Microelectrodes To Probe Spatially Heterogeneous Concentrations

Chapter 8

Microelectrodes To Probe Spatially Heterogeneous Concentrations

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R. Mark Wightman, Leslie J. May, John Baur, David Leszczyszyn, and Eric Kristensen Department of Chemistry, Indiana University, Bloomington, IN 47405 Electrodes with micrometer dimensions can be used to probe chemical concentrations which are spatially heterogeneous. With the use of a micromanipulator, the electrode can be rastered through solutions, and chemical information can be obtained with a resolution of a few micrometers. One example where such experiments provide unique information includes the examination of band broadening effects in flow injection analysis and liquid chromatography. In these experiments, the concentration of pulses of chemical substances is examined as a function of radial position in the transport tubing. Another type of experiment is the measurement of secretion of chemical substances from living cells. In this case, the measurements are made as a function of distance from the site of secretion. In analytical measurements of the chemical composition of a sample, the values that are normally measured are the average concentrations of the constituents. Indeed, the sampling process is usually designed to ensure that the analyte reflects the overall composition of the material. Most samples are normally not homogeneous in their composition, and in some cases, i t is of interest to examine these inhomogeneities. Chemical sensors with miniature dimensions provide a unique way to make measurements of chemical inhomogeneitles. The sensor can be attached to a micromanipulator and rastered through solution regions where heterogeneous concentrations are expected to be found. In this way a concentration and composition map can be generated as a function of sensor position. This approach has been pioneered by Engstrom who has used sensors to define the chemical heterogeneities that exist in the diffusion layer at large size electrodes (1,2,3). One sensor which we have developed, the carbon-fiber microelectrode, has been shown to be especially useful to obtain information on chemical heterogeneities (4). Carbon fibers are highly conductive and are commercially available in a variety of diameters ranging from 5 to 30 p . The sensor is prepared by sealing a single fiber with epoxy into a glass pipette which is pulled to a tip diameter comparable to that of the fiber. The sensing area of the 0097-615^/04(B~0114S06.0Q/0 © 1989 American Chemical Society

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

8. WIGHTMAN ET AU

Probing Spatially Heterogeneous Concentrations

s e n s o r can have the geometry o f a d i s k i f the f i b e r i s c u t f l u s h w i t h the g l a s s , a c y l i n d e r i f the f i b e r i s a l l o w e d t o p r o t r u d e from the g l a s s ( 5 ) , o r an e l l i p s e i f the e l e c t r o d e i s b e v e l l e d on a p o l i s h i n g wheel ( 6 ) . Methods t o c o a t the m i c r o e l e c t r o d e s w i t h polymer f i l m s have been d e v e l o p e d t o p r o v i d e a d d i t i o n a l s e l e c t i v i t y (7). When c o u p l e d w i t h a s e n s i t i v e p o t e n t i o s t a t , t h e s e e l e c t r o d e s can sense submicromolar c o n c e n t r a t i o n s w i t h a subsecond r e s p o n s e time. I n t h i s r e v i e w we w i l l d e s c r i b e s e v e r a l a p p l i c a t i o n s where we have e x p l o r e d d i f f e r e n t s p a t i a l c h e m i c a l h e t e r o g e n e i t i e s w i t h t h i s sensor. These a p p l i c a t i o n s i n c l u d e the c o n c e n t r a t i o n p r o f i l e a t the o u t l e t o f a c h r o m a t o g r a p h i c l o o p i n j e c t o r and a c h r o m a t o g r a p h i c column, and c h e m i c a l h e t e r o g e n e i t y o f n e u r o t r a n s m i t t e r s t o r a g e and r e l e a s e i n the r a t b r a i n . D i s p e r s i o n a t the O u t l e t o f a Loop I n j e c t i o n . I d e a l l y the o u t p u t o f a l o o p i n j e c t o r used i n l i q u i d chromatography o r f l o w i n j e c t i o n a n a l y s i s w o u l d be a sharp c o n c e n t r a t i o n p u l s e . However, t h i s i s u n l i k e l y t o be the case because o f v a r i o u s d i s p e r s i v e f o r c e s w h i c h a c t on the c o n c e n t r a t i o n p l u g . C o n v e c t i v e f l o w under l a m i n a r c o n d i t i o n s i n a c i r c u l a r tube, such as the o u t l e t tube o f a l o o p i n j e c t o r , tends t o be much s l o w e r n e a r the w a l l s o f the tube t h a n i n the c e n t e r and t h i s w i l l d i s t o r t the i n i t i a l shape o f the I n j e c t e d m a t e r i a l s ( 8 , 9 ) . I n a d d i t i o n , r a d i a l and a x i a l d i f f u s i o n o f m a t e r i a l i n the tube can a l t e r i t s i n i t i a l shape. The degree o f d i s p e r s i o n can be e v a l u a t e d (9) by the P e c l e t number ( P ) and the r e d u c e d time ( r ) . These v a l u e s a r e d e f i n e d as c

