Biomolecular Engineering 23 (2006) 135–147 www.elsevier.com/locate/geneanabioeng
Review
Organic phase enzyme electrodes M. Sa´nchez-Paniagua Lo´pez b, E. Lo´pez-Cabarcos b, B. Lo´pez-Ruiz a,* a
Seccio´n Departamental de Quı´mica Analı´tica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain b Departamento de Quı´mica-Fı´sica II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain Received 23 December 2005; received in revised form 23 March 2006; accepted 10 April 2006
Abstract In the development of biosensors, organic phase enzyme electrodes (OPEEs) have received considerable attention for the detection of substrates in organic media. This article reviews different enzymes, transductors and immobilization methods used for the preparation of OPEEs in the last decade. # 2006 Elsevier B.V. All rights reserved. Keywords: Enzyme electrode; Non-aqueous medium
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . Amperometric biosensors . . . . . . . . . . . . 2.1. Monoenzyme electrodes . . . . . . . . 2.1.1. Tyrosinase . . . . . . . . . . . . 2.1.2. Catalase . . . . . . . . . . . . . 2.1.3. Peroxidase . . . . . . . . . . . . 2.1.4. Glucose oxidase . . . . . . . . 2.1.5. Other enzymes . . . . . . . . . 2.2. Bienzyme electrodes . . . . . . . . . . . 2.3. Enzyme inhibition based electrodes 2.4. Tissue biosensors . . . . . . . . . . . . . Enzyme reactors . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Abbreviations: AChE, acetylcholinesterase; AOT, dioctylsulfosuccinate; BChE, butylcholinesterase; ChOx, choline oxidase; COx, cholesterol oxidase; CVR, current variation rate; GDE, gas diffusion electrode; GOx, glucose oxidase; HRP, horseradish peroxidase; OPEEs, organic phase enzyme electrodes; PLD, phospolipase D; PPO, tyrosinase; PVA-SbQ, polyvinyl alcohol styryl pyridinium groups; SOD, superoxide dismutase; SPP, sweet potato peroxidase * Corresponding author at: Seccio´n Departamental de Quı´mica Analı´tica, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria s/n, 28023 Madrid, Spain. Tel.: +34 91 394 1756; fax: +34 91 394 1754. E-mail address:
[email protected] (B. Lo´pez-Ruiz). 1389-0344/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2006.04.001
Enzyme catalysis has been investigated traditionally in aqueous medium, however, pioneers in biosensor research verified that biocatalysis can work not only in aqueous media, but also in organic media (Saini et al., 1991). It seems that some enzymes retain their activity in non-aqueous media whenever the microenvironment of the enzyme has a suitable level of hydration offering new opportunities in biosensor technology (Iwuoha et al., 1997; Campanella et al., 2001a). A basic requirement for the enzyme catalytic activity is the selection of
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a compatible organic phase which does not react with the hydration layer. The disruption of the hydration layer would induce conformational changes and hence enzyme denaturation. The development of organic phase enzyme electrodes has attracted considerable interest due to the potential advantages of determining compounds that are poorly soluble in water but soluble in non-aqueous solvents (Campanella et al., 1998c) broadening the useful analytical field of biosensors to hydrophobic substrates and samples. Also, the use of organic solvents decreases the interferences arising from hydrophilic ionic species, prevents microbial contamination improving the operational lifetime of the sensor (Saini et al., 1991) and simplifies immobilization techniques. In fact, when working in non-aqueous solvents, simple adsorption of the enzyme onto a solid support is often a good immobilization method due to the insolubility of enzymes in organic solvents. However, a wide variety of enzyme immobilization strategies have been reported to be applied in organic solvents, including adsorption (Kro¨ger et al., 1998; Morales et al., 2005a,b) and entrapment within polymeric and inorganic matrices (Andreescu et al., 2002; Stanca and Popescu, 2004). Furthermore, it may increase the operational stability of the biosensor due to the increased thermal stability of some enzymes in organic solvents (DiazGarcia and Valencia-Gonzalez, 1995). The lifetimes of the OPEEs comprises from few days to several weeks depending on the nature of the immobilization system. The key component of biosensors is the enzyme that is responsible for the specific recognition of the analyte. At the moment, organic phase enzyme electrodes have been prepared with some few proteins that after immobilization preserve functional activity and some biosensors working in organic media have been successfully applied in real samples. A lot of the work has been performed using tyrosinase, but other enzymes have also been immobilized in monoenzymatic and bienzymatic systems. Regarding the working electrodes, graphite, glassy carbon, and oxygen gaseous diffusion electrodes are the most usual. Concerning the solvents, acetonitrile, chloroform, dioxane and hexane have been generally used. In order to dissolve the analyte in the organic solvent reversed micellar systems (consisting of an organic continuous phase, an aqueous dispersed phase and a surfactant) have been reported (Reviejo et al., 1994). The activity of enzymes in organic media is strongly dependent on their hydration layer, which is essential for their conformational flexibility. A method for measuring solvent hydrophobicity is using the value of log P (P is the distribution coefficient of the solvent in a standard octanol/water system) (Laane et al., 1987). Solvents with log P > 4 exhibit hydrophobic properties and their interaction with the water layer are practically negligible. Organic solvents with log P between 2 and 4, interact moderately with the hydration layer of the enzyme. Solvents having log P < 2 are hydrophilic and the enzyme activity can be strongly affected by the removal of the hydrated water. This rule has some exceptions and certain enzymes show a high surprising activity in some organic solvents. This effect is attributed either to a strongly attached hydration layer that is not disrupted by the hydrophilic solvent,
or it can be also due to the partition of the substrate to the enzyme active centers which can be so favored that other detrimental effects appear insignificants. Besides, the substrate diffusion is influenced by the viscosity (h) and the dielectric constant (e) of the solvent. The higher 1/eh factor, the greater is the diffusion constant of the analyte (Adeyoju et al., 1995). 2. Amperometric biosensors 2.1. Monoenzyme electrodes 2.1.1. Tyrosinase Tyrosinase (polyphenoloxidase, PPO, E.C. 1.14.18.1) is a copper-containing enzyme that catalyses the production of pigments such as melanin and is widely distributed in plants and animal tissues. The active site of PPO consists of two copper atoms and three states: ‘‘met’’, ‘‘deoxy’’ and ‘‘oxy’’ (Rodrı´guez-Lo´pez et al., 2001). The enzyme catalyses the hydroxylation of monophenols to o-diphenols (monophenolase activity), and their subsequent oxidation to o-quinones (diphenolase activity) both using molecular oxygen (Sa´nchez-Ferrer et al., 1995). PPO enzyme electrodes employ the electrochemical reduction of these quinones to monitor the enzymatic reaction which is affected by the pH of the assay medium. Another way to determine polyphenols is based on the determination of oxygen consumption in the enzymatic reaction by electrochemical reduction of the dissolved oxygen (see Scheme 1). One important feature of oils and fats is their instability against autooxidation phenomena. Many oils of vegetal origin contain natural substances with antioxidant properties which can prevent rancidification. Polyphenol compounds have very strong natural antioxidizing properties and therefore the finding of an analytical method for these compounds with sufficient level of accuracy, precision, reliability and low response time is of practical importance. Tyrosinase is the enzyme used as biological material in organic phase biosensors for polyphenol determination in matrices such as oils and foods. Different strategies have been reported to immobilize enzymes and to improve the characteristics of the biosensors. Electropolymerized pyrrole and derivates of this polymer have been used as matrices for PPO immobilization. Cosnier et al. (1998) fabricated a biosensor for detecting catechol in chloroform by electrochemical polymerization of amphiphilic
Scheme 1. Overall process of biosensor based on PPO.
