Electrochemical Telomerase Assay with Ferrocenylnaphthalene

Md. Monirul Islam , Shinobu Sato , Shingo Shinozaki , Shigeori Takenaka. Bioorganic .... Xia Chu , Ru-Qin Yu. Chemical Communications 2012 48 (47), 59...
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Anal. Chem. 2005, 77, 7304-7309

Electrochemical Telomerase Assay with Ferrocenylnaphthalene Diimide as a Tetraplex DNA-Specific Binder Shinobu Sato,† Hiroki Kondo,† Takahiko Nojima,‡ and Shigeori Takenaka*,§

Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka 820-8502, Japan, Department of Applied Chemistry, Kyushu University, Fukuoka 812-8581, Japan, and Department of Materials Science, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan

Recently, telomerase has been attracting attention as a cancer marker.1 It is known that telomerase activity is found in human germ cells but not in normal somatic cells, and it becomes detectable in tumor cells.2 Hence, the detection of telomerase activity may be useful for cancer diagnosis just like other makers such as the carcinoembryonic antigen,3 which have been widely used for early-stage diagnosis of cancer.4 Simple and quick detection of telomerase activity will be an alternative or new addition to this approach. The detection of telomerase activity was established first by Kim et al. as a telomeric repeat amplification protocol (TRAP) assay, including the extension reaction of a telomerase substrate (TS) primer carrying a sequence extendable by telomerase, PCR amplification of its product, and analysis by gel electrophoresis.5

When the TS primer immobilized on the electrode can be extended by telomerase, an electrochemical assay of telomerase activity will be realized without PCR. Willner and co-workers6-8 reported for the first time an electrochemical telomerase assay by using the catalytic DNAzyme, which consists of hemin and single-stranded guanine-rich nucleic acids and possesses peroxidase-like activities. Once an electrochemical ligand that is concentrated on the telomeric DNA can be developed, a simpler electrochemical telomerase assay will be achieved. Unfortunately, however, no one has yet succeeded in direct extension reaction with a TS primer-immobilized electrode and its electrochemical detection, to the authors’ knowledge. In the meantime, such a telomeric DNA sequence tends to assume a tetraplex structure under ordinary electrochemical measurement conditions of a high concentration of KCl.9,10 Once an electrochemically active ligand showing preference for the tetraplex DNA is developed, the electrochemical detection on the electrode coupled with the extension of the TS primer on the electrode will become feasible. We have been studying the electrochemical DNA detection by using ferrocenylnaphthalene diimide (1, Figure 1A) coupled with a DNA probe-immobilized electrode.11,12 The electrochemical signal is based on the DNA duplex formation between target DNA and a DNA probe-immobilized electrode, deriving from the concentration of 1 by the threading-type intercalation into doublestranded DNA. In the meantime, many researchers have been studying several kinds of ligands that can bind to tetraplex DNA with intercalation-like binding.13-15 Since some of these ligands intercalate into DNA by the threading mode, it seemed possible for 1 to bind to tetraplex DNA.

* Corresponding author. Phone: +81-93-884 3322. Fax: +81-93-884 3322. E-mail: [email protected]. † Department of Biochemical Engineering and Science, Kyushu Institute of Technology. ‡ Department of Applied Chemistry, Kyushu University. § Department of Materials Science, Kyushu Institute of Technology. (1) Hiyama, E.; Hiyama, K. Cancer Lett. 2003, 194, 221-233. (2) Dhaene, K.; Van Marck, E.; Parwaresch, R. Virchows Arch. 2000, 437, 1-16. (3) Thomas, P.; Toth, C. A.; Saini, K. S.; Jessup, J. M.; Steele, G., Jr. Biochim. Biophys. Acta 1990, 1032, 177-189. (4) Saji, M.; Westra, W. H.; Chen, H.; Umbricht, C. B.; Tuttle, R. M.; Box, M. F.; Udelsman, R.; Sukumar, S.; Zeiger, M. A. Surgery 1997, 122, 11371140. (5) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L. C.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W.; Science 1994, 266, 2011-2015.

