Electrochemical DNA Detection Based on the Polyhedral Boron

Publication Date (Web): December 15, 2008 ... E-mail, [email protected]; fax, +48-42-2723630; phone, +48-42-2723629 (Z.J.L.)., † ... Selective...
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Anal. Chem. 2009, 81, 840–844

Correspondence Electrochemical DNA Detection Based on the Polyhedral Boron Cluster Label Frantisek Jelen,*,† Agnieszka B. Olejniczak,‡ Alena Kourilova,† Zbigniew J. Lesnikowski,*,‡ and Emil Palecek† Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-612 65 Brno, Czech Republic, and Institute of Medical Biology, Polish Academy of Sciences, Laboratory of Molecular Virology & Biological Chemistry, 106 Lodowa Street, PL-93232 Lodz, Poland Polyhedral boron clusters are proposed as new, chemically and biologically stable, versatile redox labels for electrochemical DNA hybridization sensors. Selective and sensitive detection of the redox labeled DNA-probe was achieved by means of covalently attached electroactive marker 7,8-dicarba-nido-undekaborate group. A nanomolar concentration of boron cluster-labeled DNA was recognized. High specificity of the analysis with the boron cluster-labeled DNA probe, including detection of singlebase mismatch, was demonstrated. The above findings, together with proposed earlier use of metallacarboranes as an electrochemical label for biomolecules opens the door for a “multicolor” electrochemical coding of DNA with boron clusters and simultaneous detection of several DNA targets. Electrochemical detection of DNA hybridization is frequently used in biosensor development. Two types of biosensors have been developed.1 In one of them, both the DNA hybridization and the detection are performed at the electrode surface.2-4 In the other, DNA is hybridized at one surface (optimized for the hybridization, usually magnetic beads) and electrochemical detection is performed at the working electrode. Previously, we demonstrated that the latter method can be conveniently used in label-free DNA hybridization sensors.3,5,6 Here, we introduce a new polyhedral boron cluster-based DNA electroactive label for DNA hybridization sensors. DNA labeled with 7,8-dicarba-nidoundekaborate was used either as a target or a signaling probe, and formation of a DNA duplex was monitored using differential * To whom correspondence should be addressed. E-mail, [email protected]; fax, +420 541211293; phone, +420 541517213(F.J.). E-mail, [email protected]; fax, +48-42-2723630; phone, +48-42-2723629(Z.J.L.). † Academy of Sciences of the Czech Republic. ‡ Polish Academy of Sciences. (1) Palecek, E.; Jelen, F. Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics. In Perspectives in Bioanalysysis, Vol. 1; Palecek, E., Scheller, F., Wang, J., Eds.; Elsevier: New York, 2005; pp 74-173. (2) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 74A–83A. (3) Palecek, E.; Fojta, M.; Jelen, F. Bioelectrochemistry 2002, 56, 85–90. (4) Wang, J.; Xu, D. K.; Erdem, A.; Polsky, R.; Salazar, M. A. Talanta 2002, 56, 931–938. (5) Palecek, E.; Fojta, M. Talanta 2007, 74, 276–290. (6) Palecek, E.; Jelen, F. Crit. Rev. Anal. Chem. 2002, 32, 261–270.

