Search PubMed⌕ Search

Biomedical subjects

G M Whitesides

Publications and source records attributed to G M Whitesides.

At least 37 records · Page 2Linked to original sources
PubMed2001

Soft lithography in biology and biochemistry.

Soft lithography, a set of techniques for microfabrication, is based on printing and molding using elastomeric stamps with the patterns of interest in basrelief. As a technique for fabricating microstructures for biological applications, soft lithography overcomes many of the shortcomings of photolithography. In particular, soft lithography offers the ability to control the molecular structure of surfaces and to pattern the complex molecules relevant to biology, to fabricate channel structures appropriate for microfluidics, and to pattern and manipulate cells. For the relatively large feature sizes used in biology (> or = 50 microns), production of prototype patterns and structures is convenient, inexpensive, and rapid. Self-assembled monolayers of alkanethiolates on gold are particularly easy to pattern by soft lithography, and they provide exquisite control over surface biochemistry.

Animals↗
PubMed2000

Measuring the forces involved in polyvalent adhesion of uropathogenic Escherichia coli to mannose-presenting surfaces.

Mechanisms of bacterial pathogenesis have become an increasingly important subject as pathogens have become increasingly resistant to current antibiotics. The adhesion of microorganisms to the surface of host tissue is often a first step in pathogenesis and is a plausible target for new antiinfective agents. Examination of bacterial adhesion has been difficult both because it is polyvalent and because bacterial adhesins often recognize more than one type of cell-surface molecule. This paper describes an experimental procedure that measures the forces of adhesion resulting from the interaction of uropathogenic Escherichia coli to molecularly well defined models of cellular surfaces. This procedure uses self-assembled monolayers (SAMs) to model the surface of epithelial cells and optical tweezers to manipulate the bacteria. Optical tweezers orient the bacteria relative to the surface and, thus, limit the number of points of attachment (that is, the valency of attachment). Using this combination, it was possible to quantify the force required to break a single interaction between pilus and mannose groups linked to the SAM. These results demonstrate the deconvolution and characterization of complicated events in microbial adhesion in terms of specific molecular interactions. They also suggest that the combination of optical tweezers and appropriately functionalized SAMs is a uniquely synergistic system with which to study polyvalent adhesion of bacteria to biologically relevant surfaces and with which to screen for inhibitors of this adhesion.

Bacterial Adhesion↗
PubMed2000

Fabrication of topologically complex three-dimensional microfluidic systems in PDMS by rapid prototyping.

This paper describes a procedure for making topologically complex three-dimensional microfluidic channel systems in poly(dimethylsiloxane) (PDMS). This procedure is called the "membrane sandwich" method to suggest the structure of the final system: a thin membrane having channel structures molded on each face (and with connections between the faces) sandwiched between two thicker, flat slabs that provide structural support. Two "masters" are fabricated by rapid prototyping using two-level photolithography and replica molding. They are aligned face to face, under pressure, with PDMS prepolymer between them. The PDMS is cured thermally. The masters have complementary alignment tracks, so registration is straightforward. The resulting, thin PDMS membrane can be transferred and sealed to another membrane or slab of PDMS by a sequence of steps in which the two masters are removed one at a time; these steps take place without distortion of the features. This method can fabricate a membrane containing a channel that crosses over and under itself, but does not intersect itself and, therefore, can be fabricated in the form of any knot. It follows that this method can generate topologically complex microfluidic systems; this capability is demonstrated by the fabrication of a "basketweave" structure. By filling the channels and removing the membrane, complex microstructures can be made. Stacking and sealing more than one membrane allows even more complicated geometries than are possible in one membrane. A square coiled channel that surrounds, but does not connect to, a straight channel illustrates this type of complexity.

Dimethylpolysiloxanes↗
PubMed2000

Patterning electro-osmotic flow with patterned surface charge.

