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Langmuir 1997, 13, 6342-6344
Modification of Electrodes with Dicarboxylate Self-Assembled Monolayers for Attachment and Detection of Nucleic Acids Mary E. Napier and H. Holden Thorp* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290 Received July 16, 1997. In Final Form: September 2, 1997
The formation of self-assembled monolayers on surfaces1 has enabled the design of new interfaces for the study of specific redox-active analytes,2 solar energy conversion,3 and fundamental electrochemistry.4 Such monolayers can be formed via the familiar alkanethiol-gold linkage but also via related linkages between carboxylates and phosphonates and metal oxide surfaces, such as tin-doped indium oxide (ITO).3,5 Self-assembled monolayers have been particularly useful in the study of biomolecules; particularly relevant examples to that described here are the formation of alkanethiol monolayers on gold with pendant carboxylate groups that electrostatically bind the redox-active cytochrome c protein2 and the attachment of DNA oligonucleotides to gold electrodes via alkylthiol linkers.6,7 Here we report the formation of monolayers of 1,12-dodecanedicarboxylic acid (DDCA) on ITO, using the known affinity of carboxylate for metal-oxide surfaces.3,5 The modified electrodes can be further derivatized with DNA via reaction of the pendant carboxylate with endogenous amines of the nucleobases following activation with water-soluble carbodiimide. The electrochemistry of Ru(bpy)32+ is unaffected by the formation of the monolayer (bpy ) 2,2′-bipyridine); however, the attachment of DNA to the electrode leads to a large catalytic enhancement due to the oxidation of guanine by the oxidized metal complex.8,9 This arrangement can be used to detect the hybridization of poly[G] to an electrode modified with poly[dC], providing a new type of DNA hybridization sensor. Importantly, the carboxylate-ITO interface is compatible with the electrochemistry of Ru(bpy)32+ at E1/2 ) 1.05 V (vs Ag/AgCl), which would not be the case with gold-thiol monolayers. Experimental Section Reagents and DNA. Inorganic reagents used in these experiments were of analytical grade or higher. Water-soluble carbodiimide (WSC, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride), 1,12-dodecanedicarboxylic acid (DDCA), and hexadecane were obtained from Aldrich (Milwaukee, WI). Na2HPO4, NaH2PO4, NaCl, and hexane were obtained from Maillinckrodt (Phillipsburg, NJ). Highly polymerized calf thymus DNA, poly(deoxycytidylic acid) (Poly[dC]), and poly(guanylic acid) (Poly[G]) were obtained from Sigma (St. Louis). Water was obtained from a Milli-Q Plus purification system (Millipore, (1) Laibinis, P. E.; Hickman, J. J.; Wrighton, M. S.; Whitesides, G. M. Science 1989, 245, 845-847. (2) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 18471849. (3) Pechy, P.; Rotzinger, F. P.; Nazeeruddin, M. K.; Kohle, O.; Zakeeruddin, S. M.; Humphry-Baker, R.; Gratzel, M. J. Chem. Soc., Chem. Commun. 1995, 65-66. (4) Terrettaz, S.; Becka, A. M.; Traub, M. J.; Fettinger, J. C.; Miller, C. J. J. Phys. Chem. 1995, 99, 11216-11224. (5) Gardner, T. J.; Frisbie, C. D.; Wrighton, M. S. J. Am. Chem. Soc. 1995, 117, 6927-6933. (6) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 38303833. (7) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Bioconjugate Chem. 1997, 8, 31-37. (8) Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933-8938. (9) Napier, M. E.; Loomis, C. R.; Sistare, M. F.; Kim, J.; Thorp, H. H. Bioconjugate Chem., in press.
S0743-7463(97)00796-8 CCC: $14.00
Bedford, MA). ITO electrodes were obtained from Delta Technologies (Stillwater, MN). Electrode Modification. ITO-coated sheets were cut into 1.5 cm × 1.5 cm squares. The ITO electrodes were cleaned using the following procedure: sonication in Alconox in water for 15 min, sonication in 2-propanol for 15 min, and sonication twice in water for 15 min each cycle. Monolayers were self-assembled by immersion of the ITO electrodes in 5 mM 1,12-dodecanedicarboxylic acid in hexadecane for 36-48 h. The electrodes were thoroughly rinsed with hexane to remove any physically adsorbed 1,12-dodecanedicarboxylic acid. Formation of dicarboxylic acid monolayers was confirmed by X-ray photoelectron spectroscopy (XPS) analysis. The activation of the surface carboxylates was performed by placing 40 mL of a freshly prepared 10 mM water soluble carbodiimide (WSC) in 20 mM sodium phosphate buffer (pH 7.0) onto the electrode. The carbodiimide was allowed to dry on the electrode surface. The electrode was thoroughly rinsed with 20 mM sodium phosphate buffer and 200 mM NaCl. The DNA was coupled to the activated electrode surface by application of 20 mL of 2.0 mM calf thymus DNA in water or application of 20 mL of a 1 mg/mL poly[dC] in water. Hybridization of poly[dC] and poly[G]. The ITO electrodes with poly[dC] attached were exposed to a single application of 90-120 mL of 1.4 mg/mL poly[G] in 50 mM sodium phosphate buffer at room temperature for 3 h. The electrodes were thoroughly rinsed with 20 mM sodium phosphate and 200 mM NaCl prior to electrochemical analysis. Electrochemical Analysis. Cyclic voltammograms were collected using a PAR 273A potentiostat/galvanostat with a single compartment voltammetric cell equipped with an indium tin oxide (ITO) working electrode (area 0.32 cm2), a Pt-wire counter electrode, and a Ag/AgCl reference electrode. In a typical experiment, 200 mL of 200 mM Ru(bpy)32+ in 50 mM sodium phosphate buffer (pH 7.0) was placed into the voltammetric cell containing the modified ITO working electrode, and cyclic voltammograms from 0.0 to 1.3 V were taken at a scan rate of 25 mV/s.
