Anal. Chem. 1990, 62, 2347-2351
2347
Stearate-Modified Carbon Paste Electrodes for Detecting Dopamine in Vivo: Decrease in Selectivity Caused by Lipids and Other Surface-Active Agents P a u l D. Lyne' and Robert D. O'Neill* Chemistry Department, University College, Belfield, Dublin 4, Ireland Electrochemicalcharacteristicsof dopamine, ascorbic acid, and ferrocyanide measured with carbon-Nujoi paste electrodes (CPEs) and stearatemodifiedcarbon paste electrodes (SMEs) before and after treatment with either surfactant (triton-X), lipid (phosphatidylethanolamine), or brain tissue indicate that the lipophilic nature of the brain destroys the selecthrlty of SMEs for dopamine by removing the hydrophobic elements from the electrode surface. Measurements of the degree and time-course of changes in surface capacitance of SMEs following contact with surface-active agents support this conclusion. The results suggest that SMEs cannot be used to detect dopamine unambiguously in vivo and emphasize the need to characterize electrochemicalsensors in an environment similar to that of intended applications.
INTRODUCTION A variety of voltammetric techniques have been developed to monitor changes in the concentration of endogenous electroactive compounds in the intact central nervous system ( I , 2). In particular, attention has been focused on the detection of dopamine, a neuromodulatory catecholamine, in the extracellular fluid (ECF) of mammalian brain. The two main problems associated with measuring levels of this monoamine in vivo have been (i) the very low baseline concentration of dopamine (0.02-0.05 pmol/L) in the ECF (3-6) and (ii) the presence of large excesses of other substrates, such as ascorbic acid a t 200-500 pmol/L (7) and 3,4-dihydroxyphenylacetic acid (DOPAC) a t about 10 pmol/L ( 5 ) ,which oxidize a t potentials similar to that of dopamine on many electrode materials. As a result of these adverse conditions, extracellular dopamine has been reliably detected with electrochemical techniques only in situations of strong physiological perturbation, such as electrical stimulation (8,9)or high potassium ion concentration (IO, 11), or with severe pharmacological manipulation of dopaminergic systems, i.e., total inhibition of transmitter metabolism (12). In contrast, the detection of dopamine in vivo using stearate-modified carbon-Nujol paste electrodes (SMEs) has been claimed extensively for a wide variety of mild challenges to dopamine neurons (13-28). The basis of the modification is that the presence on SMEs of unprotonated carboxylic moieties a t physiological p H retards the electrooxidation of anionic species, such as ascorbate and DOPAC, to such an extent that the cationic dopamine species can be detected in their presence. Apart from the complication of the electrocatalysis of dopamine oxidation by ascorbate already investigated (29),the major inconsistency posed by this technique (2, 30) is the large size of the putative dopamine signal recorded in vivo which corresponds to a concentration greater than 1 pmol/L ( I 3 ) ,some 2 orders of magnitude greater than that estimated by using other electrochemical (3, 4 ) and microdialysis ( 5 , 6) techniques. Since a recent report has shown that the response of unmodified carbon paste electrodes Present address: Department of Inorganic Chemistry, South Parks Rd., Oxford, U.K.
