442
Anal. Chem. 1988, 60,442-446
Surface-Enhanced Raman Spectroscopy of the Catecholamine Neurotransmitters and Related Compounds Nam-Soo Lee,'You-Zung Hsieh, Richard F. Paisley, and Michael D. Morris* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109
The swface-enhanced Raman spectra (SERS) of dopamine, norepinephrlne, epinephrine, eplnlne, isoproterenol, 3methoxytyramlne, and oatechd In pH 7.2 buffers on a silver ektrod@are reported. Catechol and the ca-t are shown to be COordlMted to diver through both oxygens. The methoxylated derlvatlve Is a monodentate complex. Intenskies maximize near -0.9 V vs saturated calomel electrode. The strongest bands In the spwtra are phendic carbon-oxygen stretches and the vlOb modes around 1270 and 1480 cm-l, respectively. Ascorbate, acetylchdlne, glutathione, L-Dopa, and the catecholamine acetlc acid metabolites are SERShracHveunderthemeasuementcondltknr. Dopamkre detectlon lM for the vlOb band Is 3 X lo-' Y wlth a 10-s measurement tlme.
The facile oxidation of catecholamines and their metabolites on carbon electrodes has made anodic voltammetry a major measurement technology in neurochemistry (1-3). Both direct voltammetry of the catecholamines and voltammetric detection of molecules eluted from a lipid chromatographare widely employed. Voltammetric detection of chromatographic eluates (3)is a conventional finish to brain extract procedures and to perfusion isolation (2) of catecholamines from living animals or tissue slices. Perfusion, although widely employed, has well-known limitations. Replacement of the extracellular fluid by Ringer's solution or any other synthetic approximationmay itself perturb neuronal activity and cause systematic errors in neurotransmitter assay. Push-pull cannulae, dialysis perfusion apparatus, and cortical cups are all bulky devices. Their size limits spatial resolution and may cause serious tissue damage. The time constant of push-pull perfusion is about 10 s, but the time constant of dialysis perfusion is 1 min or longer. The extended time averaging makes relation of analytical results to physiological processes difficult. Anodic voltammetry at a carbon electrode can be performed on tissue slices or in vivo and circumvents some of these limitations. The measurement time constant is 10 s or less ( 4 ) . The results correlate well with those of independent chemical measurements (5). The probe is compact. Commonly employed electordes range in size from about 5 pm to about 50 pm in diameter (2). Recently, dopamine voltammetry with 1-2 pm diameter carbon fibers has been demonstrated (6). These fibers have been shown to penetrate the synapse of a large neuron in Aplyasia californiea successfully. Anodic voltammetry has interference problems. Of these, perhaps the most serious is ascorbate. Ascorbate is oxidized at a less anodic potential than dopamine on carbon-epoxy or carbon paste electrodes. The oxidation is retarded to more anodic potentials on graphite fibers, however. Alternatively, electrodes coated with Ndion (4)or impregnated with stearate can be used to measure dopamine in the presence of ascorbate. Present address: Department of Chemistry, Syracuse University, Syracuse, NY 13244. 0003-2700/88/0360-0442$01.50/0
Nafion also rejects anionic metabolites, further enhancing selectivity. Liquid chromatography eliminates the selectivity problems which plague direct voltammetry, of course, but with the loss of real-time response. Not counting sample preparation time, the chromatographicmeasurement time is typically 5-15 min, dependmg on the compounds sought and the column and flow rate employed. Interference problems might be absent or greatly reduced in Raman spectrometric measurements. Vibrational spectroscopy easily distinguishes among substituted benzenes, such as catecholaminesand their metabolites. Band