Electrochemical Studies of Biological Systems

10 Rotating Ring Disk Enzyme Electrode for Biocatalysis Studies Downloaded by UNIV OF MASSACHUSETTS AMHERST on September 10, 2015 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0038.ch010

1

RALPH A. KAMIN, FRANK R. SHU, and GEORGE S. WILSON Department of Chemistry, University of Arizona, Tucson, Ariz. 85721

In recent years there has been considerable interest i n catalytic surface reactions particularly those of biological interest. This has been manifested, for example, i n the rapid development of immobilized enzyme technology (1,2) and electrochemical sensors based on electroactive product formation within an enzyme layer (3,4). In comparing the kinetic behavior of an immobilized enzyme with i t s soluble counterpart, it is necessary to establish that the overall reaction rate i s catalysis rather than mass transport limited. It has been shown, for example, that immobilized enzymes i n flowing streams give apparent Michaelis constants K ', that are flow rate dependent (5). Under conditions where the overall reaction i s limited by mass-transport supply of substrate to the catalytic surface, K ' i s larger than expected. One i s then tempted to conclude that the properties of the enzyme have been modified by immobilization. On the contrary, increasing flow (mass transport) rates may lead to a limiting value for K ' essentially identical to that of the soluble enzyme (6). The rotating disk electrode as described by Levich (7) appears to offer an experimentally facile means for varying the rate of substrate mass transport. The addition of a concentric ring (rotating ring disk electrode) (8) permits independent monitoring of the reaction at the disk surface. We have recently (9) derived the theory describing the response of the rotating disk enzyme electrode. In the present work we report further experimental studies in support of this theoretical model. The system selected for study i s the glucose/glucose oxidase reaction: M

M

M

Glucose + 0 2 o

l u c

s e

g ° ) oxidase

Gluconic Acid + H 0 22 o

o

(1)

The peroxide produced i s either monitored directly or coupled 1

Present address: Smith-Kline Instruments, 880 W. Maude Ave., Sunnyvale, CA 94086 170

In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

10.

KAMIN

ET

Rotating

AL.

Ring

Disk Enzyme

171

Electrode

with the i n d i c a t o r r e a c t i o n : H 0 2

2

+ 2H

+

+ 2Γ

m

o

l

r

b

d

a

t

e

>

I

2

+ 2H 0 2

(2)

Downloaded by UNIV OF MASSACHUSETTS AMHERST on September 10, 2015 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0038.ch010

Experimental Instrumentation, The f our^ e l e c t r o d e p o t e n t i o s t a t used i n these s t u d i e s was s i m i l a r to that described by Shabrang and Bruckenstein (10). The r o t a t i n g d i s k e l e c t r o d e , Model DT-6, was purchased from the Pine Instrument Co., Grove C i t y , PA. The d i s k was a 0.5 cm deep c a v i t y with a radius of 0.382 cm ac cording to the manufacturer's s p e c i f i c a t i o n s . When f i l l e d with carbon paste the c a l c u l a t e d d i s k area was 0.46 cm^. The width of the platinum r i n g e l e c t r o d e was 0.024 cm and was separated from the d i s k by a 0.016 cm wide epoxy gap. The c o l l e c t i o n e f f i c i e n c y measured experimentally i n the usual way as a carbon paste e l e c t r o d e (8) was 0.18 and was i n good agreement with experimental r e s u l t s . A platinum wire counter e l e c t r o d e and a Ag/AgCl reference e l e c t r o d e ( E = 0.200 v) were employed. A Pine Instruments Model PIR r o t a t o r was used to c o n t r o l e l e c trode r o t a t i o n speed. o f