(6) Xiao, Y.; Pavlov, V.; Gill, R.; Bourenko, T.; Willner, I. ChemBioChem 2004, 5, 374-379. (7) Pavlov, V.; Xiao, Y.; Gill, R.; Dishon, A.; Kotler, M.; Willner, I. Anal. Chem. 2004, 76, 2152-2156. (8) Xiao, Y.; Pavlov, V.; Niazov, T.; Dishon, A.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2004, 126, 7430-7431. (9) Hardin, C. C.; Henderson, E.; Watson, T.; Prosser, J. K. Biochemistry 1991, 30, 4460-4472. (10) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 14286. (11) Takenaka, S.; Yamashita, K.; Takagi, M.; Uto, Y.; Kondo, H. Anal. Chem. 2000, 72, 1334-1341. (12) Sato, S.; Maeda, Y.; Nojima, T.; Kondo, H.; Takenaka, S. Nucleic Acids Res. Suppl. 2003, 3, 169-170. (13) Guo, Q.; Lu, M.; Marky, L. A.; Kallenbach, N. R. Biochemistry 1992, 31, 2451-2455. (14) Haq, I.; Trent, J. O.; Chowdhry, B. Z.; Jenkins, T. C. J. Am. Chem. Soc. 1999, 121, 1768-1779.

Spectroscopic studies revealed that ferrocenylnaphthalene diimide (1) can bind to tetraplex DNA at high potassium ion concentration. The tetraplex DNA was stabilized by the binding of 1, and this effect was larger than that of any other tetraplex stabilizers, which are known as a telomerase inhibitor. Quantitative analysis with circular dichroism and a quartz crystal microbalance strongly suggested a 3:1 binding stoichiometry of 1 to the tetraplex DNA. The telomere sequence could be extended by telomerase with the telomerase substrate primer on the surface of an electrode as proven by an increased current signal of 1 bound to the tetraplex DNA formed on the electrode. This is the first example of electrochemical detection of telomerase activity without relying on PCR.

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10.1021/ac0510235 CCC: $30.25

© 2005 American Chemical Society Published on Web 10/20/2005

Figure 1. (A) Structure of ferrocenylnaphthalene diimide (1). (B) Principle of the electrochemical assay of telomerase activity. Telomerase extension is allowed to proceed on a TS primer-immobilized electrode. The extended telomere DNA assumes a tetraplex structure under electrochemical conditions containing excess potassium ion and an electrochemical signal from 1 bound to this tetraplex DNA on the electrode is collected.

In this paper, we report that ferrocenylnaphthalene diimide (1) can bind to tetraplex DNA and can be applied to the electrochemical telomerase assay by using telomerase extracted from HeLa cancer cells. The principle of this system is depicted in Figure 1B. Where the sample contains telomerase, the TS primer immobilized on the electrode may be extended in proportion to the amount of the primer. Under conditions of electrochemical measurement containing a large amount of potassium ion, tetraplex DNA is formed on the electrode and 1 may be concentrated on the tetraplex DNA region. The activity is then estimated from a current response based on 1. EXPERIMENTAL SECTION Materials. Oligonucleotides were custom-synthesized by Genenet Co., Fukuoka, Japan. 5′-CAT GGT GGT TTG GGT TAG GGT TAG GGT TAG GGT TAC CAC-3′ was used as a model sequence of telomere DNA. The biotinyl oligonucleotide used for quartz crystal microbalance (QCM) experiments were as follows: B-TS, 5′-biotinyl AAT CCG TCG AGC AGA GTT; B-TS5, 5′-biotinyl AAT CCG TCG AGC AGA GTT AAG GTT AGG GTT AGG GTT AGG GTT AGG G. The thiolated oligonucleotides used for electrochemical experiments were HS-T8TS, 5′-HS-TTT TTT TTA ATC CGT CGA GCA GAG TTA GGG TTA GGG. Telomerase used was TeloChaser (Toyobo, Osaka, Japan), which is the supernatant of a lysis solution of HeLa cells (2.5 × 104 cells/µL), and its activity was confirmed by the conventional TRAP assay.5 Telomerase Inhibitors I and V, PIPER, and DODC were purchased from Calbiochem (San Diego, CA). 1 was synthesized according to the route described previously.16 Water was purified by a MilliQ system Gradient AIO coupled with Elixs3 kit (Millipore, Billerica, MA). RNase-free Gengard water was obtained by filtering MilliQ water with a Gengard Cartridge (Millipore). General Physicochemical Methods. Absorption spectra were recorded with a Hitachi 3300 spectrophotometer equipped with an SPR temperature controller. Circular dichroism (CD) spectra were recorded on a Jasco J820 spectropolarimeter under the following conditions: response, 2 s; sensitivity, 100 mdeg; speed, 20 nm min-1; resolution, 0.1 nm; bandwidth, 2.0 nm; integration time, 4 s; temperature, 25 °C. To obtain the melting (15) Perry, P. J.; Reszka, A. P.; Wood, A. A.; Read, M. A.; Gowan, S. M.; Dosanjh, H. S.; Trent, J. O.; Jenkins, T. C.; Kelland, L. R.; Neidle, S. J. Med. Chem. 1998, 41, 4873-4884. (16) Sato, S.; Nojima, T.; Waki, M.; Takenaka, S. Molecules 2005, 10, 693-707.

