Immobilization-Free Sequence-Specific Electrochemical Detection of

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Anal. Chem. 2008, 80, 7341–7346

Immobilization-Free Sequence-Specific Electrochemical Detection of DNA Using Ferrocene-Labeled Peptide Nucleic Acid Xiaoteng Luo,† Thomas Ming-Hung Lee,‡ and I-Ming Hsing*,†,§ Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, and Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong An electrochemical method for sequence-specific detection of DNA without solid-phase probe immobilization is reported. This detection scheme starts with a solutionphase hybridization of ferrocene-labeled peptide nucleic acid (Fc-PNA) and its complementary DNA (cDNA) sequence, followed by the electrochemical transduction of Fc-PNA-DNA hybrid on indium tin oxide (ITO)-based substrates. On the bare ITO electrode, the negatively charged Fc-PNA-DNA hybrid exhibits a much reduced electrochemical signal than that of the neutral-charge FcPNA. This is attributed to the electrostatic repulsion between the negatively charged ITO surface and the negatively charged DNA, hindering the access of FcPNA-DNA to the electrode. On the contrary, when the transduction measurement is done on the ITO electrode coated with a positively charged poly(allylamine hydrochloride) (PAH) layer, the electrostatic attraction between the (+) PAH surface and the (-) Fc-PNA-DNA hybrid leads to a much higher electrochemical signal than that of the Fc-PNA. The measured electrochemical signal is proportional to the amount of cDNA present. In terms of detection sensitivity, the PAH-modified ITO platform was found to be more sensitive (with a detection limit of 40 fmol) than the bare ITO counterpart (with a detection limit of 500 fmol). At elevated temperatures, this method was able to distinguish fully matched target DNA from DNA with partial mismatches. Unpurified PCR amplicons were detected using a similar format with a detection limit down to 4.17 amol. This detection method holds great promise for single-base mismatch detection as well as electrochemistry-based detection of post-PCR products. Sequence-specific detection of DNA is an important tool in medical diagnostics, environmental and food safety monitoring, * To whom correspondence should be addressed. Tel: (852) 23587131. Fax: (852) 31064857. E-mail: [email protected]. † Bioengineering Graduate Program, The Hong Kong University of Science and Technology. ‡ The Hong Kong Polytechnic University. § Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology. 10.1021/ac8010236 CCC: $40.75  2008 American Chemical Society Published on Web 09/05/2008

pathogen identification, and forensic sciences.1 Electrochemical DNA detection has the advantages of high sensitivity, compatibility for miniaturization, easy operation, and low cost.2-5 In a typical electrochemical DNA detection scheme, three main steps are involved.6 First, a single-stranded DNA probe is immobilized onto an electrode. Then, a target DNA is hybridized with the immobilized DNA probe. Finally, the hybridization between the immobilized DNA probe and the target DNA is transduced into an electrochemical signal. However, the probe immobilization step is usually laborious and time-consuming, and efficiency of hybridization between the immobilized probe and the target DNA is usually quite low due to the steric hindrance effect on the electrode surface. Therefore, it is desirable to develop a DNA detection scheme that requires no probe immobilization, in which the hybridization between the probe DNA and the target DNA occurs in solution phase instead of on the surface of the electrode. Tamiya and co-workers7 reported an immobilization-free electrochemical DNA quantification method based on aggregation induced by Hoechst 33258. Hoechst 33258 was found to form an aggregate in the presence of DNA, which led to a decrease in the voltammetric signal in proportion to the quantity of DNA. However, this DNA quantification method is not sequence-specific, unless it is combined with allele-specific primer polymerase chain reaction (PCR).8 Peptide nucleic acid (PNA) is an analogue of DNA composed of repeating N-(2-aminoethyl)glycine units linked by peptide bonds, with the four purine and pyrimidine bases linked to the backbone by methylene carbonyl bonds.9 While DNA’s backbone is negatively charged, PNA has a neutral backbone. As a result, (1) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev. 2008, 108, 109– 139. (2) Wang, J. Anal. Chim. Acta 2002, 469, 63–71. (3) Lee, T. M. H.; Hsing, I. M. Anal. Chim. Acta 2006, 556, 26–37. (4) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192–1199. (5) Kerman, K.; Kobayashi, M.; Tamiya, E. Meas. Sci. Technol. 2004, 15, R1– R11. (6) Gooding, J. J. Electroanalysis 2002, 14, 1149–1156. (7) Kobayashi, M.; Kusakawa, T.; Saito, M.; Kaji, S.; Oomura, M.; Iwabuchi, S.; Morita, Y.; Hasan, Q.; Tamiya, E. Electrochem. Commun. 2004, 6, 337– 343. (8) Ahmed, M. U.; Idegami, K.; Chikae, M.; Kerman, K.; Chaumpluk, P.; Yamamura, S.; Tamiya, E. Analyst 2007, 132, 431–438. (9) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497–1500.

