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Effect of Tin-Doped Indium Oxide Electrode Preparation Methods on the Mediated Electrochemical Detection of Nucleic Acids Natasha D. Popovich,* Brian K. Yen, and Sze-Shun Wong Xanthon, Inc., 104 T.W. Alexander Drive, P.O. Box 12296, Research Triangle Park, North Carolina 27709 Received August 7, 2002. In Final Form: November 3, 2002 Oxidation of guanine residues in nucleic acids using a metal mediator, tris(bipyridyl) ruthenium(II) (Ru(bpy)32+), provides a sensitive and simple method for detection of unmodified nucleic acids. This method can be implemented in a hybridization-based assay, in which nucleic acid probes are immobilized at the electrode surface to capture the nucleic acid target of interest. In this paper, the effect of the tin-doped indium oxide (ITO) surface preparation on the immobilization of nucleic acid probes and electron-transfer kinetics of Ru(bpy)32+ was examined. ITO preparation methods have a profound effect on the amount of material immobilized on the electrode surface via either phosphonate or silane linkage. Common cleaning procedures that utilize phosphate-containing detergents cannot be employed in this system because of the strong adsorption of phosphate to the ITO surface, which was shown to reduce the amount of nucleic acid bound to the ITO surface. It was also found that a negative charge at the electrode surface significantly enhances the electron-transfer kinetics between Ru(bpy)32+and the ITO electrode. Phosphate ions adsorbed at the ITO surface were found to be the most effective way to enhance the electron-transfer kinetics of Ru(bpy)32+. As expected for a charge-related phenomenon, phosphate ions adsorbed at the ITO surface had the opposite effect on the negatively charged redox probe, Fe(CN)64-. The presence of adventitious carbon on the electrode surface was shown to adversely impact solid-phase immobilization by decreasing the stability of both phosphonate and silane immobilized materials exposed to hybridization conditions. UV treatment of ITO surfaces prior to solid-phase immobilization results in the removal of adventitious carbon yielding a more stable solid phase.
Introduction Advances in genomics have generated a need for simple nucleic acid detection methods.1 Rapid and accurate detection and quantitation of nucleic acids is required for a variety of market applications including drug discovery, pathogen detection, forensics, etc. Thorp and co-workers have developed a nucleic acid detection system based on the catalytic oxidation of guanine residues by a tris(bipyridyl) ruthenium(II) (Ru(bpy)32+) mediator.2,3 This detection method does not require the attachment of a label to the nucleic acid target and has been shown to have high sensitivity due to the catalytic nature of the oxidation reaction.4,5 In this system, the Ru(bpy)32+ mediator transfers electrons from guanine to a tin-doped indium oxide (ITO) working electrode:
Ru(bpy)32+ f Ru(bpy)33+ + eRu(bpy)33+ + guanine f Ru(bpy)32+ + guanineox The Ru(bpy)32+/3+ couple is an excellent mediator for use in this system because its standard reduction potential * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Henke, C. IVD Technology 1998, 4, 28-32. (2) Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933-8938. (3) Thorp, H. H. Trends Biotechnol. 1998, 16, 117-121. (4) Armistead, P. M.; Thorp, H. H. Anal. Chem. 2000, 72, 37643770. (5) Popovich, N. D.; Eckhardt, A.; Mickulecky, J. C.; Napier, M. E.; Thomas, R. S. Talanta 2002, 56, 821-828.