shifts of 5-10 cm-' are expected for many ring modes and relative band intensities may be quite different. Structurally unrelated species, such as ascorbate, will have different vibrational spectra altogether. At the same time, measurement time could be reduced to about 10 s, if an array detector is used to monitor a 300-800 cm-l region. In some cases, where only one component is sought, a single-channel detector could be used to monitor one band only. Interference reduction and a shorter measurement time would be advantageous in most neurochemical studies. Application to extracts and perfusates would be desirable. If feasible, application to tissues slices or living animals would be even more welcome. Spontaneous Raman spectroscopy is inadequately sensitive for neurochemical applications. Measurements of catecholamines and their metabolites must be made at (1-100) X lo+ M concentrations. Adequate sensitivity can be obtained by resonance enhancement or by surface enhancement at silver or other coinage metals. The allowed electronic transitions of the catechol ring lie deep in the ultraviolet. Resonance enhancement using pulsed UV lasers is feasible but has apparently not yet been demonstrated. Resonance Raman spectra have been reported for metal catecholates (7,8) and for the metalloproteins enterobactin (7) and pyrocatechase (8) which contain catechol derivatives or oxidize catechols. The spectra show typical features of ortho-disubstituted benzenes and polysubstituted benzenes. The resonance Raman spectra of copper(I1) transferrin (9)and manganese(II)acid phosphatase (10)which contain phenolate linkages, have been reported. The Raman spectrum of the copper(I1) complex of poly(L-lysine-L-tyrosine) (11) is also known. From the Raman spectra, coordination to phenolate or one or both catecholate oxygens is easily identifiable. Surface-enhanced Raman spectroscopy (SERS) is potentially a useful tool for studies of biogenic amines. Although the SERS of the biogenic amines themselves has not yet been reported, SERS of many aromatic oxygen and nitrogen comM and lower. pounds is observed at For example, Nabiev and co-workers have reported good SERS from 1 X lo4 M phenylalanine, histidine, tryptophan, and tyrosine on colloidal silver (12). Kim and co-workers obtained excellent spectra of the aromatic amino acids and their glycl dipeptides at the 1 X M level on aged colloidal silver (13). Similar sensitivity has been reported by Picquart and co-workers (14). 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988 443
G b l e I. SERS Shifts (cm-') of Catecholamines and Related Compounds" bandsb DA' NOR' MTA' EPIC ISO' epinine catechol R1
R2
R3
R4
Compounds
R1
Y
Dopamlne Norepinephrine Epinephrine 3-Methoxylyrarnine Epinine
H
H
H
H
Homovanillic acid
H H
O H H OH H
H CHI
DOPAC
CH3 H
US ulDa ~19b
CH3 H
H H
H H
H CH3
lsoproterenoi
H
OH
H
CH(CH&
Flgure 1. Structures of catecholamines and thelr acetic acid metab-
olites. Koglin and SBquaris and their co-workers have extensively investigated the SERS of nucleic acid bases and their derivatives (15). Typically, excellent spectra are obtained from 1 X M solutions on silver electrodes or colloids. Recently Thierry and Leygraf (16) have reported the SERS of 5 X lo* M triazole and imidazole on copper substrates. None of these cases involves resonance enhancement, where SERS often produces excellent spectra at the lo4 to lo* M level (17) and detection limits of M (17,18).
us, Ysb
C
1150 1269 1272 1331 1329 1152
~ 1 5
Compounds
~
1424
1422
1154
1149
1273 1271 1271 1363
1269 1332
1258 1331
1441
1426
1479 1479 1572 1584
1486 1483
1482
1469
1507 1578 1601
OXe, = 514.5 nm, 30 mW, pH 7.2, E = -0.9 V vs SCE. *Assignments,from ref 7. Abbreviations: DA, dopamine; NOR, norepinephrine; MTA, 3-methoxytyramine;EPI, epinephrine; ISO, isoproterenol.
