Analytica Chimica Acta, 249 (1991) l-15

Elsevier Science Publishers B.V., Amsterdam

Review

Organic phase enzyme electrodes

Selwayan Saini

Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedford MK43 OAL (UK)

Geoffrey F. Hall

Cranfield Biotechnology Limited, Cranfield, Bedford MK43 OAL (UK)

Mark E.A. Downs

Sensors Technology Group, Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex TWII OLY (UK)

Anthony P.F. Turner *

Biotechnology Centre, Cranfield Insiitute of Technology, Cranfield, Bedford MK43 OAL (UK)

(Received 27th December 1990)

Abstract

Organic phase enzyme electrodes (OPEEs) offer new possibilities both for the design of biosensors for use in

organic solvents and in the construction of devices incorporating an organic phase for use with aqueous samples. The

advantages that accrue from performing biocatalytic and electrochemical reactions in non-aqueous systems are

discussed in relation to possible applications in medicine, pharmaceuticals, petrochemicals, the food industry,

environmental monitoring and defence. This critical but speculative review considers the detailed implications of

operating enzyme electrodes in organic solvents and briefly examines the broader issues of optical, calorimetric and

piezoelectric transduction coupled with both catalytic and affinity systems, such as nucleic acid probes and antibodies.

Keywords: Enzyme electrodes; Biosensors; Organic phase; Review

Biosensor technology has emerged as a dy- namic field of biotechnology with new methods of detecting specific chemicals at analytically useful

levels [1,2]. Its impact has been largely due to the advances made in bioelectrochemistry, microelec- tronics and micro-optic technology. Biosensor re-

search attracts scientists from far-ranging fields such as pharmacology, biochemistry, protein chemistry, electronics and physics [3]. This coher- ent multidisciplinary approach is vital for the suc- cessful introduction of biosensors in new and ex-

isting fields that once required skilled techniques,

0003-2670/91/$03.50 0 1991 - Elsevier Science Publishers B.V.

complex instrumentation and high expenditure costs and were often time consuming.

Over the last few years market researchers have

begun to evaluate potential biosensor markets. One of the most recent [4] predicts a global market of over US$l billion annually at the turn of the century. Biosensor market penetration is expected

to grow rapidly during the next 5 years so that global market sales will increase from $46 million

in 1988 to $760 million in 1995 and $1160 million per annum by 2000. If biosensors are to meet these projected targets then many of the factors

L S. SAINI ET AL.

impeding their widespread distribution at present will have to be confronted. Current problems in-

clude a frequent calibration requirement, short operating lives owing to instability of the biologi- cal component,‘narrow analyte range and interfac- ing the sensor with its environment. Placing bio- logical molecules in close association with micro-

electronics can also lead to problems with efficient mass production.

To date, the medical sector has been the major beneficiary of this technology, utilizing products such as blood glucose and blood gas analysers. Other potential applications for biosensor technol- ogy may also exist in the food and drink industry, the chemical and pharmaceutical industries, agri-

culture and in environmental and military applica- tions. This diverse range of industries requires devices that are precise, rapid, affordable and in most instances durable. These demands dominate a large portion of current research and develop- ment worldwide. One important and sometimes neglected aspect of biosensor technology is that of analyte range. If biosensors could detect a wider range of analytes, then their acceptance in many situations might be facilitated. At present, how- ever, biosensing generally only permits the mea- surement of analytes that are capable of dissolving in the aqueous phase and thereafter interacting

with the biological element. As water has conven- tionally served as the solvent base for biological interactions (because of the belief that other solvents would denature the biocatalyst), only those chemicals which are soluble in water have been amenable to bioanalysis. Contradictory to

this belief, it has been shown that biocatalysts can function in extreme environments such as super-

critical fluids [5] and organic solvents [6]. The latter has important implications for the imple- mentation of biosensor technology in formerly inaccessible environments and more importantly extends the number of detectable analytes to in- clude poorly water soluble organic species.

An enzyme electrode combines the specificity and affinity of an enzyme for its substrate with the analytical power of electrochemical devices [1,7]. In the first enzyme electrode configuration

[8], the enzyme operated in a totally aqueous environment. The effect of non-aqueous solvents

on enzyme action has been investigated since at least 1913 [9]. In 1967, Dastoli and Price [lo] demonstrated that lyophilized xanthine oxidase

and crystalline cy-chymotrypsin suspended in pre- dominantly organic media could still retain their activity, yet it has taken almost a quarter of a century for researchers to begin to use the analyti- cal utility of enzyme-based biosensors in organic environments to advantage [ll]. The increased

solubility of organic analytes in organic solvents and their subsequent detection suggest that far more industrial areas can benefit from biosensor technology. Obvious candidates for this technol- ogy include the petrochemical and chemical in- dustries in addition to those outlined above. There may also be further applications in certain clinical

analyses. This paper attempts to present the principal

features of organic phase enzyme electrodes

(OPEEs). The advantages and problems are high- lighted and potential OPEE applications dis- cussed. The present status of these instruments is also reviewed and future directions are considered.

ENZYMOLOGY IN THE ORGANIC PHASE

The knowledge that enzymes can function vigorously in some organic solvents has led to an expansion in this area of biochemistry in recent years. Several good reviews have appeared re- cently [12-U].

From a bioanalytical viewpoint, it is only nec-

essary to discuss those factors which influence the activity of an enzyme in an organic solvent and

how such parameters can be engineered so that optimum performance is achieved. It is generally accepted that enzymes retain catalytic activity in certain organic solvents as a consequence of a hydrated shell engulfing the enzyme particle. Fur- ther, it has been suggested that the amount of water required for catalytic activity is enzyme dependent [15]. Fundamental to the catalytic ac- tivity of the enzyme is the selection of a compati- ble organic phase, i.e., one that does not interact strongly with the essential hydration [16]. It has

been found that sufficiently hydrophobic, water- immiscible organic solvents are usually the best

ORGANIC PHASE ENZYME ELECTRODES. REVIEW

media for enzymatic catalysis because of their decreased ability to interact with the water closely associated with the enzyme surface [17]. When

water-miscible solvents are used, the enzyme is inactivated in most instances owing to the distor- tion of the essential water layer by the solvent. Problems with water-distorting solvents can be

overcome for some enzymes by adding small amounts of water so to satisfy a solvent’s “thirst” so that it is less likely to distort the essential hydration layer in the enzyme microenvironment.

