Anal. Chem. 1088, 6 0 , 2263-2268
Wennrich, R.; Dktrich, K.; Bonk, U. Spectrochlm. Acta, Part 8 1984, 398, 657-666. Dktrich. K.; Wennrich, R. Prog. Anal. At. Spectrosc. 1984, 7 ,
139-198.
Felske, A.; Hagenah, W.D.; Laqua, K. Spectrochim. Acta, Part B 1972, 278, 1-21. Pleameler. E. H. I n AnaMicel ADDlicetions of Lasers: PieDmeier. E. H., Ed.: bileyi -New York. 7988;Ckpter 19. I Peppers, N. A,; Scribner. E. J.; Aiterton, L. E.; Honey, R. C.; Beatrice, E. S.; Harding-Barlow, 1.; Rosan, R. C.; Glick. D. Anal. Chem. 1988, 40, 1178-1182. (12) Morton, K. L.; Nohe, J. D.; Madsen, B. S. Appl. Spectrosc. 1973, 27,
109-117. (13) Carr, J. W.; Horlick, G. Spectrochlm. Acta, Part 8 1982, 378,1-15. (14) Talmi. Y.; Sieper, H. P.; Moenke-Bankenburg, L. Anal. Chlm. Acta 1961, 127. 71-85. (15) Saffir, A. J.; Marlch, K. W.; Orenberg, J. B.; Treyti, W. J. Appl. Spectrosc. 1972, 2 6 , 489-471. (16) . . HOuk. R. S. I n AnaMlcel Appllcatlons of Lasers; Piepmeler, E. H., Ed.; Wiley: New York, 1986;Chapter 18. (17) Allemand. C. D. Spectrochlm. Acta, Part8 1972, 278, 185-204. (18) Margoshes, M.; Marcellus, D. A,; Rasberry. S. D. froceedlngs of the 13th Colloquium Spectroscoplcum Internatlonale; Hilger:
London,
ln68: D 156. (19) B&u&v, V. I. Zh. Anal. Khim. 1984. 3 9 , 909-927. (20) Bykovskii, Yu. A.; Basova, T. A.; Belousov, V. I.; Giadskol, V. M.;
Qorshkov, V. V.; Degtyarev, V. G.; Laptev, I.D.; Nevolin, V. N. Sov. Phys. Tech. Phys. (Engl. Trans/.) 1978, 2 1 , 761-763. (21) Chen, G.; Yeung. E. S. Anal. Chem. 1988, 60. 864-868. (22) Carroll, P. K.; Kennedy, E. T. Contemp. Phys. 1981, 22, 61-96. (23) Conzemius, R. J.; Capellen, J. M. Int. J. Mass Spectrom. Ion Phys. 1980, 3 4 , 197-271. (24) Fabbro, R.; Fabre, E.; Amiranoff, F.; Garban-Labaune, C.; Virmont, J.; Weinfeld, M.; Max, C. E. Phys Rev. A 1982, 2 6 , 2289-2292. (25) Browne, P. F. R o c . Phys. SOC. 1985. 8 6 , 1323-1332. (26) Piepmeler, E. H.; Osten, D. E. Appl. Spectrosc. 1971, 2 5 , 642-652. (27) Knox, B. E. I n Trace Analysls by Mass Spectrometry; Ahearn, A. J., Ed.; Academic: New York. 1972;Chapter 14. (28) Plepmeier, E. H.; Malmstadt, H. V. Anal. Chem. 1969, 4 1 , 700-707. (29) Dimitrov. G.; Zheleva, T. Spectrochim. Acta, Part B 1984. 398,
.
.-- - .- .- .
1209-1219
(30) Sucov, E. W.; Pack, J. L.; Pheips, A. V.; Engelhardt, A. G. Phys. Fluids 1987, 10, 2035-2048.