(1.1 V vs Ag/AgCl) is almost identical to that of guanine.6,7 Similarly, ITO is the preferred electrode material because it is able to support the high positive potential necessary for guanine oxidation.8,9 Because ITO is the material of choice for fabrication of a number of optoelectronic devices, such as flat panel and liquid crystal displays,10 and more recently for organic light-emitting diodes,11 a number of application-specific ITO preparation methods have been developed for these uses.12,13 However, there has been little effort directed to the development of methods to prepare an ITO surface suitable for electrochemical applications or for immobilization of biological materials.14 In an assay for the detection of nucleic acid hybridization based on the electrochemical detection of guanine, it is necessary to immobilize nucleic acid capture probes on the surface of an ITO electrode.5 In the work presented here, two attachment methods were used for probe immobilization, one based on the affinity of phosphate or (6) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617619. (7) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; von Zelewsky, A. Coord. Chem. Rev. 1988, 84, 85-277. (8) Armstrong, N. R.; Lin, A. W. C.; Fujihira, M.; Kuwana, T. Anal. Chem. 1976, 48, 741-750. (9) Popovich, N. D.; Wong, S.-S.; Yen, B. K.; Yeom, H.-I.; Paine, D. C. Anal. Chem. 2002, 74, 3127-3133. (10) Hamberg, I.; Granqvist, C. G. J. Appl. Phys. 1986, 60, R123. (11) Tang, C. W.; VanSlyke, S. A. Appl. Phys. Lett. 1987, 51, 913915. (12) Kim, J. S.; Granstrom, M.; Friend, R. H.; Johansson, N.; Salaneck, W. R.; Daik, R.; Feast, W. J.; Cacialli, F. J. Appl. Phys. 1998, 84, 68596870. (13) Christou, V.; Etchells, M.; Renault, O.; Dobson, P. J.; Salata, O. V.; Beamson, G.; Egdell, R. G. J. Appl. Phys. 2000, 88, 5180-5187. (14) Donley, C.; Dunphy, D.; Paine, D.; Carter, C.; Nebesny, K.; Lee, P.; Alloway, D.; Armstrong, N. R. Langmuir 2002, 18, 450-457.
10.1021/la026369e CCC: $25.00 © 2003 American Chemical Society Published on Web 12/20/2002
Effect of Tin-Doped Indium Oxide Electrode
phosphonate groups for ITO and the second based on the condensation of silane at the ITO surface. These methods provide stable and reproducible attachment of probe sequences to the surface while maintaining rapid electron transfer between Ru(bpy)32+ and the electrode surface. The present study evaluated various ITO preparation methods to identify processes most suitable for optimization of these two electrode functions. A common procedure used for removal of contaminants such as adventitious carbon from ITO surfaces is washing with a detergent.15 Most commercial detergents contain inorganic phosphate in some form and the affinity of phosphonate/phosphate-containing compounds for metal oxide surfaces is well documented.16-18 It has been postulated that the interaction between inorganic phosphates and metal oxides can be divided into two types: (a) chemisorption of phosphate to the metal oxide surface via surface hydroxyl groups and (b) precipitation of phosphate at the oxide surface to form phosphate “islands”. These interactions have been shown to depend on the type of metal oxide and the phosphate cation.18 A variety of factors determine the charge of a metal oxide surface. These factors include pH, the presence or absence of charged surface groups or adsorbed ions, and the amount of adventitious carbon present on the oxide surface. Nelson et al.18 showed that changes in adsorbed species, such as hydronium, hydroxide, phosphate or arsenate ions, affected the space charge layer and surface conductivity of a titanium oxide surface and determined that such changes could be used to control the adsorption of charged molecules to the surface. In the same study, the authors showed that addition of phosphate made the oxide surface significantly more negative at a given pH, which resulted in increased binding of cationic probe molecules. A similar effect could be achieved by modifying the surface charge using an increased pH. It can be predicted that the ITO surface charge will influence the adsorption of charged redox molecules such as Ru(bpy)32+ to the metal oxide surface and that this adsorption will, in turn, impact the electrochemical behavior of the redox molecules. The studies presented here