I
i
1
n
i
EXPERIMENTAL SECTION Dopamine, norepinephrine, epinephrine, 3-methoxytyramine, 3,4-dihydroxyphenylalanine(L-Dopa),3,4-dihydroxyphenylacetic acid, homovanillic acid, isoproterenol, deoxyepinephrine (epinine), acetylcholine, glutamic acid, and ascorbic acid were purchased from Sigma and used as received. The structural interrelationships among the various catecholamines and their metabolites are shown in Figure 1. The catecholamines were hydrochlorides, except epinephrine, which was used as the bitartrate or as the free base. All inorganics were ACS reagent grade materials. All solutions were prepared with type I water. All solutions were buffered to pH 7.2 with 0.1 M phosphate. Solutions contained 0.1 M poM ascorbate. tassium chloride and 4 x Electrode potentials were controlled in a conventional threeelectrode system, using a locally constructed potentiostat. The working electrode was a 2 mm diameter silver disk formed by epoxy cementing a silver (Alfa, 99.99%) wire into a J-shaped glass tube. The counter electrodewas a 7 mm length of 1mm diameter platinum wire cemented into a glass tube. The reference electrode was a saturated calomel electrode (SCE). The silver electrode was polished with 0.3-pm A1203,followed by 0.05-pm A1203. It was then electrochemically cleaned in a deaerated 0.1 M KCl solution at -0.7 V vs SCE for 20 minutes. After the cleaning step the electrode was roughened in a single oxidation-reduction cycle, which consisted of an anodic potential step from -0.2 to +0.2 V for 2 s, with return to -0.2 V. The electrode potential was shifted to the starting potential of the experiment and the electrode assembly was transferred to the cell containing the sample under study. Except as noted in the text, the electrodes was maintained at -0.9 V vs SCE during data acquisition. Complete spectra were obtained on a multichannel Raman spectrometer consisting of a Spex 1877 monochromator with an 1800 groove/mm grating in the spectrograph stage and a Tracor Northern 6122 intensified diode array detector. Except as noted, data acquisition was for 10-100 s, as needed. All spectra were normalized to the spectrum of a tungsten filament light bulb. Concentration dependence and detection limits were carried out on a single-channel instrument consisting of a Spex 1870 0.5-m monochromator, with a Hamamatsu R1527 photomultiplier, operated at -1 kV, with a 1-MQload resistor. Spectra were scanned at 3 cm-'/s. Both instruments were operated at about 5 cm-' resolution. Savitsky-Golay quadratic-cubic smoothing ( n = 9) was employed on the presentation spectra. Illumination and scattered light collection were through a fiber-optic probe (29)placed about 2 mm from the working electrode surface. Spectra were obtained with 30-mW Ar+ 514.5-nm light at the sample.
1 I
1000
I 1200
1 400
1 600
Wavenumber, cm"
Flgure 2. Surface-enhancedRaman spectra of 1 X M (A) dopamine, (B) norepinephrine,(C) catechol, and (D) 3-methoxytyramine: pH 7.2E , -0.9 V vs SCE; laser power, 30 mW; excitation wavelength, 514.5 nm; resolution, 5 cm-'; laser irradiation time, 100 s.
Peak height reproducibility measurements were made with 1 For each measurement a fresh aliquot of dopamine was used. Between measurements the apparatus was disassembled and the silver electrode was polished and conditioned by the method described above. After each measurement the acquired spectrum of dopamine was ratioed to a background spectrum of the electorde obtained without an applied potential, in order to correct for possible laser power fluctuations and differences in illumination and collection efficiency caused by changes in the placement of the electrode under the probe. The time dependence of the SERS response was obtained by using 4-min diode m a y data acquisition for 1 X IO-' M dopamine. Sequential measurements were made for periods of 1-2 h. X
lo4 M dopamine, using 100-9 diode array data acquisition.
RESULTS AND DISCUSSION The surface-enhanced Ramm spectrum in the aromatic ring stretching region of dopamine in pH 7.2 buffer is shown in Figure 2a and tabulated in Table I. The spectrum has intense bands at 1269,1331,1424, and 1479 cm-l and weaker bands a t 1152,1572, and 1584 cm-I. With our spectrograph, good signal/noise ratios are obtained from millimolar solutions at 10-5 integration times with delivered laser power of 30 mW. Norepinephrine (Figure 2b) gives a similar spectrum. Most bands are 0-3 cm-' displaced from the corresponding bands in dopamine. For comparison Figure 2 also includes the spectrum of catechol itself in the pH 7.2 buffer, and the spectrum of 3-methoxytyramine, in which one hydroxyl has been methoxylated. In Figure 3 we show the spectra of compounds containing methyl groups on the side chain. At the millimolar level only a weak epinephrine 1480-cm-' band is observed (Figure 3a). The spectrum of isoproterenol (Figure 3b) is also weak, but several bands are visible at millimolar concentration. The
444
=
._
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
1 I
A
A
1
1000
, I200
7-
1
1 400
/ _ _ I 1 SO0
Wavenumber, cm.'
