Characterization of PNA and DNA Immobilization and Subsequent

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Characterization of PNA and DNA Immobilization and Subsequent Hybridization with DNA Using Acoustic-Shear-Wave Attenuation Measurements F. Hoö¨k,*,†,‡ A. Ray,§ B. Nordeń,§ and B. Kasemo† Departments of Applied Physics and Physical Chemistry, Chalmers University of Technology and Go¨ teborg University, S-41296 Go¨ teborg, Sweden, and Department of Cell and Molecular Biology, Go¨ teborg University, S-41390 Go¨ teborg, Sweden Received May 24, 2001. In Final Form: August 15, 2001

We report here how the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, simultaneously measuring changes in the induced energy dissipation, D (cf. viscoelastic properties), and the frequency, f (cf. coupled mass), can be used to characterize the bound state of single-stranded peptide nucleic acid (PNA) and deoxyribose nucleic acid (DNA) in relation to their ability to function as selective probe(s) for fully complementary and single-mismatch DNA. The possibility to use the QCM-D technique for detection of binding kinetics and structural differences in the formed duplexes is also presented. We found that thiol-PNA and thiol-DNA attached via a sulfur group directly on a bare-gold surface are less efficient as probes for DNA than are biotin-PNA and biotin-DNA, coupled on top of a two-dimensional (2-D) arrangement of streptavidin, formed on a biotinylated phospholipid bilayer on a SiO2 surface. The fully complementary and singly mismatched DNA oligomers hybridize with the immobilized PNA and DNA. A single mismatch is discriminated via a significant difference in the binding and dissociation kinetics, demonstrating a high selectivity and thus successful immobilization of functional single strands. The observed ratios between hybridization-induced energy dissipation (∆D) and the frequency shift (∆f) made it possible to discriminate thiol-PNA directly attached to a gold surface from biotin-PNA coupled to the streptavidin 2-D arrangement, where the former were shown to be inefficient for detecting subsequent hybridization. Structural differences of the immobilized layers composed of biotin-PNA-DNA and biotinDNA-DNA were clearly reflected by the ∆D and ∆f response.

Introduction Surface-based biosensors for DNA hybridization biosensors are important in DNA sequencing, gene mapping, clinical diagnosis of inherited diseases, and the rapid detection of infectious microorganisms.1-4 In recent years, much effort has been devoted to improvements in the selectivity and sensitivity of such devices and miniaturization to allow detection in small sample volumes.3,5 In all of these sensing and diagnostic systems, one or several molecular components of the detection system must be immobilized on a sensor surface. A factor of prime importance in this context is that the influence from a solid support frequently induces conformational changes, causing a change or even loss of the desired functionality. The reason is the strong perturbation exerted by many surfaces on adsorbed molecules. Thus, proper immobilization strategies that minimize any negative influence from the surface on the functionality of the biomacromolecules, combined with measurement techniques that * To whom correspondence should be addressed. E-mail: fredrik@ fy.chalmers.se. Tel: +46-31-7723464. Fax: +46-31-7723134. † Department of Applied Physics, Chalmers University of Technology and Go¨teborg University. ‡ Department of Cell and Molecular Biology, Go ¨ teborg University. § Department of Physical Chemistry, Chalmers University of Technology and Go¨teborg University. (1) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (2) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1993, 21, 46634669. (3) Ziegler, C.; Gopel, W. Curr. Opin. Chem. Biol. 1998, 2, 585-591. (4) Luther, A.; Brandsch, R.; von Kiedrowski, G. Science 1998, 396, 245-248. (5) Niemeyer, C. M.; Blohm, D. Angew. Chem., Int. Ed. 1999, 38, 2865-2869.

improve the understanding of both the immobilization and the subsequent biorecognition process, are of utmost importance. The challenge is to find the right balance between sufficiently strong surface binding to achieve immobilization and sufficiently weak surface-induced perturbation to maintain the functionality of the biomolecules on a surface that is also inert toward unspecific binding. The most commonly used immobilization strategies are direct, on-chip syntheses of nucleic acids1 or attachment of chemically modified oligonucleotides for specific surface immobilization, using, for example, thiol-gold chemistry6-9 or biotin-streptavidin/avidin coupling.10,11 The success of these various immobilization strategies is generally tested by the ability of the surface-bound recognition layers to hybridize with complementary strands, in measurements using some type of surface sensitive method, where surface plasmon resonance (SPR) is by far the most frequently applied technique, see, for example, refs 6-12. However, since SPR basically measures the adsorbed amount (molar or dry mass) versus time via changes in the interfacial optical properties, it gives no explicit information about (6) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (7) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 89168920. (8) Georgiadis, R.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166-3173. (9) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044-8051. (10) Ijiro, K.; Ringsdorf, H.; Birch-Hirschfeld, E.; Hoffmann, S.; Schilken, U.; Strube, M. Langmuir 1998, 14, 2796-2800. (11) Niemeyer, C. M.; Boldt, L.; Ceyhan, B.; Blohm, D. Anal. Biochem. 1999, 268, 54-63. (12) Liebermann, T.; Knoll, W.; Sluka, P.; Herrmann, R. Colloids Surf., A 2000, 169, 337-350.