2263
(31) Treytl, W. J.; Marich, K. W.; Gllck, D. Anal. Chem. 1975, 47, 1275-1279. (32) Archbold, E.; Harper, D. W.; Hughes, T. P. 8 r . J. Appl. Phys. 1984, 15, 1321-1326. (33) Lltvak, M. M. Edwards, D. F. I€€€ J. Ouantum Electron. 1988, E - 2 , 486-492. (34)Scott. R. H.; Strashelm, A. Smctrmhlm. Acta, Part 8 1970, 2 5 8 , 311-332. (35) Treyti, W. J.; Orenberg, J. B.; Marich, K. W.; Glick, D. Appl. Spectrosc. 1971, 25. 376-378. (36) Minck, R. W. J. Appl. Phys. 1984, 35, 252-254. (37) Adams, M. J.; King, A. A,; Kirkbright, G. F. Analyst (London) 1978, 101.73-85. (38) Treyti, W. J.; Marich, K. W.; Orenberg, J. 8.; Carr, P. W.; Mllier, D. C.; Glick, D. Anal. Chem. 1971, 43, 1452-1456. (39) Beenen, G. J.; Piepmeier, E. H. Appl. Spectrosc. 1984, 3 8 , 851-857. (40) Antlpov, A. B.; Kapitanov, V. A,; Nikiforova. 0. Yu.; Ponomarev. Yu. N.; Sapozhnlkova, V. A. J. Photoacoust. 1983-1984, 1 , 429-443. (41) Kriege, 0.H.; Marks, J. Y.; Welcher, 0. 0. I n Flame Emission and Atomic Absorption Spectroscopy, Vol. 3 , Elements and Matrlces ; Dean, J. A., Rains, T. C., Eds.; Dekker: New York. 1975;Chapter 7. (42) Conzemius, R. J.; Zhao, S.; Houk, R. S.; Svec, H. J. Int. J. Mass Spectrom. Ion Processes 1984, 6 1 , 277-292. (43)Zharov, V. P.; Letokhov, V. S. I n Laser Ontoacoustlc Spectroscopy; Tamir, T., Ed.; Springer Series in Optical 7 :iences, vol. 37;SpringerVerlag: New York, 1984;Chapter 6. (44) Wagner, R. E. J. Appl. Phys. 1974, 45, 4631-4637. (45) Manabe, R. M.; Plepmeler, E. H. Anal. Chem. 1979, 51, 2066-2070. (46) Measures, R. M.; Kwong, H. S. Appl. Opt. 1979, 18, 281-286. (47) Kahtor, T.; Bezur, L.; Pungor, E.; Fodor, P.; Nagy-Balcgh, J.; Heincz, 0. Spectrochlm. Acta, Part 8 1879, 348, 341-357. (48)Rudnevsky, N. K.; Tumanova, A. N.; Maximova, E. V. Spectrochlm. Acta, Part B 1984, 398, 5-11.
RECEIVED for review May 20,1988. Accepted July 13, 1988. The Ames Laboratory is operated by Iowa State University for the U.S.Department of Energy under Contract W-7405Eng-82. This work was supported by the Director of Energy Research, Office of Basic Energy Sciences, Division of Chemical Sciences.
Catalytic Reduction of Myoglobin and Hemoglobin at Chemically Modified Electrodes Containing Methylene Blue Jiannong Ye and Richard P. Baldwin* Department of Chemistry, University of Louisville, Louisville, Kentucky 40292
Chemically modified electrodes (CMEs) exhibiting eiectrocatalytic response toward myoglobin and hemogiobln were constructed by adsorbing the phenothiazine mediator titrants methylene blue and thionine onto spectroscopic graphite. These CMEs, which were prepared by a rapid (60 s) and reproducible (3.2 % relative standard deviatlon) dip-coating procedure, permilted the hemoprotein electroreductionto take place at the reduction potential of the mediator molecule. For neutral and slightly acidic solutions, this corresponded to very modest negative potentials (vs Ag/AgCi). When used in flow injectkm and iiquld chromatography detection, with an applied potential of -0.12 V, the methylene blue CMEs gave detection ilmits of 10 and 20 pmol Injected for myoglobin and hemoglobin, respectively, with linear response extending 2 to 3 orders of magnitude higher. After a brlef equilibration period, the CME retained more than 90% of its inltlai myoglobin response over several hours of contlnuous exposure to the chromatographic flowstream.
