1874
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
Enzyme-Bound Thermistor as an Enthalpimetric Sensor Canh Tran-Minh" and Didier Vallin Ecole Nationale Superieure des Mines de Saint-Etienne, 158, cours Fauriel
The construction of a new type of immobilized enzyme sensor for the determination of chemical species is described. The sensitive part of a thermistor is coated with an artiiicial enzyme membrane obtained by the cross-linking of the enzyme together with an inert protein, using glutaraldehyde as a bifunctional reagent. The enthalpy change during the enzymatic reaction induces a production or absorption of calories which can be taken as a reaction product and depends then upon the reaction parameters (K,, V,, [SI). This type of enzyme-bound thermistor is used for the determination of hydrogen peroxide, glucose, and urea. The pH and temperature dependence of the hydrolysis of urea by urease given by this sensor are compared to the results previously obtained by other methods.
During the past years, extensive research has been conducted on the construction and the use of new types of matrix-bound enzyme systems (1-3). A large number of those studies have been concerned with the construction of enzyme electrodes for t h e determination of organic and biological compounds (urea, uric acid, aminoacids, etc.). In t h a t type of sensor, a n enzymatically active membrane is bound t o a specific electrode such as a gas electrode: 02,C 0 2 (4-7), or a monovalent cation electrode: H+, NH4+ (7-11). T h e electrode is sensitive either to the substrate or to the product of t h e reaction. Although this type of sensor offers many advantages, it depends essentially on t h e basic electrode, t h e detection of which is often restricted to a single chemical species whatever the immobilized enzyme may be. T h e specificity of the electrode (02, C02, H+, NH4+)implies that the enzymatic reaction gives rise to species detectable by the sensitive part of t h e electrode. In t h e present work, we describe the construction of an enzymatic sensor using the detection of an entity belonging to all t h e enzymatic reactions: t h e enthalpy change of the reaction. Several authors have applied microcalorimetry to t h e determination of many chemical and biochemical species (12 14). However, this technique demands an expensive and cumbersome apparatus. From the principle of microcalorimetry, some authors have constructed measuring cells with thermistors, using a flow system, to measure the temperature change of the substrate solution once it passed through a bed of immobilized enzyme (15-18). These cells allowed the determination of several substrates, but they need a large quantity of enzyme, and their response time is still long. Our aim is t o construct a new type of enzymatic sensor which could be applied to all the enzymatic reactions provided that their enthalpy change is not equal to zero and large enough to be detected by the thermistors. We use two thermistors, both are coated with crosslinked proteins on their sensitive parts. The first one-the measuring thermistor-is covered with a mixture of albumin and enzyme crosslinked together by a bifunctional reagent: glutaraldehyde. The second one coated only with albumin is used as a reference. For measurements, both thermistors are dipped into a stirred 0003-2700/78/0350-1874$0 1. O O / O
- 42023 Saint-Etienne
CBdex, France
solution of substrate. The differential device eliminates the effect of temperature fluctuations induced by the thermostat. During the exothermic reaction, one part of the calories produced inside the enzyme active layer diffuses toward the solution, and the other toward the thermistor. In order to favor the second way of exhausting calories, we surrounded t h e measuring thermistor with a glass jacket. This one decreases the liquid flow velocities (forced convection) along the active membrane, and then reduces the amount of calories lost by diffusion toward the solution. It also allows sufficient supply of the substrate for the enzymatic reaction. The same technique has been applied to the reference thermistor to have a symmetrical device (Figure 1). Both thermistors are parts of a Wheatstone bridge. Electric zero is established by using a solution without substrate. As soon as the substrate is introduced into the solution, the temperature changes because of absorption or production of calories. It induces a variation in the measuring thermistor resistance which unbalances the h'heatstone bridge. An electrical signal is sent to an amplifier and then to a recorder. For a given concentration of substrate, a steady state of temperature can be achieved quickly all over the active layer. This type of sensor has been used for the determination of hydrogen peroxide, glucose, and urea, with a response time lower than 10 s (Figures 2-4). For a given enzyme membrane and a given substrate concentration, the precision on the repeatability of t h e sensor response is approximately 3 7 ~ .
