Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane).

This paper describes a procedure that makes it possible to design and fabricate (including sealing) microfluidic systems in an elastomeric material [Formula: see text] poly(dimethylsiloxane) (PDMS) [Formula: see text] in less than 24 h. A network of microfluidic channels (with width >20 μm) is designed in a CAD program. This design is converted into a transparency by a high-resolution printer; this transparency is used as a mask in photolithography to create a master in positive relief photoresist.

Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors.

Found throughout biology, polyvalent interactions are characterized by the simultaneous binding of multiple ligands on one biological entity to multiple receptors on another (top part of the illustration) and have a number of characteristics that monovalent interactions do not (bottom). In particular, polyvalent interactions can be collectively much stronger than corresponding monovalent interactions, and they can provide the basis for mechanisms of both agonizing and antagonizing biological interactions that are fundamentally different from those available in monovalent systems.

Design and fabrication of topologically complex, three-dimensional microstructures.

Two concepts for use in the fabrication of three-dimensional (3D) microstructures with complex topologies are described. Both routes begin with a two-dimensional (2D) pattern and transform it into a 3D microstructure. The concepts are illustrated by use of soft lithographic techniques to transfer 2D patterns to cylindrical (pseudo-3D) substrates.

Regulation of protrusion shape and adhesion to the substratum during chemotactic responses of mammalian carcinoma cells.

We report here the first direct observation of chemotaxis to EGF by rat mammary carcinoma cells. When exposed to a gradient of EGF diffusing from a micropipette, MTLn3 cells displayed typical ameboid chemotaxis, extending a lamellipod-like protrusion and moving toward the pipette. Using a homogeneous upshift in EGF to model stimulated lamellipod extension (J.

Micropatterned surfaces for control of cell shape, position, and function.

The control of cell position and function is a fundamental focus in the development of applications ranging from cellular biosensors to tissue engineering. Using microcontact printing of self-assembled monolayers (SAMs) of alkanethiolates on gold, we manufactured substrates that contained micrometer-scale islands of extracellular matrix (ECM) separated by nonadhesive regions such that the pattern of islands determined the distribution and position of bovine and human endothelial cells. In addition, the size and geometry of the islands were shown to control cell shape.

Fabricating large arrays of microwells with arbitrary dimensions and filling them using discontinuous dewetting.

This paper describes the fabrication of large (up to 45 cm(2)) arrays of microwells, with volumes as small as ∼3 fL/well and densities as high as 10(7) wells/cm(2). These arrays of microwells are formed by casting an elastomer, poly(dimethylsiloxane) (PDMS), against "masters" prepared by photolithography; arrays of microwells in other polymers can be formed by using a master consisting of posts in PDMS. A straightforward technique, discontinuous dewetting, allows wells to be filled rapidly (typically on the order of 10(4) wells/s) and uniformly with a wide range of liquids.

A trivalent system from vancomycin.D-ala-D-Ala with higher affinity than avidin.biotin.

Tris(vancomycin carboxamide) binds a trivalent ligand derived from D-Ala-D-Ala with very high affinity: dissociation constant (Kd) approximately 4 x 10(-17) +/- 1 x 10(-17) M. High-affinity trivalent binding and monovalent binding are fundamentally different. In trivalent (and more generally, polyvalent) binding, dissociation occurs in stages, and its rate can be accelerated by monovalent ligand at sufficiently high concentrations.

Affinity capillary electrophoresis: a physical-organic tool for studying interactions in biomolecular recognition.

Affinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor-ligand complex.

Soft Lithography.

Elastomeric stamps and molds provide a great opportunity to eliminate some of the disadvantages of photolithograpy, which is currently the leading technology for fabricating small structures. In the case of "soft lithography" there is no need for complex laboratory facilities and high-energy radiation. Therefore, this process is simple, inexpensive, and accessible even to molecular chemists.

A miniaturized arrayed assay format for detecting small molecule-protein interactions in cells.

Background: Two complementary approaches to studying the cellular function of proteins involve alteration of function either by mutating protein-encoding genes or by binding a small molecule to the protein. A mutagen can generate millions of genetic mutations; correspondingly, split-pool synthesis can generate millions of unique ligands attached to individual beads. Genetic screening of mutations is relatively straightforward but, in contrast, split-pool synthesis presents a challenge to current methods of screening for compounds that alter protein function.

Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver.

This paper describes a convenient methodology for patterning substrates for cell culture that allows the positions and dimensions of attached cells to be controlled. The method uses self-assembled monolayers (SAMs) of terminally substituted alkanethiolates (R(CH2)11-15S-) adsorbed on optically transparent films of gold or silver to control the properties of the substrates. SAMs terminated in methyl groups adsorb protein and SAMs terminated in oligo(ethylene glycol) groups resist entirely the adsorption of protein.

On-line detection of nonspecific protein adsorption at artificial surfaces.

A detailed understanding of the interaction of proteins with artificial surfaces is essential for many applications in medicine and biochemistry. The affinity of surfaces toward proteins may, for instance, remove pharmacological proteins from media or control the adherence of pathogenic bacteria to protheses. Only a few analytical techniques now exist that can be used to study the binding process in real time, using unlabeled proteins.

