Micro carbon electrode for intracellular voltammetry - American

melting point as compound A. A mixture melting point was identical with that of the individual materials. A literature search revéaled that compound ...
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Anal. Chem. 1986, 58.2088-2091

elemental analysis and the mas8 spectrum with an apparent molecular ion at nupi 170 suggesteda compound with the formula C6H1&02 (C, 42.39; H, 5.93;N, 33.H). This combined with the IR data suggested structure I for the compound: 3a,6a-dimethyltetrahydroimidazo[4,5-d]imidazole-2,5-dione. Structure

imately 2-3, the hydrolysis of urea will rapidly increase the pH to 4.5 and Ni-DMG will be precipitated quantitatively without being contaminated with compound A (even if a small amount of A is produced, it is soluble enough to stay in solution). If the initial solution is more acidic than pH 2-3, then both compound A and Ni-DMG will be formed; if the solution is very acidic, only compound A will be formed. Each of these results has been observed. CONCLUSION We attribute this problem to an inadequate control of the initial pH of the Ni2+solutions prior to addition of urea and DMG. We suggest that when the homogeneous precipitation method in the determination of Ni is used,the pH be carefully adjusted to lie between 2 and 3. Obviously the problem does not arise if urea is omitted from the analysis. Registry No. I, 28115-25-5;Ni, 7440-02-0;dimethylglyoxime, 95-45-4;urea, 57-13-6;butane-2,3-dione, 431-03-8.

I suggested ari alternative synthetical route using butane-2,3-dione and urea in an aqueous acidic solution. This reaction was extremely facile and produced a crystalline proddct that had ideritical mass spectra, IR spectra, C, H and N analysis and melting point as compound A. A mixture melting point was identical with that of the indivitld kterials. A literature search revealed that compound A was a known compound, with it and ita near derivatives having been breRafed from the corresponding diketones and substituted ureas (4-9). We conclude that our compound A has structure I. This was further confirmed by comparing the proton N M R of compourid A and the synthesized material run in deuterated trifluoroacetic acid using a JEOL FXSOQ NMR spectrometer. DISCUSSION The acidic solution chemistry of DMG in the presence of urea is obviously more complex than the hydrolysis of urea to ammoriia and formation of Ni-DMG. We suggest that the formation of compound A occurs as a result of the slow hydrolysis of DMG to butane-2,3-dione and its subsequent reaction with urea. If the initial pH of the solution is approx-

LITERATURE CITED (1) Tschugaeff, L. BBr Dtsch. Chem. as.1905, 3 8 , 2520. (2) Tschugaeff, L. 2.Anorg. Chem. 1905. 4 6 , 144. (3) Brunck, 0.Z . Angew. Chem. 1907, 20,824. (4) Butler, A. R.; Hassain, I J. Chem. Soc., Perkin Trans 2 1981, , 310-16. (5) Biltz, H. Ber. Dtsch. Cbem. Ges. 1908, 41, 167-173. (6) Seekles, L. Red. Trav. Chim. Pays-Bas 1927, 48, 77-84. (7) Imidazole and Its Derlvatives ; Hofmann, K., Ed.; Interscience: New York, 1953;pp 227-232. (8) Kuhling, D. Justus Liebigs Ann. Chem. 1973, 263-277. (9) Suvorova, L. 1.; et al. Izv Akad. Nauk SSSR, Ser. Khim. 1979, 6 , 1306-13 13.

J e n s Hemmingsen David Larkin* Thomas Martin Department of Chemistry Towson State University Towson, Maryland 21204

RECEIVED for review January 13, 1986. Accepted April 14, 1986.

