Characterization of DNA Immobilization and Subsequent Hybridization

Anal. Chem. 2003, 75, 5080-5087

Characterization of DNA Immobilization and Subsequent Hybridization on a 2D Arrangement of Streptavidin on a Biotin-Modified Lipid Bilayer Supported on SiO2 Charlotte Larsson,*,† Michael Rodahl,‡ and Fredrik Ho 1o 1 k*,†

Department of Applied Physics, Chalmers University of Technology and Go¨teborg University, S-41296 Go¨teborg, Sweden, and Q-Sense AB, Redegatan 13, S-426 77 Va¨stra Fro¨lunda, Sweden

We show how the water content (and effective density) of thin adsorbed films composed of biomolecules can be determined using combined quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) analysis. In particular, these techniques, combined with theoretical treatment using a Voigtbased viscoelastic model, were applied to analyze the state of surface immobilized single stranded biotin-modified probe DNA (b-DNA) coupled via streptavidin to a biotindoped supported phospholipid bilayer (b-SPB)). From a proper analysis, it is demonstrated how changes in effective thickness, δf, and the viscoelastic components (shear viscosity, ηf, and shear elasticity, µf)) can be obtained during both DNA immobilization and hybridization with single stranded fully complementary target DNA. In particular, it is demonstrated how this type of analysis can be used to control the state of streptavidin arrangement for improved measurements of DNA hybridization kinetics. The latter is demonstrated by identifying a surface-coverage dependent viscoelastic behavior of immobilized b-DNA, which is shown to influence the hybridization efficiency.

Much effort is currently concentrated on improvements in the selectivity and sensitivity of surface-based DNA hybridizationdetection systems using novel detection schemes and miniaturization allowing hybridization and single-base mutation detection of low quantities in small sample volumes.1,2 The most commonly used immobilization strategies are direct on-chip synthesis3,4 and * Corresponding author. Tel: +46-31-7723464. Fax: +46-31-7723134. E-mail: [email protected] (F.H). Tel: +46-31-7723368. Fax: +46-31-7723134. Email: [email protected] (C.L.). † Chalmers University of Technology and Go ¨teborg University. ‡ Q-Sense AB. (1) Niemeyer, C. M.; Blohm, D. Angew. Chem. Int. Ed. 1999, 38, 2865-2869. (2) Pirrung, M. C. Angew. Chem. Int. Ed. 2002, 41, 1277-+. (3) 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. (4) Butler, J. H.; Cronin, M.; Anderson, K. M.; Biddison, G. M.; Chatelain, F.; Cummer, M.; Davi, D. J.; Fisher, L.; Frauendorf, A. W.; Frueh, F. W.; Gjerstad, C.; Harper, T. F.; Kernahan, S. D.; Long, D. Q.; Pho, M.; Walker, J. A.; Brennan, T. M. J. Am. Chem. Soc. 2001, 123, 8887-8894.

5080 Analytical Chemistry, Vol. 75, No. 19, October 1, 2003

coupling chemistries based on thiol-gold5-7 and silane-silica8-10 or various biotin-streptavidin/avidin coupling strategies.11-13 Efficient protocols for DNA hybridization detection measurements rely, however, not only on sufficient surface-coupling chemistries but also on the lateral packing density. This is because steric hindrance and/or electrostatic repulsion may at high coverage influence the hybridization efficiency.14 Unfortunately, there exist a severe competition between packing density and detection sensitivity, since the detected signal (number density per area) essentially decreases with the square of the lateral separation distance, thus putting high demands on the applied mode of detection. Analytical approaches proven versatile to evaluate DNA immobilization protocols and subsequent hybridization efficiency utilizes either fluorescence for end-point measurements,1 surface plasmon resonance (SPR) for kinetic evaluation,5 or even a combination thereof.12 Recently, the quartz crystal microbalance (QCM) technique has also been proven a promising tool for DNA hybridization kinetic evaluations,11,15,16 especially in situations when information about changes in the structure and/or water content of the immobilized DNA films is analyzed.17,18 (5) Georgiadis, R.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166-3173. (6) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (7) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (8) Balladur, V.; Theretz, A.; Mandrand, B. J. Colloid Interface Sci. 1997, 194, 408-418. (9) Chrisey, L. A.; Lee, G. U.; Oferrall, C. E. Nucleic Acids Res. 1996, 24, 30313039. (10) Halliwell, C. M.; Cass, A. E. G. Anal. Chem. 2001, 73, 2476-2483. (11) Caruso, F.; Rodda, E.; Furlong, D. F.; Niikura, K.; Okahata, Y. Anal. Chem. 1997, 69, 2043-2049. (12) Liebermann, T.; Knoll, W.; Sluka, P.; Herrmann, R. Colloids Surf., A 2000, 169, 337-350. (13) Niemeyer, C. M.; Boldt, L.; Ceyhan, B.; Blohm, D. Anal. Biochem. 1999, 268, 54-63. (14) Shchepinov, M. S.; CaseGreen, S. C.; Southern, E. M. Nucleic Acids Res. 1997, 25, 1155-1161. (15) Zhou, X. C.; Huang, L. Q.; Li, S. F. Y. Biosens. Bioelectron. 2001, 16, 8595. (16) Okahata, Y.; Niikura, K.; Furusawa, H.; Matsuno, H. Anal. Sci. 2000, 16, 1113-1119. (17) Hoö ¨k, F.; Ray, A.; Norden, B.; Kasemo, B. Langmuir 2001, 17, 8305-8312. (18) Pope, L. H.; Allen, S.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 2001, 17, 8300-8304. 10.1021/ac034269n CCC: $25.00

