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Robert T. Kennedy is an Assistant Professor of Chemistry at the Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611, USA. He earned a B.S. in chemistry at the University of Florida in 1984. He received a Ph.D. in chemistry at the University of North Carolina at Chapel Hill in 1988 under the direction of Professor James W. Jorgenson. He spent two years as an NSF Post-Doctoral Fellow with Professor R. Mark Wightman, also at North Carolina, before returning to Florida in August of 199 7. His research at Florida concerns the development of microelectrodes and microcolumn separations to monitor neurotransmitters and hormones, especially peptides, in biological microenvironments. Lan Huang is a graduate student in chemistry beginning her fourth year under the direction of Robert T. Kennedy at the University of Florida. She earned a B.S. in chemistry from Nanjing University (P.R. China) in 7986. Her Ph.D. research concerns the development and application of microelectrodes for the detection of insulin secreted from single pancreatic p-cells.

Enzyme-modified electrodes for peroxide, choline, and acetylcholine

Michael G. Garguilo, Adrian C. Michael * Pittsburgh, PA, USA

Enzyme-modified electrodes for peroxide, choline, and acetylcholine have been devel- oped. Horseradish peroxidase, choline oxidase, and acetylcholinesterase were immobilized within a cross-linkable, redox polymer deposited onto the surface of car- bon electrodes. The amperometric, enzyme-

* Corresponding author.

based sensors were developed for use in the extracellular fluid of brain tissue and for sin- gle-cell analysis. The sensitivity, selectivity, size, and response time of the sensors were addressed.

1. Introduction

It is now well established that voltammetric tech- niques employing microelectrodes [ l-71 are extremely useful in research aimed at understand- ing biochemical processes in single cells. The key benefits of microelectrodes to single-cell research

trends in analytical chemistry, vol. 14, no. 4, 7995 165

are derived from their small size which permits them to be positioned very near, or even inside, cells without destroying them. Furthermore, micro- electrodes are well suited to high-speed voltam- metric measurements which have permitted, for example, the observation of individual exocytotic events [ 1,6]. Despite these advantages, microelec- trode-based measurements have been mainly lim- ited in scope to the intrinsically electroactive neurotransmitters (i.e. catecholamines and indole- amines) since these compounds are readily oxi- dized at electrodes. The immobilization of enzymes onto the surface of electrodes has often been used to prepare electrochemical sensors for analytes that are not intrinsically electroactive [ 8-

141. In this paper, we will describe our efforts to prepare enzyme-modified microelectrodes for cho- line and acetylcholine that, while intended for measurements in brain tissue, will also be valuable for the study of cholinergic neurotransmission at the single-cell level.

The principles underlying the development of enzyme-based electrochemical sensors are quite well known and many strategies for incorporating enzymes into electrochemical measurements have been established. Our work has taken advantage of the existence of the enzyme choline oxidase, a fla- vin-adenine dinucleotide (FAD) enzyme, which catalyses the two-electron oxidation of choline to betaine. We plan to co-immobilize acetylcholin- esterase and choline oxidase onto electrodes in order to produce sensors for acetylcholine. To this point, however, we have mainly focussed our atten- tion on optimizing and validating the performance of choline sensors [ 15-171. Choline oxidase exhibits extremely high co-substrate specificity for O,, so an electrochemical sensor based on choline oxidase must either monitor the O2 consumed or the H202 produced concomitantly with the oxida- tion of choline. The sensors described in this paper monitor the H20, produced. Incorporating an oxi- doreductase enzyme into the electrochemical scheme certainly provides sensitivity towards the enzyme substrate but it provides only partial selec- tivity. The biological environment in which these sensors will eventually be used contains high levels of ascorbate [ 181 (vitamin C) which is electroac- tive and a potent reducing agent. Thus, a useful sensor for choline must not only be sensitive to choline but also insensitive to ascorbate.

2. Immobilization of enzymes on microelectrodes

Not all strategies for making enzyme-modified electrodes are adaptable to microelectrodes. One strategy that has proven suitable for microelec- trodes is the use of a cross-linkable polymer that can be mixed with the enzyme, deposited onto the electrode by dip coating or painting and then cured in place. Recently, Adam Heller’s group showed that a cross-linkable redox polymer could be used to both immobilize enzymes onto microelectrodes and shuttle electrons between the entrapped enzymes and the electrodes [ 19,201. The redox polymer consists of poly( vinylpyridine) derivati- zed at the nitrogen atoms with a combination of cross-linking and redox functionalities. The particular advantage of this approach to electrode modification is that all reagents required for oper- ation of the sensor are immobilized, including the enzyme and the electron transfer mediator. Thus, this strategy is suitable for microelectrodes because no additional containment membranes are required. In addition, this strategy is also suitable for use in biological systems since the sample solu- tion need not be flooded with the mediator in order for the enzyme-modified electrode to function. In other words, sensors prepared in this manner are fully self-contained.

