Chemical Sensors and Microinstrumentation - American Chemical

Chapter 7

Bioelectrochemistry at Microelectrodes H. Allen O. Hill, Napthali P. Klein, A. Surya N. Murthy, and Ioanna S. M. Psalti

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Inorganic Chemistry Laboratory and Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QR, England

The successful detection of glucose, up to 15mM, employing disc microelectrodes (25μm to 6Ομm diameter) and 8mM, with dual microband gold electrodes (1μm thick), is described. The studies made use of the coupling of the glucose-glucose oxidase reaction to electro-generated ferricinium species. Detection of the analyte by the dual electrode is dependent upon interelectrode distance. The electrochemistry of biological molecules has been studied thoroughly in recent years; that of simple redox proteins is now (1^ 2) well-understood. Two new features have recently come to promin ence: the re-interpretation of some aspects of the electrochemistry of proteins in terms (Armstrong, F.A.; Bond, A.M.; Hill, H.A.O.; Oliver, B.N.; Psalti, I.S.M. J. Amer. Chem. Soc, 1989, in press) of the behaviour at multi-microelectrodes and the dynamics of movement both of, and within, protein-protein complexes when at the electrode surface (3-4). The latter came to the fore when the elec trochemistry of protein-protein complexes was investigated. Re versible electron transfer of both proteins was observed at an electrode at which one component of the complex separately gave good electrochemistry and the other none. It was at first thought that some form of mediation of electron transfer was occuring, as indeed seems to be the case (Barker, Ρ.D.; Hill, H.A.O.; Walton, N.J. J. Electroanal. Chem., in press) when a modified gold electrode is used. However, when a complex i s prepared between zinc (II) cytochrome c, which is redox-inactive, and cytochrome b , the electrochemistry of the latter was observed. N.m.r. spectroscopic investigations have suggested (Driscoll, P.C.; Goodall, K.; Hill, H.A.O.; Redfield, C. University of Oxford, unpublished results) that the two proteins, within the complex, move with respect to one another. This implies that motion, both within the complex and of the complex at the electrode surface, is possible. The rate of elec tron transfer between electrode and protein, or protein-protein 5