EXPERIMENTAL Apparatus. Thermistors provided by Feenwall Electronics were used. The main characteristics are: R:j, resistance at 25 "C: 2000 (1 and CY, percent resistance change per "C at 25 "C: -3.9. Thermistors were connected to a Wheatstone bridge (temperatur-Messgerat from Knauer). As the fluctuations of temperature inside the active membrane occur in a small range, a linear variation of R,, with temperature can be assumed. Reagents. Enzymes are provided by Sigma. Enzyme activities are given by Sigma. Crease. Powder from Jack beans, type IX, 5.1 Sigma units/mg. One unit of urease activity is the amount of enzyme that will liberate 1 mg of ammonia nitrogen from urea in 5 min at pH 7 at 30 "C. Glucose Oxidase. From Aspergillus niger, type 11, 18.9 Sigma units/mg, catalase impurity 2% by weight (800 Sigma units/mg). One unit of glucose oxidase activity is the amount of enzyme that will oxidize 1.0 pmol of b-D-glucose to D-glUconiC acid and H20, per minute at pH 5.1 at 35 "C. Catalase. From bovine liver 15000 Sigma units/mg. One Sigma unit will decompose 1 pmol of H202per minute at pH 7.0 at 25 "C, while H 2 0 2concentration falls from 10.3 to 9.2 pmol per mL of reaction mixture. Procedure. The enzyme was dissolved in 200 pL of 17.5% human albumin solution. Then 20 p L of 25% glutaraldehyde solution was added. The solution was stirred for 30 s. A drop of this solution ( e 2 0 mg) was deposited on the sensitive part of the thermistor and left for crosslinking at 25 "C during approximately 15 min. Then a glass jacket could be fitted if necessary. Before use in biological media, the sensor must be rinsed for a few hours with a phosphate buffer solution (P,) at pH 7.0, and then with a glycine solution, in order to elute or neutralize the excess of bifunctional agent. All measurements t 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
1875
c
F v
LOO
1
'hermistor
-gloss jacket
- i mrno bi I ized enzvrne
Figure 1. Enzyme-bound thermistor
were carried out in phosphate buffer solutions (0.1 M) and in thermostated cells.
RESULTS The response of the above-mentioned sensor has been studied with catalase, glucose oxidase, and urease. Enzyme Thermistor Using Immobilized Catalase. The enzymatic reaction is:
-
HzO2
H 2 0 + Y2O2 + AH
T h e enthalpy change of this reaction is: AH = -23.44 kcal/mol-'. The enzymatic membrane was obtained by dissolving 5 mg of catalase according to the above-described procedure. The amount of catalase immobilized on the sensitive part of the thermistor was approximately 10 000 Sigma units. Because of this amount of catalase, it was not necessary to use the glass jacket. For much greater amounts of catalase, the temperature increase inside the active layer is large enough to give a partial and irreversible destruction of enzyme activity (19, 20). T h e assays were performed in buffer solution (PI,0.1 M, p H 7.0) in thermostated cells (25.0 & 0.1 O C ) for various concentrations of hydrogen peroxide between lo-' M and 7 x lo-' M. T h e steady-state response curve of the sensor as a function of substrate concentration is shown on Figure 2. For concentrations ranging from lo-' M to 5 X lo-' M, the sensor response is proportional to the substrate Concentration. For higher concentrations ( > 5 x lo-' M) the response curve tends towards a plateau corresponding to the maximum rate of the enzymatic reaction. The precision of the sensor response is approximately 5% when hydrogen peroxide concentration is lower than lo-, M and 3% for higher substrate concentration. Enzyme Thermistor Using Immobilized Glucose Oxidase (GOD). The glucose oxidase used for these assays contains catalase impurity (800 Sigma units/mg). Then the catalyzed reaction implies two consecutive steps.