Preparation of Glucose Oxidase E l e c t r o d e . The carbon paste was prepared i n the usual manner from 5 g of graphite powder #38 ( F i s h e r S c i e n t i f i c Co.) and 3 ml of Nujol except that 10 mg (except where otherwise s p e c i f i e d ) of n-octadecylamine ( t e c h n i c a l grade, A l d r i c h ) was a l s o added. The carbon paste was packed f i r m l y i n t o the d i s k c a v i t y of the DT-6 e l e c t r o d e which was then p o l i s h e d with a piece of weighing paper. A f t e r the r i n g and gap were c a r e f u l l y cleaned, the e l e c t r o d e was allowed to r o t a t e i n a 12.5% glutaraldehyde s o l u t i o n f o r 10 - 15 min. followed by a 1 minute washing with c o l d 0.2M phosphate b u f f e r pH 6.5. (Glutaraldehyde must be f r e s h l y p u r i f i e d and s t o r e d below 0°C as i t r e a d i l y polymerizes (11))· The r o t a t i n g e l e c t r o d e was dipped i n t o a bovine serum albumin s o l u t i o n (0.1 g/ml) (BSA F r a c t i o n V 96-99%, Sigma Co.). After 2 - 3 minutes the e l e c trode was washed f o r 1 minute i n c o l d phosphate b u f f e r . The e l e c t r o d e was then removed from the r o t a t o r and p o s i t i o n e d with the e l e c t r o d e surface f a c i n g up. A glucose oxidase s o l u t i o n prepared by d i s s o l v i n g 0.3 g of the enzyme (Glucose Oxidase E.C. 1.1.3.4 Sigma Type II 15,000 units/g) i n 1 ml. of 5% g l u t a r a l dehyde s o l u t i o n (buffered with phosphate at pH 6.5) was a p p l i e d to the d i s k s u r f a c e . A f t e r standing at room temperature f o r 5 min., the excess enzyme s o l u t i o n was discarded and the gap and r i n g were c a r e f u l l y cleaned. Rotating the e l e c t r o d e i n c o l d phosphate b u f f e r at 2500 rpm f o r 5 min. aids i n removing phys i c a l l y entrapped or weakly bonded enzyme. When not i n use the e l e c t r o d e was stored i n phosphate b u f f e r at 5°C.


172

ELECTROCHEMICAL

STUDIES O F

BIOLOGICAL

SYSTEMS


S o l u t i o n s and Reagents Unless otherwise mentioned, a l l chemicals used were reagent grade. The stock s o l u t i o n of 0.1 M glucose was allowed to mutar o t a t e at room temperature f o r at l e a s t 24 hr. before using. When the course of the r e a c t i o n was measured by f o l l o w i n g I2 formation (Reaction 2) a K I - b u f f e r c a t a l y s t described p r e v i o u s l y (12) was employed. Where d i r e c t monitoring of peroxide format i o n (Reaction 1) i s p o s s i b l e the glucose i s d i s s o l v e d i n a 0.05 M phosphate b u f f e r pH 6.5. Procedure. The enzyme e l e c t r o d e was allowed to r o t a t e f o r about 30 sec. i n the glucose s o l u t i o n at which time a p o t e n t i a l was a p p l i e d to the r e s p e c t i v e i n d i c a t i n g e l e c t r o d e . The i o d i n e formed i n Reaction 2 was monitored at the d i s k by applying a p o t e n t i a l of -0.2 V vs Ag/AgCl r e f e r e n c e . D i r e c t peroxide f o r mation (no i o d i d e present) was monitored at the platinum r i n g by h o l d i n g the p o t e n t i a l at -0.2 V followed by a step to 0.75 V at which p o i n t the current t r a n s i e n t was measured. The p o t e n t i a l was then returned to -0.2 V u n t i l the next measurement. Enzyme E l e c t r o d e T h e o r e t i c a l Model. The d e t a i l s of the d i g i t a l s i m u l a t i o n c a l c u l a t i o n s f o r t h i s e l e c t r o d e have been presented elsewhere (9). Our model assumes the existence of an enzyme l a y e r extending i n t o s o l u t i o n from the e l e c t r o d e surface (X=0). This uniformly d i s t r i b u t e d t h i n enzyme l a y e r i s assumed not to i n t e r f e r e with d i f f u s i o n of species to or from the e l e c t r o d e s u r f a c e . The enzyme l a y e r l i e s w i t h i n the minimum hydrodynamic l a y e r j u s t i f y i n g the assumption that s o l u t i o n flow i n the e l e c t r o d e v i c i n i t y i s a l s o unaffected by the immobilization process. Michaelis-Menten k i n e t i c theory i s assumed to describe the enzymatic r e a c t i o n . Figure 1 i l l u s t r a t e s the nature of the concentration gradi e n t s at the e l e c t r o d e surface f o r a p a r t i c u l a r set of condit i o n s . The steady s t a t e product (or coupled product) concentrat i o n gradient i s f i r s t simulated f o r the r o t a t i n g e l e c t r o d e at open c i r c u i t . Product concentration increases as substrate pene t r a t e s the enzyme l a y e r from the s o l u t i o n s i d e . I f a p o t e n t i a l i s a p p l i e d to the d i s k i n a region where the product i s e l e c t r o a c t i v e , i t s concentration at the e l e c t r o d e surface drops to zero. E v e n t u a l l y the steady-state c o n d i t i o n shown i n Figure 1 i s a t t a i n e d . I t w i l l be noted that the concentrations i n the outer p o r t i o n of the enzyme l a y e r are r e l a t i v e l y unaffected by the pot e n t i a l perturbation. The r a t e of product formation i s given by Michaelis-Menten theory d

