Construction and Study of Electrodes Using Cross-Linked Enzymes Canh Tran-Minh’ and Georges Broun Department de Genie Biologique, Universite de Technologie,B.P. 233-60206 Compiegne, France
The construction of electrodes, the behavior of which could be accurately analyzed and optimized? is described. Two methods were used to construct such enzyme electrodes. A direct binding method, where a cation electrode was dipped into a solution of enzyme, albumin, and glutaraldehyde: a thin layer of cross-linked protein coated the electrode bulb. A two-step method, where an active membrane was first made by Cross-linking these proteins by glutaraldehyde on a glass plate, then fitting with a silicone membrane onto a gas electrode (02 or C02). Both methods were used to construct glucose and urease electrodes measuring either NH4+ ions or COP. Glucose oxidase and L-amino acid oxidase-p02 electrodes were also developed. The theoretical and experimental behavior of these enzymic membrane electrodes were compared using the analysis of product concentrations resulting from the association of diffusion with enzyme reaction in the active layer, and the optimum operating conditions were determined.
Enzymes have recently appeared as possible mediators between chemical determinations of metabolic compounds and their electrical measurements. This specific and rapid procedure involves the use of efficient techniques for immobilizing enzymes in artificial membranes. When available, they can be applied t o the determination of numerous biochemical species. In 1966, Updike and Hicks (1-3) included glucose oxidase in polyacrylamide gels, and applied these gels onto oxygen electrodes. The decrease of 02-one substrate of the enzyme oxidation-provided a means of measuring glucose concentration. Guilbault and Montalvo (4-7) applied several physical entrapment enzyme immobilization techniques to the measurement of substrate concentrations by electrodes responding to one product of the enzyme-catalyzed reaction, ammonium ions. In particular, the entrapment of enzymes in a polyacrylamide gel was successfully applied by Guilbault e t al. (8, 9) and by several authors to the assay of urea, glucose, amino acids, ethanol, and so on. This physical method of immobilization retards leaching of enzymes. The resulting while nonhomogeneous systems are compatible with accurate and even reproducible results under well specified conditions. They hardly can be used for a methodic analysis of the enzyme membrane electrode system. The substrate profile analysis, which appears to be an efficient approach, cannot be securely applied t o membrane entrapped enzymes, but needs the use of homogeneous films containing strictly immobilized enzymes. Enzyme electrodes using chemically bound enzyme have been described by Anfalt and Granilly ( 1 0 ) using glutaraldehyde and polyacrylic acid. Such electrodes possess greater stability. Our aim was to obtain such stable homogeneous layers of Present address, Ecole Nationale Supbrieure des Mines-
42100 Saint Etienne, France. Reprints are to be requested at this
address.
covalently bound enzymes on the surface of electrodes. Two methods were used: i) a direct binding by the use of a proteic feed and a cross-linking agent ‘(14), ii) a membrane synthesis by the cross-linking of the enzyme together with an inert protein: this membrane was then applied onto the electrode. Both procedures were used to quickly and reproducibly obtain enzyme electrodes starting with standard commercial glass electrodes and polarographic electrodes. Electrodes sensitive to glucose? urea, and amino acids were systematically assayed. The parameters controlling their response were determined. Theoretical analysis was carried out using the analysis of substrate or product concentration profiles inside the active layer, in order to better understand the kinetics of such electrodes, their possible optimization, and their limitations of use. This paper concerns the enzymic membrane kinetics which control the behavior of those various enzyme electrodes.
