Organic Phase Enzyme Electrode Research Articles

Bioethanol in Biofuels Checked by an Amperometric Organic Phase Enzyme Electrode (OPEE) Working in "Substrate Antagonism" Format.

The bioethanol content of two samples of biofuels was determined directly, after simple dilution in decane, by means of an amperometric catalase enzyme biosensor working in the organic phase, based on substrate antagonisms format. The results were good from the point of view of accuracy, and satisfactory for what concerns the recovery test by the standard addition method. Limit of detection (LOD) was on the order of 2.5 × 10−5 M.

Reliable new immunosensor for atrazine pesticide analysis

The aim of the present research was to develop a highly selective and sensitive amperometric immunosensor for atrazine based on a “competition” assay procedure. An immunosensor device for the determination of triazinic pesticides based on two different competition procedures is therefore described. The immunosensor developed uses an amperometric electrode for hydrogen peroxide as transducer and the peroxidase enzyme as marker. The results demonstrate the full validity of this immunosensor method which was optimized by comparing two different “competition” operating procedures. The results obtained using the new immunosensor were then compared with those found using an inhibition OPEE (Organic Phase Enzyme Electrode) for triazinic pesticide detection, which showed no selectivity toward different classes of triazinic, carbamate or organophosphate pesticides. The immunosensor developed displayed on the contrary an appreciable selectivity both toward different classes of pesticides and toward triazinic or benzotriazinic products. In addition, the K aff value was evaluated. Lastly, the immunosensor was also used to test triazinic pesticide recovery from common real matrices such as milk and vegetal samples, obtaining good recoveries.

An innovative organic phase enzyme electrode (OPEE) for the determination of ethanol in leadless petrols

A new biosensor for the determination of the ethanol content of liquids or organic solvents, such as leadless petrols has been developed. The new biosensor is also quite peculiar: it is a, “competitive substrate” organic phase enzyme electrode (OPEE), in which the enzyme, catalase, is, coupled to an amperometric gaseous diffusion Clark type oxygen electrode. The biosensor was, optimized to operate in decane solvent. The new OPEE was applied to the analysis of ethanol in, “leadless petrol”.

A novel hemin-based organic phase artificial enzyme electrode and its application in different hydrophobicity organic solvents

Based on the newly discovered artificial enzyme formed by mixing hemin with supramolecular hydrogels via the self-assembly of amphiphilic oligopeptides, we prepared a novel organic phase artificial enzyme electrode by coating the artificial enzyme on an electrode which was then covered with sodium alginate for protection. Scanning electron micrograph showed that the supramolecular hydrogel kept its nanofibers structure on the electrode surface. Hemin dispersed in the supermolecular hydrogel as monomer greatly promotes its direct electrochemistry behavior in organic solvents. At the same time, this electrode exhibited higher electrocatalytic ability to tert-butyl hydroperoxide (TBHP) than free hemin modified electrode (free hemin mainly present as dimer). As low as 27μM TBHP could be detected with a linear range from 6.6×10−5 to 1.27×10−2M via amperometric method. The biosensor can reach 95% of the steady-state current in about 10±2s. More importantly, it can be applied in both hydrophilic and hydrophobic solvents without adding extra buffer or mediators to them that cannot be received by most traditional organic phase enzyme electrodes. This unique property greatly promotes the development of the organic phase enzyme electrodes by facilitating the detection of different kinds of substrates of the hemin-based artificial enzyme soluble in hydrophilic and hydrophobic solvents. The artificial enzyme electrode was successfully used to determine organic peroxides in body lotion samples.

Investigation of Interfering Species in Phytodrug Analysis Using an Inhibition Tyrosinase Enzyme Electrode Working Both in Water and in Organic Solvent

In recent years several inhibition biosensors have been proposed for the analysis of aqueous solutions of phytodrugs. We recently built an inhibition OPEE (organic phase enzyme electrode) based on the inhibition of tyrosinase for the analysis of triazine, carbamate, and organophosphate pesticides operating in water-saturated chloroform. It was possible to determine the concentration of these pesticides contained in lipophilic or aqueous samples by relating it to the inhibiting action measured directly in water, or in water-saturated chloroform, after using the same solvent to extract the pesticides themselves. In the present investigation, attention was focused above all on two points of particular interest: on the study of potential interferents, i.e., of other inhibitors of the tyrosinase enzyme consisting above all, when operating in aqueous solution, of different heavy metal ions or several carboxylic acids, such as cinnamic, sorbic, or benzoic acids, which can apparently interfere in inhibition analysis of pesticides in aqueous matrixes; in the second place, on a detailed comparison of the results of the analysis of triazine, organophosphate, and carbamate pesticides in the presence of the above-mentioned interferents operating both in aqueous solution and in water-saturated chloroform.

