Unlabeled Hairpin-DNA Probe for the Detection of Single-Nucleotide

Department of Chemistry, Beijing Normal University, Beijing, China, 100875, Department of Chemistry, University of Western. Ontario ... Science, Insti...
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Anal. Chem. 2008, 80, 2255-2260

Unlabeled Hairpin-DNA Probe for the Detection of Single-Nucleotide Mismatches by Electrochemical Impedance Spectroscopy Ying Wang,† Congjuan Li,† Xiaohong Li,*,† Yongfang Li,‡ and Heinz-Bernhard Kraatz*,§

Department of Chemistry, Beijing Normal University, Beijing, China, 100875, Department of Chemistry, University of Western Ontario, 1151 Richmond Street, London, N6A 5B7 Canada, and Key Laboratory of Organic Solids, Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China

An unlabeled hairpin-DNA probe was used for the detection of eight single-nucleotide mismatches by electrochemical impedance spectroscopy (EIS). Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened and forms a duplex DNA. Accordingly, the film thickness is increased, which causes differences in the electrical properties of the film before and after hybridization. Randles equivalent circuits were employed to evaluate the EIS result. The differences in the charge-transfer resistance ∆RCT between hairpin DNA (before hybridization) and duplex DNA (after hybridization) shows the consequence of a large structural rearrangement from hairpin to duplex. If a single-nucleotide mismatch is present in the center of the duplex, the difference in charge-transfer resistance ∆RCT between B-DNA in the absence and presence of Zn2+ allows the unequivocal detection of all eight single-nucleotide mismatches. The detection limit was measured, and ∆RCT allows the discrimination of a single-nucleotide mismatch with the concentration of the target strand as low as 10 pM. Human genome research has uncovered a large number of genetic diseases that are caused by gene mutations. The largest degree of sequence variation in human DNA is attributed to singlenucleotide polymorphisms (SNPs) induced by environmental factors1,2, occurring as often as every few hundred to few thousand base pairs in genomic DNA. SNPs are associated with many diseases and individual variations in response to therapeutics3-6, * To whom correspondence should be addressed. E-mail: Xiaohong Li, [email protected], and Heinz-Bernhard Kraatz, [email protected]. ‡ Institute of Chemistry, Chinese Academy of Sciences. † Beijing Normal University. § University of Western Ontario. (1) Kong, A.; Gudbjartsson, D. F.; Sainz, J.; Jonsdottir, G. M.; Gudjonsson, S. A.; Richardsson, B.; Sigurdardottir, S.; Barnard, J.; Hallbeck, B.; Masson, G.; Shlien, A.; Palsson, S. T.; Frigge, M. L.; Thorgeirsson, T. E.; Gulcher, J. R.; Stefansson, K. Nat. Genet. 2002, 31, 241-247. (2) Tomita-Mitchell, A.; Muniappan, B. P.; Herrero-Jimenez, P.; Zarbl. H.; Thilly, W. G. Gene 1998, 223, 381-391. (3) Wang, D. G.; Fan, J. B.; Siao, C. J.; Berno, A.; Young, P.; Sapolsky, R.; Ghandour, G.; Perkins, N.; Winchester, E.; Spencer, J. Science 1998, 280, 1077-1082. (4) Brookes, A. J. Gene 1999, 234, 177-186. (5) Schafer, A. J.; Hawkins, J. R. Nat. Biotechnol. 1998, 16, 33-39. 10.1021/ac7024688 CCC: $40.75 Published on Web 02/22/2008

© 2008 American Chemical Society

and the identification of these polymorphisms provides an opportunity in both the diagnosis and treatment of diseases. Therefore, SNP detection and genotyping assays are of fundamental importance in the early identification of numerous genetic and hereditary diseases even if a few genetic alterations are not necessarily harmful. Current optical assays are based on the hybridization of a labeled DNA target strand to a complementary or mutated capture strand.7-10 In order to address sensitivity issues and boost the optical output, the polymerase chain reaction has been integrated into optical sensors to amplify the target DNA.11,12 Alternatively, structured DNA probes such as hairpin DNA have been used to recognize specific sequences.13-18 Among these assays, hairpin DNA was functionalized at one terminus with a fluorophore and at the other with a quenching agent. Subsequently, in order to develop “label-free” optical biosensor, hairpin DNA was functionalized only by a fluorophore and the immobilized substrate19 or nucleotide base20 served as a quenching agent. Although some of these approaches exhibit superb sensitivity and achieve singlebase mismatch discrimination,17,18 hairpin DNA still requires specific labeling with probes, such as fluorescent dyes. Electrochemical detection has allowed the simplification of the detection and the increase of the sensitivity of the measure(6) (7) (8) (9) (10) (11)

