David L. Kaplan

Molecular assembly of multilayer enzyme on fiber optic surface: toward the development of chemiluminescence-based fiber optic biosensors

We report here on a technique to immobilize a multilayer enzyme assembly on an optic fiber surface. A multilayer of an enzyme, alkaline phosphatase, was successfully immobilized on an optical fiber surface. Chemiluminescence, ellipsometry, and surface plasmon resonance were used to characterize the structure and activity of the assembly. A chemiluminescence-based fiber optic biosensor utilizing this immobilization technique has been developed for the detection of organophosphorous-based pesticides. Detection of pesticide at sub-ppm level has been achieved for paraoxon.

Langmuir films of amino acid-modified diacetylenes as organic templates for biomimetic mineralization

Amino-acid modified diacetylenes were used as organic templates for controlled biomineralization. Macromolecular changes in the monolayers have been observed upon application of pressure or polymerization. The degree of film compression and film polymerization influenced calcium carbonate polymorph selectivity. The desired optimization of the physical properties of the biomineral product is approached via structural control of the organic monolayer.

Integrating biotinylated polyalkylthiophene thin films with biological macromolecules: biosensing organophosphorus pesticides and metal ions with surface immobilized alkaline phosphatase utilizing chemiluminescence measurements

We describe a methodology for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer poly (3-undecylthiophene-co-3- thiophenecarboxaldehyde) 6-biotinamido hexanohydrazide attached hydrophobically to silanized glass. The biotin-streptavidin protein interaction is used to carry out this immobilization. Alkaline phosphatase catalyzes the dephosphorylation of a class of macrocyclic compounds: including CSPD {chloro 3-[4-methoxy spiro(1,2 dioxetane-3-2-trichloro-(3.3.1.1)-decan]-4 yl}phenyl phosphate to a product species which emits energy by chemiluminescence. We can detect this chemiluminescence signal with a photomultiplier tube for both enzymatic catalysis in solution and the surface immobilized enzyme (streptavidin conjugate). This enzyme is inhibited by the organophosphorus class of pesticides as well as nerve agents. The enzyme is also inhibited by Be(II), Bi(III) as well as excess Zn(II), while the apoenzyme is reactivated by Zn(II). We demonstrate in this study that two representative organophosphorus pesticides inhibit the enzymatic production of chemiluminescent products. This is true for the enzyme conjugate both free in solution and immobilized. We can detect pesticides down to about 50 ppb for the enzyme in solution and 500 ppb for surface immobilized enzyme in a 100 (mu) l capillary. Detection of Zn(II) by apoenzyme reactivation occurs down to 3 ppb. Be(II) and Bi(III) are detected by inhibition down to 1 ppm.

Ivermectin Promotes Peripheral Nerve Regeneration during Wound Healing

Peripheral nerves have the capacity to regenerate due to the presence of neuroprotective glia of the peripheral nervous system, Schwann cells. Upon peripheral nerve injury, Schwann cells create a permissive microenvironment for neuronal regrowth by taking up cytotoxic glutamate and secreting neurotrophic signaling molecules. Impaired peripheral nerve repair is often caused by a defective Schwann cell response after injury, and there is a critical need to develop new strategies to enhance nerve regeneration, especially in organisms with restricted regenerative potential, such as humans. One approach is to explore mechanisms in lower organisms, in which nerve repair is much more efficient. A recent study demonstrated that the antiparasitic drug, ivermectin, caused hyperinnervation of primordial eye tissue in Xenopus laevis tadpoles. Our study aimed to examine the role of ivermectin in mammalian nerve repair. We performed in vitro assays utilizing human induced neural stem cells (hiNSCs) in co-culture with human dermal fibroblasts (hDFs) and found that ivermectin-treated hDFs promote hiNSC proliferation and migration. We also characterized the effects of ivermectin on hDFs and found that ivermectin causes hDFs to uptake extracellular glutamate, secrete glial cell-derived neurotrophic factor, develop an elongated bipolar morphology, and express glial fibrillary acidic protein. Finally, in a corresponding in vivo model, we found that localized ivermectin treatment in a dermal wound site induced the upregulation of both glial and neuronal markers upon healing. Taken together, we demonstrate that ivermectin promotes peripheral nerve regeneration by inducing fibroblasts to adopt a glia-like phenotype.