© 2003 American Chemical Society Published on Web 08/26/2003

MATERIALS AND METHODS Solution and Surface Preparations. Water was deionized and filtered (MilliQ, Millipore). DNA strands (5′-CCC-CCT-GTACGT-CAC-AAC-TA-3′ (b-DNA15); 5′-CCC-CCT-GTA-CGT-CAC-AACTAT-CCA-GTC-ACA-GTA-AT-3′ (b-DNA30); 5′-TAG-TTG-TGA-CGTACA-3 (fcDNA15); 5′-ATT-ACT-GTG-ACT-GGA-TAG-TTG-TGACGT-ACA-3′ (fcDNA30)) were purchased from MedProbe AS, Norway. b-DNA15/30 are derivatized at the 5′-end with biotin. The five cytosines in the 5′-end of DNA15 and DNA30 were used as spacers. Stock solutions of DNA strands (20 µM in 10 mM Tris, 1 mM EDTA, pH 8.0) and streptavidin (Sigma, lyophilized from potassium phosphate buffer, pH 6.5, dissolved in Milli-Q to 1 mg/ mL) were stored at -20 and -80 °C, respectively. Egg yolk phosphatidylcholine (PC) (Sigma) (obtained in chloroform) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) (Avanti Polar Lipids) (dissolved in chloroform) were stored at -20 °C. Lipid vesicles were prepared in buffer 1 (10 mM Tris (tris(hydroxymethyl)aminomethane), 100 mM NaCl, pH 8) (8 mg/ mL) using a lipid mixture (95% PC and 5% biotin-PE) following the protocol by Barenholtz et al.23 The vesicle solution was stored

under nitrogen atmosphere at 4 °C. The lipid bilayer formation, streptavidin binding, and b-DNA coupling was done in buffer 1, while the DNA hybridization experiments were done using buffer 2 (10 mM Tris, 200 mM NaCl, pH 8). The SiO2 coated24 QCM crystals (Q-Sense AB, Go¨tebrog, Sweden) and Biacore sensor chips were cleaned in 0.4% SDS (>60 min), followed by rinsing with Milli-Q water, drying (N2), and UVozone treatment (15 min). QCM-D and SPR Measurements. All QCM-D measurements were done under a nonflowing condition, that is, in batch mode, in a cell designed to provide a fast nonperturbing exchange of a stagnant liquid (Q-Sense AB). The measurement chamber was temperature-stabilized to 22 ( 0.05 °C. All substrates (AT-cut quartz crystals, f0 ) 5 MHz, with SiO2) and the QCM-D instrument (Q-Sense D300) were from Q-Sense AB. All SPR measurements were performed using a BIAcore 2000 system (Biacore AB, Uppsala, Sweden) at a flow rate of 30 µL/min, using gold chips coated with a 3-nm adhesion layer of Ti and 30 nm of SiO2. In the present study a multichannel mode was used, and the injection volume was always 325 µL. Modeling of SPR and QCM-D Data. In this work we have exploited the different measurement principles of QCM-D and SPR. For the latter, a well-established model (eq SI1 in Supporting Information) has been proposed for translation of changes in the SPR response to the mass of adsorbed biomolecules.25 The assumptions of this model: (i) the sensed film is much thinner than the decay length of the evanescent field (∼200 nm) and (ii) the refractive index increment with concentration is identical for biomolecules in bulk and in the adsorbed state, are sufficiently met under our experimental conditions. The QCM-D response (i.e., changes in f and D at various overtones) is, in contrast to SPR, sensitive to the viscoelastic properties, thickness, and density of any mass coupled to the mechanical oscillation of the quartz crystal. Out of several proposed models, the most used (and simplest) is the Sauerbrey equation.26 However, the assumption that the adsorbed film is rigid with no internal energy losses is not met in the present case (see below). We have therefore used a Voigt-based model,27,28 where the adsorbed layer is represented by a homogeneous viscoelastic film being characterized by a shear viscosity, a shear modulus, a thickness, and a density. In the present situation the film is composed of biomolecules adsorbed on the sensor surface from an aqueous solution. Thereby water (and other constituents in the buffer) will get acoustically “trapped” between adsorbed biomolecules (see schematic representation in Figure 1). This “trapped” water is by no means fixed to the film, but as probed by the crystal’s oscillation (i.e., hydrodynamically) it does not behave as the bulk liquid above the film. It is important to note that the Voigt-model parameters are effective parameters of the whole film (just like the SPR model

