glucose oxidase electrode for determination of glucose

Analytica Chimica Acta, 187 (1986) 39-45 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

LACCASE/GLUCOSE OF GLUCOSE

U. WOLLENBERGER,

OXIDASE

F. SCHELLER*

Central Institute of Molecular Biology, Buch (German Democratic Republic) V. A. BOGDANOVSKAYA A. N. Frumkin (U.S.S.R.)

Institute

ELECTRODE

FOR DETERMINATION

and D. PFEIFFER Academy

of Sciences

of the GDR, 1115 Berlin-

and M. R. TARASEVICH

of Electrochemistry,

Academy

of Sciences

of the USSR, Moscow

G. HANKE District

Policlinic

Schwerin,

27 Schwerin

(German Democratic

Republic)

(Received 24th February 1986)

SUMMARY The electrode involves a layer of co-immobilized glucose oxidase and lactase in a gelatin membrane placed over a modified oxygen electrode. Hexacyanoferrate(II1) is added to the samples to oxidize reductive interferents such as ascorbic acid, and the hexacyanoferrate(I1) formed is re-oxidized by a lactase-catalyzed reaction. Ascorbic acid is completely eliminated up to a concentration of 20 mM in the sample.

A well established principle of blood glucose measurement with enzyme electrodes is the combination of an immobilized glucose oxidase membrane and a hydrogen peroxide electrode [ 1, 21. However, amperometric detection of hydrogen peroxide suffers from interferences from other oxidizable substances present in biological samples, e.g., ascorbic acid, glutathione, cysteine, uric acid and bilirubin. In order to eliminate these substances, hydrogen peroxide-permselective membranes have been used [3, 41. In addition, electrochemical interferences have been compensated by simultaneous use of a non-enzymatic membrane electrode [5]. In spectrophotometric assays of blood glucose and cholesterol, samples have been pretreated with soluble bilirubin oxidase, ascorbate oxidase and lactase to remove their respective substrates [6]. However, this method is expensive because of the high consumption of enzymes. Recently, multilayer enzyme electrodes for eliminating interfering substances have been described for sucrose measurements in glucose-containing solutions [ 7, 81. This paper describes interference-free glucose measurements with a bienzyme electrode bearing a layer of co-immobilized glucose oxidase and lactase. The value of this method is exemplified for glucose determinations in glucose/ascorbic acid mixtures.

40 EXPERIMENTAL

Chemicals Catalase-free glucose oxidase (GOD) from Penicillium no tatum (E.C. 1.1.3.4., 46 U mg-‘) was purchased from VEB Arzneimittelwerk Dresden. Lactase from Polyporus uersicolor (diphenol: oxygen oxidoreductase; E.C. 1.10.3.2.; 180 000 U ml-‘) was obtained from the Institute of Biochemistry, Academy of Sciences, Armenian SSR. Other reagents used were of analytical grade. The background solution was a Sorensen phosphate buffer of pH 5.0, with or without 5.0 mM potassium hexacyanoferrate(II1). Membrane preparation Glucose oxidase was immobilized alone or co-immobilized with lactase by entrapment in gelatin [9]. Acid photogelatin (50 ng; VEB Gelatinewerk Calbe) was suspended in 0.5 ml of double-distilled water and allowed to swell for 60 min at room temperature; 0.5 ml of 0.05 M phosphate buffer, pH 6.5, was added, and the solution was thoroughly mixed and heated in a water bath at 40°C for another 60 min. Glucose oxidase (2800 U) alone, or with 5600 U of lactase, was added and the mixture was gently stirred and cast on a flat polyvinyl chloride support. The liquid was spread over an area of 50 cm* with a glass rod. The enzyme layer was allowed to dry at room temperature for 6 h, and then removed from the support. The layer had a thickness of 20-30 pm. The bienzyme membrane contained 56 U cm-* glucose oxidase and 112 U crne2 lactase. The single-enzyme membrane contained 56 U cm-* glucose oxidase. Apparatus and procedure For preparation of the sensor, the mono- or bi-enzyme layer was fixed on the dialysis membrane (Nephrophan, 17 pm thick; VEB CK Bitterfeld, GDR) of the modified oxygen electrode, which comprised a platinum indicator electrode of 0.5 mm diameter (VEB Metra Radebeul, GDR) covered by a dialysis membrane held by an O-ring. For glucose determination by hydrogen peroxide detection, the platinum electrode was polarized at +600 mV vs. Ag/AgCl (0.1 M KCl). In the lactase reaction, all reactant are electrochemically measurable if the appropriate potential is applied. Thus, hexacyanoferrate(II1) reduction can be monitored, at +150 mV, hexacyanoferrate(I1) oxidation at + 600 mV and oxygen reduction at -600 mV. Each electrode was inserted into a measuring cell containing 2 ml of stirred Sorensen phosphate buffer, pH 5.0, at room temperature. Current/time curves obtained with the new electrode were measured with the GWP 673 polarographic system (ZWG Berlin, GDR) and a chart recorder. Sample volumes added to the buffer were
41 RESULTS