(CPEs) is significantly altered by contact with brain tissue (3I),we felt it necessary to reexamine the properties of SMEs in vitro following tissue contact. A preliminary report of this work showed a loss in selectivity of the SMEs for dopamine over ascorbate following implantation in the brain (32). In order to elucidate the processes leading to this loss of selectivity, the electrochemical behavior of ascorbic acid, dopamine, and ferrocyanide a t CPEs and SMEs following treatment with a variety of surface-active agents, and a t carbon powder electrodes, is described here. The results obtained emphasize the need to consider electrode-environment interactions when developing new sensors for use in biological systems. EXPERIMENTAL S E C T I O N Electrodes. Stearate-modified carbon paste electrodes (SMEs) were manufactured from Teflon-coated silver wire as previously reported (13). Briefly, stearate-modified paste was prepared by thoroughly mixing 1.5 g of carbon powder (UCP-1-M, Ultra Carbon Corp.) with 100 mg of stearic acid (octadecanoic acid, Sigma Chemical Co.) dissolved in 1mL of Nujol (Aldrich Chemical Co.). The stearic acid did not dissolve easily in the oil, and the mixture was warmed to approximately 55 "C for some time to achieve a solution that was very viscous. Unmodified carbon paste electrodes (CPEs) were prepared in an identical manner, omitting stearic acid. The carbon powder electrodes (CPWEs) were made by packing unpasted UCP-1-M into the Teflon-insulated cavity. The diameter of the active surface of each electrode type was 250 pm; the external diameter was 350 pm. CPEs and SMEs were subjected to different treatments. SMES were exposed for 24 h to either surfactant solution (0.1% Triton-X loo), SMESwf,or a lipid suspension (phosphatidylethanolamine, PEA, 100 mg/mL), SMElipid.CPEta,, and SMEtissuerepresent CPEs and SMEs inserted into brain tissue for a 24-h period. The tissue in this study was removed from the skull of rat before use, and either used immediately or stored frozen before being defrosted; the effect of fresh and stored tissue was similar. The relevance of the state of the tissue to the modification of electrodes is discussed later. Because of the relative importance of the tissue modification of SMEs to our final conclusions, eight such electrodes were treated in this way. Apparatus. Cyclic staircase voltammograms were recorded at 25 f 1 "C in solutions containing either ascorbate, dopamine, or ferrocyanide, using a microcomputer-based three-electrode system similar to that reported previously (33),at 50 mV/s with a saturated calomel electrode as reference and silver wire in an isolated compartment as auxiliary electrode. For the scan rate and electrode dimension used, this corresponds adequately to cyclic voltammetry (34, 35). Analysis. The peak separation was calculated as the difference between the anodic and cathodic peak potentials, AE = E,, and used as an index for the rate of electron