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Hongkai Wu

Publications and source records attributed to Hongkai Wu.

16 recordsLinked to original sources
PubMed2007

Counting low-copy number proteins in a single cell.

We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta2 adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells (Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.

Animals↗
PubMed2006

Controlling electroosmotic flow in poly(dimethylsiloxane) separation channels by means of prepolymer additives.

The electroosmotic flow (EOF) in a poly(dimethylsiloxane) (PDMS) separation channel can be altered and controlled by adding a carboxylic acid to the prepolymer prior to curing. When the prepolymer is doped with 0.5 wt % undecylenic acid (UDA), the electroosmotic mobility in a modified PDMS channel rises to (7.6 +/- 0.2) x 10(-4) cm(2) V(-1) s(-1) (in HEPES buffer at pH 8.5), which is nearly twice that in the native PDMS channel. Because this modification does not significantly change the hydrophobicity of the PDMS surface, it is possible to combine the modified PDMS with a dynamic coating of n-dodecyl beta-d-maltoside (DDM), which prevents protein sticking (see Huang, B.; Wu, H. K.; Kim, S.; Zare, R. N. Lab Chip 2005, 5, 1005-1007). The modified PDMS channel with a dynamic coating of DDM generates an electroosmotic mobility of (5.01 +/- 0.09) x 10(-4) cm(2) V(-1) s(-1), which shows excellent reproducibility both in successive runs and during storage in water. Combining this surface modification and the dynamic coating of DDM is an effective means for both providing stable EOF in the PDMS channels and preventing protein adsorption on the channel walls. To demonstrate these effects, we show that the electrophoretic separation of immunocomplexes in free solution can be readily accomplished in a microfluidic chip made of UDA-doped (0.5 wt %) PDMS with a dynamic coating of DDM.

Dimethylpolysiloxanes↗
PubMed2006

Generation of complex, static solution gradients in microfluidic channels.

A microfluidic device is used to generate a complex gradient of diffusible molecules in a static solution. The gradient is precise and steady both in space and in time. This device, made from poly(dimethylsiloxane), consists of three layers. The molecules in reservoirs on the top layer diffuse through the flat middle layer of hydrogel and reach an equilibrium distribution. Microfluidic channels on the bottom layer that are in close contact with the hydrogel contain free solution that has concentration gradients based on the gradient in the gel. The gradient profile in the channel can be designed to have an arbitrary form (within the range of the existing gradient in the hydrogel) by controlling the local direction of the channel at each point.

Dimethylpolysiloxanes↗
PubMed2006

Phospholipid biotinylation of polydimethylsiloxane (PDMS) for protein immobilization.

Polydimethylsiloxane (PDMS) surfaces can be functionalized with biotin groups by adding biotinylated phospholipids to the PDMS prepolymer before curing. The addition of beta-D-dodecyl-N-maltoside (DDM) in the solution blocks non-specific protein binding on these functionalized PDMS surfaces. We characterize the surface by measuring fluorescently labeled streptavidin binding. Single molecule tracking shows that the phospholipids are not covalently linked to PDMS polymer chains, but the surface functionalization is not removed by washing. We demonstrate the immobilization of biotinylated antibodies and lectins through biotin-avidin interactions.

Avidin↗
PubMed2005

Construction of microfluidic chips using polydimethylsiloxane for adhesive bonding.

A thin layer of polydimethylsiloxane (PDMS) prepolymer, which is coated on a glass slide, is transferred onto the embossed area surfaces of a patterned substrate. This coated substrate is brought into contact with a flat plate, and the two structures are permanently bonded to form a sealed fluidic system by thermocuring (60 degrees C for 30 min) the prepolymer. The PDMS exists only at the contact area of the two surfaces with a negligible portion exposed to the microfluidic channel. This method is demonstrated by bonding microfluidic channels of two representative soft materials (PDMS substrate on a PDMS plate), and two representative hard materials (glass substrate on a glass plate). The effects of the adhesive layer on the electroosmotic flow (EOF) in glass channels are calculated and compared with the experimental results of a CE separation. For a channel with a size of approximately 10 to 500 microm, a approximately 200-500 nm thick adhesive layer creates a bond without voids or excess material and has little effect on the EOF rate. The major advantages of this bonding method are its generality and its ease of use.