(19) Hillman, A. R.; Jackson, A.; Martin, S. J. Anal. Chem. 2001, 73, 540-549. (20) Ho ö ¨k, F.; Kasemo, B.; Nylander, T.; Fant, C.; Mjo ¨rn, K.; Elwing, H. Anal. Chem. 2001, 73, 5796-5804. (21) Ho ö ¨k, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloids Surf., B 2002, 24, 155-170. (22) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143, 513-526. (23) Barenholz, Y.; Gibbes, D.; Litman, J.; Goll, J.; Thompson, T. E.; Carlson, F. D. Biochemistry 1977, 16, 2806-2810.

(24) Keller, C. A.; Glasmastar, K.; Zhdanov, V. P.; Kasemo, B. Phys. Rev. Lett. 2000, 84, 5443-5446. (25) Lofas, S.; Malmqvist, M.; Ronnberg, I.; Stenberg, E.; Liedberg, B.; Lundstrom, I. Sens. Actuators, B 1991, 5, 79-84. (26) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (27) Bandey, H. L.; Hillman, A. R.; Brown, M. J.; Martin, S. J. Faraday Discussions 107: Acoustic waves and Interfaces; Lester, U.K., 1997; Vol. 107, pp 105122. (28) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391-396.

We have recently shown that one very reproducible immobilization strategy is to make use of biotin-modified supported lipid bilayers on SiO2, which allows for sequential coupling of streptavidin and single stranded biotin-modified DNA for subsequent DNA hybridization detection.17 Using QCM-D, we were in this way able to demonstrate single-mismatch discrimination for 15-mer DNA strands. In this work we extend our previous efforts by focusing on how combined frequency (f) (cf. adsorbed mass) and energy dissipation (D) (cf. rigidity) QCM-D measurements combined with a theoretical viscoelastic representation can be used to further improve the characterization of surface immobilization DNA for hybridization kinetics measurements. The theoretical modeling of the QCM-D response is carried out using a Voigt-based representation, in which each layer is represented by four unknown parameters: an effective density, Ff; thickness, df; shear elastic (storage) modulus, µf; and shear viscosity (loss modulus), ηf. The nonuniqueness problem originating from a model containing four unknown parameters whereas only two parameters (∆f and ∆D) are simultaneously measured19 is taken care of by QCM-D measurements at multiple harmonics, as described previously.20,21 The analysis is further combined with parallel SPR measurements, where the latter yields an independent measure of the coupled mass22 and thus an independent mean to determine the effective film density and film thickness. It is also demonstrated how this combined analysis improves the precision by which the viscoelastic components of the probed films can be separated and quantified. The use of the so achieved information for improved DNA coupling and hybridization efficiency is demonstrated.

Analytical Chemistry, Vol. 75, No. 19, October 1, 2003

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Figure 1. Schematic representation (top) of the immobilization strategy and the Voigt-based model used to represent the QCM-D response during subsequent formation of (i) a biotin-modified supported lipid bilayers, (ii) a 2D arrangement of streptavidin, (iii) coupling of biotin-DNA, and (iv) subsequent hybridization. Note that during hybridization, the changes are assumed to occur within the same layer resulting in changes in F, δ, η, and µ. (a) ∆fn)3 (cf. mass uptake) and (b) ∆Dn)3 (cf. rigidity) versus time upon sequential exposure of a SiO2 coated QCM crystal to (i) biotin-doped lipid vesicles (0.16 mg/mL) and (ii) streptavidin (0.2 µM) followed by superimposed responses observed upon addition of (iii) (open squares) b-DNA15 (0.25 µM) and (open circles) b-DNA30 (0.25 µM) and finally (iv) DNA hybridization with a fcDNA15 (0.25 µM) and fcDNA30 (0.25 µM). Steps i-iv were done in buffer 1, while step iv was done in buffer 2. The buffer exchange has been corrected for in the graph. All changes in frequency have been divided by overtone number.

