www.elsevier.com/locate/geneanabioeng

Biomolecular Engineering 23 (2006) 135–147

Review

Organic phase enzyme electrodes

M. Sanchez-Paniagua Lopez b, E. Lopez-Cabarcos b, B. Lopez-Ruiz a,*a Seccion Departamental de Quımica Analıtica, Facultad de Farmacia,

Universidad Complutense de Madrid, 28040 Madrid, Spainb Departamento de Quımica-Fısica 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. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

2. Amperometric biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

2.1. Monoenzyme electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

2.1.1. Tyrosinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

2.1.2. Catalase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

2.1.3. Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

2.1.4. Glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

2.1.5. Other enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

2.2. Bienzyme electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

2.3. Enzyme inhibition based electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

2.4. Tissue biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

3. Enzyme reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

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 electro-

des; PLD, phospolipase D; PPO, tyrosinase; PVA-SbQ, polyvinyl alcohol styryl

pyridinium groups; SOD, superoxide dismutase; SPP, sweet potato peroxidase

* Corresponding author at: Seccion Departamental de Quımica Analıtica,

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: bealopru@farm.ucm.es (B. Lopez-Ruiz).

1389-0344/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.bioeng.2006.04.001

1. Introduction

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

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147136

Scheme 1. Overall process of biosensor based on PPO.

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 (Kroger

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 (Diaz-

Garcia 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’’

(Rodrıguez-Lopez 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 (San-

chez-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

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147 137

pyrrole lactobionamide–enzyme mixtures previously adsorbed

on a glassy carbon electrode. The electrochemical immobiliza-

tion 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), dichlor-

omethane (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 electro-

polymerized 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’’.

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 M�1) and in chloroform (149 mA M�1). The biosen-

sor 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 cross-

linked structure of poly(byspirrole) facilitate enzyme entrap-

ment, as compared with polypyrrole and poly(pyrrole-

ammonium). The biosensor was used to detect catechol in

anhydrous chloroform at �0.2 V versus Ag/AgCl with a

sensitivity of 15.6 mA�1 cm�2 (Cristea et al., 2005). Further-

more, it was reported that the electroanalytical parameters

strongly depend on the hydration state of the enzyme matrix.

Besides polypyrrole, other matrices for enzyme immobili-

zation 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

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147138

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) gas-

permeable 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 kappa-

Carrageenan 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 electroche-

mical 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 ampero-

metric phenol biosensor, which can detect phenols in both

aqueous and organic phases. A planar three-electrode electro-

chemical 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

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147 139

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’’.

Fig. 4. Structure of (a) tert-butylhydroperoxide (b) and cumene hydroperoxide.

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 determina-

tion of hydrogen peroxide is based on the measurement of the

oxygen produced during the enzymatic reaction according to

the accepted mechanism:

H2O2þCat-FeðIIIÞ ½catalase�! H2O þ Cat-FeðIVÞ¼O ½compound I�

H2O2þCat-FeðIVÞ¼O ! H2O þ O2þCat-FeðIIIÞ

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 pharma-

ceutical 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 tert-

butylhydroperoxide (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 polytetra-

fluroethylene 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).

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147140

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 O-

ring. 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 hemopro-

teine 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 H2O2 ! HRP�� ½compound I�

AH2 ½substrate� þ HRP��

! AH� ½oxidized product� þ HRP� ½compound II�

AH2þHRP� ! AH� þHRP

Enzyme electrodes based on HRP monitor the electro-

chemical 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 phenylenedia-

mine film on a glassy carbon electrode (GCE). The HRP/

poly(m-aminoanilinomethylferrocene)-modified GCE reagent-

less 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 t-

butyl 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-methyl-

pyrrole 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 non-

aqueous 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 poly-

urethane 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 mea-

surements 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-FADH2þb-d-gluconolactone

enzyme-FADH2þO2 ! enzyme-FAD þ H2O2

Frequently, GOx is selected as a model enzyme to essay new

immobilization systems for application in glucose ampero-

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147 141

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-Carragee-

nan 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).

Kroger et al. (1998) employed a solvent resistant screen-

printed 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 (Kroger

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-

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 H2Oþ O2 �!xanthine oxidaseuric acidþ 2Hþ þ O2

��

O2�� þ O2

�� þ 2Hþ�!SODH2O2 þ O2

The addition of any sample possessing antioxidant proper-

ties 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) immobi-

lized 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:

lecithin�!PLDcholineþ phosphatidic acid

cholineþ 2O2 þ H2O�!ChOxbetaineþ 2H2O2

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 chloro-

form–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 water-

saturated 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 con-

tent in food (egg yolk, mil chocolate, soya seed oil), diet

products (Campanella et al., 1998c), as well as in pharmaceu-

tical 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 conductiv-

ity of the non-aqueous solvent. The latter were necessary when

amperometric electrodes were used in organic polar solvents.