EXPERIMENTAL Construction of Enzyme Electrodes. Direct Binding Method. The binding of urease on a monovalent cation electrode is given as as example. A Beckman cation glass electrode was used for urea measurement. When using glass pH electrodes, the same procedure could be applied. Urease (600 I.U., Worthington) was dissolved in 1 ml of 0.02M, pH 6.8 phosphate buffer. One ml of 17.5% bovine albumin solution was added, then 0.07 ml of 25% glutaraldehyde solution (to obtain a final concentration of 0.8%).The solution was stirred for 2 minutes. The preparation of the active coatings is described in Figure 1. The cation electrode was first rinsed with distilled water, then dried with filter paper. Once dipped into the active mixture so that the whole bulb was coated (Figure la), the electrode was gently rotated around its axis for 15 minutes, in order to obtain an even coating on the bulb as a result of the cross-linking (Figure lb). An “0” ring could be fitted (Figure IC)in order to keep the membrane adherent to the electrode bulb. The electrode was rinsed with distilled water (Figure Id), with a glycine solution then with water again, in order to elute or neutralize the excess of bifunctional agent. The correct binding time is to be strictly observed: after too
(C
I
(d I
Figure 1. Direct coating of the bulb of a glass electrode for the construction of an enzyme electrode
(a)The glass electrode is dipped into the active solution.(b) The electrode is rotated around its axis during the cross-linking.(c)An 0 ring is pulled on the coating.(d) The electrode is rinsed in glycine solution,then in water ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1359
e Flgure 2.
c
b
COP electrode adapted to urea measurements
Electrode inner solution. (B) Glass membrane. (C) Hydrophobic Silicone sheet. (D) Urease-plasmalbumin film.
(A)
\
ill"
I
/-Figure 4. Steady-state responses of L-amino acid-oxidase coated cation electrode to concentrations of various aminoacids
(0) Tryptophane:(A)Tyrosine: (0)Methionine: (9) Leucine; (V)Arginine: ( 0 ) Histidine: ( W ) Phenylalanine.C A =~ Concentration of amino acids L lc-j
upep
,,-4
1:--
Response of t h e monovalent cation electrode coated with urease to urea solutions
Figure 3.
urease per cm3 of coating solution. ( W ) 60 Units of urease per cm3 of coating solution. (A)20 Units of urease per crn3 of coating SOIU-
( 0 )300 Units of
tion
short a time, the coating was fragile and easily torn; if too long, it was less active and adherence was poor. An appropriate binding time had to be chosen for each albumin and glutaraldehyde concentration. Once the right conditions of immobilization were determined, less than 30% of the initial enzyme activity was lost during insolubilization. Active Membrane. Enzymic membranes were easily applied onto electrodes which usually require a membrane, such as CO2 or 0 2 electrodes (Radiometer E 5036 and E 5046). These electrodes are equipped with hydrophobic gas permeable membranes (Silicone or Teflon). An urease membrane on a pC02 electrode is taken as an example. Sixty IU of enzyme were dissolved in 0.56 ml of O.O2M, pH 6.8 phosphate buffer; 0.4 ml of 1.5%albumin solution and 0.04 ml of 25% glutaraldehyde were added (for a final concentration of 1%). This mixture was spread over a perfectly flat glass plate. The area ( 2 5 cm2) was limited by a glass-marking pencil. Cross-linking was performed at 4 "C until complete solidification. The membrane was then rinsed and peeled off from the glass plate. It was divided into squares of 2 X 2 cm, one of which was fitted together side by,side with the special silicone membrane of the gas electrode. Both were applied to the electrode, the enzyme membrane on the side towards the solution (Figure 2). The electrode was then ready for use. The monovalent cation electrode coated with urease was connected t o a pHM27 Radiometer pH-meter. A calomel electrode was used as a reference. The gas electrode equipped with the enzymic membrane was connected to the pHM27 Radiometer pH-meter fitted with p02 and pC02 measuring units. By recording the response curve, the attainment of a steady state was easily detectable. In all cases, the measurements were performed at 25 "C, both electrodes being dipped into the unknown solution. Between measurements, the electrodes were rinsed with distilled water and kept in Tris-HC1 buffer.