uses an effective thickness and an effective refractive index for the biomolecules in the film). This means that within the frame of this model, it is not possible to separate what part of the film’s mass that originates from adsorbed biomolecules and what part that originates from water: The mass sensed is the mass of biomolecules plus “trapped” water. Unfortunately, several QCM studies have treated the mass measured with QCM as being equal to the number of adsorbed molecules (per surface area) times their molecular weight. This has, in turn, caused a lot of confusion when comparing that data to that obtained with other techniques (such as SPR). In this work it is demonstrated that by combining SPR and QCM-D data it is possible to obtain several synergistic effects, mainly by being able to extract more information out of measured QCM-D data and thereby making more precise predictions about not only structural properties of the adsorbed films but also the water content (“trapped” water). The latter is extracted by simply taking the difference between measured QCM-D mass and SPR mass. A less obvious benefit by combining SPR and QCM-D is also the possibility to separate the film mass into effective film density 5082


and effective film thickness. The problem in doing that from QCM-D data alone originates from the fact that the response, to a first order, only depends on the product of thickness and density (i.e., film mass). This is indeed similar to the ellipsometry/SPR case, where it is hard to separate the contribution from changes in refractive index, ∆n, and thickness, ∆d. However, in agreement with ellipsometry/SPR analysis, where the obtained mass (being proportional to ∆n × ∆d) is not influenced by this uncertainty,29 the obtained mass from a Voigt-based analysis of the QCM-D response is conserved independent of which density (or thickness) that is chosen as a “known” input in the modeling. Thus, given the total mass obtained from the Voigt-based analysis, combined with the optical mass obtained from SPR analysis, the thickness and density can indeed be separated. The way by which this is done can be described as follows. Note first that the film consists of biomolecules and water (we are for simplicity ignoring ions and other constituents of the buffer). This means that the total volume of the film is the sum of the volume occupied by the biomolecules and the volume (29) Tiberg, F. J. Chem. Soc., Faraday Trans. 1996, 92, 531-538.

occupied by water. Using that a volume can be expressed as its mass divided by its density we arrive at the following equation:

∆mSPR ∆mQCM - ∆mSPR ∆mQCM ) + Feffective Fbiomolecule Fwater

(1)

In eq 1, we have omitted to multiply ∆mSPR and ∆mQCM with the area of the film since it cancels out. The effective thickness, deffective, can thus be defined as

deffective )

∆mQCM Feffective

(2)

where ∆mSPR and ∆mQCM are the changes in mass due to the biomolecules adsorbing as measured by SPR and QCM, respectively. Fbiomolecule and Fwater are the densities of water (∼1 g‚cm-3) and biomolecules, being 1.1, 1.35, and 1.7 g‚cm-3 for the lipid bilayer, streptavidin, and DNA, respectively. By using the Voigt model (for details, see Supporting Information) to extract the total mass of the film, eqs 2 and 3 can then be used to obtain the film density and the film thickness. RESULTS AND DISCUSSION The formation of the senor template is schematically illustrated in Figure 1, also displaying a typical QCM-D measurement (∆f and ∆D versus time) upon exposure of a SiO2 surface to (i) a biotin-doped lipid-vesicle solution (t ∼ 5 min) followed by (ii) subsequent addition of (ii) streptavidin (t ∼ 30 min), (iii) b-DNA15/30 (t ∼ 62 min), and finally (iv) fc-DNA15/30.30 At saturated binding after each exposure, the solutions were exchanged to pure buffer.31 A typical fit between the QCM-D response and the Voigt-based model is shown in Figure 2 for coupling of 15-mer biotin-DNA and subsequent hybridization (cf. Figure 1) for n ) 3 (15 MHz) and n ) 5 (25 MHz). Equally good fits were obtained for the b-SPB formation and SA binding (not shown). From Figure 2 it is clear that the agreement between the fit and the measured data is within 2% for all harmonics. Even though the good agreement shows that the Voigt-based model can reproduce the measured QCM-D data, it does not prove that the model is a true representation of the measured system. The model is a simplistic representation of the real system (e.g., immobilized DNA strands together with acoustically coupled water are modeled as a homogeneous film, cf. Figure 1) which simply means that the obtained parameters from the modeling should be seen as effective parameters. (30) The biotin-modified strands are assigned 15-mer and 30-mer, with reference to the complementary part of the sequence, thus excluding the linker composed of 5 cytosine bases. (31) In separate experiments it was shown that DNA without biotin induces no change in f or D when added to SA or b-SPB (not shown). It was also demonstrated that fcDNA15 strands carrying two mismatch base-pairs (mm2) or more did not display detectable hybridization to b-DNA15, whereas single mismatch discrimination between fc and mm1 could be made from variations in the rate of binding and the total coupled amount (not shown). A similar discrimination for fcDNA30 could not be made between fc and mm1, but discrimination could be made for mm2 or more (not shown).