Figure 2. Two possible conformations of the telomere DNA used here: (I) hairpin and (II) tetraplex structures.

temperature (Tm), the CD spectral change of model telomere DNA (2.2 µM) was measured in the presence of various amounts of 1 in 10 mM morpholinoethanesulfonic acid (MES) buffer (pH 6.2) and 1 mM EDTA containing 50 mM KCl. QCM experiments were performed on AffinixQ (Initium Co., Tokyo, Japan) by using the gold surface (2.5 mm in diameter and 4.9 mm2 in area) of 27 MHz and an AT-cut QCM sensor chip (Initium Co.). A frequency change of 1.0 Hz is equivalent to absorption of 30 pg on the surface in aqueous solution.17 Equilibrium Binding Study. Spectrophotometric titration of 5 µM 1 with various amounts of telomere DNA was carried out in 0.1 M AcOK-AcOH buffer (pH 5.6) containing 0.1 M KCl, and an absorption change at 383 nm based on 1 was monitored. The binding affinity of 1 for the telomere DNA was determined from the data obtained by Scatchard analysis shown below:14 ν/L ) K(n - ν), where ν is the moles of 1 bound per base pair, L is the free concentration of 1, K is the observed binding constant, and n is the maximum number of 1 bound per telomere DNA. Electrochemical Measurements. Immobilization of DNA on the gold electrode was carried out by the following procedures. A gold electrode having 2.0 mm2 in area was polished with 6 and 1 µm of diamond slurry and 0.05 µm of an alumina slurry in this order and washed with MilliQ water. The electrode was soaked (17) Okahata, Y.; Niikura, K.; Sugiura, Y.; Sawada, M.; Morii, T. Biochemistry 1998, 37, 5666-5672.

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Figure 3. Absorption spectra of 5.0 µM 1 in the presence of 0, 2.0, or 4.0 µM telomere DNA at 25 °C in 10 mM MES buffer (pH 6.2) and 1 mM EDTA (A) or in the presence of 0, 0.2, 0.4, 0.7, 1.0, 1.4, 2.0, 2.7, 3.7, or 4.0 µM 1 from bottom to top of the traces at 383 nm in 0.1 mM AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl (B).

in boiling 2 M NaOH for 1 h and then washed with MilliQ water. This electrode was then soaked in concentrated nitric acid, washed with MilliQ water, and dried. One microliter of 0.5 M NaCl solution containing 1 pmol of a DNA probe was placed on the gold electrode, held upside down, and kept in a closed container under high humidity for 2 h at room temperature. After the electrode was washed with MilliQ water, 1 µL of 1 mM 6-mercaptohexanol was placed on the electrode for 1 h at 45 °C. The electrode was kept in MilliQ water for 30 min at room temperature. After electrochemical measurements of HS-T8TS immobilized on the electrode as a TS primer, the electrode was washed with Gengard water, 2 × SSC (0.03 M sodium citrate buffer containing 0.3 M NaCl), 70% ethanol, and Gengard water in this order and dried by blowing with a dry nitrogen gas. Two microliters of 20 mM Tris-HCl buffer (pH 8.5), 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1.0 mM EGTA, and 1 mM dNTP mix containing a proper amount of telomerase were dripped on the electrode and kept for 1 h at 20 °C to allow the extension reaction by telomerase to proceed on it. Electrochemical measurements were carried out with an ALS model 600 electrochemical analyzer (CH Instrument, Austin, TX). Differential pulse voltammogram (DPV) measurements were performed in 0.1 M AcOH-AcOK buffer (pH 5.6) containing 0.1 M KCl and 0.05 mM 1 at 25 °C with a three-electrode configuration consisting of an Ag/AgCl reference electrode, a Pt counter electrode, and a DNA-immobilized electrode as the working electrode. Electrochemical telomerase assay was also carried out with an electrochemical array (ECA) chip composed of 25 electrodes with a diameter of 1.0 mm (TUM-gene Inc., Chiba, Japan) in a way similar to those described above. One microliter of 0.5 M NaCl solution containing 0.3 pmol of a DNA probe was used in this case and DPV measured to obtain an i0 value. After extension reaction by telomerase, the ECA chip was washed with a PBST solution (0.05% Tween 20 and 1 × PBS (137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4) buffer) and MilliQ water three times in this order. Electrochemical measurement was conducted in 0.1 M AcOH-AcOK buffer (pH 5.6) containing 0.1 M KCl and 0.05 mM 1 on the ECA chip reader STR3000 (TUMgene Inc.) after immersing in the electrolyte for 15 min at 45 °C. Assay solutions contained the following amount of cancer cells 7306 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