(12) (13) (14) (15) (16) (17) (18) (19) (20)

McCarthy, J. J.; Hilfiker, R. Nat. Biotechnol. 2000, 18, 505-508. Skogerboe, K. J. Anal. Chem. 1995, 67, 449R-454R. Southern, E. M. Trends Genet. 1996, 12, 110-115. Eng, C.; Vijg, J. Nat. Biotechnol. 1997, 15, 422-426. Okamoto, A.; Kanatani, K.; Saito, O. J. Am. Chem. Soc. 2004, 126, 4820. Reed, R.; Holmes, D.; Weyers, J.; Jones, A. Practical Skills in Biomolecular Sciences; Addison-Wesley Longman Ltd.: Edinburgh Gate, Harlow, England, 1998. Walker, M. J.; Rapley, R. Molecular Biology and Biotechnology; The Royal Society of Chemistry: Thomas Graham House, Cambridge, U.K., 2000. Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303. Kostrikis, L. G.; Tyagi, S.; Mhlanga, M. M.; Ho, D. D.; Kramer, F. R. Science 1998, 279, 1228. Fang, X. H.; Liu, X. J.; Schuster, S.; Tan, H. W. J . Am. Chem. Soc. 1999, 121, 2921. Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365. Wang, H.; Li, J.; Liu, H. P.; Liu. Q. J.; Mei, Q.; Wang, Y. J.; Zhu, J. J.; He, N. Y.; Lu, Z. H. Nucleic Acids Res. 2002, 30, e61. Ramachandran, A.; Flinchbauch, J.; Ayoubi, P.; Olah, G. A.; Malayer, J. R. Biosens. Bioelectron. 2004, 19, 727. Du, H.; Strohsahl, C. M.; Camera, J.; Miller, B. L.; Krauss, T. D. J. Am. Chem. Soc. 2005, 127, 7932. Knemeyer, J. P.; Marme, N.; Sauer, M. Anal. Chem. 2000, 72, 3717.