Figure 2. ∆f and ∆D versus time at n ) 3 (dashed lines) and n ) 5 (filled lines) for the QCM-D data on b-DNA15 coupling and fcDNA15 hybridization shown in Figure 1. The data are normalized to the starting value prior to addition of b-DNA. The best fit between the viscoelastic model (see Supporting Information) and the experimental data are shown as open circles (∆fn)3), open squares (∆Dn)3), open diamonds (∆fn)5), and open triangles (∆Dn)5). All changes in frequency have been divided by overtone number. Table 1. Analysis of the SPR and QCM-D Responsese ηf immobilization ∆mSPR ∆mQCM a,b µf deffective a (ng/cm2) (ng/cm2) (mPa‚s) (MPa) (eq 2) (nm) step supported lipid bilayer streptavidin b-DNA c 15-mer 30-mer DNA duplex c,d 15-mer 30-mer

320

442 (442)

>20

200

447 (442)

82

23 32

329 (245) 437 (326)

36 54

477 (354) 693 (536)

>1

4.1

0.28

3.4

1.8 1.8

0.19 0.19

3.1 4.1

2.0 2.0

0.19 0.2

4.5 6.4

a The mass and thickness was obtained using an effective density of 1.06 g/cm3 (eq 1). However, identical mass values were obtained from the Voigt modeling if the density was varied between 1.0 and 1.7 g/cm3. b Values within parentheses refer to the Sauerbrey relation using n ) 3, i.e., 15 MHz. c These values were obtained using a multilayer modeling, in which the rigid bilayer (D < 10-7) and the SA layer were also included. The results were, however, identical if the bilayer and SA were treated as perfectly rigid. d The data refers to the total mass-uptake, density, and effective thickness (i.e., layer 3 in Figure 1) after DNA hybridization, excluding the b-SPB and SA. e Summary of the analysis of the SPR and QCM-D data upon the sequential additions shown in Figure 1. All values refer to saturated binding using the same batch of surfaces, lipids, proteins, and DNA, for which the experimental variation was (1%. The variation in thickness, taking this experimental variation into account, yields a similar variation in df, whereas it is increased to (5% for ηf and µf. The latter variation increased, however, to ∼(10% if the variation from measurements using different batches were taken into account.

The fit shown in Figure 2 was equally good independent of the density chosen as input parameter, even though different choices of the density produced variations in the set of output parameters (viscosity, elasticity, and thickness). However, the coupled mass (density × thickness) was in this particular case shown to be independent of density (within a reasonable interval), which thus verifies the use of eqs 1 and 2 to estimate film density and thickness. The sequence of events shown in Figure 1 were also analyzed with SPR using an identically treated SiO2-coated SPR sensor chip as summarized together with the output parameters from the Voigt-based modeling in Table 1. Also shown is the mass obtained from a simplified analysis using the classical Sauerbrey relation to convert changes in f into coupled mass (using n ) 3, i.e., 15 MHz). Analytical Chemistry, Vol. 75, No. 19, October 1, 2003