According to the above argument, a solvent that is sufficiently hydrophobic (non-water dis- torting) should allow the enzyme to function. However, how does one go about assessing which of the 107 commonly used organic solvents are hydrophobic enough to sustain enzyme activity? The hydrophobicity of an organic solvent can be expressed as the logarithm of the partition coeffi-

cient (log P) of the test organic medium in a standard octanol-water two-phase system. It has been suggested [lg] that solvents with log P < 2 are not suitable as they strongly distort the bio- catalyst-water interaction. Solvents with log P values between 2 and 4 are relatively weak dis- torters, affecting the activity in an unpredictable way. Those solvents with log P > 4 are normally

biocompatible. This trend, however, has been established with relatively few systems. Moreover, the model fails to account for the surprisingly high activity for certain enzymes in some organic solvents, e.g., subtilisin in dimethylformamide (log P = - 1.0) or porcine pancreatic lipase in pyridine (log P = 0.71). A possible explanation for this observation is that these enzymes retain their

hydration shell so strongly that even hydrophilic solvents cannot strip it [15] or that the partitioning of the substrate to the enzyme’s active site is sufficiently favoured to outweigh the other detri- mental effects. Despite these anomalies, the model has at present been generally accepted as the best current guide in predicting biocatalytic activity in a given organic solvent.

Other factors that should also be considered in the optimization procedure are pH and activation

by certain ligands. An enzyme functioning in the organic phase “remembers” the pH of the last aqueous solution to which it has been exposed

3

[15]. The ionization states of the charged groups of a catalytic protein are affected by the pH of the solution. In the organic phase there is no driving force to alter this charge, so the enzyme remains

unchanged in the organic environment. Therefore, the charged groups of the enzyme retain their existing ionizations from the last solution to which

they have been exposed [15]. Second, it has been reported that lyophilization of chymotrypsin from aqueous solutions containing certain ligands (e.g., N-acetyl+phenylalanine) has significant effects

on the activity of the enzyme in the organic phase. In this case, chymotrypsin had a 35-fold increased activity when lyophilized in the presence of the above ligand [19]. Similar ligand effects have also been documented for subtilisin 1201 and again more recently for chymotrypsin [21]. This en- hanced activity is thought to be a result of the ligand “locking” the enzyme into a more active

conformation. If this ligand activation is a general effect then it may be useful in the preparation of an enzyme for analysis. An alteration for an en- zyme’s specificity to favour a desired analyte may

be possible if the enzyme is lyophilized in the presence of the intended analyte.

ENZYME-BASED BIOSENSORS IN ORGANIC PHASES

The application and advantages of bioanalyti- cal devices based on enzymes functioning in organic phases have only recently been realized [11,22,23,]. Before this exploration, only a few conventional enzyme-based sensors in which the

enzyme was maintained in an aqueous compart- ment were able to operate in non-aqueous en- vironments. These biosensors relied on the parti- tioning of an organic analyte from a non-aqueous phase across a permeable selective membrane which retained the enzyme in an aqueous phase.

One example of this approach is the alcohol sensor devised by Phillips Petroleum [24]. The enzyme alcohol oxidase was immobilized onto the tip of an oxygen electrode and kept suitably hydrated by an outer semi-permeable membrane. Alcohol in the non-aqueous media could penetrate the outer

S. SAINI ET AL.

membrane and enter the hydrated enzyme layer where it was biocatalytically oxidized. The concur- rent fall in the oxygen concentration was mea- sured amperometrically at the sensing electrode. This sensor was potentially useful in the de- termination of the alcohol content in gasoline. In other cases, the biosensor is incapable of operating in an organic phase but instead relies on a poorly water-soluble analyte in an aqueous sample to pass through a selective barrier and into an inner hydrated enzyme-containing chamber. In some in- stances, the insoluble analyte’s solubility in water is increased by the use of detergents. However, if the organic analyte is very poorly soluble in water or the use of detergents is undesirable, then the sensor will lack sensitivity. If, however, the en- zyme was able to function in a predominantly organic phase, then poorly water soluble analytes would be present at high enough concentrations for bioanalysis. Apart from extending the analyte range, organic phase biosensors could offer other advantages as outlined in Table 1.

Flygare and Danielsson [25] took advantage of some of the points outlined in Table 1 by operat- ing an enzyme thermistor in various non-aqueous media. They correctly predicted an increase in

TABLE 1

Advantages of organic phase biosensors

sensitivity due to the lower heat capacities of organic solvents compared with water. Further, the enzymes used remained active over several weeks. Organic solvents were also used for en- zymic assays involving photoacoustic spectrome- try. Again, an increase in sensitivity (compared with operation in water) was obtained by oper- ation in organic solvents.

ORGANIC PHASE ENZYME ELECTRODES

Organic phase enzyme electrodes (OPEEs) can be used for the specific detection of poorly water- soluble, hydrophobic analytes that become acces- sible to an enzyme’s microenvironment via an organic phase. The enzyme, in turn, interacts di- rectly or indirectly with an electrode surface. The organic phase can refer to a mobile liquid phase or a gel. The enzyme microenvironment should con- tain a sufficient level of hydration such that en- zyme activity will be near optimum. The extent of hydration will depend on the enzyme, its support and the organic environment.

The first report of a biosensor in which the biocatalyst was in direct contact with the organic phase appeared in 1988, when Hall et al. [22,23]

Feature Advantage

Biocatalysis in non-aqueous media

Change in substrate specificity for some enzymes leading

to new biocatalytic reactions

Increased thermal stability for some enzymes

Decreased microbial contamination

Resistance to oxidation and reduction in organic solvents

(see later)

Low solubility of aqueous electroactive species

Ability of enzymes to remain active in essentially

dehydrated states

Altered solubility of sensor reagents

Lower heat capacities of organic solvents compared with water

Extended analyte range of biosensors

Novel detectors

Increased operational stability of a sensor

Improved operational lifetime of a sensor

Wider potential window for electron transfer reactions

Decreased interference of hydrophilic ionic species

Absence of water may allow easier fabrication of sensors

New options for the design of stable devices

Increased sensitivity of enzyme thermistors

ORGANIC PHASE ENZYME ELECTRODES. REVIEW. 5

described an amperometric enzyme electrode for the determination of phenols in chloroform. In this solvent, polyphenol oxidase catalyses the con- version of p-cresol to 4-methyl-l,Zbenzoquinone, which can then be electrochemically reduced at a carbon working electrode [at - 275 mV versus a

saturated calomel electrode (SCE)]. Using this method of detection, a linear response for p-cresol

concentrations up to 0.1 M resulted. The enzyme was retained in a sufficiently hydrated environ- ment by adsorption onto a nylon membrane which

was held in close contact with the carbon elec- trode. Because of the highly resistive nature of

organic solvents, a supporting electrolyte, tetra- butylammoniumtoluene-4-sulphonate was used in all work. The enzyme electrode was responsive to a range of phenols, indicating its potential use as a phenolic contamination sensor. It is particularly applicable to the detection of phenols in waste water following an organic extraction procedure.

Conner et al. [26] recently reported a simple

method of immobilizing an enzyme into the pores of a graphite disc electrode. By mixing tyrosinase with silicone grease and using the resulting paste to “fill” the micropores of a graphite electrode, a self-supporting enzymatic layer in close associa- tion with the electrode was obtained. Phenolic compounds that entered the enzyme layer from

aqueous solution were converted to their quinone products, which were subsequently reduced at the graphite working electrode.

