Inhibition Biosensor Research Articles

Inhibition biosensor for determination of nicotine

An amperometric nicotine inhibition biosensor has been substantially simplified and used for determination of nicotine in tobacco sample. Besides the use of single enzyme choline oxidase to replace bienzyme, the use of 1,4-benzoquinone as an electron mediator makes it possible to avoid the use of oxygen or hydrogen peroxide sensor as the internal transducer. Choline oxidase was immobilized on the carbon paste electrode through cross-linking with bovine serum albumin (BSA) by glutaraldehyde. In the presence of choline oxidase and its endogenous cofactor flavin-ademine dinneleotide (FAD), choline was oxidized into betaine while FAD was reduced to FADH 2 which subsequently reduced 1,4-benzoquinone into hydroquinone. The later was finally oxidized at a relatively low potential of +450 mV versus saturated calomel electrode (SCE). Nicotine inhibits the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to choline within 1.25×10 −4 to 1.25×10 −3 mol l −1. The nicotine measurements were carried out in 0.067 mol l −1phosphate buffer of pH 7.4 at an applied potential of 450 mV versus SCE. The electrode provided a linear response to nicotine over a concentration range of 2.0×10 −5 to 9.2×10 −4 mol l −1 with a detection limit of 1.0×10 −5 mol l −1. The system was applied to the determination of nicotine in tobacco samples.

Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted skimmed milk. Four aminoglycosides (gentamicin, neomycine, kanamycin and a streptomycin derivative) were immobilised onto the sensor surface of a biosensor chip (CM5) in the four Fcs of the biosensor system by amine coupling. In the Biacore, milk (reconstituted from skimmed milk powder) was 10 times diluted with a mixture of the four specific antibodies and injected through the four serially connected Fcs (1 min at a flow rate of 20 μl min −1). The responses measured just prior to the injection (20 μl at a flow rate of 20 μl min −1) of the regeneration solution (0.2 M NaOH + 20% acetonitril) were indicative for the presence or absence of the aminoglycosides in reconstituted milk. The limits of detection were between 15 and 60 ng ml −1, which was far below the maximum residue limits (MRLs) (varying from 100 to 500 ng ml −1) and the total run time between samples was 7 min.

A Theoretical Model for Immobilized Enzyme Inhibition Biosensors

The development and experimental verification of a theoretical model for immobilized enzyme inhibition biosensors is reported. The model predicts that the percentage of inhibited enzyme (I %), after exposure to an inhibitor, is linearly related to both the inhibitor concentration ([I]B) and the square root of incubation time (t1/2): I%=Kt1/2[I]B×100 The model is based on diffusion limited inhibitor transport and takes into account the heterogeneous nature of enzyme inhibition that results from immobilization of an enzyme at the surface of a transducer. Experimental verification of the model was achieved using an electrochemical (amperometric) acetylcholine sensor. The immobilized enzyme used in this study was acetylcholinesterase, while the organophosphorus pesticide, paraoxon, was employed as the inhibitor. A second enzyme, choline oxidase, was co-immobilized to facilitate electrochemical detection of the substrate. To satisfy the conditions required by the proposed model a specially designed biosensing device was fabricated employing a microdisk array as the transducer. Acetylcholinesterase (0.00025 units) and choline (1.0 units) were co-immobilized onto the array using a water-based polyurethane/polyethylene oxide colloidal dispersion matrix. A dialysis membrane was used as an active barrier to prevent fouling of the array and to restrict enzyme leaching. The device was used for both batch and flow injection analysis and experimental results verifying the proposed model are presented using both modes.

Development of a quantitative relationship between inhibition percentage and both incubation time and inhibitor concentration for inhibition biosensors—theoretical and practical considerations

Theoretical and practical insights into the design and development of immobilised enzyme inhibition biosensors are reported. A general mathematical expression relating the percent of enzyme inhibition (i.e. the analytical signal) to both the inhibitor concentration and the incubation time is presented. The relevant physical, chemical and biochemical parameters required by the model are developed and discussed in terms of the inhibition of acetylcholinesterase by the organophosphorous pesticide, paraoxon. A second enzyme, choline oxidase and an amperometric transducer are used to facilitate the determination acetylcholinesterase inhibitor.