Although many enzymes and proteins possess functional 0003-2700/86/0360-2263$01.50/0
groups that can be readily oxidized or reduced by chemical redox agents, it is rare for these same compounds to undergo facile oxidation or reduction at electrodes. Rather, for reasons ascribed either to their extended three-dimensional structure and the resulting inaccessibility of the eledroactive center or to their adsorption onto and subsequent passivation of the electrode surface, most biological macromolecules exhibit such slow rates of electron transfer that no useful currents are observed at conventional electrodes, even with the application of relatively large overpotentials. To date, only a few exceptions to this behavior have been demonstrated, with these occurring primarily when rather specialized electrode materials were employed. For example, reversible or quasi-reversible electrolysis of cytochrome c has been reported at indium oxide (1-3), tin oxide (3), and edge-graphite (4), and the use of ruthenium oxide has been shown to facilitate the electrode reactions of several proteins including cytochrome c , azurin, rubredoxin, ferredoxin, and plastocyanin (5). However, even in the most favorable of these cases, the observed electrochemistry was strongly dependent on pH, electrolyte, and other solution conditions. In a few instances, bare gold ( 3 ) , platinum ( 3 ) ,and silver (6) have been reported to give rela0 1988 American Chemical Soclety
2284
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15. 1988
tively rapid electron transfer for cytochrome c; but the response a t these electrodes was usually short-lived and depended on very specific electrode pretreatment and protein purification procedures. An alternative approach that has been successfully applied to obtain electrochemical information concerning enzymes and proteins has involved the use of low molecular weight “mediator titrants” (7-9). These species, which themselves exhibit ideal reversible electrode behavior, are added to the protein solution in low concentration and serve as electron transfer intermediaries by carrying out the chemical oxidation or reduction of the macromolecule away from the electrode surface. Thus, the mediator is electrolyzed at its characteristic potential and, in a succeeding chemical step, effects the desired redox conversion of the protein. At this point in time, dozens of mediator compounds, possessing redox potentials spanning the range from +LO to -0.8 V vs NHE, have been characterized electrochemically, and their applications with a wide variety of proteins and enzymes have been summarized (9). In light of the recent developments that have occurred in the chemical modification of electrode surfaces, a logical extension of the mediator titrant approach would be the construction of CMEs using proven solution-phase mediators as the electrode-modifying entities. If properly designed, such electrodes could provide a mediated or electrocatalytic response toward a target protein a t the redox potential of the mediator but without requiring the actual addition of the mediator to the sample solution. Furthermore, incorporation of a single mediator might produce a CME active toward several different proteins possessing similar redox potentials. A few CMEs designed with such a function in mind have recently been reported. The most frequent target of these studies has been the 12000 molecular weight redox protein cytochrome c for which reversible and quasi-reversible response has been obtained at several different CMEs. In this regard, Lewis and Wrighton (10) and Elliott and Martin (11) devised viologen-modified silicon and glassy carbon electrodes, which showed electrocatalytic response toward cytochrome c a t -0.5 V vs. SCE, and CMEs formed by adsorption of numerous bipyridyl “promoters” onto gold and platinum (12-16) have been shown to exhibit activated response toward this protein. In addition, electropolymerization of viologens onto gold ( I 7-19) yielded electrodes that possessed quasireversible activity toward cytochrome c , myoglobin, and ferredoxin. The functioning of these latter CMEs, however, was thought to involve adsorption-related mechanisms rather than direct electron transfer mediation. In this work, we consider the construction of a new mediator titrant-based CME intended to provide an electrocatalytic response to myoglobin, hemoglobin, and perhaps other hemoproteins as well. The modifiers used for this purpose were the phenothiazine methylene blue and several related mediator titrant compounds that are well-known for their capability to effect the reduction of these proteins in solution. (See Figure 1for structures.) Characterization of the performance of these CMEs in both static and flow experiments was carried out for the purpose of evaluating their potential as analytical sensors for the above group of redox proteins. EXPERIMENTAL SECTION Reagents. Myoglobin (Type I, from horse skeletal muscle) and hemoglobin (from human blood), both in oxidized form, were obtained from Sigma Chemical Co. as were cytochrome c (Type VI, from horse heart), glucose oxidase (Type X, from Aspergillus niger), catalase (from bovine liver), ferredoxin (Type VI, from red marine algae), and ceruloplasmin (Type X, human). Methylene blue, thionine, Meldola Blue, and phenazine methosulfate were purchased from Mallinckrodt or Aldrich Chemical Co. and were used as received without further purification. All buffer/ electrolyte components were reagent grade or equivalent, and
Methylene Blue
Thion ine
Meldola Blue
PMS C H,