Results and Discussion Electrode Fabrication. The ITO electrodes were modified by formation of monolayers of DDCA. The monolayers could be used any time within a week of formation with no change in the electrochemical response (stability beyond 1 week was not evaluated). The terminal carboxylate was linked to the exocyclic amines of DNA nucleobases via carbodiimide-catalyzed reaction of the amine group to form amide linkages.9 The DNA attachment reaction was followed by X-ray photoelectron spectroscopy. Indium, tin, oxygen, and carbon XPS peaks were observed for the unmodified ITO electrode; the carbon is due to contamination from the ambient laboratory environment. Following self-assembly of the DDCA monolayers, increases in both carbon and oxygen were observed in the appropriate C/O ratio of 4:1. The appearance of a modest amount of nitrogen was observed following the activation by carbodiimide, and a much larger (4-fold) increase in nitrogen was observed followed DNA attachment. Phosphorus could not be used as an indicator of DNA attachment, because sodium phosphate buffer was used to wash the electrode after each step in the coupling reaction. DNA Detection. We have shown previously that nucleic acids can be detected in solution via the catalytic oxidation of guanine using Ru(bpy)32+ as the mediator.8-11 In solution, Ru(bpy)32+ exhibits a reversible redox couple at 1.05 V (all potentials given versus Ag/AgCl), which is very similar to the oxidation potential of guanine.12 Addition of DNA to a solution of Ru(bpy)32+ leads to a (10) Johnston, D. H.; Welch, T. W.; Thorp, H. H. Met. Ions Biol. Syst. 1996, 33, 297-324. (11) Johnston, D. H.; Thorp, H. H. J. Phys. Chem. 1996, 100, 1383713843.
© 1997 American Chemical Society
Notes
Langmuir, Vol. 13, No. 23, 1997 6343
Figure 1. Cyclic voltammograms (25 mV/s) of 200 mM Ru(bpy)32+ at a (A, solid) bare ITO working electrode, (B, dotted) DDCA-modified ITO working electrode, and (C, dashed) DDCAmodified electrode following attachment of calf thymus DNA: reference electrode, Ag/AgCl; auxiliary electrode, Pt wire; supporting electrolyte, 50 mM phosphate buffer, pH 7. Preparation of the DDCA and DDCA/DNA electrodes is described in the Experimental Section. Voltammograms in A and B are not distinguishable.
catalytic enhancement in the oxidation current according to a two-step mechanism
Ru(bpy)32+ f Ru(bpy)33+ + e-
(1)
Ru(bpy)33+ + DNA f DNAox + Ru(bpy)32+
(2)
where DNAox is a DNA molecule where guanine has been oxidized by one electron. We have shown previously that the rate constant for eq 2 in 50 mM phosphate buffer (pH 7) with 700 mM added NaCl is 1.0 × 104 M-1 s-1 for double stranded DNA; at lower ionic strength (50 mM phosphate with no added NaCl), the rate constant for eq 2 increases to 1.4 × 105 M-1 s-1 because of increased binding of the metal complex to the DNA and a consequent increase in the local concentration.11 The simulations and kinetic analyses for these scenarios have been described in detail elsewhere.11 We have reported elsewhere that this detection scheme can be applied to ITO electrodes modified with DNA-coupled poly(ethylene terephthalate) for detection of products of the polymerase chain reaction.9 In the both the poly(ethylene terephthalate) films and those described here, maximum catalytic current is obtained only on the initial voltammetric or amperometric scan, because while the reaction is catalytic in Ru(bpy)32+, guanine is consumed by the electrolytic process. In fact, maximum sensitivity will be obtained when all of the guanines are oxidized in a single scan. The detection of nucleic acids coupled to a monolayermodified electrode surface was initially performed with double-stranded calf thymus DNA that was coupled to the terminal carboxylic acid groups via the endogenous amines of the nucleobases using the carbodiimide reaction. Figure 1 shows cyclic voltammograms of Ru(bpy)32+ at an unmodified ITO electrode (Figure 1A), an electrode modified with DDCA only (Figure 1B), and a modified ITO electrode following coupling to calf thymus DNA (Figure 1C). Modification of the electrode with the dicarboxylic acid has a negligible effect on the electrochemistry of Ru(bpy)32+, probably due to defect sites in the monolayer on the amorphous surface that are accessible to the small metal complex but apparently not accessible to DNA itself (vide infra). A significant increase in current was observed following attachment of calf (12) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617-618.
