Langmuir 2008, 24, 5155-5160
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Scanning Electrochemical Microscopy Imaging of DNA Microarrays Using Methylene Blue as a Redox-Active Intercalator Andrew J. Wain and Feimeng Zhou* Department of Chemistry and Biochemistry, California State UniVersitysLos Angeles, Los Angeles, California 90032 ReceiVed December 14, 2007. In Final Form: February 4, 2008 Scanning electrochemical microscopy (SECM) has been employed in the imaging of DNA microarrays fabricated on gold substrates using methylene blue (MB) as a redox-active intercalator and ferrocyanide as the SECM mediator in solution. MB intercalated between base pairs of immobilized ds-DNA is electrochemically reduced via electron transfer from the underlying gold substrate, and the product is reoxidized in solution by SECM tip-generated ferricyanide. The resulting feedback current allows a heterogeneous electron-transfer rate constant for the MB-intercalated DNA to be deduced. Moreover, DNA microarray spots can be imaged at a detection level of 14 fmol/spot for ds-DNA consisting of 15 base pairs. Microarrays prepared using 20 µM DNA solutions are easily visualized, and the feasibility of detecting base pair mismatches is also demonstrated.
Introduction The study of electrodes modified with self-assembled monolayers (SAMs) of double- and single-stranded (ds- and ss-) DNA has long been established as an effective means of developing electrochemically based biosensors.1,2 With the aim of determining DNA concentration through hybridization or detecting base pair mismatches in ds-DNA, common practice involves the use of either an exogenous electroactive label or the intrinsic redox activity of DNA bases (adenine and guanine) to permit interrogation using amperometric, coulometric, or impedance measurements at the DNA-modified surface.1,3-12 A range of signal transduction methodologies have been proposed with varying complexity, the desirable features being low cost, scope for miniaturization, and ease of operation and data analysis. One of the many challenges in biosensor development is the extension of such detection schemes to the high-throughput screening of numerous samples for them to rival fluorescence-based multiplexing techniques.13-15 Scanning electrochemical microscopy (SECM) offers a unique means to visualize surfaces by monitoring the limiting current at a microelectrode tip immersed in an electrolyte solution containing a suitable redox species. Restricted diffusion at short * Corresponding author. E-mail:
[email protected]. Tel: 323-3432390. Fax: 323-343-6490. (1) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323. (2) Napier, M. E.; Loomis, C. R.; Sistare, M. F.; Kim, J.; Eckhardt, A. E.; Thorp, H. H. Bioconjugate Chem. 1997, 8, 906-913. (3) Kelley, S. O.; Boon, E. M.; Jackson, N. M.; Hill, M. G.; Barton, J. K. Nucleic Acids Res. 1999, 27, 4830-4837. (4) Li, X.; Lee, J. S.; Kraatz, H.-B. Anal. Chem. 2006, 78, 6096-6101. (5) Lucarelli, F.; Marrazza, G.; Turner, A. P. F.; Mascini, M. Biosens. Bioelectron. 2004, 19, 515-530. (6) Takagi, M. Pure. Appl. Chem. 2001, 73, 1573-1577. (7) Tansil, N. C.; Xie, H.; Xie, F.; Gao, Z. Anal. Chem. 2005, 77, 126-134. (8) Wang, J. Biosens. Bioelectron. 2006, 21, 1887-1892. (9) Campbell, C. N.; Gal, D.; Cristler, N.; Banditiat, C.; Heller, A. Anal. Chem. 2004, 76, 4093-4097. (10) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134-9137. (11) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770-772. (12) Wang, J.; Li, J.; Baca, A. J.; Hu, J.; Zhou, F.; Yan, W.; Pang, D.-W. Anal. Chem. 2003, 75, 3941-3945. (13) Kricka, L. J. Clin. Chem. 1999, 45, 453-458. (14) Epstein, J. R.; Biran, I.; Walt, D. R. Anal. Chim. Acta 2002, 469, 3-36. (15) Wolcott, M. J. Clin. Microbiol. ReV. 1992, 5, 370-386.
