Novel Electrochemical Detection Scheme for DNA Binding Interactions

Apr 18, 2003 - This work demonstrates a label-free strategy for probing the interaction of DNA with small organic molecules. A solid-phase monolayer o...
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Novel Electrochemical Detection Scheme for DNA Binding Interactions Using Monodispersed Reactivity of Silver Ions Isaac O. K’Owino, Rahul Agarwal, and Omowunmi A. Sadik* Department of Chemistry, State University of New York at Binghamton, P.O. Box 6000, Binghamton, New York 13902-6016 Received September 9, 2002. In Final Form: January 15, 2003 This work demonstrates a label-free strategy for probing the interaction of DNA with small organic molecules. A solid-phase monolayer of silver deposited on a gold quartz-crystal electrode that is modified with DNA is used as the probe template. Results indicate that, by oxidizing the silver monolayer and in the presence of immobilized DNA molecules, highly reactive oxides of silver ions are generated in-situ, causing a change in the electronic properties of the immobilized dsDNA molecules. By scanning in the reverse direction, current is measured which is attributed to the reduction of the oxide layers. If a low molecular weight, organic DNA binding molecule is introduced into the medium, a structural change in the DNA may occur that is evidenced by a corresponding change in the redox properties of the silver monolayer. This ultimately presents a barrier to the interfacial charge transfer and related “site-blocking effects” of the organic molecules. The variation in the redox current is proportional to the concentration of the DNA binding molecule. The limit of detection was on the order of parts per trillion, which is remarkably lower than previously reported in the literature. Experimental evidence is provided from cyclic voltammetry, differential pulsed voltammetry, scanning electron microscopy, and energy-dispersive X-ray spectroscopy.

Introduction The unique complementary recognition features of nucleic acids can provide a key to understanding their specific binding properties with ligands, metals, and proteins. This knowledge can also provide insights into the actions of anticancer drugs and the mechanisms by which chromosomal damage by contaminants and toxins occurs.1,2 The signal transduction approaches for studying the unique nucleic acid interactions with small molecules include electrochemical, optical, piezoelectric, and acoustic wave transducer principles.3-5 These principles rely on the immobilization of a single-stranded DNA probe onto the different transducers, which convert the hybridization events into electrical or optical signals. Current signaling approaches for probing nucleic acid duplex formation, intercalation, or groove-binding are still largely dependent on fluorescent, electroactive, or radioactive labels.6-10 The exceptional simplicity provided by electrochemical signals of small molecules with DNA has * Corresponding author. Fax: (607) 777-4478. E-mail: osadik@ binghamton.edu. (1) (a) Mecklenburg, M.; Danielsson, B.; Boije, H.; Surugiu, I.; Rees, B. In Chemical and Biological Sensors for Environmental Monitoring; Mulchandani, A., Sadik, O. A., Eds.; ACS Symposium Series 762; American Chemical Society: Washington, DC, 2000; pp 299-301. (b) Heller, A. Faraday Discuss. 2000, 116, 1-13. (2) Dandliker, P. J.; Dandliker, R. E.; Barton, J. K. Angew. Chem., Int. Ed. Engl. 1997, 36, 2715-2730. (b) Marshall, A.; Hodgson, J. Nat. Biotechnol. 1998, 16, 27-32. (3) Kelley, S.; Boon, E.; Barton, J.; Jackson, N.; Hill, M. Nucleic Acid Res. 1999, 27 (24), 4830-4837. (4) Maryyama, K.; Mishima, Y.; Minagawa, K.; Motonaka, J. Anal. Chem. 2002, 74, 3698-3703. (5) Hashimoto, K.; Ito, K.; Ishimor, Y. Anal. Chem. 1994, 66, 3830. (6) Korri-Youssoufi, H.; Garnier, F.; Srivtava, P.; Godillot, P.; Yassar, A. J. Am. Chem. Soc. 1997, 119, 7388. (7) Bauerle, P.; Emge, A. Adv. Mater. 1998, 10, 324. (8) Fan, C.; Song, H.; Hu, X.; Genxi, L.; Jianquin, Z. Anal. Biochem. 1999, 271, 1-7. (9) Yan, F.; Sadik, O. A. J. Am. Chem. Soc. 2001, 123, 11335-11340. (10) (a) Tan, W.; Fang, X.; Li, F.; Liu, X. Chem. Eur. J. 2000, 6, 1107-1111. (b) Berney, H.; West, J.; Haefele, E.; Alderman, J.; Lane, W.; Collins, J. Sens. Actuators, B 2000, 68, 100-108.