0097-6156/89ΑΜ03-Ο105$06.00/Ό © 1989 American Chemical Society

In Chemical Sensors and Microinstrumentation; Murray, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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complex, w i l l o b v i o u s l y depend on b o t h t h e r a t e o f s u c h d y n a m i c motion and on t h e r e s u l t a n t d i s t a n c e from t h e e l e c t r o d e s u r f a c e . L a r g e r p r o t e i n s , e s p e c i a l l y enzymes, s t i l l cause many problems though t h e r e have been some e n c o u r a g i n g r e p o r t s (5-6) and r e c e n t l y (Guo, L.H.; H i l l , H.A.O.; Hopper, D.J.; Lawrence, G.A.; Sanghera, G.S. J . E l e c t r o a n a l . Chem., s u b m i t t e d ) e x c e l l e n t e l e c t r o c h e m i s t r y o f t h e enzyme, p - c r e s o l m e t h y l h y d r o x y l a s e , has been a c h i e v e d . The p r o b l e m o v e r t h e y e a r s seems t o have been t h a t , even where m o d i f i e d e l e c t r o d e s were used, s u f f i c i e n t c a r e was not t a k e n t o e n s u r e t h a t the enzyme was s t i l l i n t a c t , i . e . , t h a t t h e p r o s t h e t i c group had not been r e l e a s e d f r o m t h e enzyme. I n such c a s e s , i t was d i f f i c u l t t o d i s t i n g u i s h between t h e e l e c t r o c h e m i s t r y o f t h e ' f r e e ' p r o s t h e t i c group and the i n t a c t p r o t e i n . T h i s i s p a r t i c u l a r l y t r u e when a p p l i e d t o f l a v o e n z y m e s s i n c e t h e p r o s t h e t i c group i s o f t e n not c o v a l e n t l y bound t o t h e enzyme. A t u n m o d i f i e d e l e c t r o d e s , t h e p r o b l e m i s more i n t e n s e and i r r e v e r s i b l e a d s o r p t i o n w i t h , o r w i t h o u t , c o n c o m i t a n t d e n a t u r a t i o n i s o f t e n e n c o u n t e r e d . A d d i t i o n a l problems concerned t h e presence of a d v e n t i t i o u s mediators or those r e l e a s e d from the e l e c t r o d e m a t e r i a l i t s e l f , e.g., i n t h e use (2) o f conducting o r g a n i c s a l t s as e l e c t r o d e s . Now, with well-defined electrode s u r f a c e s , a b e t t e r a p p r e c i a t i o n o f t h e n e c e s s i t y o f u s i n g p u r e and s t a b l e enzymes and t h e c o r r e c t c r i t e r i a f o r a s s e s s i n g t h e s u c c e s s f u l a t t a i n m e n t of e l e c t r o n t r a n s f e r , we may e x p e c t an i n c r e a s i n g number of r e p o r t s i n t h e near f u t u r e . ANALYTICAL BIOELECTROCHEMISTRY There has l o n g been an i n t e r e s t i n t h e use o f e n z y m a t i c methods, t o g e t h e r w i t h e l e c t r o c h e m i s t r y , as a way o f c o m b i n i n g t h e power and c o n v e n i e n c e o f t h e l a t t e r w i t h t h e e l e g a n c e and e f f i c i e n c y o f t h e f o r m e r . F o l l o w i n g t h e p i o n e e r i n g work o f C l a r k (£), most o f t h e methods employed made use o f t h e e l e c t r o c h e m i s t r y o f one o f t h e enzyme's n a t u r a l s u b s t r a t e s o r p r o d u c t s , o f t e n d i o x y g e n o r hydrogen peroxide. Replacing e i t h e r of these w i t h a l t e r n a t i v e mediators, l e d to c o n s i d e r a b l e problems w i t h dioxygen i n t e r f e r e n c e . Ferrocene d e r i v a t i v e s were i n t r o d u c e d as m e d i a t o r s (!) s i n c e i t was w e l l known t h a t they were o n l y s l o w l y o x i d i s e d . The e x i s t e n c e of a huge v a r i e t y o f f e r r o c e n e s i s a n o t h e r major a d v a n t a g e . A p a r t i c u l a r ferrocene can be chosen on account o f i t s redox p o t e n t i a l , s o l u b i l i t y , charge, e t c . One can r e a d i l y make f e r r o c e n e d e r i v a t i v e s o f d r u g s (10) f o r immunoassays o r even of p r o t e i n s (11-12). BIOELECTROCHEMISTRY AT MICROELECTRODES M i n i a t u r i z a t i o n o f enzyme e l e c t r o d e s (13) has a l w a y s been r e l e v a n t f o r i n v i v o a p p l i c a t i o n s . A new g e n e r a t i o n o f m i c r o e l e c t r o d e s has a l r e a d y b e e n e m p l o y e d (14) f o r i n v i v o e l e c t r o a n a l y s i s . The d i m i n i s h e d s u r f a c e area of a m i c r o e l e c t r o d e r e s u l t s i n reduced c a p a c i t a n c e , r e d u c e d ohmic l o s s e s as a consequence o f t h e s m a l l e r c u r r e n t and i n c r e a s e d r a t e s o f mass t r a n s p o r t t o and f r o m t h e e l e c t r o d e r e s u l t i n g i n a steady s t a t e response. T h i s has l e d (15) t o i n v e s t i g a t i o n s of e l e c t r o c h e m i s t r y i n h i g h l y - r e s i s t i v e s o l u t i o n s , s t u d i e s of f a s t r e a c t i o n s w i t h e l e c t r o n t r a n s f e r r a t e s greater than