AND DISCUSSION

The principle of glucose measurement with the lactase/glucose oxidase electrode is shown in Fig. 1. It is based on the amperometric detection at +600 mV of hydrogen peroxide produced in the enzymatic conversion of glucose by the immobilized glucose oxidase: glucose + O2 + gluconolactone

+ HzOz

HzOz --f O2 + 2H’ + 2eAt +600 mV various substances besides hydrogen peroxide, e.g., ascorbic acid, are oxidized. These interfering substances are converted by reaction with hexacyanoferrate(II1) in the measuring cell to their respective oxidation products, which do not affect the hydrogen peroxide oxidation at the electrode. However, the hexacyanoferrate(I1) formed is also oxidized at +600 mV. In the bi-enzyme membrane, lactase catalyzes the re-oxidation of hexacyanoferrate(I1) by dissolved oxygen. In this way, consumption of oxygen is the only result of the removal of the electrochemically interfering reducing substances. Glucose oxidase electrode The relations between the response of the glucose oxidase electrode and the concentrations of glucose and ascorbic acid are shown in Fig. 2. The electrode has a linear concentration dependence in the range O-50 mM glucose in the sample. The signal for anodic oxidation of ascorbic acid is about 30% higher than the peroxide oxidation current for the same glucose concentration. The presence of ascorbic acid in the sample therefore causes a positive error in the measured glucose concentrations. The physiological concentration of ascorbic acid in blood is only ca. 60 PM, so its effect on

Fig. 1. Principle of glucose determination ing substances.

with elimination of electrochemically

interfer-

42

glucose determination in blood can be neglected, but this may not be so for glucose measurements in urine, soft drinks and juices. In order to eliminate this and similar interferences, the glucose oxidase/laccase membrane was produced and tested. Glucose oxidase/laccase electrode Provided that the appropriate potential is applied in the investigation of the lactase-catalyzed reaction, the substrates hexacyanoferrate(I1) and oxygen, and the product, hexacyanoferrate(III), can be detected with the electrode. Initially, the effectiveness of the immobilized lactase was tested by measuring the extent of oxidation of hexacyanoferrate(I1) in the enzyme layer by detecting the residual hexacyanoferrate(I1) reaching the electrode surface (Fig. 3, curve b). When first used, at +600 mV vs. Ag/AgCl, the electrode had an oxidation current that was almost zero up to a final hexacyanoferrate(I1) concentration of 4.3 mM, indicating complete oxidation of hexacyanoferrate(I1) in the lactase layer. Higher concentrations were only partially oxidized in the lactase layer, as is reflected by the linearly increasing oxidation current (Fig. 3, curve b). The reduction current at +150 mV gives evidence of hexacyanoferrate(II1) formation by lactase (Fig. 3, curve a). It increases linearly with hexacyanoferrate(I1) concentration up to 2 mM, with a slope 3 times that of hexacyanoferrate(I1) oxidation at +600 mV. These differences might be explained by the coupling of electrochemical and enzymatic reactions in this sensor. In amperometric electrodes, the concentration gradient of the electroactive substance at the membrane/electrode

$600 / 0

Fig. 2. Concentration dependence6 of the glucose oxidase electrode: ascorbic acid. Conditions: + 600 mV vs. Ag/AgCl (0.1 M KCl).

mV

(1) glucose;

(2)

Fig. 3. Relationship between the response of the glucose oxidase/laccase electrode (on the first day after preparation) and the final potassium hexacyanoferrate(I1) concentration. Ion detected: (a) hexacyanoferrate(II1) at + 150 mV (cathodic current), (b) hexacyanoferrate(I1) at + 600 mV (anodic current).