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pyrrole lactobionamide–enzyme mixtures previously adsorbed on a glassy carbon electrode. The electrochemical immobilization of PPO was carried out in bilayers and trilayers of poly(pyrrole-amonium) and poly(pyrrole-lactobionamide) films. The amperometric response of the biosensor to catechol in chloroform was based on the reduction (at 0.25 V versus SCE) of the generated o-quinone. The immobilization of PPO in poly(amphiphilic pyrrole) films with lactobionamide as a polymeric additive produced a great enhancement of the biosensor sensitivity (+350%) in anhydrous chloroform due to the hydrophilic character of lactitol (Cosnier et al., 1998). The avidin–biotin technique has been employed as immobilization system in the development of biosensors, for its use in aqueous solution because provides both, a high degree of control over the molecular architecture of enzyme assemblies, and a large accessibility to the immobilized enzyme. The use of a biotinylated tyrosinase in combination with electrogenerated poly(pyrrole-biotin) films for the developed of an OPEEs has been reported (Mousty et al., 2001). Multilayered PPO assemblies were transferred into an organic solvent (chloroform) for the catechol detection at – 0.2 V. The catechol sensitivity and the maximum current values were lower than those recorded in Tris-buffer solution, and the authors attributed this decrease of activity to a loss of the enzyme structure. The immobilized PPO was protected against denaturation by a hydrophilic coating of alginate gel enhancing the biosensor performance (Fig. 1). Films of electrogenerated polypyrrole and hydrophilic alginate, both functionalized with biotin moieties were used to preserve PPO activity in organic media. Biotinylated PPO electrodes, based on multilayered structures, were protected at the molecular level by affinity binding of alginate as a hydrophilic additive, and then transferred into chlorobenzene, dichloromethane, chloroform, ethyl acetate or acetonitrile. The sensitivity, as well as the biosensor maximum current response were markedly higher in chlorobenzene (log P 2.84), dichloromethane (log P 2.00) and chloroform (log P 1.97), than in ethyl acetate (log P 0.66) and acetonitrile (log P 0.33) (Cosnier et al., 2004). Stanca and Popescu (2004) constructed two amperometric biosensors: one based on PPO immobilized in an electropolymerized poly(amphiphilic pyrrole) matrix, and the other obtained by enzyme cross-linking using glutaraldehyde. The analytical parameters of the resulting biosensors were compared for the detection of phenol in both chloroform
Fig. 1. Schematic representation of electrode configuration. ‘‘Reprinted Mousty et al., 2001, with permission from Elsevier’’.
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solution and phosphate buffer solution. The polypyrrole matrix shows a higher efficiency for enzyme retention resulting in higher bioelectrode sensitivity, both in aqueous buffer (690 mA M1) and in chloroform (149 mA M1). The biosensor was applied to benzoic acid determination in organic medium using a method based on the inhibition of PPO by the acid (Stanca and Popescu, 2004). Cristea et al. (2005) described the construction of an OPEE, via PPO entrapment within a hydrophilic polypyrrole film, adopting the adsorption procedure. The polymer matrix was electrogenerated from a new bispyrrolic derivative containing a long hydrophilic spacer. The hydrophilic character and a crosslinked structure of poly(byspirrole) facilitate enzyme entrapment, as compared with polypyrrole and poly(pyrroleammonium). The biosensor was used to detect catechol in anhydrous chloroform at 0.2 V versus Ag/AgCl with a sensitivity of 15.6 mA1 cm2 (Cristea et al., 2005). Furthermore, it was reported that the electroanalytical parameters strongly depend on the hydration state of the enzyme matrix. Besides polypyrrole, other matrices for enzyme immobilization have been proposed for its use in non-aqueous media. Wang and Dong (2000) reported a sol–gel composite tyrosinase biosensor for the detection of catechol, phenol and p-cresol in chloroform solution saturated with phosphate buffer. The tyrosinase retains its catalytic activity in the organic solvent and the enzyme electrode can reach 95% of steady-state current in about 18 s (Wang and Dong, 2000). Yu and Ju (2004) determined phenols in pure chloroform by immobilizing PPO in a titania sol–gel membrane obtained with a vapour deposition method. No extra water was required, because the titania sol–gel membrane retains the essential water layer needed for maintaining the enzyme activity in the organic phase, thus providing a promising platform for the construction of pure organic phase biosensors (Yu and Ju, 2004). The immobilization of tyrosinase onto a pre-activated membrane (immobilon) by covalent bonds with glutaraldehyde was achieved by Capannesi et al. (2000). The enzymatic membrane was placed on the head of an amperometric gas diffusion electrode (GDE) for oxygen between a gas permeable membrane and a dialysis membrane. The biosensor was used to evaluate the phenolic content of an extra-virgin olive oil. The method presents advantages, it is relatively inexpensive, easy to operate and prior extraction is not necessary because of the solubility of oil in n-hexane. As a result, pre-treatment of the sample is eliminated and the analysis time is reduced (Capannesi et al., 2000). Campanella et al. (2001c) have reported a number of organic phase enzyme electrodes using tyrosinase and other enzymes (monoenzymatic and bienzymatic systems), and operating in different organic solvents or solvent mixtures. As enzyme immobilisation system they propose kappa-Carrageenan gel in which enzyme was entrapped. The gel loaded with the enzyme was placed on the head of an amperometric GDE for oxygen, between the gas permeable membrane of the electrode and a dialysis membrane as is illustrated in Fig. 2. Applications performed in the field of foodstuff and cosmetic control (Campanella et al., 1999a,b) supported the correlation
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Fig. 2. Gaseous diffusion amperometric electrode and biosensor assembly: (a) electrode body; (b) internal solution (phosphate buffer); (c) reference electrode (Ag/AgCl) (anode); (d) platinum electrode (cathode); (e) teflon cap; (f) gaspermeable membrane; (g) teflon O-ring; (h) dialysis membrane; (i) enzyme immobilized in kappa-carrageenan. ‘‘Reprinted from Campanella et al., 2001c, with permission from Elsevier’’.
