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Siloxane-based biocatalytic films and paints for use as reactive coatings.

We have developed a new methodology for preparing films and paints suitable for use as biocatalytic coatings. The hydrolytic enzymes pronase and alpha-chymotrypsin were immobilized by either sol-gel entrapment or by covalent attachment into a polydimethylsiloxane (PDMS) matrix and cast into thin films or incorporated into an oil-based paint formulation. All of the coatings retained enzymatic activity and adhered to several different materials. The enzymatic films and paints also exhibited higher thermostability than enzyme free in solution or covalently attached to the outer surface of PDMS. A porous membrane based on a PDMS-immobilized enzyme was also prepared by an immersion precipitation process. Protein adsorption measurements showed that the enzyme-containing films and paints adsorbed less protein than enzyme-free controls, and that protein adsorption decreased with increasing proteolytic activity of the coating. These coatings thus provide the means to apply a stable enzymatic surface to a wide range of materials, and may be generally useful as biocatalytic paints and films.

Adsorption↗

Textured polydimethylsiloxane elastomers in the human larynx: safety and efficiency of use.

For the treatment of unilateral laryngeal paralysis with glottic insufficiency injection laryngoplasty is a popular modality of treatment. This procedure augments the volume of the paralyzed vocal fold by endoscopic injection. However, the ideal substance has not yet been found for that purpose. We report for the first time the systematic application and long-term results of vulcanized polydimethylsiloxane (PDMS) particles in the treatment of glottic insufficiency in the human larynx. Of 10 patients treated with PDMS in the early 1990s, 7 patients could be retrieved for reevaluation. Laryngeal function was assessed by videostroboscopy, expert rating, and further characterized by the objective parameters of voice profile and maximum phonation time. In a standardized questionnaire, all patients were asked for their personal impression of the results. Mean follow-up time was 88.4 months (range 69-102 months). Glottic closure was complete in 5 of 7 patients. There were no signs of granuloma formation or other pathologic changes of the injected vocal folds. In 4 cases, voices were rated normal or near-normal; 2 voices were rated as fair; 1 usable. All the patients reported significant and lasting voice improvement. None of the patients reported any problems related to PDMS. PDMS particles provide permanent augmentation of human vocal fold volume without complications. Their use is a valuable and safe alternative when a definitive one-step procedure seems advantageous. However, further studies are needed to assess voice improvement in comparison to other materials.

Aged↗

Individual plasma proteins detected on rough biomaterials by phase imaging AFM.

In the past several years, atomic force microscopy (AFM) has provided topographic images of adsorbed plasma proteins in situ at unprecedented resolution. Imaging has been limited to adsorbed protein on relatively smooth model substrates such as mica, graphite, or self-assembled monolayers on which the small height of the protein can be observed from the background. The inherent roughness of biomaterial surfaces has prevented observation of adsorbed proteins in topographic images. We report imaging isolated fibrinogen molecules adsorbed on National Heart Lung and Blood Institute (NHLBI) reference materials polydimethylsiloxane and low-density polyethylene in situ using phase imaging AFM. Fibrinogen, a plasma protein important for blood coagulation and platelet aggregation, was adsorbed from dilute solution onto reference biomaterial surfaces at sub-monolayer coverage. Tapping mode AFM was used to image the samples. For polydimethylsiloxane, the lateral size of the surface features is much greater than the dimensions of proteins. This allowed adsorbed proteins to be observed in topographic images. The phase imaging signal of tapping mode AFM provides information on differences in material properties of the surface, and was used to distinguish individual protein molecules from the underlying polymer surface. On the low-density polyethylene surface, characteristic topographical features are of the same magnitude as the protein molecules, so that protein cannot be distinguished from the surface using topographic images. However, phase images were used to successfully locate and characterize the distribution of the protein. Phase imaging was not able to distinguish fibrinogen adsorbed onto expanded polytetrafluoroethylene. The utility and limitations of the phase imaging technique for characterizing protein adsorption to rough surfaces is discussed.

Adsorption↗

Bioactivity and mechanical properties of PDMS-modified CaO-SiO(2)-TiO(2) hybrids prepared by sol-gel process.

