Electrochemical Detection of Single-Nucleotide Mismatches Using an

Anal. Chem. 2006, 78, 6096-6101

Electrochemical Detection of Single-Nucleotide Mismatches Using an Electrode Microarray Xiaohong Li,†,‡ Jeremy S. Lee,*,† and Heinz-Bernhard Kraatz*,‡

Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5, and Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada S7N 5C9

Gold electrode arrays with electrode diameters of 10 µm were used for the detection of eight single-nucleotide mismatches in unlabeled and prehybridized DNA by electrochemical impedance spectroscopy (EIS). Because of the differences in the electrical properties of films of duplex DNA (normal duplex DNA in B-form) in the presence and absence of Zn2+ at pH g 8.6, Randles equivalent circuits were employed to evaluate the EIS results. The difference in the charge-transfer resistance (∆RCT) between B-DNA (absence of Zn2+ at pH g 8.6) and M-DNA (presence of Zn2+ at pH g 8.6) allows unequivocal detection of all eight single-nucleotide mismatches within a 20-mer DNA sequence. After dehybridization/rehybridization with target DNA, ∆RCT allows the discrimination of single-nucleotide mismatches with concentrations of the target strand as low as 10 fM. Although the presence of protein impurities (bovine serum albumin, 10 µg/mL) interferes with the detection of the target strand (1 pM detection limit), the presence of nontarget DNA (calf thymus DNA, 10-8 M) does not interfere, and the detection limit for recognition of the target strand remains at 10 fM. Human genome research has revealed that gene mutations or single-nucleotide polymorphisms often indicate genetic predisposition toward diseases. A simple, fast, and high-throughput analysis method is urgently required for early diagnosis. Most of the current methods are based on spectroscopic techniques in which changes in fluorescence are observed due to hybridization of a target strand to the complementary capture strand.1-4 Electrochemical detection methods usually rely on the presence of a redox reporter, and in many cases, the label is covalently linked to the DNA.5-13 In addition, the MutS family of proteins †

Department of Biochemistry. Department of Chemistry. (1) Skogerboe, K. J. Anal. Chem. 1995, 67, 449R-454R. (2) Southern, E. M. Trends Genet. 1996, 12, 110-115. (3) Eng, C.; Vijg, J. Nat. Biotechnol. 1997, 15, 422-426. (4) Okamoto, A.; Kanatani, K.; Saito, O. J. Am. Chem. Soc. 2004, 126, 4820. (5) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 11921199. (6) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253. (7) Throp, H. H. Top. Curr. Chem. 2004, 237, 159-181. (8) 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. ‡

6096 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

have been employed to detect mismatches optically14-16 or electrochemically.17-19 In many cases, the polymerase chain reaction has been used to amplify the target in order to boost the sensitivity of the assay.20,21 In recent years, DNA biochips have emerged as a platform for the detection of DNA mutations. Their inherent miniaturization and compatibility with advanced semiconductor technologies promise to provide a simple, accurate, and inexpensive method for nucleic acid assays.22 Some of the key advantages of a chipbased detection system include reduced sample and reagent consumption, increased sensitivity, parallel analysis and high throughput, and portability. Most commonly, the format of such DNA chips makes use of immobilized DNA capture strands on the chip that are hybridized to target strands that carry a fluorescent label.23 The incorporation of nonplanar capture systems and three-dimensional elements into the sensors was reported to improve the sensitivity and selectivity;24-27 however, the hybridization efficiency remains a problem in these systems. A MutS-based (9) Kelley, S. O.; Boon, E. M.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Nucleic Acids Res. 1999, 27, 4830-4837. (10) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-774. (11) Liu, G. D.; Lee, T. M. H.; Wang, J. J. Am. Chem. Soc. 2005, 127, 38-39. (12) Millan, K. M.; Saravallo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 294348. (13) Di Guisto, D. A.; Walssoff, W. A.; Giesebrecht, S.; Gooding, J. J.; King, G. C. J. Am. Chem. Soc. 2004, 126, 4120. (14) Wagner, R.; Debbie, P.; Radman, M. Nucleic Acids Res. 1995, 11, 39443948. (15) Gotoh, M.; Hasebe, M.; Ohira, T.; Hasegawa, Y.; Shinohara, Y.; Sota, H.; Nakao, J.; Tosu, M. Genet. Anal.: Biomol. Eng. 1997, 14, 47-50. (16) Geschwind, D. H.; Rhee, R.; Nelson, S. F. Genet. Anal. 1996, 13, 105-111. (17) 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. (18) Li, C. Z.; Long, Y. T.; Lee, J. S.; Kraatz, H. B. Chem. Commun. 2004, 574575; (19) Palecˇek, E.; Masarˇ´ık, M.; Kizek, R.; Kuhlmeier, D.; Hassmann, J.; Schu ¨ lein, J. Anal. Chem. 2004, 76, 5930-5936. (20) Reed, R.; Holmes, D.; Weyers, J.; Jones, A. Practical Skills in Biomolecular Sciences; Addison-Wesley Longman Ltd.: Edinburgh Gate, Harlow, England, 1998. (21) Walker, M, J.; Rapley, R. Molecular Biology and Biotechnology; The Royal Society of Chemistry: Thomas Graham House, Cambridge, UK, 2000. (22) Campa`s, M.; Katakis, I. Trends Anal. Chem. 2004, 23, 49-62. (23) Jain, K. K. Science 2001, 294, 621-623. (24) Afanassiev, V.; Hanemann, V.; Wolfi, S. Nucleic Acids Res. 2000, 28, e66. (25) Proundnikov, D.; Timofeev, E.; Mirzabekov, A. Anal. Biochem. 1998, 259, 34-41. (26) Ferguson, J. A.; Steemers, F. J.; Walt, D. R. Anal. Chem. 2000, 72, 56185624. (27) Ali, M. F.; Kirby, R.; Goodey, A. P.; Rodriguez, M. D.; Ellington, A. D.; Neikirk, D. P.; McDevitt, J. T. Anal.Chem. 2003, 75, 4732-4739. 10.1021/ac060533b CCC: $33.50