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ments.21-29 Currently, electrochemical measurements using hairpin DNA rely on the presence of redox probes, such as ferrocene,30,31 methylene blue,32-34 and others,35 which is attached to the 5′ or 3′ terminal of the DNA strand, with the other terminus being linked to a gold surface via a thiol group. These assays focus on the detection of hybridization events and to some degree on the detection of single-nucleotide mismatches. Only recently, a report appeared about measurements involving unlabled hairpin DNA.36 Recently, we reported a detailed electrochemical impedance spectroscopy(EIS)studyinvolvingunlabeledDNAand[Fe(CN)6]3-/4and demonstrated the detection of single-nucleotide mismatches.37 This technique is based on the differences in the charge-transfer resistance between a film of duplex DNA in the presence and absence of Zn2+ at pH g 8.6. In this present article, we exploit our results for the detection of single-nucleotide mismatches in unlabeled hairpin-DNA structures by EIS. In the presence of target strand DNA, the DNA hairpin opens the stem-loop structure and hybridizes with the target strand, which leads to an increase in the charge-transfer resistance of the film, presumably due to changes in the film thickness. The presence of mismatches in the target strand is readily detected by monitoring differences in the charge-transfer resistance (∆RCT) between double stranded DNA (ds-DNA) in the presence and absence of Zn2+. Our approach allows the discrimination of single-nucleotide mismatches with the target strand at target strand concentrations as low as 10 pM. EXPERIMENTAL SECTION Materials. Ten DNA sequences were synthesized by standard solid-phase techniques using a fully automated DNA synthesizer in Shanghai (Shanghai Sangon Biological Engineering Technology & Service Co. Ltd): 5′-HO-(CH2)6-SS-(CH2)6-GCA-CGC-GT-CACGAT-GGC-CCA-GTA-GTT-GCG-TGC-3′ (1); 3′-CA-GTG-CTA-CCG(21) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192-1199. (22) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253. (23) Throp, H. H. Top. Curr. Chem. 2004, 237, 159-181. (24) 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. (25) Kelley, S. O.; Boon, E. M.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Nucleic Acids Res. 1999, 27, 4830-4837. (26) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-774. (27) Liu, G. D.; Lee, T. M. H.; Wang, J. J. Am. Chem. Soc. 2005, 127, 38-39. (28) Millan, K. M.; Saravallo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 294348. (29) Di Guisto, D. A.; Walssoff, W. A.; Giesebrecht, S.; Gooding, J. J.; King, G. C. J. Am. Chem. Soc. 2004, 126, 4120. (30) Fan, C. H.; Plaxco, K. W.; Heeger, A. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134. (31) Jenkins, D. M.; Chani, B.; Kreuzer, M.; Presting, G.; Alvarez, A. M.; Liaw, B. Y. Anal. Chem. 2006, 78, 2314. (32) Xiao, Y.; Piorek, B. D.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2005, 127, 17990. (33) Xiao, Y.; Lubin, A. A.; Baker, B. R.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16677. (34) Lubin, A. A.; Lai, R. Y.; Baker, B. R.; Heeger, A. J.; Plaxco, K. W. Anal. Chem 2006, 78, 5671. (35) Mao, Y. D.; Luo, C. X.; Ouyang, Q. Nucleic Acids Res. 2003, 31, e108. (36) Miranda-Castro, R.; de-los-Santos-Alvarez, P.; Lobo-Castanon, M. J.; MirandaOrdieres, A. J.; Tunon-Blanco, P. Anal. Chem. 2007, 79, 4050. (37) (a) Li, X. H.; Lee, J. S.; Kraatz, H.-B. Anal. Chem. 2006, 78, 6096. (b) Li, X.; Zhou, Y.; Sutherland, T. C.; Baker, B.; Lee, J. S.; Kraatz, H.-B. Anal. Chem. 2005, 77, 5766-5769.

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Table 1. Matched and Mismatched Duplex DNA Used for the Detection of Mismatches 1+2

1 + 3 1 + 4 1 + 5 1 + 6 1 + 7 1 + 8 1 + 9 1 + 10

matched C-C

C-A

C-T

G-A

G-G

G-T

T-T

A-A

GGT-CAT-CAA-CGC-ACG-5′ (2); 3′-CA-GTG-CTA-CCG-GCT-CATCAA-CGC-ACG-5′ (3); 3′-CA-GTG-CTA-CCG-GAT-CAT-CAA-CGCACG-5′ (4); 3′-CA-GTG-CTA-CCG-GTT-CAT-CAA-CGC-ACG-5′ (5); 3′-CA-GTG-CTA-CCG-GGT-AAT-CAA-CGC-ACG-5′ (6); 3′-CA-GTGCTA-CCG-GGT-GAT-CAA-CGC-ACG-5′ (7); 3′-CA-GTG-CTA-CCGGGT-TAT-CAA-CGC-ACG-5′ (8); 3′-CA-GTG-CTA-CCG-GGT-CTTCAA-CGC-ACG-5′ (9); 3′-CA-GTG-CTA-CCG-GGA-CAT-CAA-CGCACG-5′ (10) One complementary strand and eight single-nucleotide mismatched strands (mismatch in italic) are listed above. The resulting duplexes and mismatches are shown in Table 1. NaClO4, K3[Fe(CN)6], K4[Fe(CN)6], Zn(ClO4)2, Tris (Tris(hydroxymethyl)-aminomethane)), and 6-mercaptohexanol were purchased from Aldrich and used without further purification. Deionized water (18.2 MΩ cm resistivity) from a Millipore Milli-Q system was used throughout this work. As described in our previous work, the Tris buffer was adjusted to pH 8.7 with HClO4.37 An Ag/AgCl reference electrode was added to the cell through a miniature salt bridge (agar plus KNO3) to avoid any contamination due to Cl- from the reference electrode. Monolayer Preparation. The freshly cleaned gold electrodes (1.0 mm diameter) were incubated in a solution of 0.01 mM hairpin-structured strand 1 in 50 mM Tris-ClO4 buffer (pH ) 8.7) for 5 days. Then the electrodes were washed with the buffer solution and subsequently incubated in 1 mM 6-mercaptohexanol for 10 min. The electrodes were then washed with Tris-ClO4 buffer and mounted into an electrochemical cell. Hybridization was performed by incubating a film of hairpin DNA self-assembled on gold electrodes in the target strand solutions for another 10 h to form matched 1 + 2 and single-nucleotide mismatched 1 + 3 duplex DNA. First, the EIS measurements were carried out in the absence of Zn2+. Next, the electrode with the duplex DNA film was incubated for 2 h in a solution of Zn(ClO4)2‚6H2O (0.4 mM) in 20 mM Tris-ClO4 buffer (pH ) 8.7); the EIS of the films were recorded.37 Electrochemical Measurements. A conventional threeelectrode system was used, and all the measurements were carried out at room temperature in an enclosed and grounded Faraday cage. The reference electrode was constructed by sealing a Ag/ AgCl wire into a glass tube with a solution of 3 M KCl that was capped with a Vycor tip. The counter electrode was a platinum wire. Impedance spectra were measured using a potentiostat/ frequency analyzer (EG&G 2273). The ac voltage amplitude was 5 mV, and the voltage frequencies used for EIS measurements ranged from 100 kHz to 100 mHz. The applied potential was 250 mV vs Ag/AgCl (formal potential of the redox probe [Fe(CN)6]3-/4in the buffer solution). All measurements were repeated for a minimum of five times with separate electrodes to obtain statistically meaningful results. RESULTS AND DISCUSSION Hairpin-DNA films on the gold electrodes were prepared by incubating freshly cleaned gold electrodes in solutions of 0.01 mM

