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Probing of DNA and Single-Base Mismatches by Chemical Force Microscopy Using Peptide Nucleic Acid-Modified Sensing Tips and Functionalized Surfaces Oleg Lioubashevski, Fernando Patolsky, and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received April 6, 2001 DNA hybridization to peptide nucleic acid (PNA)-functionalized Au-coated tips is characterized by probing the force interactions between the PNA-functionalized tip or the DNA/PNA-functionalized tip with a hydrophobic 1-mercaptoundecane-modified Au surface, using chemical force microscopy (CFM). DNA concentrations as low as 3 × 10-13 M can be detected by probing the respective force interactions. The CFM method is sufficiently sensitive to detect a single base mismatch in the analyte DNA.
Chemical force microscopy (CFM) is a rapidly developing technique for probing affinity and recognition properties at the molecular level.1 The method is based on the detection of complementary molecular interactions between a functionalized tip and a modified surface.2 This method was applied to sense intermolecular hydrogen bonds,3 electrostatic interactions,4 ligand-receptor complexes,5 host-guest complexes,6 antigen-antibody complexes,7 and nucleic-acid hybridization processes.8 Recently, CFM was applied to characterize in situ chemical transformations.9 Peptide nucleic acids (PNAs) are DNA analogues, consisting of a peptide chain and the respective tethered purine/pyrimidine bases.10 PNAs lack the sugar/ phosphate components and, thus, in contrast to DNA, are uncharged and exhibit hydrophobic character. The PNA reveals, however, high affinity interactions to form the double-stranded assembly with the complementary nucleic * To whom correspondence should be addressed. Tel.: 972-26585272. Fax: 972-2-6527715. E-mail:
[email protected]. (1) Janshoff, A.; Neitzert, M.; Oberdo¨rfer, Y.; Fuchs, H. Angew. Chem., Int. Ed. 2000, 39, 3213-3237. (2) Noy, A.; Vezenov, D. V.; Lieber, C. M. Annu. Rev. Mater. Sci. 1997, 27, 381-421. (3) (a) Boland, T.; Ratner, B. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5297-5301. (b) McKendry, R.; Theoclitou, M. E.; Rayment, T.; Abell, C. Nature 1998, 391, 566-568. (4) (a) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353, 239-241. (b) Marti, A.; Ha¨hner, G.; Spencer, N. D. Langmuir 1995, 11, 4632-4635. (5) (a) Florin, E. L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415417. (b) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354-357. (6) Scho¨nherr, H.; Bealen, M. W. J.; Bu¨gler, J.; Huskens, J.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Vancso, G. J. J. Am. Chem. Soc. 2000, 122, 4963-4967. (7) (a) Ros, R.; Schwesinger, F.; Anselmetti, D.; Kubon, M.; Scha¨fer, R.; Plu¨ckthun, A.; Tiefenauer, L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7402-7405. (b) Willemsen, O. H.; Snel, M. M. E.; van der Werf, K. O.; de Grooth, B. G.; Greve, J.; Hinterdorfer, P.; Gruber, H. J.; Schindler, H.; van Kooyl, Y.; Figdor, C. G. Biophys. J. 1998, 75, 2220-2228. (c) Allen, S.; Chen, X. Y.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Edwards, J. C.; Roberts, C. J.; Sefton, J.; Tendler, S. J. B.; Williams, P. M. Biochemistry 1997, 36, 7457-7463. (8) (a) Lee, G. U.; Chrisey, L. A.; Colton, J. R. Science 1994, 266, 771-773. (b) Noy, A.; Vezenov, D. V.; Kayyem, J. F.; Meade, T. J.; Lieber, C. M. Chem. Biol. 1997, 4, 519-527. (c) Rief, M.; ClausenSchaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6, 346-349. (9) (a) Werts, M. P. L.; van der Vegte, E. W.; Hadziioannou, G. Langmuir 1997, 13, 4939-4942. (b) Green, J.-B. D.; McDermott, M. T.; Porter, M. D. J. Phys. Chem. 1996, 100, 13342-13345. (10) (a) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895-1897. Egholm, M.; Buchardt, O.; Christensen, L.; Bejrens, C.; Freier, S. M.; Driver, D.; Berg, R. H.; Kim, S. K.; Norde´n, B.; Nielsen, P. E. Nature 1993, 365, 566-568. (b) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. 1998, 37, 2796-2823.