Figure 4. Scatchard plot for 5.0 µM 1 with the telomere DNA in 0.1 mM AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl at 25 °C.

with telomerase activity: 15.6, 31.3, 46.9, 62.5, 81.3, 93.8, 112.5, or 125.0 cells/µL in 20 mM Tris-HCl buffer (pH 8.5), 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1.0 mM EGTA, and 0.2 mM dNTP mix. RESULT AND DISCUSSION Interaction of 1 with Tetraplex DNA. Telomeric DNA adopted in this study was a part of the human telomere DNA sequence carrying a GGG sequence four times, 5′-CAT GGT GGT TTG GGT TAG GGT TAG GGT TAG GGT TAC CAC-3′. The structure of this DNA fragment studied by Vialas et al.18 revealed a terminal duplex (I) or tetraplex structure (II) in the absence or presence of potassium ion, respectively, as depicted in Figure 2. We studied the interaction of 1 with this telomere DNA in the absence or presence of potassium ion. The absorption spectra of 1 in 10 mM MES buffer (pH 6.2) and 1 mM EDTA (no potassium ion, condition A in Figure 3A) showed large hypochromic and small red shifts upon addition of the telomere DNA. This behavior is reminiscent of a threading intercalation of 1 into doublestranded DNA19,20 deriving from the interaction of 1 with the partial duplex region of this DNA (Figure 2A). Likewise, the (18) Vialas, C.; Pratviel, G.; Meunier, B. Biochemistry 2000, 39, 9514-9522. (19) Yen, S. F.; Gabbay, E. J.; Wilson, W. D. Biochemistry 1982, 21, 20702076. (20) Tanious, F. A.; Yen, S.-F.; Wilson, W. D. Biochemistry 1991, 30, 18131819.

Figure 5. CD spectra of 1.46 µM telomere DNA in the presence of 0, 3.3, or 6.6 µM 1 at 25 °C in 10 mM MES buffer (pH 6.2) and 1 mM EDTA (A) or in the presence of 0, 1.7, 3.3, 5.0, 8.3, 14.8, or 27.5 µM 1 from bottom to top of the traces at 288 nm in 0.1 mM AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl (B).

absorption spectra of 1 in 0.1 M AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl (condition B in Figure 3B) also showed large hypochromic and small red shifts upon addition of telomere DNA. Since telomere DNA is expected to assume a tetraplex structure (Figure 2B) under the latter conditions, this result proved that 1 can bind to a tetraplex DNA structure. The large hypochromic shift observed for 1 in the presence of tetraplex DNA suggested a stacked structure between the naphthalene diimide chromophore of 1 and the nucleic bases of tetraplex DNA. Comparison of the spectra in the presence of 2.0 µM Telomere DNA in Figure 3A and B reveals that the hypochromic effect for the latter was larger than that for the former. Thus, the hypochromic effects of 5 µM 1 upon addition of 2 µM telomere DNA were 40 and 54% in the absence or presence of potassium ion, respectively. These results suggested that the telomere DNA can form a tetraplex structure under condition B and that 1 has higher binding affinity for tetraplex DNA than for double-stranded DNA. Scatchard analysis based on the absorption change upon addition of various amounts of this telomere DNA yielded a binding constant K ) 2.0 × 106 M-1 and site size n ) 3.2 in 0.1 M AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl at 25 °C (Figure 4). This binding constant was larger than that for calf thymus DNA (K ) 3.0 × 105 M-1)16 and is larger than those of cationic porphyrin (7.4 × 104 M-1)14 and anthraquinone derivatives (5 × 104-8 × 104 M-1).15 The n value of 3 is suggestive of a 3:1 binding stoichiometry of 1 to the telomere DNA and is interesting, contrasting the previous reports of a 1:1 binding of ligand to telomeric DNA.14,15 The binding constant for 1 with telomere DNA in the absence of potassium ion could not be obtained because of the appearance of precipitates during spectrophotometric titration of 1 with telomere DNA. Structural Change of Telomere DNA Induced by 1. The interaction of 1 with telomere DNA was studied also by CD spectroscopy under two different conditions: (A) 10 mM MES buffer (pH 6.2) and 1 mM EDTA (no potassium ion); (B) 0.1 M AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl (an excess of potassium ion). Under condition A, the positive and negative Cotton effects were observed around 240 and 280 nm as shown in Figure 5A, but the intensities were smaller than that with ordinary double-stranded DNA, presumably because the telomere DNA could not form a tetraplex structure as described in the