between classical indicators such as log P values of the solvent and empirical new indicators such as ‘‘maximum current variation’’ (MCV) or ‘‘current variation rate’’ (CVR) of the enzyme biosensor. A close correlation exists between the trends of log P and CVR indicator assuming that the enzyme molecules involved in the catalysis are those at the interface and neglecting any diffusion phenomena and solvent effects in the kappaCarrageenan gel. The latter assumptions are supported by the long lifetime of the biosensors and the good reproducibility of their response (Campanella et al., 1998a). The tyrosinase biosensor operates in n-hexane and was applied to rapid analysis of polyphenols in olive oils in which olive oil is highly soluble. For evaluating the progressive rancidification of olive oil they determine the peroxide number, since peroxides are typical oxidation products of fatty substances, and in their method they evaluate the progressive decrease in the content of polyphenols. A correlation between the stability of olive oil under an artificially induced process of randicification and its polyphenol content was established as well as the inverse correlation between the peroxide number and the polyphenol content during the rancidification process (Campanella et al., 1999c). Another contribution of the later authors is the development of biosensors able to operate in organic solvents for the determination of water in food fats, and pharmaceutical or cosmetic ointmens. The method is based on the increase of the 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 and catalase; the substrates were phenol or p-cresol and tert-butyl hydroperoxide respectively, and the organic solvents were acetonitrile or dioxane. An amperometric GDE for oxygen measurements was used as electrochemical transducer (Campanella et al., 2001b).
Morales et al. have reported a composite graphite–Teflon– PPO biosensor for the determination of the additive propyl gallate (phenolic antioxidant employed in foods) in dehydrated broth bars using buffer solution and in olive oil using 80:20 acetonitrile–Tris-buffer mixtures. They employed acetonitrile because this solvent is an extractive agent of polar compounds used as movile phase in HPLC. No amperometric response was observed in pure acetonitrile and in mixtures with very low water content, thus confirming the importance of this element in the catalytic activity of PPO (Morales et al., 2005a). Reversed micelles (formed with an organic solvent as the continuous phase, a phosphate buffer solution as the dispersed phase and dioctylsulfosuccinate, AOT, as the emulsifying agent) were used as a suitable working media for the determination of propyl gallate. The use of reversed micelles presents advantages such as an ease control of the optimum amount of water necessary for the hydration of the enzyme and a simplified enzyme immobilization scheme onto the electrode surface. The enzyme reaction was monitored by electrochemical reduction at 0.10 V of the corresponding o-quinone formed during the catalytic oxidation of propyl gallate. Different solvents were essayed as the continuous phase of the micelle, and the highest response was obtained using ethyl acetate, which is the solvent with lower log P value. The authors attributed this behaviour to its higher dielectric constant, thus ensuring the conductivity of the emulsion formed. Composite bioelectrodes allow the regeneration of the electrode surface by polishing and exhibit long-term operation (70 days). Moreover, the behaviour of propyl gallate as an inhibitor-like of the phenol oxidation reaction catalysed by tyrosinase was studied from an analytical point of view. This process is due to a competition of the two substrates for the enzyme active centers. The analytical characteristics were very similar using both measurement methodologies (direct amperometric and inhibition-like responses). The performance of the composite biosensor for the analysis of propyl gallate in foodstuffs was proved in lard samples. Aliquots of the analyte extracted in ethyl acetate were directly transferred to the electrochemical cell containing the reversed micellar medium for their measurement, with satisfactory results (Morales et al., 2005b). Zhang et al. developed an all-in-one dual-phase amperometric phenol biosensor, which can detect phenols in both aqueous and organic phases. A planar three-electrode electrochemical probe containing a microdisk array working electrode was employed as transducing system, and a very thin layer of hydrophilic polymer colloidal dispersion of polyurethane polyethylen oxide was used to immobilise tyrosinase on the probe tip. The entire electrochemical cell including electrodes, enzyme and buffer was retained within a hydrophilic dialysis membrane (Fig. 3). The enzymatic and also the electrochemical reaction take place behind the membrane and direct analysis in the organic phase can be performed without the need of added electrolyte. Moreover, the hydrophilic nature of both the immobilisation matrix and the membrane ensured the stability of the enzyme layer for analysis in hydrophobic organic solvents. This
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Fig. 3. The schematic diagram of the electrochemical biosensing system: (a) electrode arrangement; (b) the assembly of biosensing probe; (c) the assembly of batch measurement cell; (d) the assembly of flow injection analysis cell. ‘‘Reprinted from Zhang et al., 2001, with permission from Elsevier’’.
analysis is more complicated than in aqueous phase because the analyte have to be extracted out of the organic phase prior to the onset of the enzymatic reaction. Thus, the analyte is first extracted into this hydration layer before diffusing across the membrane where the biochemical reaction occurs. Phenol and catechol were measured in both batch and flow injection systems, in heptane, hexane, chlorobenzene, toluene and chloroform. The analytical properties such as response time, sensitivity and linear range, were found to be dependent on the degree of hydration layer as well as the relative hydrophobicity of the solvents and substrates employed. An increase in the hydrophobicity of the solvent led to higher sensitivity while sensitivity decreases in substrates with higher hydrophobicity (Zhang et al., 2001). 2.1.2. Catalase The ability to measure peroxides in non-aqueous media is of interest because the scarce solubility of these compounds in water. Catalase (hydrogen-peroxide oxidorreductase, E.C. 1.11.1.6) is a very fast biocatalyst that uses hydrogen peroxide as substrate liberating oxygen and water. The enzyme can also act on alkylhydrogen peroxides and several organic substances can replace the second hydrogen peroxide molecule as hydrogen donor (ethanol, formiate, thiol, etc.). The determination of hydrogen peroxide is based on the measurement of the oxygen produced during the enzymatic reaction according to the accepted mechanism:
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Campanella et al. (1998a) developed an OPEE based on catalase immobilized in kappa-Carrageenan gel for hydrogen peroxide. These researchers determined hydrogen peroxide in different organic solvents as chloroform, chlorobenzene, ethyl acetate and toluene. The measurements were carried out in a oxygen diffusion electrode recording the oxygen production in the enzymatic reaction. Similar to tyrosinase, a good correlation exists between log P and the CVR indicator (Campanella et al., 1998a). Attempts have been made to use a catalase biosensor operating in organic solvent to determine the hydrogen peroxide contained in cosmetic and pharmaceutical matrices in dioxane or water/dioxane mixtures. However, this biosensor has been used scarcely in systematic applications involving real matrices (Campanella et al., 1998b). The same biosensor was applied to monitor the hydroperoxide content of extra virgin olive oil during an artificial rancidification process studying, in n-decane or toluene, the response to cumene hydroperoxide and tertbutylhydroperoxide (Fig. 4). During the rancidification process an increase of peroxide concentration occurs, accompanied by a decrease in polyphenols content. A direct correlation was observed between hydroperoxide content (measured by catalase OPEE) and peroxide number (evaluated by titration). However, an inverse correlation was established with the last indicator and the polyphenol content determinate by tyrosinase OPEE (Campanella et al., 2001c). Horozova and co-workers investigated the catalytic activity of immobilized catalase in a polymeric films prepared with Nafion (mixture of polymer and enzyme solution) adsorbed on spectrographic graphite. They used two model peroxide compounds, dibenzoyl peroxide and 3-chloroperoxybenzoic acid in non-aqueous medium (acetonitrile), to prepare an OPEE. The response of the electrode was correlated with the reduction of the oxygen generated in the enzyme layer at the graphite electrode (Horozova et al., 2002). More recently, Varma and Mattiasson (2005) reported a biosensor coupled to a flow injection analysis (FIA) system for detection of hydrogen peroxide in organic solvents. Catalase entrapped in polyacrylamide gel was placed on the surface of platinum (working electrode) and fixed in a Teflon holder with Ag-wire (auxiliary electrode) (see Fig. 5). The catalase loaded gel was held on the electrode using cellulose and polytetrafluroethylene membranes. The electrode response was studied in water, dymethyl sulfoxide and 1,4-dioxane and it was found that the sensor was able to monitor very high ranges of hydrogen peroxide, although the response time depends on the concentration of the substrate as the system is mass transport limited (Varma and Mattiasson, 2005).