P

c

- auo/D

;

τ - Dt/a

2

Where a i s the r a d i u s o f the tube, UQ i s the l i n e a r v e l o c i t y o f the l i q u i d a t the c e n t e r o f the tube, D i s the d i f f u s i o n c o e f f i c i e n t o f the a n a l y t e , and t i s the t i m e . For l a r g e v a l u e s o f P (>1000) and s m a l l v a l u e s o f time ( r < 0.02) the d i s p e r s i o n s h o u l d be p r i m a r i l y a r e s u l t o f c o n v e c t i o n ( 9 ) . These c o n d i t i o n s a r e those e x p e c t e d a t a l o o p i n j e c t o r o u t l e t under normal o p e r a t i n g c o n d i t i o n s . To e x p e r i m e n t a l l y t e s t these p r e d i c t i o n s ( 1 0 ) , a d i s k shaped m i c r o v o l t a m m e t r i c e l e c t r o d e was p l a c e d i n the e x i t tube, F i g u r e 1, o f a p n e u m a t i c a l l y c o n t r o l l e d l o o p i n j e c t o r (Type 50, Rheodyne, I n c . , C o t a t i , CA). The r a d i a l p o s i t i o n o f the e l e c t r o d e was con t r o l l e d by a m i c r o m a n i p u l a t o r w i t h 0.01 cm r e s o l u t i o n , and measure ments were made a c r o s s the 0.08 cm d i a m e t e r e x i t tube. The d i s t a n c e between the r o t a r y p o s i t i o n o f the l o o p i n j e c t o r and the e l e c t r o d e was 7.5 cm. A pH 7.4 b u f f e r was pumped t h r o u g h the system a t a f l o w r a t e o f 0.93 mL min" . Under these c o n d i t i o n s P > 4x10* and r-0.02. Dopamine was used as the s u b s t a n c e i n j e c t e d t h r o u g h the l o o p i n j e c t o r a t a c o n c e n t r a t i o n o f 20 μΜ, and was d e t e c t e d a t a f i x e d applied potential. C u r r e n t - t i m e t r a c e s a f t e r i n t r o d u c t i o n o f dopamine w i t h the l o o p i n j e c t o r a r e shown i n F i g u r e 2 as a f u n c t i o n o f r a d i a l p o s i t i o n . When the sensor i s p l a c e d n e a r the c e n t e r o f the e x i t tube, the o b s e r v e d response approaches a square p u l s e . However, as the m i c r o e l e c t r o d e i s moved c l o s e r t o the tube w a l l , the time f o r the sample b o l u s t o r e a c h the i n j e c t o r i s i n c r e a s e d and an i n c r e a s e i n c

1

c


115

116

CHEMICAL SENSORS AND MICROINSTRUMENTATION

W

FIA

Computer

Potentiostot

A

System

Syringe Pump

θ

Loop Injector

Electrochemical Detector

F i g u r e 1. S t a i n l e s s s t e e l tube u s e d w i t h t h e m i c r o v o l t a m m e t r i c e l e c t r o d e : i n s e r t , b l o c k diagram o f t h e f l o w i n j e c t i o n system. (Reproduced from r e f . 10. C o p y r i g h t 1986 American C h e m i c a l Society.)


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Probing Spatially Heterogeneous Concentrations117

-0.4C

Ο T i m e

( S e c o n d s )

F i g u r e 2. Average c u r r e n t response t o a 30-s 20 μΜ dopamine p u l s e measured a t d i f f e r e n t p o s i t i o n s from t h e c e n t e r o f t h e tube. The average s t e a d y - s t a t e c u r r e n t f o r a l l t h e p o s i t i o n s was measured a t 20 s a t the s p e c i f i e d f l o w r a t e ( c i r c l e ) and a z e r o f l o w r a t e ( s q u a r e ) . (Reproduced from r e f . 10. C o p y r i g h t 1986 American Chemical S o c i e t y . )


118

C H E M I C A L SENSORS AND

MICROÏNSTRUMENTATION

dispersion i s observed. At each electrode p o s i t i o n the current reaches a l i m i t i n g , steady-state value by 20 s. The r a d i a l v e l o c i t y of the c a r r i e r stream i n the tube (V ) i s given by: r

