Anal. Chem. 2000, 72, 3191-3195
Electrocatalysis at a Conducting Composite Electrode Doped with a Ruthenium(II) Metallodendrimer Scott D. Holmstrom and James A. Cox*
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056
A pentaerythritol-based metallodendrimer with RuIIterpyridine units was synthesized and tested as a mediator for the electrochemical oxidation of methionine (L-Met), cystine (L-Cys), and AsIII. A reversible oxidation of RuII was observed with the metallodendrimer as a solute in mixed acetonitrile-water solvents and as a component of carbonbased conducting composite electrodes. Mediated oxidation of the test species was observed. In aqueous solution, the composite electrode yielded a cyclic voltammetric peak current for the oxidation of L-Met in a 0.1 M phosphate buffer (pH 7.0) at 1.1 V vs Ag/AgCl. This anodic process was employed for amperometric detection in a flow system. Linear calibration curves were obtained over the range 1.0-10 µM Met and Cys. Using the criterion of the concentration yielding a signal 3 times the uncertainty of a blank, detection limits of 0.6 and 0.5 µM were calculated for Met and Cys, respectively. The slopes with three nominally identical electrodes varied by 10%. We are investigating methods of preparing modified electrodes with a combination of high stability and activity at physiological pH toward the oxidation of biochemically important compounds. Various forms of ruthenium oxide are now well known to promote the oxidation of insulin and other sulfur-containing peptides and proteins; however, limited stability except in acidic media is a limitation on the scope of their application.1-4 The incorporation of catalytically active macromolecules in sol-gel materials is a route to meeting our objectives. In this regard, a dirhodiumsubstituted polyoxometalate that is immobilized in a sol-gel material was stable under potentiostatic conditions in flow injection amperometry and catalyzed the oxidation of L-Met at pH 7.5 Metallodendrimers have the merits for our interests of large size and of evenly distributed centers that are potential sites for mediation. To date, few electrochemical studies have been performed on these compounds. Those that have appeared are in two categories. In one case, the dendrimer serves as a complexing agent for redox-active metal ions. For example,
polyamidoamine (PAMAM) dendrimers host ions such as Mn2+ or Cu2+ in relationship to their generation. About 16 mol of Cu2+ was complexed per mole of fourth-generation (G-4) PAMAM.6 Apparently the sorption occurs by a reaction of a Cu2+ ion with two tertiary amine groups (1:2 binding) on the exterior of the G-4 PAMAM rather than of a result of 1:4 binding that involves interior sites.6 Reduction of the Cu2+ that is complexed with surfaceconfined PAMAM produces catalytically active nanoclusters. Storrier et al.7 functionalized the exterior amines of PAMAM dendrimers (dend) with bipyridyl (bpy) groups and subsequently prepared RuIIterpyridyl (tpy) complexes of PAMAM by refluxing dend-bpy with Ru(tpy)Cl3 under reducing conditions. Cyclic voltammetry in acetonitrile showed a reversible RuII,III peak at a formal potential of 1.31 V vs Ag/AgCl. Attendant experiments supported the suggestion that film deposition and stripping occurred during voltammetry. Indeed, varying the voltammetric conditions provided results that were consistent with charge trapping in multilayers as the electrode process. Constable et al.8,9 have developed strategies for spatially directed syntheses of metallodendrimers based on metal-donor atom interactions rather than carbon bonding with other carbon centers or with heteroatoms. An example was the synthesis and characterization of a metallodendrimer assembled from pentaerythritol, terpyridine, and Ru(tpy)Cl3.9 As in the PAMAM-based system,7 reversible RuII,III redox, which involved a surface process, was observed by cyclic voltammetry in acetonitrile. The data reported in the above studies allow a projection of utility of these RuII-containing metallodendrimers in electrontransfer mediation. Specifically, the reversibility of the RuII,III couple along with the surface and solubility properties of these metallodendrimers are consistent with developing stable modified electrodes that will promote oxidations when RuII is the initial oxidation state of the metal center. Here, the macromolecule is used as a dopant in a conducting carbon composite rather than as an adsorbed film in order to allow reactivation of the electrode in the event of passivation by adsorption of the analyte and/or the oxidation products. Because the long-term goal is the am-
* To whom correspondence should be addressed: (fax) 513-529-5715; (e-mail)
[email protected]. (1) Cox, J. A.; Dabek-Zlotorzynska, E. Electroanalysis 1991, 3, 239-242. (2) Cox, J. A.; Gray, T. J. Electroanalysis 1990, 2, 107-111. (3) Cox, J. A.; Gray, T. J. Anal. Chem. 1990, 62, 2742-2744. (4) Cox, J. A.; Gray, T. J. Anal. Chem. 1989, 61, 2462-2464. (5) Tess, M. E.; Cox, J. A. Electroanalysis 1998, 10, 1237-1240.
