[3] Coupled enzyme reactions in enzyme electrodes using sequence, amplification, competition, and antiinterference principles

[3]

29

COUPLED ENZYME REACTIONS IN ENZYME ELECTRODES

has instruments available for triglycerides, lipase, cholesterol, and amylase. Fuji Electric (Tokyo) has a glucose instrument, similar in design to the Yellow Springs Instrument. Self-contained electrode probes are available from two companies: Tacussel (Lyon, France), which sells a glucose electrode based on the collagen immobilization of Coulet, and Universal Sensors (P.O. Box 736, New Orleans, LA 70148), which offers probes for urea, glucose, creatinine, amino acids, alcohols, and others on request, based on pig intestine immobilization. (See also Table II in [1].)

[3] C o u p l e d E n z y m e R e a c t i o n s in E n z y m e E l e c t r o d e s Using Sequence, Amplification, Competition, and Antiinterference Principles By

FRIEDER W. SCHELLER, REINHARD RENNEBERG,

and

FLORIAN SCHUBERT

The number of substances which can be directly determined by monoenzyme electrodes is limited because in many enzyme-catalyzed reactions the cosubstrates involved and the products formed are electrochemically inactive. Therefore readily measurable substances have to be formed in coupled subsequent (sequential) or parallel (competitive) enzyme reactions. Furthermore, the sensitivity of cosubstrate-dependent electrodes is increased when the substrate is shuttled in a cyclic enzyme sequence to give multiple products at the expense of the cosubstrate excess. Interfering substrates, which particularly pose problems when multienzyme systems are used, may be advantageously eliminated in enzymatic antiinterference layers. Beside the pH and temperature dependence of the functional parameters, in the development of enzyme electrodes the coupling of substrate transport and enzyme-catalyzed reactions has to be characterized at different enzyme loadings by measuring the electrode signal, the effectiveness factor and turnover number, and the degree of substrate conversion. In this chapter the development of a family of sensors is described based on a well-characterized glucose oxidase membrane and different coupling principles. The sensors are suited for the determination of saccharides, cofactors, and peroxidase substrates as well as for activities of amylases. METHODS IN ENZYMOLOGY, VOL. 137

Copyright ~9 1988 by AcademicPress, Inc. All righlsof reproduction in any form reserved.

30

ANALYTICAL APPLICATIONS

[3]

Experimental

Preparation of Gelatin-Entrapped Enzyme Layers All enzyme layers for electrode covering are prepared according to the following procedure: 50 mg of acid photogelatin (VEB Gelatinewerk, Calbe, GDR) is suspended in 0.5 ml doubly distilled water and allowed to swell for 60 min at room temperature. Then 0.5 ml of 50 mM phosphate buffer, pH 6.5, is added; the solution thoroughly mixed and heated in a water bath at 40° for another 60 min. The powder or solution of the respective enzyme (at least 25 U) is added, and the mixture is gently stirred and cast on a plane poly(vinyl chloride) support. The liquid is spread on a total area 5 x 10 cm using a glass rod. The enzyme layer is allowed to dry at room temperature over a period of 6 hr and is then removed from the support. The layer has a thickness of 20-30/xm. It is placed between two sheets of filter paper and stored at 4°.

Preparation of Glutaraldehyde Cross-Linked Enzyme Layers To a mixture of 500 U disaccharidase (e.g., 13-fructofuranosidase, invertase, EC 3.2.1.26) dissolved in 400/x120 mM phosphate buffer, pH 6.8, is added 30/zl of 30% bovine albumin solution, followed by 60/~1 of 25% glutaraldehyde solution. The solution is stirred for 1 min. This enzyme mixture is homogenously spread on 5 x 10 cm stretched silk. After drying, a piece 2 x 1 cm is cut off and attached by two O rings to the rack under the lid of the measuring cell.