demonstrate that electrontransfer kinetics and solid-phase attachment are profoundly influenced by adsorbed species at the ITO surface, especially adsorbed phosphate ions. While phosphate ions have a detrimental effect on solid-phase immobilization via both silane and phosphonate groups, the negative surface charge created by adsorbed phosphate ions enhances the electron-transfer kinetics of Ru(bpy)32+ and hence the mediated electrochemical detection of guanine. Experimental Section Chemicals. All chemicals were used as received from the manufacturer unless otherwise indicated. Sparkleen 1, a common glassware-cleaning agent, was obtained from Fisher Scientific Co. (Pittsburgh, PA) and used in detergent treatment experiments, as were sodium phosphate (Na3PO4, “P1”), sodium pyrophosphate (Na4P2O7, “P2”), and sodium tripolyphosphate (Na5P3O10, “P3”). “Sodium polyphosphate”, a mixture of metaphosphates commonly used in detergent formulations, “Pn”, was obtained from Aldrich (Milwaukee, WI). 12-phosphonododecanoic acid (PDA) was synthesized by Research Triangle Institute (Research Triangle Park, NC) and tritium-labeled by Amersham (15) Willit, J. L.; Bowden, E. F. J. Phys. Chem. 1990, 94, 8241-8246. (16) Nooney, M. G.; Campbell, A.; Murrell, T. S.; Lin, X.-F.; Hossner, L. R.; Chusuei, C. C.; Goodman, D. W. Langmuir 1998, 14, 2750-2755. (17) Hingston, F. J.; Atkinson, R. J.; Posner, A. M.; Quirk, J. P. Nature 1967, 215, 1459. (18) Nelson, B. P.; Candal, R.; Corn, R. M.; Anderson, M. A. Langmuir 2000, 16, 6094-6101.
Langmuir, Vol. 19, No. 4, 2003 1325 Biosciences, Inc. (Piscataway, NJ). Triethoxy (3-isocyanatopropyl) silane was purchased from Aldrich and was stored under N2 when not in use. A 13-mer peptide nucleic acid (PNA) sequence containing four guanines was obtained from Boston Probes, Inc. (Bedford, MA). Ru(bpy)32+ and Fe(CN)64- were obtained from Aldrich. ITO Electrodes. Glass substrates (14 in. × 14 in.) were coated with a 200 nm thick ITO film by DC magnetron sputtering (Applied Films Corporation, Longmont, CO). Prior to use as electrodes, ITO-coated glass sheets were cut into 2.3 cm2 pieces by Delta Technologies, Limited (Stillwater, MN). Unless otherwise indicated, electrodes were cleaned prior to use by successive 10 min sonication treatments in H2O, isopropyl alcohol, H2O, and H2O. Instrumentation. Electrochemical data were obtained using a CH Instruments electrochemical analyzer. A single-compartment cell was used with Ag/AgCl/3 M KCl (Cypress Systems, Inc., Lawrence, KS) as the reference electrode and Pt wire as the auxiliary electrode. The ITO working electrode area (0.28 cm2) was defined by the size of a rubber O-ring. All potentials are reported with respect to Ag/AgCl/3 M KCl. Phosphorimager data were obtained using a Molecular Dynamics Phosphorimager SI (Amersham Pharmacia Biotech Ltd., Piscataway, NJ). Surface compositional analyses were performed using X-ray photoelectron spectroscopy (XPS) (Physical Electronics, QUANTUM 2000, Charles Evans & Associates, Eden Prairie, MN) and secondary ion mass spectrometry (SIMS) (Physical Electronics, TRIFT II, Charles Evans & Associates). DigiSim software (BAS), courtesy of Prof. Holden Thorp at UNC, Chapel Hill, was used to simulate the cyclic voltammograms. The parameters established in the Thorp group were used for the simulations. UV Treatment. UV treatment was conducted by placing ITO electrodes 5 cm from a 254 nm Spectroline model EF-140C UV lamp (Spectronics Corp., Westbury, NY) for the specified times. The intensity of UV radiation was approximately 0.5 mW/cm2 at the ITO surface. Phosphate Treatments. Phosphate treatment of ITO electrodes was typically conducted by immersion of the electrodes in the phosphate solutions for 20 h followed by rinsing with copious amounts of H2O. Electrodes were allowed to air-dry prior to use in experiments. Sparkleen solution was prepared in water at a concentration of 20 mg/mL. Solutions of P1, P2, or P3 were prepared at 12, 50, 60, or 300 mM and were adjusted to pH 7 with small amounts of HNO3. Sodium polyphosphate solution (Pn) was prepared at a concentration of 10 mg/mL in water. Phosphonate Self-Assembled Monolayers (SAMs) on ITO. A phosphonate-based self-assembled monolayer was allowed to form on ITO electrodes by immersing the electrodes in a solution of 