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the structure or properties other than mass of surfacebound biomolecules. There are, in fact, only a few reports in which the conformation of surface-attached nucleotides has been investigated. Using steady-state fluorescence energy transfer, Charreyre et al.13 investigated the influence of pH, ionic strength, and surfactants on the conformation of single-stranded DNA oligomers covalently bound to the surface of polystyrene latex microspheres. Levicky et al.6 used neutron reflectivity to compare the concentration profiles of chemically modified thiol-DNA monolayers coupled to gold with their hybridization efficiency. Here, we present how the quartz crystal microbalance with dissipation monitoring (QCM-D) technique can be used as a sensor technique that, on one hand, gives information similar to that obtained from, for example, SPR, but in addition provides additional information about both the structure of immobilized nucleotides and the kinetics of the subsequent hybridization processes through energy dissipation monitoring. The QCM-D technique measures simultaneously and in real time two properties of the surface layer. First, it measures changes in mass, m (detected via changes in its resonant frequency, f), and energy dissipation, D (cf. viscoelastic properties) (see ref 14 for technical details and ref 15 for application to biomacromolecule adsorption). The frequency shift of the QCM-D resonator yields a measure of adsorbed mass, ∆m, in some cases directly obtainable by a proportion between ∆f and ∆m, and in other cases (see below), by a nonproportional relation requiring the use of modeling. In fact, the magnitude of ∆D variations provides information about the shear-viscoelastic properties and signal if ∆f can be directly converted to mass or if modeling is required. The information contained in combined energy dissipation and frequency measurements has previously been shown to add unique information (not obtainable by, e.g., SPR) about the structure and other properties of biomolecular films, such as proteins15,16 and lipid vesicles.17,18 Past measurements in our group16-19 have shown that the real new added value of QCM-D measurements derived from the fact that the two measured quantities (mass and intralayer dissipation) give much richer information than a mass measurement alone. Of particular value is that structural changes of surface-bound molecules (e.g., upon antigen-antibody binding or vesicle decomposition into a supported lipid bilayer) yield detectable signals that via algorithms based on existing theories can be converted to well-known quantities such as film thickness, shear viscosity, and elastic modulus of the adlayers. Alternatively, one can use the measured data as just a qualitative fingerprint of the studied system. Since the technique is a real-time measurement method (∼1 s), it also provides detailed information on adsorption and recognition kinetics. One attractive route to achieve both specific immobilization and controlled spatial distribution of biomolecules, structurally and functionally unaffected by (13) Charreyre, M. T.; Tcherkasskaya, O.; Winnik, M. A.; Hiver, A.; Delair, T.; Cros, P.; Pichot, C.; Mandrand, B. Langmuir 1997, 13, 31033110. (14) Rodahl, M.; Hoö¨k, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. Rev. Sci. Instrum. 1995, 66, 3924-3930. (15) Hoö¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14 (4), 729-734. (16) Hoö¨k, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (21), 12271-12276. (17) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75 (3), 1397-1402. (18) Keller, C. A.; Glasmastar, K.; Zhdanov, V. P.; Kasemo, B. Phys. Rev. Lett. 2000, 84, 5443-5446. (19) Hoö¨k, F.; Kasemo, B.; Nylander, T.; Fant, C.; Mjo¨rn, K.; Elwing, H. Anal. Chem. 2001, in press.

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the solid support (which is not based on sulfur-gold chemistry), is to use supported phospholipid bilayer membranes20,21 preferably supported on mica or SiO2. For instance, specific binding to lipid films at interfaces has proven efficient for protein 2-D crystal growth at both the air-liquid and solid-liquid interfaces.22-26 In these cases, a fraction of the lipids is chosen such that binding occurs to a specific region of the protein, utilizing, for example, receptor-ligand interactions22-24 or binding of histidinerich regions of both wild-type and engineered proteins to metal-chelating lipids.25,26 In addition to the controlled binding, the success of this approach relies on the facts that (i) the lipids, and thus the attached proteins, have a high lateral mobility, and (ii) the nonfunctionalized lipid monomers, that constitute the majority of the lipid layer, do not cause unspecific adsorption of water-soluble biomacromolecules such as proteins or nucleotides (see, e.g., refs 27-30). In the present case, streptavidin, presumably forming a protein 2-D crystal on a biotin-doped lipid bilayer supported on a SiO2 surface21 evaporated on the quartz crystal sensor surface, has been utilized as a template for biotinylated PNA and DNA. [Note: PNA is a synthetic nucleic acid analogue in which the sugar-phosphate backbone is replaced by a synthetic pseudopeptide backbone composed of N-(2-aminoethyl)-glycine units31 capable of sequence-specific recognition of DNA and RNA obeying the Watson-Crick hydrogen bonding rules.31,32 The hybrid complexes thus formed exhibit extraordinary thermal stability and unique ionic strength properties.] While immobilized single-stranded DNA has been quite extensively studied as recognition layers for DNA, only a few reports exist where PNA has been used.33-35 Wang et al.34 used the traditional QCM technique, measuring frequency shifts only, by immobilizing 15-mer thiolderivatized PNA probes on a gold-coated AT-cut quartz crystal. According to their work, the thiol-PNA formed a dense layer of PNA associated with the gold surface via the SH group of a cystein-ethylene glycol linker. The adsorption of a 15-mer full complementary target DNA oligomer was confirmed through a decrease in frequency (increase in mass), while no frequency shift was observed upon exposure of the surface to a large excess of a 15-mer, singly mismatched DNA oligomer. Using SPR, Jensen et (20) Sackmann, E. Science 1996, 271, 43-48. (21) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706. (22) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387-396. (23) Knoll, W.; Zizlsperger, M.; Liebermann, T.; Arnold, S.; Badia, A.; Liley, M.; Piscevic, D.; Schmitt, F. J.; Spinke, J. Colloids Surf., A 2000, 161, 115-137. (24) Reviakine, I.; Bergsma-Schutter, W.; Brisson, A. J. Struct. Biol. 1998, 121, 356-362. (25) Pack, D. W.; Chen, G. H.; Maloney, K. M.; Chen, C. T.; Arnold, F. H. J. Am. Chem. Soc. 1997, 119, 2479-2487. (26) Ng, K.; Pack, D. W.; Sasaki, D. Y.; Arnold, F. H. Langmuir 1995, 11, 4048-4055. (27) Chapman, D. Langmuir 1993, 9, 39-45. (28) Buijs, J.; Britt, D. W.; Hlady, V. Langmuir 1998, 14, 335-341. (29) Malmsten, M. J. Colloid Interface Sci. 1994, 168, 247-254. (30) Glasma¨star, K.; Hoö¨k, F.; Kasemo, B. J. Colloid Interface Sci., accepted for publication. (31) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Nordeń, B.; Nielsen, P. E. Nature 1993, 365, 566-568. (32) Egholm, M.; Christensen, L.; Dueholm, K. L.; Buchardt, O.; Coull, J.; Nielsen, P. E. Nucleic Acids Res. 1995, 23, 217-222. (33) Jensen, K. K.; Orum, H.; Nielsen, P. E.; Norden, B. Biochemistry 1997, 36, 5072-5077. (34) Wang, J.; Nielsen, P. E.; Jiang, M.; Cai, X. H.; Fernandes, J. R.; Grant, D. H.; Ozsoz, M.; Beglieter, A.; Mowat, M. Anal. Chem. 1997, 69, 5200-5202. (35) Weiler, J.; Gausepohl, H.; Hauser, N.; Jensen, O. N.; Hoheisel, J. D. Nucleic Acids Res. 1997, 25, 2792-2799.