tip-to-surface distances allows topological features to be mapped, and information related to the conductivity of the surface can be acquired from the analysis of feedback currents.16,17 The imaging of immobilized DNA arrays using SECM has been demonstrated by various authors.18-26 This group used the oxidation of guanine by Ru(bpy)33+ as an indicator of surface-bound DNA24 and also made use of a silver staining process to allow the visualization of hybridization events on immobilized DNA spots.23 Other notable contributions include the work of Schuhmann and coworkers, who used the repulsion between ferricyanide and negatively charged DNA to differentiate between immobilized ss- and ds-DNA,21,22 and Kondo et al., who employed a ferrocenetagged intercalator to image DNA microarrays.26 Fortin et al. combined SECM with surface plasmon resonance imaging to deposit and image spots of oligodeoxynucleotide-modified copolymers.19 Also, Bard and co-workers used SECM to investigate electron transfer (ET) through DNA-modified surfaces in the absence and presence of metal ions.20 The practice of SECM has advantages over conventional electrochemical methods, particularly in that the use of microelectrodes under steadystate conditions eliminates problematic background currents due to double-layer charging and contributions from possible adsorbates. The extension of SECM to established electrochemical biosensing schemes should therefore prove beneficial, particularly because some such assays have been reported with nano- and even picomolar detection levels.5,8 (16) Bard, A. J.; Fan, F.-R. F.; Mirkin, M. V. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1994; Vol. 18, p 243. (17) Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1221-1227. (18) Roberts, W. S.; Lonsdale, D. J.; Griffiths, J.; Higson, S. P. J. Biosens. Bioelectron. 2007, 23, 301-318. (19) Fortin, E.; Defontaine, Y. P. M.; Livache, T.; Szunerits, S. Electroanalysis 2005, 17, 495-503. (20) Liu, B.; Bard, A. J.; Li, C.-Z.; Kraatz, H.-B. J. Phys. Chem. B 2005, 109, 5193-5198. (21) Turcu, F.; Schulte, A.; Hartwich, G.; Schuhmann, W. Biosens. Bioelectron. 2004, 20, 925-932. (22) Turcu, F.; Schulte, A.; Hartwich, G.; Schuhmann, W. Angew. Chem., Int. Ed. 2004, 43, 3482-3485. (23) Wang, J.; Song, F.; Zhou, F. Langmuir 2002, 18, 6653-6658. (24) Wang, J.; Zhou, F. J. Electroanal. Chem. 2002, 537, 95-102. (25) Komatsu, M.; Yamashita, K.; Uchida, K.; Kondo, H.; Takenaka, S. Electrochim. Acta 2006, 51, 2023-2029. (26) Yamashita, K.; Takagi, M.; Takenaka, S.; Uchida, K.; Kondo, H. Analyst 2001, 126, 1210-1211.
10.1021/la703922v CCC: $40.75 © 2008 American Chemical Society Published on Web 03/21/2008
5156 Langmuir, Vol. 24, No. 9, 2008 Scheme 1. Schematic Representation of the SECM Detection Methodologya
Wain and Zhou Table 1. Probe and Target DNA Sequences (Mismatched Bases Indicated) thiolated probe complement one-base mismatch two-base mismatch three-base mismatch
5′ HS(CH2)6-AGT ACA GTC ATC GCG 3′ 5′ CGC GAT GAC TGT ACT 3′ 5′ CGC GAT AAC TGT ACT 3′ 5′ CGC GAT AAC TAT ACT 3′ 5′ CGC GGT AAC TAT ACT 3′
Experimental Section
Barton, Hill, and co-workers have demonstrated that methylene blue (MB), a redox-active phenothiazine intercalator, may be used as an electrochemical indicator to probe the behavior of surface-bound DNA.27 MB has been shown to bind strongly to ds-DNA, inserting between base pairs and thus becoming electronically coupled to the duplex π-π stacks. Evidence suggests that the electroreduction of intercalated MB (E0 ∼ -0.2 V versus Ag/AgCl) is facilitated by base pairing in fully complementary ds-DNA but charge transfer is impeded by the presence of mismatched bases.3 Moreover, signal amplification has been demonstrated by the use of an electrocatalytic cycle; the reduced form of MB (leucomethylene blue, LB) is chemically oxidized by ferricyanide, present in excess in the electrolyte solution, thus regenerating MB and allowing the DNA-modified surface to be repeatedly probed.28 The detection of base pair mismatches was realized with this scheme, although the application of the approach to a multiplexed DNA microarray would be difficult to envisage unless different sequences were immobilized on a single chip of individually addressable electrodes using sophisticated microarrayers with precise alignments with respect to the electrodes. In this article, we demonstrate how the electrocatalytic reaction of the MB/ferricyanide scheme28 can be modified to allow SECM imaging of DNA microarray spots. Applying a suitable potential to the substrate reduces the MB molecules, intercalated into the immobilized DNA, to LB. Ferrocyanide can be oxidized at a microelectrode tip to generate ferricyanide, which, in close proximity to the microarray surface, oxidizes LB molecules back to MB (Scheme 1). Consequently, the ferrocyanide regenerated at the surface results in enhanced tip currents over DNA-covered regions and permits array visualization at a sensitive level. Such SECM imaging of DNA microarrays, fabricated without laborious labeling and expensive microarrayer devices, marks a step toward high-throughput multiplexing imaging based on electrochemical detection schemes. The capability of SECM to measure heterogeneous electron-transfer rates through the DNA molecules demonstrates the added flexibility of SECM in studying biological events in highly localized regions.