1 mM PDA in 50% DMSO/50% H2O for 16.5 h. The electrodes were removed from solution and washed with successive 10 min rinses of H2O, 1 M NaCl, H2O, and H2O. Tritiumlabeled PDA was used to form the SAMs, and the amount of adsorbed PDA was quantitated by exposing a phosphorimager screen to the monolayer-modified electrodes for 2 days and scanning with the phosphorimager. PNA-Modified ITO. PNA was coupled to triethoxy (3isocyanatopropyl) silane in DMF/HEPES buffer, pH 7, and immobilized on ITO electrodes. The terminal primary amine in the PNA reacts with the isocyanato group on the silane to form an amide bond. After the amidation was allowed to proceed for 90 min at room temperature, the reaction mixture was diluted 10-fold with 0.5 M acetate (pH 4.15). This solution was then manually spotted onto ITO electrodes (10 µL spot per electrode). The electrodes were placed into a humid environment for 17-23 h and then cured at 50 °C in air until dryness (about 90 min). The electrodes were then agitated in 10 mM NaOH for 5 min, rinsed with copious amounts of H2O, and allowed to air-dry before use. Some modified electrodes were immersed in a hybridization solution for 19 h. The hybridization solution contained 50 mM sodium phosphate buffer (pH 7), 25% formamide (by volume), 0.1% Triton X-100, and 0.5 M NaCl. The amount of PNA immobilized on the electrode surface was quantified by performing successive cyclic voltammetry (CV) scans of the electrodes in 100 µM Ru(bpy)32+ and 50 mM phosphate buffer (pH 7). As previously reported,2,3 enhanced oxidative current in the first scan (in comparison to subsequent
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Figure 1. Calculated PDA coverage on ITO electrodes treated with phosphate. Postcleaning treatment consisted of immersion of ITO electrodes in various phosphate solutions (50 mM) for 22 h followed by copious H2O rinses and by application of PDA solution. P1, P2, P3 and Pn denote monophosphate, pyrophosphate, triphosphate, and polyphosphate solutions, respectively. One set of electrodes was immersed in pure H2O for 22 h as a control, while the set labeled “cleaned” received no additional treatment following the cleaning step and prior to PDA application. scans) corresponds to the irreversible catalytic oxidation of guanine residues. This method was used to quantitate the amount of guanine-containing PNA on the electrode surface, and data were expressed as either peak current or integrated charge. Peak current or integrated charge from the third scan was typically used as background.
Results and Discussion Phosphate Treatment. The interaction between phosphate and the ITO electrode surface was first examined by SIMS analysis of ITO samples cleaned using a commercially available detergent (Sparkleen). SIMS analysis showed the presence of significant levels of phosphate on the surface following cleaning, whereas phosphate was absent from as-received ITO samples. XPS analysis of ITO surfaces immersed overnight in 50 mM phosphate buffer confirmed this observation in that cleaned surfaces showed 1.2 atom % P, which was absent from control ITO surfaces immersed in H2O only. The effect of adsorbed phosphate on the amount of PDA present on the electrode surface and on the amount of silanecoupled PNA on the electrode surface was evaluated. Figure 1 shows the impact of phosphate treatment of the ITO electrode on the amount of PDA bound to the ITO surface. 3H-labeled PDA was used to quantify the PDA present on phosphate-treated and untreated electrodes. Figure 1 shows the calculated coverage of PDA selfassembled onto ITO surfaces treated with a variety of phosphate species commonly found in detergents. PDA coverage on ITO electrodes treated with sodium phosphate (Na3PO4, P1), sodium pyrophosphate (Na4P2O7, P2), sodium tripolyphosphate (Na5P3O10, P3) or sodium polyphosphate (mixture of metaphosphates, Pn) was at least 40% lower than the PDA coverage on untreated controls. The P2 and P3 species appear to be more effective in blocking PDA attachment to the surface than the P1 or Pn species. The effect of phosphate treatment on the quantity of nucleic acid immobilized on ITO electrodes using silane attachment chemistry was also investigated. In this study, a PNA sequence containing four guanines was covalently
Popovich et al.