¥

2

- 2V (1 - r V a )

r

0

1

where V i s the average v e l o c i t y (3 cm s" ) and r i s the r a d i a l p o s i t i o n . Thus, the t r a n s i t time t from the loop i n j e c t o r to the sensor i s t - 1/V + t where 1 Is the distance from the rotary portion of the loop injector to the electrode t i p . The o f f s e t term i s the time i t takes the pneumatic activator to turn the loop i n j e c t o r (0.8 s ) . This curve i s shown i n Figure 3 and i s superimposed on the time that the microelectrode response reached h a l f of i t s l i m i t i n g value. As can be seen, a good f i t i s obtained i n d i c a t i n g that convection i s the predominant cause of the observed r a d i a l dispersion. To obtain the t o t a l dispersion i n the tube, the i n d i v i d u a l responses were weighted by the area of the concentration r i n g i n which each electrode was located, and these results were summed (10). Very good agreement i s obtained between t h i s r e s u l t and the response measured with a channel type amperometric electrode connected d i r e c t l y to the loop injector (10). These measurements c l e a r l y show that convective flow i s the major o r i g i n of the dispersion observed at the output o f a loop i n j e c t o r . However, these measurements were made i n a time domain which i s close to that where dispersion by d i f f u s i o n should s t a r t to become apparent according to hydrodynamic theory. This Is apparent i n the data obtained closest to the wall where the dispersion i s greatest. Solution l i n e a r v e l o c i t y i s lower i n t h i s region giving greater time f o r d i f f u s i o n . The small size of the detector allows the r a d i a l concentrations to be probed with l i t t l e perturbation o f the stream. This i s evidenced by the a b i l i t y to reconstruct the bulk behavior from the individual r a d i a l measurements. 0

r

r

r

o f f e e t

Dispersion i n Chromatographic Columns. Microelectrodes have also been used to examine the r a d i a l dispersion of bands e l u t i n g from commercial HPLC columns. Conventional chromatographic detectors detect the entire volume of a band e l u t i n g from a chromatographic column. For this reason, the primary figure of merit o f a chromatographic column i s the number o f chromatographic plates measured i n the a x i a l d i r e c t i o n . However, i t has been shown that r a d i a l dispersion can also occur, and t h i s may a f f e c t the a x i a l dispersion measured with a bulk detector. Previous studies have modelled the chromatographic column with columns packed with spherical beads to characterize this phenomenon (11,12). These fundamental studies have shown that r a d i a l dispersion does not exert a great e f f e c t on column e f f i c i e n c y unless dispersion i s suff i c i e n t l y great that the injected species reaches the w a l l o f the column. This "wall e f f e c t " causes a large degree o f dispersion because o f the lower e f f i c i e n c y o f packing near the wall o f the column. Thus, i t i s of interest to experimentally characterize the r a d i a l dispersion present with a commercial loop i n j e c t o r combined with a commercial chromatographic column (13).


8.

WIGHTMAN E T A L .

0.04

r


E-

(cm)

-0.04

Time (Seconds) F i g u r e 3. The time r e q u i r e d t o r e a c h h a l f o f the s t e a d y - s t a t e c u r r e n t i s p l o t t e d a g a i n s t the p o s i t i o n o f the e l e c t r o d e from the c e n t e r o f the tube. The a s t e r i s k symbol r e p r e s e n t s a s i n g l e measurement whereas the s o l i d l i n e i s t h a t p r e d i c t e d by p u r e convection.