Reagents. DNA, purchased from Integrated DNA Technologies, Inc. (Coralville, IA), was dissolved in 5 mM sodium phosphate buffer (pH 7.2) containing 50 mM NaCl (phosphate-buffered saline, PBS). The probe sequence was modified with a hexanethiol group at its 5′ end to allow tethering to the gold substrate electrodes. The 15-mer DNA sequence is the same as that in the work of Barton, Hill, and co-workers (Table 1).28 Target DNA included the fully complementary sequence and sequences with one, two, and three base mismatches. In the case of thiol-terminated DNA, disulfide bonds were reduced to thiols using excess tris(2-carboxyethyl) phosphine (Aldrich, Milwaukee, WI). Potassium ferri- and ferrocyanide, MB (chloride salt), 6-mercaptohexanol (MCH), 11mercaptoundecanoic acid, 16-mercaptohexadecanoic acid, mercaptohexadecane, and triethylene glycol mono-11-mercaptoundecyl ether (HS(CH2)11(OCH2)3OH, TEG) were all purchased from Aldrich and used without further purification. Instrumentation. Electrochemical and SECM experiments were carried out using a CHI-900 workstation (CH Instruments, Austin, TX) and an XYZ stage equipped with Burleigh piezo inchworms (Exfo Life Sciences, Ontario, Canada) to control the tip position. Polycrystalline, 2-mm-diameter gold disk working electrodes (BAS, West Lafayette, IN) were used in preliminary studies, and a 6 mm polycrystalline gold disk embedded in a flat PEEK block (BAS) served as the substrate for DNA microarray fabrication. We found that thin gold films evaporated onto glass slides according to our published procedure can also be used to produce quality DNA microarrays.29 SECM tips were made in-house by sealing a 25- or 10-µm-diameter platinum wire (Alpha Aesar, Ward Hill, MA) in soft capillary glass, as described elsewhere.16 A commercial Ag/ AgCl reference electrode (BAS) and a platinum coil auxiliary electrode were used. DNA microarrays were produced using a simple, inexpensive spotting device (SpotBot, ArrayIt, Sunnyvale, CA) employing a 100-µm-diameter pin (SMP3, ArrayIt) with a loading volume of 250 nL, which is sufficient to deposit an array of 200 spots. Typical arrays consisted of 5 spots × 5 spots separated by 100 µm. Approach curves were measured at a typical approach rate of 10 µm s-1, and a tip scan rate of 25 µm s-1 was employed for SECM imaging. Preparation of DNA-Modified Electrodes. Gold electrodes were prepared by first polishing sequentially with 1, 0.3, and 0.05 µm alumina slurries, followed by sonication in ethanol. Immediately prior to DNA immobilization, electrodes were electrochemically cleaned in 1 M H2SO4 by repetitively cycling between 0.4 and 1.4 V versus Ag/AgCl. Exposure of the cleaned surface to the air for periods of time greater than an hour prior to immobilization of DNA resulted in poor monolayer formation, presumably because of surfaceadsorbed atmospheric contaminants. For the purposes of measuring approach curves, DNA monolayers were immobilized onto gold disk electrodes by covering them with 100 µM thiolated ds-DNA in pH 7.2 PBS for approximately 12 h. DNA duplexes were formed by annealing the thiolated ss-DNA molecules with their complementary or mismatched target DNA for 2 min at 90 °C. To ensure the formation of densely packed films, excess MgCl2 (0.2 M) was dissolved in the immobilization solution.30 After incubation, surfaces were rinsed with PBS and submerged for 30 min in an aqueous solution of 100 µM MCH to remove any nonspecifically adsorbed DNA. For imaging experiments, DNA arrays were spotted onto the
(27) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Bioconjugate Chem. 1997, 8, 31-37. (28) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096-1100.
