Madhu S. Ayyagari

Enzymatic synthesis and modification of polymers in nonaqueous solvents

Enzymes catalyse several reactions that are difficult to perform with chemical catalysts and that are important in the synthesis and modification of different polymers in organic solvents. In enzyme-based synthesis, alteration of the reaction medium can have a significant influence on the molecular weight, polydispersity, yield and architecture of the polymers that are produced. Modification of these macromolecules for industrial applications requires an understanding of the different reaction strategies involved.

Solvent effects in horseradish peroxidase-catalyzed polyphenol synthesis

Abstract Enzyme-solvent-monomer molecular interactions and their effect on horseradish peroxidase-catalyzed polymerization of m-cresol in ethanol/water mixtures were studied. A mechanistic approach of the effect of reaction medium composition on the poly (m-cresol) molecular weight and polydispersity was elucidated from the standpoint molecular interactions. Solvent effects on the enzyme activity and structure were studied by reaction kinetics and spectroscopic methods including UV-vis, fluorescence, circular dichroism and electron paramagnetic spectroscopy (EPR). In view of the results from these studies, the observed polymer molecular weight profile could be deduced from the solubility of the polymer as well as the partitioning of the monomer between solvent and the enzyme active site.

Biosensors for pesticide detection based on alkaline phosphatase-catalyzed chemiluminescence

Abstract An enzyme-based detection methodology for sensing and quantitation of organophosphorus pesticides is described. The enzyme, alkaline phosphatase, is used in soluble form for reactions in bulk solutions, and in immobilized form for reactions on optical fibers or in glass capillaries. The immobilization strategy involves building a molecular assembly of the enzyme and a conjugated copolymer, poly (3-undecylthiophene-co-3-thiophenecarboxyldehyde-biotin-LC-hydrozone) on a glass surface. Hydrophobic or specific biotin-streptavidin interactions are used to immobilize the biotinylated copolymer on a silanized glass surface and attach a streptavidin conjugate of alkaline phosphatase to the copolymer. Alkaline phosphatase catalyzes the dephosphorylation of a macrocyclic compound, chloro-3-(4-methoxy spiro[1,2 dioxetane-3-2′-tricyclo-“3.3.1.1”-decan]-4-yl) phenyl phosphate and releases light; the chemiluminescence signal is detected with a simple photomultiplier tube. The enzyme activity, and proportionately the chemiluminescence signal strength, are inhibited in the presence of organophosphorus pesticides such as paraoxon. Quantitation of pesticides is thus possible from the inverse correlation between the enzyme activity or signal strength and pesticide concentration. Detection of paraoxon at a concentration of about 50 ppb is achieved with this system. Reaction kinetics are examined for the enzyme in both states. The immobilized enzyme can be reused a number of times, and there is no significant loss of enzyme activity over a period of eight weeks. The design of a flow cell for continuous analysis of the pesticides is described.

Trace Analysis of Zn(II), Be(II), and Bi(III) by Enzyme-Catalyzed Chemiluminescence

A novel technique for the trace analysis of metal ions Zn(II), Be(II), and Bi(III) in bulk solutions is discussed. This technique involves the generation of a chemiluminescence signal from alkaline phosphatase catalyzed hydrolysis of a phosphate derivative of 1,2-dioxetane. Zn(II) can be determined by two methods, reactivation of the alkaline phosphatase apoenzyme and inhibition of the native enzyme. Be(II) and Bi(III) can be determined quantitatively by inhibition of the native enzyme. Subppb to ppm level detection of Zn(II), Be(II), and Bi(III) has been achieved. Initial studies with mixed metals are also reported. The technique described is rapid and sensitive and can be readily applied to the microassay of heavy metal ions.

A chemiluminescence-based biosensor for metal ion detection

Abstract Inhibition of the native metalloenzyme, alkaline phosphatase, in the presence of some metal ions, and the reactivation of its apoenzyme by Zn(II) ions is used to determine metal ion concentrations. Alkaline phosphatase-catalysed hydrolysis of a chemiluminescent substrate, chloro 3-(4-methoxy spiro [1,2-dioxetane-3-2′-tricyclo-[3.3.1.1]-decan]-4-yl) phenyl phosphate, generates light. By measuring the chemiluminescence signal strength in the presence or absence of metal ions, this reaction can be used to detect and determine metal ion concentrations. The immobilization of alkaline phosphatase on different glass surfaces by covalent coupling using a bifunctional reagent, glutaraldehyde, was demonstrated. Using chemiluminescence measurements, Zn(II), Be(II) and Bi(III) were detected in trace levels. This technique forms the basis in the development of a metal ion-based fibre optic sensor.