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pulse voltammetry (DPV) and carbon paste electrode (CPE). A parallel label-free assay based on oxidation of guanine residues was performed. With the use of both approaches, detection of double-base mismatches was possible. For the reliable detection of a single-base mismatch, application of the labeled DNA was necessary. Many protocols have been described in which labels such as enzymes,4,7 labels in combination with nanoparticles,8-11 or covalently bound labels12,13 have been used in DNA hybridization sensors. The notable advantages of explicit electroactive labeling includes, among others, a positive detection signal, low background, and the ability to introduce several electrochemically distinguishable tags. Ferrocene is the most frequently used electroactive label in DNA hybridization systems.14-16 Another example from this group is osmium tetroxide, which is covalently bound to oligonucleotide (ODN) structures in the presence of some ligands.13,17-21 (7) Palecek, E.; Kizek, R.; Havran, L.; Billova, S.; Fojta, M. Anal. Chim. Acta 2002, 469, 73–83. (8) Cai, H.; Cao, X. N.; Jiang, Y.; He, P. G.; Fang, Y. Z. Anal. Bioanal. Chem. 2003, 375, 287–293. (9) Wang, J.; Liu, G. D.; Merkoci, A. Anal. Chim. Acta 2003, 482, 149–155. (10) Wang, J.; Liu, G. D.; Polsky, R.; Merkoci, A. Electrochem. Commun. 2002, 4, 722–726. (11) Wang, J.; Xu, D. K.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 4208–4209. (12) Palecek, E. In Methods Enzymol., Vol. 212; Abelson, J. N., Simon, M. I., Eds.; Academic Press: New York, 1992; pp 139-155. (13) Ihara, T.; Nakayama, M.; Murata, M.; Nakano, K.; Maeda, M. Chem. Commun. 1997, 17, 1609–1610. (14) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134–9137. (15) 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, 11155– 11161. (16) Lesnikowski, Z. J. Curr. Org. Chem. 2007, 11, 355–381. (17) Havran, L.; Fojta, M.; Palecek, E. Bioelectrochemistry 2004, 63, 239–243. (18) Jelen, F.; Karlovsky, P.; Makaturova, E.; Pecinka, P.; Palecek, E. Gen. Physiol. Biophys. 1991, 10, 461–473. (19) Palecek, E.; Fojta, M.; Jelen, F.; Vetterl, V. In Encyclopedia of Electrochemistry, Vol. 9, Bioelectrochemistry; Bard, A. J., Stratsman, J., Eds.; WileyVCH Verlag: Weiheim, Germany, 2002; pp 365-429. (20) Trefulka, M.; Ferreyra, N.; Ostatna, V.; Fojta, M.; Rivas, G.; Palecek, E. Electroanalysis 2007, 19, 1334–1338. (21) Trefulka, M.; Ostatna, V.; Havran, L.; Fojta, M.; Palecek, E. Electroanalysis 2007, 19, 1281–1287. 10.1021/ac801235b CCC: $40.75  2009 American Chemical Society Published on Web 12/15/2008

Table 1. Oligodeoxynucleotides Used for Strategies A and B Experimentsa

a (A)T, target ODN (oligomer containing dA25 segment complementary to T25 in the DBT and capture 18-mer part). RP-2, 18-mer reporter probe identical to the capture 18-mer part of the target sequence. RP-3, 18-mer complementary to the capture 18-mer part of the target ODN T, used as an unlabeled control. RP-4, 18-mer complementary to the capture 18-mer part of the target ODN T, containing the 7,8-dicarba-nido-undekaborane7-yl (CBM) label. RP-5, 18-mer forming 2 mismatches with the capture 18-mer part of the target ODN T, containing the CBM label. RP-6, 18-mer noncomplementary (scrambled) to the capture 18-mer part of the target ODN T, containing the CBM label. RP-7, 18-mer complementary to the capture 18-mer part of the target ODN T, containing the 3-cobalt bis(1,2-dicarbollide)-8-yl label. 2′-CBM, 2′-O-(7,8-dicarba-nido-undekaborane-10yl)methyl-group; BEMC, 3-cobalt bis(1,2-dicarbollide)-8-yl group. (B) CP1, 5′-biotynylated capture probe with a sequence complementary to the RP-5 used as the target ODN. CP2, 5′-biotynylated capture probe forming 1 mismatch with RP-5. CP3, 5′-biotynylated capture probe forming 2 mismatches with RP-5. Mismatches are highlighted.