Scheme 1. Schematic View of an Unlabeled Hairpin-DNA Probe for Hybridization and the Detection of Single-Nucleotide Mismatchesa

a (1) After deposition of the hairpin DNA, 6-mercaptohexanol is used to fill possible pinholes on the electrode surface. This will also aid in the formation of a suitably aligned and immobilized DNA film. (2) Hairpin DNA opens the stem-loop structures and hybridizes with the target strand to form the matched (a) and mismatched (b) duplex DNA.

32-mer strand 1 in 50 mM Tris-ClO4, followed by soaking in 1 mM 6-mercaptohexanol in 50 mM Tris-ClO4. Hairpin DNA has a stem-loop structure, which prevents the formation of a compact film on gold surfaces and at the same time prevents the molecule from laying flat on the surface. This made it necessary to backfill the gaps with 6-mercaptohexanol and force the immobilized DNA to align reproducibly in an orientation extending away from the electrode surface,31 which was shown in Scheme 1. Hybridization. The self-assembled hairpin DNA and 6-mercaptohexanol films on the gold electrode were evaluated by EIS. Upon hybridization with 26-mer strand 2 or 3, the hairpin DNA opened the stem-loop structure and formed a matched duplex DNA 1 + 2 or mismatched duplex DNA 1 + 3 containing a single-nucleotide mismatch in the center of the duplex as shown in Table 1 and Scheme 1. Representative Nyquist plots for films of 1 + 2 and 1 + 3 are shown in Figure 1. The impedance spectra for all the system were analyzed with the help of a modified Randles equivalent circuit, shown in the inset to Figure 1. The results of this analysis are listed in Table 2. To provide a rational explanation for the electrochemical process, the electronic elements in the equivalent circuit are described as below. The solution resistance, Rs, is the resistance between the reference electrode and the DNA-modified gold electrode. For every measurement, the position of the two electrodes and the distance between the two electrodes are kept same. All 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 6.0 to 6.8 Ω cm2.

Figure 1. Nyquist plots (-Zim vs Zre) of (a) the 32-base hairpin DNA 1 (b) and (b) hybridization with 26-base strand 2 to form matched DNA 1 + 2 (9) and (c) with strand 3 to form a C-C mismatched DNA 1 + 3 (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 the DNA monolayer; RCT, charge-transfer resistance of DNA monolayer; Rx and CPE, resistance and nonlinear capacitor accounting for the 6-mercaptohexanol film.