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the binding between PNA and DNA is stronger and with higher selectivity compared to DNA-DNA binding.10,11 Because of these attractive characteristics, PNA has been utilized in a number of DNA detection schemes.12 Aoki and co-workers13 reported the detection of DNA at femtomolar level using a gold electrode modified with a selfassembled monolayer of a 13-mer PNA probe and 8-amino-1octanethiol based on the ion channel sensor technique. They also reported a strategy for label-free and marker-free gene detection based on the hybridization-induced conformational flexibility change of the PNA probes immobilized on a gold electrode.14 Electrochemical DNA hybridization detection methods have been developed based on immobilized PNA probes, using methylene blue, [Ru(NH3)6]3+, or [Co(phen)3]3+ as the electroactive label.15-17 Electrochemical impedance sensing of DNA on PNA self-assembled monolayer has also been reported.18 In this paper, a ferrocene-labeled (Fc) PNA probe is used to electrochemically detect complementary DNA (cDNA) sequence using a bare indium tin oxide (ITO) electrode or a poly(allylamine hydrochloride) (PAH)-modified ITO electrode. It was found that hybridization with cDNA remarkably reduced the differential pulse voltammetric (DPV) peak current of the ferrocene-labeled PNA on the negatively charged bare ITO electrode, while addition of noncomplementary (noncDNA) did not change the DPV peak current. On the positively charged PAH-modified ITO electrode, hybridization with cDNA significantly increased the DPV peak current of the ferrocene-labeled PNA. Both detection platforms require no probe immobilization step and provide a method to detect DNA with high specificity in less than 10 min. The capability of this new method for single-base mismatch detection and PCR product detection is also investigated. EXPERIMENTAL SECTION Reagents and Instrumentation. Fc-PNA was purchased from Panagene, with the following structure: Fc-O-AACCACCACCANH2 (11-mer), where Fc and O denote a ferrocene moiety and an ethylene glycol linker, respectively. DNAs with a fully matched sequence of 5′-TGGTGGTGGTT-3′(11-mer), a single-base-mismatched sequence of 5′-TGGTGCTGGTT-3′(11-mer), a two-basemismatched sequence of 5′-TGGTCCTGGTT-3′(11-mer), and a noncomplementary sequence of 5′-CTCAACCTCCTGTCAATGC3′(19-mer) were purchased from Integrated DNA Technologies. The DNA template for PCR M13mp18 phage DNA was purchased from Sigma-Aldrich. The phosphorothioated forward primer (5′CTCAACCTCCTGTCAATGC-3′), the normal reverse primer (5′GTCATAGCCCCCTTATTAGC-3′), and the dNTPs were pur(10) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365, 566–568. (11) Demidov, V.; Potaman, V. N.; Frank-Kamenetskii, M. D.; Buchardt, O.; Egholm, M.; Nielsen, P. E. Biochem. Pharmacol. 1994, 48, 1309–1313. (12) Wang, J. Biosens. Bioelectron. 1998, 13, 757–762. (13) Aoki, H.; Umezawa, Y. Analyst 2003, 128, 681–685. (14) Aoki, H.; Tao, H. Analyst 2007, 132, 784–791. (15) Ozkan, D.; Erdem, A.; Kara, P.; Kerman, K.; Gooding, J. J.; Nielsen, P. E.; Ozsoz, M. Electrochem. Commun. 2002, 4, 796–802. (16) Steichen, M.; Decrem, Y.; Godfroid, E.; Buess-Herman, C. Biosens. Bioelectron. 2007, 22, 2237–2243. (17) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, N.; Luo, D.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 7667–7670. (18) Degefa, T. H.; Kwak, J. J. Electroanal. Chem. 2008, 612, 37–41.

