Langmuir 1990, 6, 66-73
66
Electrochemical and SERS Studies of Chemically Modified Electrodes: Nile Blue A, a Mediator for NADH Oxidationt Fan Ni,’ Helena Feng,$ Lo Gorton,$ and Therese M. Cotton*$’ Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, and Department of Analytical Chemistry, University of Lund, Lund, Sweden Received June 27, 1989 The electrochemical and spectroscopic behavior of the phenoxazine-type mediator Nile Blue A adsorbed on glassy carbon (GC) and Ag electrodes has been examined. The pH dependence of the formal redox potential and the pH dependence of the catalytic oxidation of NADH were found to be similar, although not exactly the same, on both types of electrodes. Those differences that were observed may be attributed at least partially to multilayer formation on GC, whereas only one monolayer is present on Ag (e.g., the catalytic current for NADH oxidation was much greater for GC than for Ag). For both electrodes, the catalytic peak was shifted several hundred millivolts more positive than the E”’ value of adsorbed Nile Blue A. This shift is attributed to the formation of a Nile Blue A/NADH complex. The RR and SERRS spectra of the mediator on both GC and Ag electrodes were very similar. Changes in the spectra as a function of pH are interpreted to reflect changes in Nile Blue A orientation at both electrode surfaces. Two distinct species were detected in the SERRS spectrum of Nile Blue A in the presence of NADH. These are assigned to Nile Blue A and a complex between Nile Blue A and NADH.
Introduction Nile Blue A, 3-amino-7-(diethylamino)-1,2-benzophenoxazin, is a phenoxazine dye that was previously shown to adsorb strongly on the surface of graphite to produce a chemically modified electrode, CME.1-4 In previous studies, it was noted that the formal potential, E”’, of Nile Blue A is shifted in the adsorbed state as compared to its value in aqueous s01utions.l’~’~ In addition, drastic changes in the pK, values of both the oxidized as well as the reduced forms of Nile Blue A occur for the adsorbed specie^."^^^ These observations suggest that a specific functional group of the molecule interacts with the electrode surface. Some phenoxazines have shown very promising properties as redox catalysts for NADH oxidation when adsorbed on graphite electrode^,'^^'^*^ but Nile Blue A in the adsorbed state reacts very slowly with NADH in the contacting s ~ l u t i o n .However, ~ Nile Blue A has served as the starting compound for the synthesis of new, very efficient mediators for NADH oxidation. If the 3-amino group of Nile Blue A is reacted with an aromatic acid chloride'^^ or an aldehyde,6 the E”’ of the adsorbed Nile Blue A moiety is shifted from -430 mV vs SCE, at pH 7.0 for the unreacted form, to near -200 mV for the reacted
* Author
to whom correspondence should be addressed. Presented a t the symposium entitled “Photoelectrochemical and Electrochemical Surface Science: Microstructural Probes of Electrode Processes”, sponsored jointly by t h e Divisions of Analytical Chemistry and Colloid a n d Surface Chemistry, 197th National Meeting of the American Chemical Society, Dallas, April 9-14, 1989. University of Nebraska-Lincoln. 5 University of Lund. (1) Huck, H. Fresenius’ Z. Anal. Chem. 1982, 313, 548. ( 2 ) Huck, H. Ber. Bunsen-Ges. Phys. Chem. 1983,87,945. (3) Gorton, L. J . Chem. Soc., Faraday Trans. 1 1986,82, 1245. (4) Gorton, L.; Persson, B.; Polasek, M.; Johansson, G. Conference report, ElectroFinnAnalysis, 1988, submitted. (5) Gorton, L.; Tortensson, A.; Jaegfeldt, J.; Johansson, G. J . Electroanal. Chem. 1984, 267, 103. (6) Persson, B.; Gorton, L.; Johansson, G.; In Proceedings of the 2nd International Meeting on Chemical Sensors; Ancouturier, J.-L., Cauhape’, J.-S., Destriau, M., Hagenmuller, P., Lucat, C., Menil, F., Porter, J., Salardenne, J., Eds.; Mprimerie Biscaye, Bordeaux, 1986; pp 584587.