This amperometric enzyme electrode displayed a detection limit for dopamine of 6 x lop6 M.

Rapid response times (95% steady-state current in 5 s) made this sensor suitable for use in flow-injec-

tion analysis (120 samples-per hour with a relative standard deviation of 2.4%). This work demon- strated the ability of enzymes to function in sig- nificantly hydrophobic gel-like environments.

Miyabayashi et al. [27] described a potentio- metric OPEE that monitored chymotrypsin-cata- lysed ester synthesis in organic media. The enzyme

was adsorbed onto magnetically precipitated iron oxide particles that were immobilized in an elec- tromagnetic field at a platinum anode immersed in a solvent within a flow cell. On addition of

substrate, the potential difference that arose from the enzyme reaction on the surface of the mag-

netic beads was monitored potentiometrically. It was found that the response was strongly depen- dent on the solvent hydration, which was also confirmed by product formation analysis using liquid chromatography. Under conditions of opti- mum water content, however, the response was stronger in a less hydrophobic solvent, diisopropyl ether, when compared with operation in toluene. The electrode system was relatively simple to con-

struct and by using magnetic particles, the enzyme could easily be exchanged. The authors high- lighted the potential use of this sensor in the monitoring of bioorganic processes.

In 1990, Hall and Turner [28] described an enzyme electrode for determining cholesterol con- centrations in a solvent mixture of chloroform and hexane. The enzyme cholesterol oxidase was ad- sorbed onto alumina particles that were located above the sensing element of an oxygen electrode.

This sensor displayed linearity from 1 to 10 mM cholesterol. Further work [29] demonstrated that

the limit of detection was 0.4 mM. It was observed that the enzyme associated hydration was the most critical parameter affecting the sensor response. An optimum response at 5% (v/v) water in chlo- roform-hexane (1: 1, v/v) was found. It is likely that the immobilization substrate, alumina, also bound a significant amount of water. A major advantage with this sensor was that all the electro- chemistry took place on the aqueous side of a gas-permeable membrane, allowing enzyme analy- sis in non-polar solvents that do not normally permit electrochemistry at conventional electrodes because of the insolubility of supporting electro- lytes. Hence, the sensor may provide a simple route to exploring any organic solvent-compatible oxidase on any supporting material in a range of organic media. Although the primary objective of this work was to investigate the feasibility of en- zymes in organic solvents with a view to their

incorporation into direct electrochemical sensors, the device was shown to be useful in “one-shot” cholesterol assays of butter and margarine, al- though problems with substrate interference did

arise owing to cholesterol oxidase’s wide substrate range of related sterols.

Recently, Schubert et al. [30] demonstrated that it is possible to use mediators in the organic phase

6 S. SAINI ET AL.

100 -

200 400

Hydrogen peroxide,pM

Fig. 1. Calibration graph for horseradish peroxidase-mediator

electrode in chloroform (LC grade) with 0.1 M conducting

electrolyte and 10% (v/v) buffer [30].

by inverting the conventional concept of im- mobilization of water insoluble mediators by ad- sorption on electrodes. Utilizing a water soluble mediator in a predominantly organic phase, there

was little tendency for the mediator (or enzyme) to detach from the electrode under conditions of low water content. This method allowed the de- termination of peroxide levels in organic solvents by co-adsorption of potassium hexacyanofer- rate(I1) and horseradish peroxidase on a carbon electrode. The electron mediator, potassium hexa- cyanoferrate(II), was enzymatically converted to potassium hexacyanoferrate(II1) in the presence of hydrogen peroxide. The adsorbed mediator was then reduced at the electrode at - 20 mV vs. SCE. In chloroform, the enzyme-mediator electrode gave a stronger signal than operation in dioxane.

A linear response for hydrogen peroxide con- centrations up to 0.6 mM was observed (Fig. 1). The increase in activity in chloroform may have been due to the solvent’s inability to distort the essential water layer on the enzyme surface according to Laane et al. [18]. However, in hydro- phobic solvents such as octanol (log P = 2.9) little or no activity was recorded. Only chloro- benzene (log P = 2.8) facilitated activity at a higher rate than in chloroform (log P = 2.0). In

other work [16], horseradish peroxidase has been observed to function in toluene (log P = 2.0). Schubert et al. [30] however, were unable to obtain activity in toluene with their system although they

did note that toluene is a poor solvent for dissolv-

ing the supporting electrolyte. This work demonstrated the feasibility of using

mediators with enzymes in organic phases. One notable potential application of this device is in the measurement of organic hydroperoxides, which can indicate rancidity levels in edible oils. The development of elegant and effective bioelectro- chemical systems, however, will rely on an in- creased understanding of the underlying biochem- istry and electrochemical principles. These are dis-

cussed further in the following sections.

ELECTROCHEMISTRY IN THE ORGANIC PHASE

Before 1960, little work had been carried out in organic solvents, mainly because there was no

particular advantage in replacing water as the solvent for most electrochemical applications. During the 1960s and 1970s electroanalytical chemistry using conventional electrodes expanded into non-aqueous solvents such as dimethylform- amide, acetonitrile, propylene carbonate and di- methyl sulphoxide. It was soon realized that the analytical possibilities of non-aqueous electron transfer systems held considerable promise. The characteristics of these solvents included low di-

electric constants, the ability to solubilize organic analytes and certain inorganic compounds and usually good storage characteristics. A prominent feature was the resistance to oxidation and reduc- tion, which allows a wide “potential window” (scan) to be employed. Water as the electrochem- ical medium does not have this broad potential

range and therefore has a restricted potential window in which electron transfer of analytes is possible. Another important property is the ability to dissolve hydrophobic non-polar analytes or samples that react with water through hydrolysis or redox processes.

Electrochemistry in organic solvents at present is routinely carried out in extremely polar solvents

such as acetonitrile and dimethylformamide [31]. Tetraalkylammonium salts are excellent support- ing electrolytes in such solvents and there are no excessive solution resistance problems to over- come. However, as was discussed earlier, enzymes have been found to be generally more stable in

ORGANIC PHASE ENZYME ELECTRODES. REVIEW. 7

TABLE 2

Novel features of electrochemistry at microelectrodes

Feature

Sigmoidal voltammograms

Applications

Diffusion coefficient and n determination a [33]

Insensitive to solution movement

Ultra-fast voltammetry

Detectors in flow streams

1341

Cyclic voltammetry at lo6 V s-i; study of intermediates [35,36]

Studies in resistive media Non-polar solvents [37] Gases [38] Supercritical fluids [39]

a n = Number of electrons transferred per molecule.

less polar solvents in which electrochemistry at conventional electrodes is impossible owing to the lack of suitable electrolytes and hence high solu- tion resistances. The flow of current through the

solution creates a potential difference between the electrodes that opposes the applied potential. This potential drop is the product of the current and solution resistance (iR) and results in meaningless

distorted voltammograms. One solution is the use of microelectrodes. The

theoretical advantages of electrodes with micro- metre dimensions have been realized for some time. However, it is only recently that suitable fabrication materials have become readily availa- ble and measurement of the small currents in-

volved has become routine. There are several sig- nificant changes that occur when the critical di-

mension of an electrode is reduced to less then 50 pm. These have been reviewed extensively [32,33] and are outlined in Table 2. The theory of voltam- metry at microelectrodes is well developed [40]. Discussion here will be restricted to their applica- tion to electrochemical studies in resistive solu-

tions. The distribution of current through the solution

at the surface of the electrode, rather than the

small current values, results in much diminished iR potential losses at the surface of microelec- trodes compared with conventional electrodes [41].