Adhesiveness↗
PubMed2004

Chemical cytometry on a picoliter-scale integrated microfluidic chip.

An integrated microfluidic device has been fabricated for analyzing the chemical contents of a single cell (chemical cytometry). The device is designed to accomplish four different functions: (i) cell handling, (ii) metering and delivering of chemical reagents, (iii) cell lysis and chemical derivatization, and (iv) separating derivatized compounds and detecting them by laser-induced fluorescence. These functions are accomplished with only two valves, formed by multilayer soft lithography. A new kind of three-state valve and a picopipette are described; these elements are crucial for minimizing the reaction volume and ensuring optimal shape of the channel for electrophoresis injection. By using these valves, a reaction volume of approximately 70 pl is achieved for the lysis and derivitization of the contents of a single Jurkat T cell (approximately 10 microm diameter). As a demonstration of the use of this integrated microfluidic device, electropherograms of amino acids from individual Jurkat T cells are recorded and compared with those collected from a multiple-cell homogenate.

Electrophoresis, Capillary↗
PubMed2003

Two-dimensional colloid crystals obtained by coupling of flow and confinement.

This Letter describes the generation of 2D colloidal lattices in microchannels by coupling the laminar flow of dispersions of spherical colloids and geometrical confinement. We describe a nonequilibrium, convective, mechanism leading to formation of ordered 2D structures of both closed-packed hexagonal and non-closed-packed rhombic symmetries. The number and types of possible lattices is determined by the ratio of the width of the channel to the diameter of the particle. The structures tend to return to a regular lattice after a defect is introduced; that is, for example, they tend to self-repair disorder induced by particle polydispersity, contaminants, and flow instabilities. The stability of different lattices is analyzed numerically for particles with different polydispersity.

Journal Article↗
PubMed2003

Rapid prototyping of 2D structures with feature sizes larger than 8 microm.

This paper extends rapid prototyping for several types of lithography to the 8-25-microm size range, using transparency photomasks prepared by photoplotting. It discusses the technical improvement in photomask quality achieved by photoplotting, compared to the currently used image setting, and demonstrates differences in the resolution that can be obtained with photomasks with features in the 8-100-microm size range. These high-resolution photomasks were used to microfabricate microelectrodes, microlenses, and stamps for microcontact printing, following methods described previously.

Biological Assay↗
PubMed2003

Fabrication of complex three-dimensional microchannel systems in PDMS.

This paper describes a method for fabricating three-dimensional (3D) microfluidic channel systems in poly(dimethylsiloxane) (PDMS) with complex topologies and geometries that include a knot, a spiral channel, a "basketweave" of channels, a chaotic advective mixer, a system with "braided" channels, and a 3D grid of channels. Pseudo-3D channels, which are topologically equivalent to planar channels, are generated by bending corresponding planar channels in PDMS out of the plane into 3D shapes. True 3D channel systems are formed on the basis of the strategy of decomposing these complex networks into substructures that are planar or pseudo-3D. A methodology is developed that connects these planar and/or pseudo-3D structures to generate PDMS channel systems with the original 3D geometry. This technique of joining separate channel structures can also be used to create channel systems in PDMS over large areas by connecting features on different substrates. The channels can be used as templates to form 3D structures in other materials.

Dimethylpolysiloxanes↗
PubMed2002

Using hierarchical self-assembly to form three-dimensional lattices of spheres.

This paper describes an approach to the fabrication of three-dimensional (3-D) structures of millimeter-scale spherical beads having a range of lattices-tetragonal, cubic, and hexagonal-using hierarchical self-assembly. The process has five steps: (i) metal-coated beads are packed in a rod-shaped cavity in an elastomeric polymer (poly(dimethylsiloxane), PDMS); (ii) the beads are embedded in a second polymer (PDMS or polyurethane, PU) using a procedure that leaves the parts of the beads in contact with the PDMS exposed; (iii) the exposed areas of the beads are coated with a solder having a low melting point; (iv) the polymer rods-with embedded beads and exposed solder drops-are suspended in an approximately isodense medium (an aqueous solution of KBr) and allowed to self-assemble by capillary interactions between the drops of molten solder; and (v) the assembly is finished by several procedures, including removing the beads from the polymer matrix by dissolution, filling the voids left with another material, and dissolving the matrix. The confinement of the beads in regular structures in polymer rods makes it possible to generate self-assembled structures with a variety of 3-D lattices; the type of the lattice formed can be controlled by varying the size of the beads, and the size and shape of the cross-section of the rods.