Figure 1. Structures of methylene blue, thionine, Meldola Blue, and phenazine methosulfate (PMS). deionized water was used for all solutions. All solutions were deaerated prior to use by thorough degassing with prepurified nitrogen. Working Electrodes. The glassy carbon working electrode (3-mm diameter) used in some cyclic voltammetry experiments was purchased from Bioanalytical Systems,Inc. (West Lafayette, IN). Graphite electrodes were prepared in-house by press-fitting 5 mm diameter rods of spectroscopic graphite (Grade AGKSP, Fischer Scientific) into a Teflon or Plexiglas holder so that, upon immersion, only the flat end of the rod contacted the solution. When used in LCEC, the graphite rod was sanded down to 3-mm diameter and placed in the top half of a conventional thin-layer flow cell assembly. Before use in electrochemical experiments and also before the start of modification procedures, the graphite surface was polished on fine emery paper and then rinsed thoroughly with water. Modified graphite electrodes were then prepared by a dip-coating approach similar to that suggested by Gorton et al. (20). The polished graphite surface was simply immersed for 60 s in a stirred 0.01% (w/v) solution of the modifying agent in 0.10 M pH 5.3 phosphate buffer and then rinsed thoroughly with water. Apparatus. Cyclic voltammetry was performed with a Bioanalytical Systems Model CV-1B potentiostat and a Hewlett-Packard Model 7035B X-Y recorder. All experiments employed a three-electrode cell with a saturated Ag/AgCl reference electrode and a platinum wire auxiliary electrode. Flow injection and high-performance liquid chromatography experimentswere conducted with a Beckman Model llOB pump, a Rheodyne (Berkeley, CA) Model 7125 injector with a 20-pL sample loop, and an IBM Model EC/230 amperometric detector connected to a Bioanalflical Systems thin-layer cell. Occasionally, detection was also carried out by monitoring the absorbance at 254 nm with a Waters Associates Model 440 UV-visible absorbance detector. Chromatographywas performed with a 250 mm long, 4.6 mm i.d. SynChrom, Inc. (Linden, IN), GPC-100 size exclusion column containing 5-pm glycerolpropylsilane-bonded silica particles with 100-8, pores. The mobile phase for all chromatographywas 0.20 M sodium acetate adjusted to pH 5.45 with acetic acid. Unless indicated otherwise, the mobile phase flow rate was always 0.5 mL/min. RESULTS AND DISCUSSION Myoglobin Electrochemistry. Myoglobin (molecular weight 16 700) is found primarily in mammalian muscle tissue and is the protein responsible for O2 storage and transport within these tissues. The molecule contains a single iron protoporphyrin or heme moiety and, because of the different redox states available to this group, has frequently been the object of electrochemical investigation. The most successful previous studies have employed mercury electrodes (21), indium oxide electrodes (22), and viologen/gold CMEs (18,19). Typical behavior obtained a t a conventional glassy carbon electrode is illustrated in Figure 2 (curve b) by the cyclic
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
2265
e
a
b
T
\
I
V
I
0.4
I
0.2
0
-0.4
E / V v s Ag/AgCI Figure 3. Cyclic voltammograms of methylene blue CME in 0.10 M HCI solution for scan rates of (a) 12.5 mV/s, (b) 25.0 mV/s, (c) 50.0 mV/s, (d) 100 mV/s, and (e) 200 mV/s. 1 " " " " " ~ 0.4
0.2
EiV
0
-0.2
-0.4
-0.6
VS Ag/AgCI
Flgure 2. Cyclic voltammograms obtained at glassy carbon for (a)pH
M 5.45 acetate buffer, (b) 0.1 mM myoglobin in buffer, (c) 2.0 X methylene blue in buffer, (d) 0.05 mM (-) or 0.2 mM (- - -) myoglobinl2.0 X loJ M methylene blue in buffer. Scan rate was 20 mV/s.
voltammogram (CV) for a 0.10 mM myoglobin solution. Only a single wave, a broad reduction centered at -0.4 V vs. Ag/ AgCl, was observed. In view of the fact that an identical wave was also seen for CV scans run in blank buffer with no myoglobin present (curve a), the reduction was attributed to the glassy carbon background and not to any redox process involving the protein itself. The effect of adding a mediator titrant species such as methylene blue to the myoglobin solution is also shown in Figure 2. Methylene blue alone in solution gave rise to a reversible pair of redox waves with a cathodic peak potential E , of -0.11 V and an anodic peak potential E,, of -0.07 V vs. Ag/AgCl (curve c). This behavior is similar to that which has previously been reported for methylene blue (23) and corresponds presumably to the following reaction:
Addition of myoglobin to the methylene blue solution and subsequent scanning over the same potential range (curve d) produced no new CV waves but only an increase in the height of the methylene blue reduction and a correspondingdecrease in the accompanying oxidation-both of which effects were directly dependent on the myoglobin concentration added. Note that this is exactly the CV behavior to be expected for a catalytically mediated electrode reaction. CME Construction. Initial efforts aimed at immobilizing methylene blue onto an appropriate electrode substrate in a
