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Biochemical fuel cells
ARCHIVES
OF BIOCHEMISTRY
AND
169, 146-151 (I9751
BIOPHYSICS
Biochemical Demonstration
of an Obligatory
Enzymatically MICHAEL Department
of Biochemistry,
Fuel Cells
Pathway
Involving
Catalyzed
Aerobic
K. WEIBEL
AND
of Medicine,
University
School
an External
Oxidation
CHRISTOPHER
Received January
of Pennsylvania,
Circuit
of Glucose’,
for the
2
DODGE Philadelphia,
Pennsylvania
19174
10, 1974
An in vitro biochemical fuel cell based upon the enzymatically catalyzed aerobic oxidation of glucose is described. The anodic half-reaction employs an electron transfer sequence consisting of the glucose oxidase reductive half-reaction and dichloroindophenol. The cathodic half-reaction involves reduction of molecular oxygen. A high Faradic efficiency for the intact cell approaching 100% has been experimentally demonstrated. The steady state current is exponentially related to the concentration of the terminal electron transfer species in the anodic chamber. The behavior is consistent with application of the Nernst relationship to define the cell potential and a simple resistance circuit. The discharge profile of the cell after complete oxidation of the primary fuel, glucose, can be modeled as a capacitor discharging through a resistor.
The aerobic oxidation of glucose is extremely favorable from a thermodynamic assessment. In point of fact, however, virtually no reaction occurs under in vitro physiological conditions due to severe kinetic contraints for the uncatalyzed aerobic oxidation. Several reports have appeared concerning biochemical fuel cells based upon noble metal catalysts such as platinum black and polymeric redox dyes (1). The explicit half-reaction electrode chemistry for these systems has not yet been unequivocally identified. An enzymatically catalyzed oxidation represents a molecular battery operating at short. The internal resistance is defined by intrinsic rate limiting intra- and intermolecular processes of the catalytic pathway. The cell potential at any given time is thermodynamically defined by the difference in free energy of products and substrates participating in the redox reaction. Since the process proceeds in a highly irreversible manner, no useful work is ob’ Supported by NSF GI 32498X2. *This paper is the first in a series, “Biochemical Fuel Cells.” 146 Copyright All rivhtc
0 1975 by Academic nf wnrodnrtinn
Press, Inc. in nnv form reserved.
tained and the available chemical energy is dissipated in the form of reaction enthalphy to the environment. If an external circuit could be inserted between the reductive and oxidative half-reactions of the enzymatically catalyzed redox reaction and that circuit represent an obligatory pathway for electron transfer, then the opportunity exists for transduction of chemical energy from the reaction into useful work. Similarly, the cell may be electrochemically driven in reverse utilizing the tightly coupled external circuit to provide energy for an enzyme catalyzed reduction or oxidation. Two reports concerning a biochemical fuel cell employing the enzymatically catalyzed aerobic oxidation of glucose have appeared (2, 3). In both cases only empirical characterization was reported. The absence of a systematic analysis and elucidation of the half-cell chemistry have made it difficult to interpret prior work and formulate basic principles concerning this type of biochemical fuel cell. We report here the first of several studies on the glucose oxidase fuel cell employing bright platinum electrodes.
