Chip-Based Microelectrodes for Detection of Single-Nucleotide

Anal. Chem. 2005, 77, 5766-5769

Chip-Based Microelectrodes for Detection of Single-Nucleotide Mismatch Xiaohong Li,†,‡ Yinglin Zhou,†,‡ Todd C. Sutherland,†,‡ Brian Baker,§ Jeremy S. Lee,*,† and Heinz-Bernhard Kraatz*,‡

Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5, Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9, and Microfabrication Laboratory, Department of Engineering, University of Utah, 495 East 100 Street, Salt Lake City, Utah 84112

Microelectrode arrays having eight 10-µm-diameter gold microelectrodes arranged on a gold-covered Si chip were designed and characterized. The chips prove useful for the detection of single-nucleotide mismatches in unlabeled and prehybridized DNA by electrochemical impedance spectroscopy. A large number of human genetic diseases are caused by gene mutation. Single-nucleotide polymorphisms are point mutations, which constitute the most common genetic mutation. The detection of these point mutations is useful for the early identification of defective genes and may ultimately lead to “personalized medicine”. To realize this goal, techniques that are able to detect point mutations in a reliable, fast, and cost-effective fashion are required. Present optical methods rely on the hybridization of a labeled DNA target strand to a complementary capture strand.1 Hybridization for mismatched strands is less effective allowing the detection of matched DNA sequences. It has to be stressed that the hybridization conditions strongly affect these results. At the present time, DNA targets require amplification using the polymerase chain reaction (PCR).2 This contributes to the cost and can introduce additional sources of error. Electrochemical detection has been the intense focus of various groups, aiming at increasing the sensitivity of the measurement.3-6 DNA mismatch detection usually relies on the presence of redox reporters, and in many cases, the label is covalently linked to DNA. * To whom correspondence should be addressed. E-mail: kraatz@ skyway.usask.ca; [email protected]. † Department of Biochemistry, University of Saskatchewa. ‡ Department of Chemistry, University of Saskatchewan. § University of Utah. (1) (a) Skogerboe, K. J. Anal. Chem. 1995, 67, 449R-454R. (b) Southern, E. M. Trends Genet. 1996, 12, 110-115. (c) Fodor, S. P. A. Science 1997, 277, 393. (d) Eng, C.; Vijg, J. Nat. Biotechnol. 1997, 15, 422-426. (2) (a) Reed, R.; Holmes, D.; Weyers, J.; Jones, A. Practical Skills in Biomolecular Sciences; Addison-Wesley Longman Ltd.: Edinburgh Gate, Harlow, England, 1998. (b) Walker, M, J.; Rapley, R. Molecular Biology and Biotechnology; The Royal Society of Chemistry: Thomas Graham House, Cambridge, U.K., 2000. (3) Yu, C. J.; Wan, Y. J.; Yowanto, H.; Li, J.; Tao, C. L.; James, M. D.; Tan, C. L.; Blackburn, G. F.; Meade, T. J. J. Am. Chem. Soc. 2001, 123, 1115511161 (4) Kelley, S. O.; Boon, E. M.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Nucleic Acids Res. 1999, 27, 4830-4837. (5) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-774. (6) (a) Liu, G. D.; Lee, T. M. H.; Wang, J. J. Am. Chem. Soc. 2005, 127, 3839. (b) Millan, K. M.; Saravallo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 2943-2948.