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Figure 1. Schematic showing the chip for electrochemical measurements. The chip has four ITO working electrodes (diameter 1 mm), a Pt counter electrode, and a Pt pseudoreference electrode.

chased from Invitrogen. The VentR (exo-) DNA polymerase, T7 exonuclease, and the hybridization and PCR buffer were purchased from New England Biolabs. All other chemicals used were of analytical reagent grade. All aqueous solutions were prepared with deionized water (specific resistance >18.2 MΩ/cm) obtained with a Milli-Q reagent grade water system (Millipore). Electrochemical measurements were performed with an Autolab PGSTAT30 potentiostat/galvanostat (Eco Chemie) controlled by the General Purpose Electrochemical System (GPES) software (Eco Chemie). Polymerase chain reaction (PCR) was performed with a C1000 thermal cycler (Bio-Rad). Procedure. 1. Fabrication and Preparation of Electrodes. The electrochemical measurements were conducted on an ITOcoated glass chip (Figure 1), which is similar to a DNA microchip previously reported by our group for the multiplexed detection of Escherichia coli and Bacillus subtilis.19 The chip has four patterned circular ITO spots serving as working electrodes, a Pt counter electrode, and a Pt pseudoreference electrode. Each of the four ITO working electrodes has an active surface area of 7.85 × 10-3 cm2. For electrochemical measurements, 2 µL of sample is enough to cover one of the ITO working electrodes, the Pt counter electrode, and the Pt pseudoreference electrode. The potential of the Pt pseudoreference electrode in the hybridization buffer was determined to be +0.36 V with respect to a Ag/AgCl reference electrode. The chip was fabricated by microfabrication. Briefly, photoresist was coated onto the ITO-coated glass (Delta Technologies, Stillwater, MN) and patterned by photolithography. After the selective etching of ITO, the desired working electrode pattern was formed. Then, a second photolithographic step was performed, and Pt was sputtered onto the patterned photoresist. After a liftoff process, patterned Pt was obtained. Before each electrochemical measurement, the chip was cleaned according to a previously reported procedure20 to obtain (19) Yeung, S. W.; Lee, T. M. H.; Cai, H.; Hsing, I. M. Nucleic Acids Res. 2006, 34, e118. (20) Napier, M. E.; Thorp, H. H. Langmuir 1997, 13, 6342–6344.