been used to design selective biosensors.3,11 Such electrochemical strategies use either direct or indirect procedures.12-14 For the direct approach, the electrochemical oxidation of guanine or adenine in the targeted DNA is used for the detection of mismatches.12,13 The indirect approach, however, uses the ability of electroactive species (e.g. the intercalating organic compounds, metal complexes, anticancer drugs, and probes) to transfer electrons from solution to the electrode surface. Signal generation involving electroactive species has also been achieved by associating ligand species with DNA through purely electrostatic interactions.3,10 The electron transfer between the electroactive species and the solid electrode surface has been used extensively to detect the hybridization of a DNA probe with the target.10-14 In a typical reaction, single-stranded nucleic acid probes are immobilized on an electrode, which is subsequently exposed to the target molecules containing the complementary species. The duplex formation (or hybridization) of the complementary probe to the target molecule is indicated by an increase or decrease in the electrochemistry of the charged, redoxactive reporter molecule. Indirect electrochemical detection shows a weak dependence on distance and provides advantages such as reversibility of the redox reaction, chemical stability, and simple functionalization.11 However, because they interact purely by electrostatic interactions, they do not show a significant measurable response in the presence of mismatches, thus resulting in very low binding for DNA (11) Wang, J. Chem.sEur. J. 1999, 5, 1681-1685. (12) (a) Wetmur, J. G. Crit. Rev. Biochem. Mol. Biol. 1991, 26, 227. (b) Anderson, M. L. Nucleic Acid Hybridization, 1st ed.; SpringerVerlag: New York, 1998. (13) (a) Patolsky, F.; Filanovesky, B.; Katz, E.; Willner, I. J. Phys. Chem. B 1998, 102, 10359. (b) Patolsky, F.; Zayats, M.; Katz, E.; Willner, I.; Palecek, E. Nature 1960, 188, 656. (c) Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H.; Farias, P. A. M.; Dontha, N. Anal. Chem. 1996, 68, 2629. (14) (a) Mikkelsen, M. S. R. Anal. Chem. 1994, 66, 2943-2948. (b) Liu, S.; Ye, J.; He, P.; Fang, Y. Anal. Chim. Acta 1996, 335, 239. (c) Carauna, D.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769. (d) Hall, D. B.; Barton, J. J. Am. Chem. Soc. 1997, 119, 5045-5046. (e) Fojta, M.; Kubicarova, T.; Palecek, E. Biosens. Bioelectron. 2000, 15, 107-115.

10.1021/la0265272 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/18/2003

Novel Electrochemical Detection Scheme

recognition reactions.15 Moreover, some electroactive species are especially difficult to use as electrochemical probes because they require high redox potentials, and this often destroys the complementary DNA, which is immobilized on the electrode surface.14 Others such as transition metal complexes result in relatively low binding affinity.15,16 Since DNA is practically insulating, electric currents can be carried through it by the deposition of a silver monolayer on an electrode surface. The redox behavior of the surface bound silver at assessable potentials then provides a convenient interface between immobilized nucleic acids and solution bound analytes. Although silver in its various forms has been used to modify electrodes for a host of reasons, no work has been done in which monodispersed silver ions are used as an analytical tool, and comparatively little has been done for monitoring biomolecular reactions. For example, changes in the extinction spectrum of microscopic metal particles have been used to measure low concentrations of specific analytes using UV/vis spectroscopy.17 Silver ions were used too to instill electrical functionality along the DNA molecule and have been shown to form complexes with DNA bases.18 Moreover, silver provides stability for the self-assembled monolayers of thiols on gold and for detecting DNA hybridization, based on the precipitation of silver on gold nanoparticle tags.19 Ag(I) has been shown to promote pyridine nucleobase pairing (p-p) inside the DNA even at the micromolar order despite low affinity between Ag(I) and pyridine in aqueous media and has also been used to provide signal enhancements in genebased detection.20 It appears that no prior work exists in which monodispersed silver ions are used for direct electrochemical detection of DNA binding to ligands. Our previous works9,21 illustrated a supramolecular design of dsDNA biosensors for the study of DNA-organic molecule interactions using Ag-Au coated quartz crystal, and these utilized solution-based ferricyanide as mediator. The approach involved nonhybridization based DNA immobilization chemistry and was subsequently demonstrated for the supramolecular design of biosensors for low molecular weight organics including metals, polychlorinated biphenyl, and chlorophenols.21,22 In this work, we report a novel electrochemical scheme for probing the electronic properties of DNA binding with small molecules. The strategy for direct signaling using the reactivity of silver ions is illustrated in Figure 1. A monolayer of silver is deposited on a gold electrode or other conducting substrates (e.g. platinum, silver, or glassy carbon). The silver deposition can also be achieved by incubating silver compounds for approximately 5 min in the dark at room temperature. The electrochemical (15) (a) Carter, M. T.; Rodriguez, M.; Bard, A. J. Am. Chem. Soc. 1989, 111, 8901-8911. (b) Dandliker, P. J.; Nunez, M. E.; Barton, J. K. Biochemistry 1998, 37, 6491-6502. (16) (a) Friedman, A. E.; Chambron, J. C.; Sauvagae, J. P.; Turro, N.; Barton, J. K. J. Am. Chem. Soc. 1990, 112, 4960-4962. (b) Sharon, T. S.; Jamil, S. S.; Franesco, P. F.; Luigi, G. M. J. Am. Chem. Soc. 2002, 124 (8), 1558. (17) Van Duyne, R.; Duval-Malinsky, M. J. Am. Chem Soc. 2001, 123, 1471. (18) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775-778. (19) (a) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739-5741. (b) Wang, J.; Xu, D.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 42084209. (20) (a) log K(ML/M*L) ) 2; log K(ML2/L*L2) ) 4. Smith, R. M.; Martell, A. E. Critical Stability Constants; Plenum Press: New York and London, 1975; Vol. 2, Chapter IV. (21) Yan, F.; Sadik, O. A. Anal. Chem. 2002, 73, 5272-5280. (22) Breimer, M.; Yevgheny, G.; Sadik, O. A. Nucleic Acid Res. (in press).

