Sandwich-Type Amperometric Enzyme Electrodes for Determination of

Chapter 6

Sandwich-Type Amperometric Enzyme Electrodes for Determination of Glucose 1

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S. Mutlu , M . Mutlu , P. Vadgama , and E. Pişkin 1

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Department of Chemical Engineering, Department of Food Engineering, University of Hacettepe, 06530 Ankara, Turkey Department of Medicine, Clinical Biochemistry Section, University of Manchester, Salford M6 8HD, United Kingdom 3

Sandwich type amperometric enzyme electrodes for glucose based on oxidases were studied. Polyethersulphone (PES) and polycarbonate (PC) membranes were used as inner and outer membranes, respectively. Glucose oxidase was entrapped between these two membranes. The selectivity of the inner membrane against a group of electroactive compounds in blood were tested. The PES membrane fulfilled all the requirements of a permselective membrane. The outer surface of PC membranes were further modified by deposition of hexamethyldisiloxane (HMDS) in a glow-discharge reactor or by coating with polyvinylalcohol (PVAL) to increase the linear range of the electrode while keeping or improving the blood-compatible properties of membrane. Linearity of these enzyme electrodes were investigated in different media (i.e., buffer solutions, blood plasma and blood). Blood-compatibilities of the PC membrane and its modified forms were obtained in in-vitro experiments. HMDS treatment significantly increased the linearity of enzyme electrode but did not affect their blood-compatibilities or significantly change the selectively. Also, the P V A L coating, considerably, extended the linear range of the electrode, slightly improved blood-compatibility but did not change the selectivity. In the last decade, a rapid technological evolution took place in the field of chemical sensors in general, and enzyme based sensors in particular (7). This progress has been at least partly due to the commercial interest in sensor technology for diagnostic medicine, both for real-time analysis of metabolites in metabolically unstable patients and rapid bed-side monitoring (2-3). Sandwich type amperometric enzyme electrodes are amongst the most widely studied electrodes for diagnostic medicine. A typical sandwich type enzyme electrode consists of three major layers, i.e., outer membrane, enzyme layer and inner membrane. The outer membrane, where the analyte diffuses through should have diffusion-limiting property which provides a means of extending linear range through their reduction in local substrate concentration while maintaining sufficient oxygen 4

Current address: P.K. 716, Kizilay, 06420 Ankara, Turkey

0097-6156/94/0556-0071$08.00/0 © 1994 American Chemical Society

Usmani and Akmal; Diagnostic Biosensor Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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DIAGNOSTIC BIOSENSOR POLYMERS

for the enzymatic reaction. In the middle, the enzyme layer accepts the analyte and necessary oxygen to achieve the enzyme-substrate reaction and to produce electrochemically detectable species. The immobilization of the enzymes is generally maintained by gel-entrapment. The inner membrane with permselective property prevents the interference by other electrochemical species on electrode (4-5). A number of techniques to improve linearity and selectivity, and also biocompatibility and enzyme utility of sandwich type electrodes have been described elsewhere (6). However, There are only a few approaches in use in screening new electrodes for linearity, selectivity and biocompatibility that were proven to be of value and accepted by majority of researchers. Recently, we have studied a variety of sandwich type electrodes for development of enzyme electrodes based on oxidases. In this paper, we focused on extending linearity and blood-compatibility of the outer membrane, i.e., polycarbonate (PC) membrane, without affecting the selectivity. We modified the outer surface of this membrane by plasma polymerization of hexamethyldisiloxane (HMDS) in a glowdischarge reactor or by coating with polyvinylalcohol (PVAL). The P V A L coated surfaces were further treated with an anticoagulant, i.e., heparin. We studied the linearity, selectivity and blood-compatibility of these membranes and related electrodes. Here, we present the results of these studies. Construction of the Electrodes Materials. Polycarbonate (PC) membranes (i.e., outer membrane) with nominal pore size ranging from 0.01 to 0.05 μπι (rated by manufacturer) were supplied by Poretics (USA), hexamethyldisiloxane (HMDS) was obtained from Wacker (Germany). The enzyme, i.e., glucose oxidase (E.C.I.1.3.4. from Aspergillus Niger: 292IU mg" protein) was purchased from Sigma (UK). Polyethersulphone (PES) and polyvinylalcohol ( P V A L , with an average molecular weight of 50000, 100% hydrolysed) were supplied by ICI (UK) and by Aldrich (Germany), respectively. Gluteraldehyde (25% aqueous solution), bovine serum albumin (fraction V), glucose, solvents, buffer components and other standard reagents were obtained from Sigma (UK). Cellulose acetate (CA; 39.8% acetyl content) was obtained from Aldrich (Germany). Blood plasma was donated by the "Kizilay Central Blood Bank" (Ankara, Turkey). Human blood were taken from volunteers. 1