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These labels range from polymers and fluorescent dyes to semiconductor nanoparticles. However, there are reports of hybridization detection using solution probes only.6b In addition, some groups have made use of the Mut family of proteins to detect mismatches electrochemically.7 In recent years, research on DNA biochips has advanced rapidly.8 Most current technology still relies on chemically labeled DNA,9-11 in which DNA capture probe is supported on the chip and then hybridized under stringent conditions with the target strand. Either the target or the capture strands can be chemically labeled. The single-nucleotide mismatch is then detected by a change in the optical or electrochemical signal. But hybridization efficiency is an unfavorable factor for these detective applications. Previously, we reported on a label-free method for mismatch detection using electrochemical impedance spectroscopy (EIS).12 Briefly, the unlabeled probe DNA was attached to a gold working electrode through a thiol linkage and the target, also unlabeled, was hybridized to it. We focused on the differences in the impedance between double-stranded DNA (B-DNA) and M-DNA, which is formed after incubation with Zn2+ at pH g8.6. The difference in the charge-transfer resistance (∆Rct) before and after formation of M-DNA allows the unequivocal detection of a singlenucleotide mismatch in various positions within a synthetic DNA 20-mer. In this technical note, we now report on the design and use of an eight-electrode microarray that has 10-µm-diameter exposed gold disks as working electrodes. The chip is simple to manufacture yet gives reliable electrochemical measurements even for such small electrodes. (7) (a) Boon, E. M.; Livingston, A. L.; Chmiel, N. H.; David, S. S.; Barton, J. K. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12543-12547. (b) Li, C. Z.; Long, Y. T.; Lee, J. S.; Kraatz, H. B. Chem. Commun. 2004, 574-575. (c) Palee`ek, E.; Masaøı´k, M.; Kizek, R.; Kuhlmeier, Dirk.; Hassmann, J.; Schu ¨ lein, J. Anal. Chem. 2004, 76, 5930-5936. (8) Campa`s, M.; Katakis, I. Trends Anal. Chem. 2004, 23, 49-62. (9) (a). Umek, R. M.; Lin, S. W.; Vielmetter, J.; Terbrueggen, R. H.; Irvine, B.; Yu, C. J.; Kayyem, J. F.; Yowanto, H.; Blackburn, G. F.; Farkas, D. H.; Chen, Y. P. J. Mol. Diagn. 2001, 3, 74-84. (b) CMS-DNA chips are manufactured by Motorola Clinical Micro Sensors (Pasadena, CA) and sold under the eSensor trade mark. (10) Ali, M. F.; Kirby, R.; Goodey, A. P. Rodriguez, M. D.; Ellington, A. D.; Neikirk, Dean P.; McDevitt, John T. Anal. Chem. 2003, 75, 4732-4739. (11) Zhang, Y. C.; Kim, H. H.; Heller, A. Anal. Chem. 2003, 75, 3267-3269. (12) Long, Y.T.; Li, C. Z.; Sutherland, T. C.; Kraatz, H. B.; Lee, J. S. Anal. Chem. 2004, 76, 4059-4065. 10.1021/ac050741o CCC: $30.25

© 2005 American Chemical Society Published on Web 07/28/2005

persistent over the potential range from -0.2 to 1.5 V versus Ag/ AgCl. An atomic force microscopy (AFM) image of one of the electrode wells is shown in the Supporting Information. AFM was measured using a Quesant Universal SPM in the “intermittent mode”. Materials. Three DNA sequences were synthesized by standard solid-phase techniques using a fully automated DNA synthesizer at the Plant Biotechnology Institute (PBI-NRC, Saskatoon, SK, Canada).

5′-HO-(CH2)6-SS-(CH2)6-GTC-ACG-ATG-GCCCAG-TAG-TT-3′ (1) 5′-AAC-TAC-TGG-GCC-ATC-GTG-AC-3′ (2) 5′-AAC-TAC-TGG-ACC-ATC-GTG-AC-3′ (3)

Figure 1. Schematic view of the design of the microelectrode array showing the eight microelectrodes in the center of the chip. Each electrode has a diameter of 10 µm and is separated from the other electrodes by a distance of 50 µm. Each microelectrode is connected by an individually addressable wire to a connector pad (1 mm2) at the edge of the chip.

EXPERIMENTAL SECTION Chip-Based Microelectrodes. A schematic view of the electrode array is shown in Figure 1. An 800-nm silicon dioxide insulating layer was thermally grown on a p-type silicon wafer. An electrode layer consisting of a 20-nm titanium adhesion layer and a 200-nm gold conductive layer was sputter deposited on the silicon dioxide. The metal layers were photolithographically patterned using Shipley 1813 photoresist as a mask layer. The gold layer is etched in an iodine solution (I2/KI/H2O 1 g/4 g/40 mL), and the titanium layer was etched in dilute hydrofluoric acid (10:1 H2O/HF). As a result, eight gold wires (50-µm width) remained. One end of this gold wire was centered on the chip; the other end was connected with one of eight gold pads (1 mm2). The microwires are each separated by 50 µm. Finally, an insulating layer of Shipley 1813 photoresist was spin-deposited over the entire wafer to a thickness of 2.2 µm. The 10-µm-diameter electrodes are exposed and developed through the photoresist layer down to the gold conductive electrode. Similarly, the gold pads (1 mm2) were exposed as connections for electrochemical measurements. The photoresist was hard baked on a hot plate for 30 min at 120 °C. To prevent contamination of the electrodes, a protective photoresist layer was spin-deposited over the entire wafer and soft baked at 95 °C for 2 min. The wafer, containing 20-30 individual arrays, was diced into chips, and the top photoresist layer was removed, leaving the insulating photoresist layer and electrode layers intact. The photoresist was not sensitive to the reagents for the electrochemical measurements. In addition, the photoresist is also