The electrochemical activity of simple boron compounds, including tetrafluoroborate (-1) salts, which are widely used as supporting electrolytes, has been known for a long time.22 With the discovery of boron clusters in the 1960s, the electrochemical properties of caged boranes and carboranes, and their complexes with metals, metallaboranes, and metallacarboranes, have been the subject of extensive studies.23,24 Despite the vast knowledge about the electrochemistry of boron clusters that has accumulated during past decades, their potential as electrochemical labels for DNA has not, thus far, been explored due to the lack of methods for tethering the boron clusters to nucleic acids and their components. Recently, we developed several chemical approaches for the synthesis of boron clusters bearing DNA-oligomers containing a carborane cage within the internucleotide linkage,25 attached to the nucleobase,26 with the carborane cage linked to the sugar residue at the 2′ position,27 as well as with metallacarborane.28,29 Progress in the synthesis of boron cluster modified DNAoligomers was presented in recent reviews.16,30,31 (22) Bellavance, M. I.; Miller, B. In Encyclopedia of Electrochemistry of the Elements, Vol. II; Bard, A. J., Ed.; Dekker: New York, 1974; pp 1-19. (23) Morris, J. H.; Gysling, H. J.; Reed, D. Chem. Rev. 1985, 85, 51–76. (24) Geiger, W. E. In Metal Interactions with Boron Clusters; Grimes, R. N., Ed.; Plenum Press: New York, 1982; pp 239-268. (25) Lesnikowski, Z. J.; Schinazi, R. F. J. Org. Chem. 1993, 58, 6531–6534. (26) Fulcrand-el-Kattan, G.; Lesnikowski, Z. J.; Yao, S. J.; Tanious, F.; Wilson, W. D.; Schinazi, R. F. J. Am. Chem. Soc. 1994, 116, 7494–7501. (27) Olejniczak, A. B.; Corsini, M.; Fedi, S.; Zanello, P.; Lesnikowski, Z. J. Electrochem. Commun. 2007, 9, 1007–1011. (28) Olejniczak, A. B.; Mucha, P.; Gruner, B.; Lesnikowski, Z. J. Organometallics 2007, 26, 3272–3274. (29) Olejniczak, A. B.; Plesek, J.; Kriz, O.; Lesnikowski, Z. J. Angew. Chem., Int. Ed. 2003, 42, 5740–5743. (30) Lesnikowski, Z. J. Eur. J. Org. Chem. 2003, 4489–4500.

The availability of methods for the attachment of boron clusters to DNA-oligomers opened the way to study the applicability of the carborane label for electrochemical detection of DNA. Preliminary observation was performed under HPLC conditions using a standard electrochemical detector Coulochem II (Esa, Inc.) equipped with an coulometric analytical cell model 5010 (Esa, Inc.). This observation has shown that the ODN probe, complementary to a fragment of the US14 gene of human cytomegalovirus (HCMV) and labeled with the 7,8-dicarba-nido-undekaborate (CBM) group, provided an intense diagnostic peak at potential 0.9 V at the porous graphite electrode (vs Pd).32 The findings above prompted us to study the 7,8-dicarba-nido-undekaborate cluster as a redox label in more detail. In this work, we utilized differential pulse or square-wave voltammetry in combination with an adsorptive transfer stripping technique for measuring ODN hybridization. The whole procedure involves ODN hybridization magnetic beads with subsequent detection of the CBM label and the oxidation guanine signal at a CPE. EXPERIMENTAL SECTION Materials. 2′-O-(7,8-Dicarba-nido-undekaborane-7-yl)methyluridine (1) was prepared from 2′-O-(1,2-dicarba-closo-dekaborane1-yl)methyluridine33 as previously described.34 Modified ODNs containing the 7,8-dicarba-nido-undekaborane-7-yl label, RP-4, RP(31) Lesnikowski, Z. J.; Shi, J. X.; Schinazi, R. F. J. Organomet. Chem. 1999, 581, 156–169. (32) Olejniczak, A. B. Ph.D. Thesis, 2002, University of Lodz, Poland. (33) Tjarks, W.; Anisuzzaman, A. K. M.; Liu, L.; Soloway, A. H.; Barth, R. F.; Perkins, D. J.; Adams, D. M. J. Med. Chem. 1992, 35, 1628–1633. (34) Olejniczak, A. B.; Koziolkiewicz, M.; Lesnikowski, Z. J. Antisense Nucl. Acid Drug Develop. 2002, 12, 79–94.

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Scheme 1. 2′-O-(7,8-Dicarba-nido-undekaborane-7-yl)methyluridine (1) and the 18-mer Complementary to the Capture 18-mer Part of the Target ODN T, Which Contains Two 7,8-Dicarba-nido-undekaborane (CBM) Labels (RP-4)