DIRECT DETERMINATION OF NICOTINE IN ANTISMOKING PHARMACEUTICAL PRODUCTS AND IN TOBACCO USING AN INHIBITION BIOSENSOR

A bienzymatic inhibition biosensor, consisting of a hydrogen peroxide amperometric electrode coupled to a functionalised nylon membrane chemically bonding the enzymes butyrylcholinesterase and choline oxidase and using butyrylcholine as substrate, was prepared and tested for the analysis of nicotine. The system was applied to the analysis of several pharmaceutical formulations containing nicotine used to treat addiction and to determine the nicotine content of different types of commercial tobacco.

Inhibition‐based biosensors for the detection of environmental contaminants: Determination of 2, 4‐dichlorophenoxyacetic acid

AbstractIn this work data are reported on the possibility for detection of environmental contaminants endowed with toxic effects by specifically designed electrochemical biosensors. A new bienzymatic inhibition biosensor based on the combined catalytic activity of the enzymes alkaline phosphatase and glucose oxidase is proposed and discussed for the direct determination of 2, 4‐dichloro‐phenoxyacetic acid (2, 4‐D), one of the most powerful and diffused defoliants, which also is endowed with estrogenic properties. A discussion on the different approaches followed to manage the experimental data obtained by enzymatic inhibition biosensors is also presented.

Remote Biosensor for In-Situ MOnitoring of Organophosphate Nerve Agents

A remote electrochemical biosensor for field monitoring of organophosphate nerve agents is described. The new sensor relies on the coupling of the effective biocatalytic action of organophosphorus hydrolase (OPH) with a submersible amperometric probe design. This combination results in a fast, sensitive, selective, and stable response at large sample-instrument distances. Such attractive performance is illustrated for direct measurements of micromolar levels of paraoxon and methyl parathion in untreated river water samples. Unlike multi-step inhibition biosensors, the remote OPH probe offers single-step direct measurements, and is thus highly suitable for the continuous monitoring task. Variables relevant to field operations are discussed, along with prospects for remote monitoring and early detection of nerve agents.

Amperometric Thick-Film Strip Electrodes for Monitoring Organophosphate Nerve Agents Based on Immobilized Organophosphorus Hydrolase

An amperometric biosensor based on the immobilization of organophosphorus hydrolase (OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and rapid anodic detection of the enzymatically generated p-nitrophenol product at the OPH/Nafion layer immobilized onto the thick-film electrode in the presence of the OP substrate. The amperometric signals are linearly proportional to the concentration of the hydrolyzed paraoxon and methyl parathion substrates up to 40 and 5 μM, showing detection limits of 9 × 10(-)(8) and 7 × 10(-)(8) M, respectively. Such detection limits are substantially lower compared to the (2-5) × 10(-)(6) M values reported for OPH-based potentiometric and fiber-optic devices. The high sensitivity is coupled to a faster and simplified operation, and the sensor manifests a selective response compared to analogous enzyme inhibition biosensors. The applicability to river water sampling is illustrated. The attractive performance and greatly simplified operation holds great promise for on-site monitoring of OP pesticides.

Multienzyme Inhibition Biosensor for Amygdalin Measurement

The multienzyme system, containing β′-glucosidase, mandelonitrile lyase and horseradish peroxidase, crosslinked in BSA-gel on the surface of an ion sensitive field effect transistor (ISFET) was used for measuring a cyanogenic glycoside amygdalin which is potentially present in the foods of plant origin. Hydrogen cyanide, produced from amygdalin as a result of β′-glucosidase + mandelonitrile lyase catalyzed reaction, suppressed the activity of horseradish peroxidase, which resulted in a reduction of the biosensor response to hydrogen peroxide. The resulting biosensor can measure amygdalin concentration in the range of 100–300 μM, and can thus be used for the routine amygdalin measurements in food samples where the tolerated concentration of cyanogenic glycosides should not exceed 7 mM.