manner that retained the molecule's mediating capabilities focused on the carbon paste electrode modification approach. However, the aqueous solubility of methylene blue was sufficiently high that CMEs formed by simply mixing the modifier into carbon paste were too unstable for practical long-term usage. Thus, even though carbon paste surfaces containing methylene blue initially exhibited a CV response characteristic of the modifier, the paste rapidly lost methylene blue upon immersion into aqueous buffer solution and continued scanning. This was evidenced not only by a marked decrease in the modifier redox current upon continued CV scanning but also by the formation of the intense blue color of the modifier in the solution surrounding the CME surface. An alternative modification strategy involved chemisorption of the methylene blue onto graphite. Such an approach possessed some appeal for use because of the success reported by Gorton et al. in immobilizing somewhat similar polyheterocyclic aromatic compounds-most notably, 7-(dimethylamino)-1,2-benzophenoxazine,commonly called Meldola Blue-by means of a simple dip-coating procedure (20). The resulting CMEs were quite stable under a variety of solution conditions and mediated the oxidation of the nicotinamide coenzymes NADH and NADPH at very modest overpotentials. It was our hope that a similar immobilization, presumably arising from interaction between the aromatic rings of the modifier and the graphitic structure of the electrode, would occur for the phenothiazine methylene blue. In fact, a CME prepared (as described in detail in the Experimental Section) by briefly exposing a polished graphite surface to an aqueous solution of the methylene blue did exhibit stable cathodic and anodic waves that behaved exactly as expected for a reversible, surface-immobilizedredox couple. This behavior is illustrated in Figure 3 by typical CVs obtained at several potential scan rates for the CME immersed in 0.10 M HCl. Both the cathodic and anodic waves were nearly symmetrical in shape and, at slow scan rates, occurred at nearly the same potentials, E,, = +0.06 V and E,, = +0.07 V vs. Ag/AgCl. These waves were shifted to positive potentials
2286
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
compared to those seen in Figure 2 for solution-phase methylene blue because of the pH difference of the two electrolysis media employed. Upon investigation, increases in solution pH were found to result in a negative shift of 59 mV/pH unit for both the cathodic and the anodic waves up to pH 4.4 but of only 29 mV/pH unit for higher pH values. Finally, peak currents observed for both processes increased linearly as the potential scan rate was increased over the accessible range from 10 to 200 mV/s. The CME used to obtain the results shown in Figure 3 had been prepared with a 60-s dip-coating exposure to a 0.01% methylene blue solution. The use of longer exposures produced no further increases in the modifier CV currents and therefore gave no apparent advantage. Integrating the area under the methylene blue redox waves CVs in Figure 3 and taking into account the geometric surface area of the electrode gave an approximate surface coverage of 8.8 nmol/cm2. Presumably, this coverage corresponds to what can be considered a saturated graphite surface. The stability of the methylene blue CMEs was tested in a variety of ways. Extended cycling of the potential across the modifier redox wave was possible in numerous electrolyte/buffer systems, ranging from 0.10 M HC1 to pH 8.0 phosphate buffer. In pH 5.45 acetate buffer, for example, 10 h of scanning between +0.30 and -0.36 V vs. Ag/AgCl produced no significant decrease in cathodic and anodic peak currents; this included more than 500 complete CV cycles. Also, CV experiments could be suspended and the electrode then held continuously at either +0.30 or -0.12 V with no observable decrease in peak heights seen upon resumption of CV scans. Furthermore, the CME appeared to possess good mechanical stability as rotating the electrode or stirring the solution with a magnetic stirbar had little effect on the magnitude or longevity of the CME response. Finally, in cases where a fresh electrode surface was desired, the old surface could be removed by simply polishing the old CME surface on fine emery paper for 30 s and then repeating the 60-9 dip-coating procedure. New surfaces usually exhibited very reproducible methylene blue currents; a series of eight CMEs generated in this manner and immersed in 0.10 M HC1 gave an average cathodic peak current of 38.9 pA with a relative standard deviation of only 3.2%. In addition to methylene blue, the potential mediators thionine, Meldola Blue, and phenazine methosulfatecould also be immobilized on spectroscopic graphite by the same dipcoating method. The resulting CMEs were stable and behaved roughly the same as the methylene blue CME had except, of course, that the resulting redox waves occurred at the potentials characteristic of the specific modifier used: thionine, E , = -0.19 V and Ew = -0.14 V; phenazine methosulfate, E, = -0.04 V and E , = +0.03 V; and Meldola Blue, E, = -0.08 V and Ew = -0.04 V (all in pH 5.45 acetate buffer at 20 mV/s). CME Electrocatalysis. Once the construction of stable CMEs containing surface-immobilizedmediator titrants had been clearly demonstrated, it was of interest to determine whether or not these electrodes exhibited the electrocatalytic capabilities of the solution-phase forms of the mediators. In these studies, both CV and liquid chromatography/electrochemical detection (LCEC) techniques were employed to confirm and characterize the resulting CME behavior first toward myoglobin and then toward a few other redox proteins. CVs obtained with a methylene blue CME and run in pH 5.46 acetate buffer both with and without added myoglobin are shown in Figure 4. The voltammogram obtained in the blank buffer exhibited cathodic ( E , = -0.20 V vs. Ag/AgCl) and anodic ( E , = -0.17 V) redox waves at potentials approximately 100 mV more negative than those seen in Figure 2 for solution-phase methylene blue under identical pH con-
I
0.3
- 0.2
0
I
-0.4
E / v vs Ag/AgCI Figure 4. Cyclic voltammograms of CME in (a)pH 5.45 acetate buffer, (b) 0.05 mM myoglobin in buffer, and (c)0.20 mM myoglobin in buffer. Scan rate was 20 mVls.