Electrode. The main body of the sandwich type of electrode that we used in this study was a Rank oxygen electrode system (Rank Brothers, Bottishom, Cambridge), which is schematically described in Figure 1. The electrode consisted of a central 2 mm diameter platinum working electrode with an outer 12 mm diameter silver ring as the reference. The electrode was polarized at 650 mV (versus Ag/AgCl) for hydrogen peroxide detection. The meter was linked to a strip-chart recorder. Outer Membrane. The outer membrane was a track-etched polycarbonate membrane, which was purchased from Poretics. PC membranes with three different nominal pore sizes of 0.01, 0.03 and 0.05 μπι were tested. The outer surfaces of these membranes were further modified by following methods: (1) glow-discharge treatment and (2) chemical coating. G l o w - D i s c h a r g e Treatment. H M D S was deposited by plasma polymerization on the PC membranes in a glow-discharge reactor system shown schematically in Figure 2. The reactor was a glass tube (inner diameter: 6.4 cm and length: 35 cm) with two copper electrodes (surface area of each electrode: 140 cm ) mounted on the outside. 2


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Sandwich-Type Amperometric Enzyme Electrodes

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Membrane layer Platinum working electrode Ag/AgCl reference electrode

[ZzD ο ο Measuring device

Figure 1. Schematic description of amperometric electrode system. Radiofrequency generator (Tasanm Ltd. Co.-Turkey, frequency: 13.6 MHz and power range: 0-100 W) was used to form the plasma in the reactor. Power losses were kept to a minimum by a matching network. In a typical glow-discharge treatment, two membranes placed back to back (outer surfaces facing the plasma medium) were placed in the middle of the reactor. The reactor was evacuated to 10-MO mbar. The monomer (i.e., HMDS) was allowed to flow through the reactor at a flow rate of 30 ml/min. The PC membranes were exposed to plasma medium for 5 min and 20 min at a discharge power of 10 W. -4

Electrode

Needle Valve

r-xh-d

Needle Valve

/

1 ' Membrane

1 1

1 . Plasma Reactor Vacuum Pump

—

Monomer Tank

CD

Matching \-TQ I network — Watt Meter 1

R.F.G.

Mains

Electrode

Figure 2. Glow-discharge apparatus. Chemical Coating. The PC membranes were also coated with P V A L by a two-step procedure. Details of this procedure have been described previously (7). Briefly, a monolayer of P V A L molecules was first deposited on the outer surface of the PC membrane by a simple adsorption process. The P V A L concentration in the adsorption medium was 4 x l 0 g/ml. The PC membrane (16 cm ) was incubated with 130 ml of this solution for 2 h at room temperature (20°C). Next, P V A L molecules -3

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adsorbed on the PC membrane surface were crosslinked in order to have a non-watersoluble P V A L coating. Crosslinking was achieved chemically either by using terephtalaldehyde (at 80°C) or by exposing to γ -irradiation (0.2864 Mrad). In order to include an anticoagulant activity on these P V A L coated membranes, these membranes were activated (through hydroxyl groups) by using C N B r as an activating agent, and then heparin was attached through the active sites, covalently. Details of this procedure have been described previously (8). Briefly, an activating solution containing CNBr (CNBr concentration: 0.0166 g/ml) was prepared and its pH adjusted to 11-12 with 2M NaOH. The PC membrane (surface area: 32 cm ) was then added to the activation solution and incubated for 90 minutes. After completion of the activation step, the membrane was incubated with a heparin solution (heparin concentration: 3.33 mg/ml) at room temperature for 4 h. 2

