate. Separations of small hydrophobic molecules and peptides (trial molecules) have efficiencies of 24,000-33,000 theoretical plates m{sup -1}. The combination of a highly non-polar stationary phase and a mobile phase where the polarity can be controlled provide for excellent separation.