I
Figure 5. Stability of CME in flow injection. Each peak represents injection of 1 nmoi of myoglobin. ditions. In addition, both waves were much smaller in magnitude for the CME and were superimposed on a relatively large cathodic background. Of course, as would be expected for a reversible surface-bound redox process, both the oxidation and the reduction at the CME occurred at nearly the same potential. The voltammograms obtained upon addition of myoglobin showed an increase in the height of the methylene blue reduction current and a decrease in its oxidation current, with no change in the potentials of the two electrode processes. The extent of both the reduction increase and the oxidation decrease was proportional to the myoglobin concentration employed. Again, such behavior is exactly what would be anticipated for an electrocatalytic CME reduction. The myoglobin electrocatalysis could also be carried out under flow conditions. As shown in Figure 5 for a series of flow injection experiments performed in a conventional thin-layer LCEC cell (but with no chromatographic column in place), distinct myoglobin peaks were observed at the CME as long as the applied potential was maintained in the range of the methylene blue reduction. For the signals obtained in this figure, the applied potential was -0.12 V vs. Ag/AgCl. The peaks themselves exhibited a moderate amount of tailing and therefore were slightly broader than would have been dictated by simple diffusional band spreading of the sample plug during the flow process. Presumably, the broadening was due to a rather slow interaction between the myoglobin and the somewhat porous CME surface. Furthermore, following a brief initial period in which the CME response de-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
2267
Table I. Response of Redox Proteins at Methylene Blue CME redox protein myoglobin hemoglobin cytochrome c catalase glucose oxidase ferredoxin ceruloplasmin
ic lnAl
E0’(vs NHE),’ 0.04V 0.17’ 0.25c -0.4Ie 0.49, 0.5ad
CME response?
i,,b nA
Yes yes yes Yes no no no
110 87 47 5.8
“At pH 7.0. bThese are flow injection currents observed for injection of 1.0 nmol of each protein. CME was maintained at E = -0.12 V vs. Ag/AgCl. CFromref 25. dFrom ref 26.
I
0.2
0.1 0 POTENTIAL, v
VS
-0.1 -0.2 Ag/AgCI
-0.3
Flgure 6. Hydrodynamic voltammograms obtained at the methylene blue CME for Injections of (a) 0.5 nmol of myoglobin and (b) 0.5 nmol of hemoglobin.