120


MICROINSTRUMENTATION

I n t h i s case t h e e l e c t r o d e had a c y l i n d r i c a l geometry ( t o im prove s e n s i t i v i t y ) and was p l a c e d a t t h e e x i t f r i t o f a 10-cm l o n g r e v e r s e - p h a s e column packed w i t h 3-/im p a r t i c l e s (Brownlee L a b s , S a n t a C l a r a , CA). The column h a d a d i a m e t e r o f 3.2 mm, and t h e e x i t f r i t was c r i m p e d i n t o p l a c e so t h a t a column end f i t t i n g was n o t r e q u i r e d . The geometry o f t h e e l e c t r o c h e m i c a l c e l l i s shown i n F i g u r e 4. The c h r o m a t o g r a p h i c system i n c l u d e d a p n e u m a t i c a l l y o p e r a t e d l o o p i n j e c t o r (Model 3XL, S c i e n t i f i c Systems, I n c . , S t a t e C o l l e g e , P A ) . The i n j e c t i o n l o o p had a 10 j*L volume, and m i x t u r e s o f c a t e c h o l a m i n e s were used as t e s t compounds. The e l e c t r o d e c o u l d be p o s i t i o n e d w i t h 100 μη r e s o l u t i o n i n t h r e e d i m e n s i o n s w i t h a m i c r o m a n i p u l a t o r ( N a r i s h i g e Co., L t d . , Tokyo, J a p a n ) . The v e r t i c a l r e s o l u t i o n was enhanced w i t h a p i e z o e l e c t r i c p o s i t i o n e r w i t h 0. 5-/im r e s o l u t i o n ( B u r l e i g h I n s t r u m e n t s , I n c . , F i s h e r s , NY). R e p r e s e n t a t i v e chromatograms measured a t two r a d i a l p o s i t i o n s w i t h t h e m i c r o e l e c t r o d e a r e shown i n F i g u r e 5. The t r a c e r e c o r d e d a t t h e c e n t e r o f t h e e x i t column e x h i b i t s s h a r p e r peaks t h a n when the chromatograms a r e r e c o r d e d a t the edge. The c h r o m a t o g r a p h i c peaks r e c o r d e d a t t h e edge a r e b r o a d e r , a t t e n u a t e d , s i g n i f i c a n t l y t a i l e d , and have l o n g e r r e t e n t i o n t i m e s . F i g u r e 6 shows t h e n o r m a l i z e d c o n c e n t r a t i o n o b s e r v e d f o r one peak as a f u n c t i o n o f r a d i a l p o s i t i o n . Superimposed on t h e c u r v e i s the d i s p e r s i o n e x p e c t e d f o r a c e n t r a l l y i n j e c t e d s u b s t a n c e c a l c u l a t e d f o r t h e column c o n d i t i o n s and f l o w r a t e (1.0 mL min" ) em ployed. I t i s c l e a r t h a t t h e d i s p e r s i o n i s much g r e a t e r f o r t h e experimental data. I t i s l i k e l y that the observed r a d i a l concentra t i o n p r o f i l e i s a consequence o f t h e sample i n t r o d u c t i o n p r o c e s s e s as p r e v i o u s l y p r o p o s e d by K i r k l a n d ( 1 4 ) . The reduced p l a t e h e i g h t ( h - H /dp, where H i s t h e a x i a l p l a t e h e i g h t and dp i s t h e p a r t i c l e d i a m e t e r ) and r e t e n t i o n time a r e p l o t t e d i n F i g u r e 7 as a f u n c t i o n o f r a d i a l p o s i t i o n . I n contra d i c t i o n w i t h t h a t w h i c h i s e x p e c t e d from t h e w a l l e f f e c t , t h e r e t e n t i o n time i s g r e a t e r near t h e w a l l . T h i s t o o may be a r e s u l t o f t h e sample i n t r o d u c t i o n because a s i m i l a r p r o f i l e was o b s e r v e d a t t h e o u t l e t o f t h e l o o p i n j e c t o r . However, t h e d a t a c l e a r l y show t h a t the s e p a r a t i o n e f f i c i e n c y d e c r e a s e s as t h e w a l l o f t h e column i s ap proached. The s e p a r a t i o n e f f i c i e n c y was e v a l u a t e d u s i n g t h e e x p o n e n t i a l l y m o d i f i e d G a u s s i a n model. W i t h t h i s p r o c e d u r e t h e peak i s c h a r a c t e r i z e d b y σ, t h e s t a n d a r d d e v i a t i o n o f t h e peak, and r , t h e expo n e n t i a l m o d i f i e r . F o r measurements made i n t h e c e n t e r o f t h e column, τ and σ b o t h i n c r e a s e d f o r l o n g e r r e t a i n e d compounds. The v a l u e o f r i n c r e a s e d as t h e column w a l l was approached w i t h l i t t l e change i n σ. Comparison was made between t h e r a d i a l measurements and t h a t made w i t h a b u l k d e t e c t o r , a commercial amperometric d e t e c t o r . When the i n d i v i d u a l r a d i a l measurements were w e i g h t e d and summed as des c r i b e d e a r l i e r , t h e r e c o n s t r u c t e d chromatogram superimposed on t h a t measured w i t h t h e b u l k d e t e c t o r . S e p a r a t i o n e f f i c i e n c i e s measured i n t h e c e n t e r o f t h e column were a t l e a s t 20% h i g h e r t h a n those measured w i t h t h e b u l k d e t e c t o r . Most n o t a b l e was t h e v e r y h i g h e f f i c i e n c y o b t a i n e d w i t h u r i c a c i d (k' - 0.33) where a r e d u c e d p l a t e h e i g h t o f 1.5 was o b t a i n e d a t a f l o w r a t e o f 0.56 mL min" . This r e p r e s e n t s a 90% improvement o v e r t h e s e p a r a t i o n e f f i c i e n c y o b t a i n e d w i t h the bulk detector. 1

a

a

a

1


8. WIGHTMAN ET AL

121 Probing Spatially Heterogeneous Concentrations

PP MM

{

Figure 4. Column end-assembly configured f o r microvoltammetric electrochemical detector: AE, a u x i l i a r y electrode; CA, cartridge holder; CH, column holder; CM, Column; EH, electrode holder; FR, f r i t ; MM, micromanipulator; PP, p i e z o e l e c t r i c posi tioner; RE, reference electrode; SC screw cap; WE, working electrode. (Reproduced from r e f . 13. Copyright 1988 American Chemical Society.)