Cmonolayer accounts for the capacitance of the DNA film on gold electrodes. Cmonolayer is larger for films of 1 + 2 and 1 + 3 compared to hairpin DNA (strand 1). This can be rationalized by an increased film thickness upon opening of the stem-loop structure and hybridization with target strands 2 and 3. In addition, Cmonolayer is larger for films of 1 + 3 containing a C-C Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

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Table 2. Equivalent Circuit Element Values for Unhybridized Strand 1 and Hybridized DNA Filmsa Cmonolayer circuit Rs elements (Ω cm2) (µF cm-2) 1 1+2 1+3

6.0 (0.1) 6.8 (0.1) 6.1 (0.1)

12.7 (1.2) 9.2 (1.0) 11.5 (1.8)

RCT (Ω cm2)

Rx (Ω cm2)

CPE (µF cm-2)

1099 (79) 25.9 (3.5) 24.2 (3.9) 7096 (641) 27.4 (4.2) 13.8 (2.3) 4318 (393) 24.0 (2.1) 25.5 (2.2)

n 0.7 (0.05) 0.7 (0.04) 0.7 (0.02)

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

mismatch compared to the matched films of 1 + 2. Structural changes in films containing a single-nucleotide mismatch, for example due to kinking of the DNA,38 contribute to the increased Cmonolayer for mismatched films 1 + 3. The changes might be caused by differences in the dielectric constants due to structural variations. The combination of Rx and the constant phase element (CPE) accounts for the 6-mercaptohexanol films on the electrodes. CPE acts as a nonlinear capacitor accounting for the inhomogeneity of the film and the electrode surface with the exponential modifier n ) 0.7.39 6-Mercaptohexanol present in the film does not interfere with the detection of DNA hybridization or with the detection of mismatches. 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. Charge-transfer resistance, RCT, is the result of resistance to charge transfer from the [Fe(CN)6]3-/4- redox probe to the electrode surface through the DNA film. As shown in Table 2, RCT values for the hybridized DNA film are larger than that of films of hairpin DNA 1, presumably due to changes in the film thickness for films of 1 + 2 or 1 + 3, which will increase the distance for electron transfer through the film and hence increase RCT. Importantly, in the presence of a single-nucleotide mismatch in the center of the duplex as the case for a film of 1 + 3, the charge- transfer resistance RCT is significantly reduced. Because 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. This initial finding promoted us to evaluate the effect of other mismatches. Detection of Single-Nucleotide Mismatches. To explore the performance of this approach for mismatch detection, the hybridized ds-DNA films were evaluated by EIS in the absence and presence of Zn2+ at pH g 8.6. Representative Nyquist plots for the matched ds-DNA 1 + 2 and for a ds-DNA 1 + 3 containing a C-C mismatch in the center of the duplex are shown in Figure 2. The measured data were analyzed using an equivalent circuit described above. The fitted results for matched and all mismatched DNA films are list in Table 3. As expected, Rx is almost the same in the absence and presence of Zn2+, which further confirms that 6-mercapohexanol films on the gold electrodes are not affected by Zn2+ Cmonolayer is smaller for ds-DNA in the absence of Zn2+ compared to when Zn2+ is present. This is rationalized by an enhanced (38) Long, Y. T.; Li, C. Z.; Sutherland, T. C.; Kraatz, H. B.; Lee, J. S. Anal. Chem. 2004, 76, 4059. (39) Dijksma, M.; Boukamp, B. A.; Kamp, B.; van Bennekom, W. P. Langmuir 2002, 18, 3105.

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Figure 2. Nyquist plots (-Zim vs Zre) of the 32-mer hairpin DNA 1 hybridized with the 26-mer strand 2 to form fully matched film of 1 + 2 in the absence (O) and presence of Zn2+ (b) and hybridized with 26-mer strand 4 to form a C-C mismatched film 1 + 4 in the absence (0) and presence of Zn2+ (9). Measured data are shown as symbols with the calculated fit to the equivalent circuit shown as solid lines.