Apparatus To prepare the enzyme electrode 4 x 4 mm of the enzyme layer is sandwiched between two cellulose membranes 15-30 /~m thick (Nephrophan, VEB CK, Bitterfeld, GDR). This layered membrane is held by an O ring on the tip of the electrode holder of the oxygen electrode SMZ (VEB Metra, Radebeul, GDR). One-half milliliter of 0.1 M KCI solution is inserted into the holder to assure electrolytic contact between the Pt indicator electrode (diameter 0.5 mm) and the Ag/AgC1 counter electrode. The electrode body is then placed in the holder and the indicator electrode pressed against the enzyme membrane. The complete enzyme electrode is introduced in a horizontal position into the cell of the G L U K O M E T E R GKM 01 (Zentrum for Wissenschaftlichen Ger~itebau der Akademie der Wissenschaften, Berlin, GDR). Two milliliters of the appropriate air-saturated buffer solution is filled into the cell which is

[3]

COUPLED ENZYME REACTIONS IN ENZYME ELECTRODES

31

equipped with a magnetic stirring bar and thermostated at 25 --- 0.1 °. The sample volume varies between 10 and 100/zl. The indicator electrode is poised at a potential of +600 mV versus the Ag/AgC1 electrode in the H2Oz indicator setup or at -600 mV for an oxygen consumption measurement. The dialysis membrane system is also used for oxygen measurement. At -600 mV potential almost no interfering electrode reactions have been found. Either the current-time curves are recorded (stationary measuring principle) or the numerical value of the maximum of the first derivative of the current-time curve is displayed digitally (kinetic measuring principle). Characterization of the Enzyme Membrane

Recovery of Enzyme Activity Determination of the intrinsic activity of the immobilized enzyme is complicated since diffusion processes and distribution equilibria may influence the measured (apparent) value. These effects were eliminated by measuring the remaining glucose oxidase (GOD, EC 1.1.3.4) activity after resolubilization of a definite part of the enzyme membrane by heating in 50 mM phosphate buffer, pH 5.5, to 40 °. The initial rate of HzO2 formation in the measuring cell after addition of a portion of the resolubilized enzyme membrane containing about 1 U of GOD to the air-saturated 5 mM glucose solution in 50 mM phosphate buffer, pH 5.5, was measured at 37°. In these experiments the platinum indicator electrode set at +600 mV was covered only by two Nephrophan membranes. Its sensitivity was calibrated using H202 standard solutions. About 90% of the original GOD activity was recovered after the immobilization and resolubilization steps? This very high activity recovered may be ascribed to the mild conditions of the immobilization procedure and the "native milieu" of the gelatin matrix.

Characterization of Reaction-Transport Coupling In homogeneous solutions the initial rate of substrate conversion rises linearly with increasing concentration of the enzyme. However, because of diffusion the overall rate with immobilized enzymes is constant at high enzyme loadings. Thus only part of the enzyme is used in accelerating the F. Scheller, D. Pfeiffer, I. Seyer, D. Kirstein, T. Schulmeister, and J. Nentwig, Bioelectrochem. Bioenerg. 11, 155 (1983).

32

ANALYTICAL APPLICATIONS

~

M glucose 2~

==

[3]

~---e --

270 fE

r

2.5

5.0 GOD [Ulcm 2]

50

FIG. 1. Enzyme loading test: Influence of GOD concentration on the signal of the GOD sensor at 0.14 and 5 mM glucose. Buffer: 66 mM phosphate buffer, pH 7.0. Polarization voltage: +600 mV.

substrate conversion. The effectiveness factor "0 reflects the influence of transport processes. It is defined by the ratio of the measured (apparent) activity and the reaction rate in the presence of the same amount of enzyme in homogeneous solution. Using the GOD-gelatin membrane, different parameters characterizing the reaction-transport coupling have been studied. Enzyme Loading Test. The steady-state current at both 0.14 and 5 mM glucose increases linearly from 0.046 U GOD/cm 2 to about 1 U/cm 2 (Fig. 1). At higher GOD loadings the electrode current reaches a saturation value. Using the following parameters for the GOD-gelatin membrane, the enzyme loading factor fE was calculated according to Carr and Bowers2:

fE = k2cL2/Km/D where k2 is the rate constant, c the enzyme concentration, L the membrane thickness, Km the Michaelis constant, and D the diffusion coefficient. 2 p. Carp and L. D. Bowers, Chem. Anal. (N.Y.) 56, 218 (1980).