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oxidation of silver produces silver ions and electrons accompanied by a reversible redox signal (Figure 1a). If dsDNA is present at the surface, the silver ions are dispersed and are held electrostatically. Upon reduction, the silver ions return to the surface and a reduction current is measured (Figure 1b). If a DNA binding low molecular weight organic molecule is introduced into the solution, structural change in the DNA molecule occurs, which is signified by a simultaneous change in the redox currents from silver, and a decrease in current is measured (Figure 1c). This decrease is proportional to the concentration of the DNA binding molecule. The underlying signal transformations produced here are believed to result from the DNA conformational or structural changes in the presence of the analyte molecules, which ultimately hinder the flow of electrons. The proposed signaling strategies described are monitored using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) and are demonstrated for PCBs and cisplatin as model compounds. Experimental Section Reagents and Stock Solutions. Avidin and biocytin hydrazide were obtained from Pierce Chemicals while PCBs (Aroclor standards 12221,1242, 1254, and 1260) were purchased from Ultra Scientific, North Kingstown, RI. cis-Platinum(II) diammine dichloride (cisplatin) was obtained from Sigma. In addition, silver nitrate (AgNO3) was purchased from Aldrich Chem. Co. All chemicals were used as received from the vendors without further purification. 17-base pair oligonucleotide sequences (probe A, biotin-5′-TAA-GCA-ACC-TGA-TTT-GAA-3′; target B, 5′-TCAAAT-CAG-GTT-GCT-TA-3′) were obtained from Synthegen (Houston, TX). Buffers used were as follows: 50 mM Hepes buffer (pH 7.3); 0.2 M ammonia buffer (made from 0.2 M ammonia sulfate and pH adjusted to 7.3 using concentrated ammonia); 70 mM Tris-HCl (pH 7.3), and 0.1 M sodium acetate buffer (pH 7.3). A 1 nM standard solution of cisplatin was made and diluted to the required concentrations using phosphate buffer (pH 7.3). Instrumentation. An EG&G potentiostat/galvanostat was used for electrochemical experiments. All microgravimetric QCM experiments were performed using a QCA 917 quartz crystal analyzer (Seiko EG&G). An open-circuit system was used for QCM measurements in which only the gold coated quartz crystal working electrode was connected but in the absence of auxiliary and reference electrodes. The Au coated Quartz crystals (ATcut, 9 MHz) of 0.2 cm2 geometric area per face were obtained from EG&G instruments (Princeton Applied Research). The resonant frequency was determined using a QCA 917 quartz crystal analyzer (Seiko EG&G). The AT-cut crystals were purchased from International Crystal Manufacturers Co., OK. The electrode consists of 1000 Å of gold film on polished quartz with a texture of e1 µm and a 50 Å chromium adhesion layer between the electrode and quartz. An HP 8453 UV-visible spectroscopy system was used for absorption measurements. Surface Microscopy. The surface morphology and qualitative analysis of the DNA-modified electrode were determined using a scanning electron microscope (SEM, Phillips-Electroscan, model 2020) equipped with a Link ISIS EDS analyzer. Following underpotential deposition of silver on quartz crystal electrodes, the surface was rinsed with Nanopure water and then blowdried in a stream of nitrogen. It was mounted with a resin onto the sample stage of the SEM instrument, and the surface morphology was subsequently recorded. Similar surface topography and elemental analysis were performed after every immobilization step involving the analytes. Short Sequence Biotinylated dsDNA. A 1.8 × 10-5 M stock solution of each sequence was obtained by dissolving the pure sample in 0.1 M phosphate saline buffer (pH 7.3) to a total concentration of 100 µg/mL. The biotinylated double stranded DNA was generated by heating a mixture of equal volumes of the two sequences at 70 °C for 30 min and cooling to room temperature. This was confirmed by the decrease in the absorbance at λ260 of the mixture after hybridization reaction relative to that of each sequence before the experiment. The double stranded DNA formed was used without further purification,