M.S

.-P.

Lop

ezet

al./B

iom

olecu

lar

En

gin

eering

23

(20

06

)1

35

–1

47

14

2Table 1

Monoenzyme electrodes

Enzyme Electrode Immobilization system Solvents Determination Reference

PPO Glassy carbon Entrapment in poly(pyrrole-ammonium)

and poly(pyrrole-lactobionamide)

Chloroform Catechol Cosnier et al. (1998)

PPO Oxygen amperometric

GDE

Entrapment in kappa-Carrageenan gel n-Hexane Polyphenols in olive oil Campanella et al. (1999c)

PPO Oxygen amperometric

GDE

Covalent union with glutaraldehyde to

pre-activated membrane

n-Hexane Phenol Capannesi et al. (2000)

PPO Glassy carbon Silica sol–gel Chloroform Catechol, phenol, p-cresol Wang and Dong (2000)

PPO Platinum microdisk array Entrapment in polyethylene oxide Heptane, hexane, chlorobenzene,

toluene, chloroform

Catechol and phenol Zhang et al. (2001)

PPO and

catalase

Oxygen amperometric

GDE

Entrapment in kappa-Carrageenan gel Acetonitrile or dioxane Water content in food fats

(butter, margarine) or ointments

Campanella et al. (2001b)

PPO Glassy carbon Polypirrol and alginate film with

avidin–biotin interactions

Chloroform Catechol Mousty et al. (2001)

PPO Glassy carbon Polypirrol and alginate film with

avidin–biotin interactions

Chlorobenzene, dichloromethane,

chloroform, ethyl acetate and

acetonitrile

Catechol Cosnier et al. (2004)

PPO Platinum Entrapment in polypyrrole matrix or

cross-linked with glutaraldehyde

Chloroform Polyphenolic compounds Stanca and Popescu (2004)

PPO Glassy carbon Titania sol–gel Chloroform Phenol, catechol, p-cresol Yu and Ju (2004)

PPO Glassy carbon Entrapment in hydrophilic polypyrrole film Chloroform Catechol Cristea et al. (2005)

PPO Graphite–Teflon composite Adsorption on graphite–Teflon composite Acetonitrile–tris buffer mixtures Propyl gallate in dehydrated

broth bars and olive oil

Morales et al. (2005a)

PPO Graphite–Teflon composite Adsorption on graphite–Teflon composite Reversed micelles Propyl gallate in lard samples Morales et al. (2005b)

HRP Glassy carbon Entrapment in ferrocene-modified

phenylenediamine film

Acetonitrile–buffer mixture Hydrogen peroxide and other

organic peroxides

Mulchandani and Pan (1999)

HRP Platinum Entrapment in N-methyl-pyrrole Reversed micelles Organic peroxide content in body

lotion samples

Garcia-Moreno et al. (2001)

HRP Graphite Adsorption on graphite Acetonitrile Lauroyl peroxide Ramirez-Garcia et al. (2001)

HRP and SPP Graphite Cross-linked to a redox hydrogel Acetonitrile Hydrogen peroxide Castillo et al. (2003)

SPP Graphite Adsorption on graphite Methanol–phosphate buffer solution Hydroquinone in cosmetic creams Vieira and Fatibello-Filho (2000)

SPP Graphite Adsorption on graphite Methanol–phosphate buffer solution Hydroquinone in cosmetic creams Fatibello-Filho and Vieira (2000)

Catalase Oxygen amperometric

GDE

Entrapment in kappa-Carrageenan gel Dioxane and water–dioxane mixtures Hydrogen peroxide in cosmetic and

pharmaceutical formulations

Campanella et al. (1998b)

Catalase Oxygen amperometric

GDE

Entrapment in kappa-Carrageenan gel n-Decane, toluene or chloroform Hydroperoxide content of olive oil Campanella et al. (2001c)

Catalase Graphite Adsorption on polymer film with nafion Acetonitrile Dibenzoyl peroxide and

3-chloroperoxibenzoic acid

Horozova et al. (2002)

Catalase Platinum Entrapment in polyacrilamide gel Dimethylsulfoxide, dioxane Hydrogen peroxide Varma and Mattiasson (2005)

GOx Screen-printed electrode:

rhodinised-carbon

Adsorption Methanol, ethanol and isopropanol

aqueous buffer mixtures

Glucose Kroger et al. (1998)

SOD Oxygen amperometric

GDE

Entrapment in kappa-Carrageenan gel Dimethylsulfoxide Hydrophobic compounds showing

radical scavenging

Campanella et al. (2001d)

SOD Oxygen amperometric

GDE

Entrapment in kappa-Carrageenan gel Dimethylsulfoxide and glycerine or

dimethylsulfoxide, glycerine and

Tween 20, in ‘‘crown ether’’

Antioxidant capacity of integrator-

phytotherapeutic products

Campanella et al. (2004)

AChE Screen-printed electrode Entrapment in polyvinyl alcohol

styrylpyridinium groups polymer

Acetonitrile and ethanol p-Aminophenol Andreescu et al. (2002)

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147 143

The only disadvantage of a GDE for oxygen, namely inter-

ference 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 analy-

tical techniques require long time. Other oxygen diffusion

electrode based on peroxidase and tyrosinase working in par-

allel 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 hydro-

peroxide 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.

catechol þ 12O2�!

