Biomedical subjects

Edward S Yeung

Publications and source records attributed to Edward S Yeung.

At least 19 recordsLinked to original sources

PubMed2006

Adsorption of single DNA molecules at the water/fused-silica interface.

We applied total internal reflection fluorescence microscopy (TIRFM) to study intermolecular interactions at the water/fused-silica interface at the single-molecule level. Real-time molecular motion at the interface was recorded to reveal adsorption behavior and conformational dynamics of three DNAs with sticky ends of different numbers of unpaired bases. Features of DNA motion at the interface, such as evanescent-field residence time, linear velocity and frequency of adsorption/desorption events were measured to assess the relative affinities of the oligonucleotides for the surface. The general trend of stronger interaction with the surface for longer sticky ends confirmed hydrophobic interaction and hydrogen bonding as the driving forces of DNA adsorption to fused-silica at pH 5. For DNAs of different sizes, different conformational dynamics and the accessibility of sticky ends give rise to a nonlinear relationship with respect to affinity. Such information may prove valuable for chromatography studies as well as for the design of DNA microarrays and drug delivery systems.

Adsorption↗

PubMed2006

Quantitative screening of single copies of human papilloma viral DNA without amplification.

We describe a novel quantitative viral screening method based on single-molecule detection that does not require amplification. DNA of human papilloma virus (HPV), the major etiological agent of cervical cancer, served as the screening target in this study. Eight 100-nucleotide single-stranded DNA probes were designed complementary to the E6-E7 gene of HPV-16 DNA. The probes were covalently stained with Alexa Fluor 532 and hybridized to the target in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell and had a linear dynamic range of over 6 orders of magnitude. In the dual-color mode, we employed fluorescence resonance energy transfer and added YOYO-3 dye as the acceptor. The two colors from Alexa Fluor 532 and YOYO-3 were dispersed by a transmission grating located in front of the ICCD. With this reinforced criterion for identifying the hybridized molecules, zero false-positive count was achieved. We also showed that DNA extracts from Pap test specimens did not interfere with the measurements.

Cell Line, Tumor↗

PubMed2006

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits.

Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.

Apoproteins↗

PubMed2006

Single molecule adsorption at compositionally patterned self-assembled monolayers on gold: role of domain boundaries.

This paper examines the single-molecule adsorption of YOYO-I-labeled lambda-DNA at compositionally patterned self-assembled monolayers (SAMs). The interactions of fluorescently labeled lambda-DNA molecule with the patterned SAMs, which are comprised of different functional groups (i.e., amine-, alcohol-, and acid-terminated thiolates), were monitored at optically transparent gold films using total internal reflection fluorescence microscopy. The role of solution pH, lambda-DNA concentration, and domain size was investigated. In addition to delineation of the relative adsorption strength as a function of terminal group identity (NH2 > COOH > OH), the potential importance of structural defects was also revealed. The latter result, found both at the disordered boundaries between domains and at adlayers in which structural order was affected by the length of the alkyl chain, points to the subtle but preferential adsorption of the "sticky ends" of lambda-DNA. These experiments also detected an intriguing dependence of adsorption with respect to domain size.

Adsorption↗

PubMed2006

Real-time dynamics of label-free single mast cell granules revealed by differential interference contrast microscopy.

We demonstrate the capability of differential interference contrast (DIC) microscopy as a simple and useful tool for studying cellular events without fluorescence labeling. By coupling an advanced DIC microscope to a computer-controlled motorized vertical stage and a high-speed, high-resolution CCD camera, real-time three-dimensional monitoring is possible in a high-throughput manner. The performance among three modes of microscopy, bright-field, dark-field and DIC, in terms of horizontal resolving power and vertical sectioning was investigated. As a model, exocytosis of rat peritoneal mast cells was recorded on the subsecond time scale. Three-dimensional tracking of granules during degranulation was achieved and granule-granule fusion before plasma membrane fusion was recorded.

Animals↗

PubMed2006

Fabrication of optically transparent carbon electrodes by the pyrolysis of photoresist films: approach to single-molecule spectroelectrochemistry.

