Fibroblast extracellular matrix and adhesion on microtextured polydimethylsiloxane scaffolds.

The immediate physical and chemical surroundings of cells provide important biochemical cues for their behavior. Designing and tailoring biomaterials for controlled cell signaling and extracellular matrix (ECM) can be difficult due to the complexity of the cell-surface relationship. To address this issue, our research has led to the development of a polydimethylsiloxane (PDMS) scaffold with defined microtopography and chemistry for surface driven ECM assembly. When human fibroblasts were cultured on this microtextured PDMS with 2-6 µm wide vertical features, significant changes in morphology, adhesion, actin cytoskeleton, and fibronectin generation were noted when compared with cells cultured on unmodified PDMS. Investigation of cellular response and behavior was performed with atomic force microscopy in conjunction with fluorescent labeling of focal adhesion cites and fibronectin in the ECM. Changes in the surface topography induced lower adhesion, an altered actin cytoskeleton, and compacted units of fibronectin similar to that observed in vivo. Overall, these findings provide critical information of cell-surface interactions with a microtextured, polymer substrate that can be used in the field of tissue engineering for controlling cellular ECM interactions.

Cell behavior on surface modified polydimethylsiloxane (PDMS).

Designing complex tissue culture systems requires cell alignment and directed extracellular matrix (ECM) and gene expression. Here, a micro-rough, polydimethylsiloxane (PDMS) surface, that also integrates a micro-pattern of 50 µm wide lines of fibronectin (FN) separated by 60 µm wide lines of bovine serum albumin (BSA), is developed. Human fibroblasts cultured on the rough, patterned substrate have aligned growth and a significant change in morphology when compared to cells on a flat, patterned surface. The rough PDMS topography significantly decreases cell area and induces the upregulation of several ECM related genes by two-fold when compared to cells cultured on flat PDMS. This study describes a simple surface engineering procedure for creating surface architecture for scaffolds to design and control the cell-surface interface.

Super-hydrophobic, highly adhesive, polydimethylsiloxane (PDMS) surfaces.

Super-hydrophobic surfaces have been fabricated by casting polydimethylsiloxane (PDMS) on a textured substrate of known surface topography, and were characterized using contact angle, atomic force microscopy, surface free energy calculations, and adhesion measurements. The resulting PDMS has a micro-textured surface with a static contact angle of 153.5° and a hysteresis of 27° when using de-ionized water. Unlike many super-hydrophobic materials, the textured PDMS is highly adhesive, allowing water drops as large as 25.0 µL to be inverted. This high adhesion, super-hydrophobic behavior is an illustration of the "petal effect". This rapid, reproducible technique has promising applications in transport and analysis of microvolume samples.

Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor.

Acetylcholinesterase (AChE) inhibitors are potentially lethal but also have applications as therapeutic drugs for neurodegenerative diseases such as Alzheimer's. Enzyme inhibitor binding are difficult to be detected directly by surface plasmon resonance (SPR) due to their small molecular weight. In this article, we describe the detection of AChE inhibitor binding by SPR without the use of competitive binding or antibodies. AChE was immobilized on the gold surface of an SPR sensor through covalent attachment to a self-assembled monolayer (SAM) of a COOH-terminated alkanethiol. The activity of the immobilized protein and the surface density were determined by using a standard photometric assay. Binding of two reversible inhibitors, which are used as therapeutic drugs, was detectable by SPR without the need to further modify the surface or the use of other reagents. The binding affinities (K(A)) obtained from the fits were 3.8 × 10(3)M(-1) for neostigmine and 1.7 × 10(3)M(-1) for eserine, showing a higher affinity of the sensor for neostigmine. We believe that the SPR sensor's ability to detect these inhibitors is due to conformational changes of the enzyme structure on inhibitor binding.

SPR-based single nucleotide mismatch biosensor.

