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Technical Notes
Carbon Electrodes Modified with Ruthenium Metallodendrimer Multilayers for the Mediated Oxidation of Methionine and Insulin at Physiological pH Long Cheng, Gilbert E. Pacey, and James A. Cox*
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056
The electrochemical oxidation of insulin is of interest because it can serve as a basis for amperometric detection coupled to flow systems1 and can provide a means of monitoring cellular processes.2 Perhaps the first reported study in aqueous solution was one in which a mixed-valent ruthenium oxide with cyano crosslinks, mvRuOx, in the form of a film on glassy carbon electrode served as the catalyst.1 The surface modification was by cyclic voltammetry of a pH 2 solution of 2 mM RuCl3/2 mM K4Ru(CN)6 over the range 0.5-1.1 V versus Ag|AgCl.1,3 Flow injection amperometry (FIA) at pH 2.0 permitted determination of insulin over the range 0.2-2.0 µM at a sensitivity of 21 nA µM-1; the
detection limit (k ) 3 criterion) was 47 nM. This catalyst was used subsequently on a fiber electrode to electrochemically monitor insulin release from β-pancreatic cells;2 however, the study was limited by the instability of the mvRuOx film at pH values above ∼5.2,3 Apparently the oxidized form of mvRuOx slowly dissolves in aqueous solution except at low pH values. Kennedy and co-workers4 investigated potential catalysts for the oxidation of insulin at physiological pH. Polynuclear hexacyanometalates, binary metal oxides, and metallophthalocyanines with various metal centers were tested using Ru, Cr, Fe, Co, and Os alone or in combination with Pb, Pd, and Ir. Films originating from the oxidation of RuCl3 or (NH4)RuCl6 promoted the reaction. A ruthenium oxide film deposited on glassy carbon from a 0.2 mM RuCl3/10 mM HClO4 mixture permitted the amperometric determination of insulin at pH 7.4 at the submicromolar level over a 4-day period during which the electrode was stored in 0.1 M KNO3 between experiments. The detection limit of an FIA experiment with this electrode was 23 nM insulin with a pH 7.4 phosphate buffer as the carrier solution. Recently, an iridium-based catalyst for the electrochemical oxidation of biochemical compounds was reported.5 An iridium oxide film, IrOx, that is formed on glassy carbon during cyclic voltammetry of 0.20 mM IrCl63- in 0.10 M KNO3 mediated the oxidation of insulin at pH 7.4 at 0.70 V versus Ag/AgCl. An FIA calibration curve was linear over the range 0.05-0.5 µM. The sensitivity and detection limit were 35 nA µM-1 and 20 nM, respectively. We are exploring the use of the layer-by-layer (LBL) method6 of immobilizing catalytic centers on electrodes. An important attribute of the LBL method is that it provides a rapid route to forming extended arrays. Commonly, polyelectrolytes are employed as molecular spacers. Representative examples include the immobilization of phosphomolybdate in alternating layers with either polypyrrole7 or a bipolar pyridine salt.8 The former system
* To whom correspondence should be addressed: (phone) 513-529-2493; (fax) 513-529-5715; (e-mail)
[email protected]. (1) Cox, J. A.; Gray, T. J. Anal. Chem. 1989, 61, 2462-2464. (2) Kennedy, R. T.; Huang, L.; Atkinson, M. A.; Dush, P. Anal. Chem. 1993, 65, 1882-1887. (3) Gorski, W.; Cox, J. A. J. Electroanal. Chem. 1995, 389, 123-128.
(4) Gorski, W.; Aspinwall, C. A.; Lakey, J. R. T.; Kennedy, R. T. J. Electroanal. Chem. 1997, 425, 191-199. (5) Pikulski, M.; Gorski, W. Anal. Chem. 2000, 72, 2696-2702. (6) Decher, G. Science 1997, 277, 1232-1237. (7) Sun, C.; Zhao, J.; Xu, H.; Sun, Y.; Zhang, X.; Shen, J. J. Electroanal. Chem. 1997, 435, 63-68.