GLUCOSE METHODS
OXIDASE
oxidase
CELL
AND MATERIALS
1.1.3.4) from Aspergillus from Sigma Chemical Co. The enzyme was purified to homogeneity by chromatography on DEAE cellulose (4). Anhydrous and ultra pure glucose (Ultrex) was obtained from J. T. Baker Chemical Co. Solutions of glucose were allowed to obtain anomeric equilibrium prior to use. Mutarotation rates within the anodic chamber under the experimental conditions employed were fast relative to the steady state oxidation rates of P-o-glucose. The redox dye mediator, 2,6-dichlorophenolindophenol (Cl, indophenol or Cl, Ind)s, was obtained from Sigma Chemical Co. and used as obtained. All other chemicals were of “certified” grade. The simple cell design is shown in Fig. 1. The half-cell chambers were glass cylinders, 16 mm diam by 80 mm high, with a standard Luer connecter on the side for attachment of the salt bridge. The top of the half-cell chamber was capped with a thin septum 17 mm diam serum bottle plug which allowed easy access to the electrode chamber for material additions, gas flushing and electrode leads. An electrode material was chosen which does not significantly serve as a direct electron acceptor for glucose under near physiological conditions. The solid electrode employed for this study was bright platinum foil (0.025 mm thick) with a surface area of 3 cm*. A salt bridge was constructed from a 2 mm i.d. glass T joint and possessed a longitudinal path length of 3 cm. The bridge matrix material was 1% agar (wt/vol) in the buffer medium. The buffer for both half cells was 0.1 M potassium phosphate, pH 6.0 containing 0.5 M KCl. Anerobic conditions in the anode half cell were obtained by purging the chamber solution for 2 h with oxygen free argon and maintained after start up of the cell by continuously flushing of the space above the liquid phase. Argon was a commercial grade and traces of oxygen were removed by passing the gas through a gas scrubbing train of four Dreschsel units containing vanadous sulfate (5). The cathode half-cell chamber solution was saturated with oxygen by continuous purging with water saturated oxygen. A small Teflon stir bar was placed in the anode half-cell and operated by an external rotating magnet. This arrangement was necessary for rapid mixing of added constituents such as glucose or electron transport components to the solution within the chamber. Integrated amperometric measurements in the external cell circuit were obtained with a model 153, Kiethly microvolt-ammeter. Current-time profiles of the full cell were recorded with a model EU-POB Health recorder. Integration of the trace was performed manually employing finite interval areas and is within *50/o of the exact integral. Glucose
FUEL
(EC
niger (type VI) was obtained
‘Abbreviations used are: Cl, Ind and Cl, indophenol, 2,4-dichloroindophenol; and FAD, Flavin adenine dinucleotide.
ARGON-
ANODE
CATHODE
Fig. 1. Schematic
of cell system.
RESULTS
Figure 2 shows a typical current-time profile for the fuel cell upon addition of the anode substrate glucose. Three distinct regions are apparent. The presteady state behavior consists of two distinct phases. The early increase in current reflects rate limiting solution kinetics of the anode electron transport sequence and correlates with the formation of reduced ClJnd. The decrease in current from the peak value to the plateau region probably reflects an internal resistance change associated with the attainment of a steady state ion gradient in the salt bridge. The plateau region of the trace represents a steady state corresponding to complete reduction of the anode electron transport sequence. This condition represents the maximum current value for the experimental conditions employed and it appears to be limited by the high internal resistance of the charge transport bridge between half-cells. This high resistance is deliberately introduced to minimize internal chemical shorting between half-cells. The duration of the plateau region depends upon the concentration of glucose added to the anodic chamber as the steady state region is basically defined by maintaining [glucose] > [ClJnd]. The third region represents an exponential decay of current versus time. The onset and extent of the current decline is associated with the appearance of the oxidized form of Cl, indophenol. This portion of the curve represents a discharge profile for the cell associated with reoxida-
148
WEIBEL
AND DODGE
tion of the fully reduced electron transport sequence after the primary fuel, glucose, has been completely oxidized. The internal resistance of the cell appears constant during at least the first half-life of this region as evidenced by the linearity of the first order plot in Fig. 2. A control experiment with glucose in the anodic chamber under anaerobic conditions and oxygen in the cathodic chamber produced no current under direct short conditions in the external circuit signifying that bright platinum does not serve as a catalyst for the glucose oxidation half-reaction. Appropriate control experiments involving all possible paired combinations of the three components for the anode half-reaction displayed no measurable current in the external circuit. The cell demonstrated delivery of current to the external circuit only when all three anodic constituents (glucose, glucose oxidase, and Cl, indophenol) were present indicating the obligatory nature of the electron transport scheme. Figure 3 shows a plot of the integrated current values obtained for several glucose concentrations. In this set of experiments the same electrode assembly and constituents were used. After each complete con-
5
GLUCOSEikles
FIG. 3. Integrated discharge profiles: The ordinate is the integrated value of the entire discharge profile for several of glucose concentrations and the abscissa represents the glucose equivalents introduced into the anodic chamber for each experiment. Experimental values are represented by the symbol (- -o- -o- -1 and the theoretical curve by the solid line. The slope of the experimental line is 90% of the theoretical indicating a high Faradic efficiency. The experimental conditions are the same as in Fig. 2 with the exception of variation in glucose for separate experiments.
IO TIME,
-
15 hr.