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Figure 1. Concept of the electrochemical detection scheme using a monodispersed Ag/Ag+ couple (a) in buffer only, (b) at an immobilized dsDNA electrode in buffer, or (c) at an immobilized dsDNA electrode in buffer and analytes. since the purity was sufficiently high, as determined from the optical measurements (OD260/OD280 was larger than 1.8, where the OD represents absorbance). Electrochemical Measurements. All electrochemical measurements utilized a deep-well 600 µL, three-electrode electro-

chemical Teflon cell. The working electrode was a gold coated quartz crystal while the auxiliary electrode was a platinum wire (0.5 mm diameter). All potentials were measured relative to an aqueous, saturated Ag/AgCl double junction serving as reference electrode unless otherwise noted. The potentials were applied

Novel Electrochemical Detection Scheme and measured using an EG&G 263A potentiostat that was equipped with EG&G M270 software for data acquisition. The solutions were purged with nitrogen for 10 min before any measurement. The underpotential deposition of silver (upd-Ag) was performed under the following conditions: initial potential of 503 mV, potential step 1 of 303 mV (49.90 s), and potential step 2 of 503 mV (10.11 s). CV experiments were performed at a scan rate of 50 mV/s with a potential range of -200 to 400 mV. Immobilization of DNA on the Gold Quartz Crystal and Signal Generation. Underpotential Deposition of Silver. Silver was electrochemically deposited onto the gold quartz crystal using a 1 mM silver nitrate solution. This surface is designated as upd-Ag. Prior to silver deposition, the gold quartz crystal was cleansed in freshly prepared piranha solution. It was subsequently rinsed in water and ethanol followed by blow-drying in a stream of nitrogen. This was later referred to as upd-Ag in the text. Short Sequence DNA Modified Electrode. The modification of the upd-Ag surface with synthetic dsDNA was performed according to the work of Osa et al.23 with some modifications. 50 µL of streptavidin solution was left undisturbed for 30 min on the upd-Ag surface fitted in a 600 µL well type Teflon cell. The well was thereafter rinsed with copious amounts of Nanopure water. 500 µL of phosphate buffer was left undisturbed on the electrode surface for 20 min to remove weakly adsorbed avidin molecules. In a similar manner, the avidin-modified surface was treated with 50 µL of biotinylated dsDNA from the hybridization reaction to give a short sequence DNA modified surface. The cyclic voltammetry experiment with 500 µL of 0.1 M phosphate buffer in this well served as a blank. Cisplatin Analysis Using Short Sequence Biotinylated dsDNA. The DNA modified surface was treated for 30 min with 50 µL of 1 pM cisplatin and thereafter rinsed with phosphate buffer to remove unreacted cisplatin molecules. This was followed by a CV experiment using 0.1 M phosphate buffer as a supporting electrolyte. This electrode surface was rinsed again with phosphate buffer before spiking the DNA bound cisplatin concentrations with 50 µL of 10 pM cisplatin solution in a similar 30-min incubation process. The procedure was repeated for higher cisplatin concentrations up to 105 pM. A reproducibility test (for n ) 6) was carried out on different sensor surfaces for all the analyte concentrations used. As a control, the CV experiment was repeated on a bare upd-Ag for the different cisplatin concentrations. Variation of Peak Current with the Scan Rate. After the immobilization of the short sequence biotinylated dsDNA and the incubation of the sensor surface with 50 µL of 104 pM cisplatin solution as described earlier, CV measurements were carried out at different scan rates (20, 30, 40, 50, 70, and 90 mV/s). Effect of Buffer Ions. CV experiments on the upd-Ag surface were performed with different buffers as supporting electrolytes to determine optimum buffer conditions for sensor signal generation. The buffers thus tested included ammonia, Hepes, sodium acetate, and Tris-HCl, all at pH 7.3.

Results and Discussion As described in the Experimental Section, chronoamperometry was used to produce a uniform coverage of silver. Controllable formation of a silver submonolayer was achieved by monitoring both the time of deposition and the applied potential steps. To avoid possible anion discharge problems, up to one monolayer of silver was electrodeposited onto the gold surface at potentials positive of bulk electrodeposition. This procedure generates fresh surfaces while simultaneously increasing the reproducibility of DNA immobilization, which is a key factor for an efficient signal transduction. A charge of 48 µC for the silver monolayer was calculated on the basis of the geometric area of the electrode used (i.e. 0.20 cm2) and the area occupied by a silver atom (6.61 Å2). The texture of the electrode was e1 µm, and the charge recorded is in (23) Hoshi, T.; Anzai, J.-I.; Osa, T. Anal. Chem. 1995, 67, 770-774.