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Analysis of Coupled Water. The initial exposure of lipid vesicles results in spontaneous lipid-bilayer formation,24,32 and the subsequent binding of streptavidin is in good agreement with previous results.17 The slow decrease in D observed at about half streptavidin coverage is tentatively attributed to protein-2D crystal formation.33 The observed difference between ∆mQCM and ∆mSPR (Table 1) for these two steps is attributed to the fact that the mass uptake estimated from the QCM technique includes water hydrodynamically coupled to adsorbed biomolecules,21 whereas the SPR response, which originates from changes in the refractive index as water is replaced by biomolecules, is proportional to the molecular weights of adsorbed biomolecules.22 Accordingly, while the percentage of water coupled to the planar SPB is, as expected, relatively low (∼25%), it is ∼55% for streptavidin, in good agreement with that expected for a densely packed 2D arrangement of a symmetric protein assuming that most water between the proteins is sensed. Considering the DNA coupling, a comparison between ∆mQCM and ∆mSPR shows that the DNA duplexes form very water-rich films containing up to ∼90% water. In combination with the large energy dissipation per coupled mass (cf. Figure 1), this demonstrates that the DNA layers are composed of DNA strands/duplexes adopting elongated and flexible structures on top of the 2D arrangement of streptavidin. We emphasize this as a most important observation, pointing toward the large error introduced (up to 1 order of magnitude) when converting the frequency change into molecular mass, which has been done so far in most QCM studies involving immobilized DNA. Also note that for the viscolestic DNA films, the obtained masses from the Voigt modeling are about a factor of ∼1.3 larger than those obtained using the classical Sauerbrey relation, whereas there is very good agreement for the more rigid films (b-SPB and SA). This emphasizes the importance of using a viscoelastic representation to estimate the coupled mass for viscoelastic films (inducing significant changes in D), even though this error is, in this particular case, less than that introduced due to coupled water. One should be aware, however, that given that the amount of coupled water per DNA molecule does not vary with coverage (see below), kinetic constants can still be estimated with good accuracy.16,34 For instance, from the data shown in Figure 1, it can be concluded that the rate of hybridization with fc-DNA30 is significantly more rapid than that of fc-DNA15. Since the diffusion constant decreases with increased length (hydrodynamic radius) of DNA, the higher rate of hybridization for the 30-mer strand compared with the 15-mer strand demonstrates that for fc-DNA15 the actual hybridization reaction cannot be limited by masstransport but corresponds to the rate of the actual hybridization reaction. It is also interesting to note that the sensitivity of the QCM-D system increases significantly compared to SPR (or any other optical technique based on detection of changes in interfacial refractive index) in situations when a large amount of water is coupled (or, for that reason, if the amount of water changes significantly during a reaction20). In this particular case, QCM-D beats the sensitivity (signal-to-noise) of the SPR system by (32) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397-1402. (33) Reviakine, I.; Brisson, A. Langmuir 2001, 17, 8293-8299. (34) Okahata, Y.; Kawase, M.; Niikura, K.; Ohtake, F.; Furusawa, H.; Ebara, Y. Anal. Chem. 1998, 70, 1288-1296.


Figure 3. The time evolution of the changes in the effective thickness, df (red), and the shear viscosity, ηf (blue), obtained from the fit shown in Figure 2 (and Table 1).

approximately a factor of 3. Note, however, that the QCM response is not quantitative in the same respect as SPR, since the latter (for thin films) gives a response being proportional to the molecular weight of the adsorbed biomolecules.22 The information from SPR can thus be used to quantify the number density of different types of adsorbed biomolecules. In relation to previous applications of the QCM technique to probe DNA mediated reactions, for example, polymerase-induced elongation of DNA,35 this must be considered a most relevant conclusion, since proper quantification of the relative amount of protein and DNA can hardly be made from a simplified QCM analysis. In our particular case it is interesting to note that the mass uptake of streptavidin and b-DNA, given the molecular weights of streptavidin (60 kD) and b-DNA15 (6.5 kD) signals that approximately one b-DNA is coupled per streptavidin based on SPR. Intuitively, one might expect a 2:1 DNA/streptavidin ratio, since two of the four biotin-binding sites of streptavidin are expected to be exposed to solution in a planar 2D arrangement of streptavidin.36,37 However, a closer inspection of the anticipated 2D arrangement of streptavidin on SPBs shows that the distance between the binding sites on one molecule is close to the hydrodynamic diameter of DNA, whereas the distance between DNA duplexes on adjacent molecules is likely to be larger. A combination of electrostatic repulsion and steric hindrance may thus prevent efficient coupling of two biotin-DNA per streptavidin (see further below). In contrast, if hydrodynamically coupled water is not considered, the QCM data suggests ∼5 DNA per streptavidin. The latter is obviously not reasonable. Evaluation of the Complex Shear Modulus. One set of obtained parameters from the modeling is shown in Figure 3, displaying the time evolution of changes in shear viscosity and thickness (obtained using eq 1 using a density of ∼1.06 g/cm3 which was obtained from eq 2) versus time. (The variations in the elastic modulus was qualitatively similar to that of the viscosity, but the noise is too big to draw detailed conclusions about variations versus time or coverage, see Table 1.) During b-DNA15 coupling and subsequent hybridization, the effective viscosity is ∼1.2 mPa‚s at low coverage, a value being relatively close to that of water (∼1 mPa‚s) as expected for a film that couples a substantial amount of water. As the coverage increases the shear (35) Niikura, K.; Matsuno, H.; Okahata, Y. J. Am. Chem. Soc. 1998, 120, 85378538. (36) Lindqvist, Y.; Schneider, G. Curr. Opin. Struct. Biol. 1996, 6, 798-803. (37) Diamandis, E. P.; Christopoulos, T. K. Clin. Chem. 1991, 37, 625-636.