Tyrosinase inhibition organic phase biosensor for triazinic and benzotriazinic pesticide analysis (part two)

Several triazine pesticides, such as atrazine, are much more soluble in several organic solvents, such as chloroform, than in water. Our recent research was aimed at analyzing this class of pesticides using tyrosinase OPEE (organic phase enzyme electrodes), exploiting their inhibiting action on the tyrosinase enzyme when operating in water-saturated chloroform medium. In this work we studied the response of a tyrosinase inhibition enzyme sensor to several triazinic (simazine, propazine, terbuthylazine) and benzotriazinic (azinphos-ethyl and azinphos-methyl) pesticides (LOD=0.5x10(-9) mol l(-1)). Recovery trials were also performed in vegetal matrixes (corn, barley, lentils). Lastly, the effect of the solvent (chloroform or water) on the inhibition process was investigated via Hill's equation and the diffusion of analyte from the solvent to the enzyme membrane.

Determination of polyphenol ‘pool’ in olive oil mill waste water using a tyrosinase biosensor operating in aqueous solution or in organic solvent

Biosensors are very versatile devices that can be used to solve various kinds of problem that are increasingly found in the various branches of chemistry, particularly in the fields of foodstuffs and the environment. In recent years, there has been considerable development in a new biosensor sector, that of OPEEs (organic phase enzyme electrodes). These are biosensors able to function also in organic solvents or in mixtures of several different organic solvents. One of these enzymatic biosensors that has recently proved to be particularly versatile is the tyrosinase biosensor, of which several different versions have been developed. Our group in particular has in recent years developed both a version that operates in aqueous solutions and one suitable for organic solvents. These tyrosinase biosensors are essentially made up of an amperometric transducer for oxygen (Clark type), coupled with the tyrosinase enzyme, which is suitably immobilized according to the solvent in which it must operate. In this article the possibility was assessed of using them to determine the polyphenol ‘pool’ in olive oil mill wastewater. The method has been optimized as regards both the solvent and the type of enzymatic immobilization to be used. Results have been compared with those obtained using the Folin–Ciocalteau method, which is chosen as reference method.

Horseradish peroxidase-based organic-phase enzyme electrode

An organic-phase enzyme electrode (OPEE) based on horseradish peroxidase (HRP) immobilized within Nafion on spectroscopic graphite was investigated in acetonitrile. The amperometric electrode response to hydrogen peroxide and cumene hydroperoxide present was found to be the result of the reduction of oxygen, produced upon enzymatic decomposition of both hydroperoxides (i.e., by the catalase-like activity of HRP). The electrode response was found to depend linearly on the hydroperoxide concentration up to 700 microM within the range of potentials from -200 to -400 mV (versus Ag|AgCl). Detection limits of approximately 45 microM for H2O2 and 100 microM for cumene hydroperoxide were determined under the selected experimental conditions. Nernstian dependence (the open circuit voltage of HRP-based electrode versus logarithm of H2O2 concentration) was obtained between 0.2 and 2.0 mM, with a slope of approximately 23 mV per logarithmic unit, suggesting a catalase-like, two-electron disproportionation of the substrate in acetonitrile.

Determination of triazine pesticides using a new enzyme inhibition tyrosinase OPEE operating in chloroform

A biosensor method for the determination of triazine pesticides based on an inhibition organic phase enzyme electrode (OPEE) is described. The OPEE was developed using a tyrosinase biosensor assembled in the version operating in organic phase and used to determine triazine pesticides by exploiting their power to inhibit the tyrosinase enzyme. The results show the full validity of the method, which has also been optimized by comparing two different operating procedures. Lastly, the tyrosinase OPEE was also used to test triazine recovery from common vegetal samples, obtaining recoveries always >90%.