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agreement with the formation of a submonolayer deposit of silver on a gold substrate under upd conditions.24 A scanning electron micrograph of silver deposition onto the quartz crystal electrode is shown in Supplementary Information Figure 1. Two surfaces are shown, one in which only a bare gold electrode is exposed to the SEM and the other after the monolayer of silver has been deposited. In the former, no silver peaks could be seen. The latter SEM, however, revealed the distribution of silver islands on the gold quartz crystal electrode. An additional EDS spectrum confirmed that the islands are indeed made of silver (Supplementary Information Figure 1b). To function as a solid-state probe, the silver layers must be evenly dispersed along the length of the DNA molecule while simultaneously retaining its stability (Figure 1). We tested the validity of this concept by exposing the silver surfaces to repeated electrochemical cycling. The results showed that the silver layers are indeed stable even after repeated electrochemical cycling up to 400 mV. The EDS results shown in Supplementary Information Figure 2 displayed silver peaks after UPD deposition and exposure to the PCB solutions. These results are also accompanied by the SEMs, revealing the continued presence of the silver islands. These findings are in agreement with the work of Jennings et al.,24 in which XPS studies illustrated that silver coverage on gold was not affected by redox cycling. The noticeable strong Au peak is due to the relatively higher concentration of gold on the quartz crystals. This Au peak masked the S, C, and P peaks that signify the presence of biotinylated DNA on the electrode surface (Supplementary Information Figure 1). These peaks were notably absent before the immobilization of avidin and DNA on the sensor surface (Supplementary Information Figure 2). Moreover, no interference is expected from the gold substrate during the electrochemical oxidation of silver due to its relative chemical inertness.24 The persistence of the Ag peak indicates that, during anodic oxidation of the silver monolayer and subsequent reduction in the cathodic scan, fairly reversible redox peaks are expected. Once silver is oxidized, silver ions are formed and electrons produced will generate the anodic current. A portion of the silver will be reduced to produce the reverse cathodic peaks. Some, however, may be available for binding to the nucleophile rich regions on the DNA. It is also feasible that not all the silver ions are recovered during the reverse scan. The CV data, however, confirmed that some of the silver ions are returned to produce the cathodic currents. Thus, the redox behavior of the Ag0/Ag+ couple could provide an indicator of conformational or structural changes in DNA upon exposure to the analyte solutions. In that case, the stability of the DNA modified electrode is maintained. Further electrochemical characterization of the Ag modified surface was carried out in different buffer solutions. Figure 2 also shows results obtained in 50 mM Hepes buffer (pH 7.3), sodium acetate buffer (pH 7.3), 70 mM Tris-HCl (pH 7.3), and 0.2 M ammonia buffer (pH 7.3). These CV studies revealed that the redox behavior of the Ag0/Ag+ couple is dependent on the nature of the buffer system. Besides, they illustrate that the oxidation/ reduction of the silver ions is measurable in buffers acting as the supporting electrolyte. The summary of the electrochemical characterization is reported in Table 1. The observed shifts in peak potential using different (24) (a) Jennings, K. G.; Laibinis, P. E. J. Am. Chem. Soc. 1997, 119, 5208-521427. (b) Yan, F.; Sadik, O. A. J. Am. Chem. Soc. 2001, 123, 11335. (c) Burgess, J. D.; Hawkridge, F. M. Langmuir 1997, 13, 3781.

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K’Owino et al. Table 1. Summary of the Cyclic Voltammetry Data in Buffers sample

Figure 2. Cyclic voltammograms of the Ag0/Ag+ couple in various buffers (pH 7.3): (i) 0.1 M sodium acetate buffer; (ii) 70 mM Tris-HCl buffer; (iii) 0.1 M phosphate buffered saline; (iv) 50 mM Hepes buffer; (v) 0.2 M ammonia buffer.

buffers may be attributed to the formation of chargetransfer complexes, for example, charge transfer to solvent (CTTS) and metal charge transfer to ligand (MCTL) by Ag+ ions.25 The signal generation mechanism is attributed to the oxidation/reduction of the Ag0/Ag+ couple and is accompanied by the formation of oxide layers (eq 1).26 Other possibilities were considered such as the role of chloride and the precipitation of insoluble Ag3PO43- ions. Silver chloride and Ag+ may be formed at about 0.222 and -0.779 V, respectively. The potential region recorded in this work indicates that this is not the case. We believe that the most significant chemistry is the formation of oxides. The presence of an oxide film on noble metals considerably influences the mechanism and kinetics of anodic processes.27,28 This submonolayer oxide is believed to cause or enhance the catalytic properties of noble metals.29 The oxide film may present a barrier to the interfacial charge transfer.28,29 Although silver oxide forms in all aqueous solutions at varying pH, it is most easily formed in alkaline solution, and under such conditions, secondary silver oxides are formed at more anodic potentials.29c