[3]

C O U P L E D E N Z Y M E R E A C T I O N S IN E N Z Y M E E L E C T R O D E S

1

4.6 U/cm 2

2

0.46 U/crn 2 - - o

3

0.0.4.6 U / c m 2

33

400

300o~~

o~

200

//

G

.

loo ,//~ , , j ~ /

~

....__~A

1'0

0.046 U/cm e

2'0 cErnN]

3tO

¢0

FIG. 2. Concentration dependence of the glucose signal at different GOD loadings. Conditions are as in Fig. 1. Plots 1-3, oxygen-saturated solution; plots 4-6, air-saturated solution.

The parameters are L = 30/zm, k2 = 320 s e c - I , 3 and KM (glucose) = 10-2 M. In accordance with theoretical considerations,4 the transient from the linear region to the saturation proceeds at about fE = 10 (Fig. 1). Dependence of Current on Substrate Concentration. When plotting the values of anodic steady-state currents obtained for different substrate concentrations, Michaelis-Menten-type curves are obtained at different enzyme loadings (Fig. 2). The glucose concentration leading to half-maximal current was estimated to be between 1.4 and 1.8 mM when airsaturated. The linear region extends up to 2 mM glucose in the measuring solution, the slope of the linear part being 1.5/~A/mM/cm2. With oxygen saturation of the solution, both linearity and sensitivity are considerably extended. The linear dependence of the reciprocal of the current plotted against the reciprocal of the bulk glucose concentration (Fig. 3) at low enzyme loading gives evidence that the current is controlled by the enzyme reac3 W. K0hn, D. Kirstein, and P. Mohr, Acta Biol. Med. Germ. 39, 1121 (1980). 4 D. A. Gough and J. K. Leypoldt, Appl. Biochem. Bioeng. 3, 181 (1981).

34

ANALYTICALAPPLICATIONS

0.25

[3]

/ *

0.20

/~~6U/cm 2

~-0.15 c;

•

o

0.~'6U/cm2 ~ x

0.10, ~ j o ~ / ~ i

~

0.05 /I

0

-

1 I

2 1/cE1/mN] I

3 I

FIG. 3. ElectrochemicalLineweaver-Burkplot of steady-statecurrents at differentenzyme loadings. Conditions are as in Fig. 1.

tion. The small curvature at extremely high glucose concentrations is due to limitation by consumption of the cosubstrate, oxygen. The apparent Km value of glucose (when air-saturated) determined by these "electrochemical Lineweaver-Burk plots" is 7.5 mM. The corresponding value of soluble GOD was determined to be 3.8 mM. At high enzyme loading the electrochemical Lineweaver-Burk plot is curved, and extrapolation passes through the origin. It should be noted that the Lineweaver-Burk plot of the kinetic signal (di/dt) yields a considerably higher Km value, i.e., 22 mM, as expected for this measuring system. 5 Effectiveness Factor. In order to characterize the apparent activity of the GOD membrane, the initial rate of H202 accumulation or the rate of

5G. G. Guilbaultand G. J. Lubrano, Anal.

Chim. Acta

64, 439 (1973).