(6) Zhao, M. Q.; Sun, L.; Crooks, R. M. J. Am. Chem. Soc. 1998, 120, 48774878. (7) Takada, K.; Storrier, G. D.; Moran, M.; Abruna H. D. Langmuir 1999, 15, 7333-7339. (8) Constable, E. C.; Harverson, P. Inorg. Chim. Acta 1996, 252, 9-11. (9) Constable, E. C.; Housecroft, C. E.; Cattalini, M.; Phillips, D. New J. Chem. 1998, 22, 193-200.
10.1021/ac0002137 CCC: $19.00 Published on Web 05/19/2000
© 2000 American Chemical Society
Analytical Chemistry, Vol. 72, No. 14, July 15, 2000 3191
perometric determination of proteins, the primary test compound in the present study is L-Met, which is electrochemically silent in aqueous solution at unmodified electrodes. A secondary analyte is AsIII, which also is not oxidized under the test conditions at carbon-based electrodes and has the additional attribute of not causing electrode passivation under a wide range of conditions. EXPERIMENTAL SECTION Reagents and Synthesis. All chemicals were of reagent grade ACS unless otherwise stated. Acetonitrile (HPLC grade), potassium hydrogen phosphate monobasic, and sodium arsenite were from Fisher Scientific (Fair Lawn, NJ). Ruthenium chloride hydrate, 4′-chloro-2,2′:6′,2′′-terpyridine, L-Met, L-Cys, ammonium hexafluorophosphate, and 2,2′:6′,2′′-terpyridine were obtained from Sigma (St. Louis, MO); methyltrimethoxysilane (MTMOS) at 95% purity, was from Aldrich Chemical Co. (Milwaukee, WI); and Ultra “F” carbon was from Ultra Carbon Corp. (Bay City, MI). All solutions were prepared from house-distilled water that was further purified with a Barnstead NANOpure II system. The trichloroterpyridinylruthenium10 and the metallodendrimer9 were prepared by published methods that provide a final product, RuIIDen, with four RuIItpy subunits. The RuIIDen was purified using a column (length, 25 cm; diameter, 2 cm) consisting of 230-400-mesh silica gel. The mobile phase was a mixture comprising 82% acetonitrile, 12% saturated (aqueous) KNO3, and 6% water (v). Carbon-composite electrodes were prepared by a procedure based on previous reports.5,11-13 The binder was a sol-gel material prepared from a mixture comprising 1.0 mL of MTMOS, 1.0 mL of methanol, 1.5 mL of deionized water, and 10 µL of HCl. This mixture was magnetically stirred for 5 min, after which 2 g of carbon powder was added. Doping with the catalyst was done by coating the carbon with RuIIDen by the following procedure: the carbon was mixed with 40 mL of 0.625 mM RuIIDen in acetonitrile; the solvent was evaporated from a stirred solution at 75 °C. The slurry of carbon in the sol-gel precursor was mixed with a microspatula and packed into a plastic mold. Electrical contact was made by placing a copper wire into contact with the wet composite. All composite electrodes were polished with 360-grit sandpaper prior to use. Apparatus and Methods. Characterization of the metallodendrimer was primarily by NMR and mass spectrometry. A Bruker (Billerica, MA) Reflex II time-of-flight mass spectrometer with a reflectron mass analyzer was used for the matrix-assisted laser desorption/ionization (MALDI) experiments. The accelerating voltage was 25 kV, and the effective flight path was 290 cm. The desorption/ionization was achieved with a Nd:YAG laser, operated at 355 nm, using R-cyano-4-hydroxycinnamic acid (CHCA) or gentisic acid as the matrix. The measurements were in the positive ion mode. Proton NMR was performed on a Bruker Advance 300-MHz instrument. The solvent consisted of 99.8% deuterated acetonitrile from Cambridge National Laboratories, Inc. (Andover, MA). Spectra were obtained with 512 scans. Interroga(10) Adcock, P. A.; Keene, F. R.; Smythe, R. S.; Snow, M. R. Inorg. Chem. 1984, 23, 2336-2343. (11) Tsionsky, M.; Gun, G.; Glezer, V.; Lev, O. Anal. Chem. 1994, 66, 17471753. (12) Sampath, S.; Lev, O. Anal. Chem. 1996, 68, 2015-2021. (13) Pankratov, I.; Lev, O. J. Electroanal. Chem. 1995, 393, 35-41.