This means that voltammetry can be carried out in a range of media that were not amenable to use with conventional electrodes. These range from

acetonitrile with no added electrolyte [37] to gases [38], toluene [42] and benzene [43]. The radial diffusion observed at microelectrodes results in better mass transfer to the electrode surface than that observed when planar diffusion predom- inates. This means that the phenomenon of “cata-

lytic wave” voltammograms at macroelectrodes is impossible to observe at microelectrodes. Any catalytic effect is hidden by rapid diffusion from

the bulk solution to the electrode surface. There are also a few problems that are not

generally significant when carrying out electro- chemistry in conventional solutions. Firstly, a reli- able reference potential needs to be established. One method is to use aqueous reference electrodes dipped into the solvent being used. Whereas this might be adequate for amperometry, it does not provide a defined potential owing to unknown liquid junction potentials and is therefore not suit- able for accurate electrochemical analysis. Suit- able electrodes have been developed for use in polar organic solvents [44]. One approach is to use a quasi-reference such as silver wire and refer all

potentials to a standard test couple [45]. Another factor that comes into play when the concentra- tion of electroactive species is greater then that of the inert electrolyte is the effect of migration on the mass transport of one of the species in the

redox reaction. Finally, the solubility of some charged species in organic solvents can be so low that film deposition on the electrode surface may occur [45]. The use of microelectrodes may also provide useful information on the electrochem- istry of putative mediator molecules that are re- quired for use in organic phases.

IMMOBILIZATION OF THE ENZYME

At present, direct adsorption of enzymes onto inert supporting materials or directly onto elec- trodes is favoured. This technique is simple and rapid and higher enzyme loadings are usually achieved compared with covalent immobilization

techniques. However, adsorption suffers when the water content of the organic solvent is high.

Chemical immobilization is used extensively in enzymological studies and should be compatible for use with OPEEs, although care must be taken

8 S. SAINI ET AL.

as some solvents will attack certain immobilizing chemicals. Different types of gel materials, such as

polysaccharides, proteins and synthetic polymers, are commonly, used to entrap biocatalysts [46]. The physico-chemical properties of synthetic gels such as hydrophobicity-hydrophilicity balance and ionic nature can be modified by selecting suitable prepolymers. These and other properties have been discussed by Fukui and Tanaka [46]. Synthetic polymers that will allow enzyme activity

in the presence of water-saturated organic two- phase systems and in pure solvents such as ben- zene and heptane may be used for entrapping

enzymes onto electrodes. Recently, redox gels such as cross-linkable

polyvinylpyridine have found increased applicabil-

ity in bioelectrochemistry, allowing oxidoreduc- tases such as glucose oxidase [47] to pass its elec- trons through the polymer and to the electrode efficiently. Conducting polymers such as polypyr- role [48,49] which are stable in non-aqueous media

may also prove useful. In aqueous systems, immobilization may alter

the thermal and pH properties of an enzyme so that in some instances enhanced resistance to ex- tremes of pH and temperature results. Equally, in the organic phase, similar properties of enzymes may be manipulated to advantage. Another sup- port characteristic, polarity, will affect the parti- tioning of analytes and products (and mediators where applicable). The possible role of the support

on enzyme activity needs to be explored further.

ROLE OF MEDIATORS

In most instances the electron transfer region of an enzyme is buried deep within the enzyme structure. This means that direct electron transfer with an electrode is difficult. However, this prob- lem may be alleviated by introducing a small compound to act as an electrochemical shuttle, transferring electrons from the enzyme to the elec-

trode surface [50]. The use of these low-molecu- lar-weight mediators eliminates the dependence on

natural electron acceptors such as oxygen. With oxidases, the mediator will replace oxygen as the electron acceptor and allow reoxidation of the

enzyme. This may be important in analysis where the oxygen tension varies. Another useful feature of the mediator compound is that the potential at which electrochemical detection is carried out is determined by the redox potential of the mediator, which can be selected to avoid interference from other electroactive species in the sample. The

properties of mediators have been discussed previ- ously [50,51]. As mentioned above, Schubert et al. [30] have demonstrated that solvent-insoluble mediators and enzymes can exchange electrons in organic environments. In some sensors, however, it is desirable to utilize a soluble mediator. One example of this approach is the capillary fill de-

vice developed at Unilever [52]. All the necessary components (enzyme, mediator, buffer salts) are contained within a small reaction chamber in the working cell. The sample liquid is drawn into the

device by capillary action and dissolves the test reagents. The subsequent enzyme and mediator reaction can then be monitored coulometrically. Adapting such a system for use in organic analysis will be challenging, especially in view of the in- solubility of enzymes in organic phases. However, with the application of solvent soluble polyethyl- ene glycol-modified enzymes [53] and the diverse range of solvent soluble mediators, one could en- visage such devices where solvent is introduced

into the reaction chamber containing the test re- agents.

An interesting and flexible technique of study- ing mediators of different solubility and their cou-

pling with enzymes has been described by Hall [54]. In this work, a bienzymic reaction was car- ried out in a microemulsion system formed by

Triton X-100 in butyl acetate. The enzymes used were alcohol dehydrogenase, which reduces NAD on reaction with primary alcohols and diaphorase, which reoxidizes reduced nicotinamide adenine di- nucleotide (NADH) and reduces the dye 2,6-di- chlorophenolindophenol (DCPIP). The accumula- tion of DCPIP can be followed electrochemically by its reoxidation at a glassy carbon electrode

(Fig. 2). DCPIP is soluble in both aqueous and organic phases, but this is not a prerequisite for the operation of this system. A similar reaction

was demonstrated using dimethylindoaniline (DMIA), which is only soluble in the organic

ORGANIC PHASE ENZYME ELECTRODES. REVIEW 9

160 1 I

140

pzo

> 100 c $ 60.

’ 60. ::

i? 40.