*
0743-7463 /90/2406-0066$02.50/0
and the rate constant with NADH is raised from 10 M-l s-l (ref 3) to about 104-105 M-l s-l (ref 4 and 6). The phenoxazine mediators incorporating a charged p-phenylenediamine functionality are particularly appealing for preparing CMEs for electrocatalytic NADH oxidation for the following reasons: (i) the reaction rate with NADH is high, approaching mass-controlled values; (ii) NADH oxidation does not appear to foul these CMES,~” contrary to bare electrodesg and CMEs based on other mediating functionalities;lOv’l and (iii) the mediating properties seem to be selective for NADH, which is not the case for other electrodes incorporating different catalytic molecules.1°J2 Previous electrochemical investigations of a series of adsorbed phenoxazines showed that the reaction rate with NADH varied with the concentration of NADH in the contacting solution. Rotating disk electrode experiments gave credence to the belief that an intermediate charge-transfer complex is formed in the redox reaction between NADH and the adsorbed m e d i a t ~ r . ~ sThe ~*~,~~ reaction rate between the mediator and NADH was also found to be strongly pH-dependent, being lower at more alkaline pHs.3,6,s,13These two observations suggest that the electron transfer from NADH to the oxidized form of the mediator occurs in several steps rather than a single hydride transfer, as is generally believed to occur in biological charge-transfer reactions from NADH.14 Thus far, electrochemical results have not lead to a consensus regarding the mechanism of catalytic NADH oxidation by phenoxazine derivatives. The coupling of spectroscopic and electrochemical meth(7) Appelqvist, R.; Marko-Varga, G.; Gorton, L.; Torstensson, A.; Johansson, G. Anal. Chem. Acta 1985, 169, 237. (8) Polasek, M.; Gorton, L.; Appelqvist, R.; Marko-Vargo,G.; Johansson, G. In Thesis of R. Appelqvist, Lund University, 1987. (9) Samec, 2.; Elving, P. J. J. Electroanal. Chem. 1983, 144, 217. (10) Chi-Sing Tse, D.; Kuwana, T. Anal. Chem. 1978,50, 1315. (11) Jaegfeldt, H.; Kuwana, T.; Johansson, G. J . Am. Chem. Soc. 1983,105,1805. (12) McKenna, K.; Boyette, S. E.; Brajter-Toth, A. Anal. Chim. Acta 1988, 206, 75. (13) Gorton, L.; Torstensson, A,; Johansson, G. J . Electroanal. Chem. 1985, 196, 81. (14) Powell, M. F.; Bruice, T. C. J . Am. Chem. SOC.1983,105, 7139.
0 1990 American Chemical Society
Electrochemistry of Chemically Modified Electrodes ods has been used successfully to characterize chemically modified electrodes. Electronic absorption spectroscopy has been used to examine the polymer complex polyviologen-poly(styrenesu1fonate)on SIIO,'~ and PtI6 electrodes. This technique does not provide information about molecular structure, however. Vibrational spectroscopy is ideally suited for this purpose, and there has been a burgeoning interest in applying various forms of IR and Raman spectroscopy to the study of chemically modified electrodes and electrode processes. Examples include the characterization of a poly(pheny1ene oxide)modified metal electrode by multiple-reflection IR spectro~copy.'~ Modulated specular reflectance spectroscopy [MSRS] in the IR has also been used to study adsorbed species on Pt.16 Thionine-modified electrodes have been examined by FT-IRl' and the combination of FT-IR, UV-vis, and Raman s p e c t r o s c ~ p y . ' These ~ ~ ~ techniques have also been used to study silanized electrodes.'l In each of the examples cited, detailed molecular information was obtained from the combined use of vibrational spectroscopy and electrochemical methods.l6Pz0 However, both IR and Raman spectroscopies have serious drawbacks for the in situ characterization of electrode surfaces. The high absorbance of IR radiation by water is a problem in the former, whereas the extremely small Raman cross section of most compounds limits the use of the later technique. In contrast to conventional FT-IR and Raman spectroscopy, surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) spectroscopy provide high sensitivity and low detection limit^.^^,^^ As a result, detailed molecular information can be obtained regarding the mode of interaction between an adsorbate and an electrode surface. The orientation of the adsorbed species on the electrode surface can also be ascertained from its SERS or SERRS spectrum in many case^.^'-'^ Therefore, a SERRS investigation of Nile Blue A was undertaken to confirm and clarify some of the previous electrochemical observations and also to determine whether this technique could provide information regarding the mechanism of NADH oxidation. Nile Blue A is a very good candidate for this study, because it is the basic structure used to prepare new efficient mediators and also because of its slow reaction rate with NADH. As a result of the latter property, the putative chargetransfer complex may be present in sufficient concentration for its spectrum to be observed at the electrode surface.