Micro Carbon Electrode for Intracellular Voltammetry Sir: Since the earlier studies of Adams’ group (1,2)electrochemistry has proved to be a powerful method for the determination of amine neurotransmitters and ascorbic acid in vivo ( 3 , 4 ) . The most frequently used microelectrodes are constructed from carbon paste or a single carbon fiber enclosed in a glass micropipet. The carbon fiber either protrbdes forming a cylindrical electrode of 250-500 pm length and 8 pm diameter or is cut on the end of the glass coat so that the active surface is a single disk of 20-500 pm diameter (5-8). The size and form of these dlectrodes make them inadequate for intracellular measurements. Yet there is no doubt that much could be learned if measurements could be performed inside individual cells. In this paper we report a technical method to prepare a needle-tip micro carbon electrode with a tip diiuneter ranging f r o b 0.5 to 2 pm which works in oxidation as well as in reduction. This microelectrode could be implantd for several hours into identified neurons of Aplysia witbout damaging them or affecting their electrophysiological properties. We tested this new microelectrode by studying, by differential p& voltammetry (DPV), the penetration into a neuron of two electroactive drugs (antipyrine and metronidazole, respectively, in oxidation and reduction) and by measuring endogenous intracellular ascorbic acid. 0003-2700/86/0358-2088$01 S O / O

EXPERIMENTAL SECTION Working and Reference Microelectrodes. The working microelectrodes were prepared with carbon fibers (external diameter 5-12 pm, Le Carbone Lorraine) sealed to a copper wire with graphite powder in polyester resin (Figure 1A). The sharpening and insulation of the carbon fiber bear some similarities to the preparation of microplatinum electrodes used in oxygen determination (9, IO). The carbon fiber was electropolished (Figure 1A) by dipping the extreme tip onto the surface of an half-saturated solution of sodium nitrite containing detergent, TWEEN 40 (Sigma). A similar technique as been described earlier (11). An alternating current (50 Hz, 2-6 V) was passed through the solution between the carbon fiber and a platinum macroelectrode. Under these conditions, the carboli dissolved away slowly. At the end of this treatment, the carbon fibei. had a very sharp tip in the range of 0.4-1.5 p m diameter. A soda glass tube, 1 mm o.d., was heated locally and pulled down to a diameter slightly larger t h d that of the carbon fiber. A hook was made on the end of the pipet. The carbon fiber was pushed down inside the tube uhtil the end of the copper wire was blocked against the taper on the inside of the capillary tube (Figure 1B). The copper wire was fixed to the glass with adhesive and the glass tube was placed in a de Fonbrune microforge, point downward, and a small weight (about 10 mg) was hung on the 0 1986 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 58, NO. 9, AUGUST 1986

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Figure 1. Preparation of the working microelectrode: (A) electro-

palishing; (B) carbon fiber inside a glass capillary tube; (C) covering the carbon taper with a very thin glass coat; (D) complete carbon microelectrode.