Ags + H2O (l) h AgOHads + H+aq + e-

(1)

AgOHads + e- f AgOHads-

(2)

Equation 2 is expected for the redox reactions of silver at higher potentials, whereas the CV results and additional characterization, using differential pulse voltammetry (DPV), in phosphate buffer produced peaks at ∼102 mV. The oxide formation is actually composed of two emerging peaks (Figure 4, discussed below), which may be attributed to transition of Ag(I) to Ag(III). The appearance of the separation becomes more apparent with successive incubation in cisplatin because the formation of silver species with a δ+ charge facilitates adsorption of negatively (25) Kapoor, S. Langmuir 1999, 15, 4365-4369. (26) Hecht, D.; Frahm, R.; Strehblow, H. H. J. Phys. Chem. 1996, 100, 10831. (27) Asha, C.; Malhotra, B. D. Biosens. Bioelectron. 2002, 17, 441456. (28) Jerkiewicz, G. In Interfacial Electrochemistry; Wieckowski, A., Ed.; Marcel-Dekker: New York, 1999. (29) (a) Griffith, B. R. P. J. Electrochem. Soc. 1962, 109, 1005. (b) Avramov-Ivic, M.; Jovanovic, G.; Vlanic, J.; Popic, G. J. Electroanal. Chem. 1994, 364, 265. (c) Pourbaix, M. Atlas of Electrochemical Equilibria in Aqueous Solutions; Pergamon Press: New York, 1966. (d) Mansour, A. N. J. Phys. Chem. 1990, 94, 1006-1010.

Ip(ox) Ip(red) Ip(ox)/Ip Ep(ox) Ep(red) δE (mV) (mV) (mV) (µA cm-2) (µA cm-2) (red) Buffers (pH 7.3) 74 152.0 -30.9 -2.0 148.0 -779 122.0 118.0 -278.4 -6.0 310.0 -179.5 32.0 190.0 -245.5

ammonia phosphate acetate Hepes Tris

226.0 146.0 240.0 304.0 226.0

31.8 449.0 273.0 179.5 209.8

∼1.0 1.7 1.0 1.0 1.2

1221 1242 1254

PCB Aroclors in Phosphate Buffer 158.0 -35.1 193.0 -1.8 1.1 172.0 -34.0 206.0 -2.4 1.9 152.0 -23.5 178.0 -1.7 1.2

1.6 1.3 1.4

charged species. This peak separation will become obvious in the next section, where the voltammograms of silver in the presence of small organic molecules are discussed. The low potential recorded, comparable to that of ferrocene and its derivatives,27 would minimize the interference from other electroactive species if present.27 A small peak-topeak separation (δE) and higher current values were used in the evaluation of optimum characterization in buffer conditions. Due to the need to maintain both the pH and stability of the nucleic acid structure, we selected phosphate buffer for subsequent reactions even though the acetate buffer gave lower δE values. In addition, voltammograms obtained in both CV and DPV analyses were fairly symmetric and reproducible. The average standard deviation obtained for cisplatin using the anodic peak current was 2.37 µA. Probing DNA-Small Molecule Interactions. We postulate that the monolayer silver surface formed via upd may fall under heterogeneous mediated bioelectrochemistry in which the Ag+ ions diffuse into the solution during the application of electrical potential.27 This is also compatible with observations in biological systems for drug development.30,31 The multiplicity of binding sites exists on the adenine and guanine rings, and the potential for the formation of different complex types with different metals is feasible.30 The N7 of adenine appears to be frequently useful as an electron donor in many complexes involving metals.30b Not all the silver ions formed during the cathodic sweep are used in complex formation. Some silver ions may use the available empty 5s orbital to form π-bonds. This process may impede the electron transfer between the metal ions and the electrode surface and, inherently, be reflected in the observed changes in the redox peaks. DNA was immobilized as described in the Experimental Section. With immobilized dsDNA on the electrode surface, any Ag+ generated is expected to bind the negatively charged sugar-phosphate backbone and N7 of guanine. Given that the DNA bases may be oriented toward the electrode while the sugar-phosphate backbone is inclined toward the solution phase,30-32 organic molecules may interact with the DNA through intercalation or binding to either the minor or major groove.30-32 Simultaneously, a potential driven cathodic reduction of the silver ions and the oxides or hydroxides may compete with the Ag+-DNA complexation. This mechanism in the presence of organic molecules is illustrated in Figure 1b (30) (a) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215-2235. (b) Eichhorn, G. L. In Organic Biochemistry; Eihhorn, G. L., Ed.; Elsevier: New York, 1973; Vol. 2, pp 1210-1240. (31) (a) Trnkova´, L. Talanta 2002, 56, 887-894. (b) Tepperman, K.; Elder, R. C.; Jones, W. M.; Ameson, L.; Taryer, M. L. J. Inorg. Biochem. 1991, 42, 291. (c) Oakley, G. G.; Devanaboyina, U.; Robertson, L. W.; Gupta, R. C. Chem. Res. Toxicol. 1996, 9, 1285-1292. (32) Curtis, W.; Johnson, S.-H. P. In Principles of Physical Biochemistry; van Holde, K. E., Eds.; Prentice-Hall: New York, 1998; p 57.

