Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

The surface coating properties of enzymatically polymerized decyl esters of d-tyrosine from aqueous solutions onto gold surfaces have been investigated utilizing the complementary techniques of Potentiometeric Titration, UV-VIS Spectroscopy, and X-ray Photoelectron Spectroscopy (XPS). The aqueous-based coatings are of interest as environmentally friendly and cost-effective replacements for epoxy-based coatings currently employed in the microelectronics industry for both chemical protection and electrical insulation of gold-covered metallic conductors. Experimental results with respect to polymerization pH, immersion pH, and immersion time are presented and compared to the ionization behavior of the monomers in solution. Optimum processing conditions have been established, which yield uniform aqueous-based polymeric coatings on gold conductor surfaces.

A Quartz Crystal Microbalance (QCM) was used to create a biosensor utilizing living adherent endothelial cells (ECs) as the biological sensing element. This EC QCM biosensor detected the effect of varying concentrations of nocodazole, a microtubule binding and disrupting drug, on the adherent cells as they altered the underlying QCM device state frequency, Δf, and motional Resistance, ΔR, shift values. Over the dose range 0.11-15 μM nocodazole, the Δf shift values decreased significantly in magnitude in a dose dependent fashion over a 5-6 hr incubation period following drug addition to a limiting value, with a 900 nM midpoint. This effect is consistent with nocodazole's known dose dependent effect on the disruption of microtubules. At all drug concentrations, the relative Δf decrease with time was found to be very similar and well fit by a single exponential decay equation. For all nocodazole doses, t 0.5 was invariant, averaging t 0.5 = 0.83 ± 0.14 hr. These data demonstrate that a single dynamic sensing system within the cell, the microtubules, responds to the addition of nocodazole and its response can be quantified by the biosensor. These results indicate that the EC QCM biosensor can be used to detect EC cytoskeletal alterations and dynamics and may be a valuable screening method for all classes of biologically active drugs or biological macromolecules that affect cytoskeleton perturbations or cellular attachment.

We have applied the Quartz Crystal Microbalance (QCM) technique to continuously record the processes of endothelial cell (EC) adhesion, spreading and cellular mass distribution changes during initial cell to surface contact and homeostatic attachment. ECs (50,000) were layered onto a set volume of media in the QCM device and simultaneously in a mock cell used for photomicroscopy. As cells were observed in the mock cell device to contact and attach to the surface over 45-55 min, we measured in the QCM device a continuous decrease in frequency and continuous increase in resistance, achieving a maximum at about one hr (1400 Ω frequency change and 1400 Ω motional resistance change). These frequency and resistance values stabilized over the next 24 hrs and were unchanged out to 72 hr by QCM measurement (to ∼700 Hz, ∼700 Ω), as the cells were observed to spread in the mock device. Both bovine aortic (BAE) and bovine capillary (BCE) endothelial cells were studied and found to exhibit similar behavior. These studies demonstrate that QCM can be used to detect continuous changes in cell mass and viscoelastic behavior.

Normal endothelial cells (ECs), lining the blood vessels, are influenced by their interaction with the underlying potentially piezoelectric extracellular matrix (ECM). That this interaction may affect the EC metabolic state and functions in vivo prompted us to study the subsequent response of cultured ECs on indium-tin oxide (ITO) glass electrodes subjected to 1 hr of constant DC surface potential ranging from -0.3 to +0.6 V (vs. Ag/AgCl). We measured, relative to controls, cellular viability, growth rate and changes in actin microfilament organization in ECs over a subsequent 6 days in culture. The growth rate of ECs was stimulated by negative potential and inhibited by positive potential. Differences could be detected as early as three days post-potential. We also observed a potential dependent cellular shape change and actin microfilament rearrangement at positive potentials within four days of treatment. ECs changed in average cell surface area and assumed a polygonal cell shape in response to treatment. Using NBD-phalloidin stain for actin and fluorescence microscopy, microfilaments were observed to re-distribute to the periphery of the cell at positive potential, indicative of cellular stress.

The yeast S. cereviseae represents the first eukaryotic organism whose genome has been entirely sequenced as a result of the Human Genome Project(1). In this report we demonstrate the good agreement between an experimental high resolution melting curve of total nuclear S. cereviseae DNA and the theoretical melting calculated for the complete yeast DNA genome (12,067,277 bp: Saccharomyces Genome Database) by the statistical thermodynamics program MELTSIM, parameterized for long DNA sequences(2,3). The experimental and theoretical melting curves are both fairly symmetrical and possess nearly identical Tm values. Calculated melting of coding and flanking DNA regions indicates that flanking DNAs are more (A+T)-rich than coding sequences and account for the earliest melting DNA. Calculated melting curves of the 16 individual yeast chromosomes are very similar and with few exceptions exhibit symmetric melting curves. MELTSIM was also used to calculate a theoretical denaturation map of Chromosome III DNA. The agreement between MELTSIM calculated and experimental melting data demonstrates our ability to accurately simulate long DNA sequence melting in complex eukaryotic genomes, whose sequences are becoming increasingly available for study in public databases. This has important consequences for the understanding of sequence dependent energetic properties of DNA in their biological sequence context and also for their potential use in biomaterials applications.