hook (Figure 1C). The heated platinum loop of the microforge was brought toward the capillary until the glass around the carbon fiber began to melt (Figure 1C). The glass was pulled. With more heat the glass was broken on the extreme tip of the electropolished carbon fiber. Near the end of the carbon fiber, the glass was so thin that the addition to the diameter of the tip was negligible. Thus this needle-tip microelectrodehad an external diameter of 0.5-2 pm at the tip which gradually increased to 8-12 pm at a distance of 50-250 pm from the tip (Figure 1D). The reference microelectrode, a conventional unpolarizable electrophysiological microelectrode, was a pipet pulled on a de Fonbrune microforge and filled up with 3 M KCl into which was dipped a chlorinized silver wire (Ag/AgCl microelectrode). This glass microelectrode had an external diameter at the tip in the range of 0.5-1.5 pm and its resistance was about 1 MQ. Cellular Preparation, Chemicals, and Electrolytes. Experiments were performed on identified neurons (150-300 pm diameter) of dissected buccal ganglions of the well-known marine mollusc Aplysia californica (12-14) obtained from Pacific Biomarine, Venice, CA. The neurons had a resting potential near -50 mV and maintained normal synaptic and firing activities for 10-12 h. The experiments were performed at 25 "C. The biological preparation pinned in a 1-mL experimental chamber was continuously superfused at 1 mL-min-' with artificial seawater (ASW: NaC1,460 mM; KCl, 10 mM; CaCl,, 11 mM; MgC12,25 mM; MgS04, 28 mM; Tris-HC1, 10 mM, pH 7.8). All solutions were prepared with pyrodistilled water and ASW was prepared from reagent grade chemicals without further purification. Metronidazole (Flagyl) was a gift of Rhane Poulenc; ascorbic acid and antipyrine were obtained from Merck. Ascorbic acid (AA) (100 mgmL-l in ASW) was injected intracellularly by the air pressure method described earlier (12). Voltammetry and Electrophysiology. The voltammetric measurements were made with a commercial apparatus (PRG5 Tacussel,Lyon, France) used in the differential pulse mode (DPV). In our studies, the DPV parameters were a sweep rate of 40 mV/s and a pulse modulation 100 mV in amplitude, 88 ms in duration, and 100 ms in period. Current measurements were made for an 8-ms period immediately before a pulse and just before the end of the pulse. The initial potential was 0 V vs. Ag/AgCl. Because measurements were stopped just before the front of oxidation or reduction, final potential was f1.5 to 2 V vs. Ag/AgCl. The reference microelectrode and the working carbon microelectrode were implanted with micromanipulators (Prior) in the same neuron under microscope observation and electrophysiological control. The auxiliary microelectrode,a platinum wire (external diameter 2 mm), was bathed in the experimental chamber (Figure 2). Neuronal activity was intracellularlyrecorded by the reference and the working microelectrodesthrough cathode followers (Figure 2) with respect to an unpolarizable electrode dipped in the bath. RESULTS AND DISCU@SION Testing of Microelectrodes f o r Intracellular Use. Routine trials enabled the electrical quality of insulation of the micro carbon electrode to be evqluated, and the electrodes

Figure 2. Schematic drawing of the experimental apparatus: (1-2) potentiostat;(3)current amplifier; (4-5) cathode followers; (R) toward recorders; (r) 3 M KCI Ag/AgCI reference intracellular mlcroelectrode; (w) working intracellular carbon microelectrode; (a)auxillary platinum extracellular macroelectrode; (g)unpohrizable electrode connected to ground for electrophysiological recordings. The drawn configuration was that used for Intracellular voltammetry. By reversing the four switches (s)the apparatus was adapted to record bioelectrical actMtles of the tested neuron.

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Flgure 3. Recordlngs of spontaneous action potentials. Action POtentials of a neuron were recorded intracellularly through a classical 3 M KCI Ag/AgCi microelectrode used as reference microelectrode and thrqugh the carbon working microelectrode. had to conform to the following criteria: (1)Taking into account the resistance of the working microelectrode (0.1-0.5 MQ) the current in DPV mode a t 0 potential vs. Ag/AgCl had'to be below 500 PA; higher values indicated bad insulation usually due to cracks in the gl-s. (2) Microelectrodes, under microscope observations, submitted to a negative potential (3 V) had to have oxygen bubbling only at the extreme tip. (3) During implantatjon of $he microelectrode in a neuron, a badly insulated microelectrode, i.?., presenting a second electroactive surface shunting the intracellular signal recorded at the extreme tip or having an excessive resistance (tip cove!ed by glass), recorded neuronal activity with a markedly reduced amplitude comRared to recordings of the 3 M KCl reference electrode. (4) The time constant of the carbon microelectrodes had to be less than 0.5 ms in order to record action poteptials nearly as easily as the reference KCl microelectrode (Figure 3). ( 5 ) When the electrode was implanted into the cell, the electroactive range hqd to be as large as if it was bathed in ASW. Electrochemical Characteristics. Intracellularly the potential range in oxidation and reduction was as large as in ASW (k1.5 V vs. Ag/AgCl on 5 nA full scale) and permitted the study of either reducible or oxidizable drugs. Peaks in DPV experiments were well-defined (width 350-400 mV and half width peak = 150-160 mV). The accuracy of concentration measurements was from 5 to 10% depending on the microelectrode. In the described conditions, the limit of sensitivity was equa1 to 10 Mg/mL (5 X M)for the two compounds, metronidazole and antipyrine. The microelectrode cogd work in a neuron for 6-8 h. m e signal then began