H2 O2 þ Cat-FeðIIIÞ ½catalase ! H2 O þ Cat-FeðIVÞ¼O ½compound I H2 O2 þ Cat-FeðIVÞ¼O ! H2 O þ O2 þ Cat-FeðIIIÞ
Fig. 4. Structure of (a) tert-butylhydroperoxide (b) and cumene hydroperoxide.
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Fig. 5. Schematic representation of the indigenously designed flow cell. The flow cell comprises of a base made of Teflon which houses the inlet, outlet and the analyte solution chamber. The volume of the analyte is defined by a viton Oring. The electrodes (working and the counter electrodes) are inserted by fitting it into a jacket, which in turn can be screwed into the holder connecting to the base. ‘‘Reprinted from Varma and Mattiasson (2005), with permission from Elsevier’’.
2.1.3. Peroxidase Horseradish peroxidase (HRP, E.C. 1.11.1.1) is a hemoproteine with extensive glycosylation. Peroxidase catalyses the oxidation of a wide range of substrates including ascorbate, ferrocyanide, cytochrome C and many organic molecules. HRP can use several oxidants including hydrogen peroxide, organic peroxides, perborate ions and superoxide radicals. HRP requires two electron equivalents to recycle back to the original enzyme state. The heme group is essential for the enzyme activity and undergoes spectroscopic changes during the oxidation reaction indicating that iron atom participates in the reaction mechanism. Enzyme oxidation proceeds in two steps each of them transfers a single electron and may involve different substrate molecules. The overall reaction can be represented as follows: HRP þ H2 O2 ! HRP ½compound I AH2 ½substrate þ HRP ! AH ½oxidized product þ HRP ½compound II AH2 þ HRP ! AH þ HRP Enzyme electrodes based on HRP monitor the electrochemical oxidation of the peroxidase when it reacts with the substrate using two systems: conducting polymers o graphite/ enzyme mixtures. The electron transfer of the HRP could be direct or mediated. Mulchandani and Pan (1999) have developed an enzyme electrode based on horseradich peroxidase (HRP) incorporated in an electrically deposited ferrocene-modified phenylenediamine film on a glassy carbon electrode (GCE). The HRP/ poly(m-aminoanilinomethylferrocene)-modified GCE reagentless biosensor measured peroxides in both aqueous (citrate– phosphate buffer solution) and organic medium (90% acetonitrile–10% 2-(N-morpholino)ethanesulfonic acid buffer mixture) by reduction at a low applied potential of 0.05 V
(versus Ag/AgCl) without interference from molecular oxygen. The current response of the enzyme electrode, based on ferrocene-conjugated polymer for electron transfer from HRP to the GCE, compared with an electrode based on HRP entrapped in poly(phenylenediamine) on the GCE showed a 5000-fold higher sensibility. This is a significant advantage of the mediated electron transfer over the direct electron transfer from HRP. However, the electrode was more sensitive for tbutyl peroxide and lauroyl peroxide in aqueous medium. Despite the low solubility of peroxides in aqueous medium this should be expected since HRP conspicuously decreases its activity in organic medium (Mulchandani and Pan, 1999). Another peroxidase biosensor fabricated by immobilization of the enzyme during the electropolymerization of N-methylpyrrole was reported by Garcia-Moreno et al. The biosensor was used in the determination of organic peroxides (2-butanone peroxide and tert-butylhydroperoxide) in a predominantly nonaqueous medium such as reversed micelles and successfully determined the organic peroxide content in body lotion samples employing 2-butanone peroxide as a standard (Garcia-Moreno et al., 2001). Ramirez-Garcia et al. (2001) investigated the behaviour of composites based on silicone, epoxies, polyester and polyurethane in organic solvents (acetone, acetonitrile, ethanol, chloroform and tetrahydrofuran). Composites with silicone or epoxy have a homogeneous surface and showed high stability and reproducibility while performing electrochemical measurements in organic solvents. The surface of the resulting device can be renewed by a simple polishing procedure. The plastic nature of these materials makes them modifiable, permitting the incorporation of fillers before they are cured. To test their capacity for biosensing in organic media, the matrix of one transducer was biologically modified with HRP producing a graphite–epoxy–HRP biocomposite. The enzyme remained stable in the biocomposite and the biosensor built with this material showed a linear response to lauroyl peroxide in acetonitrile (Ramirez-Garcia et al., 2001). Castillo et al. (2003) developed two biosensors based on sweet potato peroxidase (SPP) and horseradish peroxidase (HRP) included within redox hydrogels of polyethylene glycol diglycidyl ether. The HRP electrode displayed twice sensitivity in aqueous phase than the SPP electrode, but in non-aqueous medium (acetonitrile) the SPP biosensor performs better (Castillo et al., 2003). 2.1.4. Glucose oxidase Glucose oxidase (GOx, E.C. 1.1.3.4) from Aspergillum niger is a flavoenzyme that has been used since 1956 to determine glucose. GOx catalyses the oxidation of D-glucose to generate H2O2 in the general reaction: b-d-glucose þ enzyme-FAD ! enzyme-FADH2 þ b-d-gluconolactone enzyme-FADH2 þ O2 ! enzyme-FAD þ H2 O2 Frequently, GOx is selected as a model enzyme to essay new immobilization systems for application in glucose ampero-