Strand 3 contains a mismatched base in position 11 beginning from the 3′-end (A instead of G). The oligonucleotides were purified by two-step reversed-phase HPLC and then characterized by MALDI-TOF MS as reported before.13 NaClO4, K3[Fe(CN)6], and K4[Fe(CN)6] were purchased from Aldrich and used without further purification. Zn(ClO4)2 and Tris-ClO4 were purchased from Fluka Co. Deionized water (18.1 MΩ‚cm resistivity) from a Millipore Milli-Q system was used throughout this work. Monolayer Preparation. Films of a 20-base-pair doublestranded (ds) DNA on gold were formed by incubating the microelectrode chip for 5 days in ds-DNA solutions hybridized from equimolar amounts of strands 1 + 2 or 1 + 3 (0.1 mM ds-DNA in 50 mM Tris-ClO4 buffer, pH 8.7). The 1 + 2 ds-DNA is fully matched, whereas the 1 + 3 ds-DNA contains a single C-A mismatch. B-DNA was converted to M-DNA on the chip by the addition of a 0.4 mM solution of Zn(ClO4)2‚6H2O in 20 mM Tris-ClO4 buffer (pH 8.6), followed by incubation of the chip for 2 h. Electrochemical Measurements. A conventional threeelectrode system was used as shown in Figure S5 (Supporting Information). All the measurements were carried out at room temperature (22 °C) in an enclosed and grounded Faraday cage. A 20-µL aliquot of redox solution (4 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1) in 50 mM Tris-ClO4 buffer pH 8.6) was placed on the chip, which covers all eight electrodes. To avoid evaporation of the solvent, the Faraday cage was maintained humid (humidity >96%). The reference electrode was an Ag/AgCl electrode, connecting the Fe(CN)63-/4- solution through a miniature salt bridge (agar plus KNO3). The counter electrode was a platinum wire. The counter and reference electrodes were positioned over the electrode array using a micropositioning device (World Precision Instruments model M3301R). Cyclic voltammetry was measured using a CHI 440 instrument, while EIS was measured using an EG&G 1025 frequency response analyzer interfaced to an EG&G 283 potentiostat/galvanostat. The ac voltage amplitude is 10 mV, and the voltage frequencies range from 100 kHz to 0.1 Hz; The applied potential was 250 mV versus Ag/AgCl (formal potential of the redox probe [Fe(CN)6]3-/4- in the buffer solution). (13) (a) Li, C. Z.; Long, Y. T.; Kraatz, H. B.; Lee, J. S. J. Phys. Chem. B 2003, 107, 2291-2296. (b) Long, Y. T.; Li, C. Z.; Kraatz, H. B.; Lee, J. S.. Biophys. J. 2003, 84, 3218-3225.

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Chart 1. Schematic Representations of the Two Films Prepared on Gold Microelectrode Arrays: (a) Film of the Complementary 20-Mer ds-DNA 1 + 2; (b) Film of the Mismatched 20-Mer ds-DNA Containing a C-A Mismatch 1 + 3a

a

The mismatch is in the center of the double helix.

All the measurements were conducted on all eight microelectrodes. Results were repeated for 10 separate electrodes to get statistically meaningful results.

Figure 2. Nyquist plots (-Zim vs Zre) of the 20-base pair matched B-DNA 1 + 2 (b), C-A mismatched B-DNA 1 + 3 (9), C-A mismatch M-DNA 1 + 3 (0), and matched M-DNA 1 + 2 (O). Measured data are shown as symbols with calculated fit to the equivalent circuit as solid lines. Inset: the measured data are fit to the equivalent circuit: RS, solution resistance; Cmonolayer, capacitance of monolayer; RCT, charge-transfer resistance; Rx and CPE, defects in the monolayer. The data are summarized in Table 1. Table 1. Equivalent Circuit Element Values for Matched and Single C-A Mismatched DNA Monolayera matched

RESULTS AND DISCUSSION The design objectives of the chip had to meet several criteria. First, the process had to be simple to reduce costs. In practice, this requirement can be met by using as few layers as possible. Second, the surface of the gold must be as flat as possible to allow the formation of a uniform self-assembled monolayer This was achieved by sputter depositing gold. Third, the topology of the gold electrode must be a uniform circle and the area must be reproducible to within (10%. Thus, for example, designs in which a gold lead was attached to an isolated disk of gold were precluded. Similarly, designs in which the working electrode is attached through a connection underneath the chip were eliminated because of manufacturing problems. Finally, the photoresist insulating layer must be stable for storage (at least six months) and not be degraded by aqueous solvents. The final design (Figure 1) meets these criteria. Although, only eight electrodes are present on this test chip, the identical manufacturing steps could be used for larger arrays. Characterization of the microelectrodes is available in the Supporting Information. Solutions of prehybridized ds-DNA were prepared from strands 1 and 2, giving the matched combination 1 + 2, and from strands 1 and 3, giving the mismatched combination 1 + 3. These solutions were used to prepare DNA films on the microelectrode arrays by self-assembly of a 20-mer duplex with a thiol-terminated aliphatic linker at the 5′-end. The properties of the films of 1 + 2 and 1 + 3 compare well with those previously reported as shown in Chart 1.12,13 After film assembly, the DNA films on the electrode microarrays were evaluated by EIS. Representative impedance spectra for ds-DNA in the presence and absence of Zn2+ for 1 + 2 and 1 + 3 are shown in Figure 2. The impedance spectra for all systems are evaluated with the help of a modified Randles equivalent circuit, as shown in the inset. The analyzed results are listed in Table 1. The equivalent circuits compares well to what was reported before for 1.6-mm gold electrodes.12 The only exception is the replacement of the Warburg impedance by a constant-phase 5768 Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