Inner Membrane. Polyethersulphone (PES) membranes were prepared by a solvent casting technique (9). A 6% (w/v) solution of PES in a mixture of 2-methoxyethanol and D M F (25% + 75%, v/v) was cast on a glass plate. The polymer film (the thickness : 25 μπι) was then formed by evaporation of the solvents in a conditioned room (relative humidity: 80%, and temperature: 20°C). The membranes were stored in vacuum desiccator until use. As an alternative to the PES membranes, cellulose acetate (CA with 39.8% acetyl content) membranes (with a thickness of 25 μπι) were also prepared by solvent casting method. The composition of the casting solution was 2% C A (w/v) in acetone. Film formation was achieved by evaporation of acetone at room temperature (20°C) in 80% relative humidity environment. PC and its glow-discharge modified forms were also tested as the inner membrane. Enzyme Layer. The enzyme layer was prepared by crosslinking of glucose oxidase (GOD) with glutaraldehyde, in the presence excess bovine serum albumin (BSA). In a typical procedure, 30 mg of GOD was mixed with a 200 mg B S A in 1 ml of buffer solution. A 6 μΐ volume of this mixture was then rapidly mixed with 3 μΐ of glutaraldehyde (5%, v/v) in a Gilson pipette tip and placed between the PC and PES membranes. This sandwich was then compressed between two glass slides and held under hand pressure for 5 minutes. The glass slides were pried apart and the membrane/enzyme laminate was left to dry at room temperature for a further 5 minutes, washed with buffer and then placed over a Rank oxygen electrode. Performance of the Electrodes Three different types of tests, i.e., selectivity, linearity and blood-compatibility were investigated to ascertain the performance of the electrodes. Selectivity Tests. In these tests, a Rank electrode was used with a single membrane layer, instead of the sandwich type of laminate. The PES, PC and its modified forms, and C A membranes were used in these tests. Hydrogen peroxide ( H 0 ) , catechol, uric acid, paracetamol and ascorbate were used as the analytes. Each analyte was used separately. The initial concentration of the analyte was 0.2 mM. 2

2

Linearity Tests. In these tests, sandwich type of laminates were prepared by using the PC membrane with a 0.05 μπι pore size as the inner membrane. The GOD layer was prepared by the method described above. As the outer membrane, the PC membranes with different pore sizes (i.e., 0.01, 0.03, 0.05 μπι), the glow-discharge treated PC membrane (PC/HMDS), the polyvinylalcohol coated PC membranes (PC/PVAL) and the heparin attached P C / P V A L membranes (PC/PVAL/H) were


6. MUTLU ET AL.


tested. In these tests, the test solution was an isotonic buffer solution (pH: 7.4) containing 0.0528 moV\ N a H P 0 , 0.0156 mom N a H P 0 , 0.0051 motfl NaCl and 0.00015 mol/1 E D T A . In the tests, the enzyme electrode was first allowed to stabilize for 5-10 min in the buffer solution, before the first reading was recorded. Then, 5 μΐ of an aqueous solution of glucose was added to achieve a 2 raM glucose concentration in the medium. The response was taken. This procedure was repeated several times, without changing the solution but adding enough glucose solution to increase its concentration 2 m M each time, until the deviation from linearity was observed. 2

4

2

2

Blood-Compatibility Tests. Blood compatibility tests were carried out in three groups: (1) protein-membrane interactions; (2) blood-membrane interactions; and (3) blood cloting times on the membranes. The protein-membrane interaction tests were carried out by using plasma and plasma proteins (i.e., human serum albumin, HSA, human serum fibrinogen, HSF and human immunoglobulin, HIgG) obtained from the Kizilay Blood Bank (Ankara, Turkey). These proteins were labelled with Tc-pertechnetate as described elsewhere (10). In a typical test, a 16 c m of the membrane sample was placed into a pre-silanised petri-dish containing 5 ml of plasma with proper amounts of radiolabeled proteins. After a 30 minutes of contact time, the membrane was washed with isotonic solution, and the radioactivity of both on the membrane and the plasma sample from the petri-dish was measured by a gamma-scintillation counter (Berthold, BF 2500, Germany). In the blood-membrane interaction studies, fresh blood samples taken from healthy human donors were used. 5 ml of human blood was exposed to a 32 c m of membrane in a pre-silanised blood-sampling tube for 1 h, by continuously rotating the tubes. At the end of interaction period, the blood samples were homogenized and the blood cells were counted by using a coulter counter (Coulter T C 10, USA). Plasma of the blood samples were seperated by centrifuging at 3500 rpm for 10 minutes. The plasma samples were then analyzed by biochemical autoanalyser (Ciba-Corning, Express 550, USA) to determine the concentrations of the HSA, HSF and total protein. In the blood-clotting time experiments, fresh blood samples taken from healthy human donors were used. The membranes were contacted with the non-heparinised blood, the extrinsic and intrinsic clotting times (PTZ and PTT, respectively) were determined by using a Coagulameter (Amelung KC-10). 99m