transAr for the ferrocyanide couple at the various electrodes (36). The problem of adsorption of substrate on carbon powder-type surfaces (37) precludes a more direct comparison of heterogeneous rate constants for electron transfer. Due to the irreversibility of the oxidation of ascorbate and dopamine under the present experimental conditions, we have used the potential of maximum slope (38),E,, to characterize the waves for these compounds. Eawas used both as an index of the position of the waves on the voltage axis and as a measure of changes in rates of electron transfer for the two substrates a t the electrodes. The foot potential, Ef (the potential at which the current first reached 1 nA
0003-2700/90/0362-2347$02SO/O0 1990 American Chemical Society
2348
ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990
Table I. Potential of Maximum Slope, E,,,,, Recorded at 50 mV/s for Dopamine (DA) and Ascorbid Acid (AA) at pH 7.4, and Peak Separation, AE,,, for Ferrocyanide (pH 4) at Different Types of Carbon Paste Electrode"
e
CPE
0
-
e-
SUE
wave
separation
peak
AE,,, J
separation
DA
mV AA - DA
ferrocyanide
140 f 10 272 f 15 75 f 5 78f 9 94 f 10 75 f 5 75 f 5
425 f 56 608 f 57 113 f 28 197 f 14 108 f 45 120 f 33 67 f 11
wave position Ee,marlmV electrode*
AA
565 f 55 880 f 55 CPEti,,,,, 188 f 27 SME,iWU, 275 f 10 202 f 44 SMEiipid SME,,,,f 195 f 32 CPWE 142 f 9 CPE SME
S,/mV
202 f 31 C
108 f 2 82 f 14 114 f 11 103 f 22 77 f 7
" Means f SEM, n = 3. The AE,,,, values for all treated SMEs and CPEs were significantly less than for the unmodified CPE (P < 0.02, Student's two-tailed t test). *Electrodetypes: CPE, unmodified carbon-Nujol paste electrode; CPEti,,,,, carbon paste stearate-modielectrode after contact with brain tissue; SMEtiWU,, fied electrode after contact with brain tissue; SMEIlpid,stearatemodified electrode after contact with lipid; SMESwf, stearate-modified electrode after contact with surfactant; CPWE, carbon powder electrode. CAbsent. above the baseline), was used as a further comparison of the position of the waves due to dopamine and ascorbate oxidation. The background current, recorded in the absence of substrate, was subtracted from each voltammogram before analysis. The size of the background current recorded in phosphate buffered saline (PBS) was also used as a measure of the double layer capacitance for each electrode type (39);the charge passed was also determined in some cases by calculating the area between oxidation and reduction background currents. Chemicals. L-Ascorbic acid (BDH),dopamine hydrochloride, and PEA (Sigma Chemical Co.) and potassium ferrocyanide (Hopkinsand Williams) were used as supplied. Separate solutions of dopamine and ascorbate were made in PBS, pH 7.4: NaCl(0.15 mol/L), NaH2P04(0.04 mol/L), and NaOH (0.04 mol/L). Solutions of ferrocyanide were made in potassium chloride (1.00 mol/L) and hydrochloric acid (0.10 mol/L), diluted to pH 4. All solutions were purged with nitrogen before use.