Hydrolysis and polycondensation of poly(dimethylsiloxane) (PDMS), tetraethoxysilane (TEOS), tetraisopropyltitanate (TiPT), and calcium nitrate gave essentially pore- and crack-free transparent monolithics of PDMS-modified CaO-SiO(2)-TiO(2) hybrids, when PDMS/(TEOS + TiPT) was larger than 26:74 in weight, under constant ratios of TEOS/TiPT of 9:1 in mol and Ca/(TEOS + TiPT) of 0.15 in mol. Their apatite-forming abilities in a simulated body fluid, which is indicative of bioactivity, increased with decreasing PDMS/(TEOS + TiPT). Their extensibility and Young's modulus decreased and increased, respectively, with decreasing PDMS/(TEOS + TiPT). The hybrids with PDMS/(TEOS + TiPT) of about 30:70 in weight showed fairly high apatite-forming ability, high extensibilities, and Young's moduli almost equal to those of the human cancellous bones. These new kind of bioactive materials with unique mechanical properties may be useful as bone-repairing materials.

Apatites↗

Microfabricated elastomeric stencils for micropatterning cell cultures.

Here we present an inexpensive method to fabricate microscopic cellular cultures, which does not require any surface modification of the substrate prior to cell seeding. The method utilizes a reusable elastomeric stencil (i.e., a membrane containing thru holes) which seals spontaneously against the surface. The stencil is applied to the cell-culture substrate before seeding. During seeding, the stencil prevents the substrate from being exposed to the cell suspension except on the hole areas. After cells are allowed to attach and the stencil is peeled off, cellular islands with a shape similar to the holes remain on the cell-culture substrate. This solvent-free method can be combined with a wide range of substrates (including biocompatible polymers, homogeneous or nonplanar surfaces, microelectronic chips, and gels), biomolecules, and virtually any adherent cell type.

Animals↗

Fabrication and evaluation of a carbon-based dual-electrode detector for poly(dimethylsiloxane) electrophoresis chips.

The first carbon-based dual-electrode detector for microchip capillary electrophoresis (CE) is described. The poly(dimethylsiloxane) (PDMS)-based microchip CE devices were constructed by reversibly sealing a PDMS layer containing separation and injection channels to another PDMS layer containing carbon fiber working electrodes. End-channel amperometric detection was employed and the performance of the chip was evaluated using catechol. The response was found to be linear between 1 and 600 microM with an experimentally determined limit of detection (LOD) of 500 nM and a sensitivity of 30 pA/microM. Collection efficiencies for catechol ranged from 36.0 to 43.7% at field strengths of 260-615 V/cm. The selectivity that can be gained with these devices is demonstrated by the first CE-based dual-electrode detection of a Cu(II) peptide complex. These devices illustrate the potential for a rugged and easily constructed microchip CE system with an integrated carbon-based detector of similar scale.

Carbon↗

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip.

We report on the development of a hybrid polydimethylsiloxane (PDMS)-glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS-glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS-glass hybrid microchip with 500 bp target of lambdaDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS-glass microchip. The chip could be considered as an inexpensive single-use apparatus compared to glass or silicon-made microchips for the same purpose.

Bacteriophage lambda↗

Nanoliter capillary electrochromatography columns based on collocated monolithic support structures molded in poly(dimethyl siloxane).

Following current trends in miniaturization of analytical chemistry, an inexpensive disposable analytical tool in the form of a liquid chromatography column fabricated on a poly(dimethyl siloxane) (PDMS) chip was created. Ease of fabricating the chromatography column was demonstrated by molding collocated monolithic support structures (COMOSS) directly in the column. Positive photo-resist, SPR 220, was used to create column structures on a negative relief master providing final channel dimensions of 2.7-5.2 microm wide by 10.0 microm deep, while monolithic dimensions were 9.8 x 9.8 x 10.0 microm - 12.3 x 12.3 x 10.0 microm. The ability to separate biological samples such as peptides from a tryptic digest of fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) was shown. Separations in capillary electrochromatographic (CEC) mode were performed yielding column efficiencies of 4.0 x 10(5) plates/m.