© 2006 American Chemical Society Published on Web 07/28/2006

Table 1. Matched and Mismatched Duplex DNA Used for Preparation of Films on the Microelectrodes duplex DNA 1+2 matched

1 + 3 1 + 4 1 + 5 1 + 6 1 + 7 1 + 8 1 + 9 1 + 10 C-A

C-T

G-A

G-T

G-G

C-C

A-A

T-T

protein chip for detection of DNA mutations represents an interesting alternative. Behrensdorf et al. reported the use of fluorescence-labeled MutS on chips binding to mismatched DNA.28 Bi et al. reported surface-bound MutS proteins immobilized on the chip, which bind to fluorescence-tagged mismatch DNA.29 Unfortunately, MutS does not recognize and bind to all possible base-mismatch combinations, which is one limitation of this approach. Less attention has been paid to the development of mismatch detection systems that make use of electrochemistry-based chips. Detection of single-nucleotide mismatches using an electrochemical approach offers the benefits of an ultrasensitive detection method without any prior labeling and signal-amplification procedures. We recently reported the design of a microelectrode array and its use for the detection of a single-nucleotide mismatch in unlabeled and prehybridized DNA by electrochemical impedance spectroscopy (EIS).11 This technique relies on the difference in the charge-transfer resistance (∆RCT) between a film of B-DNA and M-DNA that is formed in the presence of Zn2+ at pH g 8.6. It was found that mismatched B-DNA has a decreased impedance as compared to a matched sequence, whereas for M-DNA, the matched sequence has the lower impedance.30,31 This approach allowed the unequivocal detection of a single-nucleotide mismatch within a synthetic 20-mer DNA. In this article, we report the application of this technology for the detection of all eight single-nucleotide mismatches located in the center of a DNA duplex. Under dehybridization-rehybridization conditions, we achieved a detection limit of 10 fM. This further confirms that a lower detection limit can be achieved by decreasing the electrode area.31 To test the robustness of this approach and its susceptibility to contaminants, we also carried out titration experiments in the presence of calf thymus DNA and bovine serum albumin (BSA) and show good behavior of the system to contamination. Although calf thymus DNA does not influence the results (10 fM in the presence of 10-8 M calf thymus DNA), BSA lowers the detection limit to 1 pM (10 µg/mL BSA). Our results show the utility of this approach and represent an important step forward toward the development of an electrochemical tool for detection of DNA mismatches. EXPERIMENTAL SECTION Materials. Ten DNA sequences were synthesized by standard solid-phase techniques using a fully automated DNA synthesizer at the Plant Biotechnology Institute (PBI-NRC, Saskatoon). (28) Behrensdorf, H. A.; Pignot, M.; Windhab, N.; Kappel A. Nucleic Acids Res. 2002, 30, e64. (29) Bi, L.-J.; Zhou, Y. F.; Zhang, J.-Y.; Zhang, Z.-P.; Xie, B.; Zhang, C.-G. Anal. Chem. 2003, 75, 4113-4119. (30) Li, X. H.; Zhou, Y. L.; Sutherland, T. C.; Baker, B.; Lee, J. S.; Kraatz, H.-B. Anal Chem. 2005, 77, 5766-5769. (31) Long, Y.-T.; Li, C.-Z.; Sutherland, T. C.; Kraatz, H.-B.; Lee, J. S. Anal.Chem. 2004, 76, 4059-4065.