RESULTS A N D DISCUSSION Oxidation of Ascorbic Acid a n d Dopamine. As expected from previous studies (13,29), incorporating the carboxylate anion of stearic acid into carbon paste electrodes increased the separation of the oxidation waves for ascorbate (AA) and dopamine (DA), quantified as E,,,(AA)-E,-(DA), from 425 f 56 mV for CPEs to 608 f 57 mV for SMEs at pH 7.4. Surprisingly, however, the modification increased E , , , for both the cationic dopamine and anionic ascorbate species (Table I). This decrease in charge transfer rate for the two substrates may be due to the approximately 12% greater hydrocarbon content of the SMEs due to the presence of the octadecanoic acid, since increasing the amount of hydrocarbon in CPEs progressively reduces the rate of electron transfer to paste electrodes of this carbon/oil ratio (37). The overall increase in the separation of the two waves is interpreted as the extra unfavorable electrostatic interaction between the ascorbate and stearate anions (13). After a 24-h period in brain tissue, the ability of SMEs to separate dopamine and ascorbate oxidation waves was reduced from 608 57 to 197 14 mV, a value significantly less than that for unmodified CPEs (Table I). The same treatment of unmodified CPEs reduced the separation from 425 f 56 to 113 i 28 mV. Thus, the effective resolution of ascorbic acid and dopamine is comparable at CPEs and SMEs after contact
*
e-0
CP'AE 0
1CC
2CC
300
400
503
6CC
i:C
e00
Sa0
mV
Figure 1. Ability of different types of carbon electrodes to separate ascorbic acid (open circles) and dopamine (filled circles) oxidation waves recorded in PBS (pH 7.4) at 50 mV/s. Mean values of E,,,, and abbrevations from Table I. with brain tissue and is insufficient to detect dopamine in the presence of large excesses of ascorbate. This conclusion is supported by the finding that the foot potential, Ef,measured using SMEsti,,,,, was 27 f 27 mV ( n = 3) for ascorbate at a concentration of 500 Kmol/L and 40 f 5 mV ( n = 3) for 100 /*mol/L dopamine. These data indicate that for a concentration ratio of ascorbate/dopamine as low as 51, the current due to ascorbate is greater than that for dopamine at 40 mV vs SCE in the absence of electrocatalytic effects (discussed below). There is a general consensus that the concentration ratio of the two compounds in rat striatum (the brain area where the majority of in vivo electrochemical recordings are made) is of the order of 1OOOO:l (2);under these conditions, the separation of dopamine and ascorbate in vivo using SMEs is clearly untenable. To investigate the possibility that lipids, which constitute about 40% dry weight of mammalian brain membranes (40), might be responsible for the loss of selectivity of the electrodes for dopamine, SMEs were treated with a suspension of PEA, a lipid abundant in brain tissue. The data in Table I show that these SMEslipidhave also lost the ability to separate ascorbate and dopamine oxidations effectively, a characteristic property of the untreated SME, and have very similar properties to the SMEtissue.The finding that a general-purpose surfactant, Triton-X 100, with a very different molecular structure to lipids also destroyed the selectivity of SMEs (Table I) suggests that a common surfactant mechanism may be responsible for the effects observed. We propose that when SMEs come into contact with surfactants, lipids, or brain tissue, the hydrophobic stearic acid is removed, together with pasting oil (31),from the exterior of the electrode to give a more powderlike surface. This interpretation of the data is supported by the measured electrochemical behavior of dopamine and ascorbate at carbon powder electrodes (Table I). The ability of the different electrodes to separate ascorbate and dopamine oxidations is illustrated in Figure 1. Oxidation of Ferrocyanide. The ferrocyanide couple was used as a probe of the electrode surface because of the relative simplicity of its one-electron transfer reaction compared with the complex oxidations of ascorbic acid and dopamine (41). In addition, the charge of -4 on the substrate makes it particularly sensitive to the presence of anions a t the electrode/solution interface. The oxidation of ferrocyanide at CPEs is irreversible at pH 7.4 due to ionized carboxylate moieties endogenous to the carbon material (42). At pH 4, however, enough of these anions are protonated to allow increaseed electron transfer a t the surface; the ferrocyanide system shifts to quasi-reversible behavior, showing both anodic and cathodic peaks with a separation greater than the characteristic value for a one-electron Nernstian system. The ferrocyanide wave recorded at 50 mV/s is a pH 4 solution using a CPE exhibited quasi-reversible characteristics (Figure
ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990 nA
50
2349
nC
50
40 -10 -20
p-..--J '
0
1OC
mV
200
300
400
500
BOG
700
30
20
10
0 100
200
300
400
500
600
700
0 0
10
mV 0
100
200
300
400
500
600
700
Figure 2. Examples of cyclic voltammograms for ferrocyanide recorded at 50 mVls and pH 4 using carbon paste electrodes: top, unmodified electrode (CPE); middle, stearate-modified CPE (SME); bottom, SME after treatment with lipid (SMEW). See Table I for electrochemical data for all electrode types.
2). The incorporation of stearic acid into the paste increased the overpotential for oxidation to the extent that hardly any faradaic current was observed up to 700 mV vs SCE. Clearly, the fraction of the surface stearic acid (pK, 4.9) unprotonated at this pH is sufficient to block electron transfer from the ferrocyanide anions to SMEs (Figure 2). After lipid treatment of the SME, however, the behavior of the ferrocyanide couple at this pH is similar to that at the unmodified CPE, again suggesting that the treatment abolishes the anion-repelling action of stearic acid at the electrode surface (Figure 2). A comparison of the ferrocyanide characteristics a t surfactant, lipid, and tissue-treated SMEs with those a t carbon powder electrodes supports this conclusion (Table I). The finding that the a,, value for the treated SMEs was significantly less than that for CPEs indicates that removal of pasting oil is an additional feature of the action of surfactants on these Nujol-paste electrodes.