H 2 0 + P-D-glucose
+ O2
+ GOD
Hzo2 D-gluconic acid H2Oz
catalase '/202
+ AHl
+ H20 + W2
(I)
(11)
These reactions can also be simplified as follows: p-D-glucose
+ 02
GOD
catalase
D-gluconic acid
+ '/202+ AH3 (111)
The total enthalpy change AH3of the reaction I11 is the sum of the two enthalpy changes referring to both steps. The production of 1 mol of D-gluconic acid from 1 mol of p-Dglucose requires 0.5 mol of oxygen. Consequently, the oxidation rate of /%D-glucose is a function of the oxygen con-
Figure 2. Response of the catalase-bound thermistor to various hydrogen peroxide concentrations
centration (7). If AH3 is taken as a product of the reaction, the rate of the reaction can be writtlen: a H -
at
3
- K [ 0 2 ] "[ g l u c o s e ] ~
For the determination of @-D-glucose,the proportionality between the reaction rate and the glucose concentration requires [O,] = constant. However, the enzymatic reaction involves a consumption of both P-D-glUCOSe and oxygen, and then a decrease of oxygen concentration. This concentration could be kept approximately constant a t the gas saturation concentration, provided that the production of oxygen is continuous. As glucose oxidase contains catalase impurity, the addition of H , 0 2 into the buffer solution gives rise to an evolution of oxygen inside the active layer up to its saturation concentration for a given temperature and a given atmospheric pressure. The excess of oxygen is removed from the solution as a gas. Under this condition, the oxidation rate is only a function of glucose concentration specially for high substrate concentrations when increasing H 2 0 2quantities are necessary to keep [O,] = constant. The assays were performed in phosphate buffer (P,, 0.1 M, pH 5.50) at 25.0 "C. For this study, the glass jacket is required. The enzyme membrane is obtained by dissolving 20 mg of glucose oxidase according to the above-described procedure. The amounts of glucose oxidase and catalase on the thermistor are approximately 60 and 2200 Sigma units, respectively. Measurements were performed with various concentrations of $-D-glucose and H 2 0 2 . The response curves of the sensor to &D-ghcose concentrations for various concentrations of hydrogen peroxide M, 2 X M, 3 X M, 4 X M, 6 X M) are shown on Figure 3. The hydrogen peroxide is added in the buffer solution before any introduction of @-D-glucose. The slight production of calories from the catalytic decomposition of added H,02 gives rise to a small electric signal which is taken as reference (sensor response 0 when [glucose] 0 ) . For a given concentration of hydrogen peroxide, the sensor response is h e a r for low P-D-glUCOSe concentrations (Figure 3). For higher substrate Concentrations, the response curve tends toward a plateau corresponding to a maximum reaction rate. For a given 8-D-glucose concentration the sensor response and then the reaction rate is a function of the hydrogen peroxide concentration. This agrees with the predicted kinetic concerning the consumption of oxygen during the enzymatic
-
-
1876
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
ioo,,pH=6 35 5
875 -
8
u 0
c
E 550
-
~
25
t I
i ~
0
0.006
OD03
0
0.010
OD14
M
1
0
004
002
006
008
0.10
M
[ureo]
blucose]
Figure 3. Response of the glucose oxidase-bound thermistor to 8c-glucose concentrations for various hydrogen peroxide concentrations
-
Figure 5. Response of the urease-bound thermistor to urea concentrations for various pH's of the buffer solution
>
4
- E
//
20-
I 01
0
001
002
003 0 0 4 0 0 5
,
006
O ' t
007 008 M [urea]
Figure 4. Response of the urease-bound thermistor to various urea concentrations for 10 Sigma units (0)and 20 Sigma units ( 0 )of immobilized enzyme on the thermistor
oxidation of 8-D-glucose. T h e experiments were carried out with a n approximate precision of 5% for [glucose] < 3 X M, and 2 % for [glucose] > 5 X M. Enzyme Thermistor Using Immobilized Urease. The enzymatic reaction is: "2,
o = c\
+ HZO + COZ + 2NH3 + A H
"2
where W = -13.17 kcal mol-'. Considering the enzymatic membrane activity, the use of the glass jacket is required. With this device, the response curve of t h e sensor to various urea concentrations has been obtained. T h e enzymatic reaction is p H and temperature
4
1
6
7
I C
8
9
PH
Figure 6.