The detection and characterization of the hybridization event of 21-base, unlabeled DNA oligonucleotides with a monolayer of complementary DNA immobilized on a gold surface, by electrochemical impedance spectroscopy and surface plasmon resonance (SPR) is presented. A thiol modification on the probe DNA strand allowed for its attachment to the surface via self-assembly. For the hybridization of full match target DNA a detection limit of 20 pM was determined. RNA hybridization was also detectable with the same sensor, with a similar detection limit. The SPR signal generated upon hybridization of the full match was always distinguishable from the single mismatch target DNA oligonucleotides when the mismatch was in the middle or at the proximal end of the target DNA sequence. However, the response of the sensor was identical for the hybridization of the full match and the distal end mismatch. The SPR sensor described is reusable over at least 20 hybridization/regeneration cycles and is insensitive to flow rate (20-800 µL min-1) or temperature (20-60 °C). Based on the SPR response, the surface density of the probe was estimated to be at least 4.3 × 1012 molecules per cm2.

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers.

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.

A multilayered approach to complex surface patterning.

A method for developing complex nanopatterns on surfaces has been developed by combining self-assembly, photolabile protecting groups, and multilayered films. An o-nitrobenzyl protecting group has been incorporated into molecular level films utilizing thiol-gold interactions. When the o-nitrobenzyl group is cleaved by ultraviolet light, a carboxylic acid terminated layer remains on the surface and is available for activation and further functionalization through amide bond formation. Using this method, multilayered films have been constructed and characterized by contact angle goniometry, cyclic voltammetry, grazing incidence infrared spectroscopy, and X-ray photoelectron spectroscopy measurements. Complex surface patterns can be achieved by creating a surface array using a photomask and then further functionalizing the irradiated area through covalent coupling. Fluorophores were attached to the deprotected regions, providing visual evidence of surface patterning using fluorescence microscopy. This approach is universal to bind moieties containing free amine groups at defined regions across a surface, allowing for the development of films with complex chemical and physicochemical properties.

Detection of oligonucleotide systematic mismatches with a surface plasmon resonance sensor.

The detection and parallel characterization of the hybridization event of 21-base, unlabeled DNA oligonucleotides with a monolayer of complementary DNA immobilized on a gold surface by surface plasmon resonance (SPR) is presented. A thiol modification on the probe DNA strand allowed for its attachment to the surface via self-assembly. For the hybridization of full match DNA a detection limit of 20 pM was determined. The change in SPR signal was always larger for the full match compared to the one-mismatch target DNA oligonucleotides when the mismatch was in the middle or at the proximal end of the target DNA. Hybridization conditions (buffer concentration, flow rate, and temperature) did not affect the ability of the sensor to discriminate for single nucleotide mismatches. To our knowledge this is the only work where a comparison on the surface hybridization efficiency is performed between proximal, distal, and middle mismatches and the effect of three hybridization parameters is studied with regard to the detection of single nucleotide mismatches using SPR.

Effect of electrode roughness on the capacitive behavior of self-assembled monolayers.

Analytical gold electrodes were polished mechanically and electrochemically and the true area of the electrode surface was measured by quantitative oxidative/reductive cycling of the electrode. A roughness factor for each electrode was determined from the ratio of the true area to the geometric area. The roughness is fully described by a combination of microscopic roughness (up to tens of nanometers) and macroscopic roughness (on the order of hundreds of nanometers) terms. The electrodes were then derivatized with a self-assembled monolayer (SAM) of dodecanethiol or a thioalkane azacrown and characterized by impedance spectroscopy. The behavior of the electrodes was modeled with either a Helmholtz or Randles equivalent circuit (depending on the SAM used) in which the capacitance was replaced with a constant phase element. From the model, an effective capacitance and an alpha factor that quantifies the nonideality of the SAM capacitance was obtained. The effective capacitance divided by the roughness factor yields the capacitance per unit true area, which is only a function of microscopic roughness. The relationship between this capacitance and the alpha factor indicates that microscopic roughness predominantly affects the nonideality of the film while macroscopic roughness predominantly affects the magnitude of the film's capacitance. Understanding the contribution of the electrode topography to the magnitude and ideality of the SAM capacitance is important in the construction of SAM-based capacitive sensors because it predicts the importance of electrode-electrode variations.

13C NMR study of the ion-binding selectivity of a new ammonium ionophore.