[3]

COUPLED ENZYME REACTIONS IN ENZYME ELECTRODES

35

oxygen or glucose consumption in the bulk solution is measured. ~For this purpose a double measuring cell containing the glucose-indicating enzyme electrode and a nonenzymatic H202- or O2-indicating Pt electrode is used. As it is less influenced by H202 destruction, the 02 consumption mode seems to be more accurate than measuring of H202 accumulation. 6 In these experiments a closed measuring cell completely filled with air-saturated 66 mM phosphate buffer, pH 7.0, is used. After obtaining a constant baseline of the oxygen electrode, 5 mM glucose is introduced into the stirred solution. The initial rate of oxygen consumption by glucose conversion catalyzed by the part of the GOD membrane contacting the glucose solution (0.14 cm 2) is measured. The initial rate of oxygen consumption (and also of glucose conversion) in the solution at saturating glucose concentrations is 14 nmol/min at 46 U GOD/cm 2. This value is below 1% of the biocatalytic activity immobilized in the membrane. This result underlines the limitation set by the internal diffusion. On the other hand, the activity of the GOD membrane containing 0.046 U/cm 2 amounts to about 70% of the original value, which is very near kinetic control. The relatively low apparent GOD activity is mainly caused by the shielding effect of the external dialysis membrane. Its diffusional resistance for glucose is almost 10 times higher than that for the cosubstrate, oxygen. Nevertheless, the apparent activity of this GOD sandwich membrane with 0.1 U/cm 2 at 25 ° compares well with GOD membranes prepared by covalent enzyme fixation. The membrane prepared by Tsuchida and Yoda, 7 who used GOD bound in pores of an asymmetric cellulose acetate layer, has an apparent activity of 0.34 U/cm 2 at 37 °. The GODcollagen membrane developed by Thevenot e t al. 8 contains an apparent activity of 0.06 U/cm 2 at 30°. On the other hand, the low apparent activity of the GOD membrane causes only very small glucose consumption during measurement. Within 10 min about 2% of the bulk glucose is converted by the GOD membrane. This amount can be neglected during the kinetic measuring time of only 10 sec. Using an enzyme concentration of 46 U/cm 2 in the measurement of a 0.14 mM glucose sample, which corresponds (at 1:40 dilution) to the normal value of blood glucose, each GOD molecule converts one glucose molecule during a measuring time of 10 sec.

6 p. R. Coulet, R. Sternberg, and D. Thevenot, Biochim. Biophys. Acta 612, 317 (1980). 7 T. Tsuchida and K. Yoda, Enzyme Mierob. Technol. 3, 326 (1981). 8 D. R. Thevenot, R. Sternberg, P. R. Coulet, J. Laurent, and D. C. Gautheron, Anal. Chem. 51, 96 (1979).

36

ANALYTICAL APPLICATIONS

elec+rodeM~

~

membrane < '.".'.."'.'.5"5"..'3".: ...... 3""

:' M

[3]

i:"':~'""""":':""

1 02

-:.'.'.'.',': ::5.:":2"5.'::::'.2.:.'.'.

iii

~

~

':'i

II maltose

FIG. 4. Enzyme sequence electrode for determination of maltose and other saccharides using a glucoamylase-GOD membrane.

Linear Enzyme Sequences

Glucoamylase-GOD Electrode One milligram glucoamylase (GA, glucan 1,4-a-glucosidase, EC 3.2.1.3) (50 U) is coimmobilized with 0.5 mg GOD (23 U) per cm 2 in gelatin according to the procedure described in the section "Experimental." GA splits Offfl-D-glucose units from saccharides; therefore the GOD can use the glucose formed directly without spontaneous or enzymatic mutarotation (Fig. 4). The respective enzyme electrode responds to glucose, maltose, and dextrins, and it indicates the sum of all these substrates. The sensitivity for maltose in comparison to glucose is about 50%. This decreased signal might be caused by the approximately doubled diffusional resistance of the membrane to maltose owing to its larger molecular cross section. The linear range extends up to 2 mM maltose. The endohydrolase, o~-amylase (EC 3.2.1.1), splits starch into maltose via dextrin fragments. These products are indicated by the glucoamylaseGOD electrode. For determining the o~-amylase activity in serum, 2 ml of 5% soluble starch in 50 mM phosphate buffer, pH 6.0, is pipetted into the measuring cell and equilibrated at 37°. When a constant baseline is reached, the reaction is started by addition of 100 txl of serum. After a rapid initial current increase corresponding to the endogenous glucose, the current continues to rise, albeit slower. The slope of this part of the current-time curve (1 min after sample addition) depends linearly on the activity of a-amylase. 9 The activity of glucoamylase can be directly deter9 D. Pfeiffer, F. Scheller, M. J~inchen, K. Bertermann, and H. Weise, Anal. Lett. 13, 1179 (1980).