Figure 3. Surface-enhanced Raman spectra of 1 X
M (A) epinephrine, (e) isoproterenol,and (C) epinine. Conditions are given in Figure 2.
bands of these compounds are included in Table 1. By contrast, the spectrum of epinine (Figure 3c) is about as intense as the spectrum of dopamine. We have been unable to obtain SERS spectra of 1 X M solutions of the acetic acid metabolites of the catecholamines, 3,4-dihydroxyphenylaceticacid, and homovanillic acid or the precursor L-Dopa a t any potential between -0.1 and -1.1 V vs SCE in neutral solution or in solutions as acidic as pH 3. We conclude that these molecules are not SERS-active on a silver electrode. Our experiments are carried out in 4 X M ascorbate. Control experiments with ascorbate blanks demonstrate that there are no ascorbate SERS bands observable under the conditions of our experiments. Similarly, control experiments show that under our experimental conditions, 1 X M glutathione and acetylcholine do not give observable SERS spectra. On a silver electrode at -0.9 to -1.0 V vs SCE, SERS spectra of catecholamines and their amine-containing metabolites are free of interference from ascorbate, glutathione, or acetylcholine. The band positions for catechol, the catecholamines, and 3-methoxytyramine are summarized in Table I. The catecholamine SERS bands are all readily assigned as catechol ring vibrations and carbon-oxygen stretches. As the figures and Table I show, the SERS spectra are quite similar to the spectrum of catechol itself, with bands displaced by no more than 10 cm-l. The spectra are also similar to resonance b a n spectra of metal catechoiates. We follow the band assignments of Salama and co-workers (7), Que and Heistand (81, and Gaber and co-workers (9) in Table I. The intense 1479-cm-l dopamine band and the corresponding norepinephrine band are easily assigned as v19b This mode occurs at 1469 cm-l in the SERS of catechol itself and in the 1480-1490 cm-* region in the resonance Raman spectrum of most metal catecholates. It is usually the most intense band in a catecholamine surface-enhanced Raman spectrum. The catechol carbon-oxygen stretch, about 1270 cm-', is almost as intense. For quantitative dopamine measurements we have used the 1479-cm-' band. The SERS spectrum of 3-methoxytyramine, Figure 2d, is quite different from the spectra of the catecholamines. The largest band is the C-0 stretch at 1273 cm-'. The q g b band is barely visible, but the u8, band at 1578 cm-' and the vsb band at 1601 cm-' are prominent. From our data we propose that the catecholamines are adsorbed on a silver electrode through metal-oxygen bonds. Methoxylation of one hydroxyl group (3-methoxytyramine) gives a spectrum with a very different intensity pattern, although the spectrum remains basically that of a 1,2,4-trisubstituted benzene. Therefore, the catecholamine/silver
Flgwo 4. Electrode potential dependence of the dopamine 1479-cm-'
band in SERS. The electrode current is also shown. complex is bidentate, as earlier proposed by Que and Heistand for metal catecholates (8). Both electromagnetic enhancement (20)and charge transfer enhancement (21) contribute to the intensity of the spectra. Charge transfer enhancement is indicated both by the perturbation from the normal Raman spectrum of a catecholamine and by the high sensitivity obtainable. As discussed below, high signal-to-noise ratio (SN) spectra for dopamine and norepinephrine are easily obtained at concentrations below M. That L-Dopa and the acetic acid metabolites of the catecholamines are not SERS-active is readily understood. The catecholamines are present as their ammonium ions at the pH of our study. Catechol itself is an uncharged molecule. In both cases, the intensity of the spectrum increases as the electrocapillary maximum of silver, about -0.9 V, is approached (22),demonstrating that catechol adsorption is weak on a positively charged silver surface. The acetic acid derivatives are anions at pH 7. They are repelled from the electrode at potentials sufficiently negative to allow catechol adsorption. The ethylamine side chain plays an important but incompletely understood role in the SERS of catecholamines. We have examined compounds with substituents on the a-carbon and on the nitrogen. Increasing substitution causes a dimunition in spectral intensity, but no sicgle substitution is sufficient to cause greatly attenuated spectra. The number of side-chain substituents appears to be as important as their location. Methylation of dopamine nitrogen to give epinine causes only slight attenuation of the spectrum. The effect is about the same as caused by hydroxylation of the dopamine a-carbon to give norepinephrine. However, methylation of the norepinephrine nitrogen to give epinephrine yields a drastically less intense spectrum. A similarly weak spectrum is obtained with isoproterenol, which has both substituents on the acarbon. The electronic structures of the benzene rings are not strongly affected by the side chain substituents of the compounds used in this study. Intramolecular hydrogen bonding between the ammonium ion and the aliphatic hydroxyl might be different among the different compounds. No consistent pattern has yet emerged. Similarly, model studies show that steric effects on the side chain should not affect adsorption through catechol oxygens on the other side of the benzene ring. The electrode potential dependence of the dopamine 1479-em-' band is shown as Figure 4. The intensities of the other bands are proportional to the 1479-cm-l band intensity at all potentials. The steady-state cathode current is also shown in the figure. The potential dependence of the catechol spectrum is essentially similar. Spectral intensity maximizes
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
apparatus was dismantled and the electrode was polished and conditioned with an oxidation-reduction cycle. In this sequence of experiments the peak height of the dopamine 1479-cm-' band varied over a range of 14%. The standard deviation was 4.7 % .