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50cm s and, o f c o u r s e , i m p l a n t a t i o n e x p e r i m e n t s . Furthermore redox p r o t e i n s were r e c e n t l y shown t o behave v e r y w e l l a t a m o d i f i e d m i c r o e l e c t r o d e (Bond, A.M.; H i l l , H.A.O.; M c C a r t h y , M.; P s a l t i , I.S.M.; Walton, N.J. J . Amer. Chem. S o c , s u b m i t t e d ) . We have made i n v e s t i g a t i o n s o f t h e p r o p e r t i e s o f two t y p e s o f m i c r o e l e c t r o d e : g o l d o r p l a t i n u m d i s c s (25μΜ t o 60|lm d i a m e t e r ) and d u a l m i c r o b a n d gold electrodes. DISC MICROELECTRODE. One d r a w b a c k o f m i c r o e l e c t r o d e s i s t h a t i n f o r m a t i o n on r e a c t i o n s t o w h i c h t h e e l e c t r o a c t i v e s p e c i e s i s c o u p l e d i s u s u a l l y l o s t due t o t h e s h o r t t i m e domain a s s o c i a t e d w i t h these e l e c t r o d e s . We were t h u s i n t e r e s t e d i n d e t e r m i n i n g t h e l i m i t i n g s i z e o f m i c r o e l e c t r o d e a t which one c a n d e t e c t t h e c o u p l i n g of t h e glucose-glucose oxidase r e a c t i o n t o the e l e c t r o c h e m i c a l o x i d a t i o n o f f e r r o c e n e as shown i n F i g u r e l a . I t was e n c o u r a g i n g t o f i n d t h a t m i c r o e l e c t r o d e s o f 25μιη i n d i a m e t e r (which i s a d m i t t e d l y r a t h e r l a r g e ) a r e s e n s i t i v e t o homogeneous c o u p l e d r e a c t i o n s as i s shown by t h e c a t a l y t i c enhancement o f t h e c u r r e n t i n F i g u r e l b . The s t u d i e s p e r f o r m e d employed a v a r i e t y o f f e r r o c e n e d e r i v a t i v e s b u t most o f t h e d e t a i l e d work i n v o l v e d 1,1'-dimethyl-3-(l-hydroxy-2aminoethyl)ferrocene ( D M H A E - f e r r o c e n e ) s i n c e i n t e r f e r e n c e by d i o x y g e n i s n e g l i g i b l e . The dependence o f t h e c a t a l y t i c c u r r e n t ( p l o t t e d as Δ ι ) on t h e g l u c o s e c o n c e n t r a t i o n u s i n g a g o l d d i s c o f d i a m e t e r 60μιη i s shown i n F i g u r e 2. I n a l l c a s e s , t h e c u r r e n t s a r e a l m o s t i n d e p e n d e n t o f t h e g l u c o s e c o n c e n t r a t i o n a b o v e 15mM. A d s o r p t i o n o f t h e enzyme o n t o t h e e l e c t r o d e s u r f a c e , p o s s i b l y combined w i t h k i n e t i c f a c t o r s , may be r e s p o n s i b l e f o r t h e absence o f l i n e a r i t y o f t h e response f o r c o n c e n t r a t i o n s g r e a t e r t h a n t h a t 6mM. (Using a c o n v e n t i o n a l e l e c t r o d e , a l i n e a r response, extending t o 30mM o f t h e a n a l y t e , was o b s e r v e d ) . BAND MICROELECTRODE. These e l e c t r o d e s have an a c t i v e s u r f a c e w i t h one d i m e n s i o n i n t h e m i c r o m e t e r r a n g e a n d t h e o t h e r s e v e r a l m i l l i m e t r e s l o n g . They r e t a i n many o f t h e f e a t u r e s o f t h e m i c r o e l e c t r o d e s b u t have two a d d i t i o n a l a d v a n t a g e s : t h e y have a much h i g h e r c u r r e n t due t o t h e i r l a r g e s u r f a c e area and i t i s r e l a t i v e l y easy t o have two (or more) c l o s e l y spaced. When t h e d i s t a n c e between t h e e l e c t r o d e s i s i n t h e m i c r o m e t e r range each e l e c t r o d e i s w i t h i n t h e d i f f u s i o n l a y e r o f t h e o t h e r , and t h e r e a c t i o n s t a k i n g p l a c e a t one i n f l u e n c e t h e a c t i v i t y o f t h e o t h e r and v i c e v e r s a (lj>) · The g l u c o s e - g l u c o s e o x i d a s e (GOD)-DMHAE f e r r o c e n e s y s t e m was employed a g a i n . The c h e m i c a l r e a c t i o n o f t h e f e r r i c i n i u m s p e c i e s g e n e r a t e d a t one e l e c t r o d e , d u r i n g t h e t i m e and space i n t e r v a l o f t h e i r m i g r a t i o n t o t h e a d j a c e n t e l e c t r o d e (scheme i n F i g u r e 3 ) , c a u s e s a change i n t h e c u r r e n t a t t h e s e c o n d e l e c t r o d e (Hill, H.A.O.; K l e i n , N.A.; P s a l t i , I.S.M.; Walton, N.J. A n a l . Chem, 1989, i n p r e s s ) . The e f f e c t on t h e s o - c a l l e d c o l l e c t i o n c u r r e n t s depends on t h e gap (W ) between t h e e l e c t r o d e s . When t h e gap i s l a r g e , most o f t h e f e r r i c i n i u m i o n s r e a c t w i t h GOD, g i v i n g a c o n s i d e r a b l e d e c r e a s e i n t h e c o l l e c t i o n c u r r e n t compared t o t h e s y s t e m w h i c h l a c k s s u b s t r a t e and/or enzyme. When t h e gap i s s m a l l , t h e r e i s no appreciable decrease i n the c o l l e c t i o n current but instead i n v e r s i o n o f t h e normal h y s t e r e s i s l o o p i s o b s e r v e d (see d i r e c t i o n