Fig. 2. Cell discharge profile: The ordinate of the graph is the measured current in the external circuit and the abscissa the time course relative to initiation of the anode reaction (s). The insert is a semilogrithmic plot of the nonlinear discharge region and the linearity reflects the first order nature of this process. The experimental conditions were as follows: anode chamber-8 ml 0.1 M potassium phosphate pH 6.0 containing 0.5 M KCl, 1 PM glucose oxidase, 50 PM Cl, indophenol and 125 PM glucose (1 pmol); cathode chamber 8 ml 0.1 M potassium phosphate pH 6.0 containing 0.5 M KCl, 50~~ Cl,Ind, and 0,saturated (approx 1.25 mM 0,). The anodic chamber was anerobic. The salt bridge was 1% (wt/vol) agar in 0.1 M potassium phosphate pH 6.0 containing 0.5 M KCl. The temperature was 22°C.
GLUCOSE
OXIDASE
sumption of glucose, the next experiment was initiated simply by addition of the appropriate aliquot of 1 M glucose to the anodic chamber. The slope is nearly 90% of the expected value based upon moles of substrate introduced and a two electron oxidation indicating a Faradic efficiency approaching that theoretically possible. The magnitude of the current in the plateau region depends upon the concentration of Cl, indophenol in the fully reduced anodic, electron transport sequence as shown in Fig. 4. The relationship is logarithmic as demonstrated in Fig. 5.
FUEL
149
CELL
1
40-
3.5z ,E * .-3.0-
DISCUSSION
Flavoprotein oxidase enzymes obey kinetic mechanism which make them attractive for application to biochemical fuel cells. The individual reductive and oxidative half-reactions on the catalytic pathway of glucose oxidase have been extensively characterized (6-8). The glucose oxidase reductive half-reaction under anerobic conditions can serve as the initial
I
2
3 TIME,
4
5
6
hour
FIG. 4. Variation of the plateau current with [ClJnd]: The ordinate represents the measured external current and the abscissa the time course of the experiment. The experimental conditions are the same as in Fig. 2 with the exceptions that [ClJnd] was omitted from the cathodic chamber, the salt bridge was 2% (wt/vol) agar and 10 rmol of glucose were added. The variation in [Cl&d] for the anodic chamber was achieved by aliquot additions of lo-’ M Cl, indophenol and are indicated in the upper portion of the figure.
2.5 1 I
2
I 3
I 4
I,,,,, 5 678910
20
log [C121nd] + 5
Fro. 5. Semilogarithmic plot of current vs [ClJnd]: The ordinate represents the plateau currents obtained from Fig. 4 and the abscissa the logarithmic of the corresponding [ClJnd].
portion of an anodic electron transport sequence. The reduced FAD (flavin adenine dinucleotide) of the resulting haloenzyme complex is not accessible for “ionizable hydrogen atom” transfer to a solid electrode due to severe steric barriers imposed by the apoenzyme. Although the most reliable median reduction potential (E,‘, pH 7 vs SHE) for FAD in the haloenzyme complex of glucose oxidase is only - 8 mV, direct electrolytic reduction at a solid electrode has never been observed (9). This fact is substantiated by our observation that production of the fully reduced flavoprotein by glucose under anerobic conditions in the anodic chamber results in no measurable current in the external circuit. Therefore an electron mediator such as Cl,Ind must be employed to provide communication between the reduced haloenzyme complex and the anodic electrode. Cl,Ind is an efficient redox acceptor for the reduced form of glucose oxidase and possesses an E,’ at pH 7.4 of 160 mV (10). The intact electron transfer sequence of the anode is shown schematically in Fig. 6. The introduction of the redox dye into the anodic chamber and the subsequent measurement of electron flow through the external circuit qualitatively correlate with
150
WEIBEL
a-D
GLUCOSE
p-0
Jr GLUCOSE
y-GLUCONOLACTONE
E. FADH,
Clelndox
FIG. 6. The anodic electron transport sequence: Electron transfer is initiated by enzymatic oxidation of &n-glucose. The oxidized and reduced forms of the enzyme are represented by E .FAD and E .FADH, respectively. C1,Ind red is the electrode active component and represents the reduced form of Cl, indophenol. With the exception of the anomeric equilibrium of glucose, each arrow pair represents an electron/proton pair transfer.