4
1
ow
12w
1600
IYH)
Wavenumber, cm"
Figure 5. Surface-enhanced Raman spectra of (A) 1 X M and (B) 2 X lo-' M dopamine. Conditions are given in Figure 2.
i
I
i -6.5
445
I -5.1
-4.5 lo.a(con..nh.llon.
-3.1
-2.5
U)
Flgure 8. Concentration dependence of dopamine 1479-cm-' SERS intensity.
near the potential of zero charge for silver -0.9 V vs SCE (22) and only declines at potentials negative enough to cause irreversible carbon reduction at the electrode (23))as well as hydrogen evolution. As is typical in SERS, once the electrode has been poised a t very negative potentials, SERS activity is lost. The electrode must be reconditioned before it can be used again. Figure 5 shows dopamine spectra at 2 X lo4 and 10 X lo4 M. The concentration dependence of dopamine SERS intensity is shown as Figure 6. The data are peak heights from scanned spectra in which the band is scanned over approximately a 10-sec period. The working curve has a log-log plot slope of 0.74, a t concentrations below M. At high concentrations the working curve approaches a limiting value, presumably corresponding to surface saturation. This kind of working curve is typically observed in SERS. With our system the detection limit (SIN = 2) is about 3 X M for a 10-5 scan. Similar detection limits require about 100-s integration with our spectrograph/array detector system. We have not carried out systematicstudies of the adsorption kinetics in the system. However, we have observed that signals in stirred sample cells grow to stable levels within 3-5 s, the approximate mixing time in our system. Thus, the time constant for catecholamine SERS is no more than a masstransport-limited time constant. We conducted a study of the long-term stability of the SERS signals at the silver electrode surface. The intensity of the dopamine 1479-cm-l band decreases with time. The relative intensity of dopamine bands remains the same, however. After 1 h, the band is about 60% of its starting intensity. The time decay is reproducible at about f 1 0 % . Construction of time calibration curves may be feasible. In reproducibility studies, we repeated SERS measurement of 1 X 10"' M dopamine seven times. Between each run, the
CONCLUSIONS Surface-enhanced Raman spectroscopy is a promising tool for neurochemical investigations. We have deliberately carried out experiments with the low laser power and short data acquisition times which might be considered realistic in a biomedical application. Of course, better detection limits could be obtained simply by increasing either laser power or data acquisition time. Even with our self-imposed limitations our current equipment and protocols give detection limits that are already low enough for many dopamine investigations. Substitution of more sensitive detectors, either photon counting or more sophisticated array detectors (24)should reduce detection limits M or below. Some improvement may also be to 1 X expected with further refinement of the optical system and electrode conditioning protocol. Very preliminary experiments in our laboratory have shown that catecholamine spectra can be obtained on copper electrodes. Copper may provde yet another route to decreased detection limits. We have not studied the effect of all possible competing absorbates. The intracellular fluid is itself a protein-poor medium. The major species are inorganic electrolytes, ascorbate, glucose, and the amino acid neurotransmitters. These are not interferences. The amino acids are not even strongly adsorbed at the negative potentials required to observed catecholamines SERS. Proteins might be expected to accompany brain extra& and are a potential problem. We have shown, however, that proteins do not perturb flavin SERS, unless the protein itself binds the flavin (25). There is no reason to expect very different behavior in the catecholamine system. The strengths of SERS are complementary to those of anodic voltammetry. SERS easily distinguishes