-
Capacitance Measurements and Electron Microscopy. It is possible that simple wetting of the oil surface could account for the increased activity of paste electrodes following contact with surface-active agents. T o distinguish between fast wetting of the oil-water interface and the physical removal of lipophiles from the electrode surface, which one might expect to occur much more slowly, the time course for changes in the total background charge for SMEs was measured after the addition of 2 mL of 0.1% Triton-X solution to 18 mL of PBS (Figure 3). The finding that the half-life of the overall process was of the order of 5 h suggests that insulating material is displaced from the surface by the surfactant. However, at higher time resolution (inset Figure 3) the initial statistically
30
20
time
/
40
h
Figure 3. Time course for changes in the integral of charging cunent (nanocoulombs,nC) recorded with a SME at 100 mV/s between -100 and +700 mV over a 40-h period in the absence of any electroactive substrate. The arrow indicates the addition of 2 mL of 0.1 % Triton-X solution to 18 mL of PBS. Mean f SEM of three measurements. See Table I1 for capacitance data for all electrode types. Inset: Changes in charge passed recorded at higher time resolution over a 40-min period during which the surfactant was added. Table 11. Capacitance Values for Different Electrode Typesa
electrode
capacitance/nF
electrode
capacitance/nF
CPE SME CPWE
SMEti,, 138 f 19 (8) 9 f 2(4) SMElipid 159 f 16 (3) 7 k l(6) SME,d 223 f 23 (5) 1250 f 600 (6) Means f SEM, number of electrodes in parentheses. See Table I for abbreviations. significant jump in the charging current, observed by 2 min, may be due to a wetting effect. A comparison of capacitance values for the different electrode types (Table 11) indicates that each treatment increased this parameter approximately 20-fold over the value for the paste electrodes. This finding is consistent with the proposal that all treatments expose additional conducting surface. The still greater capacitance value for powder electrodes suggest that the surfactant-treated pastes are not as porous as powder itself; i.e., the hydrocarbons are removed from only the outer surface of the paste electrodes. Similar conclusions have been reached previously to account for the effect of brain tissue on the electrochemical characteristics of carbon/silicone paste electrodes (31) and of surfactants on the surface resistance of three commercially available carbon pastes in addition to pastes made from mineral oil, high-vacuum grease, and hexadecane (43). Further support for these conclusions comes from electron micrographs (Figure 4) which show that the carbon platelets in paste electrodes orient themselves parallel to the disk surface, forming a relatively compressed material. The to-
2350 * ANALYTICAL CHEMISTRY, VOL. 62, NO. 2 1 , NOVEMBER 1. 1990
1
...
^.~ -T*--q
1
Flgure 4. Scanning electron micrographs of the surface of a carbon paste electrode (top)and carbon powder electrode (bottom). magnification X600. The appearance of surfactant-treated paste electrodes was Similar to that of the untreated paste electrode.
pography of powder electrodes appeared quite different with the carbon particles showing no preferential alignment and the surface being less compact. The finding that the appearance of surfactant-treated electrodes was similar to that of the untreated paste electrodes correlates qualitatively with the difference in the capacitance values (Table 11) for CPEs (compact and oil coated), treated paste electrodes (compact with reduced hydrocarbon coverage), and carbon powder electrodes (porous and oil free). General Discussion. There are two possible explanations of the large currents reported for SMEs in vivo (13) where dopamine is present with a large excess of ascorbic acid (2): either the stearic acid is still functioning to eliminate direct ascorbic acid oxidation, but electrocatalytic reduction of electrooxidizeddopamine by ascorbate increases the dopamine current (29),or the surface stearate is rendered ineffective by the brain tissue and ascorbic acid contributes directly to the faradaic current. Since the extracellular concentration of brain ascorbate shows diurnal variations (44) and can be altered by physiological (45) and pharmacological (46-51) manipulations, either of these scenarios would imply that an unambiguous dopamine signal cannot be obtained by using SMEs in vivo. T h e results reported here indicate that the latter explanation of the large current values is more justifiable and specifically that lipids present in brain tissue remove the lipophilic elements (stearate and pasting oil) from the electrode surface. The finding that a simple surfactant and an isolated lipid destroy the selectivity of SMEs suggests that its destruction by brain tissue does not depend on brain structure or function. This conclusion regarding the unimportance of the state of