Figure 2. Voltammograms of PNA-silane-modified electrodes. PNA-silane was assembled on ITO electrodes that had been treated for 23 h with (A) water, (B) 0.3 M sodium monophosphate, (C) 0.3 M sodium pyrophosphate, (D) 0.3 M sodium tripolyphosphate, or (E) 10 mg/mL polyphosphate mix. Scan rate ) 500 mV/s.
coupled to triethoxy (isocyanatopropyl) silane and applied to untreated ITO electrodes or electrodes that had been treated with P1, P2, P3, or Pn phosphates as described above. Figure 2 shows the cyclic voltammograms generated at these electrodes. The enhanced anodic peak currents reflect the catalytic oxidation of guanine residues in the PNA by the Ru(bpy)32+ mediator. Reduction in the first scan peak currents generated at treated vs untreated ITO electrodes shows that treatment with phosphate species significantly lowers PNA surface coverage. As with the PDA SAMs, the efficiency of silane-based nucleic acid surface attachment is markedly reduced by phosphate treatment. UV Treatment. Exposure to high-intensity UV radiation is commonly employed in semiconductor processing for the removal of adventitious carbon and other surface contaminants.19 Evidence of the cleaning effect of UV on ITO electrodes was provided by XPS. The XP spectrum of an as-received ITO electrode showed the presence of approximately 18.6 atom % carbon on the surface. UV irradiation of a similar electrode for 16 h in a nitrogen atmosphere did not impact this value (sample contained 21.6 atom % carbon), whereas UV irradiation for 16 h in air reduced the level of adventitious carbon on the electrode surface to 8.4 atom %. These results are consistent with previous studies in which high-intensity UV light was used in combination with O2 or O3 to clean semiconductor surfaces efficiently.13 Studies were performed to determine whether UV treatment enhanced electron-transfer kinetics or solidphase attachment at ITO electrodes. Using radiolabeled PDA and silane-coupled PNA, we first determined that UV treatment did not affect the amount of material immobilized at the ITO electrode surface using either of these attachment strategies. The stability of attachment was also determined. In these experiments, electrodes with immobilized PNA-silane were exposed to hybridization buffer solution for 18.6 h, and the amount of PNA remaining on the electrode surface following treatment was determined by CV. The results reported in Figure 3 show the effect of UV treatment on the stability of the (19) Nelson, B. P.; Candal, R.; Corn, R. M.; Anderson, M. A. Langmuir 2000, 16, 6094-6101. Kern, W., Ed. Handbook of Semiconductor Wafer Cleaning Technology: Science, Technology, and Applications; Noyes Publications: Park Ridge, NJ, 1993; Chapter 6.
Effect of Tin-Doped Indium Oxide Electrode
Figure 3. Stability of PNA-silane immobilized on ITO electrodes (A) prior to and (B) following exposure to a hybridization buffer for 18.6 h. Type I electrodes were used “asreceived”, type II electrodes were cleaned using the standard sonication procedure in water and isopropyl alcohol, and type III electrodes were UV-treated. The quantity of PNA immobilized on the electrode surface is reflected by the integrated charge of anodic peak in CV. UV treatment was accomplished by 24-h exposure to a UV lamp emitting at 254 nm.