the formation of the reduced state of the redox dye. This is visually obvious as the oxidized and reduced forms of Cl, indophenol are blue and colorless, respectively, to the eye. Since the enzymatic reduction of Cl, indophenol is significantly faster that the electrolytic oxidation of reduced Cl, indophenol, the redox status of the anodic chamber at steadystate is the fully reduced electron transport sequence. Further oxidation of glucose in this state (corresponding to the plateau region of Fig. 1) is kinetically dominated by rate limiting processes elsewhere on the cell circuit. The experimental design of the fuel cell employed for these experiments deliberately introduces a high resistance salt bridge between half-cells in order to minimize chemical shorting of the anodic halfreaction sequence by oxygen diffusion from the cathodic chamber. The small diameter agar bridge while admirably minimizing oxygen diffusion, also provides significant resistance to charge transport species of the cell. Several lines of evidence strongly suggest that a major rate limiting process of the overall cell was charge communication between half-cells. Increasing the concentration of agar in the salt bridge, decreasing the ionic strength of the cathodic and anodic buffer, and decreasing the diameter of the salt bridge caused significant decreases in the magnitude of the plateau current. The first order decay of current in the region following the current plateau can be modeled as a capacitor discharging
AND DODGE
through a resistance. Ion transport and electron transport pathways are represented by the resistor and capacitor, respectively. The charge across the capacitor represents the potential pool of electrons residing at the anode in the form of reduced Cl,Ind after all glucose has been oxidized. The discharge rate of the capacitor is a first order process whose time constant is characterized by the value of the resistance and capactance of the circuit. The experimentally obtained first order rate constant for the cell discharge rate under our experimental conditions is 0.31 h-l. The limit of minimizing internal shorting where a small, uncharged molecule such as oxygen is involved and is a cathodic constituent would result in a salt bridge of infinite resistance. A matrix to which oxygen is not permeable would also preclude ion transport and hence create an open circuit cell. The best values obtained for the Faradic efficiency were 95% theoretical and probably reflect the degree for which we were able to minimize chemical shorting between half-cells at conveniently measured current levels in the external circuit. Diffusion of glucose from anode to cathode would not contribute to chemical shorting as glucose oxidase cannot diffuse through the bridge matrix and is totally contained within the anodic chamber. Although the removal of trace oxygen from the argon used to purge the anodic chamber appeared quantitative, the cumulative effects of even extremely small quantities of oxygen in the argon may also have contributed to some shorting of the anodic electron transfer sequence as oxygen is the preferred electron acceptor for reduced glucose oxidase. The linear relationship between the measured current of the plateau region and the log [Cl, Ind] would be expected for a cell possessing a constant, high internal resistance. The chemical potential difference between the half-cells at steady state can be related by the Nernst equation to the concentrations of electroactive components. The observed current is directly related to this potential and inversely to the internal resistance by Ohm’s Law. Since the electron transport sequence in
GLUCOSE
OXIDASE
the anodic chamber is maintained in the fully reduced state by glucose at steady state and the concentration of oxygen in solution within the cathodic chamber maintained at saturation, the internal potential drop between half-cells is constant. Therefore, the plateau current of the external circuit varies linearly with log [Cl, Ind] at constant [O,].
2.
3. 4. 5. 6.
ACKNOWLEDGMENT The authors wish to thank Dr. D. Porter helpful comments and discussion.
FUEL
for his
7. 8.
REFERENCES 9. 1. Artificial Heart Program Conference, sponsored by The National Heart Institute Artificial Heart Program (Hegyeli, R. J., ed.), Chapters
10.
CELL
151
68-73, pp. 815-881, U.S. Printing Office, Washington ( 1969). Scorr, W. R., (1962). Proceedings of the Biochemical Fuel Cell Session, PIC-BAT report 209/5, p. 9-l. YAHIRO, A. T., LEE, S. M., AND KIMBEL, D. O., (1964). Biochim. Biophys. Acta 88, 375. WEIBEL, M. K. AND BRIGHT, H. J., (1971) J. Biol. Chem 246, 2734. MEITES, L. AND MEITES, T., (1948) Anal. Chem. 20, 984. GIBSON, Q. H., SWOBODA,B. E. P., AND MASSEY, V., (1964) J. Biol. Chem. 239, 3927. BRIGHT, H. J. AND GIBSON, Q. H., (1967) J. Biol. Chem. 242, 994. NAKAMURA, S. AND OGURA, Y. (1968) J. Biochem. (Tokyo) 63, 308. DUKE, F. R., KUST, R. N., AND KING, L. A. (1969) J. Electrochem. Sot. 116, 32. DIXON, M. (1971) Biochim. Biophys. Acta 226, 269.