This paper describes the preparation, physical and chemical characterization, and performance of carbon-based optically transparent electrodes (C-OTEs) fabricated by the pyrolysis of thin films of photoresist. The electrodes are prepared by spin coating dilute solutions of the positive photoresist AZ 4330 onto quartz substrates. Pyrolysis of these samples at 1000 degrees C in a reducing atmosphere yields optically transparent carbon films that have thicknesses ranging between 10 and 80 nm. Sheet resistance measurements, X-ray photoelectron spectroscopy, Raman spectroscopy, and atomic force microscopy were used to determine the physical and chemical properties of the films, and cyclic voltammetry and chronoabsorptometry were employed to delineate the electrochemical and conventional spectroelectrochemical performance of the C-OTEs. These findings showed that the transparency of this material improves as film thickness decreases, but at the expense of an increase in film resistance. At a wavelength of 500 nm, for example, 13- and 79-nm-thick films have transparencies of 47 and 10% and sheet resistances of 1100 and 210 Omega/ square, respectively. Importantly, adjusting the dilution factor allows the facile and reproducible variation of thickness and transparency. Preliminary results using these C-OTEs for single-molecule spectroelectrochemistry, which represents a new development in the merger of optical and electrochemical techniques, by probing the potential dependence of the adsorption of individual YOYO-I-labeled lambda-DNA are also presented.

Journal Article↗

PubMed2006

Ultrasensitive native fluorescence detection of proteins with miniaturized polyacrylamide gel electrophoresis by laser side-entry excitation.

Direct detection of separated proteins inside polyacrylamide gels has many advantages compared to staining methods. Ultrasensitive native fluorescence detection of proteins with miniaturized 1-D and 2-D PAGE was achieved with laser side-entry excitation. The detection limit for R-phycoerythrin protein spots in 1-D SDS-PAGE with 532 nm excitation was as low as 15 fg, which corresponds to only 40,000 molecules. The average detection limit of six standard native proteins was 5 pg per band with 275 nm excitation. The dynamic range spanned more than three orders of magnitude. By using the same detection setup, approximately 150 protein spots from 30 ng of total Escherichia coli extraction were detected on a 0.8 cm x 1 cm gel in 2-D separation. The significant improvement in sensitivity for laser side-entry excitation comes from higher excitation power and lower background level compared with other excitation modes.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Atypical mobilities of single native DNA molecules in microchip electrophoresis revealed by differential interference contrast microscopy.

A transmitted-light optical microscope using differential interference contrast (DIC) was employed to follow the real-time dynamics of different kb-sized single native dsDNA molecules without fluorescent-dye labeling. In a PDMS/glass microchip, the electrophoretic migration velocities of large dsDNA molecules are lower than small dsDNA molecules in a running buffer of 0.25% v/v nonionic polymeric surfactant C16E6 (n-alkyl polyoxyethylene ether) in 100 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer because the C16E6 behaved as a dynamic polymer. However, the order of migration reversed in 50 mM Gly-Gly buffer. The radial positions of individual DNA molecules (i.e., center or walls of the microchip) did not change the migration order. The atypical migration order correlated well with the results in CE. The alignment of the deformable molecules due to viscous drag is likely responsible for these observations.

Buffers↗

PubMed2005

Automated high-throughput nanoliter-scale protein crystallization screening.

A highly efficient method is developed for automated high-throughput screening of nanoliter-scale protein crystallization. The system integrates liquid dispensing, crystallization and detection. The automated liquid dispensing system handles nanoliters of protein and various combinations of precipitants in parallel to access diverse regions of the phase diagram. A new detection scheme, native fluorescence, with complementary visible-light detection is employed for monitoring the progress of crystallization. This detection mode can distinguish protein crystals from inorganic crystals in a nondestructive manner. A gas-permeable membrane covering the microwells simplifies evaporation rate control and probes extended conditions in the phase diagram. The system was successfully demonstrated for the screening of lysozyme crystallization under 81 different conditions.

Automation↗

PubMed2005

Prototype for integrated two-dimensional gel electrophoresis for protein separation.

Two-dimensional gel electrophoresis practitioners have long waited for a fully automated system. This article presents an integrated platform that is capable of complete automation from sample introduction to spots detection. The strip gel for the first dimensional separation is fixed on the edge of a discrete planar stage before separation. A pair of platinum pin electrodes for isoelectric focusing (IEF) makes contact from underneath the stage. IEF is performed directly after rehydration and protein loading. After the first dimensional separation, sodium dodecyl sulfate (SDS) equilibration is done on the same stage without moving the gel. The IEF stage is then moved horizontally to couple with a precast second dimensional gel. The <0.5 mm gap between the two gels is filled with poly (ethylene oxide) solution. After SDS-polyacrylamide gel electrohporesis separation, a charge-coupled device camera is used to detect spots via protein native fluorescence excited by a Hg (Xe) lamp with the gel inside the running cell. Potential for full automation is demonstrated with 0.5 microg of Escherichia coli proteins on this miniaturized platform. More than 240 spots are detected in a total experiment time of <2.5 h.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2005

Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe.