(29) Yao, X.; Li, X.; Toledo, F.; Zurita-Lopez, C.; Gutova, M.; Momand, J.; Zhou, F. Anal. Biochem. 2006, 354, 220-228. (30) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, M. J. J. Am. Chem. Soc. 2003, 125, 5219-5226.
a For clarity, the DNA molecules and the SECM tip are not drawn to scale.
SEM Imaging of DNA Microarrays
Figure 1. SECM approach curves obtained at a ds-DNA-modified gold substrate in pH 7.2 PBS containing 2 mM K4Fe(CN)6. The SECM tip was a 25-µm-diameter Pt disk held at a potential of 0.5 V. (a) Unbiased substrate in the absence of MB (-), substrate biased at -0.4 V in the absence of MB (---), and substrate biased at -0.4 V in the presence of 2 µM MB (‚‚‚). (b) Effect of substrate biasing potential in the presence of 2 µM MB (Es ) -0.1, -0.2, -0.3, and -0.4 V). Circles show theoretical curves for finite electron-transfer kinetics. iT and d are normalized with respect to the bulk solution limiting current, iT,∞, and the tip radius, a, respectively. freshly cleaned polycrystalline gold disk and isolated in a humidified environment for 1.5 h. Blocking of the unmodified gold surface was achieved by covering the array with a PBS solution containing 1 mM TEG for 1 h (vide infra). The integrity of the blocking film was verified by cyclic voltammetry in a 2 mM solution of ferricyanide.
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is unable to penetrate the negatively charged DNA monolayer such that diffusion to the SECM tip is blocked upon approach.21,22 Biasing the substrate at -0.4 V versus Ag/AgCl had little effect on the approach curve, suggesting the formation of a stable, well-packed monolayer. This can also be established by recording the cyclic voltammetry of the DNA-modified electrode in ferricyanide directly; negligible faradaic currents in the range 0.5 to -0.5 V (not shown) indicate a densely blocked gold surface. In the presence of 2 µM MB, with the DNA-modified electrode unbiased, insulating behavior was again observed (data not shown for clarity). This is in agreement with work by Horrocks and co-workers, who studied DNA monolayers immobilized on silicon substrates by SECM and observed little change in the approach curve response upon binding of MB.31 However, when the surface was biased at -0.4 V versus Ag/AgCl, a potential negative enough to reduce the intercalated MB,27 the approach curve recorded was significantly altered (dotted curve in Figure 1a). The tip current initially increased upon approach, but at very short tipsurface distances, the current rapidly dropped below the bulk limiting current (iT,∞). The effect of substrate biasing potential on the approach curves was therefore systematically investigated in 2 mM ferrocyanide and 2 µM MB (curves in Figure 1b). At a substrate potential of -0.1 V versus Ag/AgCl, negative feedback response was observed, but biasing the substrate at increasingly negative potentials results in gradually increased tip currents. The observed combination of positive and negative feedback is typical of finite heterogeneous ET kinetics, wherein charge transfer becomes limited at short tip-substrate distances.20,32,33 The following electrode reactions account for the positive feedback. (Note here that at pH 7.2 MB is cationic, but the positive charge has been omitted from the text and Scheme 1 for simplicity.)