Characterization of PNA and DNA Immobilization

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al.33 studied the effects of mismatches on the kinetics of the association and dissociation reactions of PNA-DNA hybridization. They used biotin-PNA immobilized by binding to streptavidin incorporated in a dextran-coated gold surface. In the present work, we compare thiol-based and biotinbased coupling of PNA to a bare-gold surface and to biotinlipid-immobilized streptavidin, respectively. We argue, based on the present results, that a 2-D arrangement of streptavidin on a biotin-doped supported lipid bilayer provides a more efficient coupling strategy than streptavidin coupled to a dextran gel, since likely influence from the negatively charged dextran gel on the hybridization process is reduced. The merit of combined energy dissipation, D, and frequency, f, measurements is demonstrated through (i) the differences in responses observed for thiol-PNA on gold and biotin-PNA on the streptavidin 2-D arrangement and the inability of the former to hybridize with DNA, and (ii) the structural differences, and even structural (phase) transitions, observed during formation of PNADNA and DNA-DNA duplexes. The relation of these observations to previously determined differences in solution structures of these duplexes 36,37 is discussed. Materials and Methods The QCM-D Technique. The merit of the traditional QCM technique lies primarily in the simplicity and sensitivity (below ng cm-2) by which an adsorbed mass, ∆mQCM, can be deduced from measured changes in the resonant frequency, ∆f, using the Sauerbrey relation:38

∆mQCM ) Ffilmδfilm )

CQCM ∆f n

(1)

where Ffilm and δfilm are the effective density and the film thickness, respectively, CQCM ()17.7 ng cm-2 Hz-1) is the masssensitivity constant, and n ()1, 3, ...) is the overtone number. CQCM is proportional to f0-2, where f0 is the fundamental (n ) 1) frequency of the sensor. For a given added mass, the ∆f signal thus increases linearly with n and quadratically with f0. This relation holds, however, only under certain conditions (see, e.g., refs 19 and 39-41), which qualitatively can be expressed as follows: The adsorbed film must be sufficiently thin, not too viscoelastic, and certain conditions about the coupling to the surrounding medium must be fulfilled. When the relation fails, theoretical expressions can and should be used to compensate the failure of the Saurbrey relation (see Appendix).19 The technique used here is an extension of the traditional QCM technique called QCM-D.14 The new feature in this set up is that the energy dissipation, D, is measured in addition to the frequency (mass) change. The QCM-D technique (Q-Sense AB, Go¨teborg, Sweden), described in detail elsewhere,14 measures ∆D and ∆f of up to four harmonic (for a 5 MHz crystal) with a repetition rate of about 1 Hz. In the present work, we used polished, AT-cut crystals with a fundamental resonant frequency of about 5 MHz (Q-Sense AB, Sweden) operating at its third harmonic (i.e., at 15 MHz). Combined with theoretical treatment using a model describing both the elastic and inelastic (loss) modulus of the film, entirely new information about film properties is obtained, and the influence from viscous losses on the mass uptake commonly estimated using the Sauerbrey relation can be corrected40,41 (see below). All measurements were done in a static solution, that is, in batch mode, in a cell designed to provide a (36) Eriksson, M.; Nielsen, P. E. Q. Rev. Biophys. 1996, 29, 369-394. (37) Eriksson, M.; Nielsen, P. E. Nat. Struct. Biol. 1996, 3, 410-413. (38) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (39) Zhang, C.; Schranz, S.; Lucklum, R.; Hauptmann, P. IEEE Trans. Sonics Ultrason. 1998, 45, 1204-1210. (40) Lucklum, R.; Behling, C.; Hauptman, P. Anal. Chem. 1999, 71, 2488-2496. (41) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391-396.

fast nonperturbing exchange of a stagnant liquid (Q-Sense AB, Sweden). The measurement chamber was temperature-stabilized to 24 ( 0.1 °C. Surface Preparation. The gold surfaces were cleaned in a UV/ozone chamber for 10 min. This was then followed by immersion in a 1:1:6 mixture of H2O2 (J. T. Baker, Netherlands) (30%), NH3 (Merck) (25%), and Milli-Q water (Millipore, France) for 5 min at 70 °C. The SiO2 surface on the QCM crystals was prepared by evaporation of 3 nm Ti as an “adhesive” onto the gold surface (cleaned as described above), followed by evaporation of 100 nm SiO2 (Balzers Process Systems, Sweden). The SiO2 surfaces were cleaned between each measurement in a 0.4% SDS solution for 2 h, followed by careful rinsing in Milli-Q water and cleaning in a UV/ozone chamber for 2 × 10 min. Preparation of Lipid Vesicles. Small unilamellar vesicles (SUV) were prepared according to the protocol similar to that described in refs 42 and 43, except that 5% biotin-phospholipids (Biotin-LC-DPPE, Boule) was mixed with egg-phosphatidylcholine (egg-PC) lipids (Avanti-Lipids). The lipid concentration in the SUV preparation was approximately 10 mg/mL, which was further diluted by a factor of 100 prior to surface exposure. The vesicle solution was stored under a nitrogen atmosphere at 4 °C. Preparation of Protein and Nucleotide Solutions. Streptavidin was purchased from Sigma-Aldrich and dissolved in a 10 mM TRIS, 100 mM NaCl buffer, pH 8.0. The 15-mer biotin-PNA {biotin-(egl)9-TGT ACG TCA CAA CTA-NH2 (N- to C-terminal); egl ) 8-amino-3,6-dioxyoctanoic acid}32 and cysPNA {Cys-(egl)9-TGT ACG TCA CAA CTA-NH2} (called thiolPNA below) were synthesized as described in refs 33 and 44. The same PNA sequence but without the biotin-egl and cys-egl linker was also synthesized. The 15-mer DNA, thiol-DNA, and biotinDNA oligomers (Amersham Pharmacia, Biotech, Sweden) were used as obtained. Concentration determinations of DNA and PNA were based on optical absorption at 260 nm measured at 80 °C.45,46 The buffer referred to as “buffer” is 10 mM TRIS, 100 mM NaCl buffer, pH 8.0. All aqueous media used were in Milli-Q water (Millipore, Molsheim, France). The buffer was degassed before use.

