Biomedical subjects

Gil U Lee

Publications and source records attributed to Gil U Lee.

7 recordsLinked to original sources

PubMed2006

Nanoliter-scale reactor arrays for biochemical sensing.

A general approach is described for array-based biochemical sensing that uses contact-free dispersal of compounds into addressable microfabricated reactors. The arrays are composed of 1 to 100 nL volume open reactors that have been microfabricated on quartz substrates using lithography. The open architecture of these reactors allows them to be addressed in parallel or individually with an ink-jet arrayer that is capable of distributing 0.004 to 1 nL volumes of reagents. A seven-step biochemical assay has been conducted on a small array of reactors to demonstrate how they can be integrated with an ink-jet arrayer and optical detector. This nanoreactor assay format appears to overcome several limitations that chip-based microarray technology currently imposes on protein assays: the arrays can be created in a manner that does not expose the biochemical reagents to osmotic stress, independent reactions can be conducted in individual reactors, and the conditions in all of the reactors (e.g., concentration and pH) can be rapidly scanned. We believe that these nanoreactor arrays will be useful for biochemical sensing that involves delicate proteins and protein assemblies.

Biosensing Techniques↗

PubMed2006

Properties of mixed lipid monolayers assembled on hydrophobic surfaces through vesicle adsorption.

Supported lipid films are becoming increasingly important tools for the study of membrane protein function because of the availability of high-sensitivity surface analytical and patterning techniques. In this study, we have characterized the physical chemical properties of lipid films assembled on hydrophobic surfaces through the spontaneous adsorption of large unilamellar lipid vesicles composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). The density of the lipid films was measured with surface plasmon resonance spectroscopy as the lipid composition of the vesicles and ionic concentration were varied. As expected, monolayer films were formed, but the density of the monolayers was found to be weakly dependent on the lipid composition of the vesicles and strongly dependent on the ionic concentration of the solution in contact with the monolayer. Atomic force microscopy (AFM) images of the lipid films indicate that they are composed of a homogeneous monolayer. Surface force measurements were used to determine the surface charge and DOPG density of the monolayers. The DOPG content of the films was found to be weakly dependent on the DOPG composition of the vesicles and strongly dependent on the salt concentration of the environment. A model has been developed to describe the behavior of the lipid composition of the films in terms of the hydrophobic, electrostatic, and steric forces acting on the lipid monolayer on the hydrophobic surface.

Adsorption↗

PubMed2006

Synthesis and characterization of paramagnetic microparticles through emulsion-templated free radical polymerization.

A novel method is described for the preparation of high-magnetization paramagnetic microparticles functionalized with a controlled density of poly(ethylene glycol) (PEG) and carboxyl groups. These microparticles were synthesized using four steps: (1) creation of an oil-in-water emulsion in which hydrophobic iron oxide nanoparticles and a UV-activated initiator were distributed in hexane; (2) formation of uniform microparticles through emulsion homogenization and evaporation of hexane; (3) functionalization of the microparticle with a PEG-functionalized surfactant and acrylic acid; and (4) polymerization of the microparticles. Characterization of the microparticles with electron microscopy and light scattering revealed that they were composed of densely packed iron oxide nanoparticles and that the size of the microparticles may be controlled through the pore size of the membrane used to homogenize the emulsion. The concentration of surfactant and acrylic acid used in the third processing step was found to determine the surface chemistry, iron content, and magnetization of the microparticles. Increasing the PEG surfactant to acrylic acid ratio resulted in higher PEG surface densities, lower iron content, and lower magnetization. The resulting microparticles were readily functionalized with antibodies and showed a low propensity for nonspecific protein adsorption. We believe that these microparticles will be useful for magnetic tweezers measurements and bioanalytical devices that require microparticles with a high magnetization.

Journal Article↗

PubMed2006

High-resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy.

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three-dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force-distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F-actin bundles are stiffer than surrounding F-actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions.

Actins↗

PubMed2005

Mesoporous membrane device for asymmetric biosensing.

An array of micron size reactors has been designed and fabricated on a mesoporous membrane to create a platform for asymmetric biochemical sensing. Fabrication of this device required that a technique be developed to integrate the mesoporous alumina membrane with a polymeric layer that maintains the integrity of the membrane surface and permeability. This device was used to control an enzyme reaction at the surface of the membrane through the diffusion of the substrates from the opposite sides of the membrane. Asymmetric reactions promise new modes of sensing, enhanced stability of delicate biomolecular systems, and enhanced sensitivity and speed in sensing.

Aluminum Oxide↗

PubMed2002

Dissociation of multiple protein ion charge states following a single gas-phase purification and concentration procedure.

The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone. This approach is illustrated using off-resonance collisional activation of the [M + 8H]8+ to [M + 6H]6+ precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the [M + 8H]8+ charge state. This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the [M + 12H]12+ to [M + 5H]5+ ions. Gas-phase ion/ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra. The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages. Maximum structural information is obtained by fragmentation of the intermediate-charge states.

Amino Acid Sequence↗

PubMed2002

Gas-phase concentration, purification, and identification of whole proteins from complex mixtures.

Five proteins present in a relatively complex mixture derived from a whole cell lysate fraction of E. coli have been concentrated, purified, and dissociated in the gas phase, using a quadrupole ion trap mass spectrometer. Concentration of intact protein ions was effected using gas-phase ion/ion proton-transfer reactions in conjunction with mass-to-charge dependent ion "parking" to accumulate protein ions initially dispersed over a range of charge states into a single lower charge state. Sequential ion isolation events interspersed with additional ion parking ion/ion reaction periods were used to "charge-state purify" the protein ion of interest. Five of the most abundant protein components present in the mixture were subjected to this concentration/purification procedure and then dissociated by collisional activation of their intact multiply charged precursor ions. Four of the five proteins were subsequently identified by matching the uninterpreted product ion spectra against a partially annotated protein sequence database, coupled with a novel scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential cleavage sites. The identification of these proteins illustrates the potential of this "top-down" protein identification approach to reduce the reliance on condensed-phase chemistries and extensive separations for complex protein mixture analysis.

Amino Acid Sequence↗