This Letter reports the measurement of electro-osmotic flows (EOF) in microchannels with surface charge patterned on the 200 microm scale. We have investigated two classes of patterns: (1) Those in which the surface charge varies along a direction perpendicular to the electric field used to drive the EOF; this type of pattern generates multidirectional flow along the direction of the field. (2) Those in which the surface charge pattern varies parallel to the field; this pattern generates recirculating cellular flow, and thus causes motion both parallel and perpendicular to the external field. Measurements of both of these flows agree well with theory in the limit of thin double layers and low surface potential.

Electrochemistry↗
PubMed2000

Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems.

Three-dimensional microfluidic systems were fabricated and used to pattern proteins and mammalian cells on a planar substrate. The three-dimensional topology of the microfluidic network in the stamp makes this technique a versatile one with which to pattern multiple types of proteins and cells in complex, discontinuous structures on a surface. The channel structure, formed by the stamp when it is in contact with the surface of the substrate, limits migration and growth of cells in the channels. With the channel structure in contact with the surface, the cells stop dividing once they form a confluent layer. Removal of the stamp permits the cells to spread and divide.

Animals↗
PubMed2000

Fabrication of microfluidic systems in poly(dimethylsiloxane).

Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft-lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics-derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.

Animals↗
PubMed2000

Modeling the kinetics of acylation of insulin using a recursive method for solving the systems of coupled differential equations.

This paper describes a theoretical method for solving systems of coupled differential equations that describe the kinetics of complicated reaction networks in which a molecule having multiple reaction sites reacts irreversibly with multiple equivalents of a ligand (reagent). The members of the network differ in the number of equivalents of reagent that have reacted, and in the patterns of sites of reaction. A recursive algorithm generates series, asymptotic, and average solutions describing this kinetic scheme. This method was validated by successfully simulating the experimental data for the kinetics of acylation of insulin.

Acylation↗
PubMed2000

Fabrication inside microchannels using fluid flow.

This Account summarizes techniques for carrying out microfabrication of structures with dimensions down to 10 microm in microchannels that are 0.02-2 mm wide. These methods are largely based on the exploitation of laminar flow at low Reynolds number (Re) to control the spatial delivery of reagents. These methods are illustrated by fabrication of fibers, microelectrode arrays, arrays of crystals, and patterns of proteins and cells.

Crystallization↗
PubMed1999

Nonstatistical binding of a protein to clustered carbohydrates.

Carbohydrate-derivatized self-assembled monolayers (SAMs) are used as a model system to address issues involving cell-surface carbohydrate-protein interactions. Here we examine the influence of carbohydrate surface density on protein-binding avidity. We show that the binding selectivity of Bauhinia purpurea lectin switches from one carbohydrate ligand to another as the surface density of the carbohydrate ligands increases from values of chi(sugar) approximately 0.1-1.0. Polyvalent binding is possible at all surface densities investigated; hence, the switch in selectivity is not due simply to the achievement of a critical density that permits polyvalent contacts. Instead, secondary interactions at high surface densities promote a switch in carbohydrate-binding selectivity. These findings may have implications for how changes in the composition and the density of cell-surface carbohydrates influence biological recognition processes and regulatory pathways.

Carbohydrate Metabolism↗
PubMed1999

Patterning cells and their environments using multiple laminar fluid flows in capillary networks.

This paper describes the use of laminar flow of liquids in capillary systems to pattern the cell culture substrate, to perform patterned cell deposition, and to pattern the cell culture media. We demonstrate the patterning of the cell culture substrate with different proteins, the patterning of different types of cells adjacent to each other, the patterned delivery of chemicals to adhered cells, and performing enzymatic reactions over select cells or over a portion of a cell. This method offers a way to simultaneously control the characteristics of the surface to which cells are attached, the type of cells that are in their vicinity, and the kind of media that cells or part of a cell are exposed to. The method is experimentally simple, highly adaptable, and requires no special equipment except for an elastomeric relief that can be readily prepared by rapid prototyping.

Animals↗
PubMed1999

Complexity in chemistry.