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metric biosensors because its low cost, good stability and high solubility of glucose in aqueous medium. There are few works devoted to OPEEs based on GOx, one attempt was made by Campanella et al., immobilizing GOx within kappa-Carrageenan gel in a GDE for oxygen to develop a glucose sensor. Measurements were performed in saturated chloroform containing 0.09% (v/v) water, as well as in ethyl acetate and in acetonitrile with 1.5% and 2.5% (v/v) water, respectively. The small percentage of water contained in these solvents is necessary to increase the solubility of the substrate in the solvent. Working in different organic solvents (chloroform, ethyl acetate and acetonitrile), they compared de current variation rate (CVR) indicator for the glucose sensor with log P of the solvent and found a good correlation between both parameters (Campanella et al., 1998a). Kro¨ger et al. (1998) employed a solvent resistant screenprinted three-electrode device to assess the behaviour of free and immobilised GOx in water-miscible organic solvent/ aqueous buffer mixtures. Three alcohols were examined, methanol, ethanol and isopropanol. A rhodinised-carbon electrocatalyst was used to facilitate hydrogen peroxide oxidation at a decreased operating potential. They studied the sensor response under enzyme-limiting and enzyme-excess conditions and highlighted the fact that the overall device response was influenced by the dual effect of the solvents on both, enzyme activity and on the electrochemical sensor device itself. They maintain that many sensor-based enzyme studies reported in the literature neglect enzyme loading factors which may, in part, account for the conflicting observations made regarding solvent influence on biosensor performance (Kro¨ger et al., 1998). 2.1.5. Other enzymes Andreescu et al. reported a screen-printed biosensor based on acetylcholinesterase (AChE, E.C. 3.1.1.7) immobilised in poly(vinyl alcohol) with styrylpyridinium groups, for the detection of p-aminophenyl acetate, monitoring the oxidation of p-aminophenol, product of the enzymatic reaction. As working medium, they used phosphate buffer containing water miscible organic solvent (acetonitrile and ethanol) in a concentration range from 1% to 25%. This biosensor was used for the determination of pesticides by means of inhibition measurements (Andreescu et al., 2002). A biosensor based on superoxide dismutase (SOD, E.C. 1.15.1.1) was developed by Campanella et al. Good results were obtained adopting a new way of assembling the device. The enzyme was physically entrapped, using a cellulose triacetate layer and sandwiched between two gas-permeable membranes, or using a kappa-Carrageenan gel layer entrapping the enzyme, sandwiched between an external gas permeable membrane and an internal cellulose acetate membrane, coupled in each case to the oxygen amperometric transducer. This biosensor was applied for the determination of hydrophobic compounds showing radical scavenging properties operating in dimethylsulfoxide with satisfactory results (Campanella et al., 2001d). More recently, it has been reported the measurement of the antioxidant capacity of integrator-phytotherapeutic pro-
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ducts using the second of the former immobilized systems. The superoxide radical is produced by the oxidation in aqueous solution of the xantine in the presence of xanthine oxidase. After the reaction, the superoxide radical with the superoxide dismutase releases hydrogen peroxide that is monitored by the amperometric sensor: xanthine þ H2 O þ O2
xanthine oxidase
!
uric acid þ 2Hþ þ O2
SOD
O2 þ O2 þ 2Hþ !H2 O2 þ O2 The addition of any sample possessing antioxidant properties decreases the current because the antioxidant species react with the superoxide radical, reducing its concentration in the solution and, consequently, producing a decrease in the hydrogen peroxide concentration. This measurements were carried out in a complex solvent mixture consisting of DMSO and glycerine (14.5/0.5) (v/v), DMSO, glycerine and Tween 20 (10/4/1) (v/v/v), 1% (p/v) in ‘‘crown ether’’, achieving higher sensitivity (Campanella et al., 2004). The properties of the monoenzyme electrodes described in this section are summarized in Table 1. 2.2. Bienzyme electrodes The analysis of some substances such as lecithin requires the coupling of two enzymes and due the scarce solubility in water of foodstuff containing lecithin an organic solvent is necessary. A bienzymatic OPEE using phospholiphase D (PLD, E.C. 3.1.4.4) and choline oxidase (ChOx, E.C. 1.1.3.17) immobilized in kappa-Carrageenan gel with a GDE for oxygen as transducer has been reported for the analysis of lecithin (phosphatidylcholine). The biosensor works on the basis of two coupled enzymatic reactions: PLD
lecithin!choline þ phosphatidic acid ChOx
choline þ 2O2 þ H2 O! betaine þ 2H2 O2 establishing a correlation between the substrate concentration and the oxygen consumed in the reaction catalysed by the choline oxidase and consequently, with the decrease of the current intensity measured in the device. The solvents employed in this sensor were water saturated with chloroform–hexane (1/1, v/v) and a chloroform–hexane–methanol mixture (1/1/0.02, v/v). Methanol was added to achieve a complete solubilisation of the lecithin drug matrix. A watersaturated solvent was used instead of an anhydrous solvent with the aim to increase the biosensor lifetime as much as possible. This biosensor was employed to determinate the lecithin content in food (egg yolk, mil chocolate, soya seed oil), diet products (Campanella et al., 1998c), as well as in pharmaceutical products (Campanella et al., 1998d). The use of a gas diffusion electrode (GDE) as indicator electrode instead of a voltamperometric system is dispensed by the need to introduce special electrolytes into the solution to increase the conductivity of the non-aqueous solvent. The latter were necessary when amperometric electrodes were used in organic polar solvents.