a negatively charged surface. Briefly, it was sequentially sonicated in an Alconox solution (8 g of Alconox/L of water), propan-2-ol, and twice in water. Each sonication lasted for 15 min. PAH-modified ITO electrodes were prepared by soaking the cleaned chip in a 3 mg/mL poly(allylamine hydrochloride) solution (2 M NaCl, pH 8.0, adjusted by NaOH) for 30 min to form a positively charged surface. After the electrostatic assembly of PAH, the ITO electrode was washed with distilled water and dried in a nitrogen stream. 2. Linear Sweep Voltammetry of Fc-PNA. Fc-PNA was dissolved to a concentration of 5 µM in the hybridization buffer containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Triton X-100. For each linear sweep voltammetry (LSV) scanning, 2 µL of a 5 µM Fc-PNA solution was pipetted onto the chip to cover one of the ITO working electrodes, the Pt counter electrode, and the Pt pseudoreference electrode. 3. Detection of cDNA Sequence at Room Temperature. Fc-PNA was dissolved to a concentration of 1 (for bare ITO) or 0.1 µM (for PAH-ITO) in the hybridization buffer and mixed with fully matched, single-base-mismatched, two-base-mismatched, or noncDNA. The mixture was incubated at room temperature for 5 min. Then, 2 µL of the mixture was pipetted onto the chip to cover one of the ITO working electrodes, the Pt counter electrode, and the Pt pseudoreference electrode, and DPV measurement was performed immediately. 4. Detection of Single-Base Mismatch in Target DNA at Elevated Temperature. Bare ITO electrodes were used. Fc-PNA was dissolved to a concentration of 0.45 µM in the hybridization buffer and mixed with 0.9 µM fully matched or single-basemismatched DNA. The mixture and the chip were incubated in a thermal cycler at 37 °C for 5 min. Then, 2 µL of the mixture was pipetted onto the chip to cover one of the ITO working electrodes, the Pt counter electrode, and the Pt pseudoreference electrode, and DPV scanning was performed immediately with the chip inside the thermal cycler at 37 °C. 5. Detection of Unpurified PCR Product. Bare ITO chips were used for the detection of unpurified PCR product. The PCR mixture contained 1 × ThermoPol reaction buffer (20 mM TrisHCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8), 0.2 mM dNTPs, 0.2 µM phosphorothioated forward primer, 0.2 µM normal reverse primer, 0.02 ng/µL M13mp18 DNA template, and 0.01 unit/µL VentR (exo-) DNA polymerase. The M13mp18 DNA template was omitted for the negative PCR control. The PCR master mix was subjected to the following thermal cycling profile in the thermal cycler: initial denaturation at 93 °C for 5 min, 35 cycles at 93 °C for 20 s, at 55 °C for 20 s, at 72 °C for 20 s, and a final extension at 72 °C for 5 min. After the PCR, 0.2 unit/µL T7 exonuclease was added to the PCR mixture. The mixture was incubated at 37 °C in the thermal cycler for 1 h. After that, Fc-PNA was added to the PCR mixture to a concentration of 0.45 µM. The mixture was incubated at room temperature for 5 min. Then, 2 µL of the mixture was pipetted onto the bare ITO chip to cover one of the ITO working electrodes, the Pt counter electrode, and the Pt pseudoreference electrode, and DPV measurement was performed immediately.

Figure 2. Linear sweep voltammograms of 5 µM Fc-PNA. Scan rate: 50 (s), 30 (- · -), 20 ( · · · ), and 10 mV/s (---). Inset: the relationship between peak currents (ipa) and the square root of scan rates (v1/2).

Figure 3. Differential pulse voltammograms of 1 µM Fc-PNA only (s), 1 µM Fc-PNA incubated with 2 µM noncDNA (---), 1 µM Fc-PNA incubated with 1 µM cDNA (- · -), and 1 µM Fc-PNA incubated with 2 µM cDNA ( · · · ). DPVs were carried out on bare ITO electrode using a pulse amplitude of 100 mV/s and a scan rate of 25 mV/s. Inset: plot of peak current signal in differential pulse voltammetric scans against concentration of cDNA.

RESULTS AND DISCUSSION 3.1. Linear Sweep Voltammetry of Fc-PNA. LSV of 5 µM Fc-PNA solution was conducted at scan rates from 0.01 to 0.05 V/s (Figure 2). The peak currents (ipa) were plotted against the square root of scan rates (v1/2) (inset of Figure 2). A linear relationship between ipa and v1/2 was found, indicating a diffusioncontrolled redox reaction of Fc-PNA. 3.2. Sequence-Specific DNA Detection on Bare ITO. In DPV, the peak specific to the ferrocene label on PNA was observed at ∼+0.2 V on a bare ITO electrode (versus Pt pseudoreference electrode). It was found that hybridization with cDNA remarkably reduced the DPV peak intensity of Fc-PNA, while incubation with noncDNA did not change the DPV peak intensity of Fc-PNA (Figure 3). When the concentration of the cDNA was equal to the concentration of Fc-PNA (i.e., both 1 µM), the peak intensity of Fc-PNA was reduced to one-third of the peak Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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Figure 5. Differential pulse voltammograms of 0.1 µM Fc-PNA incubated with 0.1 µM cDNA (s), 0.1 µM Fc-PNA only (---), and 0.1 µM Fc-PNA incubated with 0.2 µM noncDNA ( · · · ). DPVs were carried out on PAH-coated ITO electrode using a pulse amplitude of 100 mV/s and a scan rate of 25 mV/s. Inset: plot of peak current signal in differential pulse voltammetric scans against concentration of cDNA.