A bicyclic peptide, cyclo (L-Glu(1)-D-Leu(2)-Aib(3)-L-Lys(4)-D-Leu(5)-D-Ala(6))-cyclo-(1gamma-4epsilon) (I), was designed and synthesized to provide an ammonium ion complexation site in a tetrahedral geometry. Molecular modeling, dynamics and electrostatic studies for I indicated that it exhibits some selectivity for ammonium ions over potassium and sodium ions. NMR measurements in CDCl(3)/CD(3)OD (1:1) show that for those carbonyl groups involved in cation binding, (13)C resonances shifted downfield with increasing cation concentration. The resonance that exhibited the largest change in chemical shift between uncomplexed and complexed forms was used to determine the selectivity. Selectivity values obtained were logK(NH(4) (+), Na(+) ) = - 2.4 and logK(NH(4) (+), K(+) ) = - 0.6.

Photocurrent generation in noncovalently assembled multilayered thin films.

Multilayered photocurrent generating thin films were fabricated by templated noncovalent assembly via stepwise assembly of molecular components. Each of films I-IV contained an underlying self-assembled monolayer (SAM) consisting of an alkanethiol linked covalently to a 2,6-dicarboxypyridine ligand that served as a binding site for attaching additional molecular components. The SAM subsequently was functionalized by sequential deposition of Cu(II), Co(II), or Fe(III) ions followed by a variety of substituted 2,6-dicarboxypyridine ligands as a means to incorporate one or more layers of pyrene chromophores into the film. The films were characterized by contact angle measurements, ellipsometry, grazing incidence IR, cyclic voltammetry, and impedance spectroscopy after deposition of each layer, confirming the formation of ordered, stable layers. Following incorporation into a three-electrode system, photoexcitation resulted in the generation of a cathodic photocurrent in the presence of methyl viologen and an anodic photocurrent in the presence of triethanolamine. Using this strategy, systems were fabricated that produced up to 89 nA/cm(2) of reproducible photocurrent.

Reversible photoswitchable wettability in noncovalently assembled multilayered films.

Reversible and irreversible photoinduced changes in surface wettability were observed in noncovalently assembled multilayered films. The multilayered films studied were fabricated from a self-assembled monolayer (SAM) consisting of 4-(10-mercaptodecyloxy)pyridine-2,6-dicarboxylic acid on gold, Cu(II) ions complexed to the pyridine head group of the SAM, and either cis- (film 1) or trans- (film 2) stilbene-4,4'-dicarboxylic acid complexed to the Cu(II) ions. Irradiation of film 1 at wavelengths corresponding to the absorption band of the cis-stilbene isomer resulted in an irreversible chemical change and an irreversible increase in wettability, as indicated by surface contact angle and grazing incidence IR measurements. However, no evidence for cis-/trans-photoisomerization was observed. Films 3 and 4, similar to films 1 and 2 in that they consist of an underlying SAM, an intermediate layer consisting of Cu(II) ions, and either cis- or trans-stilbene-4,4'-dicarboxylic acid as the capping ligand, were fabricated with a mixed SAM that contained both 4-(10-mercaptodecyloxy)pyridine-2,6-dicarboxylic acid and 4-tert-butylbenzenethiol. Irradiation of these films at wavelengths corresponding to stilbene isomer absorption bands resulted in reversible cis- to trans- (film 3) and trans- to cis- (film 4) photoisomerization and reversible switching of the surface wettability between a low wetting state (cis-stilbene) and a high wetting state (trans-stilbene). The difference in observed behavior between films 1 and 2 and films 3 and 4 is attributed to the greater surface spacing afforded by the mixed monolayer, which allows greater conformational flexibility and lowers the steric barriers to isomerization.

Microscale correlation between surface chemistry, texture, and the adhesive strength of Staphylococcus epidermidis.