43

interface determines the electrode current. When hexacyanoferrate(I1) is monitored (+600 mV), there is a uniform concentration gradient from the bulk solution to the electrode. In the membrane, the concentration drops from the value at the membrane/solution interface to zero at the membrane/ electrode interface, in the diffusion-limited process, for<4.3 mM hexacyanoferrate(I1). In the product-sensitive mode, the hexacyanoferrate(II1) concentration rises from zero at the membrane/solution interface through a maximum and returns to zero at the electrode surface, so that diffusion occurs in two opposite directions [lo] . Furthermore, electrochemical regeneration of hexacyanoferrate(I1) by reduction of hexacyanoferrate(II1) at the electrode produces additional substrate formation for lactase and thus a cycling amplification [ 111. The stability of the immobilized lactase in the b&enzyme electrode was investigated by determining the maximum amount of hexacyanoferrate(I1) which is completely eliminated by lactase. Whereas this diffusion-controlled region extends to 4.3 mM on the first day of use, the limit decreases to 1.5 mM, 0.7 mM and 0.5 mM after 8, 10 and 14 days, respectively (Fig. 4). Taking into account a decrease of 65% in lactase activity over 8 days, the capacity of the lactase membrane is adequate for all practically important interfering substances if the sample is diluted 40-100 fold. Ascorbic acid, one of the potentially interfering substances in glucose measurements based on electrochemical hydrogen peroxide detection, was chosen to check the effectiveness of laccase/hexacyanoferrate(III) for removing interferences. For this purpose, the dependence of the current on the ascorbic acid concentration, both with and without 5 mM hexacyanoferrate(III), was recorded. Figure 5 shows that in the absence of hexacyanoferrate(III), at final ascorbic acid concentrations below 0.025 mM, there is

30 Id 0

25 20 -

H

2.0

4.0

6.0

K4 [F~(cN~J

[mM]

J

5

0.0 Ascorbic

ocld [mM]

Fig. 4. Dependence of the relationship between oxidation current and final measured hexacyanoferrate(I1) concentration on the time (days) after electrode preparation: (0) 1 d; (e) 8 d; (x ) 10 d; (0) 14 d. Fig. 5. Dependence of the b&enzyme electrode current on ascorbic acid concentration: (1) without hexacyanoferrate( III); (2) with 5 mM hexacyanoferrate(II1).

44

no current increase because lactase directly catalyzes the oxidation of ascorbic acid. This was proved by using a lactase/glucose oxidase electrode polarized at -600 mV. After addition of ascorbic acid to the measuring cell, oxygen consumption was observed. In the presence of 5 mM hexacyanoferrate(III), no current was observed for up to 0.2 mM ascorbic acid, giving evidence for complete oxidation of ascorbic acid by hexacyanoferrate(II1) and complete re-oxidation of the hexacyanoferrate(I1) formed by the immobilized lactase. Considering the sample dilution, 8-20 mM ascorbic acid in the sample (20-50 ~1) can be completely eliminated. Other reported electrodes, with a hydrogen peroxide permselective membrane [3] and a negatively charged membrane [4], rejected 5.68 mM and 0.0852 mM ascorbic acid, respectively. Figure 6 shows the response curves obtained by successive addition of glucose, ascorbic acid and hexacyanoferrate(II1) to the measuring cell in which the b&enzyme electrode was inserted. The introduction of 50 ~1 of 11 mM glucose produces an anodic current increase of 16.4 nA within 30 s because of the hydrogen peroxide formed in the glucose oxidase catalyzed reaction. When the current/time curve has reached a steady state, addition of 50 ~1 of 100 mM ascorbic acid causes a further current increase (29 nA) resulting from the electrochemical oxidation of ascorbic acid at the same potential as that of hydrogen peroxide. This current increase is only 15% of the response of a lactase-free electrode for ascorbic acid (Fig. 2) because

,.

