Enzyme electrodes with improved mechanical and analytical characteristics obtained by binding enzymes to nylon nets

Enzyme electrodes obtained by binding enzymes on nylon net show improved mechanical and analytical characteristics. The chemical binding process is mild so that the final membrane retains a high enzyme activity. The resulting membrane also has high mechanical strength and can be assembled on the electrode surface several times without damage. Response times without nylon net are excellent. Examples with single and dual enzymes coupled with an oxygen or ammonia electrode are discussed.     

Properties of membranes obtained by radiation castpolymerization of hydroxyalkyl methacrylate monomers

The degree of hydration and oxygen permeability of membranes obtained by radiation cast-polymerization of various hydroxyalkyl methacrylate monomers were studied. The degree of hydration of the polymeric membranes decreased with increasing numer, n, of methylene units (CH₂) in the monomers. The linear expansion of the polymeric membranes swollen by water decreased with increasing n. The oxygen permeability of the polymeric membranes increased moderately up to n = 4 with increasing n, and after that increased suddenly. This variation of oxygen permeability as a function of n was explained by the flexibility of polymer segments.     

Enzyme:nanoparticle bioconjugates with two sequential enzymes: stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles

We report the synthesis and characterization of bioconjugates in which the enzymes malate dehydrogenase (MDH) and/or citrate synthase (CS) were adsorbed to 30 nm diameter Au nanoparticles. Enzyme:Au stoichiometry and kinetic parameters (specific activity, k(cat), K(M), and activity per particle) were determined for MDH:Au, CS:Au, and three types of dual-activity MDH/CS:Au bioconjugates. For single-activity bioconjugates (MDH:Au and CS:Au), the number of enzyme molecules adsorbed per particle was dependent upon the enzyme concentration in solution, with multilayers forming at high enzyme:Au solution ratios. The specific activity of adsorbed enzyme increased with increasing number adsorbed per particle for CS:Au, but was less sensitive to stoichiometry for MDH:Au. Dual activity bioconjugates were prepared in three ways: (1) by adsorption of MDH followed by CS, (2) by adsorption of CS followed by MDH, and (3) by coadsorption of both enzymes from the same solution. The resulting bioconjugates differed substantially in the number of enzyme molecules adsorbed per particle, the specific activity of the adsorbed enzymes, and also the enzymatic activity per particle. Bioconjugates formed by adding CS to the Au nanoparticles before MDH was added exhibited higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also had 3-fold higher per-particle sequential activity for conversion of malate to citrate, despite substantially fewer copies of both enzymes present.     

A novel Mn-containing conducting metallopolymer obtained by electropolymerization in aqueous solution of a tetranuclear oxo-bridged manganese complex

The [Mn(4)(IV)O(5)(terpy)(4)(H(2)O)(2)](6+) complex shows great potential for electrode modification by electropolymerization using cyclic voltammetry. The electropolymerization mechanism was based on the electron transfer between dx(2)-y(2) orbitals of the metallic center and pπ orbital of the ligand.     

Formation and redox behaviour of polycarbazole prepared by electropolymerization of solid carbazole crystals immobilized on an electrode surface

Carbazole solid crystals have been mechanically attached to platinum or gold electrodes by two different methods and investigated by cyclic electrochemical quartz crystal microbalance measurements in the presence of aqueous acidic media. It was demonstrated that oxidative dimerization and polymerization can also be accomplished under such conditions. During electropolymerization, anions and water molecules enter the surface layer; however, these species leave the film after the reduction of the polymer formed. The mass changes observed in the course of the redox transformations of polycarbazole film can be explained by potential- and time-dependent sorption/desorption of H⁺ and ClO₄ ⁻ ions. The electroactivity of the polymer diminishes in more dilute acid media; however, it is recovered again in concentrated HClO₄ solutions. In more dilute acid solution the extent of the water sorption (film swelling) increases.     