Results and Discussion The QCM-D technique was used to explore two different immobilization strategies of single-stranded PNA and DNA using, respectively, thiol coupling to gold and biotin coupling to streptavidin. In the latter case, the platform is a 2-D arrangement of streptavidin on top of a supported biotin-doped egg-PC lipid bilayer. Biotinylated PNA or DNA is then attached to the streptavidin layer. The ability of these layers to recognize fully complementary and a single-mismatch sequence of DNA was investigated using the same technique. In the first paragraph, the results about the formation of the streptavidin layer are summarized. A more detailed account of these results will be given in forthcoming publications. In the next paragraph, results from subsequent binding of biotin-PNA to the streptavidin 2-D layer are described and compared with direct thiol-PNA binding to bare gold. In the two following paragraphs, we analyze the ability of these layers to recognize single-stranded, fully complementary, and single-mismatch DNA, with special focus on structural differences between DNADNA and PNA-DNA duplexes formed on streptavidin. Streptavidin Functionalization. Figure 1A and B shows ∆f and ∆D, respectively, versus time upon exposure (42) Barenholz, Y.; Gibbes, D.; Litman, B. J.; Goll, J.; Thompson, T. E.; Carlson, F. D. Biochemistry 1977, 16, 2806-2810. (43) Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773-14781. (44) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.; Hansen, H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.; Coull, J.; Berg, R. H. J Pept. Sci. 1995, 3, 175-183. (45) Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M. Data for Biochemical Research; Oxford Science Publications: New York, 1986. (46) Ratilainen, T.; Holmen, A.; Tuite, E.; Haaima, G.; Christensen, L.; Nielsen, P. E.; Norden, B. Biochemistry 1998, 37, 12331-12342.

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Figure 1. Changes in frequency (A), ∆f, and dissipation (B), ∆D, versus time upon the sequential exposure of a SiO2 surface to lipid vesicles and streptavidin (1 µM). Inset: A cartoon picture illustrates the sequence of events shown in Figure 1.

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Figure 2. ∆f (A) and ∆D (B) versus time for exposure of the streptavidin layer (cf. Figure 1) to biotin-PNA, followed by (superimposed) separate additions of DNAmm1 (1 µM) and DNAfc (1 µM) after saturated immobilization of biotin-PNA.

of a SiO2 surface to a biotin-doped lipid-vesicle solution (t = 500 s), followed by exposure to a streptavidin solution (t = 1500 s). At saturation of the film formation, after each exposure, the solutions were exchanged to pure buffer. The initial exposure to the vesicle solution (left arrow labeled Bilayer formation in Figure 1) results in a rapid and large decrease in f (mass uptake due to vesicle adsorption) and increase in D, reaching a minimum in f and maximum in D at t ≈ 650 s. This indicates roughly the coverage at which adsorbed unbroken vesicles start to fuse and decompose into a bilayer.17,18,47 During the decomposition of the vesicles into a bilayer (650 < t < 1100 s), f increases (caused by mass loss due to the release of water initially trapped by the vesicles on the surface) and D decreases (due to an increase in stiffness of the film as the adsorbed lipid vesicles transform into a bilayer). The changes in f and D eventually level off and stabilize at 1200-1500 s, after which the bilayer formation is complete. Thus, although the lipid vesicles contain ∼5% biotinylated phospholipids, a lipid bilayer is formed on SiO2 in the same way as nonbiotinylated vesicles do, as confirmed by a total frequency shift of ∼26 Hz at saturation.17,18 This is in good agreement with earlier results on lipid bilayer formation using the QCM-D technique.17,18 Thus, the biotinylated phospholipid bilayer provides a platform for the next preparation step: streptavidin binding in a 2-D overlayer on top of the biotin-doped bilayer. The streptavidin binding to the biotinylated-lipid bilayer (right arrow in Figure 1) results in a frequency shift of ∼-25 Hz, which, using eq 1 and an effective density of 1.15 g cm-3, corresponds to an effective thickness of 4.7 nm (see Appendix), a result which is in very good agreement with previous ellipsometric data presented by