Multienzyme Inhibition Biosensor for Amygdalin Measurement

Multienzyme Inhibition Biosensor for Amygdalin Measurement

Mediated reagentless enzyme inhibition electrodes

We have investigated the use of the copper-containing oxygenase enzymes, laccase, tyrosinase and ceruloplasmin as reagentless enzyme activity sensors. The system is based on the mediated reduction of oxygen by the enzymes co-immobilized in an osmium redox polymer hydrogel on glassy carbon electrode surfaces. Both laccase and tyrosinase present rapid homogeneous second-order rate constants for the interaction with a model monomer, [Os(2,2′-bipyridine) 2(N-methylimidazole)Cl] + (OsMeIm). Ceruloplasmin rates are several orders of magnitude slower and no catalytic currents are observed upon co-immobilization of this enzyme in the redox hydrogel. The activity of the immobilized laccase and tyrosinase sensors is shown to be influenced by the enzyme loading in the deposition solution, the electrolyte pH and ionic strenght. The immobilized sensors can be utilized for the detection of modulators of enzyme activity, such as the respiratory poison azide. Reproducible inhibition curves can be obtained by normalization of the sensor response. The resulting enzyme inhibition biosensors can detect levels of azide as low as 1 μM in solution and may be useful as an early warning sensor for the presence of such respiratory toxins.

Optimisation of a reagentless laccase electrode for the detection of the inhibitor azide.

Here we report on the optimisation of a reagentless enzyme sensor for the detection of azide based on the mediated reduction of O2 by a laccase enzyme co-immobilised in a redox hydrogel on electrode surfaces. The sensor response is shown to be influenced by the enzyme loading, the electrolyte pH and ionic strength. The response of the sensor is stable, decreasing by only 25% over a sixteen-hour period. Reproducible inhibition curves for the determination of azide levels from cyclic voltammetric scans can be obtained by normalisation of the sensor response. The resulting enzyme inhibition biosensor can detect levels of azide as low as 2.5 microM under these conditions. Constant potential amperometric detection at the laccase enzyme electrode in a flow injection set-up yields a peak current for inhibition of the mediated reduction of O2. Reproducible peak currents and areas (8.0 and 6.3% RSD, respectively, for n = 11) are obtained for repeated injections of 100 microM azide. Reproducible response curves can be obtained by injection of a 25 mM azide sample and assuming that the peak height and peak area obtained represent 100% inhibition of the enzyme.

EDTA Determination by Urease-Based Inhibition Biosensor

Urease immobilized into poly(vinylpyridine-co-styrene) film on the surface of ion sensitive field effect transistor (ISFET) has been used for determination of chelating agents (sequestrants). These species, particularly EDTA, restored catalytic activity of the enzyme if it was inhibited with cupric ions. Enzyme immobilization into a positively charged polymer seems to cause an increase of the EDTA restoration effect because of anion accumulation in the polymeric matrix. The resulting biosensor can measure EDTA concentration in the range of 5–500 ppm. The influence of other sequestrants (citrate and phosphate) were also examined.

EDTA Determination by Urease-Based Inhibition Biosensor

Urease immobilized into poly(vinylpyridine-co-styrene) film on the surface of ion sensitive field effect transistor (ISFET) has been used for determination of chelating agents (sequestrants). These species, particularly EDTA, restored catalytic activity of the enzyme if it was inhibited with cupric ions. Enzyme immobilization into a positively charged polymer seems to cause an increase of the EDTA restoration effect because of anion accumulation in the polymeric matrix. The resulting biosensor can measure EDTA concentration in the range of 5–500 ppm. The influence of other sequestrants (citrate and phosphate) were also examined.

Rapid Determination of Dimethoate with a Surface Acoustic Wave Impedance Sensor System

Based on the principle of enzyme inhibition, a novel and sensitive lipase biosensor to determine organophosphorus pesticide is presented. Contact of the enzyme with pesticide samples results in specific inhibition of enzyme activity. Sensor calibration was possible by correlating the inhibition of enzyme activity with various concentrations of pesticide compound in a buffer solution. The sensor was successfully used to determine pesticide concentrations ranging from a low of 167ng/ml to 1.34μg/ml, and the detection limit is 81ng/ml. The effects of temperature, pH value, incubation time and solvent were also investigated. The sensor was also applied to the determination of dimethoate residues in the peel and flesh of tomato.