1: 5′-HO-(CH2)6-SS-(CH2)6-GTC-ACG-ATG-GCC-CAG-TAGTT-3′ 2: 5′-AAC-TAC-TGG-GCC-ATC-GTG-AC-3′ 3: 5′-AAC-TAC-TGG-ACC-ATC-GTG-AC-3′ 4: 5′-AAC-TAC-TGG-TCC-ATC-GTG-AC-3′ 5: 5′-AAC-TAC-TGG-GAC-ATC-GTG-AC-3′ 6: 5′-AAC-TAC-TGG-GTC-ATC-GTG-AC-3′ 7: 5′-AAC-TAC-TGG-GGC-ATC-GTG-AC-3′ 8: 5′-AAC-TAC-TGG-CCC-ATC-GTG-AC-3′ 9: 5′-AAC-TAC-TGG-GCC-AAC-GTG-AC-3′ 10: 5′-AAC-TAC-TGG-GCC-TTC-GTG-AC-3′ The oligonucleotides were purified by two-step reversed-phase HPLC and then characterized by MALDI-TOF MS as reported before.31 One complementary strand and eight kinds of singlenucleotide mismatched strands (mismatch in italics) are listed above. The resulting duplexes are shown in Table 1. NaClO4, K3[Fe(CN)6], K4[Fe(CN)6], bovine serum albumin (BSA), calf thymus DNA, and NaOH were purchased from Aldrich and used without further purification. Zn(ClO4)2 and TRIS (Tris(hydroxymethyl)-aminomethane)) were purchased from Fluka Co. Deionized water (18.2 MΩ‚cm resistivity) from a Millipore Milli-Q system was used throughout this work. Monolayer Preparation. Films of a 20-base-pair doublestranded DNA (ds-DNA) were formed by incubating the fresh microelectrode chips for 5 days with ds-DNA hybridized from equimolar amounts of strands 1 + 2 or 1 + (3 - 10) (0.1 mM ds-DNA in 50 mM Tris-ClO4 buffer at pH 8.7). Only the 1 + 2 ds-DNA is fully matched, whereas other combinations contain a single-nucleotide mismatch in the center of the duplex (see Table 1 for details). Dehybridization and regeneration of the singlestranded DNA (ss-DNA) on the microelectrode was achieved by denaturing the ds-DNA by soaking in a 10 mM NaOH bath at 40 °C for 20 min followed by rinsing with 20 mM Tris-ClO4 buffer (pH 8.7) at room temperature. Rehybridization was performed by incubating the ss-DNA film with 10 µL of target strand solution (50 mM Tris-ClO4 buffer at pH 8.7) for ∼20 h. These measurements were repeated in the presence of 10 µg/mL BSA and 10-8 M calf thymus DNA, respectively. The concentration of the target strand was varied from 10-4 to 10-16 M. B-DNA was converted to M-DNA by the addition of 0.4 mM solution of Zn(ClO4)2‚6H2O in 20 mM Tris-ClO4 buffer (pH g 8.6), followed by incubation of the chip for 2 h. Electrochemical Measurements. A conventional threeelectrode system was used as before.30 All measurements were carried out at room temperature (22 °C) in an enclosed and grounded Faraday cage. A 20-µL portion of redox solution (4 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1) in 50 mM Tris-ClO4 buffer, pH 8.7) was added onto the chip to cover all eight microelectrodes. To minimize solvent evaporation, the Faraday cage was covered in plastic sheets, which maintained the humidity at >96%. The reference electrode was a Ag/AgCl electrode, connected to the [Fe(CN)6]3-/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 microelectrode array using a micropositioning device (World Precision Instrument, model M3310R). EIS was measured using an EG&G 1025 frequency response analyzer interfaced to an EG&G 283 potentiostat/galvanostat. The ac voltage amplitude was 10 mV, and the Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