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ANALYTICAL CHEMISTRY, VOL. 58, NO. 9, AUGUST 1986

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Figure 5. Penetration (continuous lines) and clearance (dotted lines) of metronidazole (0)and antipyrine (W).

peak at +450 mV. When the cytoplasm of the studied neuron was enriched by air pressure injected AA, the +450 mV peak was greatly increased. The +450 mV endogenous peak was found in all tested neurons of the buccal ganglion as was then probably due to endogenous AA. The endogenous AA concentration gave rise to a voltammogram equivalent to a M AA concentration in ASW. The AA peak was located at a different potential (+450 mV) when compared with a value obtained with carbon electrodes used by other authors (+70 mV) (3,5). This is due to our electropolishing treatment of the tip of the microelectrode. Electrolytic or chemical treatments of carbon fiber or carbon paste are indeed commonly known to move the AA peak (3,6, 7). Drug Penetration. We studied the penetration and the disappearance of metronidazole and antipyrine by DPV on the same neuron (Figure 5). None of these drugs is an endogenous compound. The preparation was perfused by ASW to which, at time 0, antipyrine and metronidazole each at a concentration of 1000 pg/mL (5 X M) were added. The outer concentration of the drugs maintained constant. The potential of the microelectrode was set at 0 V vs. Ag/AgCl and sweeps in oxidation and in reduction were performed alternately. The results in Figure 5 show that the kinetics of the penetration and of the clearance of the two drugs were similsr. These results were reproducible from one neuron to another. The maximum intracellular concentration was reached in 30-40 min. When the drugs were removed from the perfusing bath, the intracellular concentration of the drugs progressively decreased and their total elimination from the cell took place in 30-40 min. Because these two drugs are weakly ionizable species and have a good liposolubility, their ability to cross the membrane may be interpreted in terms of passive diffusion as in brain tissue where diffusion occurs through membranes of capillaries (21, 22). In conclusion, to our knowledge the microelectrode described here is the first successful attempt to insulate the polished tip of a carbon fiber. This new sensor can be used to detect electroactive drugs in oxidation as well as in reduction in an aliquot of 1 nL with a limit of detection of 5 X M using a conventional apparatus (PRG-5). Because of its shape (diameter of 0.5-2 pm at the tip), this new microelectrode can be introduced without damage into individual cells and thus enable the detection of intracellular electroactive compounds.

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0 Figure 4. Voltammograms in oxidation (A) and in reduction (B). For A and B, lower traces were voltammograms performed with the three electrodes in ASW. Upper traces: peak 1 (A), at +450 mV, was due to endogenous ascorbic acid; peak 2 (A), at 1200 mV, was antipyrhe (intracellular concentration of 675 pg/mL 25 mln after the beginning of superfusion with the two drugs, each of them 1000 w/mL in ASW); and the peak 3 (B), at -850 mV, was metronidazole (intracellular concentration of 877 pg/mL 21 min after the beginning of the superfusion).

to deteriorate as reported for other sensors in extracellular fluids of tissues in animal experiments (15, 16). Standardization. Metronidazole is a widely used antibiotic. Antipyrine is an antipyretic drug. Both were found to be electroactive compounds (I7, 18). Metronidazole was reduced at -0.85 V vs. Ag/AgCl in ASW on our electrode; antipyrine was oxidized at +1.2 V vs. Ag/AgCl in ASW on this same electrode. Experiments in neurons and calibrations were performed at 25 OC in the same experimental chamber in ASW. The calibration curves for the two products based on the peak amplitude, after subtraction of the residual current, were linear in the range studied (10-lo00 M/mL). Voltammogramswere similar in ASW and in the neuron (Figure 4). Taking into account the composition of the intracellular fluid, we considered that diffusion coefficients of drugs were similar in ASW and in the neuron and that the external calibration method could be used. Indeed, it has been shown (19) that diffusion coefficients of y-aminobutyric acid and of acetylcholine, two compounds with molecular weight in the same range of magnitude as those of metronidazole and antipyrine, were similar in neuronal axoplasm and in ASW. Measure of Endogenous Ascorbic Acid (AA). Identified cholinergic neurons in the buccal ganglion known for the absence of significant amounts of monoamines in their cytoplasm (20) were used. In the absence of any drug in the bath, characteristics of the voltammogram in the neuron and in ASW were the same, except in oxidation where a peak at +450 mV was found (Figure 4). AA diluted in ASW gave a similar