A novel CE-based noncompetitive immunoassay for prion protein (PrP) was established. Fluorescein isothiocyanate (FITC)-labeled protein A (FITC-PrA) was used as a fluorescent probe to tag monoclonal antibody through noncovalent binding of FITC-PrA to the Fc region of the antibody. The FITC-PrA-Ab was incubated with the analyte, prion protein, under optimized condition, forming the immunocomplex FITC-PrA-Ab-PrP. The complex was separated and analyzed by capillary zone electrophoresis. The addition of carboxymethyl-beta-cyclodextrin in the running buffer as dynamical coating reagent improved the reproducibility and the resolution. The complex was isolated in less than 1 min with theoretical plates of 3.8 x 10(4). Relative standard deviations of peak height and migration time for the complex were 3.46 and 1.48%, respectively. A linear relationship was established for the bovine recombinant prion protein (rPrP) concentration in the range from 0.2 to 2.0 mug/mL and the peak height. The correlation factor was r2 = 0.9969. The estimated detection limit for rPrP was approximately 6 ng/mL, which is 3 times the signal-to-noise ratio. The method was successfully applied for testing blood samples from scrapie-infected sheep.

Affinity Labels↗

PubMed2005

Direct observation of anomalous single-molecule enzyme kinetics.

We report the direct measurement of the single-molecule enzymatic cleavage rates of ApaI-DNA complex in the presence of various concentrations of MgCl2 solution with total internal reflection fluorescence microscopy. We made use of the native adsorption properties of the two 12-base sticky ends of the DNA molecules to partially immobilize and stretch out the ApaI-DNA complex onto a glass surface. Synchronous initiation of reaction was achieved by the influx of Mg2+ solution. Once the DNA was cut, the two fragments (38 and 10 kb) would either collapse or further stretch out depending on the solution flow. The time required for cleaving each ApaI-lambda-DNA complex was recorded and analyzed. At low concentrations, the higher the concentration of Mg2+, the faster the DNA was cut. However, Mg2+ ion is no longer the limiting factor when its concentration is greater than 5 mM. A surprising result is that at all concentrations the decrease in intact DNA population as a function of time is linear rather than exponential. This suggests that there exists a distribution of ApaI conformations around the restriction site.

DNA↗

PubMed2005

Single DNA molecules as probes of chromatographic surfaces.

YOYO-I-labeled lambda-DNA was employed as a nanoprobe for different functionalized surfaces to elucidate adsorption in chromatography. While the negatively charged backbone is not adsorbed, the 12-base unpaired ends of this DNA provide exposed purine and pyrimidine groups for adsorption. Self-assembled monolayers (SAMs) formed on gold substrate provide a wide range of choices of surface with well-defined and well-organized functional groups. Patterns of amino-terminated, carboxylic acid-terminated, and hydroxyl-terminated SAMs are generated by lithography. Patterns of metal oxides are generated spontaneously after deposition of metals. By recording the real-time dynamic motion of DNA molecules at the SAMs/aqueous interface, one can study the various parameters governing the retentivity of an analyte during chromatographic separation. Even subtle differences among adsorptive forces can be revealed.

Adsorption↗

PubMed2005

Motion of single DNA molecules at a liquid-solid interface as revealed by variable-angle evanescent-field microscopy.

A variable-angle total-internal-reflection fluorescence microscope (VATIRFM) capable of providing a large range of incident angles was constructed for imaging single DNA molecule dynamics at a solid/liquid interface. An algorithm using a public-domain image-processing program, ImageJ, was developed for single-molecule counting. The experimental counts at various incident angles with different evanescent-field layer (EFL) thicknesses are affected by molecular diffusion. The dynamics of molecules near the surface and the observed counts in the VATIRFM are elucidated using a limited one-dimensional random-walk diffusion model. The simulation fits well with the experimental counting results. Further analysis using the simulation reveals the details of single-molecule motion. One implication is that the measured intensities cannot be used directly to determine the distances of molecules from the surface, though the majority of fluorescence does come from the EFL. Another implication is that rather than providing molecular concentrations within EFL the experimental counting results depict the distance-dependent dynamics of molecules near the surface. Thus, the VATIRFM could be a powerful technique to study the surface repulsion/attraction of molecules within a few hundred nanometers of the surface. Further studies show that molecules at low ionic strengths experience electrostatic repulsion at distances much further away from the surface than the calculated thickness of the electrical double layer.