Experimental Section Chemicals and Supplies. Laser grade Nile Blue A perchlorate was purchased from Eastman Kodak (Rochester,NY; catalog number 11953). The highest purity grade of NADH was purchased from Sigma Chemical Co. (St. Louis, MO; catalog number N-8129) and was used as received. The supporting electrolyte solutions included 0.25 M phosphate buffer at pH 4.3(15) Akahoshi, H.; Toshima, S.; Itaya, K. J . Phys. Chem. 1981,85, 818. (16) Bewick, A.; Kunimatsu, K.; Robinson, J.;Russell, J. W. J . Electroanal. Chem. 1981,119,175. (17) Dubois, J.-E.; Lacaze, P.-C.; Pham, N. C. J. Electroanal. Chem. 1981,117,233. (18) Quickenden, T.I.; Comarmond, M. J. J . Electrochem. SOC. 1988,135,918. (19) Hutchinson, K.; Hester, R. E.; Albery, W. J.; Hillman, A. R. J . Chem. SOC.,Faraday Trans. I 1984,80,2053. (20) McQuillan, A. J.; Hester, R. E. J . Raman Spectrosc. 1984,15, 15. (21) Srinivasan, V. S.;Lamb, W. J. Anal. Chem. 1977,49,1639. (22) Ni,F.;Thomas, L.; Cotton, T. M. Anal. Chem. 1989,61, 888. (23) Ni,F.;Cotton, T. M. J . Raman Spectrosc. 1988,19,429. (24) Ni, F.;Cotton, T. M. Anal. Chem. 1986,58,3159.
Langmuir, Vol. 6, No. 1, 1990 67 9.0. Other pH values outside this range (pH 2, 3, and 10) were obtained by adding either 0.1 M HCl or 0.1 M NaOH solution. All solutions were prepared with deionized, distilled water, and the pH values were measured with a Fisher Accumet pH meter (Model 610). Instrumentation. Electronic absorption spectra were recorded on a Hewlett-Packard UV-vis spectrophotometer (Model 8450A). A BAS 100 electrochemicalanalyzer and a conventional threeelectrode cell were used for the electrochemical measurements. A saturated calomel electrode (SCE) served as a reference electrode, and all potentials are reported with respect to the SCE. A Pt wire served as the auxiliary electrode. A glassy carbon electrode (BioanalyticalSystems Inc., West Lafayette, IN) with an apparent surface area of about 6.6 mm2was used as the working electrode. The electrode pretreatment was identical with that described previ~usly.~ The electrode was polished with 600-grit emery paper, rinsed with distilled water, and sonicated to remove any loose particles. A silver electrode was used as a working electrode in some of the electrochemical experiments and as a SERS substrate. It was constructed as described previ~usly.~~ The exposed surface was rectangular with a geometrical surface area of approximately 12 mm'. The electrode was polished with a slurry of 0.05-llm alumina in water on a mechanical polishing wheel. It was then rinsed and sonicated in distilled water to remove any alumina which may have adhered to the surface. Following the cleaning procedure, the electrode was roughened by an oxidation-reduction cycle (ORC) consisting of a double potential step from an initial potential of -550 mV to +500 mV and back to -550 mV in 0.1 M Na'SO, solution. The total charge passed during the oxidation step was equivalent to about 25 mC/cm2. Following the roughening procedure, one drop of a saturated solution of Nile Blue A in water (pH 7) was placed on the electrode surface and allowed to remain in contact with the surface for 5 min. The electrode was next rinsed with buffer solution at the appropriate pH and placed into the cell for electrochemical or spectroscopic measurements. The 488-nm line of a CW argon ion laser (Coherent, Innova 90-5) was used as the excitation source for Raman spectroscopy. An Anaspec 300-S premonochromator was used to remove plasma lines. Spectra were acquired in the backscatteringgeometry. The Raman scattering light was focused onto the slits of the monochromator/spectrograph (Spex Triplemate 1877),coupled to an intensified SiPd detector (Model 1420, Princeton Applied Research Corp.). The Raman and SERS spectra were collected and processed with an optical multichannel analyzer (OMA-2, Princeton Applied Research Corp.).
Results and Discussion Electrochemical Studies. Cyclic voltammetry was used to monitor the behavior of NADH and Nile Blue A at glassy carbon (GC) and Ag electrodes. Figure 1A illustrates the typical irreversible oxidation of NADH on GC. The overpotential required to oxidize NADH is ca. 1.1 V a t pH 7 for carbon electrodes (NAD+/NADH Eo' = -560mV)25r26and 1.3on Pt.27The high overpotential limits the use of direct electrochemical detection approaches for monitoring NADH formation in enzymatic reactions. Consequently, there have been numerous attempts to enhance the electron-transfer kinetics using various types of mediator^.^ Electrodes modified with Nile Blue A have been used for this purpose. Figure 1B depicts the cyclic voltammetry response for irreversibly adsorbed Nile Blue A. The E"' is approximately -380 mV, as noted in previous s t u d i e ~ .Based ~ ~ ~ upon the geometrical area of the electrode, the surface coverage is ca. 9.5 X lo-' mol/cm2. This corresponds to ca. 50-100 monolayers of Nile Blue A if it is adsorbed flat on the ~ u r f a c e .The ~ exact num(25) Elvinp, P.J.; Schmakel, C. 0.;Santhanam, K. S. V. Crit. Reu. Anal. Chem. 1976,6 , 1. (26) Moiroux, J.; Elving, P. J. Anal. Chem. 1978,50, 1056. (27) Jaegfeldt, H.J. Electroanal. Chem. 1980,110, 295.