-50 0 50 100 150 200 250 300 350 400 450

Electrode potential vs. Ag/AgCI (mV)

Fig. 2. Response vs. electrode potential for the microemulsion

assay using 2,6-dichlorophenolindophenol (DCPIP) as the

mediator. The system contained alcohol dehydogenase (ADH,

5 ng), nicotinamide adenine dinucleotide (NAD+, 5 pg) and

diaphorase (100 pg) in buffer (125 ~1, pH 8.5) mixed with

butyl acetate (870 1.11, containing 0.4 M Triton X-100 and 64 ~1

DCPIP). The reactions were initiated by the addition of ethanol

[5 ~1, 10% (v/v) in butyl acetate, LC grade].

phase, and hexacyanoferrate(II1) (soluble in the aqueous phase) in place of DCPIP in the reaction oulined above. This work may find application in

the development of enzyme-mediator systems for use in aqueous and organic environments.

A large number of enzymes utilize cofactors such as nicotinamide adenine dinucleotide (NAD). Some enzymes which interact with this cofactor,

such as those mentioned above (alcohol dehydro- genase and diaphorase), have been shown to work in organic solvents of low water content [55]. In

the aqueous phase, direct electrochemical detec- tion of NADH is limited owing to electrode foul- ing, high detection potentials (NADH oxidation at 1.1 V vs. SCE at platinum electrodes) and slow

reaction kinetics. Modified electrodes incorporat- ing highly conducting organic metal complexes are one alternate route to achieving NADH oxidation with the added advantage of lower detection potentials [NADH oxidation at -0.2 V vs. Ag/AgCl at N-methylphenazinium 7,7,8,8,-tetra- cyano-p-quinodimethane (NMP.TCNQ) elec-

trodes]. The problem of slow reaction kinetics can be overcome by using the enzyme diaphorase to reoxidize NADH and coupling the reaction with a mediator that provides the detection signal. De-

spite these potential alternatives, a practical sensor utilizing a dehydrogenase enzyme requires ad-

dition of cofactor to the solution, and this seri- ously hinders its use as a direct or an on-line detector [50]. Retention at the electrode is possible by using selective membranes or chemical im- mobilization techniques. However, by replacing the aqueous phase with an organic phase, some of the problems outlined above may be overcome. For example, the enzyme(s) and cofactor will re- main at the electrode surface and detection may

be possible by using a water soluble mediator together with diaphorase. Apart from these fea-

tures, a number of important poorly water-soluble analytes will readily dissolve in the organic phase.

The choice of mediator linked to an enzyme or a cofactor system will be dependent on the intrin- sic properties of the redox enzyme and the accessi- bility of the mediator to the enzyme microenviron- ment. The above work suggests that enzyme- mediator detection systems are possible in organic environments. Once a better understanding of the

partitioning of mediators between the various phases has been attained, then it should be possi- ble to choose appropriate mediators for various possible sensor designs. This will mirror current

developments in aqueous mediated systems and greatly expand the range of possible options.

OPTIMIZATION OF OPEE FUNCTION

Effect of water As described earlier, the enzyme requires a

critical amount of water to ensure that catalytic activity is optimum. The extent of this hydration

will be dependent on the enzyme, the organic solvent and the affinity of the enzyme support for water. If the enzyme is adsorbed on the support or entrapped in a water-soluble polymer at the elec- trode, then high levels of water in the solvent can

result in desorption of the enzyme, as was ob- served in previous reports [29,30].

The electrochemical effect of solvent hydration remains to be studied in detail. It is clear from

preliminary experiments that increasing the level of hydration results in significant increases in background currents. It will be necessary to

elucidate the possible effects of this residual water, particularly with respect to amelioration of solu-

10 S. SAINI ET AL.

tion resistance and a restriction of the potential window. It should also be remembered that many real samples will not be rigorously anhydrous organics but instead are likely to contain signifi- cant amounts of water, although organic extrac- tion followed by solvent drying would circumvent this problem or hydrophobic synthetic polymers can be used at electrodes to exclude water con-

tamination. Preliminary experiments suggest that some en-

zymes display increased stability in low-water en- vironments [19,56]. In some situations, therefore, it may be necessary to reduce the amount of water significantly so that the enzyme exists in a near anhydrous state. By doing this, the thermal stabil-

ity of the enzyme may be elevated [19,56]. Such an approach may be useful when a sensor is required to operate at high temperatures, e.g., in the on-line monitoring of analytes in many industrial pro- cesses.

Sensor characteristics

Solvent and immobilization support properties such as polarity and matrix permeability may play important roles in determining the sensitivity and linearity of enzyme electrodes in organic solvents. It has been shown that the apparent K, values for phenols in non-aqueous media of horseradish peroxidase were higher than those in aqueous solutions [57]. The log P value of a solvent will also affect the partitioning of hydrophobic ana-

lytes from bulk solvent to the enzyme’s active site. By manipulating the polarity of the enzyme’s mi- croenvironment, the apparent K, value of an

enzyme may be altered. This in turn will affect the sensitivity and linearity of the sensor. Another parameter that should be taken into consideration is that of solvent viscosity. Some hydrophobic, long-chain hydrocarbons are viscous so that re- sponse times in these solvents may be relatively long.

AFFINITY SENSORS IN THE ORGANIC PHASE

The role of medium engineering in the design

of an enzyme electrode has been explored, but what are the possibilities for extending this phi-

losophy to other sensor systems based on, for example, affinity reactions? If the use of an organic environment can be employed to modify and im- prove an enzyme-based system, then the same may be true for systems based on antibody-antigen binding. In many ways an antibody, as a protein, will be affected by an organic environment in a similar manner to an enzyme. A crucial difference, however, is the nature of the binding reaction

itself. A substrate binds to the active site of an en-

zyme to form an enzyme-substrate complex that is unstable and transitory in nature and, prior to the formation of product, fully reversible. In the normal course of events, until an appropriate equi-

librium is reached, product will continue to be formed and released from the active site. This, of course, is the basis of the enzyme’s catalytic power. The formation of product is irreversible with en-

zymes operating down to only lop6 M concentra- tions of substrate. This situation is significantly different to that of antigen-antibody (AG-AB) binding.

The formation of the immuno-AG-AB com- plex is “permanent” and essentially irreversible (unless extreme conditions are applied), occurring with a much higher affinity than that of the en- zymatic systems (lO-‘“-lO-‘i M). To a certain

extent, the lack of product formation in an im- muno reaction simplifies its possible mode of op- eration in an organic environment by eliminating

the need for both substrate and product to be capable of rapid diffusion at the reaction site in a

soluble form. This, coupled with the knowledge that proteins can remain functional in organic

environments, suggests that there are possibilities for organic-based immunosensors. To the authors’ knowledge, no examples of immunosensors oper- ating in organic environments have so far been reported, although a small number of academic and industrial groups are interested in operating immuno systems within these more “hostile” en-

vironments. A paper describing the operation of AG-AB affinity reactions in organic solvents has

recently confirmed its potential. Russell et al. [58] raised a monoclonal antibody to 4-aminobiphenyl and investigated its binding affinity in a range of organic solvents including acetonitrile and di-

ORGANIC PHASE ENZYME ELECTRODES. REVIEW. 11

oxane. They reported that the affinity reaction proceeded well and that the binding reaction was highly specific. Unlike organic phase enzymology, however, it appears that on this limited evidence a hydrophilic environment is preferential. A clear indication of this tendency is given by the change in the dissociation constant of the complex be-

tween solvents. A hydrophilic solvent such as di- oxane (log P = - 1.1) had a dissociation constant

of 16 k 2 PM, wheras a more hydrophobic solvent such as pentan-l-01 (log P = + 1.3) had a dissoci- ation constant of only 790 * 360 PM.