PPOquinoneþ H2O

catechol þ 12H2O�!HRP

quinoneþ 2H2O

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 determi-

nation 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

Table 2

Bienzyme electrodes

Enzyme Electrode Immobilization system Solvents

PLD and

ChOx

Oxygen amperometric

GDE

Entrapment in kappa-

Carrageenan gel

Chloroform–hex

chloroform–hexa

PLD and

ChOx

Oxygen amperometric

GDE

Entrapment in kappa-

Carrageenan gel

Chloroform–hex

chloroform–hexa

PPO and

HRP

Oxygen amperometric

GDE

Entrapment in kappa-

Carrageenan gel

Decane and diox

COx and

HRP

Graphite–Teflon

composite

Adsorption on

graphite–Teflon

composite

Reversed micell

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 (Pena 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 organopho-

sphorus) 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 graphite-

disk 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 diethyl-

dithiocarbamates showed no amperometric response at the

tyrosinase electrode, except for pyrimidine-derivative carba-

mates. The developed analytical methodology was applied to

determine ziram in spiked apple samples (Perez 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

Determination Reference

ane mixture and

ne–methanol mixture

Lecithin in food samples

(egg yolk, soya flour and oil)

Campanella et al.

(1998c)

ane mixture and

ne–methanol mixture

Lecithin in drugs and

diet products

Campanella et al.

(1998d)

ane–water mixture Hydroperoxides of olive oil

and hydrogen peroxide of

cosmetic products

Campanella et al.

(2003)

es Cholesterol in butter, lard

and egg yoke

Pena et al. (2001)

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147144

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 benzotria-

zinic (azinphos-ethyl and azinphos-methyl) pesticides deter-

mination. Furthermore, the authors report recovery trials

performed in vegetal matrixes (corn, barley, lentils) (Campa-

nella 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 screen-

printed electrodes used to assess the inhibitory effect of

organophosphorus and carbamic pesticides on acetylcholines-

terase 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 butyrylcholi-

nesterase (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 kappa-

Carrageenan 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:

butyrylcholine�!BChEcholineþ butyric acid

cholineþ 2O2 þ H2O�!ChOxbetaine þ 2H2O2

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 acetylcholines-

terase biosensor based on thiocholine-hexacyanoferrate for the

analysis of organophosphate pesticides in pure organic

solvents. The strategy was based on the following signal-

amplification 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 hydro-

lysis 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 acetylcholi-

nesterase 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 (acetylthiocho-

line). The influence of miscible organic solvents was studied by

Fig. 8. Structure of aldicarb (a) and paraoxon (b).

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147 145

ctro

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nM

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sin

os

etal

.(2

00

1)

edel

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od

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ent

inp

oly

vin

yl

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ho

l

sty

rylp

yri

din

ium

gro

ups

po

lym

er

Ace

tonit

rile

Par

aox

on

and

chlo

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set

hyl

ox

on

An

dre

escu

etal

.(2

00

2)

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 reprodu-

cibility 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 p-

aminophenyl 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.

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 construc-

tion 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 electro-

chemically 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

Tab

le3

Enzy

me

inhib

itio

nbas

edel

e

Enzy

me

Ele

ctro

de

PP

OG

raph

ite

PP

OG

raph

ite–

Te

PP

OO

xy

gen

amp

PP

OO

xy

gen

amp

PP

OP

lati

nu

m

ChO

xan

d

AC

hE

Scr

een-p

rint

BC

hE

and

ChO

x

Ox

yg

enam

p

AC

hE

Gra

ph

ite

AC

hE

Scr

een

-pri

nt

AC

hE

Scr

een

-pri

nt

Fig. 9. Structure of chlorpyrifos-ethyl-oxon.

M.S.-P. Lopez et al. / Biomolecular Engineering 23 (2006) 135–147146

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).

3. Enzyme reactors

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

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 permis-

sion from Springer-Verlaq’’.

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).

Acknowledgement

The authors acknowledge financial support from DGI

(MAT2003-03051-C03-03) of the Spanish Science and

Technology Ministry.

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