acid, and the specific PNA/DNA interactions were employed to develop electrochemical DNA sensors.11 In a series of recent studies, our laboratory has reported on the development of DNA sensors by the assembly of monolayers of nucleic acids on conductive supports12 or piezoelectric crystals.13 Here we wish to report on the novel application of CFM to detect the hybridization event between PNA and DNA. We are able to detect single-base mismatches in the analyzed DNA. An Au-coated AFM tip was functionalized with the cysteine-modified PNA, (1).14,15 An Au-coated glass substrate (Berliner Glass, A45) was modified with 1-mercaptoundecane, 1 × 10-3 M, in ethanol for 12 h. Figure 1a shows a typical force-distance curve resulting from the interaction of the 1-functionalized tip with the modified surface. The retracting tip pulled off the surface with a mean adhesion force that corresponds to F ) 4.35 nN. The adhesion force is attributed to hydrophobic interactions between the 1-functionalized tip and the undecanethiolmodified surface. Using the JKR theory,16 we estimate the contact area between the tip (with radius of curvature after gold deposition of ca. 60 nm) and the surface to be ca. 2.1 × 10-13 cm2. Using independent quartz-crystal(11) Wang, J.; Nielsen, P. E.; Jiang, M.; Cai, X.; Fernandes, J. R.; Grant, D. H.; Ozsoz, M.; Beglieter, A.; Mowat, M. Anal. Chem. 1997, 69, 5200-5202. (12) (a) Patolsky, F.; Katz, E.; Bardea, A.; Willner, I. Langmuir 1999, 15, 5, 3703-3706. (b) Patolsky, F.; Lichtenstein, A.; Willner, I. Angew. Chem., Int. Ed. 2000, 39, 940-943. (c) Alfonta, L.; Singh, A. K.; Willner, I. Anal. Chem. 2001, 73, 91-102. (13) (a) Bardea, A.; Dagan, A.; Ben-Dov, I.; Amit, B.; Willner, I. Chem. Commun. 1998, 839-840. (b) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2000, 122, 418-419. (c) Patolsky, F.; Ranjit, K. T.; Lichtenstein, A.; Willner, I. Chem. Commun. 2000, 1025-1026. (14) AFM force measurements were performed on Topometrix Explorer instrument in a liquid cell filled with 5 mM Tris-HCl buffer solution, pH ) 7.4. Topometrix V-shaped premounted tips (200 µm length) were used. Tips were coated by sublayer of chromium (2 nm) followed by a gold layer (50 nm). The mean value of the spring constant of the Au-coated cantilevers corresponds to 0.04 N/m. Loading forces of 0.5-1 nN were applied, and the retraction rates used were in the range of 100-500 nm‚s-1. (15) The Au-coated tips were functionalized with 1, 24 µg‚mL, in 5 mM Tris-HCl buffer solution, pH ) 7.4, for 12 h at room temperature. Cleaned Au-coated glass slides were modified with an ethanol solution of 1-mercaptoundecane, 1 mM, for 12 h. Hybridization was performed by the immersion of the 1-modified tip in 300 µL of 5 mM Tris-HCl buffer solution, pH ) 7.4, which included the respective concentration of 2, for a period of 1 h. Buffer solution containing DNA was placed on a glass slide. The 1-mercaptoundecane-functionalized Au-coated glass support was not interacted with the analyzed DNA sample in order to avoid nonspecific adsorption of the DNA (or any residual proteins) to the monolayer interface. (16) Capella, B.; Dietler, G. Surf. Sci. Rep. 1999, 34, 1-104.
10.1021/la0105174 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/02/2001
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Figure 1. Adhesion forces generated between the PNA-modified tip or the PNA/DNA-functionalized tip and an undecanethiolmodified Au surface. (Spring-constant of the Au-coated cantilever is 0.04 N/m).