viscosity increases to 1.8 mPa‚s at saturated b-DNA binding and eventually 1.95 mPa‚s after completed DNA duplex formation. Even if the shear viscosity increases as the coverage increases, the value still remains low. For instance, the value at saturation is close to that obtained for a solvated mussel-adhesive protein adsorbed in a flexible state on a hydrophobic surface (∼1.9 mPa‚s)20 but much lower than the values for the less hydrated state of the adsorbed mussel proteins after chemical cross linking (∼6 mPa‚s) or, for that reason, that obtained for the b-SPB or streptavidin (Table 1). The observed correlation between high water content and low viscosity further signals that the viscosity can be used as a rough estimate of the water content of the films. It is also interesting to note that the hybridization demonstrates an increase in effective thickness from ∼3.1 nm for single stranded b-DNA to ∼4.5 nm for the layer composed of DNA duplexes. (For the longer DNA strands, the effective thickness increased from 4.1 to 6.4 nm upon hybridization.) This increase in effective thickness during hybridization of the 15-mer (and 30-mer) DNA strand(s) suggests that single stranded b-DNA forms a layer composed of coiled oligonucleotides, which are being stretched out during the duplex formationsan observation which is being in good agreement with previously observed thickness increase during DNA hybridization measured using SPR-induced fluorescence.12 It must be pointed out, though, that the density was kept constant throughout the modeling. It can thus be argued that the observed increase in viscosity/thickness versus time originates from a compensation of the fact that the density of the film varies as the coverage increases. However, changing the density to a lower value, being expected at lower coverage, will primarily effect the obtained thickness (see above), whereas the resulting decrease in viscosity and increase in elastic modulus turns out to be less than 3%. Furthermore, since this analysis critically relies on an independent mean for determination of the effective density (eq 1), it is also relevant to analyze the error, or even error propagation, that is introduced if an independent mean to determine the density is not available. Assume, for instance, that the effective density was initially chosen to be 1.7 g/cm3 (i.e. the density of pure DNA). This will not effect the quality of the fit at all (not shown), but the output values will be different: (i) the thickness will be lowered by a factor corresponding exactly to the ratio between the two densities ()1.7/1.06), that is, the coupled mass is conserved, and (ii) the shear viscosity and shear elastic modulus will both be a factor of about 2 lower. This thus illustrates the typical sensitivity of the modeling to our main assumption. One should be aware, however, that this sensitivity to the input parameter(s) should not be taken as a general rule but must be carefully evaluated in each particular case. Evaluation of the Frequency Dependence of the Complex Shear Modulus. One must be aware that our means of applying the Voigt-based model also relies on the assumption that the viscoelastic components (ηf and µf) are frequency independent. To test this assumption, we made use of the fact the thickness and density are not expected to be frequency dependent. Thus, the presence of an explicit frequency dependence of the viscoelastic components was possible to test by keeping the density and

Figure 4. Shear viscosity (a, c) and shear elastic modulus (b, d) versus overtone number (n) for immobilized single stranded b-DNA (a, b) and the DNA duplexes (c, d) formed after hybridization with complementary sequences (cf. above). The error bars originates from the noise in ∆f and ∆D from three separate measurements.

thickness (Table 1) fixed, followed by a fitting procedure utilizing individual harmonics, that is, two measured parameters (∆f and ∆D) at one harmonic. Figure 4 shows the obtained variation in viscosity and shear modules versus overtone number, n, for single stranded b-DNA15 and subsequently formed DNA duplexes (cf. Table 1).38 It is clear from this analysis that both the shear viscosity and shear elastic modulus exhibit a weak frequency dependence, where the former decreases by ∼20% and the latter increases by ∼50% over the whole range of harmonics analyzed (15-65 MHz). First, this demonstrates that the different states of DNA exhibits molecular relaxation in the low MHz region (10-100 ns), which certainly points toward a new phenomenon valuable to explore in greater detail. In addition, this analysis gives a number on the uncertainty introduced by the use of two nearby harmonics to create an overdetermined system. This uncertainty was further evaluated by introducing a weak frequency dependence in the viscoelastic components mimicking the behavior seen in Figure 4 (see Supporting Information). Indeed, introducing a frequency dependency significantly improved the fit (if more than two harmonics were utilized in the fitting procedure) and nicely confirmed the variation in absolute numbers shown in Figure 4. More important, however, was the observation that the relative variations (e.g. changes in viscosity/elasticity versus time and/ or coverage) were shown to be independent of frequency (not shown), thus strongly supporting our way of treating the QCM-D response. Temporal Variation of the Shear Viscosities. Given this information, we now turn to a more detailed analysis of the (38) The thickness determined using higher harmonics did not vary by more than 5%, which, when used to determine the viscosity and elastic modulus vs n, resulted in variations in the absolute values of less than 3%, while the relative variations versus overtone number remained essentially identical to those shown in Figure 4.


