Electrochemical transducers: P. Rea and P. Rolfe

the reactants, its availability may limit the rate of formation of X and H,Oz and the rate of consumption of oxygen itself. The measured elec- trode current, whether it be a cathodic oxygen current or anodic peroxide current, will con- sequently be limited by the availability of oxygen under some circumstances. The rate of the enzymically-mediated two-substrate reaction in equation (l), above, is defined by an expression of the type:

1. COMPUTERISED ENZYME ELECTRODES

Romette, J.L. and Boitieux, J.L.

1.1 Introduction One of the most interesting applications of immobilised enzymes has been their use as the active elements of electrochemical transducers’. Enzyme transducers, which generally consist of an electrochemical detector in close association with an immobilised enzyme, have several advantages over other methods of analysis. In the discussion that follows oxidase-based enzyme transducers will be examined with regard to their application to computerised measurements on samples of variable, or unknown, ~0,.

1.2 Enzyme transducers Classical analytical methods require the precipitation or dialytic separation of proteins and particulate matter from the compounds to be measured. Electrochemical monitoring with enzyme transducers, on the other hand, can be performed on whole blood or biological media, thus eliminating the requirement for sometimes lengthy sample purification procedures.

The use of an enzyme as the functional element of an electrochemical device was first suggested by Clark and Lyons2 but the first transducer incorporating an immobilized enzyme was described by Updike and Hicks in 19673 to be followed by the extensive work of Guilbault and coworkers4. Although the literature contains many methods of construction of enzyme transducers, it is noteworthy that only a few of these are commercially available. The paucity of commer- cially available devices reflects the difficulties of translating the experimentally possible into the commercially viable. For this reason, the methods examined in our laboratory have focussed on the use of oxidase enzymes which are very rugged, very substrate-specific and do not necessitate the addition of cofactors to the sample before analysis. Except for one notable problem, the influence of the oxygen content of the sample on the measured current, the oxidases represent excellent biological transducers for the fabrication of enzyme transducers.

1.3 Oxygen-dependence Oxidases, the most commonly employed enzymes for the fabrication of enzyme transducers, mediate redox reactions of the type:

XH, + O2 -X + H,O, (1)

where XH, is the reduced form of the substrate and X the oxidised form. Because oxygen is one of

Laboratoire de Technologie Enzymatique, Uniuersite de Technologie de Campi@e (UTC) BP 233, 60206 Cnm~~&ne, France

71 = Vm, [l/(1 + KxHI/[XH,] + Ko,/P,l)l (2)

where [XH,] and [0,] denote the concentrations of substrate and oxygen, respectively, within the vicinity of the enzyme, KXH2 and KOp the Michaelis constants for substrate and oxygen and V,, the maximum velocity of the reaction. For samples of unknown or variable PO,, where [0,] cannot be defined, at least in the vicinity of the immobilised enzyme, [XH,] is impossible to enumerate. Oxidases are generally favoured, therefore, over [nicotinamide adenine dinucleotide (NAD+) linked] dehydrogenases for the construction of enzyme transducers, firstly because one of the reactants (oxygen) and one of the products (hydrogen peroxide) can be readily measured electro- chemically and secondly because the cofactor for the reaction, flavin adenine dinucleotide (FAD+), is covalently attached to the enzyme, thereby obviating the need for the addition of exogenous cofactor to the bulk medium or an additional immobilisation reaction. However, the oxygen- dependence of oxidase-mediated reactions is a problem that must be overcome before they can be used in a wide range of measurement situations.

1.4 Oxygen-rich membranes We have tried to solve the problem of the oxygen- sensitivity of oxidase-based transducers by using enzyme membranes in which the solubility of oxygen is much greater than the aqueous bulk medium5. This ensures that the oxygen consumed during the measurement is the oxygen dissolved in the membrane, not the oxygen dissolved in the sample. On the condition that [0,] >> Kol, equation (2), above, reduces to one of the form:

7,~ = (vmax [XH~l)/[XH~l + KXH2)

so that reaction rate and measured current are independent of sample PO,.