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Figure 1. (a) Scheme f o r t h e r e a c t i o n s e q u e n c e o f t h e electrochemically coupled enzymatic o x i d a t i o n of glucose, (b) The e f f e c t o f t h e e n z y m a t i c r e a c t i o n on t h e e l e c t r o c h e m i s t r y of f e r r o c e n e u s i n g a g o l d d i s c o f 25|im d i a m e t e r a t 5mV s


HILL E T AL.

Bioelectrochemistry at Microélectrodes


7.


109


lia

C H E M I C A L SENSORS AND MICROINSTRUMENTATION

•GLUCOSE PRODUCT

GENERATOR

POTENTIAL

E

G

. V

vs

S C E

F i g u r e 3. E f f e c t o f t h e e n z y m a t i c r e a c t i o n w i t h accompanying schemes on t h e c o l l e c t i o n c u r r e n t o f a d u a l microband g o l d e l e c t r o d e ( l u m t h i c k ) as a f u n c t i o n o f W


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Bioelectrochemistry ai Mkrûdectrwks


of arrows i n F i g u r e 3b). The l a r g e gap a l l o w s more t i m e f o r t h e e n z y m a t i c r e a c t i o n t o t a k e p l a c e a n d t h e r e f o r e t o be m o n i t o r e d s u c c e s s f u l l y . The minimum c o n c e n t r a t i o n o f enzyme r e q u i r e d f o r t h e c a t a l y t i c r e a c t i o n t o be d e t e c t e d u s i n g t h i s d u a l e l e c t r o d e c o n f i g u r a t i o n i s about 0.05μΜ when used w i t h 5mM g l u c o s e and 0.2mM DMHAE-ferrocene. F i g u r e 4 shows t h e dependence o f t h e c o l l e c t i o n

G L U C O S E CONCENTRATION (mM)

F i g u r e 4. D e p e n d e n c e o f t h e c o l l e c t i n g c u r r e n t on g l u c o s e c o n c e n t r a t i o n f o r a d u a l m i c r o b a n d g o l d e l e c t r o d e o f W =82jxm a t d i f f e r e n t DMHAE-ferrocene and GOD c o n c e n t r a t i o n s . gap


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c u r r e n t ( A i ) on t h e a n a l y t e c o n c e n t r a t i o n . E x p e r i m e n t s i n p r o g r e s s seek 'to f i n d t h e d i m e n s i o n s o f t h e d u a l e l e c t r o d e s w h i c h may r e s u l t i n s i g n i f i c a n t e x t e n s i o n o f t h e range. cG


