Anal. Chem. 1994,66, 205-210
Glucose-Sensitive Enzyme Field Effect Transistor Using Potassium Ferricyanide as an Oxidizing Substrate Alexandre A. Shul’ga,’ Mllena Koudelka-Hep, and Nlco F. de Roolj Institut de Microtechnique, Universit6 de Neuchatel, Breguet 2, CH-2000 Neuchatel, Switzerland Larlssa I. Netchlporoukt LPCI LIRA CNRS 404, Ecole Central8 de Lyon, B.P. 163, 69131 Ecully Cedex, France
A glucose-sensitive field effect transistor was fabricated by immobilizing glucose oxidase on the gate of a pH-sensitive field effect transistor. Calibration curves of the biosensor were measured in phosphate and TRIS buffers in the presence of potassium ferricyanide. The use of the latter as an oxidizing substrate in the biocatalytic oxidation of glucose leads to an increase of the acidification rate of the solution inside the enzymatic layer, because three protons are now generated per one molecule of glucose instead of only one when the natural oxidizing cosubstrate, oxygen, is used. Depending on the concentrationof ferricyanide weobservea 10-100timesincrease of the biosensor response in concentrated buffer solutions and a substantial extension of its dynamic range. At sufficiently high concentrations of ferricyanide, the calibration curves in both buffers have a sigmoidal shape in linear coordinates with local pH changes on the surface of the field effect transistor reaching about two pH units in the saturation range. The resulting saturation of the curves at higher glucose concentrations is due to the inhibition of the activity of glucose oxidase at acidic pH by CI- ions present in the solution. The proposed approach may be extended to allow the detection of a wide range of analytes using enzyme field effect transistors based on the enzymes for which reoxidation of the cofactor (coenzyme) leads to a liberation of H+ ions. Since the introduction of the enzyme field effect transistor for penicillin (ENFET) in 19801 the ENFETs have become a typical example to which one refers when speaking about integrated biosensors. In the ENFETs ion-sensitivefield effect transistors (ISFET), mainly H+-sensitive, are used to monitor a localvariation of pH resulting from the enzymatically driven conversion of a specific substrate. A differential pair of the ISFETs (one covered with an enzyme-containing membrane and the other with a “blank” one) is usually employed to compensate for common interferences and bulk pH changes. The underlying detection principle limits the number of enzymes suitable for application in the ENFEiTs to the enzymes catalyzing the reactions that change the acid-base equilibrium of a medium. The practical importance of glucose assays in clinical diagnostics and biotechnology and the availability and low cost of glucose oxidase and its established role as a model * Present address: Institut filr Chcmo- und Bioaensorik,Wilhelm-Klemm-Strassc 8, D-4400 Milnster, Germany. t On leave from the Sector of Bioelectronics, Kiev University, Kiev, Ukraine. (1) Caras, S.;Janata, J. Anal. Chem. 1980, 52, 1935-1937. 0003-2700/94/03680205%04.50/0 0 1994 American Chemical Society
system in the field of the enzyme biosensors2 were the reasons that about one-third of the publications on the ENFETs deal with the sensors for the detection of glucose. However, compared to amperometric glucose sensors, little work has been done on the ENFETs and their real potential in general and for the assay of glucose in particular seems to remain unknown . Meanwhile, the major obstacles preventing rapid progress in practical applications of the glucose ENFETs have been recognized and may be reduced to the following: (1) a dramatic decrease of the biosensor response with the increase of buffer capacity of the test