"Complexity" is a subject that is beginning to be important in chemistry. Historically, chemistry has emphasized the approximation of complex nonlinear processes by simpler linear ones. Complexity is becoming a profitable approach to a wide range of problems, especially the understanding of life.

Biochemistry↗
PubMed1999

A strategy for the generation of surfaces presenting ligands for studies of binding based on an active ester as a common reactive intermediate: a surface plasmon resonance study.

This paper describes the immobilization of ten proteins and two low-molecular-weight ligands on mixed self-assembled monolayers (SAMs) of alkanethiolates on gold generated from the tri(ethylene glycol)-terminated thiol 1 (HS(CH2)11(OCH2CH2)3OH) (chi(1) = 1.0-0.0) and the longer, carboxylic acid-terminated thiol2(HS(CH2)11(OCH2-CH2)6OCH2CO2H) (chi(2) = 0.0-1.0). The immobilization was achieved by a two-step procedure: generation of reactive N-hydroxysuccinimidyl esters from the carboxylic acid groups of 2 in the SAM and coupling of these groups with amines on the protein or ligand. Because this method involves a common reactive intermediate that is easily prepared, it provides a convenient method for attaching ligands to SAMs for studies using surface plasmon resonance spectroscopy (and, in principle, other bioanalytical methods that use derivatized SAMs on gold, silver, and other surfaces). These SAMs were resistant to nonspecific adsorption of proteins having a wide range of molecular weights and isoelectric points. The pH of the coupling buffer, the concentration of protein, the ionic strength of the solution of protein, and the molecular weight of the protein all influenced the amount of the protein that was immobilized. For the proteins investigated in detail--carbonic anhydrase and lysozyme--the highest quantities of immobilized proteins were obtained when using a low ionic strength solution at a value of pH approximately one unit below the isoelectric point (pI) of the protein, at a concentration of approximately 0.5 mg mL-1. Comparisons of the kinetic and thermodynamic constants describing binding of carbonic anhydrase and vancomycin to immobilized benzenesulfonamide and N-alpha-Ac-Lys-D-Ala-D-Ala groups, respectively, on mixed SAMs (by methods described in this paper) and in the carboxymethyl dextran matrix of commercially available substrates yielded (for these systems) essentially indistinguishable values of Kd, koff, and kon.

Adsorption↗
PubMed1999

Geometric control of switching between growth, apoptosis, and differentiation during angiogenesis using micropatterned substrates.

Past studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 microm2 spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 microm2 failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-microm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 microm2. Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-microm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 microm2), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis-growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.

Animals↗
PubMed1999

Probing the energetics of dissociation of carbonic anhydrase-ligand complexes in the gas phase.

This paper describes the use of electrospray ionization-Fourier transform ion cyclotron mass spectrometry (ESI-FTICR-MS) to study the relative stabilities of noncovalent complexes of carbonic anhydrase II (CAII, EC 4.2.1.1) and benzenesulfonamide inhibitors in the gas phase. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) was used to determine the energetics of dissociation of these CAII-sulfonamide complexes in the gas phase. When two molecules of a benzenesulfonamide (1) were bound simultaneously to one molecule of CAII, one of them was found to exhibit significantly weaker binding (DeltaE50 = 0.4 V, where E50 is defined as the amplitude of sustained off-resonance irradiation when 50% of the protein-ligand complexes are dissociated). In solution, the benzenesulfonamide group coordinates as an anion to a Zn(II) ion bound at the active site of the enzyme. The gas phase stability of the complex with the weakly bound inhibitor was the same as that of the inhibitor complexed with apoCAII (i.e., CAII with the Zn(II) ion removed from the binding site). These results indicate that specific interactions between the sulfonamide group on the inhibitor and the Zn(II) ion on CAII were preserved in the gas phase. Experiments also showed a higher gas phase stability for the complex of para-NO2-benzenesulfonamide-CAII than that for ortho-NO2-benzenesulfonamide-CAII complex. This result further suggests that steric interactions of the inhibitors with the binding pocket of CAII parallel those in solution. Overall, these results are consistent with the hypothesis that CAII retains, at least partially, the structure of its binding pocket in the gas phase on the time scale (seconds to minutes) of the ESI-FTICR measurements.