Performance of amidocoumarins as probes for monitoring of cationic photopolymerization of monomers by fluorescence probe technology

The performance of 7‐benzamido‐4‐methylcoumarin ( B ) and 7‐acetamido‐4‐methylcoumarin ( C ) as fluorescent probes for monitoring of progress of cationic photopolymerization of monomers by fluorescence probe technology has been evaluated in comparison with 7‐diethylamino‐4‐methylcoumarin ( A ). It has been found that the probes B and C do not cause such a delay of the polymerization start as the probe A does, but in the case of B and C , the cure monitoring using the fluorescence intensity ratio at two wavelengths as an indicator of the polymerization progress is not applicable. An alternative monitoring parameter has been applied successfully and a method for compensation of the effects of probe photolysis is proposed. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Preparation and characterization of porous polymeric microspheres obtained from multifunctional methacrylate monomers

Synthesis and properties of the new difunctional methacrylate monomer 2‐hydroxy‐3‐methacryloyloxypropoxybenzene are presented. This monomer was applied for the synthesis of porous microspheres. It was copolymerized with trimethylolpropane trimethacrylate in the presence of two pairs of pore‐forming diluents dodecane and toluene, and n‐decanol and toluene. Influence of diluents composition on their porous structures was studied. Thermal resistance and tendency to swell in different organic diluents for a chosen sample were also determined. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6165–6174, 2008     

Copolymers of pyrrole and bithiophene by oxidative electropolymerization

Pyrrole (PY) and 2,2′-bithiophene (BT) have been electropolymerized at various feed ratios in LiClO₄/Propylene carbonate (PC) and the mer ratios in the copolymer determined by N and S. microanalysis. A strong correlation was observed between the copolymer compositions and the monomer feed ratios using the copolymer equation, and reactivity ratios were determined by a nonlinear error-in-variables approach. Although PY predominates in these copolymers, the proportion of BT increases with increasing applied potential. It is shown that reactivity ratios do not have to be understood in the traditional sense, but may be simply a measure of the relative ease of oxidation of the monomers at the electrode.     

Kinetic determination of l-alanine and l-alanine dehydrogenase with an ammonia gas-sensing electrode

The ammonia gas-sensing electrode is used to assay l-alanine and l-alanine dehydrogenase. Alanine is de-aminated by bacterial alanine dehydrogenase in the presence of β-NAD⁺. The initial rate of ammonia release is proportional to alanine concentration or the enzyme activity. Optimal conditions for the assays are specified. Alanine (1.0 × 10^?4-1.0 × 10^?3 M in a 1-ml sample) and enzyme (0.181-0.181 U in a 0.1-ml sample) can eb determined. Application to the determination of alanine in human serum gave results that compared well with values obtained by the Yoshida method.     

Immobilization of enzymes by radiation-induced polymerization of glass-forming monomers. double entrapping of enzymes in the presence of various additives

Immobilization of enzymes by a double entrapping method was investigated, α-Amylase was trapped by radiation-induced low temperature polymerization of 2-hydroxyethyl methacrylate after being absorbed by various inorganic additives. Drierite showed the most remarkable effect by increased enzymatic activity and decreased activity loss in batch enzyme reactions. Double entrapping of cellulase was tried by preparing an entangled mixture of enzyme and various natural polymers as additives and then polymerizing with the monomeric system at low temperatures. The composite, including enzyme-additive mixture prepared by coprecipitating and coevaporating methods, showed a considerable enhancing effect on enzymatic activity and durability in batch enzyme reactions.     

Enzyme electrode studies of glucose oxidase modified with a redox mediator

Glucose oxidase modified by the covalent attachment of ferrocenecarboxylic acid or ferrocene-acetic acid groups undergoes direct oxidation at metal electrodes. Studies of the comparative stability of the two modified enzymes on storage and on electrochemical cycling show that the material modified with ferroceneacetic acid is the more stable. Amperometric studies of enzyme electrodes based on these modified forms of glucose oxidase show that their application in practical biosensors is severely limited by the poor stability of the oxidized form of the covalently attached ferrocene mediator. A comparison of the results obtained with the native enzyme and with that modified with ferroceneacetic acid, for the oxidation of glucose, d-mannose, 2-deoxy-d-glucose, d-xylose and d-galactose, suggests that the modification procedure has little effect on the selectivity of the enzyme.     