68 Langmuir, Vol. 6, No. 1 , 1990 A
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T
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Figure 2. Differential pulse voltammograms of Nile Blue A modified Ag electrode in 3.1 mM NADH solution, pH 6.1. Experimental conditions were as follows: initial potential, -500 mV; scan direction, positive; scan rate, 4 mV/s; pulse amplitude, 50 mV.
Figure 1. Cyclic voltammograms of (A) NADH (3.1 mM) on glassy carbon electrode, (B) Nile Blue A modified glassy carbon electrode,and (C) Nile Blue A modified glassy carbon electrode in the presence of 3.1 mM NADH solution. All solutions were at pH 6.1. Scan rate: (A) 100, (B) 5, and (C) 20 mV/s. ber of monolayers is difficult to determine because the actual surface area is not known. More than a monolayer seems likely as a roughness factor of 50-100 seems improbable. Figure 1C shows the response of this electrode in the presence of NADH. A catalytic peak is present near 0.0 V. Thus, the CME decreases the overpotential required for NADH by ca. 600 mV. This is comparable to values observed for CMEs employing other phenoxazine derivative^.^ The intensity of the catalytic oxidation peak is several times greater than the peak for NADH oxidation at a bare electrode. The overall mechanism for the mediated reaction involves the following two steps:5 NBH
NB'
+ H' + 2eNAD' + NBH
(1)
NADH + NB' (2) In the first step, Nile Blue A undergoes a two-electron oxidation and loses a proton to form the positively charged species. The oxidized Nile Blue A then abstracts two electrons and a proton from NADH. The net reaction is the electrocatalytic oxidation of NADH to NAD+: NADH + NAD' + H' + 2e(3) From Figure 1, it can be seen that the catalytic peak is not coincident with the E"' value for Nile Blue A oxidation. This has been observed in other catalytic systems as well." In some cases, the shift in potential can be attributed to the slow reaction between the mediator and NADH (reaction 2). However, in the case of Nile Blue A, the rate constant for this reaction is known and is near 10 M-' s-1.3 This should not produce such a large shift in potential according to the relationship developed by Andrieux and Sav6ant2' relating peak potential to other experimental parameters. Another possible explanation for the difference between the mediator potential and that for catalytic oxidation of NADH is that an intermediate charge-transfer complex is formed between NB+ and NADH. The catalytic (28) Andrieux, C. P.; Saveant, J. M. J.Electroanal. Chem. 1978,93, 163.
10
20
30
40
Time (min)
Figure 3. Time-dependent current for Nile Blue A modified Ag electrode. Axis on the left refers to current at -110-mV peak. Axis on the right refers to current at -340-mV peak. peak represents the oxidation potential of the complex. There is other evidence for the existence of charge-transfertype complexes between NADH and various adsorbed mediators on the electrode ~ u r f a c e . ~ + . 'This ~ possibility will be considered further in the discussion of the surface Raman spectra. The CV behavior of adsorbed Nile Blue A on a Ag electrode is similar to, but not identical with, that shown for GC. A distinct but much weaker set of peaks is observed for the adsorbed species with an E"' near -340 mV at pH 6.1. Integration of the current under the peaks indicates that only about one monolayer is adsorbed. When Ag electrodes are modified with Nile Blue A and the catalytic oxidation of NADH is examined, a more poorly defined peak is observed in the cyclic voltammogram as compared to GC electrodes. Differential pulse voltammetry (DPV) provided better defined peaks. Two studies were performed: a time study at constant NADH concentration and a concentration study using increasing concentrations of NADH. Figure 2 shows the effect of increasing time on the DPV of a Nile Blue A modified Ag electrode in the presence of 3.1 mM NADH. The voltammograms after 9 and 44 min are displayed. Two peaks are observed at -110 and -320 mV. The latter peak is close to the E"' value for adsorbed Nile Blue A. Figure 3 is a plot of time versus peak current for these two peaks. As can be seen, the peak near -320 mV decreases with time, whereas that near -110 mV increases.