Figure 4. Working principle of immobilization-free, sequence-specific DNA detection based on negatively charged bare ITO electrode (A) or positively charged PAH-modified ITO electrode (B).

intensity before hybridization. When the concentration of the cDNA was twice that of Fc-PNA, the peak of Fc-PNA was completely eliminated. The peak intensity of Fc-PNA decreases as the concentration of cDNA increases. A linear relationship between the peak intensity of Fc-PNA and the concentration of the cDNA could be obtained in the range of 0.25-2 µM, equivalent to 0.5-4 pmol (inset of Figure 3). The reason for the peak reduction caused by the hybridization of Fc-PNA with cDNA can be attributed to the negatively charged surface of the ITO electrode21 (Figure 4A). As PNA has a neutral backbone, Fc-PNA can approach the ITO electrode and produce an oxidative peak. After hybridization with cDNA, the hybrid is electrostatically repelled from the ITO electrode surface because of the negative charge of the DNA backbone. Therefore, peak intensity of Fc-PNA is significantly reduced after hybridization with DNA. 3.3. Sequence-Specific DNA Detection on PAH-ITO. On the PAH-modified ITO electrode, which has a positively charged surface, the hybridization with cDNA gives an increase to the peak of the Fc on the PNA (Figure 5). The peak intensity has a linear relationship with the concentration of the cDNA in the range of 0.02-0.1 µM, equivalent to 40-200 fmol (inset of Figure 5). The reason why hybridization with cDNA increases the peak intensity of Fc-PNA is attributed to the positively charged PAH layer coated onto the ITO electrode (Figure 4B). After hybridization, the negatively charged Fc-PNA-DNA hybrid is attracted to the electrode surface and Fc is accumulated on the electrode surface. Therefore, the peak intensity of Fc is increased. It should be noted that Fc-PNA does not produce a peak on PAH-modified ITO electrode. This is probably because of the blocking effect of the PAH layer. The PAH may hinder the FcPNA from approaching the ITO electrode surface and block the (21) Guo, Z.; Shen, Y.; Wang, M.; Zhao, F.; Dong, S. Anal. Chem. 2004, 76, 184–191.

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electron transfer from Fc to the ITO electrode. Therefore, the peak intensity of Fc-PNA is reduced to an undetectable level. This blocking effect is overcome by the Fc-PNA-DNA hybrid because the concentration of Fc-PNA-DNA near the PAH-ITO electrode surface is high, due to the attraction between the positively charged PAH and the negatively charged Fc-PNA-DNA. Therefore, in spite of the blocking effect of PAH, the peak of Fc can still be detected. The detection limit for the cDNA on the PAH-modified ITO is more than 10 times better than that on the bare ITO. This may be attributed to the fact that, on the bare ITO, the oxidation of Fc is diffusion controlled, while on the PAH-ITO, the Fc-PNA-DNA is adsorbed on the electrode surface. We tested the performance of this detection method upon a mixture of complementary and noncDNAs. Fc-PNA was incubated with a mixture of complementary and noncDNAs, with the ratio of noncDNA to cDNA ranging from 0 to 15. As the results showed (Figure 6A), on PAH-coated ITO electrodes, the peak intensity of Fc decreases as the concentration of noncDNA increases (the concentration of cDNA remained unchanged). This is attributed to the fact that both cDNA and noncDNA can be attracted by the positively charged PAH and there is competition between complementary and noncDNAs to be attracted to the PAH layer. As the amount of adsorbed noncDNA increases, there is a corresponding decrease in the amount of complementary PNA/DNA hybrid that can be bound to the PAH. However, this problem is not present when using bare ITO electrodes. As demonstrated in Figure 6B, on bare ITO electrodes, the presence of noncDNA, even at a concentration 15 times higher than that of cDNA, does not affect the detection. 3.4. Detection of Partial Base Mismatches in Target DNA at Room Temperature and Elevated Temperature. Experiments were conducted to evaluate the ability of our detection method to distinguish mismatched target DNA from fully matched ones. At room temperature (20 °C) (Figure 7 A), our method can distinguish fully matched target DNA from target DNAs with two mismatched bases but cannot distinguish fully matched target DNA from target DNA with single mismatched base. When the