electron transfer in films in the presence of Zn2+.40 There is also evidence from the measurements in the solid-state that the conductive properties of DNA are affected by the presence of Zn2+.41 On the basis of these two facts, the dielectric constant of the DNA film is slightly higher, which led to the increased Cmonolayer. For films of 1 + 6 having a G-A mismatch and of 1 + 8 having a G-T mismatch, these changes are very small. For RCT, there are two major observations. First, for any given film, ds-DNA has a higher RCT in the absence of Zn2+. As discussed previously, the results can be rationalized by improved electrontransfer kinetics or potentially by enhanced penetration of the redox probe into the film in the presence of Zn2+.38,40 Second, upon the addition of Zn2+, the matched DNA has a lower RCT as compared to mismatched DNA. The higher RCT can be rationalized by a significant disruption of the base pair π-stack in the DNA helix, which is capable of mediating charge transport.42,43 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.40,44-46 As far as mismatch detection is concerned, ∆RCT, the RCT difference between ds-DNA in the presence and absence of Zn2+ for a given film, is an important parameter, which allows us to distinguish a fully complementary duplex from one containing a single-nucleotide mismatch. For a film of the matched pair 1 + (40) Liu, B.; Bard, A. J.; Li, C. Z.; Kraatz, H. B. J. Phys. Chem. B 2005, 109, 5193. (41) Rakitin, A.; Aich, P.; Papadopoulos, C.; Kobzar, Y.; Vedeneev, A. S.; Lee, J. S.; Xu, J. M. Phys. Rev. Lett. 2001, 86, 3670. (42) Liu, T.; Barton, J. K. J. Am. Chem. Soc. 2005, 127, 10160. (43) Kelley, S. O.; Jackson, N. M.; Hill, M. G.; Barton, J. K. Angew. Chem., Int. Ed. 1999, 38, 941. (44) Li, C. Z.; Long, Y. T.; Kraatz, H. B.; Lee, J. S. J. Phys. Chem. B 2003, 107, 2291. (45) Lee, J. S.; Latimer, L. J. P.; Reid, R. S. Biochem. Cell Biol. 1993, 71, 162. (46) 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.

Table 3. Equivalent Circuit Element Values for Matched and Eight Mismatched DNA Duplex Filmsa circuit elements

match C-C C-A C-T G-A G-G G-T T-T A-A a

B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA

Rs (Ω cm2)

Cmonolayer (µF cm-2)

RCT (Ω cm2)

Rx (Ω cm2)

CPE (µF cm-2)

n

6.8 (0.1) 6.3 (0.1) 6.1 (0.1) 6.2 (0.1) 6.2 (0.1) 6.0 (0.1) 6.0 (0.1) 6.0 (0.1) 6.2 (0.1) 6.3 (0.1) 6.3 (0.1) 6.2 (0.1) 6.2 (0.1) 6.1 (0.1) 6.4 (0.1) 6.2 (0.1) 6.1 (0.1) 6.1 (0.1)

9.2 (1.0) 14.0 (1.2) 11.5 (1.8) 22.9 (2.1) 12.2 (1.4) 12.7 (1.1) 13.4 (1.1) 15.3 (1.2) 11.2 (1.0) 11.7 (1.3) 10.4 (1.0) 11.5 (1.1) 12.1 (1.0) 12.3 (1.1) 9.9 (1.0) 10.9 (1.0) 11.5 (1.2) 12.3 (1.3)

7096 (641) 1335 (136) 4318 (393) 2826 (214) 1884 (93) 1531 (101) 6516 (157) 5181 (126) 3376 (234) 2434 (154) 5574 (236) 2748 (79) 2120 (143) 1570 (89) 6044 (259) 3140 (178) 3532 (156) 1806 (137)

27.4 (4.2) 25.7 (3.9) 24.0 (2.1) 26.3 (2.3) 19.3 (2.1) 17.1 (1.4) 24.0 (2.1) 28.6 (2.9) 21.0 (1.9) 18.9 (1.7) 20.0 (1.3) 18.5 (1.1) 19.1 (1.7) 16.2 (1.4) 21.7 (1.9) 23.3 (2.0) 19.3 (1.8) 20.9 (1.7)

13.8 (2.3) 40.7 (3.4) 25.5 (2.2) 34.4 (3.0) 20.3 (1.7) 17.8 (1.9) 21.7 (1.8) 20.4 (2.0) 20.3 (2.2) 15.3 (1.3) 25.5 (1.9) 24.2 (2.0) 28.0 (2.7) 36.9 (2.9) 14.0 (1.3) 25.4 (1.8) 22.9 (2.1) 25.5 (2.3)