Novel Electrochemical Detection Scheme

and c. The model compounds used to test these reactions are the nonelectroactive cisplatin and PCBs. We believe that the change in the redox current observed upon DNA-cisplatin binding might also be attributed to electron transfer resistance. For example, the electron transfer at the electrode interface is expected to change upon the assembly of the DNA and further insulation after binding with cisplatin. This behavior is not limited to DNA, any electrode modifier of insulating features should retard the electron transfer and ultimately change the redox characteristic of the silver indicator. In addition, the observed change in the redox currents may be attributed to a multiple of fundamental microscopic processes such as the flow of charged atoms or atom agglomerates in the electrolytes, charge transfer reactions resulting from the diffusion features of the redox silver indicator, and specific adsorption. Contributions from any of these, however, are easily confirmed or eliminated by changes in the ionic strength, pH, or concentrations of the analytes and/or buffers. Current flow may be further affected by band structure anomalies at any grain boundaries present, especially due to the presence of additional phases provided by the avidin-biotin layers. pH change, however, has been known to affect the stability of the DNA molecule; consequently, the most commonly used pH is pH 7. That is also what is used in this work. Cisplatin-DNA Interactions. Cisplatin, also known as cis-platinum(II) diammine dichloride or cis-DDP, is a widely used anticancer drug with therapeutic activity for effective management of testicular, ovarian, and head and neck cancers.33,34 Cisplatin is believed to act by inhibiting both DNA replication and RNA transcription. Evidence points to DNA as the main target of cisplatin in the tumor cell.33,34 Cisplatin concentrations as low as picogram levels can cause DNA damage.34 Hence, there is a need for the design of ultrasensitive biosensors for detection of cisplatin-DNA interactions. We previously reported the use of electrochemical impedance spectroscopy (EIS) for the detection of cisplatin.9,21 However, the sensitivity obtained was 1 nM. Although this is comparable to those recorded for fluorescent assays, a higher sensitivity is required to directly monitor the interactions of nucleic acids with these molecules. The CV signals obtained using the short sequence DNAimmobilized electrode in the presence of cisplatin are shown in Figure 4. The large (216 µC at 1 pM cisplatin concentration) and sharp anodic peaks are indicative of rapid charge transfer during which silver atoms are oxidized to silver(I) ions.35 A significant change of ∼101 µC was observed in Qan as concentrations were increased from 1 pM to 105 pM on the DNA modified surface (Table 2). A similar trend that is attributed to the binding between the surface confined DNA and cisplatin is also shown by the E1/2 values. In addition, the cathodic peaks are not as sharp as the anodic ones. The width of the peaks was observed to change significantly with the increase in cisplatin concentration (Table 2). This change reflects differences in the effective diffusion of ions to the electrode surface as a result of binding.36 Besides, linearity (33) (a) Hacker, M. P., Douple, E., Krakoff, I. H., Eds. Platinum Coordination Compounds in Cancer Chemotherapy; Nijhoff: Boston, 1984. (b) Sherman, S.; Gibson, D.; Wang, A.; Lippard, S. Science 1985, 230, 412. (34) (a) Bancroft, D.; Lepre, C. A.; Lippard, S. J. J. Am. Chem. Soc. 1990, 112, 6860. (b) Neidle, S. DNA Structure and Recognition; Oxford University Press: Oxford, U.K., 1994. (35) Nelson, P. A.; Elliot, J. M.; Attard, G. S.; Owen, J. R. Chem. Mater. 2002, 14, 524-529. (36) (a) Pang, D.-W.; Abruna, H. D. Anal. Chem. 1998, 70, 31623169. (b) Pang, D.-W.; Abruna, H. D. Anal. Chem. 2000, 72, 4700-4706.

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Figure 3. CV of the Ag0/Ag+ couple at different scan rates [(i) 20 mV/s; (ii) 50 mV/s; (iii) 90 mV/s)] after immobilization of short sequence biotinylated dsDNA and incubation with 105 pM cisplatin. Inset: A graph showing the linear relationship between the scan rate and the peak current.