The detector in the enzyme transducer is a Clark electrode by means of which oxygen concentration is measured amperometrically. The hydrophobic gas permeable membrane is made from poly- propylene. Our experience, and that of others6T7, is that gas membranes yield better enzyme trans- ducers because they exclude all electroactive species apart from gases from the detector. Hydrophilic membranes, by contrast, allow a wide range of compounds, in addition to the analyte, to

J. Biomed Eng. 1984, Vol. 6, July 171

Electrochemical transducers: P. Rea and P. Rolfe

I I I

l /

/

/

0

0

/ 0

/

1 2 3 4

Glucose,g/l

Figure 1.1 Calibration curve of glucose oxidase electrode bearing 0.25 U of enzyme per cm* of membrane. The enzyme membrane was prepared by the coreticulation of glucose oxidase and gelatin with glutaraldehyde8~9.

Table 1.1 Influence of p0, of sample on measured slope of response of glucose transducer after exposure to 2 g/l D-glucose. The enzyme membrane was prepared by cross- linking glucose oxidase and gelatin with glutaraldehyde. Values shown are mean * SE for n = 5

po, sample, mm Hg Slope, mm Hg/s

0 25.1 f 0.3 160 23.9 f 0.5 760 24.9 f 0.5

contact the electrochemical surface. The enzyme membrane is prepared by the corecticulation of the oxidase with gelatin using glutaraldehydes~. In order to achieve good mechanical stability, the active film containing the oxidase is coated onto the gas membrane directly and then cross-linked with glutaraldehyde89. In

A typical calibration curve for a glucose transducer prepared in this manner is shown in Figure 1.1. When using enzyme transducers, transient or steady-state responses can be used. However, in order to shorten the analysis time, we generally record the derivative in time of the current given by the oxygen electrode. As intramembrane oxygen is consumed during the measurement, the signal is expressed as the rate of change of pOr (mm Hg/s).

The results in Tabb I. I demonstrate the indepen- dence of the measured rate of decrease of the oxygen current of the electrode on the p0, of the sample. The exact reason why enzyme membranes containing gelatin should have a greater oxygen carrying capacity than similar membranes employing albumin as the support is not known but they have estimated oxygen concentrations of 6.5 mM after exposure to air in comparison to the

0.2 mM found in air-equilibrated buffers or albumin-based membranes under similar conditions.

Examples of other compounds that have been measured according to the same principle are listed below together with the component enzymic reactions involved.

Sucrose8 . Sucrose -a-D-glucose + fructose . . using invertase (4) o-D-glucose - /3-D-glucose . . using mutarotase (5) /?-D-glucose + O2 - gluconic acid + HrOr . . using glucose oxidase (6)

Lactose*. Lactose - galactose + /3-D-glucose . . using lactase (7) /l-D-glucose + 02- gluconic acid + HzOz . . glucose oxidase (8)

Ethunollo. Ethanol + 0,~ acetaldehyde + H,O, . . using ethanol oxidase (9)

L-lysine II. L-lysine + 0, + H,Or- cr-keto- &-aminocaproate + NH, + H202 (IO)

1.5 Transducer stability A remarkable characteristic of enzyme membranes prepared in this manner is their stability to long- term storage and repeated operation. The mem- branes retain their activity for at least 6 months when stored at 5°C in buffer. Their operational stability, while being good, is determined by the concentrations of substrate to which the membrane is exposed, as indicated by the results shown in Figure 1.2 for a lysine oxidase-based transducer (equation 10 above). When the device is exposed to substrate every 2 min, the rate of deterioration of

10 - I

i 0.5mM LYS 1

z 0

iii SC O.lmM LYS

\

OJ 1 I I I I 0 100 200 300 400 500

Number of measurements

Figure I.2 Relationship between transducer signal and number of measurements. The transducer, a lysine oxidase- based device, was exposed to substrate every 2 min and the signal recorded. Three concentrations of L-lysine were employed: 1.0, 0.5 and 0.1 mM.