Effect of buffer solution pH on the response of the urease-bound thermistor for various urea concentrations
dependent, so the influence of these two parameters on the catalytic decomposition of urea can also be determined by means of the sensor. Figure 4 shows the response curve of the sensor t o various urea concentrations for 20 Sigma units and 10 Sigma units of immobilized enzyme on the thermistor. The experiments were carried out with an approximate precision of 5% for [urea] < 5 X M, and 2 % for higher substrate concentration. These curves are quite similar to those previously obtained with an urease-coated glass electrode (7). The response time is below 10 s with buffer solutions containing urea as well as with plasma samples. Figure 5 shows the sensor response to urea concentrations for various p H values of t h e buffer solution. The response curves are quite normal, except for p H 6.35 and p H 5.52. In these two cases, the response curves have a sigmoid shape that can be explained by an autocatalytic effect of the reaction. As the decomposition of urea increases the p H inside the active
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
0 1 0
I
002
,
004
1
l
010
008
006
1877
-
M
[urea]
O
0
Figure 7. Response of the urease-bound thermistor to urea concentrations for various temperatures of the buffer solution layer, the activity of the enzyme increases (Figure 6) and so does the reaction rate. This phenomenon can also be found with allosteric enzymes. For higher p H values, as the enzymatic reaction proceeds, there is an increase of the p H inside the active layer which induces a decrease of the urease activity and then a decrease of the reaction rate. No sigmoid curves were then obtained in this range of pH. From the previous curves, Figure 6 reports the effect of pH on the sensor response for various urea concentrations (2.5 X lo-* M, 5.0 X M, 7.5 X lo-* M). The sensor response tends toward a maximum value when the p H of the buffer solution is 6.75. Below and above this value, the sensor response and then the reaction rate decreases. These results agree with those already known concerning enzyme kinetics. Figure 7 represents the sensor response to urea concentrations for various temperatures of the buffer solution (P, 0.1 M, p H 6.80). T h e sensor response increases with temperature when ranging from 15 to 35 "C, and then decreases for higher temperatures. From the previous curves, Figure 8 reports the effect of temperature on the sensor response for various urea concentrations (2.5 X lo-* M, 5.0 X lo-* M, 7.5 X lo-' M). This response and then the reaction rate tend toward a maximum value at approximately 38 "C. Below this value, the increase in sensor response with temperature can be explained by the variation of the kinetic parameters according to the Arrhenius law (20). Above 38 "C, the sensitivity of the enzyme thermistor decreases as the enzyme itself is affected by an irreversible denaturation of its active sites (20).
M 10
20
-
30
LO
50
60
temperalure
'C
Figure 8. Effect of temperature on the response of the urease-bound thermistor, for various urea concentrations where V , is the maximum reaction rate and K , the Michaelis constant. Inside the active layer, concentrations of S and P are controlled by diffusion coupled with the enzyme reaction. This effect can be illustrated by the foll.owing equations:
where t is the time of the reaction, Ds and Dp effective diffusion coefficients of S and P inside the active layer, and x the distance of each point of the membrane to the external surface of this membrane. The enzymatic reaction gives rise to an enthalpy change AH which induces an increase (AH < 0) or a decrease ( A H > 0) of temperature per unit of time:
_ a0 ---A H xv,-- [SI at pC K m + [SI
where C is the thermal capacity and p the specific gravity of the layer. The change in temperature inside the active layer induces a heat transfer controlled by the equation:
(4)
DISCUSSION I n the simplest case where the enzyme irreversibly transforms one substrate into one product, the reaction rate can be written:
E
+S
k+i
k2
F?
ES + E
+P
where X is the specific heat conductivity. The temperature change inside the active layer is then given by the sum of the diffusion term in Equation 4 and the reaction term in Equation 3:
k-1
where E is the free enzyme, S the substrate, P the product, and ES the enzyme/substrate complex. The reaction rate can be written (21, 22)
[SI
d[PI - --d[SI -
u=--
dt
dt
vm
(3)
K,
+ [SI
A t the steady state, we have & / a t = 0, so we can write:
1878
ANALYTICAL CHEMISTRY, VOL 50,
NO 13,
NOVEMBER 1978
Equation 6 shows the relationship between the temperature inside the active layer and t h e reaction rate, and then with t h e substrate concentration. When [ S ] / K , 0, u = ( V , [ S ] ) / ( K ,+ [SI) (V,/K,)[S] (first-order kinetics for S). The increase of t h e temperature at the interface active layer/thermistor becomes proportional to the substrate concentration. When K,/ [SI 0, u V , (zero-order kinetics for S),t h e reaction rate V , becomes proportional to the number of active sites of bound enzyme. The response curve of the sensor tends toward a plateau. This type of curve is obtained with the catalase-bound thermistor (Figure 2), with the urease-bound thermistor (Figure 5 ) and with t h e glucose oxidase-bound thermistor (Figure 3), although in this latter case, the presence of oxygen as a cosubstrate changes the enzyme kinetic. For these enzyme-bound thermistors, lifetimes of two or three weeks were obtained, without any detectable change in the response curves.