between any catecholamine and its 3-methoxy metabolite. Dopamine and norepinephrine can be distinguished by peak height ratio measurements, although their major bands are at similar wavelengths. Significantly, SERS has no ascorbate interference problems. In fact, we always prepare catecholamine solutions with ascorbate to achieve the same antioxidation protection that is conferred in vivo. However, SERS does lack the generality of catecholamine voltammetry. The anionic metabolites of the catecholamines are not active, and epinephrine signals are weak. Our macroelectrode system can be used for measurements in brain extracts or perfusates with little modification. The long-term decay of electrode response is not a serious impediment. In any case, the method of standard additions can be used. Over the short time required for a standard addition sequence, the response decay would be less than 1%. SERS microscopy has already been reported (26,27). SERS signals are independent of the illuminated surface area, unless power densities are high enough to cause rapid surface atom rearrangement (27). In general, silver electrodes do have surface rearrangements (28). However, for our electrodes the SERS response decay over periods of 1-2 h appears to be reproducible enough to allow construction of timecalibration curves. Thus, the prospects are good for construction of a micro-SERS system suitable for brain-slice studies. However, with currently known conditioning techniques, silver electrodes are probably not stable enough to warrant use in vivo measurements. Our results suggest that SERS may also prove promising for study of the other biogenic amine neurotransmitters,
Anal. Chem. 1988, 60, 446-450
448
serotonin and histamine. Like the catecholamines, these molecules are cationic derivatives of the aromatic amino acids. Therefore, these neurotransmitters are candidate molecules for SERS with strong charge transfer and electromagnetic enhancement. As described earlier, the chemistry underlying the weak SERS signals from epinephrine and isoproterenol is incompletely understood. A definitive explanation awaits more complete investigation of the SERS of substituted phenols and o-cresols. At present, however, practical catecholamine SERS must be considered limited to dopamine, norepinephrine, and some of their metabolites. Experiments under way in our laboratory are aimed a t systematic development of SERS as a tool for the analysis of biogenic amines and at a more complete understanding of the unique strengths and weaknesses of this probe. These results will he communicated at a later date.
LITERATURE CITED (1) Adams, R. N.; Marsden, C. A. I n Handbook of Psycopharmacology: Iversen, L. L., Iversen, S. D., Snynder, S. H., Eds.; Plenum: New York, 1982; Vol. 15, pp 1-74. (2) Measurement of Neurotransmitter Release In Vivo; Marsden, C. A., Ed.; Wiley: Chichester. 1984. (3) Causon, R. C. I n Research Methods In Neurochemistry; Marks, N.. Rodnight, R., Eds.; Plenum: New York, 1985, Vol. 6, pp 211-241. (4) Gerhardt, A. F.; Oke, G.; Nagy, B.; Moghaddam, B.; Adams, R. N. Braln Res. 1984, 290, 390-395. (5) Rice, M. E.; Oke, A. F.; Bradberry, C. W.; Adams, R. N. Brain Res. 1985, 340, 151-155. (6) Meulemans, A.; Poulain, B.; Baux, G.; Tauc, L.; Henzel, D. Anal. Chem . 1988, 58,2088-209 1. (7) Salama, S.;Stong, J. D.; Neilands, J. B.; Spiro, T. G. Biochemistry 1978, 17, 3781-3785.