solution, preventing applications of the biosensors in highly buffered media; (2) limitation of the dynamic range of the biosensor response because of the insufficient concentration of dissolved oxygen in real samples (oxygen solubility in water: 1.4 mM, 1 atm, 20 “C). Concerning the first point, a differential setup making use of a pair of pF- and pH-sensitive ISFETs was p r ~ p o s e d . In ~ order to reduce the effect of the buffer concentration on the glucose ENFET response, the application of semipermiable additional membranes (cross-linked bovine serum albumin, poly(vinylbutyral), Nafion films, etc.) on top of the enzymatic layer was also suggested.G The same membranes may also result in an extension of the dynamic range of the ENFET response, so reducing the “oxygen deficit” within the enzyme layer due to their greater resistance to the diffusion of glucose compared to oxygen.’’ In this paper we investigate the behavior of glucose ENFETs in the presence of potassium ferricyanide as an alternative to oxygen. The use of oxidizing cosubstrates in place of oxygen for enzyme-based8 and, in particular, glucose oxidase-based2 amperometric biosensors is well-known. However, to the best of our knowledge, it has not been tested with ENFETs. (2) Wilson, R.; Turner, A. P. F. Biosew. Bioelectron. 1992, 7, 165-185. (3) Hintsche, R.; Darnsfcld, I.;Scheller, F.; Pham, M. T.; Hoffmann, W.; Hucller, J.; Moritz, W. Biosens. Bioelectron. 1990, 5, 327-334. (4) Shul’ga, A. A.; Strikha, V. I.; Soldatkin, A. P.; El’skaya, A. V.; Maupas, H.; Martelet, C.;Clechet, P. Anal. Chim. Acta 1993, 278, 233-236. (5)Soldatkin, A. P.; El’skaya, A. V.; Shul’ga, A. A.; Nechiporouk, L. I.; NyamsiHcndgy, A.; Martclct, C. Anal. Chim. Acta 1993, 283, 695-701. (6)Soldatkin, A,; Shul’ga, A.; Martclct, C.; Jaffrczic, N.; Maupas, H.; El’skaya. A.V. French Patent, 93 OS 941, May 12. 1993. (7) Saito, A.; Miyamoto, S.;Kimura, J.; Kuriyama, T. Sew. Actuators 1991, B5, 231-239. (8) Bartlctt, P. N.; Tebbutt, P.;Whitakcr, R. G. Prog. React. Kinel. 1991, 16, 55-155. (9) Kulys, J. J.; Cenas, N. K. Biochim. Biophys. Acta 1983, 744, 57-63. (IO) Schlaepfer, P.; Mindt, W.; Racinc, Ph. Clin. Chim. Acta 1974,57, 283-289. (1 1) Alcxandrovskii, Y.A.; Bczhikina, L. V.; Rodionov, Y .V. Biokhimiycl (USSR) 1981, 4, 708-716.
Analytical Chemistry, Vol. 66, No. 2, January 15, 1994 205
The properties of potassium ferricyanide as an electron acceptor in the process of glucose oxidation catalyzed by glucoseoxidase havebeenstudied in homogeneousmedium,"" and it was used as a soluble mediator in the commercial glucose analyzer of F. Hoffmann-La Roche & Co. Ltd. (Bazel, Switzerland) for the assay of glucose in cultivation media." However, its role in the glucose-sensitive ENFETs differs substantially from that with amperometricenzymeelectrodes. Asitisknown, theoxidationofglucosecatalyzed by glucose oxidaseis a two-substrate reaction. Normally, the mechanism of the reaction include3 the following: (I) formation of an enzyme-glucose complex; (11) reduction of the coenzyme flavine adenine dinucleotide (FAD) (one molecule of glucose oxidase contains two molecules of FAD) according to
C,H,,O, P-D-gkucose
+
FAD = C6H,,,06 +FADH, oxidized o-ghcono-6-lactone reduced form form (1)
(111) fast oxidation of the reduced form of the enzyme by oxygen, which produces hydrogen peroxide and restores the initial state of the enzyme molecule
FADH,+O,= FAD +H,O, reduced oxidized form form
(2) ENZYMATlC LAYER
-
In the meantime, gluconolactone generated in reaction 1 is hydrolyzed spontaneously to gluconic acid (pK. 3.8):