CONCLUSIONS I t w i l l be a p p a r e n t t h a t t h e a n a l y t i c a l u s e o f b i o e l e c t r o c h e m i c a l methods depends on many f a c t o r s i n c l u d i n g t h e u s e o f t h e n o v e l d e s i g n o f e l e c t r o d e s and t h e employment o f t h e d i r e c t e l e c t r o c h e m i s t r y o f enzymes, whether m o d i f i e d o r n o t . P r o b a b l y t h e d e f e c t o f a l l t h e p r e s e n t methods i s t h e modest s e n s i t i v i t y o f b i o e l e c t r o c h e m i c a l methods. T h i s i s o b v i o u s l y i m p o r t a n t i f t h e s e techniques a r e going t o give r i s e t o devices capable o f sensing components o f t h e immune s y s t e m , t o s a y n o t h i n g o f DNA a n d RNA analyses. Obviously i ti s possible t o provide considerable a m p l i f i c a t i o n from t h e e l e c t r o n i c apparatus a s s o c i a t e d w i t h t h e b i o c h e m i c a l m a t e r i a l s employed b u t t h e l a t t e r w i l l have t o be made more s e n s i t i v e , p e r h a p s , f o r e x a m p l e , by a s e r i e s o f c o u p l e d e n z y m a t i c r e a c t i o n s , b e f o r e t h e f u l l advantages o f t h e s e t e c h n i q u e s can be e x p l o i t e d . ACKNOWLEDGMENTS We thank MediSense I n c . f o r generous

support.

LEGEND OF SYMBOLS Δί

c a l c u l a t e d as Δί

= i

c

- i

0

where i i s t h e c u r r e n t a t c o n c e n t r a t i o n C, and i zero c o n c e n t r a t i o n , o f t h e a n a l y t e . c

Δί

0

G

Q

the current a t

c a l c u l a t e d as

where i i s t h e c u r r e n t d e t e c t e d by t h e c o l l e c t i n g e l e c t r o d e a t c o n c e n t r a t i o n C. Gc

LITERATURE CITED 1. 2. 3.

4. 5.

Frew, J.; H i l l , H. A. O. Eu. J. Biochem. 1988, 172, 261. Armstrong, F. A.; H i l l , H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407, and references therein. Barker, P. D.; H i l l , H. A. O. In Oxidases and Related Redox Systems; King, T. E.; Mason, H. and Morrison, Μ., Eds.; M. Prog. Clinical Biological Research No. 274 Alan Liss Inc.: New York, N.Y., 1988; pp 419-432. Barker, P. D.; Guo, L. H.; H i l l , H. A. O.; Sanghera, G. S. Biochem. Soc. Trans. 1988, 16, 957-58. Armstrong, F. A.; Lannon, A. M. J. Amer. Chem. Soc. 1987, 109, 7221.


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11. 12. 13. 15. 16.

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Assefa, H.; Bowden, E. F. Biochem. Biophys. Res. Commun. 1986, 139, 1003. Kulys, J. J. Biosensors 1986, 2, 3-13. Clark, L. C.; Lyons, C. Ann. N.Y. Acad. Sci. 1962, 102, 29. Cass, A. E. G.; Davis, G.; Francis, G. D.; H i l l , H. A. O.; Aston, J.; Higgins, I. J.; Plotkin, Ε. V.; Scott, L. D. L; Turner, A. P. F. Anal. Chem. 1984, 56, 667. Di Gleria, K.; H i l l , H. A. O.; McNeil, C. J.; Green, M. J. Anal. Chem. 1986, 58, 1203. H i l l , H. A. O. European Patent Application No. 84303090.9, 1986. Dagani, Y.; Heller, A. J. J. Phys. Chem. 1987, 91, 1285. Carr, P. W.; Bowers, L. D. In Immobilised Enzymes i n Analytical and C l i n i c a l Chemistry; Wiley: New York, 1980; p197. 14. Wightman, R. M. Anal. Chem. 1981, 53, 1125A-34A. Pons, S.; Fleischmann, M. In Ultramicroelectrodes; Datatech Systems, Inc. 1987; pp 1-11. Bard, A. J.; Crayston, J. Α.; Kittleson, G. P.; Varco Shea, T.; Wrighton, M. S. Anal. Chem. 1986, 58, 2321.

RECEIVED March 17, 1989


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