microbalance measurements on Au-quartz crystals, we estimate the surface coverage of 1 on an Au surface to be 6 × 10-10 mol‚cm-2 and, accordingly, the number of PNA molecules interacting with the alkanethiol-modified surface is ca. 100. Treatment of the 1-functionalized tip with the complementary nucleic acid (2),14 5 × 10-13 M, yields the doublestranded PNA/DNA assembly on the tip. Figure 1b shows a typical force-distance curve resulting from the formation of the hybridized PNA/DNA-functionalized tip and the modified surface. All force-distance curves were measured in Tris-HCl buffer solution, 5 mM, pH ) 7.4 in order to retain the helical structure of the double-stranded PNA/ DNA. A substantial decrease in the resulting adhesion force, F ) 1.13 nN, is observed after hybridization. The decrease in the adhesion force is attributed to the hydrophilic nature of the double-stranded assembly formed between 1 and the DNA (2). That is, the negatively charged phosphorylated DNA turns the double-stranded assembly to exhibit hydrophilic properties. Further support that the decrease in the adhesion force between the DNA/PNA-functionalized tip and the modified surface originates from hydrophilic interaction is obtained by a control experiment, which examined PNA/PNA binding interactions on the tip. The PNA (3), complementary to the PNA assembled on the tip, was interacted with the modified tip. The resulting double-stranded PNA/ PNA-modified tip yields an adhesion force with the functionalized surface that is ca. 1.8-fold higher than the adhesion force of the PNA-functionalized tip. Thus, the enhanced hydrophobicity of the PNA/PNA-functionalized tip leads to the increase in the adhesion force. In a control experiment the PNA-functionalized tip was interacted with a polymerase solution (Klenow fragment, 5 µ‚mL-1, and the adhesive force interactions between the resulting tip and the alkanethiol-functionalized monolayer were analyzed. No differences in the adhesive forces of the PNAmodified tip with the hydrophobic interface were detected before and after treatment with polymerase solution, implying that no nonspecific adsorption of the enzyme on the tip occurred. This control experiment is specifically important as residual concentrations of polymerase may be present in future “real” samples of analyzed DNA. Figure 2A shows the adhesion force histograms corresponding to the PNA-functionalized tip (a) and the PNA/
Figure 2. (A) Adhesion-force histograms corresponding to (a) the PNA-modified tip, (b) the PNA/DNA-modified tip obtained after hybridization of the PNA-modified tip with 2, 5 × 10-13 M; (c) after treatment of the PNA-modified tip with the mutant 2a, 5 × 10-10 M. Each of the histograms was derived from 100 to 200 force measurements of adhesion events measured at different spots. The presented histograms represent data acquired with a single tip. (B) Concentration dependence of the adhesion forces between the PNA/DNA-functionalized tip and the alkanethiol-modified surface observed upon the hybridization of the PNA-functionalized tip with different concentrations of 2. Adhesion forces are expressed as the ratio FPNA/DNA/FPNA (for the same tip) to normalize the results for different tips. Experimental points at the same concentrations of 2 correspond to results of two different tips.
DNA-functionalized tip (b). Treatment of the PNA/DNA assembly with 4 M urea, followed by rinsing the tip, leads to the separation of the double-stranded complex, and the force-distance curve characteristic to the 1-functionalized tip, F ) 4.1 nN, is restored. The urea treatment regenerates
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the functionalized tip for the sensing of (2). Interaction of the 1-functionalized tip with a foreign DNA (2b), at a concentration of 5 × 10-7 M, yields an adhesion force almost identical to the nonhybridized PNA-modified tip, indicating that no nonspecific binding of DNA on the tip takes place. Interestingly, treatment of the 1-functionalized tip with the mutant (2a), which includes a single base mismatch as compared to (2), at a concentration of 5 × 10-10 M, higher than the concentration used for the hybridization of (2), leads to an adhesion force that is identical to the nonhybridized PNA-functionalized tip, F ) 4.04 nN (Figure 2A, histogram c). Thus, the PNAfunctionalized tip reveals specificity and does not hybridize with the mutant (2a). The CFM method is sufficiently sensitive to detect the single-base mismatch. It should be noted that the 1-modified tip that is inactive in the detection of 2a is, however, active in the sensing of 2, 5 × 10-13 M, in a secondary cycle, indicating that the PNAmodified tip retained its activity. The decrease in the adhesion forces between the PNA/ DNA-modified tip and the hydrophobic surface is controlled by the hydrophilicity of the double-stranded assembly or the content of hybridized DNA. The content of hybridized DNA is, however, controlled by the concentration of DNA in the analyzed samples. Figure 2B shows the adhesion forces between the PNA/DNA-modified tip and the alkanethiol-functionalized surface obtained upon
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the hybridization of the PNA-functionalized tip with different concentrations of 2. At concentrations of 2 higher than 3 × 10-13 M, a constant FPNA/DNA/FPNA value of ca. 0.25 is observed, indicating that the tip is saturated with 2. At concentrations of 2 lower than 1 × 10-16 M, the adhesion force characteristic to the nonhybridized PNAmodified tip is observed, with FPNA/DNA/FPNA ≈ 1. The force ratio FPNA/DNA/FPNA recorded upon interacting of the tip with different bulk concentrations of DNA differs for a series of tips by (8%. It should be noted that we analyze the hybridization of DNA to the PNA-functionalized tip in the form of a force ratio, FPNA/DNA/FPNA, and thus variations in the spring constants, tip diameter, and differences in the Au-coating are screened for different tips. We thus conclude that the PNA-modified tip acts as a nanotool for probing PNA/DNA interactions with singlebase mismatch diagnostic abilities. The present study reveals a general and novel methodology to probe specific molecular affinity interaction by measuring nonspecific force interactions between the tip and a chemically modified support. Acknowledgment. This research is supported by the Israel-Japan cooperation Program, The Israel Ministry of Science. LA0105174