C,H,,O, +H,O= C,H,,O, +H' (3) D-ghWnO-8-1aCtOne D-gluconate The rate of hydrolysis of gluconolactone depends on pH, e.&, at pH 8.0 it has a half-life of -10 min, which decreases at lower pH.l3 It is the protons produced in reaction 3 which aredetected in theglucoseENFET. The presenceof the slow rate-determining process of gluconolactone hydrolysis in the chain of the events leading to the ultimate response of the ENFET imposes a number of limitations on the thickness and morphology of a layer of immobilized glucose oxidase.14 The place of oxygen as an oxidizing agent in reaction 2 can be taken by a large number of other electron acceptors.z.8-'2 So with oxygen substituted with ferricyanide as the electron acceptor, the reaction may be rewritten as
+
= FAD + 2[Fe(CN),IC Oxidized reduced form form 2H+ (2') Thus substitution of oxygen by ferricyanide as the electron acceptor in the enzymatic oxidation of glucose would lead to asubstantial increaseof theENFET response, because in this case, three protons are generated per glucose molecule, one in reaction 3 and two in reaction 2', instead of only one in the case of oxygen. Also, one should expect an extension of the biosensor dynamic range because the limitation of the reaction rate due to a low concentration of the dissolved oxygen in the sample can be reduced by adjustment of the concentration of the alternative oxidizing substrate. FADH, reduced form
+ Z[Fe(CN),]'oxidized form
(12)Mn. J.-R.:Gusmda. R. And. Biochmm. 1977. 79. 319-328. (13)Talrahashi. T.; Matsumoto. M. Nature 1963, 199. 765-767. (II)Hanarata.Y.;Shiono,S.;Macda,M.Anoi. Chinr.Acra1990.23I.213-220.
200 A n a W a I chemlsby. Vol. 66, No. 2,January 15, 1994
Flgure 1. Schematicrepresen~tkmofmerea~muslonprocesses
withinthelayerof immobilizedglucoseoxidaseleadlngtothegeneratkm of theENFET response when both oxygen and ferricyankleare present In the solution. The case of a basic (pK. > 7) buffer, 8. Is Illustrated. The decrease of the grey color intensity Corresponds to the decrease of the solution pH. FAD Is for coenzyme flavin adenine nucleotide. Chemical processes are represented by solid arrows while diffusional mass transfer Is shown by dashed arrows. The general scheme of the diffusion-reaction processes taking place in the layer of immobilized glucose oxidase deposited on top of the gate insulator of the ISFET is shown in Figure 1. It comprises diffusion of substrates and products of the enzymatic reaction as well as mobile buffer species in and out of the enzymatic layer. The blocks of the processes involved in the oxidation of FAD by (I) oxygen and (11) ferricyanide as well as (111) a schematic representation of the mechanism of a buffer-mediated transport of H+ ions within the membrane can be easily recognized on the given scheme. The steady state pH-value at the enzymatic layer/ISFET interface is a result of the balance between the rates of generation of H+ ions due to reactions (2') and (3) inside the film and their removal from within the membrane. EXPERIMENTAL SECTION Materials. In the present study the reagents used were glucose oxidase (EC 1.1.3.4 type VII) from Aspergillus niger having an activity of 168 units/mg from Sigma, bovine serum albumin and glutaraldehyde (25% aqueous solution) from Fluka,and K,[Fe(CN)6] 0fp.a. grade from Merck. Allother reagents were of p.a. grade. SensorDesign. The used ISFET transdusers weresupplied by Emokon Ltd. (Kiev, Ukraine). Each sensor chip (3 mm X IO mm) contained two identical ISFETs. These were n-channel depletion-mode pH-sensitive SilN4 ISFETs fabricated on p-Si of orientation (100) and resistivity 7.5 R cm. Inour experiments, ISFETs witha tbresholdvoltageofabout