0

20

40

60 Time (s)

80

100

Figure 5. Chromatograms of 5 nmol each NE, Ε, and DHBA with microvoltammetric electrode near the center of the column and near the edge of the column. Flow rate - 1.0 mL min" . (Re produced from r e f . 13. Copyright 1988 American Chemical Society.) 1


122


r


(cm)

F i g u r e 6, N o r m a l i z e d r a d i a l c o n c e n t r a t i o n d i s t r i b u t i o n f o r NE. Solid line: t h e o r e t i c a l f o r c e n t r a l i n j e c t i o n w i t h σ - 0.188 mm. Circles: e x p e r i m e n t a l . The dashed l i n e s a r e t h e e s t i m a t e d p o s i t i o n o f the column w a l l s , (Reproduced from r e f . 13. C o p y r i g h t 1988 American C h e m i c a l S o c i e t y . ) κ

1.03

-0.1 0.0

0.1

r (cm) F i g u r e 7. N o r m a l i z e d r e t e n t i o n time and r e d u c e d a x i a l p l a t e h e i g h t o f 5 nmol NE as a f u n c t i o n o f r a d i a l p o s i t i o n f o r a new column. Flow r a t e - 1.0 mL min" . The dashed l i n e s a r e t h e e s t i m a t e d p o s i t i o n o f the column w a l l s . (Reproduced from r e f . 13. C o p y r i g h t 1988 American C h e m i c a l S o c i e t y . ) 1


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123 Probing Spatially Heterogeneous Concentrations

The m i c r o e l e c t r o d e i n t h i s a p p l i c a t i o n i s a b l e t o p r o v i d e c l e a r i n s i g h t i n t o r a d i a l d i s p e r s i o n processes i n l i q u i d chromatographic columns. The e x p e r i m e n t s show t h a t d i s p e r s i o n i s g r e a t e r n e x t t o the column w a l l as p r e d i c t e d from fundamental s t u d i e s . However, the o b s e r v a t i o n s t h a t the r e t e n t i o n time i s s l o w e r n e a r the w a l l s , and t h a t the i n j e c t e d b o l u s i s d i s t r i b u t e d o v e r the e n t i r e column d i f f e r from p r e v i o u s model s t u d i e s . These d i f f e r e n c e s a r e b o t h l i k e l y t o be due t o the i n t r o d u c t i o n o f sample w i t h the l o o p i n j e c t o r . These o b s e r v a t i o n s a r e s i g n i f i c a n t because t h e y i n d i c a t e t h a t a c e n t r a l l y p l a c e d d e t e c t o r w i l l r e c o r d chromatograms o f v e r y h i g h e f f i c i e n c y i n commercial columns. However, the i n c r e a s e i n e f f i c i e n c y may be c o u p l e d w i t h a d e c r e a s e i n s e n s i t i v i t y because a l a r g e p o r t i o n o f the e l u t i n g band i s d i s c a r d e d . P r e l i m i n a r y d a t a i n d i c a t e t h a t t h i s i s n o t a c o n c e r n w i t h amperometric d e t e c t i o n . T h i s i s because the n o i s e i n amperometric d e t e c t o r s I s p r o p o r t i o n a l t o the a r e a , w h i l e the s i g n a l i s p r o p o r t i o n a l t o the l a r g e s t d i m e n s i o n . Thus, a c y l i n d r i c a l electrode exhibits superior signal-to-noise characteristics o v e r a b u l k amperometric d e t e c t o r u n t i l the fundamental l i m i t o f a m p l i f i e r n o i s e i s reached. Chemical Heterogeneity

i n the B r a i n .