SECM tip (0.5 V): Fe(CN)64- - e- f Fe(CN)63DNA surface (-0.4 V): MB+ + 2e- + H+ f LB overall: 2Fe(CN)63- + LB f 2Fe(CN)64- + MB+ + H+
Results and Discussion Optimization of SECM Experimental Conditions and Measurement of ET Rate Constants through DNA. The viability of employing SECM to detect surface-bound DNA using the ferrocyanide/methylene blue scheme was first tested by measuring approach curves at polycrystalline gold surfaces modified with monolayers of ds-DNA. A thiolated 15mer DNA sequence was hybridized to its complementary strand (Table 1) and immobilized onto a gold substrate to form a densely packed film. Although less common than the in-situ probe-target capture method,8 the prehybridization approach has the advantage of high hybridization efficiency, shortening the overall electrode preparation time and ensuring optimal performance. The dsDNA substrate was immersed face up in a 2 mM solution of potassium ferrocyanide in pH 7.2 PBS, and a 25-µm-diameter Pt tip electrode was positioned close to the surface. The tip was held at a potential of 0.5 V versus AgCl, which is sufficient to bring about the diffusion-controlled oxidation of ferrocyanide to ferricyanide, and the limiting current, iT, was measured as a function of distance, d, from the surface in the absence and presence of MB (Figure 1a). In the absence of MB, with the DNA-modified substrate at open circuit, the approach curve exhibits negative feedback, as evidenced by the drop in normalized current as the tip reaches the surface. Such behavior is characteristic of an insulating surface, verifying that ferrocyanide
Because the magnitude of the SECM tip current is controlled by MB-mediated ET through the DNA duplexes, theoretical approach curves were generated using well-established analytical equations for finite heterogeneous kinetics.16,17 A standard heterogeneous rate constant of 1.0 × 10-5 cm s-1 was calculated using a known procedure.20,32,33 We note the similarity in shape of the above approach curves to those reported by Bard and co-workers for DNA-modified electrodes in the presence of metal ions (metalated DNA).20 Interestingly, the apparent MB-mediated rate constant estimated above is faster than those determined by Bard for immobilized native and metalated ds-DNA (5.2 × 10-7 and 3.9 × 10-6 cm s-1, respectively),20 highlighting the efficiency of MB at facilitating the ET process and suggesting that a less disrupted π-π stack (with respect to the metalated DNA system) may conduct current more effectively.3,28 Some studies have indicated that the enhanced ET observed through metalated DNA may result from charge compensation of the DNA polyionic phosphate backbone, allowing the penetration of negatively charged redox markers, such as ferri/ferrocyanide, into DNA (31) Lie, L. H.; Mirkin, M. V.; Hakkarainen, S.; Houlton, A.; Horrocks, B. R. J. Electroanal. Chem. 2007, 603, 67-80. (32) Liu, B.; Bard, A. J.; Mirkin, M. V.; Creager, S. E. J. Am. Chem. Soc. 2004, 126, 1485-1492. (33) Mirkin, M. V.; Arca, M.; Bard, A. J. J. Phys. Chem. 1993, 97, 1079010795.
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monolayers.20,34,35 Although such electrostatic effects cannot be excluded for the cationic MB, any contribution to the positive feedback observed is unlikely to be significant for a number of reasons. Earlier work has shown that, on average, approximately one MB molecule intercalates per 15 base pair duplexes, and thus the resulting charge neutralization is expected to be negligible.27 Furthermore, the ET rate constant reported by Bard and co-workers was measured using Zn2+ as the incorporated metal ion. Given that the electrostatic influence for such a dication is expected to be greater than for the monocationic MB, one would predict a slower ET rate constant for the latter. Because this is not the case, we conclude that these ionic effects are not dominant here. Finally, the notable difference between the metalated-DNA and MB-intercalated DNA is that in the latter case the intercalating species is electroactive and at substrate potentials more negative than -0.2 V versus Ag/AgCl the positively charged MB is reduced to neutral LB, for which the electrostatic argument does not apply. Although the negative feedback behavior observed at short tip-substrate distances is expected for finite heterogeneous ET kinetics,32 it is possible that an additional factor may operate. At short distances, the direct oxidation of LB to MB by the SECM tip could deplete the local LB concentration, preventing ferrocyanide from being regenerated in the solution. However, given that the high ionic strength of the solution facilitates the approach of ferricyanide to the DNA film, the LB conversion back to MB is rapid, and because MB is a strong DNA intercalator,36 we believe that the substrate-generated LB does not diffuse far (on the order of micrometers under the current experimental conditions) away from the DNA film. This contention is supported by (1) the excellent agreement between experimental and theoretical approach