ACKNOWLEDGMENT We thank Chloe Brown for helpful comments. Registry No. HzO, 7732-18-5;antipyrine, 60-80-0;metronidazole, 443-48-1; ascorbic acid, 50-81-7. LITERATURE CITED (1) Adams. A. N. Anal. Chem. 1976. 4 8 , 1126A-1137A. (2) Wightman, R. M.; Strope, E.: plotsky, P. M.; Adams, R. N. Netwe (London)1976, 262, 145-146.

Anal. Chem. I988, 58. 2091-2092 (3) Hutson, P. H.; Curzon, 0. Blochem. J . 1983. 211, 1-12. (4) Stamford, J. A. h l n Res. Rev. 1985, IO, 119-135. (5) Ponchon, J. C.; Cespuglb, R.; Gonon, F.; Jouvet, M.; Pujol, J. F. Anal. Chem. 1979, 51, 1483-1486. (6) Kovach, P. M.; Ewlng, A. Q.; Wllson, R. L.; Wightman, R. M. J . NeurOSCi. Methods 1984, 10. 215-227. (7) Gonon, F.; Fombarlet. C. M.; Bude, M. J.; Pujol, J. F. Anal. Chem. 1981, 53, 1386-1389. (8) Dayton, M. A.; Brown, J. C.; Stutts. K. J.; Wlghtman R. M. Anal. Chem. IgSO. 52, 946-950. (9) Slhrer, I . A. Med. Electron. Biol. Eng. 1965, 3 , 377-387. (IO) Meulemans, A.; Henzel, D.; Tran-Ba-Huy, P.; Silver, I . A. Innovations Tech. Blol. Med. 1985, 6, 353-362. (11) Armstrongdames, M.; Fox, K.; Mlllar, J. J . Neuroscl. Methods 1980, 2 , 431-434. (12) Tauc, L.; Hoffmann, A.; Tsuji, S.; Hlnzen, D. H.; Faille, L. Nature (London)l974, 250, 498-498. (13) Baux, 0.; Tauc, L. R o c . Natl. Aced. Scl. U . S . A . 1983, 8 0 , 5126-5 128. (14) Poulaln, B.; Baux, G.; Tauc, L. Proc. Natl. Aced. Sci. U . S . A . 1988, 83. 170-173. (15) Marsden, C. A. Measurement of Neurotransmiifer Re/ease In Vlvo; Wiley: 1984; Chapter 6. (16) Kreuzer, F.; Klmmlch, H. P.; Brezlna, M. Med/cal and Blobgicel Applications of Electrochembal Devlces; Wiiey: 1980 Chapter 6. (17) . . Knox. R. J.: KnMt. R. C.: Edwards, D. I. 6iochem. Pharmacal. 1983. 32, 2149-2156 (18) Yamamoto, B. K.; Lane, R. F.; Freed. C. R. Life Scl. 1982, 30, 2155-2162. (19) Kolke, H.; Negata, Y . J . Physlol. (London) 1979, 205, 397-417.

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(20) Ono, J. K.; McCaman, R. E. Nevosclenoe 1984, 1 1 , 549-560. (21) Ekbf, B.; Lessen, N. A.; N ~ w , L.; Norberg, K.; SiesJo, B. K.; Torlof, P. Acta physbl. Sand. 1974, 01, 1-10. (22) Levln, V. A. J . Med. Chem. 1980, 23, 682-884.