The mammalian b r a i n i s an e x t r e m e l y heterogeneous s t r u c t u r e on the c e l l u l a r l e v e l . W h i l e o n l y 10% o f the t o t a l c e l l p o p u l a t i o n i s c o m p r i s e d o f neurons, these are the a l l i m p o r t a n t c e l l s w h i c h c o l l e c t , i n t e g r a t e , and r e l a y i n f o r m a t i o n ( 1 5 ) . Neurons a r e a r r a n g e d i n a v a r i e t y o f n e t w o r k s , and, s i n c e t h e r e a r e o v e r 10 m i l l i o n neurons i n the human b r a i n , the degree o f h e t e r o g e n e i t y can be seen t o be e x t r e m e l y complex. Most neurons communicate w i t h one a n o t h e r by the s e c r e t i o n o f c h e m i c a l s u b s t a n c e s known as n e u r o t r a n s m i t t e r s . Neurotransmission o c c u r s i n a r e g i o n known as the synapse w h i c h c o n s t i t u t e s the i n p u t o f one neuron and the o u t p u t o f the neuron s e c r e t i n g the t r a n s m i t t e r . S i n c e neurons form synapses t h r o u g h o u t the b r a i n , one w o u l d e x p e c t t h a t the c e l l u l a r h e t e r o g e n e i t y w o u l d a l s o be r e f l e c t e d I n a c h e m i c a l h e t e r o g e n e i t y o f n e u r o t r a n s m i t t e r c o n c e n t r a t i o n s w i t h i n the b r a i n . T h i s h y p o t h e s i s can be t e s t e d f o r c a t e c h o l a m i n e neurotransm i t t e r s because c a r b o n f i b e r m i c r o e l e c t r o d e s i m p l a n t e d i n the b r a i n can d e t e c t them v o l t a m m e t r i c a l l y . In vivo v o l t a m m e t r y has now become a r e l i a b l e t e c h n i q u e f o r the d e t e c t i o n o f dopamine dynamics i n the b r a i n ( 1 6 ) . Dopamine, a c a t e c h o l a m i n e , i s found i n g r e a t e s t c o n c e n t r a t i o n i n a r e g i o n o f the b r a i n known as the s t r i a t u m . T h i s r e g i o n , w h i c h i s p r e s e n t i n a l l mammals, i s i m p o r t a n t f o r normal c o o r d i n a t e d movements. I t has been shown t h a t p a t i e n t s w i t h P a r k i n s o n ' s d i s e a s e have a s e v e r e l o w e r i n g o f t h e i r dopamine c o n t e n t i n t h i s b r a i n r e g i o n . T h i s r e g i o n i s heterogeneous a t the c e l l u l a r l e v e l because many n e u r o n a l f i b e r s p a s s t h r o u g h i t i n d i s t i n c t t r a c t s w i t h o u t f o r m i n g s y n a p t i c cont a c t s . I n a d d i t i o n , i t has been shown t h a t the dopamine n e r v e t e r m i n a l s i n the s t r i a t u m a r e a r r a n g e d I n d i s t i n c t p a t t e r n s (17,18). Thus, h e t e r o g e n e i t y o f dopamine r e l e a s e from n e r v e t e r m i n a l s w o u l d be e x p e c t e d i n t h i s r e g i o n as w i t h o t h e r n e u r o t r a n s m i t t e r s i n o t h e r b r a i n regions. The c a r b o n f i b e r e l e c t r o d e u s e d i n t h i s work i s b e v e l l e d so t h a t the a c t i v e s e n s i n g a r e a i s an e l l i p s e w i t h a minor r a d i u s o f 5