-
-
- -
CONCLUSION A new type of immobilized enzyme sensor can be obtained by coating the sensitive part of a thermistor with an enzymatic membrane. The immobilization of the enzyme and an inert protein by the use of glutaraldehyde gives chemical covalent bonds which result in a good stabilization of the enzyme molecules. The temperature change inside t h e active layer resulting from a production (AH < 0) or an absorption ( A H > 0) of calories during the reaction can be explained in terms of a n equation which includes both diffusion and chemical reaction. When the substrate concentration is lower than the K,, of the immobilized enzyme, the enzyme-bound thermistor gives a linear response and may then act as a sensor. When t h e production of calories during t h e enzymatic reaction is small, the use of a glass jacket around the thermistor decreases the calories lost by diffusion toward the external solution and then increases the sensor response for a given substrate concentration. In the case of urease and glucose oxidase-bound thermistors, only a very small electric signal could be obtained without this glass jacket, even for large substrate concentrations.
The use of thermistors with a higher (Y (percent resistance change per "C) and an improvement of the sensor technology should allow the detection of lower substrate concentrations with low enzyme activities, and the use of enzymatic reactions with lower enthalpy changes (L-amino acid oxidase, acetylcholinesterase, etc ...). Miniaturization of such enzyme-bound thermistors may allow them to be used in vivo in the future, since very small thermistors are easily constructed and the assays carried out with plasma give similar results.
ACKNOWLEDGMENT We express our appreciation to J. Fraisse and Melle Croze from Centre de Transfusion Sanguine de Saint-Etienne for providing calibrated plasma samples. LITERATURE CITED (1) 0. R . Zaborsky, "Immobilized Enzymes", CRC Press, Cleveland, Ohio, 1973. (2) M. Salmona, C. Saronio, and S. Garattini. "Insolubilized Enzymes", Raven Press, New York, N.Y., 1974. (3) R. A. Messing, "Immobilized Enzymes for Industrial Reactors", Academic Press, New York, N.Y., 1975. (4) G. P. Hicks and S. K . Updike, Anal. Chem., 38, 726 (1966). (5) S. J. Updike and G. P. Hicks, Nature (London),214, 936 (1967). (6) S. J. Updike and G. P. Hicks, Science, 270, 158 (1967). (7) C. Tran Minh and G. Broun, Anal. Chem., 47, 1359 (1975). (8) G. G. Guilbault and J. G. Montalvo, J , Am. Chem. Soc., 92, 2533 (1970). (9) G. G. Guilbault and J. G. Montalvo, Anal. Len., 2, 289 (1969). (10) G. G . Guilbault, R. K. Smith, and J. G.Montalvo, Anal. Chem., 41, 600 (1969). (11) G. G. Guilbault and E. Hrabankova, Anal. Chem., 42, 1779 (1970). (12) A. Johansson, 6.Mattiasson, and K. Mosbach, Methods Enzymol., 44, 659 (1976). (13) A. Johansson, Protides Biol. Fluids, Proc, Colloq., 20, 567 (1973). (14) A. Johansson, J. Lundberg, B. Mattiasson, and K. Mosbach, Biochim. Biophys. Acta, 304, 217 (1973). (15) B. Danielsson and K . Mosbach, Methods Enzymol., 45, 666 (1976) (16) 8. Danielsson, K . Gadd, 6.Mattiasson, and K. Mosbach, Anal. Left, 9. 987 - - .11976) ~_ . _ , . (17) B. Mattiasson, B. Danielsson,and K. Mosbach, Anal. Lett., 9 , 217 (1976). (18) B. Mattiasson. FEBS Len., 77, 107 (1977). 1191 V. Bilhou-Bouanol. Thesis. Saint-Etienne. 6 C.I. 119761. (20j I. H. Segel, "fnzyme Kinetics", John Wiley and Sons, New York, N.Y., 926 (1975). (21) C. Tran-Minh and G. Broun, C. R . Hebd. Seances Acad. Sci., Ser. D . , 276, 2215 (1973). (22) D. Thomas, G. Broun, and E. Selegny, Biochmie. 54, 229 (1972).
RECEIVED for review March 20,1978. Accepted June 15, 1978.