-4 V were employed. Their pHsensitivity in pH range 2-10 was 52-54 mV/pH. The design and mode of operation15made the sensors insensitive to light and allowed them to be used when the silicon substrate was in direct contact with the test solution. Enzyme Immobilization. Ten percent solutions of glucose oxidase and bovine serum albumin were prepared in 20 mM phosphate buffer (KHZPOd-NaOH, pH 7.4). Prior to deposition onto the gate area of the ISFETs, these solutions were mixed to final concentrations of 5% glucose oxidase and 5% albumin and glycerol was added to a concentration of 10%. As a differential experimental setup is used, one drop of the enzyme-containing mixture was deposited on the measuring ISFET, while on the reference ISFET, only a mixture containing 10% albumin and 10% glycerol was deposited. Then the sensor chips were placed in a saturated glutaraldehyde vapor and were kept there for 30 min. After the exposure to glutaraldehyde vapor, the membranes were dried at room temperature for 10-15 min. Measurements. The sensor was immersed in a measurement cell filled with -1.5 mL of vigorously stirred buffer solution, and a steady-state differential output between the measuring and reference ISFETs was recorded. The ISFETs were operated in a constant source current and drain-source voltage mode (Is= 100 PA, Vh = 1 V) using two EM1 ISFET amplifiers (Enschede, The Netherlands). The bare substrate of the sensor chip was used as a quasi-reference electrode. The concentration of glucose was varied by addition of aliquots of 0.15 or 0.75 M stock solutions of glucose in a 5 mM phosphate buffer (pH 7.4) containing 140 mM NaCl. The experiments were carried out at room temperature.
RESULTS AND DISCUSSION Oxygenas an OxidizingSubstrate. In the first experiment, the calibration curves of the fabricated glucose ENFET were measured under aerobic conditions in 10and 50 mM phosphate andTRIS buffers (pH 7.4) containing 140 mM NaCl (Figure 2). It is worth noting that the biosensor response time (the time needed to reach 95% of the steady-state response amplitude) decreases substantially with the increase of the buffer capacity of the test solution and is 10 s in a 50 mM buffer vs 90 s in a 1 mM buffer solution. The reason for the observed decrease of the response time at more concentrated buffer solution may be faster establishment of the steadystate gradient of proton concentration within the enzyme layer due to the greater efficiency of the buffer-mediated transport of H+ ions inside the membrane (a schematic presentation of this transport mechanism is shown in the lower part of Figure 1). When themeasurements are performed at a concentration of dissolved oxygen corresponding to the partial pressure of oxygen in the air, the dynamic range of the biosensor response extends only up to 1-1 $5 mM glucose due to limitation of the rateof the enzymatic reaction by an insufficient concentration of oxygen.16 Under anaerobic conditions, when before the experiment the test solution was deaerated by purging with pure nitrogen for at least 30 min, no response of the biosensor to glucose was observed.
-
(15) Rolov, 0.S.; Shul'ga, A. A.; Abalov, A. A.; Kononenko, Y. G;Netchiporuk,
L.I.; Sandrovsky, A. K.; Strikha, V. H.; Jaffrezic-Renault,N.; Martelet, C. French Patent, 93 07 340, June 14, 1993. (16) Shul'ga, A. A.; Sandrovsky,A. C.; Strikha, V. I.; Soldatkin,A. P.; Starodub, N.F.; El'skaya, A. V. Sew. Acruorors 1992, BlO, 41-46.
I"
10 mM TRIS
t
a 7
>
6
E =
5
xo
4
0
1.o
0.5 Cglucose,
1.5
mM
Flgure 2. Calibration curves of the glucose ENFET in 10 and 50 mM TRIS and phosphate buffers (pH 7.4, 140 mM NaCI). 100
ao > E
60
L
a
50 mM phosphate buffer
2 40
20
0 0
5
10 Cgl"cosa#
15
f-flM
Figwe 3. Calibration curves of the glucose ENFET in 50 mMphoephete (1 and 2) and TRIS (3-5) buffers (pH 7.4, 140 mM NaCi). Different curves correspond to the test solutions containing also (1) 80, (2) 165, (3) 40, (4) 80, and (5) 120 mM potassium ferricyanlde.