5, and RP-6, were synthesized according to the described procedure.34 ODN containing the 3-cobalt bis(1,2-dicarbollide)-8yl label, RP-7, was synthesized as described.29 Unmodified ODNs, T, RP-2, and RP-3, and biotinylated ODNs, CP1, CP2, and CP3, were purchased from Thermo Fisher Scientific, Inc. (Ulm, Germany). ODNs used in this study are shown in Table 1. The ODN concentration was determined spectrophotometrically using an HP 8452 spectrophotometer (Hewlett-Packard). Dynabeads Oligo(dT)25 (DBT), Dynabeads M-280 Streptavidin (DB-STV), and magnetic particle concentrator MPC-S were products of Invitrogen Dynal AS, (Oslo, Norway). Other chemicals were from Sigma Chemical Co. (St. Louis, MO). Dynabeads Biomagnetic Separation Procedure. ODN samples for voltammetric measurements were prepared as described previously.35 Briefly, 60 µL of a known concentration of target ODN T (20 µM) containing a (dA)25 adenine track were added to the DBT. The resulting mixture was placed on a shaker for 30 min at room temperature to allow hybridization between the probe and the target chains [(dA)25 and (dT)25] on the bead surface. The magnetic separation of DBT was followed by repeated DBT washing. The second hybridization step with the RP containing 1 labeled with CBM was performed as in the previous step. Elution of the RP signaling probe and target ODNs was accomplished by heating the DBT in 20 µL of buffer 2 at 85 °C for 2 min.

Figure 1. Adsorptive transfer stripping voltammograms of 1 µM ODNs on CPE. 3, RP-3, unlabeled 18-mer signaling probe complementary to the capture 18-mer part of the target ODN T. 4, RP-4, 18-mer reporter probe complementary to the capture 18-mer part of the target ODN T, which contains the 7,8-dicarba-nido-undekaborane7-yl (CBM) label. 7, RP-7, 18-mer signaling probe complementary to the capture 18-mer part of the target ODN T, which contains the 3-cobalt bis(1,2-dicarbollide)-8-yl label (BEMC). Broken line: background electrolyte, 0.1 M potassium sulfate, pH 7.1. DPV; see Experimental Section for details. 842

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Voltammetric Measurements. Differential pulse voltammetry (DPV), square-wave voltammetry (SWV), and cyclic voltammetry (CV) were performed with an AUTOLAB analyzer (EcoChemie, The Netherlands). The standard cell with three electrodes was used. The working electrode was a CPE with a surface area of 1 cm2, the reference electrode was a Ag/AgCl/3 M KCl electrode, and the auxiliary electrode was platinum wire. Labeled ODNs were measured using the adsorptive transfer technique.1,36,37 DNA-modified CPE was washed and transferred into the blank background electrolyte. All measurements were carried out under argon at room temperature using the following parameters: DPV, starting potential (Ei) 0.18 V; accumulation time (tA) 3 min, scan rate 50 mV/s, step potential 5 mV, amplitude 50 mV; SWV, Ei 0.2 V; tA 3 min, frequency 380 Hz, modulation amplitude 25 mV; CV,

Figure 2. Schematic presentation of hybridization at the paramagnetic beads DBT (A) and DB-STV (B). After DBT activation, two-step (Strategy A) or one-step (Strategy B) hybridization was performed. See text for details. Application of DBT was described previously.1,3,5,6

Figure 3. Hybridization at paramagnetic beads (DBT). A volume of 60 µL of the DBT were activated and hybridized with target ODN T. In the second step, additional hybridization was performed with the unlabeled 18-mer RP-3, 18-mer sequence containing 7,8-dicarbanido-undekaborane-7-yl (CBM) label RP-4, 18-mer sequence forming two mismatches and containing the CBM label RP-5, or noncomplementary ODN containing the CBM label RP-6. (A) Differential pulse (DP) voltammograms with complementary (RP-4) and noncomplementary 18-mers (RP-6). (B and C) Peak height evaluation (in column graphs) for the DP peak of the CBM label (Ep ) 0.56 V) (B) and the guanine peak (Ep ) 0.94 V) (C). Results were recalculated to percent, peak heights in the RP-4 curves were taken to be 100%, and the background electrolyte was 0.2 M phosphate buffer, pH 6.4. For details, see Figure 2.