circuit element Rs/Ω‚cm2

Cmonolayer/µF‚cm-2 RCT/Ω‚cm2 RX/Ω‚cm2 CPE/mF‚cm-2 n ∆RCT/Ω‚cm2

mismatched

B-DNA

M-DNA

B-DNA

M-DNA

0.003 25.9 (2.5) 74.2 (5.3) 0.1 (0.01) 2.7(0.2) 0.8(0.04) 63.5 (5.5)

0.003 26.5 (2.3) 10.7 (1.4) 0.1 (0.02) 3.0 (0.3) 0.8 (0.02)

0.006 25.5(2.4) 23.9(2.8) 0.1(0.02) 4.6 (0.5) 0.8 (0.05) 8.2(1.3)

0.002 28.4(3.0) 15.7(1.7) 0.1 (0.03) 3.9 (0.4) 0.8(0.04)

a The values in parentheses represent the standard deviations from several electrode measurements (n g 10).

element (CPE). CPE acts as a nonlinear capacitor accounting for inhomogeneity on the electrode surface.14 Diffusive contributions appear negligible for the 10-µm electrodes. The distance between the Pt counter electrode and the gold microelectrode is kept approximately constant in order to minimize variations in the solution resistance Rs, which is a measure of the resistance between the gold microelectrode and the Pt counter electrode (range from 0.002 to 0.006 Ω‚cm2). Cmonolayer accounts for the DNA film capacitance. The combination of Rx and the CPE is used to account for defects in the DNA film. During the self-assembling process on the electrode, it is unavoidable that there are defects in the monolayer structure. However, for DNAmodified electrodes, Rx is small compared to RCT. Thus, we can conclude that there are fewer defects in the monolayer compared to the earlier work on larger gold electrodes. As expected, electron-transfer resistance for charge transfer through the DNA film, Rct, is lower for M-DNA than for B-DNA. The results are consistent with those from the larger electrode described previously12 and have been rationalized by improved electron-transfer kinetics for the M-DNA film or perhaps with enhanced penetration of the redox probe into the film in the presence of Zn2+.15 For a film of the matched pair 1 + 2, a value (14) Dijksma, M.; Boukamp, B. A.; Kamp, B.; van Bennekom, W. P. Langmuir 2002, 18, 3105-3112.

of 74.2 (5.3) Ω‚cm2 (standard deviation) is observed for RCT for the B-DNA film, whereas in the presence of Zn2+, this resistive term drops to 10.7(1.4) Ω‚cm2. This gives a difference term, ∆RCT, between B-DNA and M-DNA of 63.5(5.5) Ω‚cm2. For the mismatched film 1 + 3, containing a single C-A mismatch, RCT for B-DNA is 23.9(2.8) Ω‚cm2 whereas it drops to 15.7(1.7) Ω‚cm2 for M-DNA. Thus, ∆RCT for the mismatched film 1 + 3 is only 8.2 (1.3) Ω‚cm2. This effect is also illustrated in Figure 2. The Nyquist plot clearly shows that the spectrum of 1 + 2 B-DNA has higher RCT compared to the related mismatched 1 + 3 B-DNA. In contrast, 1 + 2 M-DNA exhibits a lower RCT compared with the mismatched 1 + 3 M-DNA. It is this difference in ∆RCT that allows the discrimination of matched from mismatched DNA. In this technical note, we have described the design and the use of a microelectrode array for the reliable detection of a singlenucleotide mismatch. At present, we have demonstrated the principle for a simple C-A mismatch. We are now investigating the remaining mismatches and the influence of mismatch position. (15) Liu, B.; Bard, A. J.; Li, C.-Z.; Kraatz, H.-B. J. Phys. Chem. B 2005, 109, 5193-5198.

The long-term goal is to achieve PCR less detection of DNA hybridization and of DNA mismatches. ACKNOWLEDGMENT The authors thank NSERC and UMDI for financial support. H.-B.K. is the Canadian Research Chair in Biomaterials and J.S.L. is supported by a Senior Investigators Award from the Regional Partnership Program of CIHR. The authors also thank Don Schwab, the Plant Biotechnology Institute, Canada, for the preparation of DNA samples. SUPPORTING INFORMATION AVAILABLE Representative CV, EIS, and AFM image of one of the eight microelectrodes are shown. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review April 29, 2005. Accepted June 21, 2005. AC050741O

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