124



μπι and a major a x i s o f -35 μιη. The e l e c t r o d e i s c o a t e d w i t h a t h i n f i l m o f a p e r f l u o r i n a t e d c a t i o n exchange membrane w h i c h e x c l u d e s dopamine m e t a b o l i t e s and o t h e r i n t e r f e r e n c e s . I n these e x p e r i m e n t s , i n c o n t r a s t t o those d e s c r i b e d e a r l i e r i n t h i s work, the s o l u t i o n b e i n g sampled, the e x t r a c e l l u l a r f l u i d o f the b r a i n , i s s t a t i c . T h e r e f o r e , m o n i t o r i n g the c u r r e n t a t a c o n s t a n t a p p l i e d p o t e n t i a l would a l t e r the c h e m i c a l c o m p o s i t i o n s u r r o u n d i n g the e l e c t r o d e . For t h i s r e a s o n , the t e c h n i q u e o f f a s t c y c l i c v o l t a m m e t r y has been used. Voltammograms a r e r e p e a t e d a t 100 ms i n t e r v a l s a t a s c a n r a t e o f 300 V s" ( 1 9 ) , Thus, the f l u i d i s o n l y sampled f o r 10 ms. The i n d i v i d u a l voltammograms are s u b t r a c t e d from the background s i g n a l and a r e u s e d t o i d e n t i f y dopamine as the d e t e c t e d s u b s t a n c e ( 2 0 ) , The i n t e g r a t e d c u r r e n t a t the p o t e n t i a l f o r dopamine o x i d a t i o n from each voltammogram p r o v i d e s a c o n t i n u o u s r e a d o u t o f the c o n c e n t r a t i o n . The e n t i r e experiment i s under computer c o n t r o l . The e x p e r i m e n t s d e s c r i b e d h e r e have been u n d e r t a k e n i n the b r a i n o f a n e s t h e t i z e d r a t s . The a n i m a l i s p l a c e d i n a frame w h i c h h o l d s the head a t a p r e s e t a n g l e t o ensure approximate r e p r o d u c i b i l i t y o f the e l e c t r o d e placements ( 1 9 ) . An i n c i s i o n i s made on the s c a l p , the s k u l l i s exposed, and h o l e s a r e d r i l l e d i n the s k u l l . The c a r b o n f i b e r w o r k i n g e l e c t r o d e i s l o w e r e d w i t h a m a n i p u l a t o r t o the top o f the s t r i a t u m . To s y n c h r o n i z e the r e l e a s e o f dopamine w i t h the measurement p r o c e s s , the dopamine c o n t a i n i n g neurons a r e s t i m u l a t e d e l e c t r i c a l l y . The f i b e r s o f dopamine neurons w h i c h p r o j e c t t o the s t r i a t u m are c l o s e l y packed i n a r e g i o n known as the m e d i a l f o r e b r a i n b u n d l e . Thus, a s t i m u l a t i n g e l e c t r o d e p l a c e d i n t h i s r e g i o n can be used t o d e p o l a r i z e a l l o f the dopamine neurons a t once. The s t i m u l a t i o n employed i s a 60 Hz b i p h a s i c square wave w i t h a d u r a t i o n o f 2 s. The r e s u l t s from a s i n g l e s t i m u l a t i o n a r e shown i n F i g u r e 8. The voltammogram o b t a i n e d from the d i f f e r e n c e between t h a t r e c o r d e d b e f o r e and d u r i n g s t i m u l a t i o n demonstrates t h a t the d e t e c t e d sub s t a n c e i s dopamine. The c u r r e n t from the peak o x i d a t i o n p o t e n t i a l f o r dopamine r e c o r d e d from s e q u e n t i a l voltammograms demonstrates t h a t the c o n c e n t r a t i o n o f dopamine i n c r e a s e s d u r i n g s t i m u l a t i o n , and t h e n r a p i d l y d e c r e a s e s a f t e r the s t i m u l a t i o n i s o v e r . The d i s a p pearance o f dopamine i s due t o uptake back i n t o dopamine c e l l s ( 2 1 ) . The appearance o f dopamine d u r i n g the s t i m u l a t i o n i s a c o m b i n a t i o n o f the s y n a p t i c r e l e a s e o f t h i s compound as w e l l as the c o n c u r r e n t u p t a k e . The combined e f f e c t o f b o t h o f these f a c t o r s , p l u s d i f f u s i o n , can be m o d e l l e d as i n d i c a t e d by the s o l i d l i n e i n F i g u r e 8. The c h e m i c a l h e t e r o g e n e i t y o f the b r a i n i s c l e a r l y e v i d e n c e d when the experiment i s r e p e a t e d w i t h the e l e c t r o d e p l a c e d a t succes s i v e l y lower p o s i t i o n s i n the b r a i n ( 2 2 ) . I n F i g u r e 9 the maximum a m p l i t u d e o f dopamine c o n c e n t r a t i o n i s r e c o r d e d a t 100 μπα i n t e r v a l s . R e s u l t s a r e shown from f o u r d i f f e r e n t a n i m a l s . As can be seen the p a t t e r n o f c h e m i c a l h e t e r o g e n e i t y t h a t i s seen i s d i f f e r e n t i n each animal. However, the d a t a a r e c o n s i s t e n t w i t h a n a t o m i c a l r e p o r t s o f the d i s t r i b u t i o n o f dopamine nerve t e r m i n a l s i n the s t r i a t u m (17,18). S t u d i e s a r e c u r r e n t l y underway t o e s t a b l i s h whether an exact c o r r e l a t i o n e x i s t s . The d a t a i n these e x p e r i m e n t s c l e a r l y show t h a t the s p a t i a l r e s o l u t i o n o f c a r b o n f i b e r e l e c t r o d e s i s s u f f i c i e n t t o measure c h e m i c a l i n h o m o g e n e i t i e s i n the i n t a c t b r a i n . T h i s type o f i n f o r m a t i o n w i l l be u s e f u l i n e s t a b l i s h i n g the r o l e o f d i f f e r e n t dopamine 1


8.

WIGHTMAN ET AL.


ε (ν

ν»

scE)

F i g u r e 8. Data o b t a i n e d i n the caudate n u c l e u s o f an anesthe t i z e d r a t from a N a f i o n - c o a t e d , c a r b o n - f i b e r e l e c t r o d e w i t h v o l t a m m e t r y (300 V s" ) d u r i n g s t i m u l a t i o n s o f dopamine con t a i n i n g neurons. A: Temporal changes o b s e r v e d d u r i n g e l e c t r i c a l s t i m u l a t i o n s a t 60 Hz. Each p o i n t r e p r e s e n t s the c u r r e n t from i n d i v i d u a l voltammograms i n t e g r a t e d o v e r the range 400-800 mV v s SCE. The response i s c o n v e r t e d t o c o n c e n t r a t i o n by c a l i b r a t i o n w i t h dopamine. The s o l i d l i n e s a r e the m o d e l l e d response w h i c h i n v o l v e s the use o f n e u r o c h e m i c a l k i n e t i c p a r a meters and d i f f u s i o n from a d i s t a n c e o f 10 μι. Β: A s u b t r a c t e d c y c l i c voltammogram o b t a i n e d d u r i n g the 60-Hz s t i m u l a t i o n . The shape i s i d e n t i c a l t o t h a t r e c o r d e d i n dopamine s o l u t i o n s . 1


125

126



Caudate O 5 ~ s stimulation

·

1- s stimulation

%[DA]