Animals↗

Patterned self-assembled beads in silicon channels.

A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 microm wide and 50 microm deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 microm, has also been generated on the bottom of a 500 microm wide and 50 microm deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.

Animals↗

Field-effect flow control in a polydimethylsiloxane-based microfluidic system.

The application of the field-effect for direct control of electroosmosis in a polydimethylsiloxane (PDMS)-based microfluidic system, constructed on a silicon wafer with a 2.0 microm electrically insulating layer of silicon dioxide, is demonstrated. This microfluidic system consists of a 2.0 cm open microchannel fabricated on a PDMS slab, which can reversibly adhere to the silicon wafer to form a hybrid microfluidic device. Aside from mechanically serving as a robust bottom substrate to seal the channel and support the microfluidic system, the silicon wafer is exploited to achieve field-effect flow control by grounding the semiconductive silicon medium. When an electric field is applied through the channel, a radial electric potential gradient is created across the silicon dioxide layer that allows for direct control of the zeta potential and the resulting electroosmotic flow (EOF). By configuring this microfluidic system with two power supplies at both ends of the microchannel, the applied electric potentials can be varied for manipulating the polarity and the magnitude of the radial electric potential gradient across the silicon dioxide layer. At the same time, the longitudinal potential gradient through the microchannel, which is used to induce EOF, is held constant. The results of EOF control in this hybrid microfluidic system are presented for phosphate buffer at pH 3 and pH 5. It is also demonstrated that EOF control can be performed at higher solution pH of 6 and 7.4 by modifying the silicon wafer surface with cetyltrimethylammonium bromide (CTAB) prior to assembly of the hybrid microfluidic system. Results of EOF control from this study are compared with those reported in the literature involving the use of other microfluidic devices under comparable solution conditions.

Cetrimonium↗

Poly(dimethylsiloxane) microchip for precolumn reaction and micellar electrokinetic chromatography of biogenic amines.

We have demonstrated that precolumn derivatization and capillary electrophoresis separation on a poly(dimethylsiloxane) (PDMS) microchip can be realized as efficient as those on glass microchips. In an optimized condition of micellar electrokinetic chromatography (MEKC), using 25 mM sodium borate buffer (pH 10.0) with 25 mM sodium dodecyl sulfate (SDS) and 5% v/v methanol, the electroosmotic flow in an oxidized PDMS microchip is stabilized within 3% for days. By employing a fluorometric derivatization with o-phthaldialdehyde (OPA) in an optimally designed reaction chamber, four most important biogenic amines occurring in foods, histamine, tyramine, putrescine, and tryptamine, are quantitatively determined in less than 1 min at the levels applicable to real samples. The migration behaviors of anionic OPA-derivatized biogenic amines under the MEKC conditions are analyzed, and it has been found that under our separation conditions, the electrophoretic mobility of the SDS micelles is significantly greater than those of the anions in the aqueous phase. The channel manifold in a PDMS substrate is fabricated using replica molding against a thick photoresist, SU-8, pattern generated by photolithography. The plate with the microchannel pattern is strongly, irreversibly bonded to another PDMS plate by using a new bonding technique, which employs surface oxidation by corona discharge generated from a cheap, handy source, Tesla coil.

Biogenic Amines↗

A polydimethylsiloxane/glass capillary electrophoresis microchip for the analysis of biogenic amines using indirect fluorescence detection.

A polydimethylsiloxane-glass capillary microchip is fabricated for the rapid analysis of a mixture of common biogenic amines using indirect fluorescence detection. Using a running buffer of phosphate and 2-propanol, and Rhodamine 110 as a background fluorophore, both co-ionic and counter-ionic systems are explored. Studies demonstrate the separation and analysis of cations using indirect fluorescence detection for the first time in a chip-based system. Resulting electrophoretic separations are achieved within a few tens of seconds with detection limits of approximately 6 microM. The reduced sample handling and rapid separations afforded by the coupling of indirect fluorescence detection with chip-based capillary electrophoresis provide a highly efficient method for the analysis and detection of molecules not possessing a chromophore or fluorophore. Furthermore, limits of detection are on a par with reported chip-based protocols that incorporate precolumn derivatisation with fluorescence detection. The current device circumvents lengthy sample preparation stages and therefore provides an attractive alternative technique for the analysis biogenic amines.