the tissue was also drawn from earlier studies on the effect of brain tissue on CPEs made with silicone oil (52),where an increased rate of ascorbate oxidation was observed with electrodes following either use in living brain or immersion in brain homogenate. It is likely that the introduction of the electrode into intact tissue disrupts the lipid bilayer membrane structure of cells it comes into contact with, freeing lipid molecules. The results obtained by using brain homogenates (52) and lipid suspensions also suggest that the altered response induced by contact with intact tissue is not due to a mechanical disruption of the electrode surface by the brain on removal of the electrode. Many more aspects of the electrochemical behavior of treated and untreated SMEs could have been investigated, for example, the electrocatalytic interaction of dopamine and ascorbic acid, which has already been shown to be significant a t untreated SMEs (29). Also, the limit of detection for substrates is an important characteristic of any analytical technique. However, if the aim is to develop a sensor for dopamine in vivo, discrimination against ascorbate is a prerequisite property. Since SMEs do not possess this characteristic following implantation in the brain, we feel that further studies of SMEs would be unproductive. The results highlight the need for caution when developing neurochemical sensors for use in vivo. T h e lipophilic environment of brain tissue interacts strongly with carbon paste electrodes, removing hydrophobic species that are not firmly hound to the surface. It is possible that covalently bonded modifications may prove useful in this area. We suggest, however, that it is unlikely that any manipulation of electrode characteristics which shifts ascorbate oxidation to higher potentials will be successful in detecting dopamine unambiguously, since electrocatalysis becomes a significant problem at that stage. Since the redox potential of ascorbic acid is more than 200 mV less than that of dopamine ( 4 1 ) ,devising modified electrodes to encourage ascorbate oxidation a t lower potentials, as has been achieved for carbon fiber electrodes (5.3, might be a better strategy. Depending on its size, such an electrode sampling a restricted compartment in vivo (7, 54,551 might deplete the pool of ascorbate before the dopamine signal appeared, making electrocatalytic contributions negligible. ACKNOWLEDGMENT We thank Mr. Barrv Creee .. _"for electron microscopy. Registry No. DA, 51-61-6; AA, 50-81-7; ferrocyanide, 1340863-4; Triton X, 9002-93-1. ~
LITERATURE C I T E D ( 1) Vohsmmaby in Uw Newmciences : Rhclpies , Memods and &plica
-
lions: Justice, J. 8.. Jr.. Ed.: Humam FTess: Canon. NJ. 1967. (2) Marsden. C. A,; Joseph. M H.: Kruk. 2.L.: Maldment. N. T.: ONeIll. R. 0.. Schenk. J. 0.: Stamford. J. A. Neurosc~nce1988. 25. 369-400. (3) Ganon. F. 0.; Navarre. F.; Buda. M. J. Anal. Chem. 1984. 56,
-. - -. -. 671.275
(4) Kelly. R. S.: Wightman. R. M. Brain Res. 1987. 423. 79-87. (5) Zenerslrom. T.: Sharp. T.; Marsden. C. A,: Ungerrtedt. U. J . Neuochem. 1983. 41. 1769-1773. (6) Church. W. H.: Justlce. J. E.. Jr. Anal. Chem. 1987. 59. 712-716. (7) Amatore. C.: Kelly. R. S.; Kristensen. E. W.: Kuhr. W. G.; Wightman. R. M. J . Elechoanal. Chem. Inferlacial Electrochem. 1986. 213. 31-42. (6) Slamlard. J. A,: Kwk. 2. L.: Miilar. J. 61. J . phsrmscol. 1988. 94. 924-932. (9) May. L. J.: Kuhr. W. G.: Wightman. R. M. J . Neuochem. 1988. 51. 1060-1069. (10) Rice. M. E.; me. A. F.: Bradbury. C. W.: AdBms. R. N. Brah Res. 1985. 340. 151-155. (11) Gratlon. A,: Hofler. E. J.: Oerhardl. G. A. Neuroscience 1989. 29. 57-64. Marcenac. F.: Oonon. F. Anal. Cham. 1985. 57, 1776-1779. Blaha. C. 0.; Lane. R. F. Brain Res. Bull. 1983. 10. 661-664. Blaha. C. D.: Lane. R. F. Ew. J . Phannacol. 1984, 98. 113-117. Howard-Butcher. S.; Blaha. C. 0.;Lane. R. F. J . pharmacal. Exp. mer. 1985. 233. 56-61. Lane. R. F.: Blaha. C. 0.:Phillips. A. 0. Brain Res. 1986. 397, 200-204.