curves in Figure 1b and (2) the fact that attempts to use the SECM substrate generation/tip collection mode37 to image MB-intercalated DNA-covered electrodes (i.e., using the SECM tip to collect the substrate-generated LB) did not lead to appreciable tip currents. Because SECM imaging involves progressively scanning the tip, it was necessary to determine the time dependence of the feedback response. The tip was positioned 25 µm from a DNAmodified electrode in a solution of 2 mM ferrocyanide and 2 µM MB, and the tip current was monitored as a function of time as the substrate potential was stepped from open circuit to -0.4 V versus Ag/AgCl. The response (not shown) indicated that the positive feedback current is initially quite high but falls to a constant value after a few minutes to reach a steady state (constant amount of LB in the DNA film). Taking this observation into account, the substrate electrode was preconditioned at -0.4 V versus Ag/AgCl for a few minutes prior to SECM imaging. Potentials more negative than this were avoided because the reductive desorption of thiols could take place.38 Blocking Exposed Gold Surfaces. For DNA microarray imaging, it was necessary to cover any area uncovered by DNA with a blocking monolayer to prevent the direct reduction of tip-generated ferricyanide. Mercaptohexanol is commonly used as a backfilling agent but is of little use in this case because we found that MCH monolayers, being only six methylene units long, did not sufficiently impede the ET to ferricyanide across the monolayer. Other blocking thiols were considered, including (34) Maeda, M.; Nakano, K.; Uchida, K.; Takagi, M. Chem. Lett. 1994, 23, 1805-1809. (35) Park, N.; Hahn, J. H. Anal. Chem. 2004, 76, 900-906. (36) Bhagat, V.; Nersissian, M.; Wang, W.; Hill, M. G. Langmuir 2003, 19, 9255-9259. (37) Zhou, F.; Unwin, P. R.; Bard, A. J. J. Phys. Chem. 1992, 96, 4917-4924. (38) Zhong, C.-J.; Porter, M. D. J. Am. Chem. Soc. 1994, 116, 11616-11617.
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Figure 2. Cyclic voltammetry at gold electrodes modified with SAMs of mercaptohexadecanoic acid (-) and TEG (‚‚‚) in a pH 7.2 PBS solution containing 2 mM K3Fe(CN)6 and 2 µM MB. A voltammogram recorded at a naked gold electrode in 2 mM K3Fe(CN)6 is shown for comparison (---).
longer alkanethiols such as mercaptoundecanoic acid, mercaptohexadecanoic acid, and mercaptohexadecane. They were tested by preparing SAMs on gold electrodes and carrying out cyclic voltammetry in ferricyanide solutions, with and without MB. Although the mercaptohexadecanoic acid- and mercaptohexadecane-covered electrodes demonstrated good blocking behavior in the presence of ferricyanide alone, substantial faradaic currents were observed in the presence of 2 mM ferricyanide and 2 µM MB (Figure 2, solid line). This suggests that MB is incorporated by the alkanethiol monolayers and mediates ET to ferricyanide. We found that triethylene glycol mono-11-mercaptoundecyl ether (TEG) is particularly effective in passivating the gold surface without incorporating MB molecules (Figure 2, dotted line). This is understandable because the TEG units create a hydrophilic environment39 that disfavors the penetration of MB (which is relatively hydrophobic). In addition, approach curves measured at TEG-modified electrodes demonstrated pure negative feedback, suggesting that the TEG SAM is compact and well-ordered. As will be seen below, the well-contrasted SECM images of DNA suggest that the relatively long TEG chains do not impede the efficient exchange of electrons between ferricyanide ions in solution and MB molecules in immobilized DNA. SECM Imaging. In the work of Barton, Hill, and co-workers, the DNA-mediated reduction of MB was used as an indicator of base pair mismatches in immobilized prehybridized ds-DNA.28 As mentioned, SECM imaging should afford the opportunity of discriminating between multiple immobilized ss-DNA and/or ds-DNA spots in a rapid manner. To test this and the detection of mismatched base pairs, gold electrodes were modified with arrays of 100 µm DNA spots of differing sequences. Five different columns of spots were immobilized, which comprised ss-DNA, ds-DNA in which the thiolated strand was hybridized to its complement, and ds-DNA containing one, two, and three base pair mismatches (sequences in Table 1). An alternative method of assessing ET through the various sequences would be to measure approach curves at individual electrodes modified with different DNA monolayers, but because the behavior of such electrodes is highly dependent on the initial condition of the gold surface, it is more advantageous to investigate the different behaviors on a single electrode using the microarray platform. We initially imaged DNA microarrays in which the surrounding exposed gold was covered with MCH instead of the TEG SAM. Because the positive feedback observed at DNA-modified (39) Canaria, C. A.; So, J.; Maloney, J. R.; Yu, C. J.; Smith, J. O.; Roukes, M. L.; Fraser, S. E.; Lansford, R. Lab Chip 2006, 6, 289-295.
