R. Stephen Brown

Enzyme or protein immobilization techniques for applications in biosensor design

New generations of biosensors are emerging that are based on novel and promising transducers such as miniature, reagentless-mediated electrodes, field-effect transistors, piezoelectric and optical devices. Reagentless-mediated biosensors can be constructed by co-immobilizing both enzymes and mediators onto a miniaturized electrode using electropolymerization, thus improving the sensitivity and speed of the response. Even more promising is the development of electrochemical sensors, in which electron transfer is made directly from a redox enzyme to an electrode surface via molecular wires. While this has only been reported, so far, for a specific enzyme entrapped in N-methylpyrrole under defined circumstances, the development of new oriented immobilization techniques, coupled with progress in protein engineering, may make direct electron transfer the rule rather than the exception.

Enzyme reactions in the presence of cyclodextrins : biosensors and enzyme assays

Cyclodextrins, macrocyclic carbohydrates with apolar internal cavities, can form complexes with, and solubilize many normally water-insoluble compounds. Ferrocene and its derivatives, tetrathiafulvalene and tetramethylbenzidine, can function as redox mediators, but are insoluble in water; when they are complexed with cyclodextrins, they can be used in enzymatic assays and in the construction of mediated biosensors. In addition, the solubilization of polynuclear aromatic hydrocarbons (PAHs), including the potent carcinogen benzo[a]pyrene, by cyclodextrins has enabled the detection of these important environmental contaminants.

Enzymatic oxidation of water-soluble cyclodextrin-polynuclear aromatic hydrocarbon inclusion complexes, using lignin peroxidase

Abstract α-, β-, γ-, and 2-hydroxypropyl-β-cyclodextrins were capable of forming water-soluble inclusion complexes with several polynuclear aromatic hydrocarbons (PAHs). The highest solubilities were noted for β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin (hpβCD). The solubility of PAHs in hpβCD was enhanced 224-fold and 7,500-fold for naphthalene and benzo[a]pyrene, respectively, with other PAHs yielding values between these limits. The ability of lignin peroxidase (LiP) to oxidize these cyclodextrin-included substrates was similar to that previously reported for mixed solvent systems. The enzyme oxidized anthracene, pyrene, and benzo[a]pyrene but not naphthalene, phenanthrene, chrysene, and benzo[e]pyrene. The lignin peroxidase exhibited a preference for oxidizing either anthracene or benzo[a]pyrene when mixed with pyrene. On the basis of fluorescence measurement, anthracene and benzo[a]pyrene were easily distinguished by exciting at 250 nm for anthracene and 295 nm for benzo[a]pyrene. Veratryl alcohol severely inhibited the pyrene assay, with 50% inhibition noted at 0.3 m m while veratryl alcohol activated the reactions between LiP and either anthracene or benzo[a]pyrene. Maximal activation was obtained at 1.5 m m veratryl alcohol and no inhibitory effect was detected up to 4.0 m m . Under identical conditions, the rate of reaction with veratryl alcohol (4.0 m m ) was 11- and 14-fold faster for benzo[a]pyrene and anthracene, respectively, when compared to the assays in the absence of veratryl alcohol.

Inclusion complexation of tetrathiafulvalene in cyclodextrins and bioelectroanalysis of the glucose-glucose oxidase reaction

Tetrathiafulvalene (TTF) forms water-soluble inclusion complexes with α-, hp-β- and γ-cyclodextrin (CD). Cyclic voltammetry (CV) has been performed at a stationary electrode to characterize these TTF:CD inclusion complexes. The CV analysis yielded average peak separations of 63 mV and diffusion limited currents, indicating completely reversible electron transfer. The half-wave potential (E12) of the complex shifted to more positive values with increasing CD concentration, permitting use of a modified Nernst equation to estimate the complexation ratio and the equilibrium formation constant (Kform) of the complexes. The complexation ratio was determined as 1:2, 1:1 and 1:1 for TTF with α-CD, hp-β-CD and γ-CD, respectively. The Kform for these complexes was then estimated to be 5.44 × 103 m6/kmol2, 5.40 × 103 m3/kmol and 0.141 × 103 m3/kmol. The diffusion coefficients (Do) for 1 mol/m3 TTF in α-CD varied from 4.44 × 10−11 to 0.76 × 10−11 m2/s as the α-CD concentration increased from 3 to 50 mol/m3. The Do values for hp-β-CD varied from 12.9 × 10−11 to 4.03 × 10−11 m2/s over the same cyclodextrin range whereas those of γ-CD ranged from 2.20 × 10−12 to 1.75 × 10−12 m2/s. CV curves for TTF: hp-β-CD in the presence of glucose and GOx showed a large anodic current with no discernible peaks, indicating bioelectrocatalysis. The ratio of this current to the diffusion limited current was used to determine the second-order homogeneous rate constant ks for the reaction with GOx, which demonstrated the efficiency of the inclusion complex as a mediator. The ks value for 1 mol/m3 TTF decreased from 3.84 × 105 to 1.04 × 105 m3/kmol s with an increase in hp-β-CD concentration from 3 to 15 mol/m3. Extremely high diffusion and kinetic parameters in 3 mol/m3 CD solutions were due to insolubility of TTF at low CD concentrations, and indicated that the CD concentration must be maintained above this level.

Novel Biomonitoring Techniques for Phenolics and Polynuclear Aromatic Hydrocarbons

A substrate recycling assay for phenolic compounds was developed using tyrosinase in excess NADH. The reaction of various phenols with the enzyme produced an o-quinone, which was then detected by recycling between reactions with the enzyme and NADH. Absorbance measurements of the NADH consumption rate enhanced the assay sensitivity for catechol 100-fold compared to non-recycling o-quinone detection, giving a detection limit of 240 nM. Fluorescence NADH monitoring permitted a further tenfold improvement over absorbance, with a detection limit of 23 nM. The procedure was useful for assaying catechol, several derivatives of catechols, including chloro and amine derivatives, phenols, and 4-chlorophenol, with relative sensitivity being related to substrate activity of the enzyme.