3192 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
Figure 1. MALDI mass spectrometry of RuIIDen. The sample was a solution comprising 10 µM RuIIDen, 10 mM CHCA, and 0.01 M trifluoroacetic acid in 70% (v) CH3CN, 30% H2O. The solution was evaporated to dryness on the probe.
tion of the pore structure of the sol-gel materials was by atomic force microscopy using a Digital MultiMode system (Santa Barbara, CA). All experiments were performed in the Tapping Mode with an etched silicon probe. The electron paramagnetic resonance measurements were at 95 K with a Bruker EMX EPR spectrometer. The flow injection analysis system consisted of an Eldex Laboratory pump (model A-120-S, Napa, CA), a Rheodyne 7125 injection valve (Cotati, CA), and a Bioanalytical Systems (West Lafayette, IN) three-electrode amperometric detector, in which the carbon-composite electrode (CCE) replaced the MF 1000 glassy carbon electrode. The Ag/AgCl reference electrode was placed downstream of the CCE and the stainless steel counter electrode. The flow rate was 0.5 mL‚min-1, the sample volume 100 µL, and the mobile phase 0.05 M phosphate buffer adjusted to pH 7.0 for L-Met and pH 2.0 for L-Cys. A CH Instruments model 750 electrochemical workstation (Cordova, TN) was used for the voltammetry experiments. The working electrodes were CCEs, and a Pt wire was the auxiliary electrode. All potentials were measured and reported versus a Ag/ AgCl reference electrode. RESULTS AND DISCUSSION The identify of the product of the above-described synthesis was verified and its voltammetric characteristics were determined in initial experiments. First, MALDI mass spectrometry was used to establish that the primary product was the metallodendrimer with four RuIItpy subunits. In accord with a previous report,9 the metallodendrimer was precipitated from an aqueous solution as the 1:8 hexafluorophosphate salt. With R-cyano-4-hydroxycinnamic acid as the matrix, a peak was observed at m/z of 3415.9 (Figure 1), which corresponds to within 0.04% to the theoretical molecular mass of the hexafluorophosphate salt of the four-unit dendrimer minus one PF6- group. In the original report,9 gentisic acid was the matrix; the corresponding peak with the highest mass in a MALDI experiment was at m/z of 3016. In accord with their results, the peak with the highest m/z we observed with gentisic acid as the matrix was at 3037. On the basis of a mass agreement
Figure 2. Cyclic voltammogram of 1.0 mM in CH3CN with 0.1 M tetrabutylammonium perchlorate as the supporting electrolyte. Working electrode, glassy carbon; counter and quasi-reference electrodes, Pt; scan rate, 0.2 V‚s-1.