OF BIOCHEMISTRY
AND
169, 146-151 (I9751
BIOPHYSICS
Biochemical Demonstration
of an Obligatory
Enzymatically MICHAEL Department
of Biochemistry,
Fuel Cells
Pathway
Involving
Catalyzed
Aerobic
K. WEIBEL
AND
of Medicine,
University
School
an External
Oxidation
CHRISTOPHER
Received January
of Pennsylvania,
Circuit
of Glucose’,
for the
2
DODGE Philadelphia,
Pennsylvania
19174
10, 1974
An in vitro biochemical fuel cell based upon the enzymatically catalyzed aerobic oxidation of glucose is described. The anodic half-reaction employs an electron transfer sequence consisting of the glucose oxidase reductive half-reaction and dichloroindophenol. The cathodic half-reaction involves reduction of molecular oxygen. A high Faradic efficiency for the intact cell approaching 100% has been experimentally demonstrated. The steady state current is exponentially related to the concentration of the terminal electron transfer species in the anodic chamber. The behavior is consistent with application of the Nernst relationship to define the cell potential and a simple resistance circuit. The discharge profile of the cell after complete oxidation of the primary fuel, glucose, can be modeled as a capacitor discharging through a resistor.
The aerobic oxidation of glucose is extremely favorable from a thermodynamic assessment. In point of fact, however, virtually no reaction occurs under in vitro physiological conditions due to severe kinetic contraints for the uncatalyzed aerobic oxidation. Several reports have appeared concerning biochemical fuel cells based upon noble metal catalysts such as platinum black and polymeric redox dyes (1). The explicit half-reaction electrode chemistry for these systems has not yet been unequivocally identified. An enzymatically catalyzed oxidation represents a molecular battery operating at short. The internal resistance is defined by intrinsic rate limiting intra- and intermolecular processes of the catalytic pathway. The cell potential at any given time is thermodynamically defined by the difference in free energy of products and substrates participating in the redox reaction. Since the process proceeds in a highly irreversible manner, no useful work is ob’ Supported by NSF GI 32498X2. *This paper is the first in a series, “Biochemical Fuel Cells.” 146 Copyright All rivhtc
0 1975 by Academic nf wnrodnrtinn
Press, Inc. in nnv form reserved.
tained and the available chemical energy is dissipated in the form of reaction enthalphy to the environment. If an external circuit could be inserted between the reductive and oxidative half-reactions of the enzymatically catalyzed redox reaction and that circuit represent an obligatory pathway for electron transfer, then the opportunity exists for transduction of chemical energy from the reaction into useful work. Similarly, the cell may be electrochemically driven in reverse utilizing the tightly coupled external circuit to provide energy for an enzyme catalyzed reduction or oxidation. Two reports concerning a biochemical fuel cell employing the enzymatically catalyzed aerobic oxidation of glucose have appeared (2, 3). In both cases only empirical characterization was reported. The absence of a systematic analysis and elucidation of the half-cell chemistry have made it difficult to interpret prior work and formulate basic principles concerning this type of biochemical fuel cell. We report here the first of several studies on the glucose oxidase fuel cell employing bright platinum electrodes.