Anal. Chem. 1880, 62, 2351-2357
(17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39)
Blaha, C. D.; Lane, R. F. Neurosci. Lett. 1987, 78, 199-204. Lane, R. F.; Blaha, C. D. Braln Res. Bull. 1987, 78, 135-138. Lane, R. F.; Blaha, C. D. Brain Res. 1987, 408, 317-320. Lane. R. F.; Blaha, C. D.; Hari, S. P. Brain Res. Bull. 1987, 79, 19-27. Broderick, P. A. Life Sci. 1985,36, 2269-2275. Broderlck, P. A. Neurosci. Lett. 1988,95, 275-280. Yamamoto. B. K.: Lane. R. F.: Freed. C. R. Life Sci. 1882. 30. 2 155-2 162. Broderick, P. A. NeuropeptMes 1987, 70, 369-386. Signs, S.A.; Yamomoto, B. K.; Schechter, M. D. Neuropharmacology 1887,26, 1653-1656. Yamamoto, B. K.: Spanos, L. J. Eur. J. Pharmacol. 1988, 748, 195-203. Glynn, G. E.; Yamamoto, B. K. Brain Res. 1989,487, 235-241. Phillips. A. G.; Pfaus, J. G.; Blahm, C. D.; Fibiger, H. C. J. Neurosci. Methods 1989,2 9 , 275. Gelbert, M. B.; Curran, D. J. Anal. Chem. 1988,58, 1028-1032. Crespi, F.; Martin, K. F.; Marsden, C. A. Neuroscience 1988, 27, 885-896. Ormonde, D. E.; O'Neill, R . D. J. Electroanal. Chem. InterfacialNectrochem. 1989,26 7 , 463-469. Lyne, P. D.; O'Neili, R. D. Anal. Chem. 1989,67, 2323-2324. O'Neill. R. D.; Fillenz, M.; Albery, W. J.; Goddard, N. J. Neuroscience 1983. 9 , 87-93. Saralathan, M.; Osteryoung R.; Osteryoung, J. J. Nectroanal. Chem. Interfacial Electrochem. 1986,214, 141-156. Penczek, M.; Stojek, 2 . J. €lectroanal. Chem. Interfacial Electrochem. 1987,227, 271-274. Nicholson, R. S . Anal. Chem. 1985,37, 1351-1355. Rice, M. E.; Galus, 2.; Adams, R. N. J. Nectroanal. Chem. Interfacial Nectrochem. 1883. 743, 89-102. Oldham, K. B. J. .€/echoanal. Chem. Interfacial Electrochem. 1985, 784, 257-267. Bard, A. J.: Fauikner, L. R. Nectrochemicai Methods; John Wiley & Sons: New York, 1980; p 13.