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Figure 1. Representative Nyquist plots (-Zim vs Zre) for films of 20-mer matched B-DNA 1 + 2 (b), G-G-mismatched B-DNA 1 + 7 (9), G-G-mismatched M-DNA 1 + 7 (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; Cmono, capacitance of monolayer; RCT, charge-transfer resistance; Rx and CPE, defects in the monolayer.

voltage frequencies ranged from 100 kHz to 0.1 Hz. The applied potential was 250 mV vs Ag/AgCl (formal potential of the redox probe [Fe(CN)6]3-/4- in the buffer solution). Measurements were conducted on all eight microelectrodes. Importantly, all measurements were repeated for 10 separate electrodes to get statistically meaningful results. RESULTS AND DISCUSSION Prehybridized ds-DNA solutions were prepared involving strand 1 containing a 5′-disulfide group. The combination of strands 1 and 2 results in the formation of matched ds-DNA, whereas the combination of strand 1 with strands 3-10 gives ds-DNA containing a single-nucleotide mismatch in the middle of the duplex, as shown in Table 1. The ds-DNA solutions were employed to prepare DNA films on the microelectrode arrays by self-assembly of 20-mer duplexes as described in the Experimental Section. Detection of Single-Nucleotide Mismatches. The selfassembled DNA films on the microelectrode arrays were evaluated using EIS. Representative impedance spectra in the form of Nyquist plots for ds-DNA in the absence and presence of Zn2+ for the fully matched ds-DNA 1 + 2 and for a ds-DNA containing a G-G mismatch in the center of the duplex (1 + 7) are shown in Figure 1. The measured data were analyzed with the help of a modified Randles equivalent circuit, as shown in the inset, that allowed us to interpret the impedance data in terms of electronic circuit components.32 The fits to the measured data are shown as a solid line. The fitted results for matched and all mismatched DNA films are listed in Table 2. To provide a rational explanation for the electrochemical process, the electronic elements in the equivalent circuit are described in detail below. The solution resistance, Rs, is the resistance between the reference electrode and the DNA-modified (32) Long, Y.-T.; Li, C.-Z.; Kraatz, H.-B.; Lee, J. S. Biophys. J. 2003, 84, 32183225.

6098 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

gold microelectrode. For every measurement, the distance between the two electrodes was kept approximately constant, and measurements were carried out under identical conditions of electrolyte concentration (50 mM Tris-ClO4) and at room temperature to minimize variations in Rs, which ranged from 0.001 to 0.003 Ω‚cm2. As expected, the values of Rs for ds-DNA in the presence and absence of Zn2+ are similar. Cmonolayer accounts for the capacitance of the DNA film on gold microelectrodes. There are two observations from the evaluation of Cmonolayer: (a) For all films, Cmonolayer is smaller for B-DNA as compared to M-DNA; and (b) Cmonolayer is larger for films containing a single-nucleotide mismatch as compared to a matched film. In the former case, it can be rationalized by an enhanced electron transfer in films of M-DNA as compared to B-DNA.33 There is also evidence from measurements in the solid state that the conductive properties of DNA are affected by the presence of Zn2+.34 Thus, it could be rationalized that the dielectric constant of the M-DNA film is slightly higher, as compared to films of B-DNA, which leads to the increased values in Cmonolayer. For the latter case, Cmonolayer is larger for films containing a singlenucleotide mismatch as compared to a matched film. The film thickness for films containing a single-nucleotide mismatch is decreased as compared to that of matched B-DNA films due to the kinking or other structural changes in the helix caused by the mismatch in the center of the duplexes.31 This contributes to the increased Cmonolayer in the B-DNA forms for mismatched films. For films of 1 + 4 having a C-T mismatch and of 1 + 6 having a G-T mismatch, these changes are very small. The combination of Rx and the constant phase element (CPE) accounts for possible pinholes in the film structure. CPE acts as a nonlinear capacitor accounting for inhomogeneity of the film and the electrode surface.35 The values for CPE range from 1.8 to 6.6 mF‚cm-2, with the exponential modifier n ) 0.8, and the value for Rx for all films is 0.1 Ω‚cm2. Thus, from comparison of RCT for the bare electrode (0.7 Ω‚cm2) with Rx, we can see that the resistance of the defect sites is small. Although a number of pinholes may be present in the film, potentially large pinholes, they do not interfere with the detection of mismatches. From comparison of the RCT for the assembled electrode with the Rx, we can deduce that the films formed on the microelectrode arrays are more homogeneous as compared to conventional BAS gold electrodes (diameter ) 2 mm).31 Diffusion of the redox probe from the solution to the DNA film is not important in this system, as is apparent from the absence of any Warburg impedance, as shown in Figure 1. The charge-transfer resistance, RCT, is the key feature that allows us to discriminate matched from mismatched DNA. RCT is the result of resistance to charge transfer from the [Fe(CN)6]3-/4redox probe to the electrode surface through the DNA film.31,33 There are two major observations related to RCT. For a given film, RCT is lower for M-DNA (in the presence of Zn2+) as compared to B-DNA in the absence of Zn2+. As discussed previously, the results can be rationalized by improved electron-transfer kinetics for the (33) Liu, B.; Bard, A. J.; Li, C.-Z.; Kraatz, H.-B. J. Phys. Chem. B. 2005, 109, 5193-5198. (34) Rakitin, A.; Aich, P.; Papadopoulos, C.; Kobzar, Y.; Vedeneev, A. S.; Lee, J. S.; Xu, J. M. Phys. Rev. Lett. 2001, 86, 3670. (35) Dijksma, M.; Boukamp, B. A.; Kamp, B.; van Bennekom, W. P. Langmuir 2002, 18, 3105-3112.