Reiter et al.,48 who estimated the thickness to 4.72 nm. The rapid initial rise in ∆D, followed by a slow decrease during the adsorption of streptavidin, is peculiar and interesting. It indicates formation of an initial nonrigid dissipative protein arrangement followed by formation of a more rigid layer. The observed decrease in D is interesting in itself, as it is actually consistent with protein 2-D crystal formation as previously reported on fluid biotin-doped lipid films.22 In the present work, the slow decrease in D was used as a “fingerprint” for successful formation of the 2-D streptavidin layer. The 2-D streptavidin layer was found to be an excellent platform for very reproducible (error of less than 2%) immobilization of biotin-PNA and biotin-DNA. PNA Immobilization and PNA-DNA Hybridization. Figure 2A and B shows ∆f and ∆D, respectively, versus time for exposure of a streptavidin layer (cf. Figure 1) to biotin-PNA (1 µM), followed by exposure to fully complementary DNA (DNAfc) (1 µM). Also shown in Figure 2 is a separate exposure of an identically prepared streptavidin plus biotin-PNA layer to a single-mismatch DNA (DNAmm1) (1 µM). [Note: In the eighth position of the DNAfc sequence, a “G” has been replaced by an “A”.] Note that DNAfc and DNAmm1 give different f and D responses. For comparison with the data in Figure 2, Figure 3A and B shows ∆f and ∆D, respectively, versus time for exposure of a bare-gold surface to a 1 µM solution of thiol-PNA, followed by addition of a 1 µM solution of complementary DNAfc. Note the lack of distinguishable binding (recognition) in the latter case. Addition of biotin-PNA to the streptavidin layer (left arrow labeled PNA binding in Figure 2) and of thiol-PNA to the bare-gold surface (left arrow in Figure 3) results in both cases in a decrease in the frequency. These changes have similar rates and reach values at a saturation of ∼-10 and ∼-13 Hz, respectively. Binding of biotin-PNA

(47) Zhdanov, V. P.; Keller, C. A.; Glasma¨star, K.; Kasemo, B. J. Chem. Phys. 2000, 112, 900-909.

(48) Reiter, R.; Motschmann, H.; Knoll, W. Langmuir 1993, 9, 24302435.


Figure 3. ∆f (A) and ∆D (B) versus time for exposure of a bare-gold surface to thiol-PNA, followed by separate addition of DNAfc (1 µM) after saturated immobilization of thiol-PNA.

to the lipid-streptavidin layer is accompanied by a relatively large dissipation shift of ∼0.6 × 10-6, while essentially no additional energy dissipation is induced during binding of thiol-PNA to gold. On the basis of the theory for QCM-D response and previous accumulated data for adsorbed biomolecule films from an aqueous phase,16-19 we suggest that the layer of biotin-PNA on streptavidin is composed of elongated and flexible molecules primarily oriented outward from the surface, while thiol-PNA immobilized on bare gold forms a significantly more compact structure. Levicky et al.6 made a similar observation for thiol-coupled single-stranded DNA to bare gold using neutron reflectivity. By concentration-profile determinations of thiol-coupled DNA on bare gold, they found that the formed layer was compact, suggesting the presence of multiple contacts between each DNA strand and the surface. Thus, the very low induced energy dissipation observed in the present case for binding of thiol-PNA to gold strongly suggests that PNA is rigidly attached with several unspecific contact (binding) points to the surface. This suggestion is supported by the observation that PNA without a thiol end binds in a similar way to bare gold as thiol-PNA (not shown). This picture of a PNA film formed on gold via binding with several PNA-gold contact points also explains the lack of binding of DNA to this PNA layer (Figure 3). We also varied the salinity of the buffer (from 0 to 200 mM NaCl) and incubation time (from 15 to 90 min) for the thiol-PNA immobilization, which did not significantly influence the ∆D and ∆f response nor the actual hybridization efficiency (not shown). In a separate experiment, we also showed that PNA without biotin induced no change in frequency or dissipation when added to the streptavidin layer (not shown) or to the biotinylated phospholipid bilayer (not shown). In contrast to the bare-gold results, the biotin-PNA layer, bound to the 2-D streptavidin layer, creates specific binding sites for DNA. In conclusion, PNA or thiolPNA on gold does not create as good a platform for

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recognition of DNA as does biotin-PNA immobilization on streptavidin. In the Appendix, we attempt a quantitative analysis of the biotin-PNA layer thickness (which is complicated by the role of trapped water and the relatively large energy dissipation). The conclusion from that analysis is that the layer of PNA is very flexible (large energy dissipation) and extends as an elongated molecule out in the solution approximately ∼2 nm, causing a considerable amount of trapped water between the chains (see schematic cartoon in Figure 2). This conclusion is further supported by the fact that biotin-PNA coupled to streptavidin hybridizes readily with DNA, as discussed below. In contrast, a similar analysis of thiol-PNA on bare gold suggests a compact layer (see schematic cartoon in Figure 3) primarily hidden from hybridization reactions with complementary strands. Levicky et al.6 concluded that post-treatment with mercaptohexanol (a short alkanethiol with a terminal hydroxy group) is required to transform thiol-coupled DNA into an elongated and extended conformation, which, in contrast to the native thiol-coupled DNA strands, hybridizes readily to its complementary sequence. Addition of DNAfc to the thiol-PNA bound to the gold surface (right arrow in Figure 3) induces essentially no change in frequency or dissipation. In contrast, both singly mismatched DNAmm1 and fully complementary DNAfc (middle arrow in Figure 2) hybridize with the biotinPNA bound to streptavidin, while DNA with more than one mismatch (DNAmm>1) does not associate with the PNA (not shown) to give a measurable signal at the concentrations and temperature used. As seen in Figure 2, the initial rate of binding is about 1.5 times higher for DNAfc than for DNAmm1, and the frequency shift at saturation is 1.4 times higher, reaching ∼-7 and ∼-5 Hz, respectively. Moreover, exposure to pure buffer after the saturated binding of DNAmm1 results in a decrease in mass uptake (dissociation/desorption), demonstrating that the binding is, in this case, reversible under our experimental conditions. In contrast, DNAfc is essentially irreversibly bound on the time-scale of the experiment, independent of whether it is exposed directly to freshly formed PNA layers or after an initial DNAmm1 exposure to the PNA layers followed by desorption of DNAmm1 by subsequent rinsing. When the concentration of DNAmm1 was varied between 0.5 and 2 µM, the rate of binding and the frequency shift (mass uptake) at saturation increased monotonically with DNA concentration (not shown). The increase in saturation uptake is a consequence of the reversibility of adsorption; the equilibrium coverage is shifted to higher values at higher bulk concentrations. In contrast, for DNAfc, only the rate of binding increased with increased concentration, while the bound amount at saturation was constant (∼-7 Hz). This number is, in fact, about 2 times larger than expected if DNA saturates the immobilized biotin-PNA strands. Binding of two biotinPNA (mw ≈ 5.5 kD) molecules per streptavidin (where the unit cell of the latter is ∼5.8 × 5.8 nm2) would, according to eq 1, yield a frequency shift of about -3.1 Hz (see Appendix), and the additional change at saturated hybridization would thus yield a quite similar number. These larger than expected mass uptakes observed upon both biotin-PNA binding (see above) and DNA-PNA hybridization can, however, be explained by taking into account the special way the QCM-D measures mass.19,49-51 In brief, the method measures the sum of the molar (dry) (49) Rickert, J.; Weiss, T.; Gopel, W. Sens. Actuators, B 1996, 31, 45-50. (50) Fawcett, N. C.; Craven, R. D.; Zhang, P.; Evans, J. A. Anal. Chem. 1998, 70, 2876-2889.

