591
Anal. Chem. 1984, 56,591-593
LITERATURE CITED (1) Kratochvil, B.; Makra, C. Am. Lab. (Falrfkkf, Conn.) 1983, 15(1), 22, 24. 26. 28. 29. (2) Biler,'E. A.; Swift, E. H. J . Chem. Educ. 1972, 49, 425. (3) Thoburn. J. M. J . Chem. Educ. 1959, 38,616. (4) Bishop, E.; Cofr6, P. Anakst ondo don) 1978, 703, 162. and refer-
ences therein.
(5) Bishop, E.; Dhaneshwar, R. G. Analyst (London) 1982, 87, 207. (6) Cofr6, P. J . Chem. Educ. 1983, 60, 421.
(7) Hartshorn, L. 0.;Blshop, E. Analyst (London) 1971, 96, 885. (8) Bishop, E. Anal. Chim. Acta 1959, 20, 315. (9) Bishop, E. PrOC. SAC COnf. 1965, 416-429.
RECEIVED for review September 21,1983. Accepted November 21, 1983. This work was supported by the Direccidn de Investigacidn de la Pontificia Universidad Catdlica de Chile, under Grant No. 4/82.
Air-Dried Enzyme Electrodes Gilbert Bardeletti a n d Pierre R. Coulet*
Laboratoire de Biologie et Technologie des Membranes d u CNRS, Universitt? Claude Bernard, Lyon I , 43, Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cgdex, France Performance and characteristics of immobilized enzyme electrodes appear strongly dependent on the type of enzyme, the support, the immobilization procedure, and the conditions of utilization, namely, industrial or clinical (1-3). Their use still appears generally confined to the laboratory and is very often impaired by the conditions of storage. In our laboratory collagen films were chosen for enzyme immobilization and a mild method of general use has been previously developed for the covalent binding of numerous enzymes ( 4 , 5 ) . Beside polymembrane bioreactors, enzyme electrodes have been designed with either mono-, bi-, or multienzyme active membranes (6, 7). The aim of the work reported here was to test the performance of an air-dried enzyme electrode, to widen its field of usefulness, and to make it more easy to handle. EXPERIMENTAL SECTION Apparatus. The polarograph supplied by Solea-Tacussel, Villeurbanne, France, was similar to that used with our previously described glucose electrode (6). The sensor consisted of a silver cathode covered with silver chloride and a platinum anode on which the enzymic collagen membrane is maintained in close contact by a screw cap. The potential of the platinum anode was fixed at +650 mV vs. Ag/AgCl/Cl- and the anodic current was proportional to the amount of oxidized peroxide generated during assays at the enzymic membrane level. Reagents. Glucoamylase (GA, EC 3.2.1.3, 1,4-a-D-glUCan glucohydrolase) lyophilized powder, 50 U/mg, was purchased from Merck. Glucose oxidase (GOD, EC 1.1.3.4) grade I lyophilized powder, from Aspergillus niger, 210 U/mg, peroxidase (POD, EC 1.11.1.7) grade I lyophilized powder, from horseradish, 250 U/mg, and ABTS (2,2'-azinqbis(3-ethyl-6-benzothiazolesqlfonate))were supplied by Boehringer, France. The 80-100 pm thick collagen films (F61 and F70 type) were provided by the Centre Technique du Cuir, Lyon, France. All other reagents were pf the highest grade commercially available. Procedure. Enzyme Immobilization. Collagen films were activated by using the acyl-azide process previously developed in our laboratory. For glucoamylase (GA) and glucose oxidase (GOD) two coupling procedures, random and asymmetric were used ( 4 , 5 ) . Disks of the chosen area adapted to the electrode tip were cut out of these films and mounfed on the sensor. Electrodes were filled with 0.2 M acetate buffer and 0.1 M KCl pH 5.5. Measurements. Enzyme activities for free or immobilized GOD and GA were determined at 30 "C following the H202formation using either the POD-ABTS reagent (8) or electrochemical detection (9). For substrate determination, the enzyme electrode is immersed in 20 mL of 0.2 M acetate buffer and 0.1 M KCl, pH 5.5, at 30 "C. The electrode could be used either with glucose alone or with maltose according to the reactions leading to H2OP maltose
+ H20
2P-D-glucose
(1)
(GA)
0003-2700/84/0356-059 1$01.50/0
P-D-glucose + HzO + 0
D-gluconic acid
2
+ H202
(GOD)
(2)
(3) After equilibration the sample is added and then the current increases and reaches a new stable level from which the steadystate response time can be deduced (Figure 1). For further assays, the sample can be added successively after obtaining each plateau provided the final concentration in the reaction vessel is kept M. below
RESULTS AND DISCUSSION Among the characteristics of our immobilized enzyme electrode, the activity of the surface-bound enzyme related to the sensitivity and the thickness of the inner space between the membrane and the transducer are two important factors. In the work presented here the search for new conditions of storage, namely, to keep the enzyme electrode dry in the air a t room temperature, led us to focus on the influence of the thickness of the inner space on the electrode response. Prior to the air-drying procedure, a control experiment is performed. When glucose or maltose is added to the buffer solution into which the enzyme electrode is dipped, a current vs. time plot can be recorded and reaches a maximum value Io after a few minutes corresponding to the steady-state response time to (Figure la). Then the enzyme electrode is air-dried and kept at room temperature. To study its behavior after this treatment and a chosen period of storage, the enzymic sensor is reimmersed in buffer and tested after 1h of equilibration. When the electrode is kept plugged to the potentiostat a 10 min equilibration is sufficient to obtain a low and stable base line current allowing the best detection. After this dry storage an identical measurement is performed with the same setting of the device and the transient signal recorded (Figure lb). Comparing the curves, it can be seen that for the same variation of substrate concentration, I,, is lower than Io, showing that the probe sensitivity I / C is affected, but the time necesary to reach the plateau is strongly lowered, thus improving likewise the response time t,. These two factors were then more carefully studied for either glucose or maltose determinations as a function of dry storage. Furthermore the influence of asymmetric and random immobilization on the performances of the probe was investigated. Sensitivity. As already reported for enzyme collagen membrane electrodes the sensor responds to glucose or maltose within 4 to 5 concentration decades (5, 6). To estimate the evolution of sensitivity with dry storage, we have determined the value of I / C for the electrode which 0 1984 American Chemical Society
592
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
I
I P I
t
I
0 2 5
I
I
I
I
I
I
I
,
0
1
2
3
4
5
6
7
IminI
TIME
Figure 1. Transient electrode signal before (a) and after (b) airdrying procedure: s.i., sample Injection.