2

2

Result and Discussion Description of the Electrode. Sandwich type amperometric enzyme electrodes are amongst the most widely studied electrodes for diagnostic medicine (77). The working principle of a typical sandwich type glucose electrode is schematically described in Figure 3. The outer membrane mainly controls the substrate (i.e., glucose) diffusion and therefore provides the linearity. This membrane should also allow sufficient oxygen transfer which is vital for the enzymatic reaction. It should be noted that the blood-compatibility of the outer membrane is one of the most, important considerations. If it is non-compatible, blood proteins and cells (mainly platelets) are accumulated at the blood-contacting side of the membrane, and may diminish very significantly the transport of the substrate and therefore cause the failure of the electrode (72). In the middle of the sandwich laminate, an enzyme layer is located which reacts with the substrate by using oxygen and produces electrochemically detectable species (e.g., H2O2). Note that the enzyme is used in the immobilized (crosslinked) form in order to prevent the enzyme leakage and to increase the enzymatic stability.


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To control the enzymatic conversion rate, the enzyme concentration in this layer is controlled by adding diluting agents. In the case of the glucose electrode constructed in this study, GOD molecules were co-crosslinked with B S A molecules in a proper ratio (i.e., 30/200) to control the enzymatic activity.

Blood Interferences

Silver Cathode

Blood Glucose Red Blood Cells Clark Oxygen Electrode Inner Membrane | Outer Membrane (CA or PES) (PC or Modified PC) Immobilized Glucose Oxidase

Figure 3. Configuration of a biosensor. In the glucose electrode, the reaction product is hydrogen peroxide. H2O2 molecules pass through the inner membrane, reach to the electrode surface, and create the electrode response by oxidation. The most important property of this inner membrane is it's permselectivity. This membrane should only permit the H2O2 molecules, but not the interfering molecules (e.g., catechol, uric acid, paracetamol and ascorbate) which may be present in blood (73). Selectivity Tests. As described above, in this group of studies the selectivity of some candidate membranes (PES, C A , PC and PC/HMDS) were studied. Note once again that each membrane was tested as a single layer, not a whole sandwich. Electrode responses in nanoampers were measured for each electroactive analytes (i.e., hydrogen peroxide, catechol, uric acid, paracetamol and ascorbate) with an initial concentration of 0.2 mM. Figure 4 shows permeabilities of some selected membranes tested in this study. Notice that the H 0 permeabilities of all the membranes presented here were approximately equal except PC (pore size: 0.05 μπι). However, the permeabilities of the other electroactive compounds were significantly different. The original polycarbonate (PC) membrane allowed transport all of the analytes mentioned above. The glow-discharge treatment diminished the respective permeabilities but considerable permeations of the electroactive compounds were still observed. Therefore, it can be concluded that, the PC based membranes cannot be used as the inner selective membrane due to the strong interference of the potential electroactive compounds which would exist in the direct blood applications. Cellulose acetate (CA) membranes with different pore structures were prepared by solvent casting and tested here as potential inner membranes. Permeabilities of different analytes through a typical C A membrane were given in Figure 4. Note that this membrane give the best performance of all C A membranes tested in this study, 2

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6. MUTLU ET AL.

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with respect to interférences. As shown here, the H 0 permeability was high, it was not permeable for most of the electroactive compounds tested here, except catechol ions. It was not possible to prevent the passage of ascorbate ions through any of the C A based membranes that we have prepared. Therefore, we decided not to consider the C A based membranes as the inner membrane of our sandwich laminates. Very promising results were obtained with the polyethersulphone (PES) membranes. It is clearly observed from Figure 4 that, the PES membrane prepared by us following the method described above fulfilled the all requirements from a permselective membrane. Namely, the PES membrane allowed the H 0 transport but not to the other interfering compounds (i.e., electroactive analytes). Therefore, we decided to use this membrane as the inner membrane for the construction of our sandwich type of glucose electrode. 2

2

2

2

1400 Electroactive Compounds 1200

• E9 H

1000

•

800

Peroxide Ascorbate Paracetamol Catechol Uric acid

600 400 200 0 PC(0.05)

PC(0.01)