GLUCOSE METHODS
OXIDASE
oxidase
CELL
AND MATERIALS
1.1.3.4) from Aspergillus from Sigma Chemical Co. The enzyme was purified to homogeneity by chromatography on DEAE cellulose (4). Anhydrous and ultra pure glucose (Ultrex) was obtained from J. T. Baker Chemical Co. Solutions of glucose were allowed to obtain anomeric equilibrium prior to use. Mutarotation rates within the anodic chamber under the experimental conditions employed were fast relative to the steady state oxidation rates of P-o-glucose. The redox dye mediator, 2,6-dichlorophenolindophenol (Cl, indophenol or Cl, Ind)s, was obtained from Sigma Chemical Co. and used as obtained. All other chemicals were of “certified” grade. The simple cell design is shown in Fig. 1. The half-cell chambers were glass cylinders, 16 mm diam by 80 mm high, with a standard Luer connecter on the side for attachment of the salt bridge. The top of the half-cell chamber was capped with a thin septum 17 mm diam serum bottle plug which allowed easy access to the electrode chamber for material additions, gas flushing and electrode leads. An electrode material was chosen which does not significantly serve as a direct electron acceptor for glucose under near physiological conditions. The solid electrode employed for this study was bright platinum foil (0.025 mm thick) with a surface area of 3 cm*. A salt bridge was constructed from a 2 mm i.d. glass T joint and possessed a longitudinal path length of 3 cm. The bridge matrix material was 1% agar (wt/vol) in the buffer medium. The buffer for both half cells was 0.1 M potassium phosphate, pH 6.0 containing 0.5 M KCl. Anerobic conditions in the anode half cell were obtained by purging the chamber solution for 2 h with oxygen free argon and maintained after start up of the cell by continuously flushing of the space above the liquid phase. Argon was a commercial grade and traces of oxygen were removed by passing the gas through a gas scrubbing train of four Dreschsel units containing vanadous sulfate (5). The cathode half-cell chamber solution was saturated with oxygen by continuous purging with water saturated oxygen. A small Teflon stir bar was placed in the anode half-cell and operated by an external rotating magnet. This arrangement was necessary for rapid mixing of added constituents such as glucose or electron transport components to the solution within the chamber. Integrated amperometric measurements in the external cell circuit were obtained with a model 153, Kiethly microvolt-ammeter. Current-time profiles of the full cell were recorded with a model EU-POB Health recorder. Integration of the trace was performed manually employing finite interval areas and is within *50/o of the exact integral. Glucose
FUEL
(EC
niger (type VI) was obtained
‘Abbreviations used are: Cl, Ind and Cl, indophenol, 2,4-dichloroindophenol; and FAD, Flavin adenine dinucleotide.
ARGON-
ANODE
CATHODE
Fig. 1. Schematic
of cell system.
RESULTS
Figure 2 shows a typical current-time profile for the fuel cell upon addition of the anode substrate glucose. Three distinct regions are apparent. The presteady state behavior consists of two distinct phases. The early increase in current reflects rate limiting solution kinetics of the anode electron transport sequence and correlates with the formation of reduced ClJnd. The decrease in current from the peak value to the plateau region probably reflects an internal resistance change associated with the attainment of a steady state ion gradient in the salt bridge. The plateau region of the trace represents a steady state corresponding to complete reduction of the anode electron transport sequence. This condition represents the maximum current value for the experimental conditions employed and it appears to be limited by the high internal resistance of the charge transport bridge between half-cells. This high resistance is deliberately introduced to minimize internal chemical shorting between half-cells. The duration of the plateau region depends upon the concentration of glucose added to the anodic chamber as the steady state region is basically defined by maintaining [glucose] > [ClJnd]. The third region represents an exponential decay of current versus time. The onset and extent of the current decline is associated with the appearance of the oxidized form of Cl, indophenol. This portion of the curve represents a discharge profile for the cell associated with reoxida-
148
WEIBEL
AND DODGE
tion of the fully reduced electron transport sequence after the primary fuel, glucose, has been completely oxidized. The internal resistance of the cell appears constant during at least the first half-life of this region as evidenced by the linearity of the first order plot in Fig. 2. A control experiment with glucose in the anodic chamber under anaerobic conditions and oxygen in the cathodic chamber produced no current under direct short conditions in the external circuit signifying that bright platinum does not serve as a catalyst for the glucose oxidation half-reaction. Appropriate control experiments involving all possible paired combinations of the three components for the anode half-reaction displayed no measurable current in the external circuit. The cell demonstrated delivery of current to the external circuit only when all three anodic constituents (glucose, glucose oxidase, and Cl, indophenol) were present indicating the obligatory nature of the electron transport scheme. Figure 3 shows a plot of the integrated current values obtained for several glucose concentrations. In this set of experiments the same electrode assembly and constituents were used. After each complete con-
5
GLUCOSEikles
FIG. 3. Integrated discharge profiles: The ordinate is the integrated value of the entire discharge profile for several of glucose concentrations and the abscissa represents the glucose equivalents introduced into the anodic chamber for each experiment. Experimental values are represented by the symbol (- -o- -o- -1 and the theoretical curve by the solid line. The slope of the experimental line is 90% of the theoretical indicating a high Faradic efficiency. The experimental conditions are the same as in Fig. 2 with the exception of variation in glucose for separate experiments.
IO TIME,
-
15 hr.