Staphylococcus epidermidis is among the most commonly isolated microbes from medical implant infections, particularly in the colonization of blood-contacting devices. We explored the relationships between surface wettability and root-mean-square roughness (Rq) on microbial adhesive strength to a substrate. Molecular-level interactions between S. epidermidis and a variety of chemically and texturally distinct model substrata were characterized using a cellular probe and atomic force microscopy (AFM). Substrata included gold, aliphatic and aromatic self-assembled monolayers, and polymeric and proteinaceous materials. Substrate hydrophobicity, described in terms of the water contact angle, was an insufficient parameter to explain the adhesive force of the bacterium for any of the surfaces. Correlations between adhesion forces and Rq showed weak relationships for most surfaces. We used an alternate methodology to characterize the texture of the surface that is based on a fractal tiling algorithm applied to images of each surface. The relative area as a function of the scale of observation was calculated. The discrete bonding model (DBM) was applied, which describes the area available for bonding interactions over the full range of observational scales contained in the measured substrate texture. Weak negative correlations were obtained between the adhesion forces and the area available for interaction, suggesting that increased roughness decreases bacterial adhesion when nano- to micrometer scales are considered. We suggest that modification of the DBM is needed in order to include discontinuous bonding. The adhesive strength is still related to the area available for bonding on a particular scale, but on some very fine scales, the bacteria may not be able to conform to the valleys or pits of the substrate. Therefore, the bonding between the bacterium and substrate becomes discontinuous, occurring only on the tops of ridges or asperities.

Surface-based lithium ion sensor: An electrode derivatized with a self-assembled monolayer.

Self-assembled monolayers (SAMs) of 21-(16-mercaptohexadecan-1-oyl)-4,7,13,16-tetraoxa-1,10,21-triazabicyclo[8.8.5]tricosane-19,23-dione were prepared on gold. Characterization of the SAMs was carried out by sessile drop contact angle, ellipsometry, grazing angle FT-IR spectroscopy, and electrochemical techniques. The cation recognition properties of the SAM were studied by cyclic voltammetry and impedance spectroscopy. The films show moderate selectivity for detection of Li+ ions in solution over K+ and Na+, with selectivity values calculated to be log K(Li+,Na+) approximately -1.30 and log K(Li+,K+) approximately -0.92. To the best of our knowledge, this is the first demonstration of a lithium sensor fabricated using self-assembled monolayer technology.

A highly selective bicyclic fluoroionophore for the detection of lithium ions.

The macrobicyclic molecule, 21-(9-anthrylmethyl)-4,17,13,16-tetraoxa-1,10,21-triazabicyclo [8.8.5]tricosane-19,23-dione, I, was designed, synthesized and characterized as a fluoroionophore for the selective, optical detection of lithium ions. Compound I is based on a bridged diazacrown structure, which provides a semirigid binding framework. Binding takes place by electrostatic interactions between the oxygen atoms of the crown and the cation and is transduced to fluorescence emission from an attached anthracene fluorophore. In a 75:25 dichloromethane/tetrahydrofuran solvent mixture, I acts as an intramolecular electron transfer "off-on" fluorescence switch, exhibiting a greater than 190-fold enhancement in fluorescence emission intensity in the presence of lithium ions. The relative selectivity of I for lithium ions over sodium, potassium and ammonium ions was found to be log K(Li+,Na+) approximately -3.36, log K(Li+,K+) approximately -1.77 and log K(Li+,NH4+) approximately -2.78.

Non-covalent assembly of a photoswitchable surface.

A noncovalently bound multilayered thin film in which individual layers are linked by metal ligand interactions undergoes a photochemically initiated permanent change in surface wettability. The film consists of three separate layers: a SAM on gold of 4-[(10-mercaptodecyl)oxy]pyridine-2,6-dicarboxylic acid, a layer of Cu(II) ions that are deposited onto the SAM and bind symmetrically in the site provided by the two carboxylate groups and the pyridyl nitrogen atom, and a layer of cis-2,2'-dipyridylethylene, which caps the Cu(II) layer by complexation through both pyridyl nitrogen atoms (Film I). Photoexcitation of the film in chloroform at 300 nm leads to substantial cis-trans isomerization as indicated by conductivity, impedance, grazing incidence IR, and contact angle measurements. The latter show a decrease in contact angle (increase in wettability) of 17 degrees , which is attributed to exposure of both the underlying Cu(II) layer and one of the pyridyl ring nitrogen atoms following isomerization to the trans isomer.