Organic phase PPO biosensor based on hydrophilic films of electropolymerized polypyrrole

We described herein, the construction of an organic phase enzyme electrode (OPEE) via polyphenol oxidase (PPO) entrapment within a hydrophilic polypyrrole film electrogenerated from on a new bispyrrolic derivative ( 1) containing a long hydrophilic spacer. The so-called “adsorption step procedure” was adopted for the preparation of the organic phase PPO biosensor. The amperometric detection of catechol was carried out in anhydrous chloroform at −0.2 V versus Ag/AgCl. The electroanalytical parameters of the biosensor strongly depend on its configuration and on the hydration state of the enzyme matrix. The best sensitivity obtained for catechol in chloroform was 15.6 mA M −1 cm −2.

The Enzyme Source Effect on the Performance of a Catalase Organic Phase Enzyme Electrode

The role of the enzyme source was studied in the reaction of hydrogen peroxide decomposition by immobilised catalase in acetonitrile. Enzymes isolated from bacterial and mammalian sources were conveniently immobilised on a spectroscopic graphite to obtain an organic-phase enzyme electrode (OPEE). Amperometry at constant potential was employed as basic analytical approach in this study.

Development of organic phase amperometric biosensor for measuring cholesterol in food samples

Previous works on organic phase enzyme electrodes (OPEE) applied methods that worked mostly under stirred conditions. The aim of our present work was to develop a flow-through measuring set-up for determination of cholesterol content in organic media. For determination of free cholesterol content cholesterol oxidase (COD) was used, while for measuring the total cholesterol content a bi-enzyme cell containing immobilised cholesterol esterase (CE) and cholesterol oxidase was developed. Enzyme immobilisation took place on a natural protein membrane, by a glutaraldehyde cross-linking method, in a thin-layer enzyme cell made from Teflon. The enzyme cell was connected into a stopped-flow injection system (SFIA), with a flow-through amperometric detector. The parameters of biochemical and electrochemical reactions were measured. The effect on amperometric detection of different organic salts as electron mediator or conducting salts was studied. The optimal concentration of (TBATS) was 2.4 mg L−1 while for (FMCA) an optimal concentration was found at 0.4 mg L−1. The minimum amount of water, necessary for enzymatic activity in the organic phase, was also determined. Changing the concentration of toluene in acetonitrile carrier solution, the peaks increased definitely in the range 10–40% toluene. Since CE and COD were immobilised together in the enzyme cell, the conversion rate was found to be about 0.7–0.8 when the toluene content was higher then 30%. The linear measuring range for cholesterol oleate and cholesterol was 0.1–0.5 mM. Total cholesterol content of lard, butter and pasta samples were determined. It is concluded that an organic phase bi-enzyme cell may be suitable for cholesterol determination in food.

Determination of hydroperoxides in nonaqueous solvents or mixed solvents, using a biosensor with two antagonist enzymes operating in parallel

The development and characterisation of a new organic phase enzyme electrode (OPEE) for hydroperoxides is described. The biosensor was obtained by combining an oxygen gas diffusion amperometric electrode and two immobilised enzymes (peroxidase and tyrosinase) working in parallel and competing for the same substrate (catechol). LOD was about 0.1×10−3moll−1. The biosensor was applied for the determination, using decane as solvent, of the “pool” of hydroperoxides released during the heating (to about 130–140°C) of extravirgin olive oil, or used to determine the hydrogen peroxide content of lipophilic cosmetic products, working in a dioxane–water mixture.

Study of catalase electrode for organic peroxides assays

The catalytic activity of immobilized catalase (EC 1.11.1.6) for two model peroxide compounds (dibenzoyl peroxide and 3-chloroperoxibenzoic acid) in a non-aqueous medium was used to prepare an organic-phase enzyme electrode (OPEE). The enzyme was immobilized within a polymeric film on spectrographic graphite. The amperometric signal of the enzyme electrode in substrate solutions was found to be due to the reduction of oxygen generated in the enzyme layer. The electrode response is proportional to peroxide concentrations up to about 40 μM within the potential range from −450 to −650 mV (vs. Ag/AgCl), and the response time is at most 90 s. The enzyme electrode retains about 35% of its initial activity after a 3-week storage at room temperature.

Two OPEEs (organic phase enzyme electrodes) used to check the percentage water content in hydrophobic foods and drugs.