172 J. Biomed. Eng. 1984, vol. 6, July

JOO-

0, 80-

60 -

40 -

0 10 20 30 40 50 60

Time,s

Figure 1.3 Response of glucose transducer as a function of time when in contact with a sample containing 0.5 g/l D- glucose. Phase 1: injection of 20 ~1 of sample; Phase 2: buffer- rinsing; Phase 3: air-rinsing. The region AB is the time interval over which data are collected for computation.

the signal increases with increase in substrate concentration. L-lysine concentrations of 1 .O, 0.5

and 0.1 mM enable 200, 400 and more than 500 measurements, respectively, before the signal is appreciably attenuated. Since similar patterns of inactivation have been obtained with glucose oxidase-based transducersi*, the substrate concentration-dependence of deterioration may be common to most FAD+-linked oxidases. The data indicate that oxidases are inactivated by the reaction they mediate so that each enzyme molecule can only transform a limited number of substrate molecules during its life-time.

1.6 Computer-controlled data acquisition and processing Recently we have developed computerised enzyme transducer systems, fabricated in the manner described above, for the routine measurement of substrate in whole blood and industrial broth samples of variable ~0,~. The process is fully automated and under software control by an Apple II microcomputer. An example of an analysis is shown in Figure 1.3.

Each analysis takes approximately 60 s and consists of three phases: (a) air-rinsing to saturate the enzyme membrane with oxygen (step 3 in Figure 1.3); (b) exposure of the transducer to the sample containing substrate (step 1); and (c) buffer- rinsing to remove substrate from the membrane (step 2) before the start of the next cycle (step 3 again). Air-rinsing was employed to restore the intramembrane oxygen consumed during the measurements since the restoration of intra- membrane oxygen with air-equilibrated aqueous media is too slow to be practicable. The valves for

Electrochemical transducers: P. Rea and P. Rolfe

air- and buffer-rinsing and sample introduction are actuated by the microcomputer.

The substrate concentration of the sample is computed from the rate of decrease of the apparent p02 of the enzyme membrane. The transient signal measured over the course of the 10 s sampling period, AB in Figure 1.3, is least- squares approximated by a third-order polynomial whose slope at inflection is characteristic of the concentration of substrate in the bulk medium.

The transducer signal, z(t), the arc AB in Figure 1.3, closely approximates the third-order polynomial:

z(t) = a, + a, t + a,t * + us t 3 (II)

the coefftcients of which, a,. . . 3, may be obtained by a least squares method consisting of minimising the function:

&,, a,, u2, a,) = l/2 - (zk - a, - u2k2 - u,k’)* (12)

where zk is the measured value at time t = k for k=l,2...M.

The necessary conditions:

-J/-ai=O; i=O, 1, 2, 3 (13)

can be written as Au = b, where a = [a,, a,, u2, u3]t and the coefficients uij of A and bi of b are given by the expressions:

uii=-k’+jandbi=--zkki (O<i;j<3) (14)

The resultant fourth-order symmetric linear system is then easily solved by a direct method. From the computed coefficients, ai, the inflection and slope, where z”(t) = 0, may be obtained from the expressions:

t = -@a, and Z’(t) = U, - U2/3U3 (15)

An automated calibration procedure has also been developed and this is based on the computerised treatment of the signal by a cubic spline function thus enabling the automatic measurement of samples.

1.7 Conclusions The transducers described can be used with samples exhibiting high variability in oxygen content, such as plasma, whole blood or culture media. The fully-computerised system in which transient transducer signals are measured allows 60 measurements per hour with a sample volume of 20 to 50 fi and an accuracy of 1 per cent. The system could be used as part of an autoanalyser or an extracorporeal device for the semi-continuous measurement of blood constituents.

Acknowledgements Professor J P Kemevez and colleagues from UTC for the development of the signal processing techniques.

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Electrochemical transducers: P. Rea and P. Rolfe

References

6

7

8

9

10

11

Fishman, M.M. Anal Chem, 1980 52, 185-199 Clark, L.C. and Lyons, C. Ann. N.Y. Acad. Sci, 1962 102, 29 Updike, S.J. and Hicks, J.P. Nature, 1967 214, 986 Guilhault, G.G. Methods in Enzymol, 1976 44, 579 Quenesson, J.C. and Thomas, D. French Patent No 7715, 616, 1977 Di Paolantonia, C.L., Arnold, M.A. and Rechnitz, CA. Anal. Chim. Acta, 1981 128, 121 Walters, R.R., Johnson, P.A. and Buck, R.P. Anal. Chem., 1980 52, 1684 Romette, J.L. PhD Thesis, University of Compiegne, France 1980 Kernevez, J.P., Konate, L. and Romette, J.L. Biotech. Bioeng. 1983 25, 845-855 Belghith, H. and Romette, J.L. Determination of ethanol by oxidase enzyme electrode, In preparation Romette, J.L., Yang, J.S., Kusakabe, H. and Thomas, D. Biotech. Biaeng., 1983 In press

OXYGEN-INDEPENDENT GLUCOSE ELECTRODE

Dr G. Davis presented an alternative strategy to that given by Romette for overcoming rate limitation by oxygen. Dr Davis and his colleagues’ strategy is to employ an artificial redox mediator which directly shuttles electrons from the reduced oxidase, to an electrode surface.