[3]

COUPLED ENZYME REACTIONSIN ENZYME ELECTRODES

37

elecJrrode M GOD

membrane

Pslice °~ra~r°I M phospha+e or fluoride

glucose 6phospha~'e

02

FIG. 5. Enzyme sequence electrode for phosphate determination using a GOD membrane and a slice of potato tissue.

mined with the GOD electrode by measuring the /3-D-glucose split off from soluble starch in the measuring solution. 9

Potato Slice-GOD Hybrid Electrode F o r the potato s l i c e - G O D hybrid sensor a slice of about 0.1 mm thickness is cut with a razor blade from potato (Solanum tuberosum) tissue and placed directly on the GOD membrane (Fig. 5). Based on the sequential action of potato acid phosphatase (EC 3.1.3.2) and GOD, this sensor responds to glucose 6-phosphate. Since the response is controlled by the acid phosphatase reaction, 10the sensor is applicable to the determination of the phosphatase inhibitor, inorganic phosphate. To obtain a linear phosphate concentration dependence, the phosphate-containing sample is added to the measuring solution (0.1 M citric acid-sodium citrate buffer, p H 6.0) prior to injection of glucose 6-phosphate (0.37 mM). The reciprocal of the kinetic electrode signal is then proportional to inhibitor concentration between 0.025 and 1.5 mM.

GOD-Peroxidase Electrode A piece of an e n z y m e layer containing coimmobilized horseradish peroxidase (HRP, EC I. 11.1.7) (32 U/cm 2) and GOD (46 U/cm 2) is sandwiched between two dialysis membranes. The sensor is applied to the 10F. Schubert, L. Kirstein, R. Renneberg, and F. Scheller, Anal. Chem. 56, 1677 (1984).

38

ANALYTICALAPPLICATIONS

[3]

determination of the substrate for HRP, bilirubin. The reactions proceed as follows: G l u c o s e + O5 2 H~O2 + bilirubin

GOD HRP

~ gluconolactone + HzO2 ~ 2 H20 + product

In contrast to the glucoamylase-GOD and potato slice-GOD electrodes, GOD is not used for glucose indication in this case but for generation of HzO2. H At 10 mM, glucose in the background solution (0.1 M phosphate buffer, pH 7.4) causes the complete conversion of dissolved oxygen in the enzyme layer to H202. Therefore the anodic current at +600 mV is not influenced by addition of a sample, e.g., serum. When the anodic current reaches a constant level, 50/.d of the bilirubin-containing sample solution is added to the measuring cell. Part of the hydrogen peroxide formed in the GOD reaction inside the membrane is consumed by the HRP-catalyzed conversion of bilirubin, and the anodic current is decreased depending on the bilirubin concentration. The slope of the calibration curve is 0.8/xA/mM/cm2 between 0.005 and 0.05 mM bilirubin. Inoertase-GOD Reactor Electrode

For successive measurements of glucose and disaccharides, the GOD electrode described may be combined with enzymatic disaccharide hydrolysis catalyzed by an immobilized disaccharidase in the measuring cell. For sucrose determination, invertase is used. The enzyme is fixed on silk which is then installed on a rack mounted under the lid of the measuring cell (Fig. 6). Before the immobilized invertase is immersed in the measuring solution, the endogenous glucose concentration of the sample is determined. Addition of the sample causes a current increase of the GOD-covered electrode which is completed after 30 sec. At this time the immobilized invertase is inserted in the stirred solution, catalyzing the splitting of sucrose into D-fructose and a-D-glucose which spontaneously mutarotates to form fl-D-glucose. The rate of fl-D-glucose formation depends linearly on the sucrose concentration in the measuring cell up to 10 mM. Glucose concentrations up to 0.5 mM do not interfere. 12 This principle of successive determination can be extended to several substances in one and the same sample by applying other immobilized disaccharidases. The procedure combines the advantages of enzyme i1 R. R e n n e b e r g , D. Pfeiffer, F. Scheller, and M. J~nchen, Anal. Chim. A c t a 134, 359 (1982). 12 F. Scheller and C. K a r s t e n , Anal. Chim. Acta 155, 29 (1983).