Langmuir, Vol. 6, No. I , 1990 69
Electrochemistry of Chemically Modified Electrodes A
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The effect of NADH concentration on the peak currents was also examined, as shown in Figure 4. Increasing the NADH concentration resulted in an increase in the peak near -110 mV and a decrease in the peak near -400 mV. The difference in peak potentials for data used to prepare Figures 2 and 4 is probably due to slight differences in the solution pH values. The results of the above DPV studies show clearly that NADH has an effect on the Nile Blue A modified electrode. The origin of the peak near -110 mV is the fundamental question. There are a t least two possible explanations for this peak. The first is that it is associated with NADH and becomes stronger with time because of displacement of Nile Blue A with NADH. Since Nile Blue A is strongly adsorbed, it seems likely that it would take time for NADH to displacs it from the electrode surface. However, no peak is present a t this potential when NADH is present in the absence of Nile Blue A. A second possibility is that the Nile Blue A and NADH form a complex having a redox potential at -120 mV. The formation of mediator-NADH complexes has been proposed in the past to explain the catalytic response of C M E S . ~ ~Evidence ,'~ for the formation of a complex will be presented later in the Raman discussion. In previous electrochemical studies of Nile Blue A at a graphite electrode, the pH dependence of the formal potential was documented. Similar but not fully identical behavior is noted a t the Ag electrode. Figure 5 displays the decrease in EO'with increasing pH values. There are two linear regions. Between pH 2.0 and 6.0, the pH decreases with a slope of ca. 60 mV/pH unit. The slope for the second linear region, or the pH range from 6.0 to 9.0, is 30 mV/pH unit. These results can be attributed to the following equilibria, as proposed by Gorton et al. for Meldola Blue:5
2
4
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The reduction involves two electrons in both reactions, but in (4),two protonsare added to NB in the reduction step, whereas in (5) only one proton is added. There is no protonation of the oxidized form of Nile Blue A in the pH range shown here. An additional protonation of the reduced formoccurs below pH 4.0 for the soluble ~ p e c i e s .A ~ proton is dissociated from the amine group of the oxidized species a t high pH values (pK, = 9.70 in solution and 13.0 in the adsorbed state). The catalytic oxidation of NADH on Nile Blue A modified carbon electrodes as a function of pH has not been studied prior to now, but other phenoxazines have been e ~ a m i n e d . ~ ~ ~The * ~ -same ~ , ' ~type of behavior is noted for Nile Blue A as described for these other phenoxazines. Figure 6 illustrates the results obtained for the Nile Blue A modified GC electrode. The catalytic current is highest between pH 5 and 6. It decreases steadily with increasing pH values between 6.0 and 9.0. There are several possible explanations for the pH-dependent behavior. First, protonation reactions may be involved.
70 Langmuir, Vol. 6, No. 1, 1990
Ni et al.
n t
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Figure 7. Raman spectra of Nile Blue A on a roughened Ag
electrode, glassy carbon electrode, and in solution. The solution pH was 4.3. The laser excitation wavelength was 488.0 nm. The laser power was 10 mW for Ag, 200 mW for glassy carbon, and 180 mW for solution spectrum. The spectral acquisition time was 33 s. As noted above, two protons are added in the reduction step a t pH values below 6. A second possibility is that a proton catalytic mechanism is involved as proposed by Gorton et al.3 Finally, it may be that the orientation of Nile Blue A changes with pH. This could affect the catalysis if the catalytic site becomes inaccessible with reorientation. Of course, this third possibility is related to the first, since protonation may affect the orientation of Nile Blue A at the electrode surface. A fourth possibility is that NB is unstable a t the more basic pH values. Evidence obtained from the Raman experiments described below supports the third explanation. Raman Studies. In order to test the possibility that orientation changes may play a role in the catalyticresponse of Nile Blue A modified electrodes, RR and SERRS spectroscopies were used to monitor the changes in the adsorbed Nile Blue A spectrum with pH. Before discussing these results, it is essential to note that the spectrum of Nile Blue A on Ag is very similar to that on GC and the solution spectrum. Figure 7 shows that only small differences are observed in the surface spectrum on roughened Ag a t pH 4.3 as compared to the solution spectrum. These involve primarily the bands a t 1440, 1354, 1179, and 1142 cm-' in the solution spectrum, which are shifted to 1435, 1349, 1170, and 1131 cm-' in the spectrum on Ag. There are also some differences in relative band intensities (e.g., the relative 1492/ 1519-cm-' band intensity is stronger in the surface spectrum). These small differences may result from direct interaction of the atoms contributing to these normal modes with the electrode surface. In contrast to the above results, the surface spectra at high pH values are quite distinct from the solution spectrum of Nile Blue A. A comparison of the Ag and GC spectra with the solution spectrum in Figure 8 shows marked differences in peak intensities. A strong enhancement in the bands a t 1490 (1500), 1377 (1382), 1199 (1205), and 1181 (1192) cm-' occurs in the Ag (the numbers in parentheses are for GC) surface spectra. It should be noted that the spectrum obtained on Ag is surface-enhanced. Only 5-10 mW of laser power was used as compared to 200 mW for the GC spectrum. A com-
Figure 8. Raman spectra of Nile Blue A on a roughened Ag
electrode, glassy carbon electrode, and in solution. The solution pH was 9.0. All other experimental parameters were the same as in Figure 7 . 0
h
I1
Figure 9. Surface-enhanced Raman spectra of Nile Blue A adsorbed on Ag as a function of pH for the range 2.0-6.1. Spectral parameters were the same as in Figure 7 .