This work clearly demonstrates that immuno- technology can be extended into non-aqueous en- vironments and suggests that immunosensors for organic environments are not unrealistic. The clear contrast, however, between the requirement for a hydrophilic as opposed to a hydrophobic environ- ment suggests that our understanding of the un- derlying chemistry of organic phase biology needs

considerable expansion. However, as with enzyme systems, the operation of immuno reactions in an organic environment could have a number of ad- vantages: increased solubility in one organic en- vironment may allow pre concentration and thus extend the already good detection limits; applica- tions of organic solvents will (depending on the

polarity, etc.) distort the quaternary structure of the AG-AB complex, which could be used to advantage to address the problem of an immuno-

sensor’s irreversibility by creating a delicate bal- ance between binding destabilization and de- naturation; and greater stability and operational temperature range could result. These possibilities have yet to be tested, however, and the applica- tions that might be envisaged remain speculative.

Other affinity-based sensors, such as those em- ploying receptors or nucleic acids, are still very much in their infancy for application in aqueous, let alone organic, environments! Possibilities do exist, however, for the modification of these types of sensor by the employment of organic solvents.

Sensing systems employing nucleic acids have only recently started to be considered [59]. Organic solvents, however, are no stranger to the molecular geneticist, where they are employed to help pre- cipitate and clean DNA (e.g., ethanol, phenol, chloroform, diethyl ether). Formamide (40%, w/v)

is widely used in hybridization systems, employing

enzyme labels, to allow the hybridization of the probe to occur at temperatures around 40°C. This avoids denaturation of the label [60,61]. Similarly, two-phase phenol systems have been employed to increase hybridization rates [62] and, therefore, to decrease assay times. These applications are well

established, as is the knowledge that hydrophobic non-polar solvents will generally interfere with base stacking in the double helix and destabilize

it. It can be seen, therefore, that organic solvents play a useful role in nucleic acid assay systems, albeit in a somewhat different guise to the enzyme and antibody applications. Novel applications of organic solvents in nucleic acid “biosensor” for-

mats based on hybridization are, however, more difficult to envisage. In contrast, if nucleic acid sensors are developed using non-hybridization-

based systems [63], organic solvents could play an important new role. Many sequence-specific

DNA-binding ligands, for example, often bind to double-stranded DNA via hydrophobic interac- tions in the major groove of the B-form molecule.

These interactions can be modified by the use of highly hydrophobic non-polar solvents [64,65] which can be applied to destabilize the binding and help create reversible systems.

The application of organic solvents for affin- ity-based sensor systems is certainly a possibility and a few of the possible applications have been highlighted. To date, however, the area has only been investigated to a very limited extent. The real benefit of the approach, if any, will only become

clear at the laboratory bench.

APPLICATIONS

Numerous potential applications for enzymatic analysis in non-aqueous environments exist for a

diverse range of industries. Already the coupling of oxidases with horseradish peroxidase in organic solvents has led to the detection of poorly water- soluble analytes such as cholesterol [16]. A tem-

perature-abuse sensor that relied on the difference in activity of horseradish peroxidase in solid and

liquid organic media has also been developed [66]. At 4”C, hexadecane was present as a solid which

12 S SAlNl ET AL

prevented the enzyme from catalytically oxidizing a chromogen owmg to high diffusional barriers.

As the temperature increased, a phase change occurred, so that around 25°C the organic medium was in a liquid form. The diffusional barriers were decreased and enzyme catalysis occurred, and the

resulting colour change was monitored optically. Enzyme electrodes can be applied under a wider

range of conditions than spectrophotometric as- says. The full breadth of applications and ad- vantages will be further realized by exploration of suitable biocatalyttc reactions and transducers. Some areas that could benefit from OPEE tech- nology are outlined below.

Clwucal

Most commercially available biosensors have been designed specifically for the clinical market

and mainly for the determination of blood glu- cose. Potential applications for OPEEs could extst where analytes of interest have poor water solubil- tty or where existing methods lack sensitivity. Clinically relevant analytes such as cholesterol and bihrubm are examples of poorly water-soluble analytes that may be amenable to detection using OPEEs.

Food and drmk mdustly Another market sector is the food and drink

Industry, where organic phases are frequently en- countered. The use of enzymes in organic media is not an unfamiliar concept m these situations

where, for example, lipases are being evaluated for inter-esterification reactions in non-aqueous sys- tems. Alteration of the reaction conditions creates the possibility of manipulating hydrolytic enzymes such as lipase in the organic phase so that they take part m, for example, triglycende conversions. Alternatively, aldehydes in fats and oils or cholesterol in butter and margarine may be de- tected by the use of appropriate oxidoreductases. Another possible application for an OPEE is in the momtoring of the quahty of edible oils. A number of off-line tests are available, but are not

conclusive in themselves [67]. Continuous moni- toring would greatly facilitate the quality control of frying oil and render oil quality maintenance more economical.

In certam instances it is important to determine the CIS/ tram ratio of products of a reaction. Some enzymes m non-aqueous media exhibit very high stereospectficity and therefore may be used to determine optically active products in food processes. Other uses for OPEEs include momtor- mg the shelf-life of certain foods, hydrophobic fermentations and other processes such as the production of steroids and steroid-based drugs in

the pharmaceuttcal industry. Any enzyme electrode used for on-line momtor-

ing m the food and drink industry is likely to face problems with vtscous liquids and htgh operating temperatures. Although recent work [19,56] sug- gests that some enzymes may increase their ther-

mal stability in virtually anhydrous organics, one is still left with the problems inherent to on-line detection in vtscous media. Such problems include insensitivity due to low partttiorung of analytes and slow response times. Under these cir-

cumstances, OPEEs may be better suited to rapid off-line testing for many analytes, where sample pretreatment may only involve an automated liquid-liquid extraction procedure.