Bioreceptor-conducting polymer multilayer assemblies for biosensing

This research focuses on the organized integration of biological receptors and polymers into thin film architectures for biosensing applications. Layer-by-layer electrostatic adsorption was used for the first time to form alternating protein-conducting polymer multilayers. The light-harvesting, phycobiliproteins and the enzyme, alkaline phosphatase were the bioreceptors investigated and sulfonated polystyrene, poly(diallyl dimethyl ammonium chloride) and a new enzymatically polymerized, water soluble, polyaniline were the polymer counterions used for deposition. Spectroscopic characterization was used to determine both multilayer formation and biosensing function of the final bioreceptor-polymer assemblies. These techniques have proven to be simple, chemically mild, and versatile and are expected to find application in the fabrication of ultrathin films for biosensors, opto- electronic devices and biomedical applications.

Streptavidin-based conditional lethal system for biological containment of Pseudomonas putida

The soil bacterium Pseudomonas putida can potentially be applied in bioremediation of areas polluted with aromatic hydrocarbon-based organic solvents and petroleum. However, its use in the open environment has been hindered so far by the lack of reliable survival-control functions. To increase the predictability of P. putida, a novel cell suicide system was constructed, based on triple control of the expression of the lethal Streptomyces avidinii streptavidin gene (stv) coupled with P. putida TOL plasmid-derived Pm/xylS regulatory circuit. In the absence of hydrocarbon pollutants (3-methylbenzoate in described in vitro studies), all but one in 107 to 108 of genetically modified bacteria commits suicide. The stv-based conditional lethal design can thus effectively limit the spread of released microorganisms strictly to polluted localities and keep them alive only as long as the amount of contaminants keeps above a level determined by the sensitivity of their interaction with the XylS protein.

Biochemically designed polymers as self-organized materials

Self assembled molecular systems are a focus of attention for material scientists as they provide an inherent molecular level organization responsible for enhanced material properties. We have developed polymeric molecular systems with interesting optical properties by biochemical engineering, which can be self assembled to thin films. Horseradish peroxidase catalyzed polymerizations of phenolic monomers: 9-hydroxyquinoline-5-sulfonic acid, acid red and decyl ester (d&l isomers) of tyrosine, have been achieved in the presence of hydrogen peroxide. The polymer of 8- hydroxyquinoline-5-sulfonic acid acts as a polymeric ligand that can be used for metal ion sensing. The polymer of acid red, with azo functional groups in the polymer backbone, shows interesting optical properties. Amphiphilic derivatives of tyrosine self assemble into tubules from micelles in aqueous solutions. These tubules have been enzymatically polymerized to polymeric tubules. The tubules are of 5 micrometers average diameter and > 200 micrometers length. The formation and properties of these tubules are discussed.

Advanced Materials from Enzymatic Polymerization of Substituted Phenols in Ordered Templates

Enzyme-catalyzed polymerization of p-substituted phenols and aromatic amines is described. The polymers were prepared as thin films at the air-water interface of a Langmuir trough, as spherical particles at the oil-water interface of reversed micelles or as amorphous material in bulk organic solvents. Horseradish peroxidase or laccase was used to catalyze the reactions. Molecular weight and size distribution of polyphenols formed with reversed micelles or in bulk solvents could be modulated by controlling solvent properties. For example, oligomers with fairly uniform size distribution could be prepared by varying the solvent polarity, p-Alkoxyphenols with long alkyl chains could be ordered into a monolayer on a Langmuir trough, and could be subsequently polymerized using enzymes. The polymers were analyzed for their thermal, mechanical, electrical and optical properties. The application potential of the polymers in electronics/photonics is discussed.