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Table 1 Monoenzyme electrodes Electrode
Immobilization system
Solvents
Determination
Reference
PPO
Glassy carbon
Chloroform
Catechol
Cosnier et al. (1998)
PPO
n-Hexane
Polyphenols in olive oil
Campanella et al. (1999c)
n-Hexane
Phenol
Capannesi et al. (2000)
PPO PPO
Oxygen amperometric GDE Oxygen amperometric GDE Glassy carbon Platinum microdisk array
Entrapment in poly(pyrrole-ammonium) and poly(pyrrole-lactobionamide) Entrapment in kappa-Carrageenan gel
Catechol, phenol, p-cresol Catechol and phenol
Wang and Dong (2000) Zhang et al. (2001)
PPO and catalase PPO
Oxygen amperometric GDE Glassy carbon
Chloroform Heptane, hexane, chlorobenzene, toluene, chloroform Acetonitrile or dioxane
Water content in food fats (butter, margarine) or ointments Catechol
Campanella et al. (2001b) Mousty et al. (2001)
PPO
Glassy carbon
Catechol
Cosnier et al. (2004)
PPO
Platinum
Polyphenolic compounds
Stanca and Popescu (2004)
PPO PPO PPO
Glassy carbon Glassy carbon Graphite–Teflon composite
Yu and Ju (2004) Cristea et al. (2005) Morales et al. (2005a)
PPO HRP
Graphite–Teflon composite Glassy carbon
HRP
Platinum
HRP HRP and SPP SPP SPP Catalase
Graphite Graphite Graphite Graphite Oxygen amperometric GDE Oxygen amperometric GDE Graphite
Phenol, catechol, p-cresol Catechol Propyl gallate in dehydrated broth bars and olive oil Propyl gallate in lard samples Hydrogen peroxide and other organic peroxides Organic peroxide content in body lotion samples Lauroyl peroxide Hydrogen peroxide Hydroquinone in cosmetic creams Hydroquinone in cosmetic creams Hydrogen peroxide in cosmetic and pharmaceutical formulations Hydroperoxide content of olive oil
PPO
Catalase Catalase Catalase GOx SOD SOD
AChE
Covalent union with glutaraldehyde to pre-activated membrane Silica sol–gel Entrapment in polyethylene oxide Entrapment in kappa-Carrageenan gel Polypirrol and alginate film with avidin–biotin interactions Polypirrol and alginate film with avidin–biotin interactions Entrapment in polypyrrole matrix or cross-linked with glutaraldehyde Titania sol–gel Entrapment in hydrophilic polypyrrole film Adsorption on graphite–Teflon composite
Chloroform Chlorobenzene, dichloromethane, chloroform, ethyl acetate and acetonitrile Chloroform Chloroform Chloroform Acetonitrile–tris buffer mixtures
Adsorption on graphite–Teflon composite Entrapment in ferrocene-modified phenylenediamine film Entrapment in N-methyl-pyrrole
Reversed micelles Acetonitrile–buffer mixture
Adsorption on graphite Cross-linked to a redox hydrogel Adsorption on graphite Adsorption on graphite Entrapment in kappa-Carrageenan gel
Acetonitrile Acetonitrile Methanol–phosphate buffer solution Methanol–phosphate buffer solution Dioxane and water–dioxane mixtures
Entrapment in kappa-Carrageenan gel
n-Decane, toluene or chloroform
Adsorption on polymer film with nafion
Acetonitrile
Platinum Screen-printed electrode: rhodinised-carbon Oxygen amperometric GDE Oxygen amperometric GDE
Entrapment in polyacrilamide gel Adsorption
Dimethylsulfoxide, dioxane Methanol, ethanol and isopropanol aqueous buffer mixtures Dimethylsulfoxide
Screen-printed electrode
Entrapment in polyvinyl alcohol styrylpyridinium groups polymer
Entrapment in kappa-Carrageenan gel Entrapment in kappa-Carrageenan gel
Reversed micelles
Dimethylsulfoxide and glycerine or dimethylsulfoxide, glycerine and Tween 20, in ‘‘crown ether’’ Acetonitrile and ethanol
Dibenzoyl peroxide and 3-chloroperoxibenzoic acid Hydrogen peroxide Glucose Hydrophobic compounds showing radical scavenging Antioxidant capacity of integratorphytotherapeutic products p-Aminophenol
Morales et al. (2005b) Mulchandani and Pan (1999) Garcia-Moreno et al. (2001) Ramirez-Garcia et al. (2001) Castillo et al. (2003) Vieira and Fatibello-Filho (2000) Fatibello-Filho and Vieira (2000) Campanella et al. (1998b) Campanella et al. (2001c) Horozova et al. (2002) Varma and Mattiasson (2005) Kro¨ger et al. (1998) Campanella et al. (2001d) Campanella et al. (2004)
Andreescu et al. (2002)
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Enzyme
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The only disadvantage of a GDE for oxygen, namely interference due to bacteria pollution, is rendered highly improbable because the difficulty of growing bacteria in organic solvents. The new OPEE proved to be a valid analytic system to detect lecithin contained in hydrophobic matrices when other analytical techniques require long time. Other oxygen diffusion electrode based on peroxidase and tyrosinase working in parallel and competing for the same compound (catechol), has been described. Measurements were performed in two stages: first, catechol was added to the solution, subsequently the reduction of dissolved O2 occurs and a current stationary state is achieved. In the second stage, a fixed quantity of a hydroperoxide was added, catechol was oxidised not only by the O2 present in solution, but also by the hydroperoxide, according to the peroxidase-catalysed reaction. This reaction led to an increase of the dissolved O2 concentration as the hydroperoxide competed with the O2 in oxidising the catechol and a partial restoration of the current is registered as a new stationary state is reached. PPO
catechol þ 12O2 !quinone þ H2 O HRP
catechol þ 12H2 O!quinone þ 2H2 O The biosensor was applied for the determination of the ‘‘pool’’ of hydroperoxides released during the heating of extravirgin olive oil using decane as solvent because of the high solubility of the olive oil in this solvent. Other application was to detect the hydrogen peroxide content of lipophilic cosmetic products, working in a dioxane–water mixture. In the latter case, the selection of the solvent was a compromise between biosensor response and the solubility of the tested product (Campanella et al., 2003). Cholesterol oxidase (COx, E.C. 1.1.3.6) and horseradish peroxidase (HRP) together with potassium ferrocyanide, as a mediator, were incorporated into a graphite–70% Teflon matrix and prepared in the form of cylindrical pellets to fabricate a bienzyme amperometric composite biosensor for the determination of free and total cholesterol in food samples. The compatibility of this biosensor design with a non-aqueous media permits to use reverse micelles as working medium. Micelles were formed with ethyl acetate as continuous phase (in which cholesterol is soluble), a phosphate buffer as dispersed phase, and AOT as emulsifying agent. The use of
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ethyl acetate presents the analytical advantage that the extraction of cholesterol from food samples is accomplished with the same solvent used to prepare the reversed micelles. The determination of free and total cholesterol in food samples (butter, lard and egg yoke) with this biosensor, gave better results than those provided by commercial test kit (Pen˜a et al., 2001). Bienzymatic OPEEs are summarized in Table 2. 2.3. Enzyme inhibition based electrodes The ability of certain substances to inhibit the catalytic action of enzymes can be exploited for their detection and quantification in the so-called enzyme-inhibition biosensor. Different type of insecticides (carbamate and organophosphorus) when present in water and foodstuffs constitute a hazard due to their high toxicity. These compounds possess low solubility in water but are highly soluble in organic solvents. Amperometric enzyme electrodes based on cholinesterases have been successfully used for the determination of pesticides since most of them act as inhibitors of this enzymes. An amperometric tyrosinase electrode has been used for biosensing of dimethyl- and diethyldithiocarbamates based on the inhibition effects of these substances on the catalytic activity of the enzyme. The working medium consisted of reversed micelles (phosphate buffer as dispersed phase, ethyl acetate as continuous phase and AOT as emulsifying agent) and phenol as substrate. The tyrosinase electrode was constructed by direct adsorption of the enzyme on the surface of a graphitedisk electrode. Carbamates such as ziram, diram (Fig. 6) and zinc diethyldithiocarbamates showed reversible inhibition processes. Following a simple regeneration of the enzyme electrode, an acceptable reproducibility for the measurements of the inhibition response was obtained. Other carbamates belonging to families different from dimethyl- and diethyldithiocarbamates showed no amperometric response at the tyrosinase electrode, except for pyrimidine-derivative carbamates. The developed analytical methodology was applied to determine ziram in spiked apple samples (Pe´rez Pita et al., 1997). Morales et al. have developed a graphite–Teflon–tyrosinase composite biosensor based on the inhibition effect of benzoic acid on the catalytic activity of tyrosinase. Taking advantage of the capabilities of reversed micelles as universal solubilization
Table 2 Bienzyme electrodes Enzyme
Electrode
Immobilization system
Solvents
Determination
Reference
PLD and ChOx PLD and ChOx PPO and HRP
Oxygen amperometric GDE Oxygen amperometric GDE Oxygen amperometric GDE
Entrapment in kappaCarrageenan gel Entrapment in kappaCarrageenan gel Entrapment in kappaCarrageenan gel
Chloroform–hexane mixture and chloroform–hexane–methanol mixture Chloroform–hexane mixture and chloroform–hexane–methanol mixture Decane and dioxane–water mixture
Campanella et al. (1998c) Campanella et al. (1998d) Campanella et al. (2003)
COx and HRP
Graphite–Teflon composite
Adsorption on graphite–Teflon composite
Reversed micelles
Lecithin in food samples (egg yolk, soya flour and oil) Lecithin in drugs and diet products Hydroperoxides of olive oil and hydrogen peroxide of cosmetic products Cholesterol in butter, lard and egg yoke
Pen˜a et al. (2001)
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Fig. 6. Structure of ziram (a) and diram (b).