Biodegradation of Polyaromatics Synthesized by Peroxidase-Catalyzed Free-Radical Polymerization

Polymers formed from peroxidase-based free-radical polymerization reactions were characterized for rates of mineralization against lignin and humic acid controls. Degradation studies were carried out in soil systems over 202 days and cumulative net CO2 was determined. Whereas mineralization of the humic acid and alkali lignin controls totaled ca. 20% at the end of the test exposure, there was essentially no net mineralization of the hydrolytic lignin control. Mineralization of the test samples totaled 5% for poly(p-ethylphenol) and 11% for poly(m-cresol). At the same time, mineralization of the poly(p-phenyl phenol) totaled 64%. Conversely, the readily biodegradable polymers cellulose and PHB reached values of 91 to 97% in less than 60 days. Our data suggest that the mineralization kinetics of the enzymatically derived polyaromatics mimic those of the naturally occurring heteropolymers.

Characterization of phenolic polymers synthesized by enzyme-mediated reactions in bulk solvents and at oil-water interfaces

Phenolic polymers are synthesized from horseradish peroxidase-catalyzed reactions in the presence of hydrogen peroxide. The reactions are carried out in homogeneous mixtures of water and polar solvents such as DMF as well as at the oil-water interface of AOT/isooctane reversed micelles and isooctane-water biphasic systems. Polymer characteristics are analyzed using thermal, spectroscopic and chromatographic techniques. Polymer molecular weight dependence on the reaction medium composition is demonstrated for poly (p-ethylphenol) and poly (m-cresol). It is shown that thermal curing of polymers leads to cross-linking, and as a result, there is a significant increase in molecular weight. Cross-linking appears to be directly to the aromatic rings, and not via ether links, as inferred from infrared spectra. A process scheme for the production, under controlled conditions, of phenolic polymers in ethanol-water mixtures is presented. The process allows enhanced monomer conversions and ability to tailor polymer molecular weight. Possible applications for phenolic polymers are discussed.

Molecular self assembly on optical fiber-based fluorescence sensor

We discuss the molecular self-assembly on optical fibers in which a novel method for protein attachment to the sensing tip of the fiber is used. Our objective is to assemble a conjugated polythiophene copolymer as an attachment vehicle. Subsequent attachment of the photodynamic phycobiliprotein serves as the fluorescence probe element. Following our earlier experiments from Langmuir-Blodgett deposition of these polymeric materials as thin films on glass substrates, we extended the technique to optical fibers. First, the bare fiber surface is silanized with a C18 silane compound. The copolymer (3-undecylthiophene-co-3- methanolthiophene, biotinylated at the methanol moiety) assembly on the fiber is carried out presumable through van der Waals interactions between the hydrophobic fiber surface and the undecyl alkyl chains on the polymer backbone. A conjugated Str-PE (streptavidin covalently attached to phycoerythrin) complex is then attached to the copolymer via the conventional biotin-streptavidin interaction. The conjugated polymer not only supports the protein but, in principle, may help to transduce the signal generated by phycoerythrin to the fiber. Our results from fluorescence intensity measurements proved the efficacy of this system. An improved methodology is also sought to more strongly attach the conjugated copolymer to the fiber surface, and a covalent scheme is developed to polymerize and biotinylate polythiophene in situ on the fiber surface.

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.

Biotinylated polyalkylthiophene thin films and monolayers that specifically incorporate phycobiliproteins: toward smart materials

We are investigating thin film and monolayer systems that involve conjugated conducting polymers and specific biological macromolecules. One class of conducting polymers, polyalkylthiophenes, are derivatized with biotin. These biotinylated polymers form the basis for a generic cassette system of attachment for any biological molecule through biotinylation or interaction with streptavidin. The high affinity of the biotin-streptavidin system, used in sequential steps, forms the basis of the cassette method. We have formed both monolayers and thin films (a few nanometers) of the cassette assembly in which phycobiliproteins are incorporated. We are investigating the optical signal transduction properties of specific phycobiliproteins (phycoerythrin, phycocyanin and allophycocyanain) using the cassette system on the inner surface of glass capillaries and on optical fiber surfaces. Phycobiliprotein photocurrent signals in conducting polymer matrices on microelectrodes are also being investigated. Our aim is to integrate the signal transduction mechanisms of the phycobiliproteins within monolayers or thin films of the conducting polymers to create biosensors and related smart materials for applications in biomedicine and biotechnology.