Journal Article↗
PubMed2002

Reduction photolithography using microlens arrays: applications in gray scale photolithography.

This paper describes the application of reduction photolithography, using arrays of microlenses and gray scale masks, to generate arrays of micropatterns having multilevel and curved features in photoresist. This technique can fabricate, in a single exposure, three-dimensional microstructures (e.g., nonspherical microlens arrays) over areas of approximately 2 x 2 cm2. The simple optical configuration consisted of transparency film (having centimeter-sized features) as gray scale photomasks, an overhead projector as the illumination source, and arrays of microlenses as the size-reducing elements. Arrays of 40- and 100-microm lenses achieved a lateral size reduction of approximately 10(3) and generated patterns of well-defined, multilevel structures; these structures may find use in applications such as diffractive optics.

Journal Article↗
PubMed2002

Connectivity of features in microlens array reduction photolithography: generation of various patterns with a single photomask.

Microlens array photolithography (MAP) is a technique in which arrays of microlenses positioned close to photoresist reduce cm-sized figures on photomasks and form mum-scale images in the photoresist. This work demonstrates that MAP, using a single photomask, can generate patterns having different symmetries and periodicities from that of the lens array. This capability of MAP depends on (i) the connectivity between the images produced by individual microlenses and (ii) the orientation of the photomask relative to the lens array prior to exposure. By changing this orientation, MAP, using a single mask and a single array of microlenses, could be used to generate patterns that (i) are separated from each other, (ii) overlap with each other, (iii) are 2D chiral, and thus different from both the lens array and the mask in symmetry, (iv) have a symmetry reduced from that of the lens array, or (v) have a smaller unit cell and smaller pitch than that of the lens array.

Biological Assay↗
PubMed2002

A prototype two-dimensional capillary electrophoresis system fabricated in poly(dimethylsiloxane).

A method for carrying out 2D gel electrophoresis in a capillary format is presented. In this method, separation in the first dimension is carried out in a 1D capillary, with this system physically isolated from the capillaries that provide the separation in the second dimension. After completion of the first separation, the 1D channel is physically connected to the 2D capillaries, and a second separation is carried out in an orthogonal set of parallel capillaries. The ability of poly(dimethylsiloxane) (PDMS) to support the fabrication of 3D microfluidic systems makes it possible to produce membranes that both enclose the gel used in the first separation in a capillary and provide passages for the proteins to migrate into the array of orthogonal capillaries. The elastomeric nature of PDMS makes it possible to make reversible connections between pieces of PDMS. The feasibility of this system is demonstrated using a protein mixture containing fluorescein-conjugated carbonic anhydrase, fluorescein-conjugated BSA, and Texas Red-conjugated ovalbumin. This work suggests one type of design that might form the basis for a microfabricated device for 2D capillary electrophoresis.

Carbonic Anhydrases↗
PubMed2002

Arrays of self-assembled monolayers for studying inhibition of bacterial adhesion.

This paper describes a simple and convenient method for the rapid screening of potential inhibitors of bacterial adhesion and for the quantitative evaluation of the efficacy of the inhibitors using arrays of self-assembled monolayers (SAMs) of alkanethiolates on gold that are presented on a 96-well microtiter plate. The SAMs present mixtures of alpha-D-mannopyranoside (a ligand that promotes the adhesion of uropathogenic Escherichia coli by binding to the FimH proteins on the tip of type 1 pili), and tri(ethylene glycol) moieties (organic groups that resist nonspecific adsorption of proteins and cells). The SAMs provide surfaces for studies of adhesion of uropathogenic E. coli to specific ligands; they also provide excellent resistance to nonspecific adhesion. Using arrays of mannoside-presenting SAMs, inhibitors of bacterial adhesion were easily screened by observing the number of bacteria that adhered to the surface of the SAMs in the presence of inhibitor. The potency of the inhibitor was quantified by measuring the percentage of inhibition as a function of the concentration of the inhibitor. The properties of SAMs, when combined with the convenience and standardization of a microtiter plate, make arrays of SAMs a versatile tool that can be applied to high-throughput screening of inhibitors of bacterial, viral, and mammalian cell adhesion and of strongly binding ligands for proteins.

Bacterial Adhesion↗