immobilized PNA as evident in the background-corrected, integrated charge from the anodic peak. Three types of ITO were used: as-received electrodes from the manufacturer (type I); electrodes cleaned by successive 10 min sonication treatments in H2O, isopropyl alcohol, H2O, and H2O (type II); and electrodes cleaned by sonication followed by exposure to UV radiation (type III). These data show that the stability of the PNA-silane follows the trend type III (23% loss) > type II (50% loss) > type I (64% loss). This trend correlates with the level of adventitious carbon on the ITO surface in that asreceived samples (type I) have the highest level of surface contaminants. These contaminants might inhibit the formation of covalent bonds between the silane and the metal oxide surface, and the resulting loosely adsorbed PNA-silane would therefore be easily removed by immersion in hybridization buffer. Sonication and UV treatments remove substantial amounts of adventitious carbon, thereby exposing more sites for covalent attachment between the silane and the surface resulting in greater stability of the surface-immobilized PNA. Phosphate and pH Effects on Electrochemistry of Ru(bpy)32+ and Fe(CN)64- at ITO. Although surfacebound phosphates negatively impact nucleic acid attachment, the adsorbed phosphate layer enhances the observed electron-transfer kinetics of the Ru(bpy)32+ mediator. Figure 4A shows a representative cyclic voltammogram of 100 µM Ru(bpy)32+ in 0.1 M KNO3 at a scan rate of 20 V/s. High scan rates were used in this study because Armistead and Thorp showed that the maximum catalytic signal from guanine is obtained at a scan rate of 10 V/s or higher4 and because high scan rates are more sensitive to kinetic phenomena. Figure 4A shows large peak splitting and small peak current, both indicative of sluggish electron-transfer kinetics. Figure 4B is a representative voltammogram obtained following the addition of phosphate buffer to the supporting electrolyte to bring the solution to 6 mM in phosphate. This voltammogram shows smaller peak splitting, larger peak current, and generally sharper peaks. It is thought that this difference in electron-transfer kinetics is the result of the anionic phosphate present in
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Figure 4. Effect of phosphate in the supporting electrolyte on Ru(bpy)32+ electron-transfer kinetics. Graph shows cyclic voltammograms of 100 µM Ru(bpy)32+ in (A) 0.1 M KNO3 and (B) 0.1 M KNO3 containing 6 mM phosphate buffer. Scan rate ) 20 V/s.
the supporting electrolyte solution forming a layer of adsorbed phosphate at the ITO surface. With this adsorption, the surface acquires more negative charge and concentrates the positively charged Ru(bpy)32+/3+ at the electrode surface, resulting in an enhancement in apparent electron-transfer kinetics. If this is the case, the opposite trend (a decrease in observed electron-transfer rate) would be observed for a negatively charged redox couple, and indeed, as discussed below, this result is observed in cyclic voltammograms of Fe(CN)64-. The presence of sulfate at the electrode surface was also found to improve the electron-transfer kinetics of Ru(bpy)32+ (data not shown), suggesting that there is some adsorption of sulfate to the ITO surface resulting in a build-up of negative charge at the surface. This effect was less pronounced than for phosphate anion. The effect of adsorbed species on observed electrontransfer kinetics is well-known and is a special case of the Frumkin or “φ2” effect.20 Figure 5 shows the effect of phosphate treatment on cyclic voltammograms of 100 µM Ru(bpy)32+/100 mM KNO3. Overnight treatment of ITO electrodes with P2 or P3 resulted in increased apparent electron-transfer kinetics as evidenced in the reduced peak splitting and larger anodic peak currents in the CV. In contrast, overnight treatment with P1 appears to slow the electron-transfer kinetics. One explanation for this unexpected trend could be the formation of multiple phosphate layers on the electrode surface, which would tend to increase the average distance of closest approach between Ru(bpy)32+ and the electrode. Multiple phosphate layers could also be responsible for the failure of the electron-transfer kinetics to increase or decrease monotonically with changes in P2 and P3 concentration. When unbuffered supporting electrolytes such as KNO3 or KCl are used in the system, the pH of the 100 µM Ru(bpy)32+ solution is 5.5. The effect of an increase in pH on electron-transfer kinetics was examined to determine whether a pH of 7 would result in a higher concentration of ionized surface hydroxyls, which would also contribute to faster kinetics. Voltammograms obtained using KNO3 at pH 7 without phosphate as the supporting electrolyte (data not shown) gave current enhancements and small peak splitting similar to that (20) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, John Wiley & Sons: New York, 1980; pp 540-546.
