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Hung-Wing Li

Publications and source records attributed to Hung-Wing Li.

18 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

On-chip titration of an anticoagulant argatroban and determination of the clotting time within whole blood or plasma using a plug-based microfluidic system.

This paper describes extending plug-based microfluidics to handling complex biological fluids such as blood, solving the problem of injecting additional reagents into plugs, and applying this system to measuring of clotting time in small volumes of whole blood and plasma. Plugs are droplets transported through microchannels by fluorocarbon fluids. A plug-based microfluidic system was developed to titrate an anticoagulant (argatroban) into blood samples and to measure the clotting time using the activated partial thromboplastin time (APTT) test. To carry out these experiments, the following techniques were developed for a plug-based system: (i) using Teflon AF coating on the microchannel wall to enable formation of plugs containing blood and transport of the solid fibrin clots within plugs, (ii) using a hydrophilic glass capillary to enable reliable merging of a reagent from an aqueous stream into plugs, (iii) using bright-field microscopy to detect the formation of a fibrin clot within plugs and using fluorescent microscopy to detect the production of thrombin using a fluorogenic substrate, and (iv) titration of argatroban (0-1.5 microg/mL) into plugs and measurement of the resulting APTTs at room temperature (23 degrees C) and physiological temperature (37 degrees C). APTT measurements were conducted with normal pooled plasma (platelet-poor plasma) and with donor's blood samples (both whole blood and platelet-rich plasma). APTT values and APTT ratios measured by the plug-based microfluidic device were compared to the results from a clinical laboratory at 37 degrees C. APTT obtained from the on-chip assay were about double those from the clinical laboratory but the APTT ratios from these two methods agreed well with each other.

Anticoagulants↗
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

Synthesis, structure and dioxygen reactivity of a bis(micro-iodo)dicopper(I) complex supported by the [N-(3,5-di-tert-butyl-2-hydroxybenzyl)-N,N-di-(2-pyridylmethyl)]amine ligand.

The air-sensitive bis(micro-iodo)dicopper(I) complex 1 supported by [N-(3,5-di-tert-butyl-2-hydroxybenzyl)-N,N-di-(2-pyridylmethyl)]amine (L) has been prepared by treating copper(I) iodide with L in anhydrous THF. Compound 1 crystallizes as a dimer in space group C2/c. Each copper(I) center has distorted tetrahedral N2I2 coordination geometry with Cu-N(pyridyl) distances 2.061(3) and 2.063(3) A, Cu-I distances 2.6162(5) and 2.7817(5) and a Cu...Cu distance of 2.9086(8) A. Complex 1 is rapidly oxidized by dioxygen in CH2Cl2 with a 1 : 1 stoichiometry giving the bis(micro-iodo)peroxodicopper(II) complex [Cu(L)(micro-I)]2O2 (2). The reaction of 1 with dioxygen has been characterized by UV-vis, mass spectrometry, EPR and Cu K-edge X-ray absorption spectroscopy at low temperature (193 K) and above. The mass spectrometry and low temperature EPR measurements suggested an equilibrium between the bis(micro-iodo)peroxodicopper(II) complex 2 and its dimer, namely, the tetranuclear (peroxodicopper(II))2 complex [Cu(L)(micro-I)]4O4 (2'). Complex 2 undergoes an effective oxo-transfer reaction converting PPh3 into O=PPh3 under anaerobic conditions. At sufficiently high concentration of PPh3, the oxygen atom transfer from 2 to PPh3 was followed by the formation of [Cu(PPh3)3I]. The dioxygen reactivity of 1 was compared with that known for other halo(amine)copper(I) dimers.

Journal Article↗
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

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↗
PubMed2004

Single-molecule spectroscopy for molecular identification in capillary electrophoresis.