SEM Imaging of DNA Microarrays
Figure 3. SECM image of a DNA microarray immobilized on a gold disk substrate (unbiased) in a pH 7.2 PBS solution containing 2 mM K4Fe(CN)6 (no MB). A cross-sectional contour is shown below. Both rows of spots correspond to (a) ss-DNA, (b) ds-DNA (complementary), (c) ds-DNA (one base mismatch), (d) ds-DNA (two base mismatches), and (e) ds-DNA (three base mismatches). The DNA spots were surrounded by MCH.
Figure 4. SECM image of a DNA microarray immobilized on a gold disk substrate biased at -0.4 V in pH 7.2 PBS solution containing 2 mM K4Fe(CN)6 and 2 µM MB. Both rows of spots correspond to (a) ss-DNA, (b) ds-DNA (complementary), (c) ds-DNA (one base mismatch), (d) ds-DNA (two base mismatches), and (e) ds-DNA (three base mismatches). The spots are surrounded by MCH.
electrodes was expected to be a function of the surface coverage, the uniformity of the DNA arrays was first ascertained using the repelling mode of SECM as described by Schuhmann and coworkers.21,22 The array was immersed in a 2 mM solution of ferrocyanide in pH 7.2 PBS, a 10-µm-diameter Pt tip was positioned 10 µm from the surface, and an image was recorded by scanning the tip across the unbiased substrate. The image, depicted in Figure 3, exhibits regions of high anodic current resulting from the positive feedback due to facile ET across the MCH film. Notice that these regions surround spots of low current, which are a consequence of negative feedback over the DNA. The negative feedback results from the electrostatic repulsion of ferrocyanide from the negatively charged DNA phosphate backbone and gives an indication of surface DNA coverage. It is clear from Figure 4 that each of the five columns of spots has similar surface densities of DNA. This finding is supported by a separate analysis using surface plasmon resonance imaging. SECM imaging was carried out using Scheme 1, with the substrate biased at a potential of -0.4 V versus Ag/AgCl, in the presence of 2 µM MB. A typical image can be seen in Figure
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Figure 5. SECM image of a DNA microarray immobilized on a gold disk substrate biased at -0.4 V in pH 7.2 PBS solution containing 2 mM K4Fe(CN)6 and 2 µM MB. Both rows of spots correspond to (a) ss-DNA, (b) ds-DNA (complementary), (c) ds-DNA (one base mismatch), (d) ds-DNA (two base mismatches), and (e) ds-DNA (three base mismatches). The exposed gold surface was blocked with TEG.
4. As above, lower currents were observed over DNA spots than over MCH-covered regions, but the current measured over the five columns of DNA now differs appreciably as a consequence of the positive feedback current. (It is likely that repulsion effects are still in operation but are swamped by the MB catalytic cycling.) The current is lowest over the ss-DNA spots (Figure 4a), which is conceivable because MB should not intercalate specifically into ss-DNA. Higher currents are observed for fully complementary ds-DNA and for the spots comprising ds-DNA with a single mismatch and double mismatches (columns b-d), but lower currents are measured at the ds-DNA containing three mismatches (Figure 4e). Such diminished currents at DNAcovered electrodes containing mismatches were observed previously and were attributed to the disruption of the π stacking of base pairs, resulting in slower ET.3,28 In this case, the slower ET kinetics are manifested as smaller positive feedback currents. Next, arrays were tested using the TEG as a blocking agent. The same procedure as above was employed, and the resulting image is depicted in Figure 5, which now exhibits negative feedback in the regions surrounding the DNA spots. As in Figure 4, there is a clear discrimination between ss-DNA and complementary ds-DNA and some sensitivity to spots containing three mismatched base pairs. The response visibly demonstrates that TEG serves as a good blocking agent for the exposed gold surrounding the DNA spots and provides a homogeneous background, relative to which the positive feedback contributions from DNA may be easily determined. Such a feature may be a requirement of an array-based sensor, particularly when high signal-to-noise ratio measurements are necessary. Control experiments carried out under similar conditions, only in the absence of MB, revealed that feedback currents measured over the DNA spots were typically 5% less than at the TEG-covered regions as a consequence of the repulsive influence (Figure 3). From this, we estimate that the percentage current enhancement due to the intercalation of MB into ds-DNA is close to 10%. On comparison of Figure 5 with Figure 4, we note that the current differentiation observed between DNA spots is smaller in the presence of surrounding TEG than that in the presence of surrounding MCH. A plausible explanation for this difference is that MB in solution is scavenged over the area covered by the MCH molecules, whose shorter chains facilitate electron tunneling between the gold film and solution species. As a result of the
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Conclusions
Figure 6. SECM image of a DNA microarray immobilized on a gold disk substrate biased at -0.4 V in pH 7.2 PBS solution containing 2 mM K4Fe(CN)6 and 2 µM MB. The concentrations of ds-DNA solution used for each row of spots were (a) 100, (b) 50, (c) 20, (d) 10, and (e) 5 µM. The exposed gold surface was backfilled with TEG.