3. Electroenzymatic detection of hydrogen peroxide

Although H202 is electroactive, i.e. it can be oxidized at electrode surfaces, electrochemical detection of H,O, requires highly positive poten- tials that are sufficient to also cause oxidation of ascorbate. Thus, sensors based on the detection of H202 by direct oxidation are not intrinsically selec- tive. For this reason, before attempting the devel- opment of choline sensors, we devised a selective electroenzymatic approach for the detection of H202. Horseradish peroxidase (HRP) was immo- bilized onto carbon electrodes by means of the cross-linkable redox polymer. Scheme 1 shows how the combination of HRP and polymeric medi- ator allow detection of H202.

Fig. 1 compares the cyclic voltammograms obtained at a carbon fiber microcylinder electrode supporting a layer of the cross-linked redox poly- mer containing HRP. In PBS the peak-shaped vol- tammogram expected for a surface-confined

166 trends in analytical chemistry, vol. 14, no. 4, 7995

2 H20 species is obtained. In 5 mM H202 the voltammo- gram changed to the sigmoidal shape expected in the event of a catalytic process such as that in Scheme 1. Fig. 1 shows that the potential required for the electrochemical detection of H202 can be shifted to any value that is negative with respect to the half-wave potential of redox polymer. These

! 202 +2H+

. . * . . sensors can be operated in an amperometric mode Scheme 1. Schematic representation ot tne sequen- tial reactions that occur at microsensors for H,O,. Horseradish peroxidase is immobilized within a cross-linkable, redox polymer deposited onto carbon fiber microcylinder electrodes.

with the applied potential set at - 0.1 V vs. SCE which is sufficiently negative to prevent oxidation of ascorbate at the carbon electrode. Although the use of HRP allows the use of negative potentials for the detection of H,02, additional steps are required to completely eliminate ascorbate inter- ferences, as described below.

-10 v PBS

(10 mV/s)

600 400 200 0

E (mV vs. SCE)

Fig. 1. Cyclic voltammograms (5 mV/s) of a H,O, microsensor before and after the addition of 5 mM H,O, to a stationary solution of PBS.

4. Detection of H,O, formed by co- immobilized choline oxidase

To prepare choline microsensors, the cross-link- able redox polymer was used to co-immobilize choline oxidase and HRP onto carbon fiber micro- electrodes. All indications are that choline oxidase, being highly co-substrate specific, does not partic- ipate in electron transfer with the redox polymer. So, the basis for the detection of choline is to elec- troenzymatically detect the H202 produced by the action of the co-immobilized choline oxidase, according to Scheme 2.

Scheme 2 also shows how ascorbate can inter- fere with the detection of choline even though the sensors are operated at a potential that is too neg- ative to cause oxidation of ascorbate. Ascorbate will react chemically with the mediator and the immobilized HRP unless it is prevented from pen- etrating the cross-linked film. To prevent ascorbate from penetrating the film it is coated with a layer of Nafion, an anionic material that is often used to

Scheme 2. Schematic representation of the sequential reactions that occur at microsensors for choline (solid lines) and of the mechanism of ascorbate interference (dashed lines). Horseradish peroxidase (HRP) and choline oxidase (ChOx) are immobilized within a cross-linkable redox polymer deposited on carbon fiber microcylinder electrodes.

trends in analytical chemistry, vol. 14. no. 4, 1995 167

60

ascorbate (,uM)

0 100 200 300 400 I 1 1 I 1 I I

0 M ----400@lAApresent(*) 0 25 50 75 100

choline (,uM)

Fig. 2. Ascorbate interference at a Nafion-coatedcho- line microsensor. The sensor was operated at - 0.1 V vs. SCE in a flow injection analysis system at 37%. The data were collected at the end of 25-s bolus injec- tions of solutions containing either 50 p.Mcholine and O-400 @Iascorbate (horizontal line with square sym- bols) or O-l 00 @I choline with (dashed line with tri- angle symbols) and without (solid line with circle symbols) 400 PMascorbate. The error bars represent the standard deviation of three injections.

reject ascorbate from electrodes [21-231. Fig. 2 shows in vitro calibration data obtained by oper- ating these modified electrodes amperometrically in a flow-injection analysis system with solutions containing mixtures of physiologically relevant levels of choline (O-100 @I) and ascorbate (O- 400 PM), which clearly demonstrate the selectivity of the microsensors for choline over ascorbate.

The previous paragraph mentions that the incor- poration of HRP into the electrodes does not, by itself, eliminate the interfering effects of ascorbate. Thus, we employ both HRP and Nafion to establish selectivity over ascorbate. There are several main reasons why both HRP and Nafion are employed. First, we have found that the rejection of ascorbate by Nafion is not complete. Thus, when Nafion- coated electrodes are operated at positive poten- tials, an ascorbate signal is usually observed. Apparently, however, Nafion rejects ascorbate suf- ficiently so that the reactions in Scheme 2 do not occur. Second, we have also found that the immo- bilized HRP prevents H202 formed in the polymer layer from escaping into the surrounding solution

[ 161. This is important because H205 is a toxic substance that would likely inflict damage on cells near the sensor. For these reasons, the use of both HRP and Nafion to establish selectivity of ascor- bate is considered essential.