media, the composite tyrosinase electrode was used for the determination of benzoic acid in two different samples: mayonnaise sauce which is a highly hydrophobic matrix and Cola soft drinks as hydrophilic matrix for which practically no sample treatment was necessary (Morales et al., 2002). An enzyme inhibition OPEE based on the tyrosinase and operating in chloroform was proposed by Campanella et al., for determination of pesticides of the triazine family. The tyrosinase was immobilized in kappa-Carrageenan gel. The current variation was measured when a phenol solution was added and, once the signal became constant, successive addition of pesticide solution was performed in order to observe the inhibition process. The detection limit for atrazine, atraton (Fig. 7) and atrazine-desethyl and was found to be 0.5 nM (Campanella et al., 2005a). This biosensor was used for triazinic (simazine, propazine, terbuthylazine), and benzotriazinic (azinphos-ethyl and azinphos-methyl) pesticides determination. Furthermore, the authors report recovery trials performed in vegetal matrixes (corn, barley, lentils) (Campanella et al., 2005b). The presence of benzoic acid induces an inhibitory effect on the response to phenol of PPO-based biosensor. The calibration curves to phenol were recorded in absence and presence of benzoic acid. In both cases, the maximum current intensity was similar suggesting that benzoic acid inhibition is competitive with the phenol response at the cresolase active site of the enzyme. The inhibition at a constant substrate concentration was estimated by the current intensity depletion due to the presence of benzoic acid in chloroform and phosphate buffer (Stanca and Popescu, 2004). Palchetti et al. reported a choline biosensor based on screenprinted electrodes used to assess the inhibitory effect of organophosphorus and carbamic pesticides on acetylcholinesterase activity. This enzyme catalyses the cleavage of acethylcholine to choline and acetate, and therefore, the amount of choline measured by the biosensor is directly related to the enzyme activity. The extent of enzyme inhibition can be used as an index of the amount of anticholinesterase pesticide present in the sample. The low stability of screen-printed electrodes in pure organic solvents was overcome by using mixtures of non-polar organic solvent with borate buffer (methanol, acetone, acetonitrile, ethyl acetate, dimethyl
Fig. 7. Structure of atrazine (a) and atraton (b).
sulfoxide and tetrahydrofuran). The best result was obtained using a mixture of acetonitrile–borate buffer (1% v/v). The method was applied to real samples (fruits and vegetables) showing its suitability as a rapid screening assay for the assessment of anticholinesterase pesticides (Palchetti et al., 1997). A bienzymatic inhibition electrode based on butyrylcholinesterase (BchE, E.C. 3.1.1.8) and choline oxidase has been developed for the detection of organophosphorus pesticides or carbamates in chloroform–hexane mixture (50%, v/v) as a satisfactory compromise between the need of appropriate solubility for both substrate and inhibitor in the solvent considered and the need to avoid possible negative effects on biosensor response and lifetime, which depend on the nature of the solvent. The two enzymes were immobilised in kappaCarrageenan gel and an amperometric GDE for oxygen was used to measure the oxygen consumed during the oxidation of the choline produced by hydrolysis of butyrylcholine: BChE
butyrylcholine ! choline þ butyric acid ChOx
choline þ 2O2 þ H2 O! betaine þ 2H2 O2 Pesticides inhibit butyrylcholinesterase, the production of choline is reduced and the outcome is a decreased of the oxygen consumed. The detection limit for pesticides such as aldicarb (carbamate) or paraoxon (organophosphorus) (Fig. 8) is about 4.5 mg/l (Campanella et al., 1999d). Wilkins et al. developed an amperometric acetylcholinesterase biosensor based on thiocholine-hexacyanoferrate for the analysis of organophosphate pesticides in pure organic solvents. The strategy was based on the following signalamplification systems: (1) the coimmobilization of redox mediator (Prussian Blue) and AChE on the electrode surfaces; (2) the accumulation of the product of enzymatic and electrochemical reactions at the membrane/electrode interface (3) the cyclic regeneration of the redox mediators at the electrode surface. Thiocholine produced by enzymatic hydrolysis of acetylthiocholine reacts stoichiometrically with hexacyanoferrate (II). Subsequently, the reduced electron mediator is reoxidized at the graphite electrode and the analytical signal is measured amperometrically (Wilkins et al., 2000). A disposable cholinesterase screen-printed electrode based on entrapped acetylcholinesterase in a polyvinyl alcohol with styrylpyridinium groups (PVA-SbQ) has also been developed. The procedure used to measure the inhibition of acetylcholinesterase is the incubation of the electrode in the buffer– solvent–pesticide solution and the measurement of the residual output current after the addition of substrate (acetylthiocholine). The influence of miscible organic solvents was studied by
Fig. 8. Structure of aldicarb (a) and paraoxon (b).