parison of the solution spectra a t pH 9.0 and 4.3 shows only small changes in band intensities with a stronger 1495-cm-' band but weaker bands a t 1443 and 1354 cm-' at the higher pH. The differences between the surface and solution spectra reflect the differences in the structure of adsorbed Nile Blue A on the GC and Ag surface. The spectrum at high pH values is almost the same as that of phenoxazine, the parent compound. The effect of pH changes on the SERS spectrum was examined to determine the values where large spectral changes occur. Figure 9 shows results obtained between pH 2.0 to 6.1. There are no major changes in the spectrum in this pH range. Figure 10 shows results obtained between pH 6.1 and 10.0. Definite differences in the spectra can be seen as the pH is changed from 6.1 to more basic values. The changes begin near pH 7.0, and these include enhancement and small shifts of the 1489-, 1375-,
Langmuir, Vol. 6, No. 1, 1990 71
Electrochemistry of Chemically Modified Electrodes
. m
C
A
RAMAN SHIFT
cm-1
Figure 11. Surface-enhanced Raman spectra of Nile Blue A modified Ag electrode at pH 4.0 (A and B) and pH 8.0 (C and D). Spectra A and C are for the reduced species (electrode potential (A) -350 and ( C ) -580 mV). Spectra B and D are of the oxidized species (electrodepotential (B)-180 and (D) -180 mV).
Figure 10. Surface-enhanced Raman spectra of Nile Blue A
as a function of pH for the range 6.1-10.0. Spectral parameters were the same as in Figure 7. Table I. SERS Bands (cm-') for Oxidized and Reduced Nile Blue A at pH 4.0 and 8.0.
pH 4.0
pH 8.0
oxidized reduced oxidized reduced tentative (-180 mV) (-350 mV) (-180 mV) (-580 mV) assignments* 1641 s 1626 m 1640 s 1636 w a&: NH def 1592 w 1588m 1586m 1586w 1543 sh 1515 m 1489 m 1461 w 1490 s 1490m 1437 m 1437 w 1433 m 1419 sh 1399 sh 1375 w 1374 m 1377 m 1374 m a&, @(CH,) 1347 m 1350 m 1347 sh aPr,tert amine 1333 sh 1281 w 1272 w 1202 sh 1202 w 1183 w 1170 w 1183 m 1132 w 1128 sh 1076 w "s
= strong, sh = shoulder, w = weak, m = medium. 'CY@? = a(CC), p(CCH),and
bAssignments are from ref 28. r(CCC).
1199-, and 1178-cm-' bands, as discussed above. These spectral changes correlate very well with the electrochemical results that indicate a change in catalytic activity of Nile Blue A at pH values greater than 6.0 (Figure 6) and a change in the protonation reaction (Figure 5). An assignment of the normal modes of Nile Blue A is needed in order to correlate the changes in Raman intensities with pH. Unfortunately, there is only a preliminary assignment available at this time.,' The bands are listed in Table I together with several of the assignments. The strong band at 1642 cm-' at low pH values is attributed to a ring mode and an NH deformation. The 1377-cm-l band is assigned to a ring mode and the CH, deformation. If these assignments are correct, the strong 1642-cm-' band may indicate the close proximity (29) Miller, S.K.; Baiker, A.; Meier, M.; Wokaun, A. J. Chem. Soc., Faraday Trans. I 1984,80, 1305.