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mass and coupled water via direct hydration or entrapment in cavities in the film. The mass increase upon hybridization can, thus, be explained as a change of conformation of the film such that the thickness increases from ∼2 nm for the PNA layer to 3.6 nm for the hybridized layer (see Appendix), which is consistent with PNA-DNA duplexes oriented essentially normal to the streptavidin plane. In other words, the hybridization causes considerable swelling and further incorporation of water in the nucleotide film. [Note: One could also argue that biotinPNA binding to streptavidin would replace water coupled to streptavidin via a similar mechanism. However, considering the fact that the quartz crystal operates under shear motion, only water between the immobilized biomolecules can be trapped as an additional mass. Since biotin-PNA are likely to bind to the biotin-binding sites exposed on top of streptavidin, we consider the effect from displaced or coupled water on the mass uptake to be significantly smaller, in this case, compared with DNA hybridization, where the DNA strands must penetrate the flexible biotin-PNA and biotin-DNA films (cf. cartoon in Figure 2).] In summary, these measurements illustrate efficient discrimination between fully complementary DNA, where the binding is irreversible and where the saturated amount is, therefore, independent of DNA concentration, and single-mismatch DNA binding, where the binding is reversible and where the saturated amount thus depends on DNA concentration. Since the measurements were done in a static solution, the association constant Ka could be determined using a classical Langmuir isotherm, giving an association constant of 1.22 × 106 M-1 for the reversible PNA-DNAmm1 system. (The fitting error is less than 1%.) This is actually a factor ∼60 larger than the value obtained by Jensen et al.33 for the same sequence. However, their measurements were done at 35 °C, while ours were done at 24 °C, and the pH and buffer systems used were not identical. Moreover, they immobilized biotin-PNA to streptavidin associated to a dextran gel carrying a negative ζ-potential. It is thus not unlikely that the negatively charged background has both a repulsive influence on the negatively charged DNA strands and a steric influence on the transport properties, thus lowering the measured association constant. In addition, the fact that they used an egl-linker between biotin and PNA 3 times shorter than that used in the present work can contribute to the observed difference. For the conditions used in the present study, the binding of DNAfc was not reversible, and the association constant could, therefore, not be calculated for this case. Our results are also interesting in light of recent results by Lieberman et al.,12 since we demonstrate that the QCM-D technique, in contrast to their results using the SPR technique on a similarly streptavidin functionalized thiol-biotin-coated gold surface, can monitor DNA hybridization for short strands without introducing fluorescent labels. A final comment is also appropriate about how our results compare with those presented by Wang et al.34 The saturation frequency shift of -7 and -5 Hz observed by us upon DNAfc and DNAmm1 hybridization to biotin-PNA on streptavidin and the absence of hybridization to thiol-PNA on gold are in strong contrast to the results by Wang et al.,34 who observed a saturation frequency shift of ∼-90 Hz (using quartz crystals with the same fundamental frequency as used in the present work) upon sequential addition of DNAfc to thiol-PNA immobilized on gold and no response upon addition of DNAmm1. The difference in the saturation frequency shift (51) Caruso, F.; Furlong, D. N.; Kingshott, P. J. Colloid Interface Sci. 1997, 186, 129-140.

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is attributed to the much higher PNA coverage obtained in their drying-based immobilization process, and the difference in DNA hybridization is attributed to the difference in the immobilization process. A proper immobilization process would preferably mimic the situation of the bulk reaction, in which quite stable duplexes are expected to be formed between PNA and single-mismatch DNA under the solution conditions used in the present work. We thus consider the biotin-based immobilization on the 2-D arrangement of streptavidin as advantageous also in this respect. PNA-DNA Versus DNA-DNA Hybridization. The binding kinetics and hybridization-induced changes in f and D of the PNA hybridization with DNAfc were compared with DNA-DNA hybridization by replacing PNA with DNA, that is, by immobilizing biotin-DNA and thiol-DNA (with the same base-pair sequence as for the biotin-PNA) on a lipid-biotin-streptavidin surface and a bare-gold surface, respectively. While immobilization of thiol-DNA binding to a bare-gold surface resulted in essentially the same result as for thiol-PNA (see above) and as those presented by Levicky et al.6 regarding both immobilization and subsequent hybridization (not shown), very interesting results were obtained upon exposure of biotin-DNA to the lipid-biotin-streptavidin surface. Figure 4A and B shows ∆f and ∆D, respectively, versus time for exposure of the streptavidin-covered surface to biotin-DNA followed by exposure to DNAfc. The previously presented results (Figure 2) for biotin-PNA and subsequent addition of DNAfc (cf. Figure 2) are also shown for comparison. There are distinct differences in the ∆f and ∆D response for the two cases. This difference is highlighted in Figure 4C, where ∆f is plotted versus ∆D for the data displayed in Figure 4A and B. This way of displaying the data eliminates time as an explicit parameter and makes possible a direct comparison of the ratio between ∆D and ∆f, that is, the induced energy dissipation per coupled unit mass. For example, a low ∂D/∂f value indicates mass addition without significant dissipation increase, which, in turn, signals a fairly rigid layer. In contrast, a large ∂D/∂f value signals a soft, dissipative film. As seen in Figure 4A and B, the rates of binding of biotin-PNA and biotin-DNA (left arrows) to streptavidin are very similar but result in somewhat different induced changes in ∆f and ∆D at saturation. The frequency shift at saturation of biotin-PNA is ∼-10 Hz, while it is ∼-13 Hz for biotin-DNA, and the corresponding ∆D values are 0.6 × 10-6 and 1.1 × 10-6, respectively. Thus, biotin-DNA forms a more expanded (higher ∆f) layer than biotin-PNA, with a thickness that can be estimated to be ∼2.5 nm (see Appendix). The ∆D versus ∆f graphs (Figure 4C) display linear regimes for both biotin-PNA and biotin-DNA with similar slopes, indicating similar flexibility of the two systems. In contrast, both the kinetics and the magnitude of the changes in f and D differ between binding of DNAfc to biotin-PNA and biotin-DNA (right arrow in Figure 4). The interesting differences displayed in the ∆D versus ∆f graphs for the two cases show that the structures of the PNA-DNA and DNA-DNA duplex layers are quite different both during formation and at saturated binding. During DNA-DNA duplex formation, the hybridizationinduced change in D is almost negligible, except for an initial small and rapid increase followed by a slower decrease, whereas for the PNA-DNA duplex formation, there is a monotonic large increase in D. From the ∆D versus ∆f graph (Figure 4B), it is clearly seen that the PNA-DNA hybridization displays a linear regime with a slope that is about 2 times larger than that of the preceding biotin-PNA binding. Thus, while the magnitude