A pentaerythritol-based metallodendrimer with ruthenium(II) terpyridine units, RuIIDen, catalyzed the oxidation of L-methionine and insulin at pH 7.0. The RuIIDen was immobilized on a carbon surface through layer-bylayer electrostatic deposition; the negatively charged polymer, poly(styrene sulfonate), was its counterpart. These bilayers were assembled on a glassy carbon electrode that was first modified by deposition of a layer of the conjugate base of sulfanilic acid and then with quaternized poly(4-vinylpyridine). Reversible voltammetry for the RuII/III redox couple was observed, the current for which increased linearly with layer number, n, of RuIIDen up to n ) 12. Cyclic voltammetry was used to demonstrate the mediation of L-methionine oxidation by a RuIIDen-containing multilayer assembly. Flow injection amperometric determination of insulin at pH 7.0 at this modified electrode yielded a calibration curve with the following characteristics: linear dynamic range, 6 nM0.4 µM; sensitivity, 225 nA µM-1; detection limit (k ) 3 criterion), 2 nM. Of particular importance was that the sensitivity was proportional to the number of RuIIDen layers.
10.1021/ac0105585 CCC: $20.00 Published on Web 09/28/2001
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was used for the FIA determination of hydrogen peroxide;7 the latter was the basis of an analogous study of the electrocatalytic oxidation of ascorbic acid8 with these modified electrodes. We recently described the preparation of multilayer nanocomposites consisting of phosphomolybdate and poly(amidoamine)9 and applied these assemblies to the reduction of nitrite and the oxidation of arsenite. The LBL method has the potential to immobilize catalytic centers in a manner that provides greater spatial integrity than bulk composite electrodes and greater utilization of metal centers than can be attained with surface-modified electrodes such as IrOx and mvRuOx. The ability to control the thickness and structure of multilayer films provides an approach to optimizing analytical methodology based on electrocatalysis at modified electrodes. In this regard, Dong and co-workers10,11 reported that the current for the electrocatalytic reduction of HNO2 at an electrode modified by LBL deposition of silicotungstate increased with the number of layers. The present study is an evaluation of the LBL method for the immobilization of ruthenium centers. Because of the importance of developing methods for insulin determination at physiological pH, it was selected as the analyte for this study. EXPERIMENTAL SECTION Chemicals. All chemicals were ACS Reagent Grade unless otherwise stated. Ethanol, acetonitrile, and monobasic potassium hydrogen phosphate were from Fisher Scientific (Fair Lawn, NJ); and sulfanilic acid (SAA) and poly(styrene sulfonic acid) (PSS) were from Alfa Aesar (Ward Hill, MA). Lithium perchlorate, poly(4-vinylpyridine), cross-linked, methyl chloride quaternary salt, and poly(dialkyldimethylammonium chloride (PDDA), low molecular weight (100 000-200 000), 20% in water, were from Aldrich Chemical Co (Milwaukee, WI). The L-methionine (>99%) and bovine insulin (5800, >27 USP units mg-1) were from Sigma (St. Louis, MO). A stock solution of 0.5 mM insulin was prepared in water, using HCl to adjust the pH to 2.0. Serial dilutions with pH 7.0 buffer (0.1 M phosphate) were performed to prepare the standards for measurement. The pH of the diluted stock was verified by measurement. The water used in this work was housedistilled that was further purified with a Barnstead NANOpure II system to a resistance greater than 17.2 MΩ cm. All solutions were sparged with nitrogen gas for at least 10 min before use. The pentaerythritol-based metallodendrimer with four ruthenium(II) terpyridine subunits, RuIIDen, was prepared by a published method.12 The purification and characterization of RuIIDen were described in a previous report.13 Instrumentation. Voltammetry experiments were performed with a CH Instruments model 750 electrochemical workstation (Austin, TX) in a conventional three-electrode cell. The working electrode was glassy carbon (3-mm diameter) from Cypress Systems (Lawrence, KS). Prior to use, the electrode surfaces were polished successively with 1.0, 0.3, and 0.05 µm R-alumina powder (Mark V Laboratory, East Granby, CT) and sonicated in water. The counter electrode was a large area platinum wire. The (8) (9) (10) (11) (12)
Sun, C.; Zhang, J. Electrochim. Acta 1998, 43, 943-950. Cheng, L.; Cox, J. A. Electrochem. Commun. 2001, 3, 285-289. Cheng, L.; Dong, S. J. Electroanal. Soc. 2000, 147, 606-612. Cheng, L.; Liu, J.; Dong, S. Anal. Chim. Acta 2000, 417, 133-142. Constable, E. C.; Housecroft, C. E.; Cattalini, M.; Phillips, D. New J. Chem. 1998, 22, 193-200. (13) Holmstrom, S. D.; Cox, J. A. Anal. Chem. 2000, 72, 3191-3195.