Andreescu et al. (2002) Paraoxon and chlorpyrifos ethyl oxon
Wilkins et al. (2000) Montesinos et al. (2001) Dichlorovos, fenthion and diazinon Chlorpyrifos-ethyl-oxon
Immobilized covalently on polyethylenimine Entrapment in polyvinyl alcohol styrylpyridinium groups polymer Entrapment in polyvinyl alcohol styrylpyridinium groups polymer Graphite Screen-printed electrode
Screen-printed electrode AChE
Ethanol Acetonitrile, ethanol and dimethyl sulfoxide Acetonitrile
Campanella et al. (1999d) Aldicarb and paraoxon Entrapment in kappa-Carrageenan gel Oxygen amperometric GDE
BChE and ChOx AChE AChE
Acetone, acetonitrile, tetrahydrofuran and ethyl acetate Chloroform–n-hexane mixture Screen-printed electrode ChOx and AChE
Chloroform
Entrapment in polypyrrole matrix or cross-linked with glutaraldehyde Adsorption Platinum PPO
Water–saturated chloroform Entrapment in kappa-Carrageenan gel Oxygen amperometric GDE PPO
Entrapment in kappa-Carrageenan gel Oxygen amperometric GDE PPO
Water–saturated chloroform
Carbofuran in fruits and vegetables
Campanella et al. (2005a) Campanella et al. (2005b) Stanca and Popescu (2004) Palchetti et al. (1997)
Morales et al. (2002) Reversed micelles Adsorption on graphite–Teflon composite Graphite–Teflon composite PPO
Reversed micelles Adsorption Graphite PPO
Immobilization system Electrode Enzyme
Table 3 Enzyme inhibition based electrodes
Fig. 9. Structure of chlorpyrifos-ethyl-oxon.
Solvents
2.4. Tissue biosensors There are several HRP-rich tissues such as those of peach, yam, manioc, artichoke, sweet potato, turnip, horseradish and zucchini. Some of them, such as the sweet potato tissue, present high specific activity, the highest storage time, and the longest biosensor lifetime. There are articles describing the construction of biosensors working in non-aqueous medium that use sweet potato (Ipomoea batatas Lam.) tissue as the enzymatic source of peroxidase. Vieira and Fatibello-Filho developed a paraffin/graphite electrode modified with sweet potato tissue, as the source of peroxidase, for determining hydroquinone. The peroxidases present in the tissue catalyse the oxidation of hydroquinone to p-quinone in the presence of the hydrogen peroxide. As usually, the p-quinone produced was electrochemically reduced to hydroquinone. They investigated the effect of different organic solvent–phosphate buffer solution (99:1% v/v) (methanol, acetonitrile, ethanol, acetone, etc.) for the determination of a standard solution of hydroquinone. With few exceptions, there was a decrease of biosensor response with the increase of organic solvent hydrophobicity and the biosensor exhibits the highest response with methanol. The biosensor was applied in the analysis of hydroquinone in cosmetic creams in this solvent. The results were in agreement with those obtained using a Pharmacopoeia procedure (Vieira
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Ziram, diram and zinc diethylthiocarbamate. Ziram in apple samples Benzoic acid in mayonnaise sauce and cola soft drinks Triazine. Pesticide recovery from vegetal samples Triazinic and benzotriazinic. Pesticide recovery from vegetal samples Benzoic acid
Determination
Reference
measuring the sensor response resulting from the inhibitory effect of organophosphorus pesticides on acetylcholinesterase activity. The measurement was performed in phosphate buffer containing acetonitrile, ethanol or dimetylsulfoxide in the range 0–30% (v/v). With 5% acetonitrile and 10% ethanol, an increase of the recorded current was observed. The reproducibility and sensitivity of the results were excellent, of the order of ppb, for chlorpyrifos-ethyl-oxon (Fig. 9) a compound widely used for agricultural purposes (Montesinos et al., 2001). Andreescu et al. described a screen-printed biosensor based on the entrapment of acetylcholinesterase in a PVA-SbQ polymer for the detection of pesticides. The substrate was paminophenyl acetate which is characterized by a good solubility in organic solvents such as acetonitrile. The oxidation of p-aminophenol, the product of the enzymatic reaction was monitored, and to preserve the enzyme activity a buffer/ acetonitrile mixture with less than 5% acetonitrile was selected. The inhibition rate was higher when working in 5% acetonitrile and detection limits of 20 nM paraoxon and 1.24 nM chlorpyrifos-ethyl-oxon were obtained (Andreescu et al., 2002). Enzyme inhibition based electrodes working in non-aqueous medium are summarized in Table 3.
Pe´rez Pita et al. (1997)
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and Fatibello-Filho, 2000). Another biosensor based on graphite powder modified with stearic acid and the same tissue was developed for the determination of hydroquinone in organic solvents. The detection of hydroquinone in cosmetic creams using methanol, provides results comparable to that of the Pharmacopoeia method (Fatibello-Filho and Vieira, 2000).
dioxane with 1% and 10% of acetate buffer. The highest signal was obtained with acetonitrile. Glucose concentration of oily food samples was measured and compared with the results obtained by the reference UV-photometric method. The correlation between the results obtained by both methods was very good (Adanyi et al., 2004b).
3. Enzyme reactors
Acknowledgement
A bioreactor is defined as a device in which a chemical conversion reaction is catalysed by an enzyme. There are some articles that describe the use of bioreactors for immobilizing enzymes and how to connect them into a flow injection analyzer (FIA) system with an amperometric detector. The following paragraphs related some articles that describe the use of enzyme reactors. Adanyi and Varadi (2004a) immobilized the catalase enzyme by glutaraldehyde on a natural protein membrane (pig’s small intestine) in a thin-layer enzyme cell, connected to a stopped-flow injection analyser system (SFIA) with an amperometric detector (see Fig. 10), for the hydrogen peroxide determination in acetonitrile. They also developed a quick analytical method to monitor the water content (activator) in various butter and margarine samples by maintaining a fixed substrate concentration. The water content of samples obtained by this method was compared with that obtained by the gravimetric reference method and the correlation coefficient was 0.993 (Adanyi and Varadi, 2004a). With the aim to develop a flow-through measuring apparatus for glucose determination as model system in organic media, GOx was immobilized by Adanyi et al. on a natural protein membrane (pigs’ small intestine) in a thin-layer enzyme cell made of Teflon. A mixture of enzyme and bovine serum albumin was dispersed on the protein membrane and covalently bonded with glutaraldehide. The enzyme cell was connected into a flow injection analyzer system with an amperometric detector. Different organic solvent were used, acetonitrile, 2-propanol, n-butanol and
The authors acknowledge financial support from DGI (MAT2003-03051-C03-03) of the Spanish Science and Technology Ministry.
Fig. 10. Measuring setup. 1: Buffer reservoir; 2: HPLC pump; 3: injector; 4: thin-layer enzyme reactor; 5: sample in; 6: amperometric cell; 7: amperometric detector; 8: recorder. ‘‘Reprinted from Adanyi and Varadi, 2004a, with permission from Springer-Verlaq’’.
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