Ferricyanideas an Oxidizing Substrate. Ferricyanide was chosen for these experiments because of its high solubility permitting rather concentrated solutions to be used, so reducing interference from dissolved oxygen by creating an excess of the alternative oxidizing substrate. As one can see from Figure 3, the biosensor calibration curves in 50 mM phosphate and TRIS buffers differ dramatically and are strongly dependent on the concentration of ferricyanide. In TRIS buffer the calibration curves already have a sigmoidal shape at 40 mM [Fe(cN)6l3-. In 80 mM ferricyanide solution the biosensor response saturates at 100 mV. This corresponds to a -100-time increase of the concentration of H+ions at the enzymatic film-transducer interface. The corresponding response time increases up to 5-10 min, an indirect indication that the buffer capacity of the solution inside the protein layer becomesvery low. Further increase of the ferricyanide concentration does not change the saturation level but increases somewhat the biosensor sensitivity.
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AnaWical Chemistry, Vol. 66, No. 2, January 15, 1994
207
concentration y , the dependence of the solution pH on the concentration of added acid, a, is given by the HendersonHasselbalch e q u a t i ~ n : ' ~
9-4 120 mM
100
-
P
p H = pK,+ log y-a a
d
I 80
-
I
where Ka is the dissociation constant of the buffer. At a = 7 1 2 , pH of the solution equals pKa. As far as the pH of the solutions used in this study is rather close to the pKa of the buffers employed, it is more convenient to make a substitution a = 7 1 2 + a,where a now corresponds to the amount of the acid produced due to the enzymatic reaction, and to rewrite eq 4 as
I
>
ai
E
= 60 -
I
I
3
3O
I 40
I
-
pH = pKa + log
20
0 0
1
2
3
4
5
6
7
8
9
10
Cglucoser mM Fburr 4. Calibration curves of the biosensor in 10 mM phosphate (soiM lines) and TRIS (dashed line) buffers (pH 7.4, 140 mM NaCI) for different concentratbnsof ferricyanide.The concentratbnsof potassium ferricyanide added to the test solution are indicated in the figure.
The calibration curves in a 50 mM phosphate buffer in the tested range of ferricyanide concentrations are apparently linear at lower and level off at higher concentrations of glucose. Saturation occurs at higher glucoseconcentrations when higher concentrations of ferricyanide are used, and at 165 mM ferricyanide, the quasi-linear range of the sensor response extends up to a glucose concentration of 10 mM. Meanwhile, as shown in Figure 4, in a 10 mM phosphate buffer the biosensor calibration curves also assume a sigmoidal shape, as in TRIS buffer, when the concentration of ferricyanide in the solution becomes as high as 120 mM. The observed abrupt increase of the amplitude of the response is also accompanied by a substantial increase of the response time up to 10 min in the saturation range, as was the case in TRIS buffer. As follows from the above results, increase of the ferricyanide concentration results at first in an extension of the dynamic range of the biosensor response. However, the extension is limited and for a certain concentration of buffer beyond a critical value of ferricyanide concentration any further growth of the rate of the biocatalytic reaction (by increasing the ferricyanide concentration or in some other way) leads to a decrease of the dynamic range, because the saturation level will be now reached at lower glucose concentrations. This problem may be partially overcome by increasing the solution buffer capacity (compare thecalibration curves in 50 and 10 mM buffers shown in Figures 3 and 4) although at the cost of a decrease in the biosensor sensitivity. In order to understand the observed phenomena, it is necessary to take into account that the production of protons inside the enzymatic layer due to reactions 2,2', and 3 results in a strong acidification of the solution inside the layer, thus dramatically affecting (a) its buffer capacity, (b) the pHdependent kinetics of the enzymatic oxidation of glucose, and, probably, (c) the rate of gluconolactone hydrolysis. Role of the Buffer Capacity. In case of, for example, an aqueous solution of a basic buffer B with an analytical
-
208
(4)
Analytical Chemistty, Vol. 66, No. 2, January 15, 1994
712-a 712 + a
(4')
A formula describing the dependence of the buffer capacity of the test solution on the amount of added acid is obtained by differentiation of the above expression.