235 1
(40) Reinis, S.;Goldman, J. M. The Chemistry of Behavior, Plenum Press: New York, 1982 p 7. (41) Deakin, M. R.; Kovach, P. M.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1988,58, 1474-1480. (42) Deakin, M. R.; Stutts, K. J.; Wightman, R. M. J. Nectroanal. Chem. Interfacial Nectrochem. 1985, 182 113-122. (43) Albahadlly, F. N.; Mottola, H. A. Anal. Chem. 1987, 59, 958-962. (44) O'Neiil, R. D.; Fillenz, M. Neurosci. Lett. 1985,60,331-336. (45) Boutelle, M. G.: Svensson, L.; Fillenz, M. Neuroscience 1989, 30, 11-17. (46) Ciemens, J. A.; Phebus, L. A. Life Sci. 1984,35, 671-677. (47) Brose, N.; ONeill, R. D.; Boutelle, M. G.; Fillenz, M. NeuroDharmacologYl989, 28, 509-514. (48) Louilot, A,; Gonon, F.; Buda, M.; Simon, H.; Le Moal, M.; Pujol, J. F. Brain Res. 253-263. . lg85. ...., 336. .- ., . - -. . (49) Wilson,. R. L.; Kamata, K.; Bigelow, J. C.; Rebec, G. V.; Wightman, R. M. Brain Res. 1986,370, 393-396. (50) Gonzalez-Mora, J. L.; Sanchez-Bruno, J. A.; Mas, M. Neurosci. Lett. 1988,86,61-66 (51) Mueller, K.; Haskett, C. Pharmacol. Biochem. Behav. 1987, 27, 231-234. (52) O'Neill. R. D.; Grunewald, R. A.; Filienz, M.; Albery, W. J. Neuroscience 1982, 7 , 1945-1954. (53) Gonon, F.;Fombarlet, C. M.; Buda, M.; Pujol, J. F. Anal. Chem. 1981, 53, 1386-1389. (54) Aibery, W. J.; Goddard, N. J.; Beck, T. W.; Fillenz, M.; O'Neili, R. D. J. Nectroanal. Chem. Interfacial Nectrochem. 1984, 767, 221-233. (55) Cheng, H. Y. J. Elechoanal. Chem. Interfacial Electrochem. 1982, 735, 145-151.
RECEIVED for review January 2,1990. Accepted July 11,1990. We thank EOLAS for a grant to P.D.L. under the Basic Research Awards scheme.
Polynomial Filters for Data Sets with Outlying or Missing Observations: Application to Charge-Coupled-Device-Detected Raman Spectra Contaminated by Cosmic Rays G. R. Phillips and J. M. Harris* Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
Occasionally data sets contain points with excess error so that no information about the underlying signal is available from these observations. Such points should be considered lost and be given no influence on the results. Polynomial filters suitable for smoothing data sets containing outlying or missing observations are presented. Outliers are first identified by their deviation from the trends of the surrounding data, relative to a robust estimate of the standard deviation. I n the vicinity of an outlying point thus identified, the missing-point filter employs a polynomial flt to the points surrounding, but not including, the outlying or missing point. I n the absence of outliers, the procedure reverts to ordinary Savitzky-Goiay polynomial filtering. The equations for generating these filters and predicting their statistical properties are derived. Their performance is evaluated on chargetoupied-device-detected Raman spectra contaminated with cosmic ray events and is compared wlth nonlinear smoothing algorithms. The linear, missing-point polynomial filters are found to be efficient, both in speed and in ability to bridge gaps in data with minimal distortion.
Least-squares polynomial digital filters, introduced by Savitzky and Golay (I), are frequently used to remove high-
* To whom correspondence should b e addressed.
frequency noise and enhance the signal-to-noise ratio of spectroscopic or time-dependent data (2). Errors in the original smoothing coefficients published by Savitzky and Golay were corrected by Steiner (3),while Madden (4) published equations for calculating the convoluting coefficients. Polynomial filters are moving weighted averages computed by convoluting a set of regression-generatedfilter coefficients with a moving data window (5). These filters can be applied to any data set of equally spaced observations with locally constant variance (i.e. within the filter width). For data with normally distributed errors, least-squares polynomial filters are maximum likelihood estimators. The efficiency of least-squares estimators is severely degraded by the presence of deviant observations, as shown by the distortion of spectra containing outliers when smoothed by Savitzky-Golay filters. Outliers are a common problem in analytical techniques such as Raman spectroscopy that utilize ultrasensitive detection methods. The high sensitivity and low noise of charge-coupled device (CCD) detectors make them ideal for the weak signal levels produced in these experiments (6-9). The characteristics of CCD detectors have been recently reviewed (10-11). A significant problem with CCD detectors is their sensitivity to cosmic rays (6-12). Cosmic ray events produce enormous charge spikes ( 103-104 photoelectrons), which cause large distortions in the spectrum when ordinary linear smoothing procedures are used. Outliers in Raman spectroscopy may also arise from particles or bubbles in the
0003-2700/90/0362-2351$02.50/00 1990 American Chemical Society