Animals↗

Indirect fluorescence detection of simple sugars via high-pH electrophoresis in poly(dimethylsiloxane) microfluidic chips.

This article describes the successful electrophoretic separation of simple carbohydrates in a polymeric microfluidic chip. The device fabricated in poly(dimethylsiloxane) (PDMS) is found to be stable in high-pH solutions. This allows sugars to be separated electrophoretically at pH values at or above their pK(a) using indirect fluorescence detection. Signal-to-noise values greater than 10:1 were obtained using a mercury arc lamp excitation source and a fluorescein-containing mobile phase for the detection of sugars at concentrations as low as 5 mM. The results obtained compare favorably with published results for the same system using a traditional fused-silica capillary. Analysis of the data revealed a significant experimental sensitivity of the migration times measured in these PDMS devices, an aging effect that leads to considerable systematic drift over the course of a series of replicate measurements. These experiments highlighted the importance of the surface chemistry of PDMS, especially as it pertains to its ability to support stable electroosmotic flow within the separation device. Channel priming at high pH provides a necessary, but by itself insufficient, means by which this instability can be minimized.

Carbohydrates↗

Components for integrated poly(dimethylsiloxane) microfluidic systems.

This review describes the design and fabrication of microfluidic systems in poly(dimethylsiloxane) (PDMS). PDMS is a soft polymer with attractive physical and chemical properties: elasticity, optical transparency, flexible surface chemistry, low permeability to water, and low electrical conductivity. Soft lithography makes fabrication of microfluidic systems in PDMS particularly easy. Integration of components, and interfacing of devices with the user, is also convenient and simpler in PDMS than in systems made in hard materials. Fabrication of both single and multilayer microfluidic systems is straightforward in PDMS. Several components are described in detail: a passive chaotic mixer, pneumatically actuated switches and valves, a magnetic filter, functional membranes, and optical components.

Dimethylpolysiloxanes↗

A dynamically modified microfluidic poly(dimethylsiloxane) chip with electrochemical detection for biological analysis.

Separation and direct detection of amino acids, glucose and peptide in a 3.1 cm separation channel made of poly(dimethylsiloxane) (PDMS) with end-column amperometric detection at a copper microdisk electrode was developed. This system is the integration of a normal sized working electrode with electrochemical detection on a PDMS microfabricated device. The PDMS channels dynamically modified by 2-morpholinoethanesulfonic acid (MES) show less adsorption and more enhanced efficiency than that of unmodified ones when applied to separations of these biological molecules. The migration time is less than 100 s and the reproducibility of migration time is satisfactory with relative standard deviation (RSD) of 2.8% in 19 successive injections. The limits of detection of arginine (Arg), glucose, and methionine-glycine (Met-Gly) are estimated to be 2.0, 8.5, and 64.0 microM at S/N = 3, approximately 0.5-16.0 fmol, respectively. Variances influencing the separation efficiency and amperometric response, including injection, separation voltage, detection potential, or concentration of buffer and additive, are assessed and optimized.

Alkanesulfonic Acids↗

Microchip capillary electrophoresis coupled to sinusoidal voltammetry for the detection of native carbohydrates.

The development of a microchip electrophoresis system involving integrated frequency based electrochemical detection is described. The use of poly(dimethylsiloxane) (PDMS) as the channel substrate greatly simplifies the fabrication process while decreasing cost and time consumption. Characterization of this system is accomplished through the detection of native carbohydrates at planar copper electrodes. Various photolithographic techniques are explored in the optimization of electrode area. Separation efficiency of 1 x 10(5) theoretical plates per meter is demonstrated. Sinusoidal voltammetry utilizes information in the frequency domain to achieve sensitive detection through either of two approaches, maximization of signal or minimization of noise. Mass detection limits (S/N = 3) of less than 200 amol have been accomplished for glucose and sucrose. Sinusoidal voltammetry also facilitated the selective isolation of an analyte signal from a pair of chromatographically unresolved species through the use of phase discrimination.

Carbohydrates↗