The rate of decrease of buffer capacity with increase of acid concentration is given by the expression dp -=--
2In loa da Y This means that the more acid is added the faster is the decrease of the buffer capacity. It follows then that when successive portions of an acid are added to the solution, in other words, when the rate of the enzymatic reaction increases, the resulting changes of solution pH grow progressively, because every added portion of the acid decreases the pH of the solution thereby decreasing its buffer capacity and thus leading to a further decrease of pH. Since our experiments are performed at pH 7.4 and in case of TRIS buffer pKa = 8.08, any decrease of pH results in a decrease of the solution buffer capacity. Then thedependence of the biosensor response on the concentration of glucose will at first be determined by the above effect, resulting in a response increasing progressively with increasing glucose concentration. This situation is clearly demonstrated by the calibration curves measured under both air- and nitrogen-saturated conditions for different concentrations of the buffer. The curves are presented in Figure 5 in parabolic coordinates and in such presentation are linear (in all cases the correlation coefficients are greater than 0.995). Observed proportionality of the biosensor response to the square of the glucose concentration in TRIS buffer under current experimental conditions is in accordance with eq 5 , thus confirming that the shape of the initial part of the calibration curves is largely controlled by the buffer capacity of the solution inside the enzymatic layer. On the contrary, in the case of phosphate buffer, pKa = 6.8, the lowering of pH inside the enzymatic layer due to the biocatalytic reaction first leads to an increase of the solution buffer capacity. However, after the intramembrane pH reaches pH 6.8, corresponding to a response of -30 mV, a further increase of the rate of H+ ion generation will cause a decrease of the buffer capacity giving, as for TRIS buffer, (17) Koryta, J.; Dvorjak, .I. Principles of EIecfrochemisfry; John Willey & Sons Ltd.: Chichester, UK, 1987: Chapter 2.
25
air saturated 10 mM TRIS
70]
0
.*
20
sat. buffer
,....."'
50 mM TRIS
m air sat. buffer
...'...'
_..
> E
1
1 4
J
...'.
I
5
6
151
600 rnM NaCl sat. buffer
)...,.....? air
0
.
0
.
.
.
,
5
.
.
.
.
I
.
.
.
.
(Cglucora.
I
.
.
.
'
l
I
2021
15
10
mM)*
0
Flgurr 5. Initial parts of the calibration curves plotted in parabolic coordinates measured under air and nitrogen saturatlon conditions In 10 and 50 mM TRIS buffer (pH 7.4, 140 mM NaCi) containing 80 mM potasslumferricyanide. The amount of separately addedsait is indicated in the figure.
1
3
2
4
Flgurr 7. Initial parts of the calibration curves measured under air (1 and 3), oxygen (21, and nitrogen (4) saturation conditions in a 50 mM phosphate buffer (pH 7.4, 140 mM NaCI) and in the presence of 150 mM potassium ferricyanide (2-4).
35 30
$
20 glucose 10
0 3.5
4.5
6.5
5.5
7.5
8.5
PH Flgurs 6. pH dependence of the biosensor response for different concentrations of glucose. The mesurements have been performed ina multicomponent buffer(lOmMTRIS, 10mMKH2P04,lOmMcitric acid. and 10 mM sodium tetraborate) having a constint buffer capacity at the studied pH range and containing 80 mM ferricyanide. The solid and dashed curves correspond to KCI concentrations of 0.01 and 0.5 M, respectively.