PC(0.01)/HMDS

PES

CA

MEMBRANE TYPES

Figure 4. Permeabilities of some selected membranes. Linearity Tests. As mentioned before, one of the most important parameters of a sandwich type of enzyme electrode is its linearity. For the glucose measurements in the blood samples (e.g., measurement of glucose level in the blood of the diabetic patients) this linearity level is expected to be extended up to about 40 m M of glucose (14). To extend the linearity range of our sandwich type glucose electrode was one of the major goals of this study. We prepared sandwich type of laminates by using different type of PC based membranes as the outer membrane. The inner membrane was the PES membrane which was described in the previous section. The enzyme layer prepared by using 6 μΐ solution of GOD with B S A and 3 μΐ of glutaraldehyde (5%). The results of the studies related to the optimization of the composition of the enzyme layer were given elsewhere in detail (75). We used these laminates with the Rank electrode described above, and measured the electrode response of the glucose in buffer solutions (5 ml) containing different amounts of glucose (up to 100 mM).


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Two groups of studies were performed. In the first group, the PC membranes with three different pore sizes, namely 0.01,0.03 and 0.05 μπι were tested. Note that these PC membranes were prepared by a tract-etching method, which leads pores as straight cylindrical tubes (turtousity is about one) with approximately equal diameters as shown in Figure 5. Therefore, one expects significant difference in the transport properties due to sharp cutoffs. The effect of pore size of the PC membranes on the linearity of the glucose electrode is given in Figure 6. It is clearly shown that the linearity increased as the pore size decreases. Therefore, we concluded that this type of tract-etched PC membranes containing pores with narrower diameter would be more suitable for the extension of the linearity range. However, it should be noted that there is no tract-etched PC membrane having pores with a diameter smaller then 0.01 μπι commercially available.

Figure 5. S E M micrograph of plain PC membrane with 0.03 μπι pore size (Mag: Χ 60K). 500

Glucose Concentration (mM) Figure 6. The effect of pore size on the electrode linearity.


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In order to increase the linearity range of the commercially available PC membranes by decreasing the pore size, we attempted to use a novel technique, which is so-called "glow-discharge treatment". Note that by this technique, it is possible to change the surface properties of a membrane without significantly changing the bulk structure (16). We deposited hexamethlydisiloxane (HMDS) on the PC membranes with different pore sizes by plasma polymerization in a glowdischarge reactor. We changed the glow-discharge conditions (i.e., discharge power and period, monomer flow rate and pressure in the reactor) in order to optimize the treatment. Details of these studies were given elsewhere (17). Here, we presented the data related to the H M D S glow-discharge treated membrane prepared at optimum conditions which are as follows: treatment time: 20 min, monomer flow rate: 30 ml/min, pressure: ΙΟ^-ΙΟ mbar, discharge power: 10 W). Our previous experience lead us to select H M D S as the monomer for the treatment. We have coated several nonblood-compatible materials by plasma polymerization of this monomer in a glow-discharge system to create bloodcompatible surfaces for diverse biomedical applications (75-27 ). We have shown that this treatment significantly increase the blood-compatibility without affecting the substrate material properties. Figure 7 illustrates the effect of glow-discharge modification on the glucose electrode linearity. As shown here the glow-discharge treatment significantly increased the linearity. For instance, the linearity range of the original PC membrane with a diameter of 0.01 μπι was 10 m M of glucose, while the linearity range was extended to 35 m M of glucose when this PC membrane was treated with H M D S (i.e., the PC/HMDS membrane) in the glow-discharge reactor. The scanning electron micrograph of the PC/HMDS membrane is given in Figure 8. As seen here, at the glow-discharge conditions applied here, there was an observable size reduction of the pores from 50 nm to 34.2 nm, which may cause the extension of the linearity range of the electrode due to the changes in the mass transfer properties of the membrane after treatment. -4

0

10

20

30

40

50

60

Glucose Concentration (mM) Figure 7. The effect of glow discharge modification of surfaces on the electrode linearity.


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In this group of experiments, linearities of two more membranes were also tested. These membranes were the polyvinylalcohol coated PC (PC/PVAL) and its heparin attached form (PC/PVAL/H). As it will be discussed in the coming section, these modifications were carried out to increase both the linearity and the blood-compatibility of the PC membranes. Figure 9 shows the linearity test results of these membranes. As seen here, the P V A L coating (both chemically crosslinked and crosslinked with γ-irradiation) extended the linearity range of the original PC membrane from 10 m M to about 30 m M of glucose. The heparin attachment did not change the linearity further. The effect of the P V A L coating may be explained by considering the change in the mass transfer properties of the original PC membrane.