DNA↗

PubMed2005

Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation.

The development of capillary electrophoresis (CE)-based competitive immunoassay for prion protein (PrP) using carboxymethyl beta-cyclodextrin (CM-beta-CD) as a buffer additive is described here. The assay was based on the competitive binding of PrP and a fluorescein-labeled peptide from the prion protein with a limiting amount of specific antibody. The amount of both free and fluorescein-labeled peptide bound to antibody (immunocomplex) were determined by CE with laser-induced fluorescence detection. In the presence of PrP, the peak height ratio of the immunocomplex and the free peptide was altered compared to the control. These changes were directly proportional to the amount of PrP present. The fluorescently labeled peptide spanning amino acid positions 140-158 of the PrP and its corresponding monoclonal antibody is reported here. The reaction times of the antibody with either the peptide or the recombinant PrP was less than 1 min and is a large improvement over the 16-18 h required to achieve equilibrium for polyclonal antibodies. CM-beta-CD was explored as a buffer additive to suppress analyte adsorption and enhance separation selectivity in the CE analysis. A fast (1.1 min), selective (resolution 4.7), and reproducible (relative standard deviations of migration time for free and bound fluorescein isothiocyanate (FITC)-peptide 0.56% and 0.64%, respectively) separation was obtained with 0.6% CM-beta-CD in 25 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) at pH 8.8. The concentration detection limit of the assay for recombinant PrP was determined to be 80 ng/mL (or mass detection limit 1 pg). When blood samples from scrapie-infected sheep and from normal sheep were tested, the results of the blood assay were consistent with scrapie status of the sheep as determined post mortem by Western blot analysis. Development of this assay will lead to a potentially robust, rapid, and specific preclinical diagnosis for transmissible spongiform encephalopathies (TSEs) in animals and humans.

Animals↗

PubMed2005

Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis.

A continuous-wave 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection of proteins in CE. The operating current and temperature of the LED were optimized in order to achieve high luminescence power. It was found that a forward current of 30 mA and a temperature of approximately 5 degrees C gave the best S/N. By using a set of two ball lenses to focus light from the LED, we achieved a spot of approximately 200 mum with a power of 0.1-0.2 mW on the detection window. Fluorescence was collected with a ball lens at 90 degrees angle through a bandpass filter onto a photomultiplier tube. In CZE an LOD of 20 nM for conalbumin was reached. In capillary gel electrophoresis all eight proteins from a commercial standard kit were detected with high S/N. For a 10 microg/mL total protein mixture, S/N was better than 3 for all proteins in solution. Further improvement in LOD should be possible on utilization of an LED with higher luminescence power.

Electrophoresis, Capillary↗

PubMed2005

In-Situ probing of the biotic-abiotic boundary of plants by laser desorption/ionization time-of-flight mass spectrometry.

Laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry was applied for the direct analysis of cuticular waxes on intact plant tissues. Cuticular wax compounds were ionized by laser desorption in the presence of colloidal silver. Silver-adduct ions were detected on samples from Arabidopsis thaliana and from maize. Good spot-to-spot reproducibility indicated homogeneous coverage of the sample by the fine colloidal material. The results were consistent with GC-MS analyses of cuticular extracts, thus confirming the feasibility of direct analysis based on this protocol. Molecular masses of the adduct ions correspond well with the known composition of cuticular waxes. Moreover, LDI-TOF gave good estimates of the relative local abundances of a given compound. However, bias was found in cases where compounds with different ionization efficiencies were analyzed.

Arabidopsis↗

PubMed2005

High-throughput single-cell fluorescence spectroscopy.

A high-throughput method for measuring single-cell fluorescence spectra is presented. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera simultaneously recorded the zero and the first orders of the fluorescence from each cell. Single-cell fluorescence spectra were reconstructed from the distance between zero-order and first-order maxima as well as the length and the pixel intensity distribution of the first-order images. By using this approach, the emission spectrum of E. coli cells expressing green fluorescent protein (GFP) was reconstructed. Also, fluorescence spectra of E. coli cells expressing non-fluorescent apo-subunits of R-phycoerythrin (R-PE) were recorded after incubation of the cells with phycoerythrobilin (PEB) chromophore. The fluorescence spectra are in good agreement with results obtained on the same cells using a fluorescence spectrometer or a fluorescence microscope. When spectra are to be acquired, this approach has a higher throughput, better sensitivity, and better spectral resolution compared to flow cytometry.

Bacterial Proteins↗