Figure 6. Change in molar ellipticity [θ] at 288 nm with the concentration of 1 under condition B in Figure 5.

previous paper.21 On the other hand, small negative and large positive Cotton effects were observed at 240 and 290 nm, respectively, under condition B (Figure 5B), and this behavior is in agreement with the tetraplex DNA structure previously described.21 Upon addition of 1 under condition A, no CD spectral change was observed. Taken together with the absorption titration experiments, it is concluded that telomere DNA assumes a partial duplex structure shown in Figure 2A under condition A and that 1 may bind to its double-stranded DNA region. Upon addition of 1 under condition B, the Cotton effect at 260 nm increased with an increase in the amount of 1. Since 1 is achiral (does not show any CD peak in this region), this change should have arisen from the interaction of 1 with telomere DNA. Figure 6 shows a CD intensity change at 288 nm for 1.46 µM telomere DNA with the concentration of 1 and the curve tended to level off at ∼5 µM 1, suggesting a 1:3 binding stoichiometry of the telomere DNA to 1. This conclusion is in agreement with the result obtained from the Scatchard analysis described above. Binding Stoichiometry of 1 to Telomere DNA. QCM measurements were carried out to ascertain the 3:1 binding stoichiometry of 1 to the telomere DNA. After preparation of a streptavidin-covered QCM sensor chip by the procedure described previously,17 biotinyl-telomere DNA and 1 were added in this order, and the amount of 1 bound to this DNA on this chip was (21) Li, W.; Wu, P.; Ohmichi, T.; Sugimoto, N. FEBS Lett. 2002, 526, 77-81.

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Table 1. Comparison of Melting Temperatures of the Telomere DNA in the Presence of Various Ligands or Ferrocenylnaphthalene Diimde 1a ligand

none

EtBr

DODC

inhibitor I

inhibitor V

PIPER

1 (1 equiv)

1 (3 equiv)

Tm/°C

42

44

43

56

57

60

56

72

a

The experiments were carried out in 10 mM MES buffer (pH 6.2) and 1 mM EDTA containing 50 mM KCl.

Figure 7. Time course of frequency change for a streptavidinimmobilized QCM chip upon addition of 10 µL of 5 µM B-TS (A) or B-TS5 (B) and 10 µL each of 0.5 mM 1 in this order in 0.1 M AcOHAcOK buffer (pH 5.6) and 0.1 M KCl.

estimated. This chip was then dipped in 8 µL of 0.1 M AcOHAcOK buffer (pH 5.6) and 0.1 M KCl, 10 µL of 5 µM B-TS being added to this solution, and the frequency change was monitored. The frequency change at the plateau was 152 Hz, equivalent to 0.77 pmol of telomere DNA formed on this chip (Figure 7A, a). No frequency decrease was observed upon serial addition of 1, showing that 1 dose not bind to B-TS having no tetraplex DNA structure (Figure 7A, b). By contrast, a frequency change of 322 Hz (0.65 pmol) was observed after addition of B-TS5 capable of forming a tetraplex DNA structure (Figure 7B, a). A frequency change of 62 Hz (2.3 pmol) was observed upon addition of 10 µL each of 0.5 mM 1 as shown in Figure 7B, b, showing that three molecules of 1 were bound to one tetraplex DNA on average. This conclusion was in agreement with the results in CD experiments. Stabilization Effect of 1 on Telomere DNA Tetraplex. The effect of 1 on the melting temperature Tm of tetraplex DNA was studied by monitoring CD spectral changes at 288 nm with temperature. The Tm curves for the telomere DNA in the presence of 1 or 3 equiv of 1 are shown in Figure 8. The Tm value of the telomere DNA was 42 °C (Figure 8a) and is in agreement with that previously reported.13 It is known that the tetraplex DNA binding ligands, such as DODC, inhibitors I and V, and PIPER, can stabilize the tetraplex structure.22,23 It was found that all of the ligands tested in this experiment could indeed stabilize the tetraplex structure as summarized in Table 1, whereas ethidium bromide (EtBr) of the classical intercalator exerted a small Tm (22) Chen, Q.; Kuntz, I. D.; Shafer, R. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2635-2639. (23) Fedoroff, O. Y.; Salazar, M.; Han, H.; Chemeris, V. V.; Kerwin, S. M.; Hurley, L. H. Biochemistry 1998, 37, 12367-12374.