High resolution melting curves of total nuclear Dictyostelium discoideum DNA (A×3 strain) are compared to theoretical melting calculated from GENBANK sequences(1.74 % of total) by the statistical thermodynamics program MELTSIM, parameterized for long DNA sequences(1,2). The lower and upper limits of simulated melting agree quantitatively with the experimental melting of total DNA. Calculated melting of coding, intron and flanking regions indicate that intron and flanking DNAs are extremely (A+T)-rich and account for the earliest melting DNA. There is no temperature overlap of these regions with coding DNA. A theoretical denaturation map of DNA containing the ribosomal DNA genes showed excellent agreement with subtransition positions of these genes in experimental curves. Agreement between these calculated and experimental melting data demonstrates our ability to accurately simulate DNA melting in complex eukaryotic genomes. This has important consequences for the understanding of sequence dependent energetic properties of nucleic acids and their potential use as biomaterials.

The conformation change of DNA fragments due to divalent counterion condensation onto DNA was investigated by pulse gel electrophoresis, and interpreted by gel models (Reptation and Henry model) and Manning's counterion condensation theory. The measured mobility reductions μ/μ0 of λ-DNA-Hind III fragments, ranging from 23.13 to 2.027 kilobase pairs, due to interaction with divalent cation Mg2+ (1–400 μμ), and Ca2+(0–40 μM) in tris-borate buffer were well fit by Manning's Counterion Condensation (CC) theory. We observed the normalized mobility reduction to be shifted by a small amount Δ(μ/μ0) relative to the CC prediction value. Δ(μ/μ0) is a function of DNA length, and the ion environment (divalent concentration C2 and ionic strength). The ‘shift’ phenomena only occurred close to where C2 began dominating the counterion binding, a condition described by the monovalent/divalent cation isocompetition point. Combining our observation with theoretical considerations, we conclude that the divalent counterion condensation changes the DNA fragments' conformation, resulting in an end-to-end distance decrease which is molecular weight dependent. The effect was enhanced by an increase of divalent ion concentration and a decrease of the ionic strength.

We have developed a technique to immobilize a multilayer enzyme assembly on an optical fiber surface. The technique utilizes a bifunctional amino coupling agent to link between different layers of enzymes. The method offers the flexibility of controlling the number of enzymes on a fiber surface. Multilayers of enzyme — Alkaline Phosphatase have been characterized using ellipsometry and surface plasma resonance. Chemiluminecence experiments have been conducted as a function of number of enzyme layers. The results indicated that at least 3 layers of enzyme can be assembled on a fiber surface. Utilising the new immobilization technique, a novel fiber optic biosensor system for the detection of organophosphorous-based pesticide has been developed. The detection mechanism is the pesticide inhibition of alkaline phosphatase catalyzed chemiluminescence. As a model analyte, paraoxon with ppb level concentration has been detected.

The electrochemical properties of o-dianisidine have been studied in both aqueous and organic solvents. This aryl diamine has been electrochemically polymerized on ITO coated glass electrodes and it was determined that the optical properties of these films are influenced by the polymerization conditions. The use of this polymer film as substrate for chemical coupling of biomaterials has been demonstrated with the light harvesting protein, phycoerythrin. Fabrication and bioanalytical application of enzyme electrodes by electrochemical copolymerization of odianisidine with the enzymes glucose oxidase and horseradish peroxidase, modified with odianisidineis described.

The creation of smart materials is a current goal of many laboratories including our own. We are involved in studying a number of thin film and monolayer systems which share the common features of involving highly conjugated conducting organic polymers and the evolved properties of specific biological macromolecules. In this report we describe a generic ‘cassette’ methodology for immobilizing biotinylated biological macromolecules to hydrophobic surfaces using a novel class of conducting copolymers of polythiophene. These copolymers are derivatized with long alkyl chains and biotin moieties to bind, respectively, to the hydrophobic surface and the biotinylated species, through the biotin - streptavidin interaction. We utilize the monolayer ‘cassette’ approach to attach a signal transducing biomolecule alkaline phosphatase to the surface of a glass capillary. This produces a flexible system in which chemiluminescence provides the basis for a useful measurement strategy. A novel technique involving the generation of chemiluminescence signal from alkaline phosphatase catalyzed dephosphorylation of a macrocyclic phosphate compound is described. Detection of paraoxon and methyl parathion, both enzyme inhibitors, has been achieved at ppb levels. The technique is rapid, sensitive, and is applicable to the detection of all organophosphorus based insecticides. The technique will be used in developing a fiber;optic biosensor for remote detection of insecticides.