The catalytic cycle of glucose oxidase comprises two main steps: (A) The oxidation of D-glucose to D-gluconolactone concomitant with the reduction of FAD+ to FADH,; and (B) the reduction of O2 to H,Or concomitant with the regeneration of reduced cofactor.

(A) FAD+ + D-glucose -FADH, + D-gluconolactone (16)

(B) FADH, + Or- FAD+ + H,02 (17)

If FADHI, the reduced form of the prosthetic group of the enzyme, could be measured directly, reaction (B), the oxygen-dependent step of the sequence, might be eliminated. Davis and his colleagues have therefore investigated the use of artificial organic electron acceptors for the direct removal of electrons from the flavin moiety of the enzyme. One class of organic redox agents, the ferrocenes, 1, I’-dimethylferrocene (1 ,I’-DMF) in particular, have been examined in some detail.

1 ,l’-DMF has four attractive features for this application: (i) Its redox potential favours electron transfer from the reduced enzyme to the ferrici- nium ion; (ii) it is well characterised electro- chemically and the reduced form will readily donate electrons to a graphite electrode at low applied potentials. A polarising voltage of +140 mV (vs SSC) is sufficient to quantitate the reduced form of l,l’-DMF whereas most of the freely diffusable electroactive compounds in blood (uric acid, cysteine and reduced glutathione) do not

oxidise at this potential; (iii) it has a low solubility in aqueous media which results in its effective confinement at the electrode surface; (iv) it does not undergo auto-oxidation thereby minimising interference from oxygen.

As subsequently outlined by Professor Silver (section 9), conventional glucose oxidase- or lactate oxidase-based transducers are unsuitable for the measurement of glucose and lactate, respectively, in hypoxic tissues although such tissues represent some of the physiologically and clinically most relevant measurement environments for both compounds. Similarly, conventional oxidase-based transducers are unsuitable for the continuous analysis of compounds in fermentation vats where anaerobiosis may be essential for appreciable synthesis of the desired product. The elimination of oxygen from oxidase-mediated reactions, as described by Davis, may soon enable the electro- chemical measurement of biological compounds under such conditions. Rometes’s strategy, by contrast, does not overcome the problem completely as the enzyme membrane must be air- equilibrated before the start of each analysis:

2. FERROCENE-BASED ELECTRODE FOR GLUCOSE

Davis, G., Higgins, I.J.’ and Hill, H.A.O.

It is important to be able to determine rapidly and accurately blood glucose levels in patients suffering from diabetes mellitus. Most conventional assays for glucose are based on the determination of the hydrogen peroxide produced in the enzymic oxidation of glucose by the enzyme glucose oxidase.

Glucose + 0 2-gluconolactone + H,02 (18)

The peroxide formed may be determined in a number of ways, including its reaction with chromogens or its potentiometrically-controlled electrochemical oxidation at a platinum electrode. However, the latter method is often susceptible to variations in oxygen tension which cause fluctua- tions in the measured current.

An amperometric enzyme transducer for the analysis of glucose in both undiluted whole blood and serum has been developed. The electrode is not dependent on oxygen as a mediator but uses a substituted ferricinium ion to shuttle electrons between immobilised glucose oxidase and a graphite electrode. The performance of the device is dependent on the rapid rate of electron transfer between the reduced enzyme and the ferricinium ion, and the rapid electrochemical oxidation of the

Inorganic Chemistry Laborotmy, University of Oxford, OXI 3QR

‘BiotechtAgv Cmtre, Cran~ld Instime of Technoiogv, Bedford, M~43 OAL

174 J. Biomed. Eng. 1984, Vol. 6, July