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reference electrode was Ag|AgCl from Bioanalytical Systems (West Lafayette, IN), against which all potentials were measured and reported. The formal potentials, Ef, of the redox couples were determined as the average of the cathodic and anodic peak potentials, Epc and Epa. The FIA experiments were conducted with an Eldex Laboratory pump (model A-120-S, Napa, CA), a Rheodyne 7125 injection valve (Cotati, CA), and a CH Instruments model 800 electrochemical detector was a glassy carbon working electrode. The Ag|AgCl reference electrode was placed downstream of the working electrode and the stainless steel counter electrode. Analytical measurements were made at a constant applied potential, 0.95 V. The flow rate was 1.0 mL min-1; the sample volume, 100 µL; and the carrier solution, 0.1 M phosphate buffer at pH 7. Modification of the Glassy Carbon Surface. Initially, a film of SAA was formed on the glassy carbon (GC) electrode surface by the amine cationic radical method.14-16 The GC electrode was continuously scanned at 0.01 V s-1 over the range, 0.0-1.2 V, in 2 mM SAA, 0.1 M LiClO4 with ethanol as the solvent. An irreversible oxidation peak was observed around 1.0 V, which is attributed to oxidation of the amino group of SAA to its cation radical. The anodic peak current gradually diminished with scan number, indicating that a SAA film was covalently grafted to glassy carbon. The modified surface was sonicated consecutively in ethanol and water. The SAA film served as a precursor to the multilayer film. The terminal sulfonate group of the SAA film provided a negatively charged surface to which quaternized poly(4-vinylpyridine), qPVP, was deposited onto the SAA film by immersion in 1 mg mL-1 qPVP for 30 min at open circuit. A PSS overlayer was deposited by ion exchange on the positively charged GC|SAA|qPVP surface by immersion for 30 min in 4 mg mL-1 PSS at pH 7.0 (phosphate buffer). After rinsing with water, the PSS-coated electrode was immersed for 30 min in 0.3 mg mL-1 RuIIDen with acetonitrile as solvent to deposit a positively charged RuIIDen layer. The electrode was dried with a stream of nitrogen after each step. Repetition of the immersions in PSS and RuIIDen yielded multilayers of these compounds on the glassy carbon electrode. The electrode assembly was stable for more than 1 week as measured by the cyclic voltammetric current for redox of the RuIIDen in 1 M H2SO4, decreasing by less than 5% over the period. RESULTS AND DISCUSSION RuIIDen (Figure 1) has properties that suggest its utility as a mediator for electrochemical oxidations. In acetonitrile, RuII undergoes a reversible, one-electron oxidation at 1.05 V versus a Pt quasi-reference electrode.13 As a component of a conducting composite electrode (CCE), it mediated the oxidation of methionine at pH 7, and in homogeneous solution (12% acetonitrile, 88% phosphate buffer at pH 11), RuIIDen mediated the oxidation of insulin.13 The ability to restore activity of CCEs by polishing the surface makes these electrode suited for many applications. However, both CCEs and homogeneous solutions of catalysts are inefficient in terms of utilization of materials such as RuIIDen that are expensive and/or difficult to synthesis and purify. The present (14) Deinhammer, R. S.; Ho, M.; Anderegg, J. W.; Porter, M. D. Langmuir 1994, 10, 1306-1313. (15) Downard, A. J.; Mohamed, A. Electroanalysis 1999, 11, 418-423. (16) Liu, J.; Cheng, L.; Liu, B.; Dong, S. Langmuir 2000, 16, 7471-7476.
Figure 3. Cyclic voltammograms of a glassy carbon electrode modified with nine bilayers of RuIIDen and PSS in the (a) absence and (b) presence of 0.2 mM L-methionine. Electrolyte, 0.2 M phosphate buffer (pH 7.0); scan rate, 0.02 V s-1. Figure 1. Structures of (A) RuIIDen and (B) PSS.
Figure 2. Cyclic voltammograms of a glassy carbon electrode modified with RuIIDen and PSS multilayers: GC|SAA|qPVP|(PSS|RuIIDen)n, n ) 2, 4, 6, 8, 10, 12. Electrolyte, 0.2 M Na2SO4 (pH 6.8); scan rate, 0.1 V s-1.