Fig. 2. Cell discharge profile: The ordinate of the graph is the measured current in the external circuit and the abscissa the time course relative to initiation of the anode reaction (s). The insert is a semilogrithmic plot of the nonlinear discharge region and the linearity reflects the first order nature of this process. The experimental conditions were as follows: anode chamber-8 ml 0.1 M potassium phosphate pH 6.0 containing 0.5 M KCl, 1 PM glucose oxidase, 50 PM Cl, indophenol and 125 PM glucose (1 pmol); cathode chamber 8 ml 0.1 M potassium phosphate pH 6.0 containing 0.5 M KCl, 50~~ Cl,Ind, and 0,saturated (approx 1.25 mM 0,). The anodic chamber was anerobic. The salt bridge was 1% (wt/vol) agar in 0.1 M potassium phosphate pH 6.0 containing 0.5 M KCl. The temperature was 22°C.
GLUCOSE
OXIDASE
sumption of glucose, the next experiment was initiated simply by addition of the appropriate aliquot of 1 M glucose to the anodic chamber. The slope is nearly 90% of the expected value based upon moles of substrate introduced and a two electron oxidation indicating a Faradic efficiency approaching that theoretically possible. The magnitude of the current in the plateau region depends upon the concentration of Cl, indophenol in the fully reduced anodic, electron transport sequence as shown in Fig. 4. The relationship is logarithmic as demonstrated in Fig. 5.
FUEL
149
CELL
1
40-
3.5z ,E * .-3.0-
DISCUSSION
Flavoprotein oxidase enzymes obey kinetic mechanism which make them attractive for application to biochemical fuel cells. The individual reductive and oxidative half-reactions on the catalytic pathway of glucose oxidase have been extensively characterized (6-8). The glucose oxidase reductive half-reaction under anerobic conditions can serve as the initial
I
2
3 TIME,
4
5
6
hour
FIG. 4. Variation of the plateau current with [ClJnd]: The ordinate represents the measured external current and the abscissa the time course of the experiment. The experimental conditions are the same as in Fig. 2 with the exceptions that [ClJnd] was omitted from the cathodic chamber, the salt bridge was 2% (wt/vol) agar and 10 rmol of glucose were added. The variation in [Cl&d] for the anodic chamber was achieved by aliquot additions of lo-’ M Cl, indophenol and are indicated in the upper portion of the figure.
2.5 1 I
2
I 3
I 4
I,,,,, 5 678910
20
log [C121nd] + 5
Fro. 5. Semilogarithmic plot of current vs [ClJnd]: The ordinate represents the plateau currents obtained from Fig. 4 and the abscissa the logarithmic of the corresponding [ClJnd].
portion of an anodic electron transport sequence. The reduced FAD (flavin adenine dinucleotide) of the resulting haloenzyme complex is not accessible for “ionizable hydrogen atom” transfer to a solid electrode due to severe steric barriers imposed by the apoenzyme. Although the most reliable median reduction potential (E,‘, pH 7 vs SHE) for FAD in the haloenzyme complex of glucose oxidase is only - 8 mV, direct electrolytic reduction at a solid electrode has never been observed (9). This fact is substantiated by our observation that production of the fully reduced flavoprotein by glucose under anerobic conditions in the anodic chamber results in no measurable current in the external circuit. Therefore an electron mediator such as Cl,Ind must be employed to provide communication between the reduced haloenzyme complex and the anodic electrode. Cl,Ind is an efficient redox acceptor for the reduced form of glucose oxidase and possesses an E,’ at pH 7.4 of 160 mV (10). The intact electron transfer sequence of the anode is shown schematically in Fig. 6. The introduction of the redox dye into the anodic chamber and the subsequent measurement of electron flow through the external circuit qualitatively correlate with
150
WEIBEL
a-D
GLUCOSE
p-0
Jr GLUCOSE
y-GLUCONOLACTONE
E. FADH,
Clelndox
FIG. 6. The anodic electron transport sequence: Electron transfer is initiated by enzymatic oxidation of &n-glucose. The oxidized and reduced forms of the enzyme are represented by E .FAD and E .FADH, respectively. C1,Ind red is the electrode active component and represents the reduced form of Cl, indophenol. With the exception of the anomeric equilibrium of glucose, each arrow pair represents an electron/proton pair transfer.