Langmuir, Vol. 17, No. 26, 2001 8311

account. Such lateral interactions may affect both the hybridization kinetics and the structure of the duplexes formed (see Appendix) and can either amplify or suppress the properties of the free duplexes. These topics will be subject to future investigations, including studies of, for example, hybridization kinetics versus coverage, different spacer groups, and the length of the nucleotide strands. Concluding Remarks We have used the QCM-D technique to characterize the successive steps of various immobilization strategies of single-stranded PNA and DNA and the subsequent hybridization reactions with fully complementary and various mismatch sequences of DNA. Through combined ∆f and ∆D measurements, information about changes in coupled mass (thickness) and viscoelastic properties is determined for the successive steps. Both thiol-PNA and thiol-DNA attached directly to a bare-gold surface form a rigid structure (low-induced ∆D) with essentially no affinity toward fully complementary DNA, probably due to multiple contact points to the surface. In contrast, biotin-PNA and biotin-DNA coupled to a 2-D arrangement of streptavidin on top of a biotinylated phospholipid bilayer are bound in a flexible, presumably elongated, state (high ∆D) with exposed binding sites and high ability to hybridize with both single-mismatch and fully complementary DNA. Single-mismatch and fully complementary biotin-PNA-DNA and biotin-DNA-DNA combinations can be discriminated both through the kinetics (e.g., reversible versus irreversible binding) and through the total binding at saturation. It is also possible to discriminate different structures of the PNA-DNA and DNA-DNA layers from the combined ∆D and ∆f measurements, tentatively attributed to original structural differences of the individual duplexes (in solution) in combination with influence from lateral interactions in the adlayer.

Figure 4. ∆f (A) and ∆D (B) versus time for biotin-DNA and biotin-PNA binding to streptavidin (cf. Figure 3), followed by exposure to DNAfc (1 µM). (C) ∆D versus ∆f for the data shown in (A) and (B).

Acknowledgment. This work was financially supported by the Swedish Research Council (No. 2000-175) and through the Swedish Biomaterials Consortium and the Biocompatible Materials program both funded from the Strategic Foundation for Research. Assistance in the lab work from Ulrika Krave and Camilla Fant is gratefully acknowledged.

of the dissipation per frequency shift (cf. bound mass) is quite similar for the initial binding of single-stranded DNA and PNA to the streptavidin layer (left part of Figure 4C), it is much larger for the PNA-DNA duplexes compared to the DNA-DNA duplexes (right part of Figure 4C), interpreted as a more flexible structure of the PNA-DNA duplexes compared to the layer composed of DNA-DNA duplexes. This observation is consistent with structure determinations of PNA-DNA and DNA-DNA duplexes in solution.36,37 While the latter displays a more compact structure manifested by 10 nucleotide pairs per helical turn and a diameter of ∼2 nm, the corresponding values for the PNADNA duplex are 13 and ∼2.3 nm, suggesting a more flexible structure in the latter case. However, to translate these results to the present measurements, it is important to keep in mind that the measured ∆D and ∆f changes reflect the properties of the whole film, rather than individual adsorbed molecules.16 Thus, the differences observed in the ∆D versus ∆f graphs between DNA-DNA and PNA-DNA duplexes may only partly be due to differences in the individual structures of the single duplexes, since possible lateral interactions in the film, composed of individual strands, must be taken into

In this appendix, the induced changes in frequency and energy dissipation for (i) streptavidin binding, (ii) nucleotide immobilization, and (iii) DNA hybridization are subject to a quantitative analysis, supporting the conclusions presented in the text. Streptavidin Binding. The streptavidin binding to the biotinylated-lipid bilayer (right arrow in Figure 1) results in a frequency shift of ∼-25 Hz. Since the frequency shift is sensitive to both protein mass and water trapped in cavities of adsorbed biomacromolecular layers (cf. the adsorption of unbroken vesicles in Figure 1A), the frequency shift should be related to a hydrodynamic mass and layer thickness rather than actual protein mass.19 Under the present conditions, it is likely that streptavidin forms a quadratic crystalline 2-D arrangement (though not proven from the QCM-D data alone) with two of its four binding sites for biotin bound to the biotinlipid bilayer, while the two remaining binding sites are facing the solution,22,52 as schematically illustrated in Figure 5.

Appendix

(52) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Zhang, X.; Angermaier, L.; Knoll, W.; Spinke, J. J. Biomater. Sci., Polym. Ed. 1994, 6, 481-495.