study was initiated to develop and evaluate a LBL method of attaching this compound to an electrode surface in a controlled, stable manner. The formation of the GC|SAA|qPVP|PSS assembly, which uses known procedures, was to provide a stable, negatively charged surface to which the cationic RuIIDen can be bound electrostatically. The direct binding of RuIIDen to SAA also is possible, but the overlayer of qPVP on the GC|SAA platform increases the binding sites and leads to a more regular assembly. The data in Figure 2 demonstrate not only that LBL accumulation of RuIIDen occurs but also that the quantity of RuIIDen, which is in alternating layers with PSS, is proportional to layer number, n, through at least 12 cycles. In turn, n is directly related to the number of applications described in the Experimental Section.9 A linear leastsquares fit of the current for the oxidation of RuII versus the number of layers of RuIIDen yielded the following: slope, 73 ( 1 nA n-1; intercept, 7 nA; and r, 0.9992. The multilayer assemblies can be represented by
GC|SAA|qPVP|(PSS|RuIIDen)n
(A1)
A multilayer assembly with n ) 12 yielded cyclic voltammetric currents for the oxidation of RuII that were not perturbed by diffusion. Over the scan rate (v) range, 0.020-1.0 V s-1 (9 points), a linear least-squares fit of peak current versus v had a slope, 11.0 ( 0.1 µA s V-1, and r, 0.9997, that demonstrated direct proportionality between current and scan rate. Apparently the supporting
electrolyte, 0.2 M Na2SO4, penetrates the assembly sufficiently to allow facile counterion mobility, and electron propagation through assembly A1 is not current-limiting over this v range. The analysis based on the data in Figure 2 provided information on the RuIIDen that is electroactive. An analogous study using spectrophotometric measurement showed the same trend. Here, a quartz platform was modified first with PDDA and then by alternating layers of PSS and RuIIDen. The UV-visible absorption spectrum showed maximums at 272, 310, and 490 nm. Over the investigated range the PSS spectrum is featureless, so these maximums are related to the RuIIDen. A linear least-squares fit of the absorbance units, AU, versus n (over the range 2-10 layers) at 310 nm yielded the following: slope, 0.0081 ( 0.0001 AU n-1; intercept, -0.0063 AU; and r, 0.9993. These data verify that the LBL method allowed systematic deposition of both nonelectroactive PSS and electroactive RuIIDen. These data were not used to estimate the amount of RuIIDen on the surface because the thickness and density of the film are unknown. An objective in our investigations of ruthenium-based films on electrodes is to develop methods for the determination of sulfurcontaining biochemical compounds. The electrode represented by assembly A1 with n ) 9 mediated the oxidation of L-methionine, which is not electroactive at a bare carbon electrode under the conditions of this study. In 0.2 M phosphate buffer at pH 7.0, the presence of 0.2 mM L-methionine amplified the anodic peak current at 1.0 V and attenuated the corresponding reduction current (Figure 3). Insulin also was used as a test compound. The FIA of 6.1 nM insulin at a RuIIDen-containing assembly with n ) 6 is illustrated in Figure 4. The corresponding calibration curve over the range 6.1-390 nM insulin has the following characteristics: slope 225 ( 12 nA µM-1; intercept, 20 ( 3 nA; and r, 0.999. The detection limit (k ) 3 criterion) is calculated to be 2 nM. Furthermore, the sensitivity is dependent on the n value of multilayer assembly A1. Under the conditions in Figure 4 but with 1.56 µM insulin, the FIA response increases with the layer number. A plot of peak current versus n (up to n ) 6) has slope, intercept, and r of 26 ( 1 nA n-1, -0.5 nA, and 0.998, respectively. This dependence of response on layer number is consistent with reports on voltammetry at multilayer assemblies that contained polyoxometalates as catalysts.10,11 The above results suggest the potential utility of the electrode represented by assembly A1 for detection of insulin and related Analytical Chemistry, Vol. 73, No. 22, November 15, 2001
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Moreover, the ability to amperometrically determine insulin at physiological pH may find application to in vivo monitoring of insulin release. However, the latter application will require extension of the described LBL method to micrometer-scale electrodes and demonstration of stability when the electrode is continuously exposed to an insulin-containing sample. Presently, these cases are being investigated.
Figure 4. Flow injection amperometry of 6.1 nM insulin at a glassy carbon electrode modified with six bilayers of RuIIDen and PSS. Carrier solution, 0.1 M phosphate buffer at pH 7.0; flow rate, 1.0 mL min-1; working potential, 0.95 V.
biochemical compounds after separation by methods such as highperformance liquid chromatography and capillary electrophoresis.
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ACKNOWLEDGMENT This work was supported in part by the American Chemical Society through grant PRF 33863-AC5. Partial support of L.C. by Miami University as part of the Ohio Micromachined Analytical Chemistry Consortium is appreciated. Received for review May 16, 2001. Accepted August 23, 2001. AC0105585