Reagentless Tyrosinase Enzyme Electrodes: Effects of Enzyme Loading, Electrolyte pH, Ionic Strength, and Temperature

We have prepared a reagentless enzyme activity sensor based on the mediated reduction of oxygen by tyrosinase coimmobilized in an osmium redox polymer hydrogel on glassy carbon electrode surfaces. The activity of this sensor is shown to be influenced by the enzyme loading, yielding an optimum activity for 41.7% (w/w) enzyme in the deposition solution. The electroyte pH, ionic strength, and temperature also affect the electrode response by altering enzyme activity, charge transport rates, and mediator concentration in the films. The response of the sensor decreases by only 25% over a 6-h period. However, reproducible inhibition curves can be obtained by normalization of the sensor response. The resulting enzyme inhibition biosensor can detect levels of the enzyme inhibitor, azide, as low as 1.0 × 10-5 mol/dm3 in solution. The immobilized sensors can be utilized for the detection of modulators of tyrosinase enzyme activity, such as respiratory poison inhibitors.

Reagentless Mediated Laccase Electrode for the Detection of Enzyme Modulators

We have investigated aerobic mediation of electron transfer to a laccase enzyme by the solution redox couples [Os(bpy)(2)Cl(2)](+/0) and [Os(bpy)(2)(MeIm)Cl](2+/+), where bpy is 2,2-bipyridine and MeIm is N-methylimidazole. The factors that influence the homogeneous mediation reaction are investigated and discussed. Investigation of ionic strength, pH, and temperature effects on the kinetics of intermolecular electron transfer elucidates the governing factors in the mediator-enzyme reactions. Coimmobilization of both enzyme and an osmium redox mediator in a hydrogel on glassy carbon electrodes results in a biosensor for the reagentless addressing of enzyme activity, consuming only oxygen present in solution. Thus, these immobilized enzyme biosensors can be utilized for the detection of modulators of laccase activity, such as the inhibitor sodium azide. The enzyme inhibition biosensor can detect levels of azide as low as 2.5 × 10(-6) mol dm(-3) in solution.

Screen-printed tyrosinase-containing electrodes for the biosensing of enzyme inhibitors

Disposable amperometric inhibition biosensors have been microfabricated by screen printing a tyrosinase-containing carbon ink. The decrease in the substrate (catechol) steady-state current, caused by the addition of various pesticides and herbicides, offers convenient quantitation of micromolar levels of these pollutants. Unlike esterasebased disposable strips, the tyrosinase thick-film devices can be fabricated by incorporating the enzyme within the carbon ink. and do not require a prolonged incubation step in the presence of the inhibitor. The effect of experimental variables, such as the enzyme loading or substrate concentration, is assessed. Applicability to an untreated river water sample is illustrated. Such use of single-use devices for monitoring toxins addresses the problem of irreversible enzyme inhibition, and holds great promise for on-site field analysis.

Hydrazine Detection Using a Tyrosinase-Based Inhibition Biosensor

An amperometric biosensor for the determination of hydrazine compounds, based on their inhibitory effect on the activity of immobilized tyrosinase, has been developed. The hydrazine-tyrosinase interaction can be modeled as a reversible competitive inhibition. Kinetic parameters (K{sub i} and I{sub 0.5}) have been determined for various hydrazine compounds. The tyrosinase-based carbon paste electrode offers sensitive measurements to the low-micromolar level and good precision. The trend in sensitivity, methylhydrazine > hydrazine >dimethylhydrazine, reflects the degree of inhibition. The applicability to assays of unaltered river and drinking water samples is illustrated. Analogous measurements at disposable thick-film sensors are also reported. These inhibitor biosensors hold great promise for field-based monitoring of various hydrazines. 13 refs., 5 figs.

Electrically ‘wired’ tyrosinase enzyme inhibition electrode for the detection of respiratory poisons

AbstractThe use of the solution redox species, [Os(bpy)2Cl2]+/0, [Os(bpy)2(MeIm)Cl]2+/+ and [Fe(CN)6]4−/3−, where bpy is 2,2‐bipyridine and MeIm is N‐methylimidazole, as electron mediators in the enzymatic reduction of oxygen by tyrosinase is investigated. Co‐immobilization of both enzyme and an osmium redox mediator in a hydrogel on glassy carbon electrodes results in a biosensor for the ‘reagentless’ addressing of enzyme activity, consuming only oxygen present in solution. Immobilized enzyme inhibition biosensors can thus be constructed for the detection of tyrosinase inhibitors, such as sodium azide, using this approach. The enzyme inhibition biosensor can detect levels of azide as low as 5 × 10−6 mol dm−3 in solution and may be useful in environmental monitoring applications and as an early warning poison sensor.