Table 2. Equivalent Circuit Element Values for Matched and Eight Mismatched DNA Filmsa circuit elements Rs (Ω‚cm2)

Cmono (µF‚cm-2)

RCT (Ω‚cm2)

Rx (Ω‚cm2)

CPE (mF‚cm-2)

N

∆RCT (Ω‚cm2)

match

B-DNA M-DNA

0.002 0.002

99.4 (10.1) 110.6 (14.6)

75.4 (5.7) 11.1 (1.3)

0.1 (0.01) 0.1 (0.02)

2.5 (0.2) 3.0 (0.3)

0.8 (0.03) 0.8 (0.02)

64.3 (5.3)

C-A

B-DNA M-DNA

0.002 0.002

105.2 (4.6) 107.0 (11.5)

19.6 (2.0) 12.9 (1.1)

0.1 (0.02) 0.1 (0.03)

6.4 (0.7) 6.6 (0.6)

0.8 (0.05) 0.8 (0.04)

6.7 (1.4)

C-T

B-DNA M-DNA

0.002 0.002

94.5 (4.3) 104.5 (3.8)

36.1 (3.9) 22.8 (2.3)

0.1 (0.02) 0.1 (0.04)

2.7 (0.3) 2.9 (0.3)

0.8 (0.02) 0.8 (0.03)

13.3 (3.1)

G-A

B-DNA M-DNA

0.003 0.002

108.8 (11.5) 121.1 (14.3)

39.1 (3.4) 24.3 (2.3)

0.1 (0.03) 0.1 (0.03)

3.8 (0.3) 4.6 (0.4)

0.8 (0.05) 0.8 (0.06)

14.8(2.6)

G-T

B-DNA M-DNA

0.002 0.002

92.1 (9.7) 133.4 (14.9)

49.1 (4.8) 12.2 (1.3)

0.1 (0.03) 0.1 (0.04)

1.9 (0.4) 2.2 (0.7)

0.8 (0.04) 0.8 (0.1)

36.9 (4.2)

G-G

B-DNA M-DNA

0.002 0.002

107.3 (14.2) 112.8 (22.6)

57.9 (5.5) 30.1 (1.0)

0.1 (0.03) 0.1 (0.04)

1.8 (0.3) 2.0 (0.2)

0.8 (0.05) 0.8 (0.04)

27.8 (5.4)

C-C

B-DNA M-DNA

0.002 0.001

100.6 (11.6) 182.9 (17.3)

25.1 (1.5) 13.2 (1.2)

0.1 (0.02) 0.1 (0.03)

2.7 (0.2) 2.9 (0.4)

0.8 (0.02) 0.8 (0.01)

11.9 (1.4)

A-A

B-DNA M-DNA

0.003 0.002

104.4 (3.4) 113.7 (12.1)