Glucose biosensors prepared by electropolymerization of p-chlorophenylamine with and without Nafion

Two different glucose biosensors for the amperometric determination of glucose, based on poly(p-chlorophenylamide) (PCPA) and bilayer film of PCPA and Nafion (PCPA/Nafion), are successfully developed. These two biosensors show linear amperometric responses to glucose ranging from 2.0×10⁻⁴ to 3.5×10⁻² mol l⁻¹ and 5.0×10⁻⁴ to 7.5×10⁻² mol l⁻¹, respectively, with the same correlation coefficient of 0.9988. Effects of polymerization potential and polymerization time on the performance of enzyme sensors are studied. It is found that PCPA, as a non-conducting polymer, can largely reduce the influence of electroactive interferents. Introduction of inner Nafion membrane not only further eliminates the influence of ascorbic acid on the sensor response but also increases electrode stability.     

Expanding the substrate scope of enzymes: combining mutations obtained by CASTing

In a previous paper, the combinatorial active-site saturation test (CAST) was introduced as an effective strategy for the directed evolution of enzymes toward broader substrate acceptance. CASTing comprises the systematic design and screening of focused libraries around the complete binding pocket, but it is only the first step of an evolutionary process because only the initial libraries of mutants are considered. In the present study, a simple method is presented for further optimization of initial hits by combining the mutational changes obtained from two different libraries. Combined lipase mutants were screened for hydrolytic activity against six notoriously difficult substrates (bulky carboxylic acid esters) and improved mutants showing significantly higher activity were identified. The enantioselectivity of the mutants in the hydrolytic kinetic resolution of two substrates was also studied, with the best mutant-substrate combination resulting in a selectivity factor of E=49. Finally, the catalytic profile of the evolved mutants in the hydrolysis of simple nonbranched carboxylic acid esters, ranging from acetate to palmitate, was studied for theoretical reasons.     

Determination of phenol and catechol concentrations with oxygen probes coated with immobilized enzymes or immobilized cells

The enzyme phenol 2-hydroxylase was immobilized on Sepharose and used in conjunction with an O₂ electrode for quantitating phenol. Similarly, catechol 1,2-oxygenase was used for quantitating catechol. A third probe was prepared by immobilization ofTrichosporon cutaneum cells rather than purified phenol 2-hydroxylase for phenol quantitation. The whole cell system gave results comparable to the immobilized enzyme system.     

Alternating copolymer of styrene–acrylonitrile obtained with vanadium-based complex catalyst in presence of monomers

Vanadium-based catalyst complexes prepared in the presence of monomers have been used for the copolymerization of styrene and acrylonitrile. VOCl₃–Al(i-C₄H₉)₃ catalyst system seems to yield an alternating copolymer. The copolymers are easily soluble in DMF and have low softening points.     

Determination of l-amino acids and alcohols with oxidase enzymes and a tubular iodide-selective electrode

L-Amino acids and alcohols (10^-2–10^-4 M) can be determined in a flow system by using a special tubular iodide-selective electrode, after reaction with L-atnino acid oxidase and alcohol oxidase, respectively. The hydrogen peroxide produced by both enzymatic reactions reacts with iodide in the presence of a molybdate catalyst, and the change in iodide concentration is monitored. The ranges of concentration that can be determined can be varied by adjusting parameters such as iodide concentration and buffer pH.     

Recent advances in activity-based probes (ABPs) and affinity-based probes (AfBPs) for profiling of enzymes

Activity-based protein profiling (ABPP) is a technique that uses highly selective active-site targeted chemical probes to label and monitor the state of proteins. ABPP integrates the strengths of both chemical and biological disciplines. By utilizing chemically synthesized or modified bioactive molecules, ABPP is able to reveal complex physiological and pathological enzyme–substrate interactions at molecular and cellular levels. It is also able to provide critical information of the catalytic activity changes of enzymes, annotate new functions of enzymes, discover new substrates of enzymes, and allow real-time monitoring of the cellular location of enzymes. Based on the mechanism of probe-enzyme interaction, two types of probes that have been used in ABPP are activity-based probes (ABPs) and affinity-based probes (AfBPs). This review highlights the recent advances in the use of ABPs and AfBPs, and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.

This review highlights the recent advances in the use of activity-based probes (ABPs) and affinity-based probes (AfBPs), and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.     

Synthesis of fluorophore and quencher monomers for use in scorpion primers and nucleic acid structural probes

We report the syntheses of monomers to incorporate fluorescein and methyl red within the sequence and at the termini of modified oligonucleotides. These monomers have been used in the solid-phase synthesis of fluorogenic oligonucleotides for genetic analysis and the study of multi-stranded nucleic acid structures.