progressively greater values of the biosensor output. The critical, in the respect mentioned above, rate of the enzymatic reaction is reached at 80 and 165 mM ferricyanide in 10 and 50 m M phosphate buffers, respectively. pH-Dependent Kinetics of Glucose Oxidase. It is known2 that ferricyanide belongs to the class of electron acceptors that oxidizeglucoseoxidaseat the highest ratein acidic media. This is usually attributed to a decrease of the electrostatic repulsion between negatively charged ferricyanide ions and the protein. The isoelectric point for glucoseoxidase has been quoted as 4.2 and the net charge that the protein molecule carries at physiological pH is about -80.2 As was shown in previous studies, the native glucose oxidase has the maximum of its catalytic activity at pH 5-6 with oxygen as an oxidizing substrate and at pH -3 with ferricyanide.2 While the effects of the solution buffer capacity allow the shape of the calibration curves at lower glucose concentrations to be interpreted (regions of linearity and progressive growth of the biosensor
output signal), it is the changing kinetics of glucose oxidase that should be invoked to deal with their saturation region after the curves assume a sigmoidal shape. In order to determine the role of different factors contributing to the saturation of the calibration curves, the pH dependence of the biosensor response has been investigated. The measurements were performed in a multicomponent buffer (the composition of the buffer was 10 mM TRIS, 10 mM KH2P04, 10 mM citric acid, 10 mM sodium tetraborate, and 10 mM KCI) that has practically constant buffer capacity at a pH range of 3-9. The use of this buffer has allowed the influence of pH-dependent variations of the buffer capacity on the ENFET response to be avoided. At such conditions, the dependenceof the latter on pH is mainly due to the variation of the kinetics of immobilized glucose oxidase. As shown in Figure 6, in the presence of 80 mM potassium ferricyanide the curves obtained have a bell shape with the maximum at pH -5.6. However, the increase of the KCl concentration to 0.5 M results in a shift of the maximum to pH -7.0. Moreover, now the 'response practically disappears by pH -4.5. The reason for theobserved suppression of the response at acidic pH is inhibition of the activity of glucose oxidase by C1-ions. While this effect is known for native glucose oxidase,* in case of the glucose ENFET it is reported for the first time. Since previous measurements of the calibration curves have been made in the presence of 140 mM KCl, it is evident that the observed saturation is chiefly due to the above effect and occurs at pH values inside the enzymatic layer of 5.5, which corresponds to the response of 100 mV, independent of the concentration of both buffer and ferricyanide. Competition between Oxygen and [Fe(CN)& Ions as Oxidizing Substrates for Glucose Oxidase. A more detailed study of the initial part of the calibration curves reveals a very peculiar feature: at lower concentrations of glucose, the linear part of the curves measured under aerobic conditions consists of two regions having different slopes. This is clearly seen from the plots presented in Figure 7. The curves obtained in the solution without ferricyanide and in that containing 150 mM ferricyanide and saturated with air or pure oxygen have
-
-
Analyticsl Chemistry, Voi. 66, No. 2, Januety 15, 1994
208
the same slope of their initial parts. For the curve measured under oxygen saturation conditions, this part extends to 3 mM, while in two other c a s e s a n l y to a concentration of glucoseof 1 mM. At higher concentrations of glucose, the curves obtained under air and oxygen saturation conditions, while remaining linear, acquire a different slope becoming parallel to the calibration curve measured under anaerobic conditions at 150 mM ferricyanide. These results make quite clear the fact that the rate of the enzymaticoxidationof glucose by immobilizedglucose oxidase is mainly determined by oxygen; Le., reaction 2 prevails, determining the slope of the calibration curves, until the conditionsofthe"oxygendeficit"arerea1izedathigherglucose concentrations. Then the reaction rate is mainly due to the oxidation of the FADH2 by [Fe(CN)# ions in accordance with reaction 2', thus leading to a new slope of the glucose calibrationcurves. Theobserved preference byglucoseoxidase of oxygen as the oxidizing substrate compared to ferricyanide is in agreement with the results of ref 10, where a number of electronic acceptors for glucose oxidase were compared by use of an amperometric enzyme electrode. An analogous effect of the increase of the rate of oxidation of glucose under anaerobic conditions was also observed with TRIS buffer (see Figure5). However, in thiscase, the regions of preferential reduction of oxygen or ferricyanide are not so clearly separated because of the crucial effect of the variation of the solution buffer capacity on the form of the calibration curves. Therathernarrow dynamicrangeofthe biosensorresponse in comparison with the concentrations of ferricyanide used may be attributed, first, to the relative inefficiency of ferricyanide as the oxidizing substrate due to its low standard redox potential (-88 mV vs SCE) and, second, to a partitioningofthe [Fe(CN)6l3-ions between the solution and the protein matrix, which being negatively charged under the experimental conditions used may result in a much lower concentration of ferricyanide within the enzymatic layer.