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Figure 5. Effect of phosphate treatment of ITO electrodes on the electron-transfer kinetics of Ru(bpy)32+. Treatments consisted of immersing ITO electrodes in solutions of P1, P2, or P3 for 22 h followed by copious H2O rinses. Supporting electrolyte was 100 mM KNO3. Scan rate: 20 V/s.
Figure 6. Anodic peak current of Ru(bpy)32+ as a function of the concentration of phosphate in the supporting electrolyte at constant ionic strength. Peak current was corrected for charging current. Scan rate: 20 V/s.
obtained in 50 mM phosphate buffer. These findings indicate that higher pH increases the negative charge at the electrode surface, resulting in enhanced electrontransfer kinetics. It was not possible to examine the impact of pH values above 7 because current due to water oxidation becomes too pronounced and interferes with the Ru(bpy)32+ current. Figure 6 shows the results of CV runs on a series of Ru(bpy)32+ solutions having different amounts of phosphate buffer in the supporting electrolyte. The ionic strength of each solution was maintained at 0.1 M by the addition of KCl, which should not interact with the ITO surface. Solutions with low phosphate concentrations were slightly acidic and pH was adjusted to neutrality by addition of small amounts of base. The magnitude of the anodic peak currents correlates with the extent to which Ru(bpy)32+/3+ is concentrated at the surface by adsorbed phosphate. Current values reach a maximum and begin to level off at relatively low phosphate concentrations (ca. 5-10 mM). At phosphate concentrations g5-10 mM, the effect of phosphate adsorption is so pronounced that the observed Ru(bpy)32+ electron-transfer kinetics become independent of pH. Figure 7 shows cyclic voltammograms of Ru(bpy)32+ in 50 mM phosphate at various pH values. The voltammograms are virtually indistinguishable over the pH range of 5.6-8.1. The effect of adsorbed phosphate is particularly noticeable when the voltammogram run in phosphate at pH 5.6 is compared to the voltammogram run in KNO3 at the same pH (Figure 4A). Markedly faster electrontransfer kinetics were observed when phosphate was used as the supporting electrolyte.
Figure 7. Cyclic voltammograms of 100 µM Ru(bpy)32+ in 50 mM phosphate at pH 5.6, 7.0, and 9.1. Scan rate: 20 V/s.
Figure 8. Cyclic voltammograms of 100 µM K4Fe(CN)64- in various supporting electrolytes: (A) 0.4 M KNO3 at pH 4.4; (B) 0.4 M KNO3 at pH 6.4; (C) 0.4 M KNO3 at pH 10.2; (D) 0.306 M KNO3 plus 0.05 M sodium phosphate at pH 7.0. The latter was prepared such that the ionic strength was 0.4 M.
As expected, pH and phosphate adsorption were found to have the opposite effect on electron-transfer rates for Fe(CN)64-, a negatively charged redox couple, indicating that the kinetic effect is indeed controlled by a chargecharge interaction. Figure 8 shows cyclic voltammograms of 100 µM Fe(CN)64-/0.4 M KNO3 solutions at various pH values. The pH of the solutions was adjusted by addition
Effect of Tin-Doped Indium Oxide Electrode Table 1. Heterogeneous Electron-Transfer Rate Constants for Ru(bpy)32+ and Fe(CN)64- in Different Supporting Electrolytes Estimated Using DigiSima analyte/supporting electrolyte
heterogeneous ET rate constant (cm/s)
Ru(bpy)32+/50 mM phosphate buffer, pH 7 Ru(bpy)32+/0.1 M KNO3, pH 5.5 Ru(bpy)32+/0.1 M KNO3, pH 7 Fe(CN)64-/pH 9 Fe(CN)64-/pH 12
0.032 ( 0.008 0.003 ( 0.001 0.018 ( 0.005 0.0025 ( 0.0006 0.0002 ( 0.0001
a Scan rate for Ru(bpy) 2+ was 20 V/s; scan rate for Fe(CN) 43 6 was 5 V/s.