27.1 (1.8) 19.6 (2.2)

0.1 (0.02) 0.1 (0.02)

1.8 (0.3) 1.8 (0.4)

0.8 (0.01) 0.8 (0.02)

7.5 (2.1)

T-T

B-DNA M-DNA

0.002 0.001

114.0 (12.8) 271.2 (18.2)

38.1 (1.3) 16.5 (0.9)

0.1 (0.01) 0.1 (0.02)

2.4 (0.2) 5.4 (0.9)

0.8 (0.02) 0.8 (0.02)

21.6 (1.1)

a

The values in parentheses represent the standard deviations from at least 10 electrode measurements.

M-DNA film or perhaps by enhanced penetration of the redox probe into the film in the presence of Zn2+.31,33 The second observation is that in the presence of a single-nucleotide mismatch, RCT for a B-DNA film is significantly reduced. Since a mismatch will introduce significant disorder into the film, the redox probe may penetrate the film to a larger extent, giving rise to a lower RCT. As shown in Figure 1, the Nyquist plot clearly showed that 1 + 2 B-DNA has a higher RCT, as compared to 1 + 7 G-G mismatched B-DNA. Upon conversion of B-DNA to M-DNA, 1 + 2 M-DNA has a lower RCT as compared to 1 + 7 G-G mismatched M-DNA. The higher RCT for a mismatched film of M-DNA can be rationalized by a contribution from the base pair π-stack in the DNA helix, which is capable of mediating charge transport.36,37 This will be affected by metal association with the bases themselves. In addition, the thickness of the duplex on the electrode can be influenced by the addition of Zn2+, which will influence the rates of electron transfer.32-33,38-40 As far as mismatch detection is concerned, ∆RCT, the RCT difference between B-DNA and M-DNA for a given film, is an important parameter that allows us to distinguish a fully complementary duplex from one containing a single-nucleotide mismatch. For a film of the matched pair 1 + 2, the value RCT for B-DNA is 75.4 (5.7) Ω‚cm2, whereas in the presence of Zn2+, the resistive term drops to 11.1 (1.3) Ω‚cm2. This gives a ∆RCT of 64.3 (5.3) Ω‚cm2. For the film 1 + 7 which contains a single G-G mismatch, the RCT for B-DNA is 57.9 (5.5) Ω‚cm2 and drops to 30.1 (1.0) Ω‚cm2 for M-DNA, with a ∆RCT of 27.8 (5.4) Ω‚cm2. It is this (36) Liu, Tao.; Barton, J. K. J. Am. Chem. Soc. 2005, 127, 10160-10161. (37) Kelley, S. O.; Jackson, N. M.; Hill, M. G.; Barton, J. K. Angew. Chem., Int. Ed. 1999, 38, 941-945. (38) Li, C.-Z.; Long, Y.-T.; Kraatz, H.-B.; Lee, J. S. J. Phys. Chem. B 2003, 107, 2291-2296. (39) Lee, J. S.; Latimer, L. J. P.; Reid, R. S. Biochem. Cell Biol. 1993, 71, 162168. (40) Aich, P.; Labiuk, S. L.; Tari, L. W.; Delbaere, L. J. T.; Roesler, W. J.; Falk, K. J.; Steer, R. P.; Lee, J. S. J. Mol. Biol. 1999, 294, 477-485.