creased rapidly, myoglobin peak heights subsequently remained nearly the same. As a result, the final CME response was still 70% of its initial level after 8 h of continuous exposure to the flowing stream. In fact, most of the decrease occurred in the first hour of the CME usage, and from the third to the eighth hour of a typical flow experiment, the CME response dropped by less than 10%. In large part, the decrease in response appeared to be caused by the slow leaching of the methylene blue modifier from the CME surface. Supporting this hypothesis were the observations that (a) the decreases in peak height were related primarily to the length of the electrode’s service in the flow stream and were not dependent on the number of myoglobin injections made and (b) the CME’s initial response could be completely regenerated simply be reimmersing the electrode into the 0.01% methylene blue solution. In any case, once allowed to equilibrate in the flow stream, the electrode stability was such that, after 3 h of continuoususe, the relative standard deviation for 10 successive myoglobin injections was only 5%. The potential dependence of the CME response toward myoglobin is summarized by the hydrodynamic voltammogram (HDV) shown in Figure 6 . This HDV, obtained from a series of flow injection experiments performed with the CME held at different applied potentials, closely matched the CVs seen earlier for the reduction of methylene blue both in solution and adsorbed onto the graphite electrode surface. Thus, myoglobin reduction was seen only at potentials more negative than 0.0 V and produced the maximum current levels at -0.16 V and beyond. Also shown in Figure 6 is the HDV resulting for hemoglobin, the 64500 molecular weight redox protein that has four heme-containing subunits and is responsible for O2uptake and transport in blood (24). In view of the similarity of the two proteins’ structures, it was expected that the methylene blue CME would exhibit very similar response toward hemoglobin as it had for the smaller heme protein. This, in fact,
turned out to be the case in both CV and flow injection experiments. The principal difference appeared to be the somewhat smaller currents elicited by hemoglobin on a permole basis. The reason for the decreased hemoglobin response, which occurred despite the fact that this protein contains four heme groups compared to myoglobin’s one, has not yet been determined. The electrocatalytic ability of CMEs containing the adsorbed mediators thionine, Meldola Blue, and phenazine methosulfate was also investigated by CV and flow injection analysis. Of these electrodes, only that made with thionine exhibited any observable activity toward myoglobin or hemoglobin. Interestingly, of the three modifiers, thionine’s redox potentials most closely matched those of methylene blue. However, as the currents obtained with the thionine CMEs for a given myoglobin concentrationwere distinctly lower than those seen with the methylene blue CMEs, virtually all of the work reported here utilized the latter electrodes. Finally, several redox proteins in addition to myoglobin and hemoglobin were also examined via flow injection experiments with the methylene blue CME. These included the hemecontaining proteins catalase and cytochrome c and the nonheme proteins ferredoxin, glucose oxidase, and ceruloplasmin. Interestingly, only the hemoproteins showed evidence of reduction at the CME, though none produced as much current per mole as did myoglobin or hemoglobin. Furthermore, the proteins that showed no activity at the CME also showed no evidence of solution-phase catalysis by methylene blue either in this work or, to our knowledge, in the earlier mediator titrant literature. The results, summarized in Table I, are currently the subject of continued investigation in our laboratory. LCEC Applications. The fact that the methylene blue containing CMEs were able to function successfully in sensing myoglobin and other redox proteins in the flow injection experiments described above offered real promise that the use of these electrodes might make LCEC of these compounds possible. In fact, when myoglobin and hemoglobin were injected onto a size exclusion column, both were able to be detected via reduction at the methylene blue CME. With a pH 5.45 acetate buffer as mobile phase, optimum response for each of the proteins was obtained at an applied potential of only -0.12 V vs. Ag/AgCl. (Although larger currents were produced at more negative potentials, the background noise was also much worse, and no net signal/noise advantage was realized as a result.) For myoglobin, the response at -0.12 V was linear from 20 pmol injected up to 2800 pmol (i = (105 pA/pmol)C + 4.8 nA for eight concentrations over this range; correlation coefficient >0.99), and the detection limit corresponding to a signal/noise ratio of 3 was 10 pmol. For hemoglobin, the linear range extended from 50 pmol injected to 1000 pmol (i = (85 pA/pmol)C + 2.5 nA for five concentrations; correlation coefficient >0.99), and the limit of detection was approximately 20 pmol.
2268
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
However, in view of the relatively modest potential required for electrochemical detection of myoglobin and hemoglobin a t the CME, far fewer sample constituents are expected to be reducible under the analysis conditions employed. One of the possible analytical applications for the methylene blue CME would involve the determination of myoglobin in blood. This determination, however, has not yet been attempted. In view of the fact that the concentration of myoglobin in normal blood smaples is only 10-100 ng/mL, detection limits improved over those currently available with the methylene blue CME in its present form would be required. Such improvements in CME performance are currently being pursued in our laboratory. In addition, possible applications involving the determination of proteins other than myoglobin and hemoglobin are also under active investigation.
2
T
0.05 AU
1
LITERATURE CITED
i" : I 0
"
" 8
"
" 16
"
"
" 24
" 32
40
48
56
64
Retention T i m e (min) F l g w 7. Chromatograms obtalned by (a)UV detection at 254 nm and (b) CME detection at -0.12 V vs. Ag/AgCI. Quantity Injected was 0.5 nmol of Mb (peak 1) and 0.5 nmol of Hb (peak 2). Mobile phase flow rate was 0.1 mL/min. A typical chromatogram obtained for a myoglobin/hemoglobin mixture subjected to size exclusion chromatography is shown in Figure 7 . Also shown for purposes of comparison is the signal obtained for the same separation via 254-nm absorbance monitoring. Although the resolution of the two proteins could have been improved somewhat by use of a different pore-size stationary phase and the fact that the smaller of the proteins actually eluted f i s t indicates that the retention mechanism in effect involved more than simple size exclusion, it is clear that the electrochemical approach offers detectability roughly comparable to that obtained via the usual absorption approach. For example, detection limits for the two methods would probably be quite similar. For determinations performed in practical physiological samples, however, the CME/LCEC method might present a distinct advantage in selectivity. Numerous high molecular weight biomolecules absorb strongly at 254 nm and would likely produce severe interference in the UV detection scheme.