[P] dt

k C /(K /[S]+l) 3

E

M


(3)

10.

Rotating

KAMiN E T A L .

Ring

Disk Enzyme

173

Electrode


where i s the rate constant f o r the i r r e v e r s i b l e conversion of the enzyme-substrate complex i n t o products and K^. the M i c h a e l i s constant. C^, i n t h i s case i s the a n a l y t i c a l concentration of a c t i v e enzyme i n the immobilized l a y e r and S the substrate con c e n t r a t i o n i n the enzyme l a y e r . In order to evaluate the r e l a t i v e e f f e c t s of c a t a l y s i s and convective mass transport a r e a c t i o n v e l o c i t y parameter, V, i s defined: = W k ^ I

v

(

4

)

The convection time constant, t ^ , has been derived p r e v i o u s l y by P r a t e r and Bard (13) and i s given by t

2

k

= (0.51)- '

3

β"

1 7 3

v

1

/

3

ω"

1

(5)

2 where ν i s the kinematic v i s c o s i t y (cm /sec) and ω the r o t a t i o n speed i n rad/sec. For a given enzyme e l e c t r o d e , V r e f l e c t s the amount of product formed i n a given time and i s dependent only on ω, to which i t i s i n v e r s e l y p r o p o r t i o n a l . For l a r g e values of V e.g. V > 10 the c a t a l y s i s rate i s extremely f a s t and the o v e r a l l r e a c t i o n becomes convection mass transport l i m i t e d . For V < 0.1 the enzymatic r e a c t i o n i s c a t a l y s i s r a t e l i m i t e d . Thus, by v a r y i n g the e l e c t r o d e r o t a t i o n speed, the f l u x of sub s t r a t e can be modulated to change the nature of the r a t e l i m i t i n g process. The r a t i o C/K^ where C i s the bulk substrate con c e n t r a t i o n a l s o serves to define the current response. We have a l s o shown (9) that an optimal r o t a t i o n speed f o r current mea surement w i l l r e s u l t from increased substrate mass transport on one hand and decreased product production due to short contact time with the c a t a l y t i c l a y e r on the other. The steady s t a t e current r e l a t i o n s h i p s are presented below: Case I - Mass Transport Limited Rate (V > 10) From s i m u l a t i o n i t can be shown (by analogy to a L i n e weave r-Burk p l o t (14)):

nFAdk C 3

1/2 E

k

2

" d ~

1.22

D t

- k_

C

t

3 E k C

(6)

where i i s the steady s t a t e current at the d i s k , d i s the en zyme l a y e r thickness; b i s a f u n c t i o n only of ω and D. A l l other parameters have the usual e l e c t r o c h e m i c a l s i g n i f i c a n c e . At low substrate concentrations the f i r s t term of Equation 6 i s much greater than b and the steady-state current becomes 0.65nFAD V 2 /

1 / 6

U)

1 / 2

C


(7)

174

ELECTROCHEMICAL

STUDIES O F

BIOLOGICAL

SYSTEMS

which i s p r a c t i c a l l y i d e n t i c a l to the L e v i c h equation (7) f o r a r o t a t i n g d i s k e l e c t r o d e as expected. Case I I - C a t a l y s i s Limited Rate (V

Electrochemical Studies of Biological Systems

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