The electrophoretic mobilities of individual DNA molecules were determined by using fluorescence imaging. The average mobility agreed well with that obtained in capillary electrophoresis (CE). The signal-to-noise ratio (S/N) did not decrease in the presence of up to 8% plasma or 8% raw blood. Single-molecule detection was still possible in the presence of 50% raw blood. Single-molecule CE of two differently labeled molecules was carried out in the presence of a transmission grating. Even when the mobility difference is not sufficient because of low S/N, identification using different fluorescence wavelengths can be performed at >99% accuracy. So, when small differences in DNA sequence due to disease or mutation can lead to hybridization to labels with different dyes, the screening of the mutated DNA will be facilitated by online spectroscopy in addition to the electrophoretic information from CE.

Electrophoresis, Capillary↗
PubMed2004

Isolation and characterization of R-phycoerythrin subunits and enzymatic digests.

Subunits and enzymatic digests of the highly fluorescent phycobiliprotein R-phycoerythrin (R-PE) were analyzed by several separation and detection techniques including HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), CE, and HPLC-electrospray ionization (ESI) MS. R-PE subunits were isolated by HPLC and detected as single molecules by total internal reflection fluorescence microscopy. The results show efficient absorption and fluorescence of the R-PE subunits and digest peptides, originating from the incorporation of phycoerythrobilin and phycourobilin chromophores in them. In addition, HPLC-ESI-MS and SDS-PAGE were optimized to determine the molecular masses of phycobiliprotein subunits and the chromophore-containing peptides, as well as the amino acid sequences of the latter. Favorable spectroscopic and structural properties of R-PE subunits and enzymatic digests, even under denaturing conditions, make these molecules suitable for use as fluorescence labels for biomolecules.

Chromatography, High Pressure Liquid↗
PubMed2003

Tuning the valence of the cerium center in (Na)phthalocyaninato and porphyrinato cerium double-deckers by changing the nature of the tetrapyrrole ligands.

A series of 7 cerium double-decker complexes with various tetrapyrrole ligands including porphyrinates, phthalocyaninates, and 2,3-naphthalocyaninates have been prepared by previously described methodologies and characterized with elemental analysis and a range of spectroscopic methods. The molecular structures of two heteroleptic [(na)phthalocyaninato](porphyrinato) complexes have also been determined by X-ray diffraction analysis which exhibit a slightly distorted square antiprismatic geometry with two domed ligands. Having a range of tetrapyrrole ligands with very different electronic properties, these compounds have been systematically investigated for the effects of ligands on the valence of the cerium center. On the basis of the spectroscopic (UV-vis, near-IR, IR, and Raman), electrochemical, and structural data of these compounds and compared with those of the other rare earth(III) counterparts reported earlier, it has been found that the cerium center adopts an intermediate valence in these complexes. It assumes a virtually trivalent state in cerium bis(tetra-tert-butylnaphthalocyaninate) as a result of the two electron rich naphthalocyaninato ligands, which facilitate the delocalization of electron from the ligands to the metal center. For the rest of the cerium double-deckers, the cerium center is predominantly tetravalent. The valences (3.59-3.68) have been quantified according to their L(III)-edge X-ray absorption near-edge structure (XANES) profiles.

Journal Article↗
PubMed2003

Cerium(III) and neodymium(III) amides derived from a chelating 2-pyridyl amido ligand.

Homoleptic Ce(III) and Nd(III) triamides [LnL(3)] [Ln = Ce(1) or Nd(2)] and the heterobimetallic amide-alkoxide derivatives [LnL(2)(mu-OBu(t))2M(tmeda)] [Ln = Ce, M = Na (3); Ln = Nd, M = Na (4); Ln = Nd, M = K (5)] supported by the bulky [N(SiBu(t)Me2)(2-C(5)H(3)N-6-Me)]- ligand (L-) have been successfully synthesized and characterized. Complexes 1-3 and 5 show a high activity toward the ring-opening polymerization of epsilon-caprolactone.

Journal Article↗
PubMed2002

Synthesis and structural characterization of imido-lanthanide complexes with a metal-nitrogen multiple bond.

Treatment of an amido-ytterbium complex with n-BuLi leads to the isolation and structural characterization of a mixed amido-imido-ytterbium or imido-ytterbium complex, respectively, depending upon the molar ratios of the reactants; the Yb-N distance and the linearity of the imido N atom strongly suggest the presence of a formal Yb=N multiple bond in these novel complexes.

Imides↗