(1) Yeh, P.; Kuwana, T. Chem. Lett. 1977, 1145. (2) Bowden, E. F.; HawkrMge, F. M.; Chlebowskl, J. R.; Bancroft. E. E.; Thorpe, C.; Blount, H. N. J. Am. Chem. Soc. 1982. 104, 7641. (3) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. J. fI8ctroanal. Chem. 1984, 161, 355. (4) Armstrong, F. A,; Hill, H. A. 0.; Ollver, B. N. J . Chem. Soc., Chem. Commun. 1984, 976. (5) Harmer, M. H.; Hill, H. A. 0. J . Elecboanal. Chem. 1985, 189, 229. (6) Reed, D. E.; HawkrMge, F. M. Anal. Chem. 1987, 59, 2334. (7) HawkrMge. F. M.; Kuwana, T. Anal. Chem. 1973, 45, 1021. (8) Szentrimay, R.; Yeh, P.; Kuwana, T. I n EieclrochemicalShrdles of B b l o g l ~ Systems; i Swayer, D. T., Ed.; ACS Symposium Serles 3 8 American Chemical Society: Washington, Dc. 1977: p 143. (9) Fuh, M. L.; Durst, R. A. Anal. Chlm. Acta 1982, 140, 1. (IO) Lewis, N. S.; Wrlghton, M. S. Sc/ence(Wesh/ngton, D.C.) 1981, 271, 944. (11) Elliott, C. M.; Martin, W. S. J . Electroanal. Chem. 1982, 137, 377. (12) Eddowes, M. J.; Hili, H. A. 0. J . Chem. Soc., Chem. Commun. 1977, 771. (13) Eddowes, M. J.; Hill, H. A. 0. J . Am. Chem. SOC. 1978, 101, 4461. (14) Tanlguchi, 1.; Toyosawa, K.; Yamaguchl. H.; Yasukouchl, K. J . Chem. Soc., Chem. Commun. 1982, 1032. (15) Tanlguchi, I.; Toyosawa, K.; Yamaguchi, H.; Yasukouchl, K. J. Eiectroanal. Chem. 1982. 140, 187. (16) Allen, P. M.; Hill, H. A. 0.; Walton, N. J. J . Elecboanel. Chem. 1984, 178, 69. (17) Landrum, H. L.; Salmon, R. T.; Hawkridge, F. M. J . Am. Chem. SOC. 1977, 99, 3154. (18) Stargardt, J. F.; Hawkrkige, F. M.; Landrum, H. L. Anel. Chem. 1978, 50, 930. (19) Castner, J. F.; Hawkrkige, F. M. J . flec~oanal.Chem. 1883. 143, 217. (20) W o n . L.; Torstensson, A.; Jaegfeldt, H.; Johansson, G. J. Electroemi. Chem. 1984, 161. 103. (21) Scheller, F.; Janchen, M.; Lampe, J.; Prumke, H.J.; Blanck, J.; Palecek, E. Blochlm. Wophys. Acta 1975, 412, 157. (22) Klng, 8. C.; Hawkridge, F. M. Book of Abstracts; 194th Natlonal Meetlng of the Amerlcan Chemical Society, New Orleans, L A AmerC can Chemical Soclety: Washington, DC. 1987; ANYL 16. (23) Wopschall, R. H.; Shain, I. Anal. Chem. 1967, 39, 1527. (24) Harper, H. A.; Rodwell, V. W.; Mayes, P. A. Review of i%yslologlcal Chemlsby, 17th ed.; Lange Medlcal Publlcatlons: Los Altos, CA, 1979 p 201. (25) Mahler, H. R.; Cordes, E. H. Bb/oghl Chemlsby, 2nd ed.;Harper and Row: New York, 1971. (26) Deinum, J.; Vanngard, T. Bkxhlm. Blophys. Acta 1973, 370, 321.
RECEIVED for review November 6,1987. Accepted July 1,1988. This work, which was presented in part at the 194th National Meeting of the American Chemical Society in New Orleans, LA, was supported by the National Science Foundation through EPSCoR Grant 86-10671-01.