-
CONCLUSIONS The glucose-sensitive enzyme field effect transistor based on immobilized glucose oxidase using ferricyanide as an oxidizing substrate was investigated. The dependence of the biosensor responseon theconcentration ofglucosewas studied in different buffers under air, oxygen, and nitrogen saturation conditions. This investigation permitted the general form of the glucose ENFET calibration curves to be understood. For themeasurements performedinsolutionsofaone-base buffer, the range of concentrations of glucose could be divided into four regions according to the prevailing physicochemical processes affecting the slope and shape of the curves (see Figure8). Themajor processesresponsiblefortheappearance of the curves in the different regions are as follows: (I) biocatalytic reaction rate is determined mainly by oxidation ofthe FADH2 moieties of the enzyme by oxygen, and protons are produced largely due to hydrolysis of gluconolactone according to reaction 3; (11) under conditions of "oxygen deficit", the reaction rate is determined by oxidation of the FADHI by ferricyanide leading to the generation of H+ ions according to reaction 2'; (111) major contribution to the amplitudeof the biosensor response comes from the decrease 210 AnaWcal Chemlsfry, Vd. 66, No. 2, January 15, 1994
Flgure 8. General appearance of the calibration curve (solid line) of the glucose ENFET in the test solutions containing a one-base buffer and potassium ferricyanide under aerobic condnions. The bkxensw response at different parts of the curve is controlled by different physlcochemical processes occurring in the enzymatic layer as is indicated in the figure. Some tlps about the nmdificatbn of the curve shape when the concantrationsof ferricyanide andfor buffer vary ara given by a set of curves (shown by dashed lines) corresponding to different measurement conditions (see also the text).
of the solution buffer capacity inside the enzymatic layer resulting from the substantial changes of the intramembrane pH due to the enzymatically produced H+ ions; (IV) the rate of the enzymatic reaction reaches saturation mainly because of the inhibition of the activity of immobilized glucoseoxidase by the present CI- ions at acidic pH. The performanceof the sensorcan beoptimized by tailoring the matrix for the enzyme immobilization, judicious choice of a suitable oxidizing substrate, and adjustment of the buffer composition. The manner in which the shape of the ENFET calibration curve is modified when the concentrations of potassium ferricyanide and/or buffer in the test solution are changed is illustrated in Figure 8. Here an arrow shows the direction in which the curves vary when the concentration of ferricyanide is increased or the concentration of buffer is decreased. The proposed application of soluble electron acceptors in ISFET-based enzyme biosensors permits, in principle, the number of analytes that can be determined using these biosensors to be greatly extended, because any enzyme, e.&, flavoprotein or quinoprotein enzymes, dehydrogenases etc., for which reoxidation of the cofactor (coenzyme) leads to a liberation of H+ ions may be employed in the ENFETs according to the detection scheme described above. ACKNOWLEDGMENT The authors are very thankful to Dr. D. J. Strike for the critical reading of the manuscript. Financial support of this research by SwissNational Science Foundation (Grant 83GU37899) is greatfully acknowledged. Recetved fw review August 13. 1993. Accepted October 25.
1993.O