Figure 4. CV of the Ag0/Ag+ couple in the presence of different concentrations of cisplatin after DNA modification: (i) 1 pM; (ii) 103 pM; (ii) 104 pM; (iv) 105 pM. Table 2. Summary of CV Data for Cisplatin-DNA Interaction at Different Concentrations conc (pM)

Qana (µC)

Qcathb Ean (µC) (mV)

1 102 104 105

216.0 175.6 147.0 114.8

199.4 148.2 106.9 101.6

a

Ecath (mV)

∆E E1/2(cath) E1/2(an) (mV) (mV) (mV)

186.5 -27.6 216.1 175.7 -1.0 176.7 168.8 -1.4 170.2 172.7 -8.0 180.7

78.9 70.1 67.7 63.7

48.8 45.0 37.4 35.2

Qan ) anodic charge. b Qcath ) cathodic charge.

of the scan rate with anodic peak current (Figure 3) is typical of most surface confined redox active species.36 As predicted by the detection strategies in Figure 1, the oxidation peaks of silver were maintained at the low potential region. Yet a decrease in the peak current was recorded as the concentration of cisplatin increased on the DNA modified electrode. A linear range of 10 to 1 × 104 pM was obtained (Figure 5). The low limit of detection is a characteristic that provides superior detection for cisplatin in comparison to other techniques such as acoustic wave,37 in which a detection limit of 105 pM has (37) Su, H.; Williams, P.; Thompson, M. Anal. Chem. 1995, 67, 1010.

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Figure 5. Current -log [pM] calibration curve for cisplatin-DNA interactions showing the linear range as ∼101 to104 pM and a lower detection limit of 10 pM for the DNA modified surface. Inset: a nonlinear relationship for the control experiment obtained after the upd Ag step and successive 30-min incubation of standard cisplatin solutions. Without the dsDNA on the electrode surface, there is an ∼30% drop in the signal between 1 and 10 pM. This change remains almost constant with continued addition of cisplatin up to 105 pM. However, with the immobilized DNA on the electrode surface, a sigmoidal response was achieved.

been reported. Suppressed peak currents may allow the in-vitro study of cisplatin-DNA complexes.38 The nonlinear trend (Figure 5 inset) obtained on the bare upd Ag electrode is evidence that the presence DNA on the sensor surface is vital for signal generation. We noticed that, with the bare upd Ag surface, the signal measured was not only nonlinear; there was also no noticeable trend with repetitive measurements. The results also agree with previous studies showing that a DNA-cisplatin complex may distort the DNA helix.38,39 Thus, as predicted in Figure 1, the observed changes in the electrochemical signal are attributed to DNA structural changes and the resulting simultaneous insulation of the electrode surface by the immobilized biomolecules. Using the proposed electrochemical detection scheme, a detection limit of 10 pM was obtained for cisplatin-short sequence DNA interactions. This represents a 100-fold improvement compared with the previous (103 pM) detection limit.9,21 This remarkable sensitivity signifies the suitability of the new scheme for monitoring DNA-cisplatin interactions. The detection may also be used to monitor other biomolecular reactions including DNA-protein, antigen-antibody, and DNA-RNA reactions. Conclusion A novel electrochemical detection principle has been reported for the detection of DNA-small molecule inter(38) Cullinale, C.; Mazur, S.; Essigman, J.; Phillips, D.; Bohr, V. Biochemistry 1999, 38, 6204-6212. (b) Zhen, W.; Evans, M.; Haggerty, C.; Bohr, V. Carcinogenesis 1993, 14, 919-924. (39) Bard, J. A.; Faulkner, L. R. Electrochemical Methods; Wiley and Sons Inc.: New York, 1980.

actions. This work is supported by data obtained using a short double-stranded DNA sequence. The current responses with and without DNA confirm that the concentration-dependent responses are mainly due to the analytes. The approach offers an alternative route to labeled reagents and solution-based mediated DNA reactions and may be used for the detection of DNA hybridization reactions, gene detection, and DNA-protein interactions. The limit of detection, in the order of parts per trillion, was remarkably lower than previously reported in the literature. Experimental evidence of the electrochemical reactivity was provided from CV, SEM, and EDS, which confirmed that silver retains stability upon electrostatic binding at DNA. Furthermore, modulating the electronic properties of DNA was possible by changing the buffer conditions. Acknowledgment. The authors express their appreciation to Joanne Pfeil of the Department of Biological Sciences, SUNYsBinghamton, for assistance in the extraction of plasmid DNA and agarose gel electrophoresis experiments and to Dr. Barbara Poliks, Department of Physics, SUNYsBinghamton, for molecular modeling. The following agencies are acknowledged for financial support: National Science Foundation (CHE-0210968) and NYS Center for Advanced Technology (IEEC). Supporting Information Available: EDS and SEM spectra of a gold quartz crystal and avidin/DNA-PCB complexes. This material is available free of charge via the Internet at http://pubs.acs.org. LA0265272