1

I

0

05

10 CmMl

Fig. 6. Response of the lactase/glucose oxidase electrode to successive additions of 50 ~1 of 11 mM glucose, 100 mM ascorbic acid and 100 mM potassium hexacyanoferrate(II1) to 2 ml of pH 5.0 Sorensen phosphate buffer at +600 mV vs. Ag/AgCl (0.1 M KCl). Fig. 7. Dependence of the maximum of the first derivative of the current/time curve displayed by the GKM 02 unit on the final concentration of: (1) hexacyanoferrate(I1); (2) glucose or glucose with ascorbic acid (2.5 mM or 5 mM) and hexacyanoferrate(II1) (5 mM); (3) glucose in the presence of 5 mM ascorbic acid; (4) ascorbic acid at + 600 mV vs. Ag/AgCl (0.1 M KCl). The GKM 02 display indicates mM glucose in the sample.

\

45

lactase catalyzes the oxidation of some of the ascorbic acid in the membrane of the bi-enzyme sensor. The addition of 50 ~1 of 100 mM hexacyanoferrate(II1) results in a rapid current decrease to the level reached with glucose in the absence of ascorbic acid, illustrating the complete elimination of the effect of ascorbic acid. Measurement of glucose in samples containing ascorbic acid The calibration graph obtained for glucose using the enzyme electrodebased analyzer with the lactase/glucose oxidase electrode, was linear up to 50 mM. The ratio between the sensitivity to glucose as measured by hydrogen peroxide oxidation and the direct oxidation of ascorbic acid is 1:1.24 (Fig. 7). Ascorbic acid (5 mM) in glucose samples simulates an increased value of glucose concentration of 6.2 mM (Fig. 7). However, in the presence of 5 mM hexacyanoferrate(III), ascorbic acid does not increase the glucose signal. The concentration dependence shown for glucose in Fig. 7 is also the same for mixtures of glucose with ascorbic acid (5 mM or 2.5 mM) in the presence of 5 mM hexacyanoferrate(II1). Therefore an interference-free glucose measurement is possible. The concentration range studied exceeds the maximum ascorbic acid/ glucose ratio in blood and urine even in the case of therapeutic vitamin C treatment. Some initial results for the determination of glucose in urine obtained with the proposed anti-interference sensor compare well with the Polamat (reference method for glucose measurement in urine [12]), which show the potential of the proposed measuring system. For normal urine samples, an average glucose concentration of 1.3 mM was found by the lactase/glucose oxidase electrode. This value is in accord with the normal physiological value whereas the lactase-free sensor indicated values an average of 4.5 mM too high. This principle of elimination of interferences may be extended to other enzyme electrodes where hydrogen peroxide is detected anodically, e.g., to lactate or cholesterol sensors with their respective oxidases. REFERENCES 1 G. G. Guilbault and G. J. Lubrano, Anal. Chim. Acta, 64 (1973) 439. 2 F. Scheller and D. Pfeiffer, Z. Chem., 2 (1978) 50. 3 T. Tsuchida and K. Yoda, Enzyme Microb. Technol., 3 (1981) 326. 4 E. Lobe1 and J. Rishpon, Anal. Chem., 53 (1981) 5. 5 D. R. Thevenot, R. Sternberg and P. Coulet, Diabetes Care, 5 (1982) 203. 6 Jpn. Pat. DE 3239236,1982. 7 F. Scheller and R. Renneberg, Anal. Chim. Acta, 152 (1983) 265. 8 R. Renneberg, F. Scheller, K. Riedel, E. Litschko and M. Richter, Anal. Lett., 16 (1983) 877. 9 F. Scheller, D. Pfeiffer and M. Janchen, GDR Pat. GO1 N/127843, 1979. 10 D. A. Gough and J. K. Leypoldt, in L. B. Wingard, Jr., E. Katchalski-Katzir and L. Goldstein (Eds.), Applied Biochemistry and Bioengineering, Vol. 3, Academic Press, London, 1981, pp. 175. 11 T. Wasa, K. Akimoto, T. Yao and S. Murao, J. Chem. Sot. Jpn., 9 (1984) 1398. Diagnostische Laboratoriumsmethoden, 12 DAB 7 (D. L.), Deutsches Arzneibuch, Akademie-Verlag, Berlin, 1968.