scan rate 0.5 V/s; step potential 5 mV. The background electrolytes were 0.2 M sodium phosphate, pH 6.4, or 0.1 M potassium sulfate, pH 7. RESULTS AND DISCUSSION Voltammetric Behavior of the Carborane Label. The 7,8dicarba-nido-undekaborate group (CBM) (C2B9H12)- used as a redox label belongs to the vast family of boron clusters. It is an open (nido-) form of an electronically neutral 1,2-dicarbacloso-undecaborane, containing 2 carbon, 9 boron, and 12 hydrogen atoms and bearing 1 negative charge (Scheme 1). Despite extensive studies of the redox properties of boron clusters and the potent electrochemical activity of 7,8-dicarba-nidoundekaborate, studies on the electrochemistry of these substances are extremely rare.38-40 Electrochemical behavior of the unsubstituted 7,8-dicarba-nido-undekaborate monoanion displayed a welldeveloped oxidation peak at CPE (see Supporting Information for details). Voltammetric Behavior of ODNs Containing the Carborane Label. We studied the voltammetric behavior of five ODNs: RP-2, RP-3, RP-4, RP-5, and RP-7. They all contained guanine (35) Jelen, F.; Yosypchuk, B.; Kourilova, A.; Novotny, L.; Palecek, E. Anal. Chem. 2002, 74, 4788–4793. (36) Palecek, E.; Postbieglova, I. J. Electroanal. Chem. 1986, 214, 359–371. (37) Palecek, E. Bioelectrochem. Bioenerg. 1992, 28, 71–83. (38) Yarosh, M. V.; Baranova, T. V.; Shiroki, V. L.; Erdman, A. A.; Maier, N. A. Russ. Electrochem. 1993, 29, 1125–1127. (39) Texidor, F.; Pedrajas, J.; Vinas, C. Inorg. Chem. 1995, 34, 1726–1729. (40) Fabre, B.; Clark, J. C.; Vicente, M. G. H. Macromolecules 2006, 39, 112– 119.

Figure 4. Hybridization at paramagnetic beads (DB-STV, Strategy B). A volume of 10 µL of DB-STV was activated to attach different biotinylated-ODN capture probes (CP, see Table 1). CP1 was an 18-mer ODN fully complementary to the labeled RP-5, CP2 was an 18-mer ODN capable of forming a single-mismatch with RP-5, and CP3 was an 18-mer ODN capable of forming a two-base mismatch with RP-5. The specific CP was hybridized using a 18-mer sequence containing 1 labeled with 7,8-dicarba-nido-undekaborane-7-yl (CBM). (A) Differential pulse (DP) voltammograms with complementary 1-MM and 2-MM hybrids. (B) DP voltammograms after baseline correction. (C) Peak height evaluation (in column graphs) for the DP peak of the same samples. Results are recalculated to a percent, peak heights from CP1 (complementary) curves were taken to be 100%, and the background electrolyte was 0.2 M phosphate buffer, pH 6.4. For details, see the Supporting Information.

residues and produced a guanine oxidation peak at about 0.95 V; peak current increased with the number of guanines in the ODN chain. From these ODNs, all oligomers, with the exception of RP-2 and RP-3, contained the CBM label in the form of 1 (Scheme 1 and Table 1) and gave a voltammetric signal at about 0.55 V, corresponding to oxidation of the CBM in 1. This peak was observable at a wide range of pHs (from pH 3 to 9.2) (not shown). ODN RP-7, which contained the 3-cobalt bis(1,2-dicarbollide)-8yl (BEMC) label, did not give any voltammetric signal connected to oxidation of the BEMC label (Figure 1) under the given conditions. The measurements above were performed in 0.1 M potassium sulfate, pH 7.1; similar results were obtained in other electrolytes, such as 0.2 M sodium phosphate, pH 6.4. Therefore, Analytical Chemistry, Vol. 81, No. 2, January 15, 2009