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Figure 8. Temperature dependence of normalized molar ellipticity [θ] at 288 nm of 2.2 µM telomere DNA in the absence (a) or presence of 1 (b) or 3 equiv (c) of 1 in 10 mM MES buffer (pH 6.2) and 1 mM EDTA containing 50 mM KCl.

increase as reported previously.13 The Tm value increased by 14 °C upon addition of 1 equiv of 1 (Figure 8b) and further increased by 16 °C in the presence of 3 equiv of 1 (Figure 8c). These results also supported the notion that 1 can bind to tetraplex DNA at three sites and stabilized its structure dramatically. It is noted that of all the tetraplex DNA-binding ligands tested 1 exerted the greatest stabilization effect. Electrochemical Detection of Telomerase Activity. Ferrocenylnaphthalene diimide could bind to the tetraplex DNA structure and the electrochemical condition adopted here contained excess potassium ion, under which telomere DNA can easily assume a tetraplex structure.21 As shown schematically in Figure 1B, the TS primer carrying thymine 8-mer at the 5′-end was immobilized on the gold electrode. Introduction of the T8 sequence allowed telomerase accessible to the TS primer DNA. After washing of the TS primer-immobilized electrode with MilliQ water, DPV of this electrode was measured in 0.1 M AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl containing 0.05 mM 1. The electrode was washed with Gengard water, 2 × SSC, 70% ethanol, and Gengard water in this order, and the extension reaction by telomerase (125 cells/µL) was carried out at 20 °C for 1 h. After washing with Gengard water, the DPV was measured under the same conditions as those before the reaction (Figure 9). A current increase of 27% is a direct indication that the telomerase reaction occurred. This is the first example of direct detection of the telomerase extension on the electrode without PCR. To analyze telomerase activity quantitatively, similar electrochemical experiments were carried out by changing the cell numbers or the amount of telomerase. The TS primer-immobilized electrodes prepared with HS-T8TS gave rise to a current increase in proportion to the amount of the cell number or telomerase. The increased currents were represented by ∆i, defined by ∆i )

Figure 9. Background-corrected DPV response of a HS-T8TSimmobilized electrode before (a) and after treatment (b) with a telomerase solution (125 cells/µL) in 0.1 M AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl at 25 °C.

(i0 - i)/i0, where i0 and i refer to the peak current of 1 before and after telomerase reaction. This ∆i value represents the amount of the DNA extended by telomerase per TS primer immobilized on the electrode. As shown in Figure 10, a good correlation was observed between the ∆i value and cell numbers per microliter over the range of 40-140 cells/µL. CONCLUSION Ferrocenylnaphthalene diimide was found to bind to the tetraplex structure of telomere DNA more tightly than to the duplex, probably in a 3:1 stoichiometry of 1 to tetraplex DNA. This observation seems to be consistent with the previous report that three molecules of a tetraplex-binding ligand bound to the bottom of a quadruplex of G-tetrads.24 Furthermore, telomerase activity was assessed successfully with 1 and a TS primerimmobilized electrode without relying on PCR. This novel method (24) Clark, G. R.; Pytel, P. D.; Squire, C. J.; Neidle, S. J. Am. Chem. Soc. 2003, 125, 4066-4067.

Figure 10. Correlation of ∆i (%) with the cell number per micoliter (telomerase activity). DPV response of a HS-T8TS-immobilized electrode on the ECA chip before and after treatment of various amounts of a telomerase solution in 0.1 M AcOH-AcOK buffer (pH 5.6) and 0.1 M KCl at 25 °C.

will enable simple and quick detection of telomerase activity, thereby facilitating diagnosis of tumor formation. ACKNOWLEDGMENT We thank Prof. Kazuhiro Mizumoto (Department of Surgery I, Faculty of Medicine, Kyushu University, Fukuoka) for technical assistance and useful suggestions. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. S.S. is also grateful for the financial support from the Japan Society for the Promotion of Science. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 9, 2005. Accepted September 14, 2005. AC0510235

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