difference in ∆RCT that allows the discrimination of G-Gmismatched from matched DNA. The values for ∆RCT for other mismatched DNA duplexes are listed in Table 2 and range from 36.9 (4.2) (Ω‚cm2) for the G-T mismatch, 21.6 (1.1) (Ω‚cm2) for the T-T mismatch, 14.8 (2.6) (Ω‚cm2) for the G-A mismatch, 13.3 (3.1) for the C-T mismatch, 11.9 (1.4) (Ω‚cm2) for the C-C mismatch, and 7.5 (2.1) (Ω‚cm2) for the A-A mismatch to 6.7 (1.4) (Ω‚cm2) for the C-A mismatch. Using this approach, we were able to detect all eight mismatches using our microelectrode array without the need for prior labeling of the DNA. Detection of a Single-Nucleotide Mismatch after in Situ Hybridization. A logical next step in the development of the microelectrodes array was to test its performance using an in situ hybridization assay to determine the detection limit. For this purpose, we deposited a film of ds-DNA onto the gold pads of the electrode arrays. The ds-DNA is first denatured by exposing the film to a solution of 10 mM NaOH at 40 °C for 20 min, followed by thorough rinsing with buffer solution (20 mM Tris-ClO4). The result is a loosely packed film of single-stranded DNA of 1. It was shown before that the direct addition of ss-DNA results in densely packed films, which interferes with subsequent binding to the complementary strand.41 After drying, the dehybridized ss-DNA film was rehybridized with the target strands 2-10 in 50 mM Tris-ClO4 buffer. The dehybridization and rehybridization procedures are shown in Scheme 1. We evaluated this process by EIS. A typical series of Nyquist plots for 1 + 2 are shown in Figure 2. All impedance spectra were analyzed using the equivalent circuit described above. The pinholes in the monolayers are not affected by this procedure because Rx is still in the range from 0.001 to 0.003 Ω‚ cm2. However, after dehybridization, RCT for the ss-DNA of 1 is decreased as compared to that of ds-DNA. After rehybridization (41) Leviky, R.; Herne, T. M.; Tarlov, M. J.; Satija S. K. J. Am. Chem. Soc. 1998, 120, 9787.

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Scheme 1. Dehybridization/Rehybridization

(1) The ds-DNA film is soaked in 10 mM NaOH at 40 °C for 20 min and then rinsed with 20 mM Tris-ClO4 at room temperature, giving a film of ss-DNA. (2) Target DNA was added in 50 mM Tris-ClO4 and to allow duplex formation at room temperature for 20 h.

Figure 3. Nyquist plots (-Zim vs Zre) of the rehybridized 20-base pair matched B-DNA 1 + 2 (b), C-A-mismatched 1 + 3 (9), C-Amismatched M-DNA 1 + 3 (0), and matched M-DNA 1 + 2 (O). Measured data are shown as symbols with the calculated fit to the equivalent circuit as solid lines. Table 3. Equivalent Circuit Element Values for Matched and Single C-A-Mismatched DNA Monolayer after the De- and Rehybridization Procedurea matched circuit element

Figure 2. Nyquist plot (-Zim vs Zre) of fully hybridized “ideal” monolayer 1 + 2 (b); ss-DNA monolayer of 1 after dehybridization (O), and rehybridized ds-DNA film of 1 + 2 (0). The impedance of the rehybridized film is lower, as compared to that of the “ideal” 1 + 2 films, indicating the heterogeneity of the monolayer as a result of incomplete hybridization.

with a complementary strand 2, RCT is increased to a level that we estimate represents ∼70-80% rehybridization efficiency. Next, we evaluated if mismatches can be detected in films obtained by the dehybridization and rehybridization procedures shown in Scheme 1. The C-A mismatch was used as a representative example. After rehybridization of the ss-DNA film with the complementary strand 2 and with target strand 3, a matched and C-A-mismatched film were formed, respectively. Representative Nyquist plots for these two films are shown in Figure 3. These are fitted to the same equivalent circuit shown in Figure 1. The analyzed results are summarized in Table 3. For the film rehybridized with complementary strand 2 giving a film of matched DNA 1 + 2, a value of 49.1 (5.3) Ω‚cm2 is obtained for B-DNA. In the presence of Zn2+ to form M-DNA, the RCT drops to 12.2 (1.4) Ω‚cm2. The ∆RCT between B-DNA and M-DNA is 36.9 (4.3) Ω‚cm2. For the rehybridized film 1 + 3 having a C-A mismatch, a value of 18.9 (2.8) Ω‚cm2 is obtained for the B-DNA. In the presence of Zn2+, the RCT drops to 14.1(1.7) Ω‚cm2. The ∆RCT between B-DNA and M-DNA is 4.8 (2.1) Ω‚cm2; thus, the difference in the ∆RCT between C-A mismatched and matched DNA still allows the discrimination of mismatched from matched DNA. Sensitivity and Specificity. To explore the detection limit of this assay, we gradually decreased the concentrations of the target strands under the dehybridization and rehybridization conditions. Taking C-A mismatch 1 + 3 as an example, the impedance 6100 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