RESULTS T h e responses obtained with the above mentioned electrodes are given below. Interpretation of these results in terms of diffusion with enzyme reaction kinetics is given in the discussion section. Enzyme Coated Glass Electrodes. Determination of Urea Concentrations. Using a monovalent cation electrode, ammonium resulting from hydrolysis of urea by urease was measured: 1360 * ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
CO(NH,),
+
2Hz0
+
+
H+-HCO,'
2NH4+
In order to avoid any interference of buffer cations, measurements were performed in 0.1M, p H 7 Tris-HC1 buffer. T h e assays performed at various ureaconcentrations between and 10-'M gave potential responses. At steady state, these responses were nearly proportional t o logarithm of concentration when ranging from t o 10-2M. This linear zone was bordered by a plateau (11) on both sides. The results were close t o those previously obtained by Guilbault e t al. using the entrapped enzyme method (4, 5 ) confirming the validity of both procedures. T h e effect of the variation of enzyme concentration in the coating could be checked (Figure 3). Determination of Amino Acid Concentrations. Amino acid oxidase was bound to a cation electrode. This enzyme catalyses the oxidation reaction of the amino acid: R-CH-COOH
NHp
+
R-C-COOH
H+
H2O
I
+
I1
0
0 2
+
NH4*
+
H20,
The cation electrode allows determination of amino acid concentrations by the assay of the ammonium ions produced. Our results confirm those of Guilbault (7). Using the same Tris-HC1 buffer, the response to lysine, tyrosine, and leucine was quite linear in the range of to 10-2M (Figure 4). After dipping the electrode into the unknown solution, one minute was necessary to reach 99% of the steady-state response. Enzyme Coated Gas Electrodes. Enzymic p 0 elec~ trodes. Consumption of oxygen resulting from the enzymic reaction was followed in various systems. GLUCOSE ELECTRODES. Glucose oxidase and catalase glutaraldehyde membranes assembled with a hydrophobic silicon membrane could be fitted onto a pOn electrode (Radiometer 5046). Glucose
+
O2 GOD, gluconic acid
H2O2
cat alas e
HzO
+
+
H202
7202
where (GOD = Glucose oxidase). T h e partial pressure of 0 2 recorded by the electrode was a linear function (Figures 5a and 5b) of substrate concentrations, identical to the result. AMINO-ACIDELECTRODES.An amino acid oxidase and catalase-bound membrane was adjusted on a p02 electrode (Radiometer 5046). T h e decrease in 0 2 pressure was mea-
G Bml-Sd.
\=
Figure Sa. Steady-state responses of glucose-oxidase coated Clark electrode to glucose concentrations for various active membrane enzyme activities (0)60 U/cm3: ((3) 120 U/cm3: (A)300 U/cm3
~-
I
4
Figure 6. L-Amino acid-oxidase coated Clark electrode response to ~ 150 rnm various concentrations of amino acids for an initial p 0 of of mercury ( 0 )Histidine (V)Arginine (0) Methionine, (A)Phenylalanine, (0) Tryptophane, (9)Leucine ((3) Tyrosine
DISCUSSION
I
The response curves of enzyme electrodes can be explained by theoretical analysis of the concentration profiles of the substrate and/or products inside the active layer. The parameters affecting the response of the electrode can be derived from this kinetic study. Concentration Profiles of Substrate and Products in the Active Layer. While the substrate diffuses in the layer containing the insolubilized enzyme, the enzymic reaction proceeds, and the resulting product appears during this diffusion. The product itself diffuses in the layer, and its concentration is measured a t the interface between electrode and active layer. In the simplest case where the enzyme irreversibly transforms one substrate into one product, the reaction can be written as normal.
\
k+ 2
k+1
Flgure 5b. Same as Figure 5a. The measurements were made with the 60 U / c m 3 membrane for various initial p 0 2 . The slope of all curves is identical.
sured (Figure 6). It took 3 minutes to reach 99% of the steady-state response in this case. Enzyme-pCO2 Electrodes. Production of COP resulting from the enzyme reaction was measured by such electrodes. For instance, urea concentration was determined using urease-bound pC0z electrode: urea
+
2H2O
+
HCOj-
H + Z 2 N H J + + HC0,%
COZ + OH-
A polyalbumin membrane including urease and carbonic anhydrase adjusted with a Teflon membrane on a pCO2 electrode (Radiometer 5036) gave a specific urea sensitive electrode. A logarithm of p C 0 ~as a function of urea concentration in Tris-HClO.lM, p H 7 buffer is given in Figure 7 . A linear response was obtained ranging from 3.10-4 to 10-2M. On both sides of this range a curvature was observed. Our results confirm those of Guilbault and Shu (12). Increasing the enzymic content in the membrane resulted in steeper linear responses and a slight increase in sensitivity. The range of linearity, which depends on CJK,, was unchanged.