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Figure 5. Top view of a C222 crystalline 2-D arrangement (a ) b ) 5.8 nm, γ ) 90°) of streptavidin, where the two assessable biotin-binding sites are indicated by biotin molecules represented with a ball-and-stick representation. The center-tocenter distance between two adjacent biotin binding sites is ∼2.5 nm. The diameter of DNA-DNA and PNA-DNA duplexes (∼2.0-2.3 nm) is indicated with dashed lines. The thin circles indicate the possible position and (potential) overlap of two adjacent bound duplexes on the same streptavidin molecule.

Accordingly, the relation between water and protein in the protein film is approximately 50:50.53 This, in turn, gives an effective density of the protein layer between that of proteins and water (∼1.3 and 1.0 g cm-3, respectively), which in the present case is ∼1.15 g cm-3. Using eq 1, the hydrodynamic thickness of the streptavidin layer is 4.7 nm. The influence of the induced energy dissipation on the linear relation between ∆f and ∆m (eq 1) was estimated to be less than 3%.19 Nucleotide Immobilization. To evaluate the observed frequency shifts in terms of mass uptake for the immobilized single-stranded PNA on streptavidin, in comparison to the induced energy dissipation, a unit cell of the streptavidin layer corresponding to 5.8 × 5.8 nm2 (cf. Figure 5) is assumed, which is reasonable for the 2-D arrangement formed on the supported lipid bilayer.22,52 With a molecular weight for biotin-PNA of ∼5.5 kD, binding of two biotin-PNA molecules per streptavidin (which is a reasonable assumption for single-stranded 15mer nucleotides) should result in a frequency shift of only ∼-3.1 Hz, according to eq 1. This is in strong contrast to the observed frequency shifts at saturation, which for biotin-PNA is ∼-10 Hz (Figure 2). This large difference between the observed and theoretically estimated frequency shift is due to the fact that water couples directly to the strands (hydration layer) and to cavities formed between the immobilized PNA strands. Assuming a diameter of the single-stranded nucleotides of ∼1 nm and that PNA binds normal to the streptavidin plane (with a streptavidin unit cell of 5.8 × 5.8 nm2) means that the naked strands occupy approximately 5% of the surface. Assuming further that all water between the strands couples as an additional mass (cf. streptavidin above), the effective density is ∼1.02 g cm-3, and the hydrodynamic thickness, estimated using eq 1, thus becomes ∼1.7 nm. This value might, at first sight, seem low, since a 15-mer strand is about 2.5 times longer. For thin films inducing additional energy dissipation, direct use of eq 1 may, however, result in an underestimation of the coupled mass or thickness.19 Using a Voight-based viscoelastic model, (53) Edwards, T. C.; Koppenol, S.; Frey, W.; Schief, W. R.; Vogel, V.; Stenkamp, R. E.; Stayton, P. S. Langmuir 1998, 14, 4683-4687.

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presented in detail in the references,41 it is estimated that none of the layers formed by the nucleotide strands investigated in this study are underestimated by more than 10%, and the effective thickness of the biotin-PNA layer is estimated to be about 2 nm using these models. Since single-stranded PNA has no ordered structure, it is rather likely that the explanation for the relatively low thickness is that the immobilized strands exist in a somewhat contracted state prior to hybridization (see further below). Additionally, nonvertical arrangement may contribute to a lower thickness. Thus, the large dissipation (per ∆f) in combination with the fact that PNA without biotin does not associate at all are consistent with biotinPNA forming a flexible and elongated layer coupled through the biotin end group to streptavidin, while contacts between the PNA backbone and streptavidin are prevented. This picture, including hydration and entrapped water, is consistent with the measured ∆f and the streptavidin surface density. DNA Hybridization. Upon binding of DNAfc to biotinPNA, approximately a 1:1 correspondence in mass uptake at saturation is observed. Since the molecular weights of biotin-PNA and DNA are fairly similar, this seems, at first sight, reasonable. However, since coupled water (and influence of ∆D on the ∆f to mass conversion) must be taken into account for mass determinations using the QCM technique (see above), the added mass due to binding of DNA should actually correspond to the density difference between bound DNA (∼1.3 g cm-3) and replaced water (∼1.0 g cm-3) and should, hence, be much less than that measured. The large observed mass uptake upon DNAPNA hybridization can, however, be explained by a conformational change of the overall layer, such that even more water is trapped after hybridization. We suggest a conformational change of the layer upon hybridization into a more stretched-out and elongated structure. The thickness, and consequently the amount of coupled water, would then exceed that of PNA alone. Using further a diameter of a PNA-DNA duplex to ∼2.3 nm, the thickness of the layer can be estimated, as described above, to ∼3.6 nm. Accordingly, the hybridization process induces almost a 2-fold increase in layer thickness and a slight increase in the overall flexibility (increase in the ∆D/∆f ratio), which is consistent with PNA-DNA duplexes being oriented essentially normal to the streptavidin plane. Interestingly, the biotin-DNADNA duplexes induce a larger change in frequency, signaling a thicker film and thus an orientation closer to the normal than that of biotin-PNA-DNA duplexes. Consider further that each streptavidin molecule can bind two biotinylated nucleotides, with a center-to-center distance of ∼2.5 nm. The diameter of DNA-DNA and PNA-DNA duplexes has from experimental54 and theoretical55 structure determinations been determined to be ∼2.0 and ∼2.3 nm, respectively, as indicated in Figure 5A. Accordingly, the distance between adjacent nucleotide duplexes on the same or adjacent streptavidin molecules may be very short, and overlap might even occur for certain orientations, as schematically illustrated in Figure 5, assuming a C222 crystalline arrangement of streptavidin. It can thus not be ruled out that lateral interactions between adjacent duplexes influence both the structure and the hybridization kinetics (see also text). LA0107704 (54) Almarsson, O.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9542-9546. (55) Sen, S.; Nilsson, L. J. Am. Chem. Soc. 1998, 120, 619-631.

Characterization of PNA and DNA Immobilization and Subsequent

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