[3]

C O U P L E D E N Z Y M E REACTIONS IN E N Z Y M E ELECTRODES

39

T~j-inverfase inverfase~!~if s-I-irrinbar g

'! enzyme elecJrrode

P

FIG. 6. Enzyme reactor electrode for measurement of sucrose and glucose using an immobilizedinvertase reactor and a GOD electrode.

membrane electrodes with those of sequential reactors, i.e., short response time for glucose measurement, simple setup of the stirred measuring cell, and subsequent interference-free sucrose determination. Substrate Amplification If very low substrate concentrations are to be determined, the sensitivity of enzyme electrodes can be enhanced by using enzymatic substrate amplification. A dehydrogenase and an oxidase, both acting on the same substrate, may be used for this purpose. Operational conditions have to be adjusted in such a way that the dehydrogenase catalyzes the regeneration of the oxidase substrate. The substrate is continuously shuttled between the two enzymes. As a consequence, the electrochemically active product is formed in much higher amount than that of substrate diffusing into the membrane. Therefore the current exceeds that of a diffusion-controlled process. Substrate amplification is exemplified by the GOD-glucose dehydrogenase (GDH, EC 1.1.1.47) system. The gluconolactone formed in the GOD-catalyzed

40

[3]

ANALYTICALAPPLICATIONS

.......

,.,::,

:-7:( ::'.i

GDH-GOD membrane

:

~

v ..'..

.'.: ~ :'.j .'.".';" : :.:.'." ."

• 02

"~.i':

i glucose

NADH

FIG. 7. Substrate amplification principle demonstrated by a G O D - G D H electrode for measurement of glucose.

reaction is reconverted to glucose in the GDH-catalyzed reaction with NADH (Fig. 7). Twenty units GOD and 10 U GDH/cm 2 of the gelatin layer are coimmobilized. The glucose measurements are performed in 0.1 M phosphate buffer, pH 7.0, containing variable amounts of NADH (Fig. 8). The electrode is polarized to -600 mV. The glucose sample is added after attaining a constant baseline. An amplification factor of 8 is obtained in the solution containing 10 mM cofactor. The limit of detection is 0.8 /~M glucose. Amplification makes use of a part of the GOD excess which is unused in the absence of NADH. The overall reaction represents the oxidation of NADH by oxygen, with glucose acting as the mediator between the two enzymes. Enzyme Competition The problem of measuring cofactor concentrations with electrochemical sensors may be solved if a cofactor-dependent enzyme is coupled with an oxidase, with both competing for the same substrate. The competition of GOD and GDH for glucose was used to develop a bienzyme electrode for determination of the oxidized cofactor, NAD ÷ (Fig. 9). The reaction between glucose and NAD ÷ is characterized by the equilibrium constant, K -- 3 × 107 M. ~3The reaction proceeds in both directions depending on the concentrations of the partners. The above-described GOD-GDH 13 C. C. Liu and A. K. Chen, Process Biochem. Sept./Oct., p. 12 (1982).

[3]

COUPLED ENZYME REACTIONS IN ENZYME ELECTRODES

41

/°'°°/

[/0

,o

.28mM/

0

~E20

0.1/+mMglucose 0

r-~

X ,s

~ose

10

ell/× 7x j

i t 5 10 CNADHIron ] FIG.8. Dependenceof the signalof the GOD-GDBelectrodeon NADH concentrationat different glucose concentrations. Conditions are as in Fig. 1.