Figure 8. S E M micrograph of glow-discharge treated PC membrane with a pore size of 0.05 μιη (Mag: Χ 60K).

Figure 9. The effect of chemical modification of surfaces on the electrode linearity.


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Sandwich- Type Amperometric Enzyme Electrodes

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Blood Compatibility Tests. The aim of this study is to develop an enzyme electrode for the measurement of glucose levels mainly in vivo. In addition to developing a system that is linear over the physiological range another goal was to develop a system that is biocompatible. For this purpose, we conducted the following standard blood-compatibility tests. In the first group of experiments, three main blood proteins, i.e., albumin (HSA), fibrinogen (HSF) and globulin (HIgG) were radiolabeled with 9 9 m . pertechnetate. These radiolabeled proteins were added within 5 ml of blood plasma samples obtained the Kizilay Blood Bank (Ankara, Turkey). The potential membranes tested in this study as the outer membrane were then incubated with the plasma samples. After 1 h of incubation period the radioactivity levels both in the plasma phase and on the membrane surfaces were counted. Figure 10 shows the protein adsorption on different membrane surfaces. Note that the values on the vertical axis in the figure are relative values in which adsorptions of each protein on the original PC membrane were assumed to be 100. As seen here, adsorption of blood plasma proteins on the P V A L coated PC membrane were considerably lower than the PC surfaces. These results are very similar to the literature stating that P V A L is a very inert surface (22 ). But, it should be noted that inert surfaces are not always blood-compatible, they may indirectly cause other incompatibility problems, such as platelet activation and agglomeration, complement activation, etc. (23). There were significantly higher level of protein adsorption on the H M D S glowdischarge treated surface. However, it should also be noted that high protein adsorption does not lead always non-compatibility. The type of the protein layer and its time depended behaviour should be considered (24 ). In the second group of experiments, membranes were incubated directly with blood in heparinized tubes. After l h incubation period, blood cells were counted by means of a coulter counter. The blood cell counts were given Table I. Note that the tube containing no membrane was called as "control". A l l of the membranes caused drop in the blood cell counts (especially in platelet counts), however,these changes may not be considered as significant. In addition, the differences between the membranes were also not very noticeable. T c

•

Albumin Fibrinogen

•

PC(0.01)

PC(0.01)/HMDS

Globulin

PC(0.01)/PVAL

MEMBRANES Figure 10. Protein adsorption on different membrane surfaces.


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Table I. The Change in the Amount of Blood Cells After 1 h of Contact with Different Membranes in Total Human Blood Membrane

Erytrocyte (10 /1)

Leucocyte (10 /1) 9

Control PC PC/HMDS PC/PVAL PC/PVAL/H

12

6.4 5.7 5.6 5.6 5.9

5.14 4.89 4.92 4.81 4.84

Thrombocyte (10 /1) 9

165 147 139 142 130

In the third group of experiments, the extrinsic and intrinsic blood clotting times were determined. Table II summarizes these data. A l l of the membranes tested here did only slightly changed the extrinsic blood clotting times (PTZ) and the intrinsic blood clotting times (PTT). The PTT value for the PC membrane was little higher than the other (better surface), but this is again not very significant. Table Π. The Extrinsic and Intrinsic Blood Clotting Times for Various Membrane Surfaces Membrane Control PC PC/HMDS PC/PVAL PC/PVAL/H

PTZ (sec)

PTT (sec)

14 16 15 15 16

45 54 50 51 50

Conclusion In this study, we aimed to develop a sandwich type of glucose electrode to measure the glucose level in blood. The results of the selectivity tests showed that the PES membrane permits only H2O2 but not the other electroactive analytes which may interfere the electrode response in blood applications. The linearity and bloodcompatibility of the outer membrane have been considered as two important parameters in blood contacting applications of enzyme electrodes. This study showed that the linearity of the original system utilizing untreated polycarbonate membranes could be extended by either glow discharge treatment with H M D S or chemical coating by P V A L . These modifications also caused different extents of adsorption of blood plasma proteins. However, if we consider the insignificant changes in the levels of the blood cells and also in the clotting times, we can say that the respective modifications do not improve the blood compatibility of the original PC membranes, which we were not expecting. If we consider both the linearity and bloodcompatibility at the same time, we may concluded as the H M D S glow-discharge treated or the PVAL-heparin coated membranes should be used as the outer membrane of the sandwich type of glucose electrode described in this study.