Envu-onmental momtormg

Over the last few years, new legislation has been introduced to control the disposal of in- dustrial waste chemicals and the use of agricul- tural chemicals. Therefore, instrumentation is re- quired to assess and monitor chemical contamina- tion in water, soil and the atmosphere. Btosensors may offer practical alternatives to conventional

time-consuming, labour-intensive, centralized lab- oratory testing 1681. As described above, Hall et al. [22,23] reported an OPEE for detecting phenol

contamination at water intakes. The determina- tion of other water contammants such as fat-solu- ble biodegradable pesticides and other organic contaminants m rivers may also be possible by the implementation of OPEE technology. In most in- stances, an organic extraction will be required, which would involve a large aqueous volume and a small organic volume to preconcentrate the organic contaminants.

Petrochemical mdustry

There are numerous possible applications for OPEEs in this industry, where the organic phase 1s

ORGANIC PHASE ENZYME ELECTRODES. REVIEW 13

ubiquitous. Here, as in all potential applications, the viability of introducing a bioanalytical device must be considered. If there is no readily available chemical technique, then a biosensor may be of practical use if a suitable biological component is available. OPEEs may be important in the moni- toring of an analyte in a conversion or production

process. Many analytes in this industry, such as methane, alcohols, aldehydes and esters, may have suitable biocatalysts for their detection in the organic phase. The harsh environmental condi-

tions of high temperature and pressure encoun- tered in this industry may limit detection to auto- mated off-line analysis.

Military applications Military applications form one of the largest

and most intense research and development areas

in the biosensor field. Obviously, for security rea- sons, biosensor developments in this area are not widely disseminated. One can suggest, however, possible scenarios for defence applications. Clini- cal monitoring or active contamination of the environment require rapid field analysis. Specifi- cally, an OPEE might be developed for chemical

TABLE 3

Possible types of organic phase biosensor

agents that exhibit high organic solubility using, for example, cholinesterase as the biological com- ponent.

FUTURE DIRECTIONS

As with conventional biosensor development, OPEEs may be followed by more complex organic

phase transducers that will further expand the number of potential applications. Table 3 lists some possible future devices.

The recent demonstration that antibodies are capable of operating in non-aqueous media [58] may lead to the development of organic phase immunosensors which could utilize a number of modes of detection, including electrochemical or optical enzyme linked immunoassay. The fact that enzymes will retain their catalytic activity in al-

most dehydrated states has interesting implica- tions for their use in other forms of biosensor, e.g., in gas-phase analysis utilizing piezoelectric crystals coated with virtually dehydrated enzymes or even antibodies. Barzana et al. [69] recently described a

method for detecting toxic gases with enzymes.

Basis Type

Electrochemical Organic phase enzyme electrodes (OPEEs)

Amperometric enzyme electrodes, e.g., phenol sensors [22,23,26], cholesterol sensor [28,29],

mediated amperometric peroxide sensor [30]

Potentiometric enzyme electrodes, e.g., detection of ester synthesis [27]

Organic phase immunosensors

Calorimetric Enzyme thermistors, e.g., peroxidase and lipase reactions [25]

Organic phase immunosensors

Optical Using chromogens with enzymes in organic media, e.g., cholesterol determination [16],

temperature-abuse sensor [66]

Using chromogens with virtually dehydrated enzymes for toxic gas analysis [69]

Organic phase immunosensors

Piezoelectric

Biomimetic sensors

Virtually dehydrated enzyme or antibody coatings on piezoelectric crystals

Chemically synthesized compounds operating in organic phases for use as sensors offering

increased stability and flexibility, e.g., cytochrome P-450 models with various

poorly water-soluble analytes such as drugs and hydrocarbons

14

Increased solubility of many gases m organic solvents may also lead to increased sensitivity for some devices.

Progress in related areas may be beneficial to organic phase biosensors. An ultimate target in the field of gel matrix technology 1s to produce polymers that can selectively accumulate analytes of interest at transducer surfaces.

Protein engineering may play an important role m the design and modtfication of enzymes that

exhibit high stabihty and activity in unconven- tional phases. The serious problem of distortion of the hydration shell in hydrophilic solvents may be overcome in the future by binding the essential water in place. Currently, the forces that contrib- ute to enzyme stability in organic solvents are being investigated with a view to achieving in- creased stability by mampulation of the ammo acid sequences of an enzyme using techniques

such as site-directed mutagenesis [70]. Solvent en- gineering and mediators which are important fac- tors at present may therefore be superseded by the products of enzyme engineering.

Advances in molecular electronics may clarify the future role of biochemicals in electronic de- vices. In the short term, however, it is clear that the compatibility with organic solvents offers op- portunities to take advantage of microfabncation technologies widely used in the electronics m- dustry to achteve high-volume, inexpensive pro- duction of integrated devices.

In principle, organic phase biosensors offer a new approach in detecting a large number of previously inaccessible analytes. In real terms,

however, biosensors in general have had limited impact on conventional analytical techniques and have delivered relatively few practical devices. The most commercially successful class of biosensors to date are printed enzyme electrodes. The appli- cation of printing technology to OPEEs may ne- cessitate some modification, e.g., organic solvents restrict the printing substrate to relatively few materials such as glass, some ceramics or solvent- re5istan.t polymers.

Organic phase biosensors are at a early stage of development and it is not yet clear which direction they will take. Certainly, enzyme electrodes have already shown promise in organic analysis at the

S SAINI ET AL

laboratory level and further research into new forms of biosensors capable of operating in orgamc environments is clearly warranted.

S.S. gratefully acknowledges financial support in the form of an SERC CASE studentship in collaboration with the Laboratory of the Govern-

ment Chemist, Teddington, Middlesex, UK.

REFERENCES

1

2

3

4

5

6

I

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

A P F Turner, I. Karube and G S W&on (Eds ), Blo-

sensors Fundamentals and Apphcatlons, Oxford Uruver-

sity Press, Oxford, 1989

A.P F. Turner (Ed ), Advances m Blosensors, Vol. 1, JAI

Press, London, 1991

S McClelland, Sensor Rev, 9 (1989) 209

Blodetector News, 2 (1989) 1

D.A. Hammond, M Karel and A M Khbanov, Appl. BIO-

them Blotechnol., 11 (1985) 393

A.M Khbanov, ChemTech, 16 (1986) 354

J. Wang, Electroanalytlcal Techniques m Chmcal Chem-

istry and Laboratory Medicine, VCH, New York, 1988.

L. Clark and C. Lyons, Ann NY Acad. SIX., 102 (1962)

29.

E Bouquelot and M. Bndel, Ann. Chlm Phys., 29 (1913)

145

F R Dastoh and S Price, Arch Blochem Blophys., 118

(1967) 163.

S Sann and A P.F Turner, Blochem. Sot. Trans., 19 (1991)

28

Y L Khmelmtsky, A V Levashov, N.L Klyachko and K

Martmek, Enzyme Mlcrob. Technol., 10 (1988) 710

A Zaks and A Russell, J. Blotechnol , 8 (1988) 259

A M Khbanov, Trends Blochem Scl , 14 (1989) 141

J.S. Dordlck, Enzyme Mlcrob Technol , 11 (1989) 194.