B-DNA

M-DNA

mismatched B-DNA

M-DNA

Rs, Ω‚cm2 0.002 0.002 0.003 0.002 Cmonolayer, 114.6 (13.4) 152.8 (14.7) 115.9 (9.8) 127.4 (10.3) µF‚cm-2 RCT, Ω‚cm2 49.1 (5.3) 12.2 (1.4) 18.9 (2.8) 14.1 (1.7) RX, Ω‚cm2 0.1 (0.01) 0.1 (0.01) 0.1 (0.02) 0.1 (0.02) CPE, mF‚cm-2 3.2 (0.2) 3.9 (0.3) 2.5 (0.3) 4.5 (0.5) n 0.9 (0.02) 0.9 (0.02) 0.9 (0.02) 0.9 (0.03) ∆ RCT, Ω‚cm2 36.9 (4.3) 4.8 (2.1) a The values in parentheses represent the standard deviations from at least 10 electrode measurements.

spectra were recorded for matched and C-A mismatched DNA in both B- and M-DNA forms and fit to the equivalent circuit shown in Figure 1. Overall, the ∆RCT between a fully matched DNA film (1 + 2) and a mismatched film (1 + 3) decreases with decreasing concentration of the target strand. The relationship between ∆RCT and the concentration of the target strands is shown in Figure 4. Our results clearly show that our approach enables us to detect the C-A mismatch with the concentration of target strand as low as 10 fM, which translates to a detection limit of ∼1000 target DNA molecules per electrode in the array. This raises the question if the assay is sensitive to impurities, such as random DNA or protein contaminants. To test the robustness of the system, we chose to test the performance in the presence of calf thymus DNA (10-8 M) and bovine serum albumin (10 µg/mL). The results of these experiments are shown in Figures 5 and 6, respectively. Calf thymus DNA does not interfere with the detection of DNA; neither does it interfere with our ability to detect a C-A mismatch. In contrast, the presence of BSA results in a reduction of the detection limit to 1 pM of target DNA. Figures 4-6 essentially show a two-state change for mismatch recognition. A possible explanation of the observed changes is related to the differences in the makeup of the film. One may speculate that at high concentrations of the target strand DNA

Figure 4. Relationship between ∆RCT and the concentration of target strand DNA. Complimentary strands (b) and C-A-mismatched strands (O). Error bars are derived from a minimum of 10 electrodes.

Figure 6. Relationship between ∆RCT and the concentration of target strand DNA in the presence of 10 µg/mL BSA. Complimentary strands (b) and C-A-mismatched strands (O). Error bars are derived from a minimum of 10 electrodes.

mismatched system because of the lack of “information” in the film as the result of insufficient rehybridization at ultralow target strand concentrations.

Figure 5. Relationship between ∆RCT and the concentration of target strand DNA in the presence of 10-8 M calf thymus DNA. Complimentary strands (b) and C-A-mismatched strands (O). Error bars are derived from a minimum of 10 electrodes.

(from 10-4 to 10-8 M), there is sufficient target DNA present in solution to rehybridize a sufficiently large number of capture strands on the microelectrode surface, resulting in a relatively well-ordered film. In the absence of a sufficient amount of target DNA in solution, a significantly lower number of capture strands will be hybridized, and the film resembles more closely a ss-DNA film. Such a film will have significantly different properties from a more structured film. On the basis of recent work,13 we can assume that these differences are caused by changes in the ability of the redox probe and other ions in solution to diffuse in and out of the film. Thus, at low concentrations of target strand DNA, it is no longer possible to distinguish between a matched and

CONCLUSION In this paper, the application of a microelectrode array for the detection of single-nucleotide mismatches by EIS is presented. All eight different mismatches are detected by exploiting the difference in charge-transfer resistance ∆RCT of B-DNA and M-DNA and does not rely on prior labeling of the DNA. Importantly, under dehybridization/rehybridization conditions, we explored the sensitivity and selectivity of the technique in the absence and presence of BSA or calf thymus DNA. ∆RCT is sufficiently sensitive to detect a single-nucleotide mismatch down to a concentration of 10 fM of target DNA (∼103 molecules per electrode) in the presence of 10-8 M calf thymus DNA. In the presence of 10 µg/mL BSA, ∆RCT still is a reliable approach that allows us to discriminate between a matched and mismatched DNA film down to a concentration of 1 pM of target DNA. We are presently working on increasing the sensitivity by decreasing the size of the individual electrodes in the microelectrode array. ACKNOWLEDGMENT This work was supported by funding through the NSERC Strategic program. 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. Received for review March 23, 2006. Accepted June 26, 2006. AC060533B

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