Figure 6. (A) Differential pulse voltammograms of 0.25 µM Fc-PNA incubated with 0.25 µM cDNA(s), 0.25 µM Fc-PNA incubated with 0.25 µM cDNA and 1.25 µM noncDNA (---),0.25 µM Fc-PNA incubated with 0.25 µM cDNA and 2.50 µM noncDNA ( · · · ), and 0.25 µM FcPNA incubated with 0.25 µM cDNA and 3.75 µM noncDNA (- · -). DPVs were carried out on PAH-coated ITO electrode using a pulse amplitude of 100 mV/s and a scan rate of 25 mV/s. Inset: plot of peak current signal in differential pulse voltammetric scans against ratio of noncDNA to cDNA. (B) Differential pulse voltammograms of 0.45 µM Fc-PNA only (s), 0.45 µM Fc-PNA incubated with 13.5 µM noncDNA (---), 0.45 µM Fc-PNA incubated with 0.9 µM cDNA ( · · · ), and 0.45 µM Fc-PNA incubated with 0.9 µM cDNA and 13.5 µM noncDNA (- · -). DPVs were carried out on bare ITO electrode using a pulse amplitude of 100 mV/s and a scan rate of 25 mV/s.

detection was conducted at an elevated temperature of 37 °C, single-base mismatched target DNA can be clearly differentiated from the fully matched one (Figure 7B). These results are in agreement with the calculated melting temperatures (Tm) of the fully matched and mismatched PNA/ DNA hybrids. The melting temperatures for fully matched, singlebase-mismatched and two-base-mismatched PNA/DNA hybrids are 43.6, 28.6, and 13.6 °C, respectively (according to the rules that Tm for PNA/DNA hybrid is 1 °C higher than DNA/DNA hybrid and that a mismatch in the PNA/DNA duplex causes a decrease of 15 °C in Tm).22 (22) Giesen, U.; Kleider, W.; Berding, C.; Geiger, A.; Orum, H.; Nielsen, P. E. Nucleic Acids Res. 1998, 26, 5004–5006.

Figure 7. (A) Differential pulse voltammograms of 0.45 µM FcPNA only (s), 0.45 µM Fc-PNA incubated with 0.9 µM DNA with 2 mismatched bases (---), 0.45 µM Fc-PNA incubated with 0.9 µM DNA with 1 mismatched base ( · · · ), and 0.45 µM Fc-PNA incubated with 0.9 µM fully matched DNA (- · -). DPVs were carried out at 20 °C on a bare ITO electrode using a pulse amplitude of 100 mV/s and a scan rate of 25 mV/s. (B) Differential pulse voltammograms of 0.45 µM Fc-PNA incubated with 0.9 µM DNA with 1 mismatched base (s) and 0.45 µM Fc-PNA incubated with 0.9 µM fully matched DNA (---). DPVs were carried out at 37 °C on bare ITO electrode using a pulse amplitude of 100 mV/s and a scan rate of 25 mV/s.

At 20 °C, which is higher than the Tm of Fc-PNA/two-basemismatched DNA (13.6 °C) but lower than the Tm of Fc-PNA/ single-base-mismatched DNA (28.6 °C), the Fc-PNA hybridized with the single-base-mismatched DNA and the fully matched DNA, but not with the two-base-mismatched one. Therefore, the Fc peak can be obtained for Fc-PNA/two-base-mismatched DNA mixture, but not for Fc-PNA/single-base-mismatched DNA hybrid and FcPNA/fully matched DNA hybrid. At 37 °C, which is higher than the Tm of Fc-PNA/single-basemismatched DNA (28.6 °C) but lower than the Tm of Fc-PNA/ fully matched DNA (43.6 °C), the Fc-PNA hybridized with the fully matched DNA, but not with the single-base-mismatched one. As a result, the Fc peak can be obtained for Fc-PNA/single-basemismatched DNA mixture, but not for Fc-PNA/fully matched DNA hybrid. 3.5. Detection of Unpurified PCR Products. In order to produce single-stranded PCR product, a phosphorothioated forAnalytical Chemistry, Vol. 80, No. 19, October 1, 2008

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produced. As a result, when Fc-PNA was added to the negative control solution, the peak of Fc could still be obtained. These results suggest that our detection method can be readily combined with PCR. With the amplification power of PCR, the detection limit of our method can be improved to 4.17 pM, or 4.17 amol in terms of amount, which is the concentration or amount of the DNA template used in the PCR.