The development and optimization of an analytical method using enzymatic biosensors able to operate in organic solvents [organic phase enzyme electrodes (OPEEs)] for the determination of the water content in food fats (butter, margarine) or pharmaceutical or cosmetic ointments is described. The method is based on the increase in enzymatic activity which is related to the increase in the percentage water content in the organic phase into which the biosensor is dipped. The enzymes used to assemble the biosensors were tyrosinase or catalase, the substrates were phenol or p-cresol and tert-butyl hydroperoxide, respectively, and the organic solvents were acetonitrile or dioxane. A gas diffusion amperometric electrode for oxygen measurement was used as electrochemical transducer. The results were compared with those obtained applying the Karl Fischer method to the same food or drug matrices. The correlations among the two methods proved satisfactory, as the difference in the computed values of water content was never higher than 7%. Also, the precision of measurements was acceptable (RSD < 6%) in all the analyses of real matrices.

Fabrication of organic phase biosensors based on multilayered polyphenol oxidase protected by an alginate coating

We describe herein, the creation of an organic phase enzyme electrode (OPEE) via avidin–biotin interactions built over an electrogenerated polymer. Multilayered polyphenol oxidase (PPO) assemblies were transferred into an organic solvent (chloroform) for the catechol detection at −0.2 V. In conjunction with an alginate gel, as a hydrophilic additive, the biosensor performance was widely enhanced. The effects of biotinylated polypyrrole film and alginate gel on the diffusion process through the biosensor coating are studied by rotating disk electrode experiments carried out in chloroform with hydroquinone as electroactive permeant.

Hydroperoxide determination by a catalase OPEE: application to the study of extra virgin olive oil rancidification process

A new application to authentic matrices of a catalase organic phase enzyme electrode (OPEE) recently developed by the present authors is described in this communication. This biosensor was obtained by coupling an amperometric gas diffusion electrode for the oxygen, made of teflon, and the catalase enzyme immobilised in kappa-carrageenan gel. The response of the catalase biosensor to cumene hydroperoxide and to terbutylhydroperoxide, working directly in n-decane or toluene, was studied. The hydroperoxide content of the extra virgin olive oil was monitored during an artificial rancidification process. One interesting result obtained is the qualitative correlation observed between the above trend of the hydroperoxide content pool and that of the peroxide number; moreover, a qualitative inverse correlation trend was found between both the last two indicators and the content of polyphenols in the olive oil which are known to be the most important natural antioxidant agents contained in olive oil.

Organic phase enzyme electrodes: applications and theoretical studies

This short review reports the main organic phase enzyme electrodes (OPEEs) described in literature and illustrates some of the results obtained by the present authors during recent research on developing mono and bienzymatic OPEEs. Several of these are already mature biosensors (e.g. tyrosinase and catalase OPEEs and those for phosphatidylcholine and butyrylcholine determination) and applications have already been implemented, while, applications by other OPEEs are still at the experimental stage. New correlations are also illustrated between classical indicators, e.g. the log P value of several organic solvents and new empirical indicators, recently proposed by the authors, the values of which may be monitored with biosensors considered dipping directly into the organic solvent or solvent mixture.

Organic-phase enzyme electrode for phenolic determination based on a functionalized sol–gel composite

An amperometric biosensor for monitoring phenols in the organic phase was constructed by the silica sol-gel immobilization of tyrosinase on a glassy carbon electrode. The organic-inorganic hybrid materials with different sol-gel precursors and polymers were optimized, and the experimental conditions, such as the effect of the solvent, operational potential and enzyme loading were explored for the optimum analytical performance of the enzyme electrode. The biosensor can reach 95% of steady-state current in about 18 s, and the trend in the sensitivity of different phenols is as follows: catechol > phenol >p-cresol. In addition, the apparent Michaelis-Menten constants (K-m(app)) and the stability of the enzyme electrode were discussed. (C) 2000 Elsevier Science S.A. All rights reserved.

Enzymatic immobilisation in kappa-carrageenan gel suitable for organic phase enzyme electrode (OPEE) assembly

Organic phase enzyme electrodes (OPEEs) are a new class of biosensors that have extended the field of application of biosensors. The use of biosensors in organic solvent actually makes it possible to analyse insoluble or scarcely soluble substrates in aqueous medium. The best approach for obtaining a new OPEE starts from the optimisation of the experimental conditions in order to obtain the maximum activity and stability of the enzyme to be coupled to the transducer. In a previous paper we therefore optimised the choice of the solvent, while in this paper a new enzyme immobilisation method in kappa-carrageenan gel and its application to the construction of four different mono- or bienzymatic OPEEs are described and evaluated.