Figure 3. Cyclic voltammogram of 40 µM RuIIDen in (A) the presence and (B) the absence of 1.0 mM L-Met. The solution was 88% (v) 0.1 M phosphate buffer (aqueous) at pH 7.0 and 12% CH3CN. Working electrode, glassy carbon electrode; scan rate, 0.05 V‚s-1.
to within 0.03%, the peak at m/z of 3270.6 in Figure 1 was presumed to result from the loss of a second PF6- group and the concomitant addition of a proton. The general features of the 300-MHz proton NMR spectrum were identical to those reported9 for RuIIDen except that a small peak appeared at δ ) 5.3 ppm. Analysis of 2D NMR data suggested that the peak may be due to the dendrimer with fewer than four RuIItpy subunits; however, the data did not allow a positive identification of the impurity. Comparison of the integrated areas indicated that the product purity exceeded 90%. Spectrophotometric measurements further supported the conclusion that the metallodendrimer was synthesized at high purity. In agreement with Constable et al.,9 measurements made on a 40 mg of RuIIDen/L solution in acetonitrile showed an absorption maximum at 480 nm was observed. Presuming that the molecular weight is 3562, the molar absorptivity determined at 480 nm, 480, was 5.6 × 104 L‚(mol‚cm)-1, which agreed well with their value, 6.0 × 104 L‚(mol‚cm)-1. A goal of the present study is to use RuIIDen as an electrontransfer mediator, which in turn requires reversible (or at least quasi-reversible) voltammetry of this compound. Figure 2 illustrates the cyclic voltammetry of 9.6 × 10-4 M RuIIDen in acetonitrile with 0.1 M tetrabutylammonium perchlorate as the supporting electrolyte. A one-step oxidation of RuIIDen is observed near 1.05 V. The difference between the anodic and cathodic peak potentials is 52 ( 7 mV (6 points) over the scan rate, v, range 0.1-1.0 V‚s-1. A plot of log ip,a vs log v, where ip,a is the anodic peak current in µA and the units of v are V‚s-1, yields the following: slope, 0.68 ( 0.03; intercept, 2.0 ( 0.02; standard deviation of the fit, 0.027; and r, 0.998. These data are consistent with voltammetry that approaches an electrochemically reversible, diffusion-controlled, one-electron oxidation of RuIIDen. The departure from the ideal behavior (peak potential difference of 59 mV and a slope of 0.50) can be attributed to some adsorption of the metallodendrimer on the electrode. On the basis of the above data and the report by Constable et al.,9 the one-electron oxidation of RuII centers was assumed as the process near 1.05 V. Supporting evidence was obtained by EPR. First, EPR spectra for a known system, 1.5 mM RuII(bpy)3 in a mixture of 50% acetonitrile and 50% H2O (v) with 0.1 M LiClO4 as the supporting electrolyte, were obtained before and after oxidative controlled-potential electrolysis at 1.2 V, which is 100 mV positive of the anodic peak potential in cyclic voltammetry. The latter spectrum showed the characteristic peak for RuIII in the 3000 G region whereas no signal in that region was observed for the RuII(bpy)3. When the experiment was repeated with 1.0
mM RuIIDen, identical results were obtained. Hence, the product of the synthesis was confirmed as RuIIDen. To determine whether RuIIDen is suited as an electrocatalyst, the oxidation of L-Met was investigated in a mixed solvent, 88% water-12% acetonitrile (v), that included 4 × 10-5 M RuIIDen. The cyclic voltammetry (Figure 3) was characterized by amplification of the current for the oxidation of RuIIDen. Diminution of the current for the reduction of RuIIIDen occurred in both the presence and absence of L-Met, presumably because the oxidation of water was catalyzed by the RuIIIDen. These trends were consistent with catalysis by a mediated electron-transfer pathway; that is, the L-Met was oxidized by the RuIIIDen generated at the glassy carbon electrode during the positive-going scan in the cyclic voltammetry. In the absence of RuIIIDen, L-Met was not oxidized under these conditions. Because RuIIDen is sparingly soluble in aqueous solution, practical applications of this compound as an electrocatalyst require its immobilization in a conducting composite or its deposition on the surface of an electrode. The focus of the present study is on CCEs in which RuIIDen is encapsulated in silica that was prepared by a sol-gel process. The choice of sol-gel processing is based on numerous reports which have demonstrated that the resulting materials have pore widths smaller than the dimensions of macromolecules such as enzymes, thereby blocking loss of the dopant by leaching while retaining access of small analytes to reactive sites. The present system fits this model in that the diameter of RuIIDen is estimated as 3.2 nm, by ChemOffice 3D, which is larger than the pore widths of silica prepared by sol-gel chemistry under the general conditions that were used in the present study (mean pore widths,