Alain Meulemans Laboratoire de Biophysique Faculte de Medecine Xavier Bichat 16 rue H. Huchard, Paris 75018, France Bernard Poulain Gerard Baux Ladislav Tauc* Laboratoire de Neurobiologie Cellulaire et Moleculaire CNRS Gif-Sur-Yvette 91190, France Daniel Henzel

U 13 INSERM Hopital Claude Bernard Paris 75019, France RECEIVED for review December 11,1985. Accepted April 15, 1986. This work was supported by AIP No. 06931 to L.T.

Comments on Improvement of the Limit of Detection in Chromatography by an Integration Method Sir: Recently, Synovec and Yeung described a method of improving the limit of detection in chromatography by numeric integration of the chromatogram (1). They showed both by theoretical means and by computer simulation that the signal-to-noise ratio is improved by integration of a chrqmatogram when the base line noise has certain characteristics. The amplitudes of both signal and noise are increased by integration, but the signal increases more than the noise. Noise Sources. The authors model the chromatographic base line, F ( t ) ,by F ( t ) = mt

+ b + D ( t ) + R(t) + Nt

(1)

where mt is a linear base line drift, b is a base line offset, D(t) and R(t)are base line fluctuations,and Nt is random detector noise. The noise, N,, at each data point is sampled from a Gawian and each sample is independent of all others. This sampling procedure is equivalent to assuming that all time constants in the system are much shorter than the sampling interval. In the frequency domain, this type of noise is uniformly distributed throughout the spectrum and is commonly known as white noise. The authors refer to it as “uncorrelated”noise because there is no correlation from point to point in the base line. In contrast, the chromatographic signal is “correlated” because a peak spreads over a large number of data points. Most of the power of white noise is at frequencies substantially higher than the frequencies of chromatographic peaks. And, as the duration of the peak increases, an ever larger portion of the white noise spectrum falls beyond the frequency range of the chromatographic signal. The base line fluctuation terms, D ( t ) and R(t),are in the same frequency range as are chromatographic peaks. These fluctuations are of the same duration as typical chromatographic peaks and so could be mistaken for peaks. Base line offset and linear drift are examples of noise at extremely low frequencies, well below those of chromatographic peaks. Offset and drift in a chromatogram may be 0003-2700/86/0358-2091$01.50/0

inconvenient, but they cannot be mistaken for peaks. Signal-to-Noise Ratio Improvement. All methods to improve the signal-to-noiseratio involve some means of distinguishing between signal and noise. In this case, the distinguishing feature is frequency. Most of the signal in a chromatogram is contained within a limited frequency range. Power outside this range is mostly noise. Accentuating signal containing frequencies while discriminating against noise containing frequencies gives an improved signal-to-noise ratio. Integration is but one of many methods of altering the power of one frequency range relative to another. It discriminates against the higher frequencies and so eliminates much of the white noise from a chromatogram. The base line offset and drift corrections applied before integration discriminate against the extremely low frequencies. Mid-range frequencies,such as those composing chromatographic peaks, pass through this integration fiiter. The method is equivalent to a Fourier transform smoothing procedure (2). Except for a normalization factor, differentiation is equivalent to multiplication by io in the frequency domain (3).Thus, an integral, I @ ) ,may be computed from a chromatogram, C ( t ) ,by I(t) = FT1[FTIC(t)]/io] where F T and FT-’ are the direct and inverse Fourier transforms, respectively. Division by the independent frequency variable, o,in eq 2 accentuates the low frequency content of the signal relative to the high frequencies. Signal-to-noise ratio and the limit of detection are improved in the integral because the signal is less affected by this division than is the noise. The Fourier transform method of computing an integral attenuates power at low frequencies less than it does power a t high frequencies. The running total method increases power a t low frequencies more than it does power at high frequencies. The difference between these two methods is just a normalization factor and both change the 0 1986 American Chemical Soclety