E + S a E S - E + P k- 1
where E is the free enzyme; s the substrate; P the product. The reaction rate can,be written (13, 14).
where V, is the maximum rate for C , >> K,; K , is Michaelis constant; C , and C, are concentrations of S and P. For a given enzyme system, the reaction rate is a function of C,/K,. 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 equations:
where t is the time of the reaction, D,*and D,* effective diffusion coefficients of S and P in the active layer, and x the distance of each point of the active membrane to the external surface of the active layer. When
C,/K,
- 0, V
(V,/K,)C,
(first o r d e r kinetics for S). ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1361
Figure 8. Calculated substrate concentration profiles of substrate and of product diffusing in an active membrane coating an electrode
Figure 7. Steady-state responses of urease coated pCO2 electrode to various urea concentrations The curves are given for various contents of enzyme in the active membrane (Urease Worthington, 60 I.U./mg). ( 0 )= 240 iU/cm3; ( A ) = 360 IU/cm3: 0 = 600 IU/cm3
When
C,/K,
is high, V
-
Vm
( z e r o o r d e r kinetics f o r S). Dimensionless reduced variables can be used:
e being the membrane thickness. Equations 1 and 2 become:
as
a2s
at'
8x'2
+
u-= 1
S
+
s
0
(3)
This formulation is more suitable for computer calculations (15-17). The interface electrode-active layer forms an impermeable wall to both substrate and product. The concentration profiles in the active layer are identical to those of half a homogeneous membrane of double thickness. This provides a n easier way of solving the system by assimilating it to a symmetrical analog ( 17). As can be seen from these equations, the concentration of product and substrate near the surface of the electrode a t the steady state depends on several constant parameters: the K , of the enzyme, the enzyme activity in the active layer, the thickness of the layer, the diffusion coefficients of substrate, and product. Figure 8 shows the calculated evolution of concentration profiles of substrate and product in an active membrane applied to an electrode when the ratio of the diffusion coefficients in nearly equal to 1. This evolution depends only on the value which takes into account all these parameters except DP*. The response time of the electrode depends on the ratio e2/D,* which is an expression of the time-lag of the attainment of a steady state inside the membrane. T o reduce the response time, the easiest way.is to reduce the thickness of the membrane. This affects its mechanicalproperties, and it is difficult to obtain appropriate membranes of less than 30 micrometers or coatings of less than 15 micrometers thick using this procedure. Another way is to attain a better mean diffusion coefficient of the substrate. This can be ob1362
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8 , JULY 1975
The Concentration are expressed in K,: time in $/I&. At t = 0, solution substrate concentration equals K,; product concentration is zero: chosen parameters of the active membrane are: V, = 1.92 X lO-'M~cm-~sec-l: K, = 2.10 X 10-5M ~ m - e~ =; 2.5 X cm; Ds .c Dp N 1.2 X om2 sec-'. Steady state is reached for t = 0.7 e*/& = 36 sec
tained by a lower concentration of glutaraldehyde. Once again, the mechanical fragility of the membrane is a limitation to the possibility of reducing e 2 / D , * . The importance of D,* is a constraining factor when the application of such enzyme electrodes to macromolecular substrates is attempted. In the case of urease coatings with cation electrodes, a steady state was reached after 36 seconds. In the case of glucose-oxidase and p 0 polarographic ~ electrodes, the diffusion of oxygen in the electrode itself adds an extra delay to the response-time of the system. A steady-state situation could be reached in less than a minute. When choosing appropriate conditions, accurate measurements could be performed without reaching steady state by means of the slope of the response curve, when choosing the same initial concentration of substrate before the determination, thus reducing response-times to values of the same range as when using urease and a glass electrode. When using silicon or Teflon membranes with pCOz electrodes, the constraining factor is diffusion of COz across the hydrophobic selective membrane. Previous works have shown that immobilization of carbonic anhydrase on the surface of such membranes facilitated transport of COz (18).The mechanism of this effect is an acceleration of the reversible transformation of HC03- toward COz OH- in the polarization layer, a t the interface of the hydrophilic-hydrophobic phase, since COz is mainly dissolved as HC03- in our buffers or in proteic solutions, while the hydrophobic phase is permeable to gaseous COz and not to HC03-. An important improvement in response-time of such electrodes was obtained by taking advantage of the enzymic catalysis by carbonic anhydrase of the time-consuming reaction of HC03- giving Con. Response-time of less than a minute could thus be obtained using pCOz enzyme electrodes with a silicon selective membrane (Radiometer). Operating Conditions of Enzyme Electrodes. As already mentioned, the rate of the product formation depends on C,/K,. If C , is high when compared to K,, the reaction rate (V,) becomes proportional to the number of active sites of bound enzyme (V, = k z E ) and independent of substrate concentration. The concentration of the product a t the interface is constant for any high substrate concentration. If C, is low when compared to K,, kinetics are closer to