R.S KazandJlan, J S Dordlck and A M Khbanov, Blotech-

nol. Bloeng., 28 (1986) 417

C H Wong, Science, 244 (1989) 1145

C. Laane, S. Boeren, K Vos and C Veeger, Blotechnol

Bloeng , 30 (1987) 81

A Zaks and A.M. Khbanov, J. Blol Chem, 263 (1988)

3194

A J Russell and A M Khbanov, J. Blol Chem , 263 (1988)

11624

M. Stahl, M Mansson and K Mosbach, Blotechnol , Lett , 12 (1990) 161

G F Ha& D I Beg_ and. bP F Tu_rni~, Enzyme &~oh

Technol , 10 (1988) 543.

GE Hall+ a I Rest_ and bP F TmmX, anal C%n .A&&

213 (1988) 113.

D 0 Hltzman and T R Hopkms, Eur. Pat Appl ,0214336,

1987

ORGANIC PHASE ENZYME ELECTRODES. REVIEW

25 L. Flygare and B. Danielsson, Ann. N.Y. Acad. Sci., 542

(1989) 485.

26 M.P. Conner, J. Sanchez, J. Wang, M.R. Smyth and S.

Manmno, Analyst, 114 (1989) 1427.

27 A. Miyabayashi, M. Reslow, P. Adlercreutz and B. Mattias-

son, Anal. Chim. Acta, 219 (1989) 27.

28 G.F. Hall and A.P.F. Turner, in Biosensors ‘90: First

World Conference on Biosensors, Singapore, 2-4 May 1990,

Elsevier Seminars, Oxford, 1990, p. 331.

29 S. Saini, A.P.F. Turner, M.E.A Downs and G.F. Hall, in

preparation.

30 F. Schubert, S. Saini and A.P.F. Turner, Anal. Chim. Acta,

(1991) in press.

31 A.J. Fry and W.E. Britton, in P.T. Kissinger and W.R.

Heineman (Eds.), Laboratory Techniques in Electroanalyti-

cal Chemistry, Dekker, New York, 1984, p. 367.

32 R.M. Wightman, Science, 240 (1988) 415.

33 J.O. Howell, Curr. Sep., 8 (1987) 2.

34 J.W. Bixler and A.M. Bond, Anal. Chem., 58 (1986) 2859.

35 D.O. Wipf and R.M. Wightman, Anal. Chem., 60 (1988)

2460.

36 C.A. Amatore, A. Jutland and F. Pflger, J. Electroanal.

Chem., 218 (1987) 361.

37 A.M. Bond and P.A. Lay, J. Electroanal. Chem., 199 (1986)

285.

38 J. Ghoroghchian, F. Sarfarazi, T. Dibble, J. Cassidy, J.J.

Smyth, A. Russell, G. Dunmore, M. Fleischmann and S.

Pons, Anal. Chem., 58 (1986) 2278.

39 A.C. Michael and R.M. Wightman, Anal. Chem., 61 (1989)

2193.

40 A.M. Bond, K.B. Oldham and C.G. Zoski, J. Electroanal.

Chem., 199 (1988) 71.

41 A.M. Bond, M. Fleischmann and J. Robinson, J. Electro-

anal. Chem., 168 (1984) 299.

42 J.O. Howell and R.M. Wightman, Anal. Chem., 56 (1984)

524.

43 R. Lines and V.D. Parker, Acta Chem. Stand., Ser. B, 31

(1977) 369.

44 J.N. Butler, in P. Delahay and C.W. Tobias (Eds.), Electro-

chemistry and Electrochemical Engineering,. Wiley, New

York, 1970, p. 17.

45 L. Geng, A.G. Ewing, J.C. Jernigan and R.W. Murray,

Anal. Chem., 58 (1986) 852.

46 S. Fukui and A. Tanaka, Adv. B&hem. Eng. Biotechnol.,

29 (1984) 1.

47 B.A. Gregg and A. Heller, Anal. Chem., 62 (1990) 258.

48 J.M. Dicks, S. Hattori, 1. Karube, A.P.F. Turner and T.

Yokozawa, Ann. Biol. Clin., 47 (1989) 607.

49 M.F. Cardosi and A.P.F. Turner, in A.P.F. Turner (Ed.),

Advances in Biosensors, Vol. 1, JAI Press, London, 1991, p.

125.

50 M.F. Cardosi and A.P.F. Turner in A.P.F. Turner, I. Karube

and G.S. Wilson (Eds.), Biosensors: Fundamentals and

Applications, Oxford University Press, Oxford, 1989, p.

257.

51 B.D. Moore, in Biosensors 1989, An International Con-

ference from the Cambridge series on Biotechnology, Cam-

bridge, U.K., 28/29th September, 1989, IBC Technical

Services, London.

52 B.J. Birch and I.W. Burns, Eur. Pat. Appl., 87306513.0,

1987.

53 H.F. Gaertner and A.J. Puigserver, Biotechnol. Bioeng., 38

(1990) 601. 54 G.F. Hall, PhD Thesis, Cranfield Institute of Technology,

1990.

55 J.S. Deetz and J.D. Rozzell, Ann. N.Y. Acad. Sci., 542

(1988) 230.

56 A. Zaks and A.M. Khbanov, Science, 224 (1984) 1249.

57 K. Ryu and J.S. Dordick, J. Am. Chem. Sot., 111 (1989)

8026.

58 A.J. Russsell, L.J. Trudel. P.L. Skipper, J.D. Groopman,

S.R. Tannenbaum and A.M. Klibanov, Biochem. Biophys.

Res. Commun., 158 (1989) 80.

59 M.E.A. Downs, S. Kobayashi and 1. Karube, Anal. Lett.,

20 (1987) 1897.

60 M.E.A. Downs, P.J.P. Warner and A.P.F. Turner, Biotech-

nol., Tech., 1 (1987) 129.

61 B. Perbal, A Practical Guide to Molecular Cloning, Wiley,

New York, 1988.

62 D.E. Kohne, S.A. Levison and M.J. Byers, Biochemisty, 16

(1977) 5329.

63 M.E.A. Downs, B&hem. Sot. Trans., 19 (1991) 39.

64 G. Lober and H. Schutz, Biopolymers, 2 (1972) 2439.

65 D.L. Ollis and S.W. White, Chem. Rev., 87 (1987) 981.

66 C.G. Boeriu, J.S. Dordick and A.M. Klibanov, Biotechnol-

ogy, 4 (1986) 997.

67 P. Smith, Sensor Rev., 9 (1989) 15.

68 W.C. Gaisford and D.M. Rawson, Meas. Control, 22 (1989)

183.

69 E. Barzana, A.M. Klibanov and M. Karel, Appl. Biochem.

Biotechnol., 15 (1987) 25.

70 F.H. Arnold, Trends Biotechnol., 8 (1990) 244.