(8) Que, L.; Heistand, R. H. J. Am. Chem. SOC. 1979, 707, 2219-2221. (9) Qaber, Bruce P.; Mlskowski. V.; Spiro, T. G. J. Am. Chem. SOC. 1974, 96, 6868-6873. (10) Slguira, Y.; Kawabe, H.; Tanaka, H. J. Am. Chem. SOC. 1980, 702, 6582-8584. (11) Tosi, L.; Garnier, A. Inofg. Chlm. Acta 1978, 2 9 , L26LL263. (12) Nabiev, I.R.; Savchenko, V. A.; Efremov. E. S.J . Raman Smctrosc. 1883, 14, 375-379. (13) Kim, S.K.; Kim, M. S.;Suh, S. W. J. Raman Spechosc. 1987, 77, 171-175 . . . . . -. (14) Picquart, M.; Lacrampe, G.; Jaffrain, M. I n Spectroscopy ofB/o/og/ca/ h41~/eCuks; Allx, A. J. P., Bernard, L., Manfait, M., Eds.; Wiiey: Chicester, 1985; pp 190-192. (15) Koglln, E.; Sgquarls, J.-M. Top. Cum. Chem. 1886, 734, 1-57. (16) Thierry. D.; Leygraf, C. J. Electrochem. Soc. 1988, 733,2236-2239. (17) Hldebrandt, P.; Stockburger, M. J . fhys. Chem. 1984, 88, 5935-5944. (18) Sheng, R.-S.; Zhu, L.; Morris, M. D. Anal. Chem. 1986, 58, 1116-1119. (19) Schwab, S. D.; McCreery, R . . Anal. Chem. 1984, 56,2199-2204. (20) Moskovits, M. Rev. Mod. Phys. 1985, 57,783-826. (21) Furtak, T. E.; Roy, D. Swf. Sci. 1985, 158, 126-146. (22) Leikis, D. I . ; Rybalka, K. V.; Sevastyanov, E. S.;Frumkin, A. N. J. Electfoanal. Chem. 1973, 4 6 , 161-169. (23) Cooney, R. P.; Mahoney, M. R.; Howard, M. W. Chem. fhys. Len. 1980, 7 6 , 448-452. (24) Murray, C. A.; Dierker, S. B. J. Opt. SOC. Am. A 1986, 3 , 2151-2159. (25) Lee, N.-S.; Hsleh, Y.2.; Morris, M. D.; Schopfer, L. M. J. Am. Chem. SOC. 1987, 709, 1358-1363. (26) de Mul, F. F. M.; Otto, C.; Mud, J.; Greve, J. R o c . Int. Conf. Raman Spectrosc., 9th 1984, 294-295. (27) Van Dwne. R. P.: Halier, K. L.; Altkorn, R. I.Chem. Fhys. Lett. 1986, 126, 190-196. (28) Wetzei, H.; Gerishcer, H.; Pettinger, B. Chem. fhys. Lett. 1981, 78, 392-397.
RECEIVED for review June 1,1987. Accepted October 26,1987. This work was sumorted bv National Science Foundation Grant CHE-8317861.
Luminol Chemiluminescent Determination of Glucose or Glucose Oxidase Activity Using an Inverted Micellar System Shukuro Igarashi' and Willie L. Hinze* Department of Chemistry, Laboratory for Analytical Micellar Chemistry, W a k e Forest University, P.O.Box 7486, Winston-Salem, North Carolina 27109
A mlcellar improved quantitatlve enzymatic assay for anatysls of glucose or the enzyme glucose oxidase Is described. The assay is based on the reactlon of glucose with glucose oxidase to generate hydrogen peroxide. The peroxide thus generated reacts wtth the chemlkmlnescent reagent, lumlnd, present to produce llght whose intensity is proportional to the glucose concentratlon or glucose oxidase activity. The presence of an inverted (or reversed) mlceiiar medlum of hexadecyltrlmethylamhm chloride (CTAC) allows one to conduct both the enzymatk and chemiluminescence detection reactlons sdmulaneous/y at mild pH (7.8) In the absence of any added catalyst or cooxldant. The advantages and limitations of employlng Inverted micellar media In such coupled enzymatic-chemiluminescent detection schemes are discussed.
with the oxidase. The peroxide thus formed is typically quantitated via use of appropriate chromogenic or chemiluminescent (CL) reagents (1-7). Of the different CL reagents available, use of peroxyoxalates (such as bis(2,4,6-trichlorophenyl) oxalate) (2) or luminol (5-amino-2,3-dihydro-1,4phthalazinedione)appears to be the most popular (2-8). With the use of luminol in such an approach, substrates such as amino acids, uric and lactic acid, hypoxanthine, cholesterol, and enzymes like uricase, xanthine oxidase, and L-amino acid oxidase have been assayed (2, 4, 5 , 8 ) . In addition, several reports demonstrate that glucose (or glucose oxidase) can also be determined in this manner (eq 1 and 2) (2, 7-13). The intensity of the CL light emission is related to the substrate (or enzyme) concentration. P-D-glucose + glucose oxidase D-gluconic acid
It is well established that oxidases or their corresponding substrates may be determined by measuring the amount of hydrogen peroxide formed during the reaction of the substrate Present address: D e p a r t m e n t of A p p l i e d Chemistry, Tohoku University, Aoba, Aramaki, Sendai, Japan 980. 0003-2700/88/0360-0446$01.50/0
hydrogen peroxide
+ luminol
+ mild pH 0 2'
hydrogen peroxide (1)
pH 10-11
3-aminophthalate + h v (2) In order to obtain analytically useful light emission from the CL detection reaction (eq 2), the presence of catalyst (such as Cu2+,Fe(CH)63-,or Cu(phen),2+)and cooxidant and fairly 0 1988 American Chemical Society