Wave-front engineering by use of transparent elastomeric optical elements.

We describe a class of devices based on elastomeric optical phase gratings. These devices operate by reversibly controlling the phase of transmitted or reflected light by mechanical compression of the transparent elastomer. An optical modulator and an element in an optical display demonstrate two possible applications.

Studies on alpha-sialylation using sialyl donors with an auxiliary 3-thiophenyl group.

Reaction of the methyl ester of 2-chloro-3-S-phenyl-3-thiosialic acid (4) with sodium thiomethoxide in acetonitrile at 0 degrees C affords the methyl ester of 2-S-methyl-3-S-phenyl-2,3-di-thio-alpha-sialic acid (6a) in quantitative yield. Sialylation of tetrahydropyran-2-methanol (7) and 2-(trimethylsilyl)ethyl 2,2'3,6,6'-penta-O-benzyl-alpha-lactoside (8) with 6a in the presence of phenylsulfenyl triflate (PST) as promotor in CH3 CN at -40 degrees C gives alpha-sialosides 9 and 10 in good yield and excellent stereoselectivity. No beta-sialosides are formed in either case.

Representing primary electrophoretic data in the 1/time domain: comparison to representations in the time domain.

A plot of absorbance vs 1/time (the "1/time domain") is a more useful representation of the primary data in capillary electrophoresis than traditional plots of absorbance vs time (the "time domain") in a wide set of circumstances, especially when comparing electropherograms in which the rate of electroosmotic flow is not precisely the same. The quantity that is of fundamental interest in capillary electrophoresis (CE) is the electrophoretic mobility of an analyte. The electrophoretic mobility of a species is nonlinearly proportional to time and, therefore, not linearly represented in the time domain: that is, the distance between two peaks along the time axis is not linearly related to the difference in their electrophoretic mobilities.

Geometric control of cell life and death.

Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion.

Self-Assembly of Mesoscale Objects into Ordered Two-Dimensional Arrays.

Regular arrays of topologically complex, millimeter-scale objects were prepared by self-assembly, with the shapes of the assembling objects and the wettability of their surfaces determining the structure of the arrays. The system was composed of solid objects floating at the interface between perfluorodecalin and water and interacting by lateral capillary forces; patterning of the wettability of the surfaces of the objects directs these forces. Self-assembly results from minimization of the interfacial free energy of the liquid-liquid interface.

Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins.

The adsorption of proteins with net positive charges (pI > pH) on the walls of fused-silica capillaries is a common problem in the analysis of proteins by capillary electrophoresis. This paper explores the use of polycationic polymers as noncovalent coatings to limit this problem. The behavior of three sets of proteins was compared using uncoated and coated capillaries: (i) a protein charge ladder obtained by acetylation of lysozyme (EC 3.

Stimulation of human cytotoxic T cells with HIV-1-derived peptides presented by recombinant HLA-A2 peptide complexes.

HLA-A2 heavy chain and beta 2-microglobulin were expressed in Escherichia coli, and refolded in the presence of peptides derived from HIV-1 RT and gag proteins. When recombinant HLA-A2 molecules were attached to cells lacking HLA-A2, the cells became susceptible to lysis by HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones specific for peptides derived from RT and gag proteins. Limiting dilution analyses of peripheral blood mononuclear cells from HIV-1-infected individuals showed that the recombinant HLA-A2 peptide complexes covalently immobilized on microspheres stimulated the development of HLA-A2 peptide-specific CTL.

Using protein charge ladders to estimate the effective charges and molecular weights of proteins in solution.

This paper describes the use of capillary electrophoresis (CE) and protein charge ladders to estimate values of effective charge (Z) and molecular weight of proteins under nondenaturing conditions. A panel of 14 proteins with a range of charges and shapes was modified by acetylation with acetic anhydride to yield protein charge ladders. A protein charge ladder is a family of derivatives of a protein that differ in integral units of charge, but minimally in hydrodynamic drag; this mixture of proteins appears in electrophoresis as a set of peaks with regular spacings.

Synthesis of C-5 Analogs of N-Acetylneuraminic Acid via Indium-Mediated Allylation of N-Substituted 2-Amino-2-deoxymannoses.

This paper presents a short synthesis of new analogs of N-acetylneuraminic acid (Neu5Ac) varied structurally at C-5. The synthetic strategy includes indium-mediated coupling reactions between ethyl 2-(bromomethyl)acrylate and N-derivatized mannosamines, and the ozonolysis of the resulting enoates. The main advantage of this indium-mediated allylation for the synthesis of neuraminic acids comes from the efficient, stereoselective C-C bond formation, which affords predominantly the correct diastereomer having a threo relationship between the newly generated hydroxyl group and the C-2 amide group of mannosamine.

Elastomeric diffraction gratings as photothermal detectors.

A photothermal detector consisting of a relief grating on the surface of an elastomer was fabricated and characterized. The detector has a sensitivity of the order of microwatts per square millimeter and has rise and fall times of the order of tens of seconds. Numerical and analytical modeling account for the behavior of the detector.

Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide.