1426
Anal. Chem. 1980, 52, 1426-1429
cm-' with Guttman's measurements (7) is difficult since his results were measured a t temperatures of 50 O C up to 100 "C. Neglecting the influence of the temperature on the absorbance a t 50 "C, our results agree well with those of Guttman (7). It should be noted that at high concentrations of nitric acid vapor (optical path length >0.003 MPaScm) in gas mixtures containing NOz and Nz04, a small overlap of the 2960 cm-I N z 0 4absorption band with a weak nitric acid band was observed. Therefore, the determination of Nz04 in the presence of high nitric acid vapor concentrations should be carried out with the 3160 cm-' N z 0 4absorption peak or calculated from the equilibrium constant at known NOz concentrations. In order to study the accuracy of this method, gas mixtures containing NO, NOz, Nz04, and nitric acid vapor were analyzed. These gas mixtures were prepared by partial oxidation of a known amount NO with air in the presence of water vapor in the gas sample cell. After the partial oxidation the gas sample cell was pressurized with nitrogen to 0.1067 MPa. The following reactions and equilibria may occur in the gas sample cell: value of the equilibrium constant at 25.0 O C , MPa-'
+ 0, 2 N 0 , 2N0, 2N,O4_, 2 N 0 , + H,O 2HN0, + HO NO + NO, Z N,O,+ NO, + NO + H,O 2HN0, 2NO
ref.
-f
0.654
11,13
0.001 30
0.0517
18 13
0.140
14-17
The concentrations of NO, NOz, Nz04,and nitric acid vapor in the gas sample cell were determined with infrared absorption using the calibration curves. The small amounts of NZO3which are present in the gas mixtures were calculated using the equilibrium constant. A representative record of the infrared absorption spectrum of such a gas mixture is given in Figure 1.
Small amounts of water vapor did not produce serious optical interference. The initial amount of NO supplied before the reaction was compared to the amount of nitrogen oxides and nitric acid vapor after reaction had occurred in the gas sample cell and equilibrium had been attained. From Table 11, it can be concluded that the deviation in the mass balance is small. In the spectra an absorption band of nitrous acid vapor at 850 cm-' (19) was sometimes found. The occurrence of the absorption band of nitrous acid can be explained by equilibrium ( 5 ) and its presence is a function of the ratio of the NO concentration and the NOz concentration in the gas sample cell. Nitric acid vapor was detected only in the gas sample at rather low NO concentrations compared to the NOz concentrations.
LITERATURE CITED Saltzman, B. E.; Cuddeback, J. E. Anal. Chem., 1975, 4 7 , 1. Saltzman, B. E.; Burg, W. R . Anal. Chem. 1977, 49, 1. Allen, J. D.; Phil, M. J . Inst. Fuel 1973, 46, 123. Lievens, F. Rapp. Cent. Etude Energ. Nucl. B.L.G. 1973, 480. Forweg, W. V . D . I . ( V e r . Dfsch. Ing.) 1974, 2 4 , 247. Campani, P.; Fang, C. S . ; Prengle, H. W. Appl. Spectrosc. 1072, 26, 372. Gunman, A. J . Quant. Spectrose. Radiaf. Transfer 1961, 2 , 1. Enghnd, C.; Cwcoran, W. H. Ind. Eng. Chem., Fundam. 1974, 13,373. England, C.; Corcoran, W. H. Ind. Eng. Chem., Fundam. 1975, 14, 55. Fontanella. J. C. Off. Natl. D'EtUdes Recherches Adrosoafiales, 1074, Note technique no. 235. Bodenstein, M.; BOBS. F. Z Phys. Chem. 1922, 100, 68. Technicon AutoAnalyzer 11, Industrial method no. 230-72AITentative 1974. Hisatsune, I. C. J . Phys. Chem. 1061, 65,2249. Ashmore, P. G.; Tyler, B. J. J . Chem. SOC. 1961, 1017. Wayne, L. G.; Yost, D. M. J . Chem. Phys. 1951, 19,41. Karavaev, M. M.; Skortsov, G. A., Russ. J . Phys. Chem. 1962. 36,566. Waldorf, D. M.; Babb, A. L. J . Chem. Phys. 1963, 39,432. Nonhebel, G. "Gas Purification Processes for Air Pollution Control"; Newnes Butterworths: London, 1972; Chapter 5 . Jones, L. H., Badger, R. M.; Moore, G. M. J . Chem. Phys. 1051, 19. 1599. Lefers, J. B. Ph.D. Thesis, Delft University of Technology, Delft, The Netherlands, 1980.
RECEIVED for review July 27, 1979. Accepted May 5 , 1980.
Mixed Immobilized Enzyme-Porous Electrode Reactor W. J. Blaedel" and Joseph Wang Department of Chemistry, University of Wisconsin-Madison,
Madison, Wisconsin 53706
A flowthrough electrochemical cell has been constructed to evaluate an enzyme-porous electrode combination, composed of a Sepharose-bound enzyme which Is packed into a Reticulated Vltreous Carbon (RVC) disk. The effects of various experimental parameters upon the electrode response are descrlbed. Rapid response (15 s) Is obtained due to the lntlmate contact between the enzyme and the electrode surface. High sensitivity accrues to the large surface area, permlttlng substrate measurements to be made at the micromolar concentration level.
T h e application of immobilized enzyme electrodes in chemical analysis systems has greatly increased during the past decade. The enzyme electrodes have generally been 0003-2700/80/0352-1426$01 .OO/O
formed by holding a thin layer of the insoluble enzyme matrix (organic gel or polymer) over the electrode, or by trapping the enzyme in or on a semipermeable membrane that covers the electrode. The principles, applications, and recent developments of enzyme electrodes have been reviewed recently (1-3). Enzyme electrodes can be classified as amperometric or potentiometric. Although the amperometric electrode was the first enzyme device reported ( 4 ) ,its development has lagged that of the potentiometric electrode until recently ( 2 ) . The amperometric device, which usually operates at a small ratio of electrode surface to solution volume, usually electrolyzes only a small fraction of the electroactive species involved in the biochemical reaction. The amperometric enzyme electrodes are usually limited by their long response time due to the thickness of the enzyme layer or the membrane through which species must diffuse. Further improvement of their 'C 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980 A
H
Flgure 1. Flow-through cell assembly (configuration shown is for the mixed enzyme-electrode reactor). A, Sample solution inlet. B, Sample solution outlet. C,RVC disk packed with Sepharose-bound enzyme. D, Reference electrode and holder. E, Lead to working electrode. F, Teflon screens and spacer washer. G, Kel-F tube. H, O-rings
performance (i.e., shortening their response time and increasing their sensitivity) is required for their application in high speed automated analysis, for measuring trace amounts of substrates, and t o reduce (by dilution) electroactive interferences. Porous electrodes provide high analytical signals due to their large surface areas, but they have seldom been used as sensors in connection with enzyme catalyzed reactions. Platinum gauze electrodes have been employed recently for regeneration of expensive coenzymes (5, 6). Reticulated Vitreous Carbon (RVC) is a relatively new electrode material which possesses many electrochemical, hydrodynamic, and mechanical advantages (7, 8). Its properties have led to its successful exploitation as an indicator electrode for use in a variety of analytical flow systems (9). In the following work, a unique design of immobilized enzyme electrode based on RVC is investigated. I t is a mixed bed reactor composed of an immobilized enzyme gel that has been packed inside the pores of a 60 pores per inch (ppi) RVC electrode. The characteristics of the reactor-electrode, packed with Sepharose-bound alcohol dehydrogenase (ADH), are evaluated with the ethanol dehydrogenase reaction, by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH) produced. The immediate proximity of the bound enzyme to the electrode results in a rapid (15 s) response. The high analytical currents permit measurements of ethanol a t the micromolar concentration level. Cell design is simple and fabrication and use are easy.
EXPERIMENTAL The cell design is shown schematically in Figure 1. The body consisted of two 0.5-inch diameter Plexiglas cylinders (Rohm & Haas Co., Philadelphia, Pa.), each about 4 cm long. A channel (0.8-cm diameter) was drilled through the lower block and through the lower part of the upper block. The two blocks Apparatus.
1427
were held together by an inner Kel-F tube (2.2 cm long, 0.79-cm o.d., 0.55-cm i,d.),furnished with O-rings to provide a leak-proof seal. The RVC disk (Fluorocarbon Co., Anaheim, Calif.) was lodged against the end of the cavity in the upper block, and held in place with the Kel-F tube and spacer washers. A 60-ppi RVC disk (2 X 1-Stype,0.75-cm diameter, 0.5 cm long, 0.22-mL volume) was employed. Based on microscopic measurements, the pore openings range around 0.4-0.6 mm, which is much larger than the diameter of the Sepharose beads (-0.07 mm). Having a void volume of about 95%, the RVC disk can accommodate about 0.21-mL of packed Sepharose gel, corresponding to 0.06 g of the dry powder. The beads penetrated into the pores, giving a mixed enzyme electrode reactor. Leakage of the beads from the electrode volume was prevented by two Teflon screens (74-pm mesh, 21% open pore, 0.75-cm diameter) and a Teflon washer spacer (0.75-cm o.d., 0.55-cm i.d., 1-mm thick) held underneath the RVC disk. The procedure for loading the enzyme column is presented below. Electrical contact to the RVC was made by pressure to one end of a short glassy carbon rod (1-mm diameter), the other end of which was connected with conducting epoxy (Epo-Tek 410E, Epoxy Technology Inc., Billerica, Mass.) to a copper wire that led to the outside. The salt bridge of the reference electrode (Ag/AgCl in 0.1 M KC1, against which all potentials were measured) was introduced to the cell through the hole at its lower end. In systems containing the enzyme column and the supporting electrolyte, the resistance between the working and reference electrodes was 600 Q. The sample solution was stored in a 250-mL Nalgene beaker fitted with a Plexiglas cover containing two holes: one for the sample inlet and another for standard additions. Connection of the cell to the sample reservoir was made by Teflon tubing (1/16-inch0.d.) and a tube-end fitting. Solution outflow was maintained through a Plexiglas tube (1.5-cm long, 0.6-cm o.d., 0.2-cm i.d.) cemented at a right angle into the lower block. Solution flow through the cell was governed by gravity. A hydraulic head of about a foot permitted flow rates up to 5 mL/min through the enzyme-packed RVC reactor. After exit from the cell, the solution passed through a calibrated flowmeter and a Teflon needle valve to control the flow rate. Potentials were applied with a simple battery-powered potentiometer, and currents were measured with a picoammeter (Model 414S, Keithley Instrument, Cleveland, Ohio). The current output was displayed with a Houston Omniscribe chart-recorder. Reagents. All solutions were prepared from deionized water (Continental Water System (charcoal bed filter, mixed bed deionizer, 0.2-fim Gelman filter)). Supporting electrolyte was 0.1 M phosphate buffer (pH 7.4) prepared from a l j 4 mixture of analytical grade KH2P04and KpHP04. Solid preparations of alcohol dehydrogenase (ADH, E.C. 1.1.1.1)and nicotinamide adenine dinucleotide @-NAD+)(both from Sigma Chemical Co., St. Louis, Mo.) were stored desiccated at -22 "C. The cyanogen bromide activated Sepharose 4B (Sigma Chemical) was stored at +4 "C. AU other chemicals were reagent grade. Stock solutions of 10 mM NAD+ and 100 mM ethanol were prepared weekly with the buffer and stored in a refrigerator. P r e p a r a t i o n of I m m o b i l i z e d E n z y m e - P o r o u s E l e c t r o d e Reactor. Sepharose-ADH gels having two enzymatic activities were prepared and studied. For the preparation of the higher activity gel, a reaction mixture containing 10 mL of 0.1 M NaHCO3 solution, 12 mg ADH, and 0.8 g cyanogen bromide-activated Sepharose was used. The lower activity gel was prepared from a reaction mixture of 10 mL 0.1 M NaHC03 solution, 1mg ADH, and 0.3 g cyanogen bromide-activated Sepharose gel. Coupling was allowed to proceed by stirring the reaction mixture in a 25-mL beaker for 16 h at 4 "C. The suspension was transferred to a Buchner funnel and washed with large amounts of 1 M NaCl solution and phosphate buffer. The immobilized enzymes were stored in the phosphate buffer at 4 "C. The enzymatic activities of the gels were estimated as 15 and 0.5 U/g, based upon batch measurements of the enzymatically produced NADH at 340 nm (5).
The enzyme column was loaded as follows. The cell inlet was disconnected from the Teflon tubing; small air bubbles inadvertently trapped in the RVC were removed by tapping gently. The Sepharose-ADH suspension was injected from a plastic syringe into the buffer solution in the cavity above the RVC disk.
1428
ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980
The enzyme column was then built inside the RVC disk by passing the solution through slowly, allowing the Sepharose-ADH beads to settle in place. In these operations, it was important always to keep the column filled with solution to avoid air entrapment. The suspension was added until the Sepharose-ADH gel filled all of the void volume of the 60-ppi RVC disk, perceived by the buildup of a gray color. After the enzyme column was built, the Teflon tubing was connected to the cell inlet, and the buffer solution was flushed slowly through the cell for 5 min, after which the reactor was ready for use. Analysis Procedure. The analysis procedure was based on providing NAD+ as a reagent, along with the substrate. The RVC was pretreated at the beginning of each day by cycling the applied potential between -1.25 and +1.25 V for 15 min, allowing 3 min at each applied potential. The pretreatment served to put the carbon surface in a clean and reproducible state prior to the analysis. Following pretreatment, the working potential for the oxidation of NADH (+0.9 V) was applied and the transient currents were allowed to decay until steady state was reached (this usually took 40 to 60 min). Then the blank solution containing NAD+ in the phosphate buffer was flushed through the cell at the desired flow rate and the background steady-state current was recorded. Aliquots of the ethanol stock solution were then added to the blank solution to give the desired substrate concentrations. With the addition of each aliquot, the current rose as the enzymatically produced NADH became oxidized electrochemically, and a new steady-state current was attained rapidly. The response of the reactor to a given ethanol concentration was taken after correction for the background steady-state current.
RESULTS AND DISCUSSION Preliminary Experiments: Separated EnzymePorous Electrode Configuration. Preliminary experiments were undertaken with a “separated” configuration, consisting of an enzyme column packed onto the RVC electrode. For this purpose two designs were employed, using the flow-cell body described before. The first design consisted of a 100-ppi RVC disk (2 X 3 S-type, 0.75-cm diameter, 0.3 cm long), with a pore size small enough to prevent penetration of the Sepharose beads, so that it served as a support for the enzyme column bed above. The second design consisted of a 60-ppi RVC disk, like that described for the “mixed” design, with two Teflon screens (74-pm mesh, 21 % open pore, 0.7-cm diameter) above it to prevent penetration of the Sepharose particles. The enzyme columns were loaded at the cavity above the RVC disk employing a procedure similar to that described for the “mixed” design. T h e mechanism of the processes occurring in the “separated” reactor is simpler than that occurring in the “mixed” design, since the biochemical and the electrochemical reactions are separated in place and time. The NADH formed a t the enzyme column is transferred downstream to the RVC electrode, where anodic oxidation and measurement take place. However, owing to flow rate limitations, only preliminary studies were made with these designs. The dependence of the response current upon the ethanol concentration was studied for various column heights (1-5 mm), NAD’ concentrations (0.02-1 mM), and the two enzyme activities. The dependence of response upon ethanol concentration for these systems was decidedly nonlinear at high enzyme activity. For the low enzyme activity, the response was linear up to about 80 pM ethanol. In general, the response increased with the enzyme column height and activity, the electrode surface area, and the NAD+ concentration. Also, after 1-2 h of continuous operation, a 2&30% decrease in response was observed. This was postulated to be due to gradual fouling of the electrode, perhaps due to adsorption of NADH oxidation products (IO), and/or instability of the enzyme. The operation of the “separated” designs was limited to low flow rates. Enzyme column heights of 1 and 5 mm allowed maximum flow rates of 2 and 0.6 mL/min, respectively. Since the mixed enzyme electrode (5-mm height) allowed flow rates
08k
0
0
50
100
I50
ETHANOL CONCENTRATION, p M
Flgwe 2. Response of mixed enzyme porous carbon reactor to ethanol concentration. Flow rate, 1.45 mL/min. 0.1 M phosphate buffer, pH 7.4. Applied potential, 4-0.9 V. NAD’ concentration: 50 p M (a, b); 500 pM (c, d). Enzymatic activity, 0.5 U / g (b, c), 15 U/g (a, d)
up to 5 mL/min for a similar hydraulic head, further work on the separated design was abandoned. Evaluation of the Mixed-Enzyme-RVC Reactor. In order to study the effect of the Sepharose-ADH packing upon the response of the RVC electrode, its behavior was compared to an unpacked 60-ppi RVC electrode. Two similar ten-point standard addition experiments were performed for the unpacked and Sepharose-packed electrodes, using reduced nicotinamide adenine dinucleotide phosphate (NADPH), and the corresponding steady-state limiting currents for the oxidation of NADPH (without any enzymatic reaction) were measured and compared. For the NADPH concentration range 0-10 pM, a least squares slope of 140 f 4 nA/pM (90% confidence limits) was obtained for the packed RVC electrode, and a slope of 127 f 3 nA/KM for the unpacked electrode (conditions: 2.25 mL/min, applied potential of +0.9 V, 0.1 M phosphate buffer (pH 7.4)). The slightly but significantly higher sensitivity of the Sepharose-packed electrode indicates better mass transport, caused perhaps by the breaking up of laminar flow streamlines by the packing. Figure 2 presents the dependence of the reactor response upon ethanol concentration for the two enzyme activity levels a t two NAD+ concentration levels. While the plots corresponding to the higher enzymatic activity (a, d) were not linear over the whole substrate concentration range that was studied, the plots for the lower enzyme activity (b, c) were linear up to ethanol concentrations around 50 p M . This linear response range is considerably smaller than that predicted on the basis of ordinary ADH kinetics in homogeneous solution, where linearity extends to concentrations around the MichaelisMenten constant, 8.9 mM ( 1 1 ) . Further examination of the data of Figure 2 shows other deviations from the MichaelisMenten behavior: a 30-fold increase in the enzymatic activity resulted in little or no increase in the reactor response (compare curves a to b and c to d). Also, the 10-fold increase in the coenzyme concentration resulted in only a 3- to 5-fold increase in response (compare curves a to d and b to c). These observations (noted also in the separated design) indicate that the current is not limited by the rate of the enzyme reaction alone. Goldstein (12) has reviewed the various effects (mass
ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980
-1300
-I: 1 2
g 401
i;. 200
0
B~ LL
I 20c
01 0
I I
I 2 FLOW RATE, mL/MIN
I
3
lo
Flgure 3. Dependences of the response time and of the response current upon volume flow rate. Applied potential +0.9 V. Enzymatic activity, 15 U/g. Ethanol concentration, 20 p M (a), 60 p M (b). NAD' concentration, 50 p M (a), 100 p M (b). 0.1 M phosphate buffer, pH 7.4
1429
Figure 3a shows the dependence of response time (determined as the time required to reach steady state) upon the volume flow rate. As expected, the response time decreases with increasing flow rate, reaching a limiting value of 15 s for flow rates above 2.9 mL/min, where mass transport through the liquid phase probably no longer limits the response. This is considerably faster than most other immobilized enzymeelectrode designs that have been described in the literature ( 2 ) , and represents a definite advantage of the mixed enzyme-porous electrode design. Figure 3b indicates that the current response of the mixed enzyme-porous electrode design is practically independent of flow rate in the range 0.5-3 mL/min. This supports the conclusion reached above, namely, that liquid phase rate processes, such as mass transport, probably do not limit the response. Figure 4 demonstrates the response of the mixed reactorelectrode to successive standard additions of ethanol, each addition effecting a 24 pM increase in concentration. The high sensitivity and low noise level of the response are apparent, when compared to other types of enzyme electrodes of smaller surface area. The high sensitivity may permit sample dilution before measurement, which may be an advantage in dealing with samples containing interferences (15).
ACKNOWLEDGMENT The valuable technical assistance of R. Lammed is highly appreciated. LITERATURE CITED (1) (2) (3) (4) (5)
BKGD
J
L 2 MIN
Flgure 4. Current-time recording obtained upon increasing the ethanol concentration in 24-pM steps. Conditions: as in Figure 2c
transport, steric, partition, and microenvironmental) of immobilization upon the kinetic behavior of the enzymes. These effects have been studied for Sepharose-bound enzymes (13, 14) and they have been interpreted to decrease the apparent reaction rate constant for the immobilized enzyme system below that observed in homogeneous systems. Also, these effects may cause nonlinearity over the whole range of substrate concentration, as observed for the high enzyme curves (a and d) of Figure 2.
(6) (7) (8)
(9)
(IO) (1 1)
(12) (13) (14) (15)
Rechnitz, G. A. Chem. Eng. News 1975, 53(4),29. Bowers, L. D.; Carr, P. W. Anal. Chem 1976, 4 8 , 545A. Gray, D. N.; Keyes, M. H.; Watson, B. Anal. Chem. 1977, 49, 1067A. Updike, S. J.; Hicks, G. P. Nature (London) 1962, 214, 986. Coughlin, R. W.; Aizawa. M.; Alexander, B. F.; Charles, M. Biotech. Bioeng. 1975, 17, 515. Jaegfekh, T.; Torstensson, A.; Johansson. G. Anal. Chim. Acta 1978, 97,221. Strohl, A. N.; Curran, D. Anal. Chem. 1979, 5 1 , 353. Blaedel, W. J.; Wang, J. Anal. Chem. 1979, 5 1 , 799. Blaedel, W. J.; Wang, J. Anal. Chem. 1979, 5 1 . 1724. Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 4 7 , 1337. Engstrom, R. C. Ph.D. thesis, University of Wisconsin-Madison, 1979. Goldstein, L. I n "Methods in Enzymology", Vol. XLIC, Mosbach, K., Ed.; Academic Press: New York, 1976; p 397. Axen, R.; Myrin, P. A.; Jansson, J. C. Siopo/ymers 1870, 9 ,401. Levi, A. S. Arch. Biochem. Biophys. 1975, 168, 115. Blaedel, W. J.; Engstrom, R. C. Anal. Chem., in press.
RECEIVEDfor review February 25, 1980. Accepted April 28, 1980. This work was funded in part by the University Sea Grant Program under a grant from the Office of Sea Grant, National Oceanic and Atmospheric Administration, U.S. Department of Commerce, and by the State of Wisconsin.