of the NH, moiety to the surface, whereas the strong 1377cm-' band suggests that the p-phenylenediimine group is close to the surface. The latter is believed to be the catalytic site for NADH oxidation. If this is the case, it is reasonable that catalysis would cease when this group is close to the surface at high pH values. The fact that the Nile Blue A spectrum is nearly the same on GC as on Ag at high pH values indicates that the same type of interaction is present on both electrodes. The possible orientation of Nile Blue A at the electrode surface as a function of pH can be inferred from the change in band intensities. It appears that a t low pH values (16.1) Nile Blue A does not adsorb in a flat configuration, as do most aromatic compounds. Rather, it adsorbs with the aromatic ring planes in a more perpendicular orientation. The SERRS spectrum on Ag is very similar to the RR spectrum on GC and in solution as would be expected. At high pH values, Nile Blue A adsorbs with the aromatic rings parallel to the surface. Under these conditions, new vibrations involving ring deformations are observed. Both the Ag and GC surface spectra are quite different from the solution spectrum, as would be expected if the m y s t e m is perturbed by its interaction with the surface. The spectra are very similar to the spectrum of phenoxazine, the parent compound, on Ag. It is expected that this compound should adsorb flat. The same type of spectral changes observed for oxidized Nile Blue A as a function of pH can also be seen in the reduced species. Figure 11shows the spectrum of reduced Nile Blue A at pH 4.0 and 8.0. Once again, the spectra are quite different. On comparing the oxidized (B) and reduced (A) spectrum at low pH, it can be seen that the 1643-cm-' band is shifted to 1626 cm-' and reduced in intensity. This might be expected if the NH, group becomes protonated upon reduction, as shown in reaction 4. Another major change is in the intensities of the 1588- and 1374-cm-' bands. Both are much stronger in the reduced spectrum. Changes in the SERS spectrum with reduction at the higher pH value are much more subtle. The major effects include a decrease in the i n t e n s i t y of t h e 1 6 4 0 - c m - l b a n d a n d t h e overall intensification of the 1586-, 1437-, and 1374-cm-l bands upon reduction. Of course, some of these changes can be expected to result from the change in the absorption spectrum with reduction, as there are no strong transitions in the visible for reduced Nile Blue A. However,
72 Langmuir, Vol. 6, No. 1, 1990
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16.012.0: RAMAN SHIFT
cm-1
Figure 12. Surface-enhanced Raman spectra of Nile Blue A modified Ag electrode in the presence of 3.1 mM NADH (A and C) and in the absence of NADH (Band D). Spectra A and B are at an electrode potential of -200 mV. Spectra C and D are at an electrode potential of -500 mV. the laser excitation wavelength used in this work is between the major electronic transitions of the oxidized form and is not strongly in resonance. The optical effects may be smaller under these conditions. RR and SERS of Nile Blue A in the Presence of NADH. It was noted in the discussion of the DPV results that NADH produced a change in the Nile Blue A modified electrode response. A peak near -110 MV was observed that increased with time a t constant NADH concentration and with increasing NADH concentration a t a fixed time. Two possible explanations were proposed. The first was that NADH displaces the Nile Blue A on the electrode surface. The second was that a complex between NADH and Nile Blue A is formed on the electrode surface, with a redox potential near -110 mV. This complex could function as the catalytic intermediate in the oxidation of NADH to NAD+. To determine which if either of these two possibilities is reasonable, the SERS spectrum of Nile Blue A was examined as a function of electrode potential in the absence and presence of NADH. Parts B and D of Figure 12 show the spectrum of the Nile Blue A modified Ag electrode a t pH 6.1 and at two electrode potentials, -200 and -500 mV, respectively. Immediately above (Figure 12A and 12C) are spectra obtained a t the same two potentials but in the presence of NADH. As can be seen, the latter are much more complex. Bands characteristic of both oxidized (strong 1638, 1490, and 1350 cm-') and reduced (strong 1372 cm-l) Nile Blue A are observed a t -500 and -200 mV. Also, the spectra are about a factor of 4 less intense than the spectra obtained for Nile Blue A alone. From the DPV results, it is obvious that there are two species present on the surface. One appears to have the redox potential of Nile Blue A, whereas the other has a more positive value. An explanation for the SERRS results is that the portion of the Nile Blue A which undergoes oxidation a t the formal potential as indicated from the DPV is responsible for the reduced bands at -500 and the oxidized bands at -200 mV. Those bands attributable to a reduced form of Nile Blue A at -200 mV must therefore arise from the species undergoing oxidation at -110 mV. This is assigned to a complex between Nile Blue A and NADH which undergoes catalytic oxidation a t -110 mV. The rate constant for this reaction is apparently slower than that for other phenoxazine-type mediators.
8.0 4.0 I
2.4 \
-500 -400 -300 -200 -100 0.00
x L
2.0
3,
POTENTIAL (mv)
Figure 13. Results for Nile Blue A on Ag in the absence (W and presence (a ) of NADH.
)
Further evidence for the presence of two distinct species in the presence of NADH may be found in a plot of band intensities versus electrode potential. With the 592cm-' band as a probe of the oxidized form, Figure 13 shows the results for Nile Blue A on Ag in the presence and absence of NADH. In the absence of NADH (squares), the band intensity increases steadily from -500 to -200 mV. When NADH (3.1 mM) is added to the electrolyte solution, the results are quite different. The 592-cm-' band intensity is maximal near -350 mV and decreases rapidly as the potential is made more positive. This indicates that either the Nile Blue A is present in the reduced form at potentials more positive than -350 mV or a new species is formed that lacks the strong 592-cm-' band. It is conceivable that a steady-state concentration of Nile Blue A is present in the reduced form because of the catalytic oxidation of NADH. However,it seems unlikely that a detectable amount would be present if the heterogeneous electron-transfer rate constant for Nile Blue A is significant. Therefore, the presence of a new species (i.e., complex between NADH and Nile Blue A) is the preferred explanation. Complex formation was proposed to explain previous rotating disk electrode studies of Meldola Blue,5 but the SERRS results are the first spectroscopic evidence obtained for such a complex. The changes that occur in the SERRS spectrum of Nile Blue A in the presence of NADH are not due to adsorption of NADH on the electrode surface. SERS spectra are much weaker for NADH and have no bands in common with the spectra observed for the Nile Blue A modified electrode in the presence of NADH. Thus, the spectral changes appear to result solely from Nile Blue A. Partial displacement of the Nile Blue A by NADH may account for the weaker spectrum in the presence of NADH. It may be that the NADH must displace some Nile Blue A to form the complex, which could explain the time dependence of the reaction.