+
first-order reaction, and concentration of the product a t the interface electrode-active layer becomes proportional t o substrate concentration in t h e solution. These are the right conditions for an electric sensor; its response has t o be proportional to the measured magnitude (19). Figure 9 gives, in log-log coordinates, the calculated variations of concentrations C, a t the electrode-active membrane interface a t steady state as a function of concentrations C, of substrate in t h e solution for different u values, using urease membranes. The resulting curves have the following characteristics: I) They are linear when substrate concentrations are lower than one tenth of K , (C,/K, = 10-l). 2) Their slope is independent of the u value. 3) Their position depends on u: the higher u, t h e closer the linear part of the curve is to a theoretical line ( m ) obtained for CPe = C,” (0 being the outer surface of the active layer, e its interface with the electrode). When values of u increase, the response becomes less dependent toward the factors affecting this parameter. This can be seen in Figure 9: the lines for increasing values of u become closer to one another, so that any localized variation either on e or on V , has a smaller relative effect when u is high than when it is low. High u can be obtained either by increasing thickness of the active layer, or by increasing V,. Thickness of the layer has an unfavorable effect on response time. On the opposite, increasing enzymic activity has no effect on response time, The most favorable results are then obtained by the highest V, values in thin layers, which favors a t the same time sensitivity of the electrode and reproducibility of the measurements. T h e stability of the active layer depends also on its enzyme activity. For high initial activities, a small decrease has no practical effect on the response of the electrode. For instance, a decrease of V, of 25% has an effect of less than 2% on the sensitivity of the electrode for high u values. Such a decrease appears after weeks or months of use of the electrodes. For instance, a glucose oxidase pO2 electrode could be used for two months with a decrease of sensitivity of less than 2%. Such a slow decrease can be easily compensated by a careful checking. Analysis of Enzyme Electrode Responses. Electrodes Measuring t h e Concentration of One Substrate b y Determ i n a t i o n of I t s “Cosubstrate” Concentration. This is the case for oxygen in glucose and amino-acid electrodes (Figures 5a, 5b, and 6). The consumption of 0 2 is proportional to substrate concentration in the unknown sample. This decrease of 0 2 concentration in the active layer can be observed down to a pO2 close to zero. Two parameters can be studied: the activity of the enzyme bound into the layer, and the partial pressure of 0 2 dissolved in the solution. For a given membrane activity, the slope of the response curve does not depend on initial oxygen pressure (Figure 5b). For various initial pO2, parallel curves are obtained showing that electrode responses cover a larger concentration range when initial pO2 is high. Electrode sensitivity defined by the slope amVlac (20), that is potential shift of the calibration curve vs. concentration increase, is independent of initial pO2. Insofar as the bound L-amino acid oxidase electrode is concerned (Figure 6), different slopes are obtained for the same initial pop with different amino acids. This is due to the fact that the K , of the enzyme varies for these various substrates. Electrodes Measuring Substrate Concentration b y t h e Determination of t h e Product Concentration. This is the case for cation electrodes measuring urea and amino acids
10-2
I
(Km)
Figure 9. Calculated steady-state concentrations of product close to t h e electrode surface as a function of substrate concentrations in the solution for various u values 0 = (V,,,/K,,,)(e2/Ds)at steady state. Theoretical maximum line (m) is reached for cpe = c,’, when all the Substrate would be degraded during the diffusion in the active membrane
and for pCO2 enzyme electrodes specifically measuring urea. Such determinations of substrate concentrations can be performed since the concentration of the reaction product near the sensitive surface of the electrode depends strictly on substrate concentration. The response curves of such electrodes agree with the predicted curves calculated from above-mentioned equations. Such electrodes can be used when kinetics are of first order (for concentrations