Animals↗
PubMed1999

Biological surface engineering: a simple system for cell pattern formation.

Biological surface engineering using synthetic biological materials has a great potential for advances in our understanding of complex biological phenomena. We developed a simple system to engineer biologically relevant surfaces using a combination of self-assembling oligopeptide monolayers and microcontact printing (muCP). We designed and synthesized two oligopeptides containing a cell adhesion motif (RADS)n (n = 2 and 3) at the N-terminus, followed by an oligo(alanine) linker and a cysteine residue at the C-terminus. The thiol group of cysteine allows the oligopeptides to attach covalently onto a gold-coated surface to form monolayers. We then microfabricated a variety of surface patterns using the cell adhesion peptides in combination with hexa-ethylene glycol thiolate which resist non-specific adsorption of proteins and cells. The resulting patterns consist of areas either supporting or inhibiting cell adhesion, thus they are capable of aligning cells in a well-defined manner, leading to specific cell array and pattern formations.

3T3 Cells↗
PubMed1999

Patterning proteins and cells using soft lithography.

This review describes the pattering of proteins and cells using a non-photolithographic microfabrication technology, which we call 'soft lithography' because it consists of a set of related techniques, each of which uses stamps or channels fabricated in an elastomeric ('soft') material for pattern transfer. The review covers three soft lithographic techniques: microcontact printing, patterning using microfluidic channels, and laminar flow patterning. These soft lithographic techniques are inexpensive, are procedurally simple, and can be used to pattern a variety of planar and non-planar substrates. Their successful application does not require stringent regulation of the laboratory environment, and they can be used to pattern surfaces with delicate ligands. They provide control over both the surface chemistry and the cellular environment. We discuss both the procedures for patterning based on these soft lithographic techniques, and their applications in biosensor technology, in tissue engineering, and for fundamental studies in cell biology.

Animals↗
PubMed1999

Binding of a dimeric derivative of vancomycin to L-Lys-D-Ala-D-lactate in solution and at a surface.

BACKGROUND: The emergence of bacteria that are resistant to vancomycin (V), a glycopeptide antibiotic, results from the replacement of the carboxy-terminal D-Ala-D-Ala of bacterial cell wall precursors by D-Ala-D-lactate. Recently, it has been demonstrated that covalent dimeric variants of V are active against vancomycin-resistant enterococci (VRE). To study the contribution of divalency to the activities of these variants, we modeled the interactions of V and a dimeric V with L-Lys-D-Ala-D-lactate, an analog of the cell-wall precursors of the vancomycin-resistant bacteria. RESULTS: A dimeric derivative of V (V-Rd-V) was found to be much more effective than V in inhibiting the growth of VRE. The interactions of V and V-Rd-V with a monomeric lactate ligand - diacetyl-L-Lys-D-Ala-D-lactate (Ac2KDADLac) - and a dimeric derivative of L-Lys-D-Ala-D-lactate (Lac-R'd-Lac) in solution have been examined using isothermal titration calorimetry and UV spectroscopy titrations; the results reveal that V-Rd-V binds Lac-R'd-Lac approximately 40 times more tightly than V binds Ac2KDADLac. Binding of V and of V-Rd-V to Nalpha-Ac-L-Lys-D-Ala-D-lactate presented on the surface of mixed self-assembled monolayers (SAMs) of alkanethiolates on gold indicates that the apparent off-rate for dissociation of V-Rd-V from the surface is much slower than that of V from the same surface. CONCLUSIONS: The results are compatible with the hypothesis that divalency is responsible for tight binding, which correlates with small values of minimum inhibitory concentrations of V and V-Rd-V.

Anti-Bacterial Agents↗