Summary and Conclusions Electrochemicalstudies of Nile Blue A adsorbed at glassy carbon and Ag electrodes have shown that it forms multilayers on the former and a monolayer on the latter. The cyclic voltammetry results show that the electron-trans-
Electrochemistry of Chemically Modified Electrodes
fer reaction is reversible on both electrodes. The relationship between the formal redox potential and pH is somewhat different, with slightly lower E"' values on Ag than on GC (-340 and -380 mV, respectively, at pH 6.1). The Nile Blue A modified GC electrode catalyzes NADH oxidation at an E"' value which is ca. 600 mV less positive than at a bare electrode. This catalytic peak is approximately 400 mV more positive than the formal potential of the adsorbed mediator. A similar catalytic response is observed for the Nile Blue A modified Ag electrode. However, the catalytic current is much less than on GC. Differential pulse voltammetry on the modified Ag electrode showed that the peak associated with NADH oxidation is dependent upon both NADH concentration and time, with increasing current resulting from an increase in either variable. As in the case of the Nile Blue A modified GC electrode, the catalytic current is also dependent upon the solution pH. The current decreases with increasing pH values between 6 and 9. Resonance Raman and SERRS spectra of the adsorbed Nile Blue A on GC and Ag were very similar to the RR spectrum of Nile Blue A in solution at pH values I6.1. At higher pH values, the spectra on both electrodes are markedly different from the solution spectrum. The difference is attributed to a change in the interaction of Nile Blue A with the electrode surface with pH. In the low pH range, the spectra suggest that Nile Blue A is adsorbed with the aromatic plane oriented in a perpendicular fashion. At high pH values, the molecule is adsorbed with the aromatic plane parallel to the electrode surface. Under the latter conditions, a strong perturbation of the molecular T system occurs. The same spectral differences are observed for both the oxidized and reduced species. Also, the pH changes correlate with the electrochemical results: the change in Raman spectrum occurs at the point where the slope of the E"' value switches from a two-electron, two-proton process to a twoelectron, one-proton process. In the presence of NADH, the SERRS spectrum of the Nile Blue A is quite different. At both positive and negative potentials, new bands are observed. The spec-
Langmuir, Vol. 6,No. I, 1990 73
tra indicate that at least two species are present. On the basis of the electrochemical results, it appears that one of these species is adsorbed Nile Blue A. It is proposed that the second species is a complex between Nile Blue A and NADH, with an oxidation potential near -100 mV. This complex is apparently quite stable, since the changes in the SERRS spectrum persist under both reducing (-500 mV) and oxidizing (0.0 V) potentials. The strong interaction between Nile Blue A and NADH would account for the poor catalytic response. Alternative explanations for the second species is that it represents an impurity in Nile Blue A or a degradation product. These possibilities are rejected on the basis of HPLC experiments, which showed no detectable impurities in the Nile Blue A, and electrochemical studies in the absence of NADH, which showed no peak in the potential region between -200 and 0 mV. The presence of both Nile Blue A and NADH is necessary for the observation of this peak. The exact nature of the Nile Blue A/NADH complex is not known. Attempts are underway to identify RR changes in the solution spectrum of Nile Blue A in the presence of high concentrations of NADH. The RR and SERRS behavior of other phenoxazine-type mediators (e.g., Meldola Blue) that exhibit a greater catalytic response for NADH oxidation is also under investigation. It may not be possible to detect the presence of a similar complex between these mediators and NADH because of the low steady-state concentration that is likely to be present.
Acknowledgment. This work was supported by the Swedish Board for Technical Development (STUF) (L.G.) and by the Swedish National Energy Administration (STEV) (L.G.) and the National Institutes of Health (GM35108, T.M.C.). T.M.C. is the recipient of a National Institute of Environmental Health Sciences Research Career Development Award (ES00169). Registry No. NADH,58-68-4;Ag, 7440-22-4;Nile Blue A, 2381-85-3;carbon, 7440-44-0;5-amino-9-N,N-diethylamino12H-benzo[a]phenoxazinconjugate acid,122906-04-1; &amino9-N,N-diethylamino-l2H-benzo[a]phenoxazin, 18493-48-8.