Figure 8. Differential pulse voltammograms of 0.45 µM Fc-PNA incubated with negative PCR control (without DNA template) (s) and 0.45 µM Fc-PNA incubated with positive PCR product (---). DPVs were carried out on a bare ITO electrode using a pulse amplitude of 100 mV/s and a scan rate of 25 mV/s.

ward primer and T7 exonuclease were used.23 T7 exonuclease is a double-strand specific exonuclease that removes the terminal 5′-nucleotides from duplex DNA, acting in the 5′ to 3′ direction. One of the PCR primers (the forward primer) contained six phosphorothioates at its 5′ end (the underlined bases of the primer sequence described in the Experimental Section), while the reverse primer is unmodified. After PCR, the double-stranded product was treated with T7 exonuclease. The phosphorothioated strand was protected from the action of T7 exonuclease, while the opposite strand was hydrolyzed. Thus, single-stranded PCR product was generated. The primers were designed to generate single-stranded PCR product containing a sequence complementary to the Fc-PNA. The detection was carried out on bare ITO electrodes. As demonstrated in Figure 8, the peak of Fc is significant for the negative PCR control (without adding DNA template) but not for the positive PCR product. The PCR amplification step and treatment with T7 exonuclease produced a large amount of singlestranded amplified DNA that contained a sequence complementary to the Fc-PNA. When Fc-PNA was added to the positive PCR mixture, it hybridized with the single-stranded PCR amplicon, and therefore, the peak of Fc did not appear (similar to the scenario of Figure 3). The negative PCR control did not contain the DNA template, and therefore, no single-stranded PCR amplicon was (23) Nikiforov, T. T.; Rendle, R. B.; Kotewicz, M. L.; Rogers, Y. H. PCR Methods Appl. 1994, 3, 285–291.

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CONCLUSIONS A novel detection method for sequence-specific DNA using ferrocene-labeled PNA is reported. Hybridization with cDNA remarkably reduces the DPV peak intensity of Fc-PNA on bare ITO, while on PAH-modified ITO, the peak intensity of Fc-PNA is remarkably increased after hybridization with cDNA. The decrease or increase of the peak intensity of Fc-PNA is proportional to the concentration of cDNA with a linear relationship in the range of 0.25-2 µM (for bare ITO) or 0.02-0.1 µM (for PAHITO). The reported Fc-PNA based DNA detection method is able to distinguish single-base mismatch at elevated temperature. With combination to PCR, the detection limit of this method can reach a concentration of 4.17 pM, or 4.17 amol in terms of amount. The detection method reported here is expected to have good compatibility with a microchip platform for simultaneous PCR and detection and electrochemistry-based real-time PCR previously developed in our group.24,25 This detection method does not need immobilization of probe on the electrode and can be carried out in less than 10 min. Therefore, it has the potential to be developed into a powerful tool for sequence-specific DNA detection and quantification. In particular, future investigations include the detection of practical samples (water and clinic specimen) in realistic conditions and the implementation of an Fc-PNA based detection platform on a microchip for point-of-use applications. ACKNOWLEDGMENT The authors thank the funding support from the Research Grants Council of the Hong Kong Special Administrative Region Government (RGC CERG Project Number: 601106). Laboratory facilities provided by the Bioengineering Graduate Program and Nanoelectronics Fabrication Facility are also acknowledged. The authors thank Professor Guizhen Yan and Dr. Zhiyong Xiao for their help with the fabrication of the chip. Received for review May 20, 2008. Accepted August 11, 2008. AC8010236 (24) Lee, T. M. H.; Carles, M.; Hsing, I. M. Lab. Chip 2003, 3, 100–105. (25) Yeung, S. W.; Lee, T. M. H.; Hsing, I. M. Anal. Chem. 2008, 80, 363–368.