0

glucose

NAD+

FIG. 9. Enzyme competition electrode for measurement of N A D ÷ using a G O D - G D H membrane.

42

ANALYTICALAPPLICATIONS

[3]

membrane is used in combination with electrochemical measurement of oxygen consumption at -600 mV. Glucose, I mM in 0.1 M phosphate buffer, pH 7.0, is used as the background solution. Glucose conversion by GOD causes a decrease in the current. The addition of the NAD+-containing sample switches on GDH, which competes with GOD for the common substrate, glucose. The degree of the NAD+-dependent reaction is reflected by a current increase based on the diminished oxygen consumption in the GOD reaction. The calibration curve for NAD ÷ is linear up to a final concentration of 1 mM NAD+; however, the sensitivity is only about 3% as compared with that of glucose, i.e., 0.045 tzA/mM/cm 2. Furthermore the functional stability is limited to 3 days of operation. An enzyme electrode based on the competition between GOD and hexokinase has also been developed, measuring ATP in the concentration range 0.05-1 mM. 9 Antiinterference Layer Interference caused by different substances (substrates, inhibitors, activators) is a serious problem for the practical application of enzyme sensors. For coupled enzyme reactions the substrate of each particular enzyme may interfere if present in the sample. These interfering substrates can be eliminated by covering the enzyme sensor with an antiinterference layer. This layer contains immobilized enzymes which convert the disturbing substances to noninterfering products. In all enzyme electrodes using GOD, endogenous glucose from biological or food samples will interfere. This problem is overcome by converting the glucose by GOD and catalase (EC 1.11.1.6) to the electrodeinactive products, gluconolactone and water. The antiinterference layer and the GOD-based enzyme membrane are two spatially separated linear enzyme sequences (Fig. I0). For the purpose of disaccharide measurement in the presence of glucose, the bienzyme layer (e.g., glucoamylaseGOD or invertase-GOD) is covered with an antiinterference layer [containing GOD (46 U / c m 2) and catalase (320 U / c m 2 ) ] , in which f l - D - g l u c o s e (up to 2 mM glucose in the measuring solution) is completely eliminated. 14 Sucrose, maltose, starch, and a-amylase ~5are directly measurable in the presence of up to 2 mM glucose in juice of sugar beets or in samples of 14 F. Scheller and R. Renneberg, Anal. Chim. Acta 152, 265 (1983). ~5 R. Renneberg, F. Scheller, K. Riedel, E. Litschko, and M. Richter, Anal. Lett. 16, 877 (1983).

[3]

COUPLED ENZYME REACTIONS IN ENZYME ELECTRODES

43

e,ec+,od, N

/

gluconolac÷one GODi nver'~ase

[3 - D " ~

membrane'

glucose

a-D-glucose

in~erence

Jl : . "- "'..." .'.'."" :

--~

'lac'~i M-

i.i ~

U

'

p - D - glucose

~ 02

"' " ' " "

_

: ...........

sucrose

FIG. 10. Antiinterference principle. Sucrose is directly measured in the presence of glucose.

instant cocoa. The linear measuring range for sucrose extends up to 15 m M with a slope of 0.15 tzA/mM/cm 2. Concluding Remarks Coupled enzyme reactions obviously simulate the metabolic situation in organelles and microorganisms 16 which are frequently employed in biospecific electrodes) 7 However, the application of mixtures of isolated enzymes offers several advantages. Because of the absence of contaminant enzyme activities, the selectivity of enzyme sensors is higher than that of organelle or microbial electrodes. Owing to the higher specific enzyme activity applicable in multienzyme electrodes, these are also superior with regard to sensitivity and response time.

t6 p. A. Srere, B. Mattiasson, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 70, 2534 (1973). ~7 M. Hikuma and T. Yasuda [10], I. K a r u b e [11], and F. S c h u b e r t and F. W. Scheller [13],

this volume.