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Acknowledgement Dr. Mehmet Mutlu gratefully acknowledges financial support from International Atomic Energy Agency (IAEA) (Grant No: C6/TUR/8813). The authors wishes to thank Abbas Yousufi Rad for his technical assistance. Literature Cited 1. Biosensors: Fundamentals and Applications; Turner, A.P.F.; Karube, I. and Wilson, G. S., Eds.; Oxford University Press, Oxford, 1987. 2. Matthews, D. R.; Holman, R. R.; Bown, E.; Steemson, J.; Watson, Α.; Hughes, S. and Scott, D. Lancet1987,vol1,778. 3. Vadgama, P.; Alberti, K.G.M.M. In Recent advances in Clinical Biochemistry ; Price, C. P. and Alberti, K.G.M.M. Eds.; Churchill Livingstone, 1985, Vol. 3. 4. McDonnell, M. B. and Vadgama, P. M. Membranes: Separation Principles and Sensing; Selective Electrode Review ; 1989; Vol. 11, 17-67. 5. Vadgama, P. In Biosensors in vitro and in vivo; Clinical Biochemistry Nearer the Patient II ; Marks, V. and Alberti, K.G.M.M. Eds.; Bailliere Tindall, 1986. 6. Tang, L.X.; Koochaki, Z.B.; Vadgama, P. Analytica Chimica Acta, 1990,vol 232, 357-365. 7. Boomgard, V.D.; King, T.A.; Tadros, F.; Tang, T.; Vincent, B. J. Colloid InterfaceSci.,1978,vol 66, 68. 8. Kohn, J.; Wilchek, M. App. Biochem. Biotech. 1983, vol 8, 227. 9. Battersby C. M. and Vadgama, P. Analytica Chimica Acta 1986, vol 183, 59-66. 10. Atkins, H.L.; Richards, P. United States Atomic Energy Committee Publication, 1990, BNL-11831, 1-19. 11. Vadgama, P.; Desai M. A. In Biosensor Principles and Applications; Blum, L. J. and Coulet, P. R. Eds.; Marcel Dekker, Inc., 1991, 303-338. 12. Woodward, S. C. Diabetes Care 1982, vol 5, 278-281. 13. The Merck Manual of Diagnosis and Therapy , Holvey, D. N. and Talbott, J. H., Eds.; Merck & Co., Inc., 1972, 1826-1830. 14. Samson Wright's Applied Physiology, Keele, C. A. and Neil E., Eds.; Oxford University Press, 1961, 461-473. 15. Mutlu, M.; Mutlu, S.; Vadgama, P. In 1991 IChemE Research Event; Kenny, C.N. Darton, R. Davidson, P.J. Seaton, N.A. and Stitt E.H. Eds.; Institute of Chemical Engineers, Rugby, UK, 1991. 16. Pişkin, E. J. Biomater.Sci.Polymer Edn. 1992, vol 4, 45-60. 17. Mutlu. S., Ph. D. Dissertation, Hacettepe University, Ankara, 1993. 18. Mutlu, M.; Mutlu, S.; Rosenberg, M.F.; Kane, J.; Jones, M.N.; Vadgama, P. J. Materials Chemistry,1991,vol1(3), 447-450. 19. Mutlu, M.; Ercan, M.T.; Pişkin, E. Clinical Materials, 1989,vol 4, 61-76. 20. Pişkin, E.; Evren, V. Life Support Systems,1986,vol4, 2-17. 21. Özdural, Α.; Hameed, J.; Bölük, M.Y.; Pişkin, E. ASAIO J, 1980, vol 3, 116119. 22. Brinkman, E.; van der Does, L. and Bantjes, A. Biomaterials 1991, vol 12, 6370. 23. Bruck, S.D. J. Biomat. Med. Dev. Artif. Org., 1973,vol 1(1), 79-98. 24. Andrade, J. D.; Hlady, V.J.Biomater.Sci.Polymer Edn. 1991, vol 2, 161-172. RECEIVED September 17, 1993


Sandwich-Type Amperometric Enzyme Electrodes for Determination of

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