10
20
50
30
CO days ( d r y storaqel
60
Figure 4. Evolution of senskkity as a function of dry storage, bienzyme electrode tested with GA/GOD asymmetric coupling: glucose (B); maltose (0).
-2 - 1 I
1
0 2 5
10
LO
30
20
50
days ( d r y storage1
Figure 5. Evolution of sensitivity as a function of dry storage, bienzyme electrode tested with GA GOD random coupling: glucose (0); maltose (0).
+
I
I
20
30
J
I
40
50
I
[Maltose] IpM)
Figure 2. Calibration curves for maltose with asymmetric bienzyme membrane electrode in the range 1-25 pM. Subscripts for t Indicate the number of days of dry storage.
I
0 2 5
10
I
I
60
days ( d r y storage)
Figure 6. Improved steady-state response time with dry storage, bienzyme electrode tested with GA/GOD asymmetric coupling: glucose (B);maltose (0).
5 ' 00
5
10
O
[Maltose]
20
30r
lvM)
Figure 3. Callbration curves for maltose with random bienzyme membrane electrode In the range 1-25 pM. Subscripts for t indicate the number of days of dry storage.
was simply kept dry in the air between assays. Experiments were conducted within 2 months in four cases: glucose and maltose determination with membranes prepared with random or asymmetric immobilization procedure. For this purpose, experiments were conducted within a linear part of the concentration range, 10*-10-3 M. For example, data obtained with maltose from 1to 25 MMare given (Figures 2 and 3) for asymmetric and random coupling showing clearly the evolution of sensitivity vs. storage. This was also done for glucose and I / C was determined from the slopes in the four cases and plotted vs. time (Figures 4 and 5). I t can be seen that the decrease of sensitivity occurred in two steps within the 2 months of experiments for both maltose and glucose. The first step is rather fast and shorter in the
case of asymmetric coupling (Figure 4) and after a 2-5 day period, differences in day-to-day calibrations are minimal for 1 month. In the case of random immobilization (Figure 5) the difference is less striking but the values of I / C are always lower than in the case of asymmetric coupling. It must be pointed out that sensitivity obtained with maltose is always higher than that obtained for glucose with either random or asymmetric coupling, the difference being more important for the latter especially for initial values. We have also checked the detection limit which depends on the noise level and corresponds to the lowest concentration yielding a deviation from the background current. With the bienzyme asymmetric membrane its initial value for glucose before drying was 5 x lo-' M;this value was still M after 2 the same after 1 month and equal to 2.5 X months. For maltose, the initial value was 2.5 X lom8M, M after 2 identical after 1 month, and equal to 1.5 X months. Results obtained with randomly immobilized enzymes were not significantly different. Incidently measurements of concentrations as low as 2.5 X M glucose were still possible with a glucose oxidase monoenzyme electrode after 160 days of dry storage. Response Time. Figures 6 and 7 show that before drying the steady-state response time in the four previously described cases ranges from 4 to 8 min. It can be seen first that the
Anal. Chem. 1984, 56,593-595
0 2
5
10
I
I
I
I
20
30
LO
50
days ( d r y storage)
Figure 7. Improved steady-state response time with dry storage, bienzyme electrode tested with GA i-GOD random coupling: glucose ( 0 ) ;maltose (0).
response time for glucose is always lower than for maltose and second that for each of them the asymmetric coupling procedure yields a better response time (Figure 6). It is also obvious that drying strongly improves the response time within 2 days for asymmetric coupling whereas at least 2 weeks are necessary in the case of random immobilization to obtain the maximal improvement (Figure 7). For example, if we compare the values of the response times measured after 2 days of drying, 80% improvement was obtained for maltose using the asymmetric bienzymic membrane and only 35% with the randomly immobilized enzymes. When the same probes are used for sole glucose measurements, an improvement after 2 days of 70% and 35% occurred with the asymmetric and random bienzyme systems, respectively. Our goal in this work has been to improve the usefulness of our enzyme electrode by storing it in the air between assays. Owing to its fibrous protein content, our enzymic membrane undergoes during its first dehydration a shrinkage which leads to a more intimate covering of the platinum tip. Even after reimmersion of the electrode for assays into the reaction vessel, the membrane keeps tightly pressed like a skin on the anode and for interchange it must be really peeled off. This proves that a reduction of the inner space occurred which facilitates the diffusion of HzOzand then a faster establishment of the steady-state current. In the experiments reported here the
593
electrode was rehydrated several times for 6-8 h periods including equilibration and glucose and maltose assays. In these conditions the improved response time was found constant during the experiment. After being washed, the electrode was allowed to dry in the air for a definite period until the next test. The first drying which creates the intimate contact between the membrane and the platinum anode can be performed faster: instead of waiting for a natural air-drying, the electrode tip can be placed in lukewarm air blowing (30-35 “C) for 1 h. This procedure leads to the same results as those obtained after 48 h in the air at room temperature, then the electrodes were used as previously described and exhibit identical performances. Results obtained with asymmetric enzymatic membranes were also better than those obtained with the random process. The improvement in sensitivity of bienzyme electrodes equipped with nylon net membranes prepared with this asymmetric coupling procedure has been recently confirmed by Mascini et al. (10). The fact that active electrodes can be kept in a dry state simplifies considerably their storage and shipment and furthermore allows to their sterilization. Registry No. Glucoamylase, 9032-08-0; glucose oxidase, 9001-37-0.
LITERATURE CITED Guilbault, G. G.; Sadar, M. H. Acc. Chem. Res. 1979, 12, 344-350. Carr, P. W.; Bowers, L. D. “Immobiilzed Enzymes in Analytical and Cllnical Chemistry”; Wiley: New York, 1980; Chapter 5. Gough, D. A.; Leypoldt, J. K. I n “Applled Biochemistry and Bioengineering”; Wingard, L. B., Katchalskl, E., Goklstein, L., Eds.; Academlc Press: New York, 1981; Vol. 3, pp 175-206. Coulet, P. R.; Julliard, J. H.; Gautheron, D. C. Biotechnol. Bioeng. 1974, 16, 1055-1068. Couiet, P. R.;Bertrand, C. Anal. Lett. 1070, 12, 581-587. Thevenot, D. R.; Sternberg, R.; Couiet, P. R.; Laurent, J.; Gautheron, D.C. Anal. Chem. 1979, 51, 96-100. Bertrand, C.; Coulet, P. R.; Gautheron, D. C. Anal. Chim. Acta 1981, 126, 23-34. Bergmeyer, H. U. I n “Methods of Enzymatic Analysis”; Bermeyer, H. U., Ed.; Veriag Chemie-Academlc Press: New York, 1974; Vol. 3, pp 1212-1213. Blum, L. J.; Bertrand, C.; Coulet, P. R. Anal. Lett. 1983, 16, 525-540. Masclni, M.; Ianello, M.; Palleschl, G. Anal. Chim. Acta 1983, 146, 135-148.
RECEIVED for review June 10,1983. Accepted November 21, 1983.
Determinatlon of Critical Micelle Concentrations by Bipolar Pulse Conductance with an Exponential Dllution Flow System Douglas W. Taylor and Timothy A. Nieman* School of Chemical Sciences, University of Illinois, 1209 West California Street, Urbana, Illinois 61801 Micellar systems find use in analytical chemistry in such applications as UV-visible absorbance determinations of metal ions with chelometric indicators ( l ) mobile , phases for chromatographic separations (2, 3), and observation of roomtemperature phosphoresence in liquids (3, 4). In all these cases, knowledge of the critical micelle concentration (CMC) is necessary. A variety of methods have been used for determining the CMC. Several of these techniques were reviewed by Mysels and Mukerjee (5). Some common methods include light scattering, dye solubilization, surface tension, adsorption, and conductance. We have previously demonstrated (6) that bipolar pulse conductance (BICON) is a simple and rapid method for precise determination of CMC’s. BICON has certain advantages over conventional conductance techniques as noted previously (7,
8). One of these is the speed of measurement. Computer-
controlled BICON instruments can make a conductance reading as often as every 30 ps. With this measurement speed, CMC determinations would be limited only by the speed of solution preparation. Exponential diluters have been shown to be useful in preparing calibration curves in a short time (9). Starting with only one stock solution, a continuous concentration gradient can be obtained. This gradient is described by eq 1 log
c, = log c, - u t / 2 . 3 0 3 v m
(1) where C, is the concentration at time t , Co is the initial concentration, U is the flow rate of diluent, V , is the volume of mixing, and t is time. It is possible to create a calibration curve over several decades of concentration.
0003-2700/84/0356-0593$01.50/00 1984 American Chemical Society