the formation of the reduced state of the redox dye. This is visually obvious as the oxidized and reduced forms of Cl, indophenol are blue and colorless, respectively, to the eye. Since the enzymatic reduction of Cl, indophenol is significantly faster that the electrolytic oxidation of reduced Cl, indophenol, the redox status of the anodic chamber at steadystate is the fully reduced electron transport sequence. Further oxidation of glucose in this state (corresponding to the plateau region of Fig. 1) is kinetically dominated by rate limiting processes elsewhere on the cell circuit. The experimental design of the fuel cell employed for these experiments deliberately introduces a high resistance salt bridge between half-cells in order to minimize chemical shorting of the anodic halfreaction sequence by oxygen diffusion from the cathodic chamber. The small diameter agar bridge while admirably minimizing oxygen diffusion, also provides significant resistance to charge transport species of the cell. Several lines of evidence strongly suggest that a major rate limiting process of the overall cell was charge communication between half-cells. Increasing the concentration of agar in the salt bridge, decreasing the ionic strength of the cathodic and anodic buffer, and decreasing the diameter of the salt bridge caused significant decreases in the magnitude of the plateau current. The first order decay of current in the region following the current plateau can be modeled as a capacitor discharging
AND DODGE
through a resistance. Ion transport and electron transport pathways are represented by the resistor and capacitor, respectively. The charge across the capacitor represents the potential pool of electrons residing at the anode in the form of reduced Cl,Ind after all glucose has been oxidized. The discharge rate of the capacitor is a first order process whose time constant is characterized by the value of the resistance and capactance of the circuit. The experimentally obtained first order rate constant for the cell discharge rate under our experimental conditions is 0.31 h-l. The limit of minimizing internal shorting where a small, uncharged molecule such as oxygen is involved and is a cathodic constituent would result in a salt bridge of infinite resistance. A matrix to which oxygen is not permeable would also preclude ion transport and hence create an open circuit cell. The best values obtained for the Faradic efficiency were 95% theoretical and probably reflect the degree for which we were able to minimize chemical shorting between half-cells at conveniently measured current levels in the external circuit. Diffusion of glucose from anode to cathode would not contribute to chemical shorting as glucose oxidase cannot diffuse through the bridge matrix and is totally contained within the anodic chamber. Although the removal of trace oxygen from the argon used to purge the anodic chamber appeared quantitative, the cumulative effects of even extremely small quantities of oxygen in the argon may also have contributed to some shorting of the anodic electron transfer sequence as oxygen is the preferred electron acceptor for reduced glucose oxidase. The linear relationship between the measured current of the plateau region and the log [Cl, Ind] would be expected for a cell possessing a constant, high internal resistance. The chemical potential difference between the half-cells at steady state can be related by the Nernst equation to the concentrations of electroactive components. The observed current is directly related to this potential and inversely to the internal resistance by Ohm’s Law. Since the electron transport sequence in
GLUCOSE
OXIDASE
the anodic chamber is maintained in the fully reduced state by glucose at steady state and the concentration of oxygen in solution within the cathodic chamber maintained at saturation, the internal potential drop between half-cells is constant. Therefore, the plateau current of the external circuit varies linearly with log [Cl, Ind] at constant [O,].
2.
3. 4. 5. 6.
ACKNOWLEDGMENT The authors wish to thank Dr. D. Porter helpful comments and discussion.
FUEL
for his
7. 8.
REFERENCES 9. 1. Artificial Heart Program Conference, sponsored by The National Heart Institute Artificial Heart Program (Hegyeli, R. J., ed.), Chapters
10.
CELL
151
68-73, pp. 815-881, U.S. Printing Office, Washington ( 1969). Scorr, W. R., (1962). Proceedings of the Biochemical Fuel Cell Session, PIC-BAT report 209/5, p. 9-l. YAHIRO, A. T., LEE, S. M., AND KIMBEL, D. O., (1964). Biochim. Biophys. Acta 88, 375. WEIBEL, M. K. AND BRIGHT, H. J., (1971) J. Biol. Chem 246, 2734. MEITES, L. AND MEITES, T., (1948) Anal. Chem. 20, 984. GIBSON, Q. H., SWOBODA,B. E. P., AND MASSEY, V., (1964) J. Biol. Chem. 239, 3927. BRIGHT, H. J. AND GIBSON, Q. H., (1967) J. Biol. Chem. 242, 994. NAKAMURA, S. AND OGURA, Y. (1968) J. Biochem. (Tokyo) 63, 308. DUKE, F. R., KUST, R. N., AND KING, L. A. (1969) J. Electrochem. Sot. 116, 32. DIXON, M. (1971) Biochim. Biophys. Acta 226, 269.