The coupling of a photodynamic protein to an electroactive polymer may be employed in opto-electronic signal transduction for optical displays, color mimicking and biosensor applications. We have synthesized a novel biomaterial, biotin-LC-hydrazide substituted poly(3-undecylthiophene-co-3-thienylcarboxaldehyde) [B-PUAT], which is capable of incorporating any biotinylated molecule through a bridging streptavidin protein. A long spacer group between the conjugated polymer backbone and the biotin side group improves monolayer formation at the airwater interface and enhances the material's ability to bind protein. The LB technique has been employed to couple this biomaterial and streptavidin through the well known biotin-streptavidin complexation. Biotinylated phycoerythrin [B-PE] is incorporated into the above monolayer by binding the unoccupied biotin binding sites on the bound streptavidin.

The polycation conducting polymer, oxidized polypyrrole (PPy), possesses the ability to form complexes with DNA. Our previously proposed diffusion limited binding model for double helical DNA was also found to be applicable to single stranded DNA in this study. Single stranded DNA was found to bind PPy at a nearly identical level to that of double helical DNA. An investigation of electropolymerized PPy film morphology using SEM revealed two distinctly differing surface morphologies for the Platinum (Pt) electrode face (smooth) and polymeric growth face (rough). The DNA uptake levels were found to be consistently different on either surface, being higher on the rough surface. DNA penetrated into the disk interior with increasing time periods of exposure while a similar phenomenon but to a lesser extent was observed for single stranded DNA.

A novel hierarchical biomaterial capable of incorporating any biotinylated biomolecule has been created. Our strategy is to biotinylate one-dimensional electroactive polymers and use a bridging streptavidin protein on Langmuir-Blodgett (LB) organized films. The following copolymeric system which enables functionalization of other molecules and formation of good monolayers was employed. Biotinylated poly(3-methanolthiophene-co-3-undecylthiophene) (B-PMUT) demonstrated a significantly better isotherm implying superior molecular packing compared to poly(3-methanolthiophene-co-3-undecylthiophene) (PMUT) on the LB airwater surface. The isotherm showed significant area expansion when streptavidin was injected below the B-PMUT monolayer in 0.1mM NaH2PO4/0.1 M NaCl buffer (pH 6.8) subphase. We then incorporated biotinylated phycoerythrin (B-PE) into this novel biomaterial by binding the unoccupied biotin binding sites on the bound streptavidin (4 sites total). The pressure-area isotherm of the protein injected monolayer showed area expansion. A characteristic fluorescent emission peak at 576nm was detected from the monolayer transferred onto a solid substrate. These observations demonstrated the function of B-PMUT in hierarchical monolayer assembly of molecules incorporating the biotin / streptavidin interaction.

Enzymatic polymerization of a number of monomers in an ordered lattice on a Langmuir trough (L-T) was investigated. The assembly and polymerization of two mixed monomeric systems consisting of 4-tetradecyloxyphenol (C14PP) with phenol, and 4-hexadecylaniline (C16PA) with aniline, in various ratios were carried out. Polymerization was obtained with C14PP and phenol (in the ratio 1:10), and C16PA and aniline (in the ratio 1:2) on buffered MilliQ water (pH 7.5) using the enzyme horseradish peroxidase (HRP) at 20°C. Polymerized monolayers were then transferred to appropriate substrates for UV-Vis, third order non linear optical (NLO) properties, and thickness measurements. Thermogravimetric analysis (TGA) was performed on the final polymers. Results suggest that the lattice controlled polymerization results in highly ordered conjugated polymers with improved functional, electronic, NLO and processability properties, which form a basis for intelligent materials design and applications.

The polycation conducting polymers, oxidized polypyrrole and polyalkylthiophene, possess the ability to form complexes with polyanionic DNA molecules through largely electrostatic interactions. This study demonstrated the solution uptake and binding of 32p radiolabeled DNA by conducting polymer thick films (50–100μm). Polypyrrole (PPy) was synthesized by electrochemical methods and poly(3-hexylthiophene) (PHT) and poly(3-undecylthiophene) (PUT) were synthesized by chemical methods. The DNA binding rates on PPy films were affected by DNA concentration and the oxidation state (measured as conductivity). The DNA kinetics support a diffusion limited model for binding. We measured DNA binding levels onto all three polymer films; PUT, PHT, and PPy. The binding levels increased in the same order as the conductivities of the polymer films. DNA binding onto oxidized PPy film was diminished upon electrochemical reduction. These observations showed, therefore, the binding may be linked with the positive charge sites responsible for conduction in the polymer films.