of small amounts of NaOH or HNO3. This figure shows that the apparent electron-transfer rate (as reflected in the peak splitting and peak current) decreases as the pH is raised from 4.4 to 6.4 to 10.2. The electron-transfer rate is further slowed when phosphate is present in the supporting electrolyte (pH 7.0) where larger peak separation and smaller peak current are observed compared to results obtained with KNO3 alone (at all pH values). The apparent heterogeneous rate constants for Ru(bpy)32+ and Fe(CN)64- in various electrolytes on ITO were estimated from digital simulation (DigiSim 3.0) of cyclic voltammetry data. To quantify the effect of pH on the observed rate, cyclic voltammetry data from a 0.5 mM Fe(CN)64-/0.4 M KCl solution at pH 9 and 12 scanned at 5 V/s were simulated. The apparent rate constant was approximately 10 times faster for the lower pH solution than for the higher pH (Table 1). This is believed to be a result of increased negative charge at the ITO surface at higher pH. The high relative standard deviations in this study are thought to be a function of the high scan rates used in the experiments. Despite the uncertainty in the actual rate constant numbers, we believe that the different supporting electrolytes can still be accurately compared. Cyclic voltammograms of 100 µM Ru(bpy)32+ in 50 mM phosphate buffer or 0.1 M KNO3 electrolyte were also simulated. The pH of the KNO3 solution was adjusted to pH 7 with a small amount of base. Fitting the CV data showed that the apparent rate constant when phosphate buffer was used as the supporting electrolyte was approximately 2 times higher compared to the rate constant when KNO3 was used in the electrolyte. The combined
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effect of phosphate adsorption and pH on the electrontransfer kinetics of Ru(bpy)32+ was quite dramatic in that the apparent rate constants obtained in phosphate buffer at pH 7 were an order of magnitude higher than the rate constants observed in KNO3 (pH ca. 5.5). The effect of the supporting electrolyte on the electrochemical signal from guanine was also examined and found to be considerably greater with phosphate buffer compared to KNO3, even when both were at pH 7. The presence of phosphate is clearly required to achieve the maximum catalytic signal, and pH adjustment alone is not sufficient to provide this enhancement. Conclusions The electrocatalytic detection of surface-immobilized guanine using a Ru(bpy)32+ mediator is highly dependent upon preparation of the ITO surface. Common ITO cleaning procedures utilizing phosphate detergents were shown to leave a passivating layer of inorganic phosphate on the surface that is detrimental to the attachment of nucleic acids via phosphonate or silane linkages. The layer of adventitious carbon present at the ITO surface was also found to have a detrimental effect on phosphonate or silane attachment, and when this layer was removed from the electrode surface using UV radiation, a more stable solid phase was achieved. To maximize electron transfer between Ru(bpy)32+ and the ITO electrode, the charge at the electrode surface needs to be negative. Phosphate adsorption was found to be an efficient way to impart this negative charge to the ITO film. Therefore, electrochemical measurements in the nucleic acid detection system described here are best performed using phosphate buffer as the supporting electrolyte. Acknowledgment. The authors gratefully acknowledge the contributions of other members of the Xanthon Research and Development team. Helpful discussions with Professors Holden Thorp, Dennis C. Johnson, and Ed Bowden are greatly appreciated. The authors also thank Dr. Carole Golden for her guidance and critical reading of this manuscript. LA026369E