Figure 2. Cyclic voltammograms (25 mV/s) of 200 mM Ru(bpy)32+ at a (A, dashed) DDCA-modified electrode to which poly[dC] has been attached and (B, dotted) after exposure of the electrode in (A) to poly[G]. Curve (C, solid) shows the voltammogram obtained at the hybridized electrode from (B) in the absence of Ru(bpy)32+. Buffer and cell conditions are as in Figure 1.
thymus DNA. This current increase can be attributed to catalytic oxidation of the guanines in the immobilized DNA to the solution-bound Ru(bpy)33+.8-11 As expected, when the modified ITO electrode was exposed to calf thymus DNA without prior activation by the water-soluble carbodiimide, no increase in current was observed. Nonspecific binding of the DNA is therefore not responsible for the oxidation current observed in Figure 1C. Nonspecific binding of DNA was not expected to be problematic since both the monolayer and the DNA are negatively charged; we have shown previously that DNA does not adsorb to unmodified ITO.13-15 The DNA-modified monolayers were stable at ambient temperature; however, the modified electrodes begin to degrade at approximately 40 °C and by 70 °C had completely decomposed. Optical spectroscopy and electrochemistry indicated that the elevated temperatures caused the carboxyl groups to dissociate from the ITO surface rather than disruption of the DNA-carboxylate linkage. Hybridization Detection. Hybridization experiments were attempted with electrodes where poly[dC] was attached to the monolayer and then exposed to a solution of poly[G] in 50 mM sodium phosphate buffer at room temperature for 3 h. Figure 2 shows cyclic voltammograms of Ru(bpy)32+ at the poly[dC]-modified electrode (Figure 2A) and following hybridization to poly[G] (Figure 2B). The significant current increase suggests that the hybridization event was successful and that electron transfer from the guanines of the hybridized strand to the Ru(bpy)32+ is responsible for the increase in the oxidation current. Figure 2C shows the cyclic voltammogram of the hybridized electrode without Ru(bpy)32+; very little direct oxidation of hybridized guanine was observed without the soluble mediator. Use of the endogenous amines for coupling of poly[dC] and calf thymus DNA to the electrode likely leads to partial denaturation of the DNA; however, the local distortions are apparently not severe enough to prevent formation of the simple poly[dC]‚poly[G] duplex. The electrochemical results show clearly that the DDCA monolayer largely protects the guanines in DNA from direct oxidation by the electrode while maintaining a high electroactivity for Ru(bpy)32+. This situation obtains (13) Johnston, D. H.; Cheng, C.-C.; Campbell, K. J.; Thorp, H. H. Inorg. Chem. 1994, 33, 6388-6390. (14) Welch, T. W.; Corbett, A. H.; Thorp, H. H. J. Phys. Chem. 1995, 99, 11757-11763. (15) Welch, T. W.; Thorp, H. H. J. Phys. Chem. 1996, 100, 1382913836.
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because there are apparently defects in the monolayer that permit access of Ru(bpy)32+ to the electrode but do not preferentially bind to DNA, which is immobilized instead on the terminus of the dicarboxylate. Thus, collection of the large majority of charge from the immobilized DNA occurs only via the Ru(bpy)32+-mediated reaction. This feature differs significantly from that of electrodes where DNA is deliberately adsorbed directly to the electrode and charge can be efficiently collected from the nucleobases16-18 and is distinct from approaches where the presence of duplex DNA on the surface increases the (16) Palecek, E.; Fojta, M. Anal. Chem. 1994, 66, 1566-1571. (17) Wang, J.; Chicharro, M.; Rivas, G.; Cai, X.; Dontha, N.; Farias, P. A. M.; Shiraishi, H. Anal. Chem. 1996, 68, 2251-2254. (18) Palecek, E. Electroanalysis 1996, 8, 7-14.
Notes
local concentration of a solution redox couple.19,20 The advantages of the mediated approach are that the kinetics of individual nucleobase-metal redox reactions can be used to obtain specificity in detection9 and macromolecular structures that might be disrupted by passive adsorption can potentially be preserved through careful engineering of the DNA attachment. Acknowledgment. Support of this research by the David and Lucile Packard Foundation and Xanthon, Inc., are gratefully acknowledged. LA970796O (19) Millan, K. M.; Saraullo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 2943-2948. (20) Xu, X.-H.; Bard, A. J. J. Am. Chem. Soc. 1995, 117, 2627-2631.