.^__..,_1_,.„. 5J

,,1 , , 5.7

, 5 3

5.7

Depth f r o m Dura ( m m ) F i g u r e 9. H e t e r o g e n e i t y o f o v e r f l o w i n t h e caudate-putaxnen. P a n e l s a, b, c, and d each r e p r e s e n t responses from i n d i v i d u a l a n i m a l s . The maximal [DA] o b s e r v e d i s e x p r e s s e d as a p e r c e n t a g e o f t h e l a r g e s t response I n t h a t a n i m a l and i s p l o t t e d v s . v e r t i c a l electrode p o s i t i o n . Error bars represent the standard e r r o r o f t h e mean f o r r e p l i c a t e measurements; when no e r r o r b a r s a r e i n d i c a t e d , t h e s t a n d a r d e r r o r i s l e s s t h a n t h e symbol s i z e . (Reproduced w i t h p e r m i s s i o n from Ref. 22. C o p y r i g h t 1989 E l s e vier. )


8.

WIGHTMAN E T AL.


neurons and the p a r t i c u l a r neuronal c i r c u i t s i n which they are involved. Conclusions The three examples shown i n t h i s paper indicate that microvoltammetric electrodes are useful tools to probe chemical heterogeneities i n solution, and furthermore to characterize these phenomena under dynamic conditions. To achieve greater s p a t i a l resolution, smaller electrodes w i l l need to be employed. Automation of t h i s type of measurement would be desirable and can r e a d i l y be accomplished with p i e z o e l e c t r i c micropositioners and other such devices which can be remotely controlled. Such developments w i l l lead to a form of dynamic chemical microscopy which would be useful to measure such events as secretion from single c e l l s , corrosion processes i n p i t s and cracks, or further studies of solution flow. Acknowledgments This research was supported by NIH and NSF.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Engstrom, R. C.; Weber, M.; Wunder, D. J.; Burgess, R.; Winquist, S. Anal. Chem. 1986, 58, 844-848. Engstrom, R. C.; Wightman, R. M.; Meaney, T.; Tople, R. Anal. Chem. 1987, 59, 2005-2010. Engstrom, R. C.; Wightman, R. M.; Kristensen, E. W. Anal. Chem. 1988, 60, 652-656. Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1980, 52, 946-950. Kovach, P. M.; Deakin, M. R.; Wightman, R. M. J. Phys. Chem. 1986, 90, 4612-4617. Kelly, R.; Wightman, R. M. Anal. Chim. Acta 1986, 187, 79-87. Baur, J. E.; Kristensen, E. W.; May, L. J.; Wiedemann, D. J.; Wightman, R. M. Anal. Chem. 1988, 60, 1268-1272. Vanderslice, J. T.; Stewart, K. K.; Rosenfeld, A. G.; Higgs, D. J. Talanta 1981, 28, 11-18. Ananthakrishnan, V.; G i l l , W. N.; Barduhn, A. J. AIChE J. 1965, 11, 1063-1072. Kristensen, E. W.; Wilson, R. L.; Wightman, R. M. Anal. Chem. 1986, 54, 986-988. Knox, J. H.; Laird, G. R.; Raven, P. A. J. Chromatogr. 1976, 122, 129-145. Eon, C. H. J. Chromatogr. 1978, 149, 29-42. Baur, J. E.; Kristensen, E. W.; Wightman, R. M. Anal. Chem. 60, 2334, 1988. Kirkland, J. J.; Yau, W. W.; Stoklosa, H. J.; Dilks, Jr., C. H. J. Chromatogr. Sci. 1977, 15, 303-316. Cooper, J. R.; Bloom, F. E.; Roth, R. H. The Biochemical Basis of Neuropharmacology, Oxford University Press: Oxford, England, 1986. Wightman, R. M.; May, L. J.; Michael, A. C. Anal. Chem. 1988, 60, 769A-779A.


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128 17. 18. 19. 20. 21. 22.

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SENSORS A N D


Gerfen, C. R.; Herkenham, M.; Thibault, J. J. Neurosci. 1987, 7, 3915-3934. Graybiel, A. M.; Radsdale, Jr., C. W. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5723-5726. Wightman, R. M.; Amatore, C.; Engstrom, R. C.; Hale, P. D.; Kristensen, E. W.; Kuhr, W. G.; May, L. J. Neuroscience 1988, 25, 513-523. Howell, J. O.; Kuhr, W. G.; Ensman, R. E.; Wightman, R. M. J. Electroanal. Chem. 1986, 209, 77-90. May, L. J.; Kuhr, W. G.; Wightman, R. M. J. Neurochem., in press. May, L. J.; Wightman, R. M. Brain Res. 487, 311, 1989.

RECEIVED March 9, 1989


Microelectrodes To Probe Spatially Heterogeneous Concentrations

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