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Figure 5. (a) Changes in shear viscosity, ηf, versus coverage (df × Ff) using the data in Figure 3. Also shown for comparison is a curve displaying ∆D vs ∆fn)3. (b) Magnification of ∆f versus time for the DNA hybridization step at full and half coverage, where the response at half coverage is normalized with respect to the changes induced by streptavidin. Also shown are least-squares fits to a single- and double-exponential curve for half- and full coverage, respectively. All changes in frequency have been divided by overtone number.

temporal variation in the shear moduli. Figure 5a shows the changes in viscosity plotted versus the time evolution of ∆mQCM. At low coverage, there is essentially a linear increase in viscosity versus coverage. If the origin of the viscosity is interpreted as resulting from viscous friction induced by the coupling between hydrodynamically entrapped water and the immobilized DNA strands, a linear increase in viscous friction versus coverage is indeed expectedsgiven that each immobilized DNA adopts the same conformation. However, at about 75% coverage, the slope of the curve clearly changes, signaling some kind of coverageinduced structural rearrangement in the film, rendering a relative increase in the effective viscosity (cf. rigidity) of the DNA film. Also note that a very similar observation is made if ∆D is plotted versus ∆f, as previously used to identify coverage-induced structural changes of immobilized biomolecules.17,39 In this type of qualitative inspection of combined ∆f and ∆D measurements, a decrease in ratio between ∆D and ∆f reflects an increase in rigidity (cf. increase in viscosity). Since the measure of ∆f and ∆D does not rely on the “constant density assumption”, the nice qualitative agreement between the ∆f vs ∆D and viscosity vs coverage curves further supports that this assumption has a negligible influence on the interpretation. One should be aware, however, that if a low density (∼1.0 g/cm3) is used at low coverage and a very high density (1.7 g/cm3) is used at high coverage, the appearance of this representation changes dramatically. Thus, for significant changes in the effective density (e.g. water content) versus coverage the analysis becomes uncertain, again pointing toward the importance of an independent determination of the effective density. (39) Ho ö ¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14(4), 729-734.


A detailed interpretation of the relation between changes in effective viscosity (and/or elasticity) and structural changes of individual DNA strands cannot be done from this type of meantheory analysis. It is still tempting to interpret the deviation from a linear slope between viscosity and coverage as originating from a coverage-induced change in lateral interaction between adjacent immobilized strands, possibly complicated by the fact that the change in lateral dimension (roughness) of the film as the coverage varies may also contribute to changes in energy dissipation.40,41 To evaluate these scenarios, the number density of the immobilized DNA strands was reduced by (i) interrupting the streptavidin binding at the peak in the D vs time curve, that is, at approximately half coverage, followed by a repetition of the steps shown in Figure 1 and (ii) coupling of 15-mer DNA duplexes preformed in solution prior to coupling to the streptavidin layer. Concerning the latter approach, the absolute mass-uptake was reduced compared with the coverage obtained upon sequential coupling of first single stranded DNA followed by hybridization with full complementary DNA, and a perfectly linear relation between ∆D and ∆f was observed (not shown). Due to the higher lateral repulsion expected for DNA duplexes, these observations strengthen that lateral interactions between single stranded DNA, induced by a strong coupling to streptavidin and weaker intermolecular repulsion, are responsible for the observed peculiar change in viscosity (Figure 5a). Also, since in both these cases a similar change in lateral dimension is expected, the linear ∆D vs ∆f curve observed for direct coupling of DNA duplexes signals that changes in roughness do not explain the trends observed in Figure 5a. This is also in agreement with previous theoretical/ experimental work evaluating the influence from roughness on changes in f and D, indicating that variations in the lateral dimensions on this length scale (few nm) has a minor influence on changes in D (∆D

Characterization of DNA Immobilization and Subsequent Hybridization

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