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it may be of interest that for thymidine monomer bearing the BEMC label, an intense peak was detected at 1.7 V with the working Pt electrode (vs the Pd reference electrode) due to redox activity of the metallacarborane (Co(III)/Co(IV).24 The CV of the nucleoside-metallcarborane conjugate with acetonitrile as the solvent, 0.1 M [NBu4]ClO4 as the supporting electrolyte, and platinum as the working electrode provided a redox signal around -1.0 V.29 At a glassy carbon electrode, as well as at a gold one, nucleoside monomers bearing the BEMC label exhibited reduction processes in the range of -1.0 to -2.2 V(vs Ag/AgCl) in the CV measurements in DMF solutions.27 DNA Hybridization Using the Double Surface Technique. In our experiments we used two hybridization strategies, A and B. In Strategy A, commercial DB containing ODN with covalently attached oligo (T)25 (DBT, surface immobilized capture probe) was used for ODN hybridization. The method involved two steps: (a) capturing of a 43-mer target ODN containing a track of 25 adenines (ODN T) with DBT, (b) hybridization of ODN T with the signaling probes, (RPs), which were labeled with CBM containing nucleoside (1). Briefly, in the first step in which we used target ODN T, we established convenient conditions for the second hybridization. In the second step, we hybridized perfectly matched complementary ODN possessing the CBM label RP-4 or ODN containing two mismatches, RP-5 (Table 1), with the target ODN T captured on DBT. Unlabeled ODN RP-3 and scrambled ODN RP-6 were used as controls. In all cases, ODNs were eluted and separated from DB and analyzed by electrochemical detection using DPV at CPE. Results are summarized in Figure 3. We used 18-mer CBM-labeled RPs, which were either fully complementary to the target (RP-4), capable of forming a twobase mismatch duplex with the 43-mer target ODN T (RP-5) or scrambled sequence (RP-6) (Table 1A). Hybridized ODNs were eluted and separated from DBT and analyzed by electrochemical detection using DPV at CPE. In the Strategy B, different unlabeled biotinylated ODNs were attached to streptavidin-coated magnetic beads (DB-STV) as capture probes (Figure 2B). These DB-STVODNs interacted with the labeled target ODN RP-5 (Table 1A). The remainder of the procedure was continued in the same way as Strategy A. As shown by the results of Strategy A (Figure 2), the hybrid formed by ODN T with RP-4 carrying the labeled nucleoside (1) gave the highest oxidation peak at 0.56 V. A lower peak was produced by the two-base mismatch containing hybrid formed with RP-5. The noncomplementary RP-6 produced a very small peak which probably resulted from a capacity current (Figure 3). To resolve a single-base mismatch, the hybridization was performed at an elevated temperature using Strategy B. After testing several temperatures (up to the DNA melting temperature), we found that 55 °C was the most suitable under the given conditions. Figure 4 shows good discrimination between the single-base mismatch and the two-base mismatch using the signals of the CBM-labeled strand (Scheme 1). Label-free determination based on the G peak (due to oxidation of guanine) recognized the two-base mismatch but not the single-base

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mismatch. This could be due to the release of a small portion of the probe DNA from the beads at an elevated temperature, which increased the background and prevented recognition of small differences in the G signal intensity. On the other hand, the presence of a small amount of the unlabeled probe ODN could not influence the intensity of the peak of the CMB label. These results show not only high electrochemical activity and efficacy of the 7,8-dicarba-nido-undekaborate cluster as a redox label, assuring high sensitivity of the detection of the hybridization event, but also high affinity toward complementary sequence and selectivity that allowed for detection of single base mutations.30 CONCLUSION The application of electrochemical techniques to DNA hybridization is preferable, due to the more rapid and simple detection of hybridization events when compared to conventional optical studies. Here, we presented an electrochemical DNA sensor for the detection of labeled ODN, after hybridization by means of the novel and versatile electroactive redox-marker, the 7,8-dicarbanido-undekaborate group, covalently attached to the singlestranded ODN. Nanomolar concentrations of CBM-labeled target ODN were recognized at carbon paste electrodes. The results above, together with the earlier proposed use of metallacarboranes as an electrochemical label for biomolecules,27 opens the door for a “multicolor” electrochemical coding of DNA with boron clusters and simultaneous detection of several DNA targets. The only recently published synthesis and electrochemical study of dodecaborane derivatives (“closomers”)41 expands the potential of boron clusters as entirely new, flexible, and adjustable electrochemical labels for biomolecules. ACKNOWLEDGMENT This manuscript is dedicated to Professor M. Frederick Hawthorne on the occasion of his 80th birthday in recognition of his outstanding contributions to the areas of carborane and metallacarborane chemistry. This work was supported in part by the Grant Agency of Academy of Sciences of the Czech Republic (Grant Numbers A100040602 and A400040804 to F.J.), by the Grant Agency of the Czech Republic Grant 301/07/0490 to E.P., by the Polish Ministry of Science and Higher Education, Grant No. 1 T09A 121 30 to A.O. and Z.J.L., institutional Grants AV0Z50040702 and AVOZ50040507 from the Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, and the institutional grant from the Institute of Medical Biology Polish Academy of Sciences. 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 17, 2008. Accepted November 29, 2008. AC801235B (41) Lee, M. W.; Farha, O. K.; Hawthorne, M. F.; Hansch, C. H. Angew. Chem., Int. Ed. 2007, 46, 3018–3022.