local depletion of MB, the contrast between the DNA spots and the surrounding areas is greater. Finally, the detection level of the methodology shown in Scheme 1 was investigated. Figure 6 shows an array in which the concentration of DNA used to modify the surface was varied in the range of 5 to 100 µM. Clearly, the 100 µM spots (column a) give the highest positive feedback with diminishing intensity visible over the 50 and 20 µM spots (b and c) as a result of decreasing DNA coverage. The 10 and 5 µM spots (d and e) provide contrast that is less obvious than that between 20 and 100 µM, although their presence is discernible from the current profile. On the basis of a typical DNA spotting solution volume of 0.7 nL/spot, the 20 µM sample concentration corresponds to 14 fmols of DNA. This is a level that compares well with other electrochemically based microarray detection techniques,23,24 suggesting that the current method can detect DNA samples at trace levels. Although the detection level is less than that of some fluorescence-based microarray detection strategies,13 the simplicity of both array fabrication and DNA labeling, together with the relatively low cost of the SECM instrument and the avoidance of photobleaching of fluorescent labels, makes the present methodology an attractive alternative for DNA hybridization assays and gene sequencing.
In this work, we have demonstrated that, via mediated reduction of methylene blue intercalated in ds-DNA, heterogeneous ET kinetics across ds-DNA may be investigated, and DNA microarray imaging can be accomplished. The positive feedback response observed is a clear indication that, in the presence of this noncovalently bound redox-active label, DNA monolayers behave as conducting surfaces with finite electrode kinetics. Moreover, by scanning a microelectrode tip across modified surfaces, microarrays of DNA spots immobilized onto a single electrode may be imaged, obviating the need for individually addressable electrodes and extensive labeling of ds-DNA molecules. The method allows discrimination between single- and doublestranded DNA, marking its potential use as a hybridization sensor for disease diagnoses, and shows significant promise in the sensing of multiple mismatches in duplexed DNA on a single metal electrode. The method does not require a large amount of DNA solution for array fabrication and imaging. The detection level, although not as low as for some reported electrochemical assays, is viable from a practical point of view, particularly considering the merits of multisequence analysis without the need for labeling. The extension to real sample analysis can thus be envisaged, especially with the aid of polymerase chain reaction amplification for cases when DNA concentrations are present at ultratrace levels. Although the sequential reading from spot to spot is slower than fluorescence or SPR microarray reading using a CCD camera, the method is simple and cost-effective. With high-throughput capability being a major prerequisite for modern screening methods, the advantages of the methodology developed, as compared to more conventional electrochemical approaches, are clearly evident. Acknowledgment. We greatly appreciate the partial support of this work by the RIMI Program at California State University, Los Angeles (P20-MD001824-01), an NIH-SCORE subproject (GM 08101), and a Dreyfus TeacherScholar Award (TH-01-25). We also thank Professor Michael Mirkin at Queens College, City University of New York, for his help with the approach curve-fitting procedure and Dr. Yong-Jun Li at California State Universitys Los Angeles for carrying out surface plasmon resonance imaging. LA703922V