5. Preliminary in vivo characterization of choline microsensors

Preliminary in vivo testing of the new choline microsensors described in the previous section has been carried out in the brain tissue of anesthetized rats. The purpose of these tests was to discover how well the microsensors could tolerate the conditions of real biological experiments. The microsensors were lowered into the animal’s brain and used to monitor exogenous choline which was injected into the tissue with a micropipet mounted adjacent to the microsensor. The reason for choosing this approach is that fairly simple equations [24,25] can be used to predict the concentration of choline at the microsensor as a function of time after the ejection of a small quantity of solution from the

20 pA

I 1 I 1 ,

0 5 10 15 20

time (min)

Fig. 3. A trace of the current measured during a local injection experiment using Nafion-coated choline microsensors implanted in the rat striatum. The trace was obtained with the microsensor after the local injection of 375 nl of a 1 0-mM choline solution. The microsensor was operated at a constant potential of - 0.1 V vs. SCE.

168

micropipet. Fig. 3 shows an example of such an experiment in which 375 nl of 10 rnA4 choline solu- tion was injected at a location approximately 1 mm away from the microsensor. The main contrast between the data in Fig. 3 and that published before is that the concentration of choline in the injected solution has been decreased by a factor of ten. This has been made possible by continued improvement and optimization of these microsensors, a process which is still underway. We have shown previously that data of the type in Fig. 3 can be converted into choline concentration by calibration of the sensor after its removal from the brain [ 161. When this is done, reasonable agreement between the data and predictions based on diffusion equations is obtained. This shows that it is possible to perform quantitative in vivo measurements with these microsensors.

One benefit of microelectrodes is that they per- mit rapid voltammetric measurements. This has allowed the monitoring of individual exocytotic events at the single-cell level. The response time of these enzyme-modified electrodes is on the scale of a few seconds, rather than a few milliseconds, so it is unlikely that they will be suitable for mon- itoring exocytosis. The temporal response of these sensors appears to be determined by the diffusion of choline into the enzyme-containing cross-linked polymer film. As such, the response time depends on the thickness of the layer. Previously, using conventional glassy carbon electrodes, we showed that if the layer was thin response times of 2 sec- onds were possible [ 111. So far, it has proven dif- ficult to produce such thin layers on carbon fiber microelectrodes so the microsensors usually have response times in the neighborhood of 15 seconds. Although decreasing the response time continues to be a subject of our research, the microsensors we have developed to date should be useful ‘for experiments involving pharmacological manipu- lations of single cells which can last for several minutes.

6. Prototype acetylcholine sensors

The work described above clearly shows that microsensors for choline can be made. Microsen- sors for choline are of interest for several reasons, one of which is that they will serve as the basis for acetylcholine sensors. The approach we are inves- tigating for the development of acetylcholine sen- sors is to add the enzyme acetylcholinesterase to

trends in analytical chemistry, vol. 14, no. 4, 1995

o.ot , , , 0 5 10

acetylcholine (@I)

Fig. 4. Acetylcholine calibration curve obtained with a prototype trienzyme sensor set at an applied potential of - 0.1 V vs. SCE. The error bars represent the stan- dard deviation of three replicate injections.

the cross-linked redox polymer films [ 111. The addition of acetylcholinesterase, which catalyses the hydrolysis of acetylcholine to choline, will cre- ate sensors that give an amperometric signal related to the sum of the choline and acetylcholine levels in sample solutions. To delineate the contribution of acetylcholine to the total signal, a second sensor will be used to monitor choline alone. To date, we have only prepared prototype acetylcholine sensors using macro-sized glassy carbon electrodes as the substrate. These sensors have been characterized by using them as detector electrodes in a conven- tional flow-injection analysis apparatus. Fig. 4 shows a calibration curve obtained in the low micromolar concentration range which confirms the viability of the trienzyme scheme for the detec- tion of acetylcholine. The sensitivity of these elec- trodes towards acetylcholine is ca. 7 times smaller than the sensitivity towards choline. Further work is called for to increase the sensitivity of the pro- totype sensors towards acetylcholine prior to min- iaturization of the sensors for in vivo experiments.

7. Conclusion

This paper has described our efforts to extend microelectrodes techniques to choline and acetyl- choline, two important substances involved in the neurochemistry of the mammalian central nervous system. These microsensors will permit new inves-

trends in analytical chemisiry, vol. 14, no. 4, 1995 169

tigations into the properties of the cholinergic sys- tems of the brain. Generating new insights into both the functional and dysfunctional aspects of cholin- ergic systems is a very important task considering their involvement in devastating diseases of the brain, namely Parkinson’s and Alzheimer’s. Although these syndromes have been studied extensively, neither is particularly well understood and neither can be cured. These microsensors, while still in the development stages, are showing considerable promise for both in vivo and single- cell experiments.

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Michael G. Garguilo and Adrian C. Michael are at the Department of Chemistry, Chevron Science Center, University of Pittsburgh, Pittsburgh, PA 15260, USA

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