0.7 (0.04) 0.7 (0.03) 0.7 (0.02) 0.7 (0.02) 0.7 (0.02) 0.7 (0.03) 0.7 (0.05) 0.7 (0.04) 0.7 (0.02) 0.7 (0.01) 0.7 (0.03) 0.7 (0.02) 0.7 (0.03) 0.7 (0.02) 0.7 (0.02) 0.7 (0.01) 0.7 (0.01) 0.7 (0.02)

∆RCT (Ω cm2) 5761 (582) 1492 (132) 353 (46) 1335 (112) 942 (98) 2826 (201) 550 (56) 2904 (207) 1727 (141)

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

Figure 3. Relationship between ∆RCT and the concentration of the target strand of 2 (9) and mismatch containing target strand 4 (b). The gold electrode was modified with hairpin DNA 1 and diluted with 6-mercaptohexanol. Error bars are derived from a minimum of five electrodes.

2, the value RCT for ds-DNA is 7096 (641) Ω cm2, whereas in the presence of Zn2+, the resistive term drops to 1335 (136) Ω cm2. This gives a ∆RCT of 5761 (582) Ω cm2. For the film 1 + 3 containing a C-C mismatch, the RCT for ds-DNA is 4318 (393) Ω cm2 and drops to 2826 (214) Ω cm2 when Zn2+ is present, with a ∆RCT of 1492 (132) Ω cm2. It is this difference in ∆RCT that allows the discrimination of C-C mismatched from matched DNA. The values for ∆RCT for other mismatched DNA duplexes are listed in Table 3 and are range from 2904 (207) (Ω cm2) for a T-T mismatch, 2826 (201) (Ω cm2) for a G-G mismatch, 1727 (141) (Ω cm2) for an A-A mismatch, 1335 (112) (Ω cm2) for a C-T mismatch, 550 (56) (Ω cm2) for a G-T mismatch to 353 (46) (Ω cm2) for a C-A mismatch. Using this approach, we were able to detect all eight mismatches using hairpin DNA without the need for prior labeling of the DNA.

Finally, from a practical perspective, the detection limit of the assay was explored. For this purpose, we evaluated a C-A mismatch. The concentrations of target strands 2 and 4 were gradually decreased from 10-5 to ∼10-13 M, then hybridized with hairpin-DNA films of strand 1 on the gold electrodes to form matched and C-A mismatched DNA. The impedance spectra were recorded in the presence and absence of Zn2+ and fit to the equivalent circuit shown in Figure 1. As shown in Figure 3, ∆RCT of a fully matched DNA film (1 + 2) and a C-A mismatched film (1 + 4), respectively, decreases with the decreasing concentration of the target strand until the concentration is as low as 10 pM. The possible reason for the changes is related to the formation of the film (from hairpin to duplex).37,47 Given a sufficiently high binding affinity of the target strand, at higher concentrations of the target strand DNA, a large number of hairpin DNA on the electrodes can open the stem-loop structure and hybridize with target strand to form the duplex. At lower concentrations, such as 10-12 M, the concentration is so low that the hairpin DNA cannot open the stem-loop structure and does not hybridize with the target strand. CONCLUSION In this paper, the application of an unlabeled hairpin-DNA probe for the detection of single-nucleotide mismatches by EIS was presented. Upon hairpin DNA hybridization with unlabeled complementary or single-nucleotide mismatch target strand, the stem-loop structure opens and matched- or mismatched-duplex DNAs are formed. In the case of the mismatched structure, the mismatch is located in the center of the duplex. Under these conditions, all eight different mismatches are detected by evaluating the difference in charge-transfer resistance ds-DNA in the presence and absence of Zn2+ at pH g 8.6. We are presently working on the performance of this assay for (47) Di Giusto, D. A.; Wlassoff, W. A.; Giesebrecht, S.; Gooding, J. J.; King, G. C. J. Am. Chem. Soc. 2004, 126, 4120.

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recognizing the specific DNA/RNA sequences containing different impurities. ACKNOWLEDGMENT We are grateful for financial support from the National Natural Science Foundation of China (Grant 20703006). H.-B.K. acknowledges the support from NSERC and the University of Western Ontario for start-up funds.

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SUPPORTING INFORMATION AVAILABLE Additional Nyquist plot and table of equivalent circuit element values for the bare electrode and the self-assembled 6-mercaptohexanol films. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 3, 2007. Accepted January 15, 2008. AC7024688