A non-covalent strategy for the assembly of supramolecular photocurrent-generating systems.

Three photocurrent-generating thin films were assembled on gold surfaces. SAM I was constructed from molecules consisting of an alkyl disulfide group linked covalently to a 12-residue helical peptide and terminated with an alanine residue containing a pyrene chromophore. SAM I served as a benchmark for multilayered films II and III in photocurrent generation experiments. Films II and III were assembled from several components that were linked noncovalently by metal-ligand complexation. Cyclic voltammetry and contact angle measurements suggest that the films consist of ordered layers with relatively few defects. Photoexcitation of SAM I by the output of a 350 nm lamp ( approximately 0.2 mW power incident on the sample) results in current generation in the range 5-10 nA/cm2. Photoexcitation of II and III yields higher current in the range 10-30 nA/cm2, representing a quantum efficiency of approximately 1%. The observation of comparable or higher current from noncovalently assembled multicomponent films indicates that this method of assembly may obviate the problems associated with the covalent assembly of devices from large molecules.

Synthesis of an ammonium ionophore and its application in a planar ion-selective electrode.

A modular technique was used to synthesize an ammonium-selective ionophore based on a cyclic depsipeptide structure. The ionophore was incorporated into a planar ion-selective electrode sensor format and the selectivity tested versus a range of metal cations in a commercial clinical diagnostic "point-of-care" instrument. Four sensor membrane formulations were tested, all of which consisted of plasticized PVC. Formulations differed as to the type of plasticizer used and whether an ionic additive was present. It was found that the membrane containing the polar plasticizer nitrophenyl octyl ether in the absence of ionic additive exhibited near-Nernstian behavior (slope, 60.1 mV/decade at 37 degrees C) and possessed high selectivity for ammonium ion over lithium and the divalent cations, calcium and magnesium (log K(POT)NH4+(j) = -7.3, -4.4, and -7.1 for lithium, calcium, and magnesium ions, respectively). The same membrane also exhibited sodium and potassium selectivity that was comparable to that reported for nonactin (log K(POT)NH4+(j) = -2.1 and -0.6 for sodium and potassium, respectively, compared to -2.4 and -0.9 in the case of nonactin). Membranes containing the less polar plasticizer, dioctyl phthalate, showed sub-Nernstian behavior (slope, <50 mV/decade at 37 degrees C). In all cases, the presence of the ionic additive potassium tetrakis(4-chlorophenyl)borate substantially reduced the selectivity observed. The flexible modular synthetic technique developed and reported here will allow the cyclic depsipeptide structure to be tuned for optimum selectivity.

Photochemistry of Aryl Vinyl Sulfides and Aryl Vinyl Ethers: Evidence for the Formation of Thiocarbonyl and Carbonyl Ylides.

Aryl vinyl thioethers 5a and 9a and aryl vinyl ethers 5b and 9b form ylide intermediates following laser irradiation at 308 nm. In benzene, the ylides possess long-lived absorption bands in the 600-800 nm region with a second weaker band at approximately 460 nm. In methanol, a known quencher of zwitterionic species, the lifetimes are reduced significantly. The decay kinetics measured within the long wavelength absorption envelope vary with wavelength, indicating the presence of more than one ylide species. Formation of the ylides occurs via a naphthalene-like triplet state in the case of aryl vinyl ethers, while for the thioethers the multiplicity of the ylide precursor could be either singlet or triplet. Product formation in benzene for 5a and 5b involves ring closure of the ylide to produce dihydrothiophene and dihydrofuran products, respectively. For short periods of irradiation (either lamps or laser) a mixture of cis- and trans-fused products is observed, while for prolonged irradiation only the cis-fused compound is detected, suggesting a photoenolization mechanism for conversion of trans to cis. In addition to products derived from ring closure, 9a provides intramolecular addition product 12. Conversely, the ylide derived from 9b gives rise to the [3 + 2] cycloaddition product 13.