Construction and Modeling of Concatemeric DNA Multilayers on a

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Construction and Modeling of Concatemeric DNA Multilayers on a Planar Surface as Monitored by QCM‑D and SPR Lu Sun,† Sofia Svedhem,‡ and Björn Åkerman*,† †

Department of Chemical and Biological Engineering and ‡Department of Applied Physics, Chalmers University of Technology, 412 96 Gothenburg, Sweden S Supporting Information *

ABSTRACT: The sequential hybridization of a 534 base pair DNA concatemer layer was monitored by QCM-D and SPR, and the QCM-D data were analyzed by Voigt viscoelastic models. The results show that Voigt-based modeling gives a good description of the experimental data but only if shear viscosity and elasticity are allowed to depend on the shear frequency. The derived layer thickness, shear viscosity and elasticity of the growing film give a representation of the DNA film in agreement with known bulk properties of DNA, and reveal a maximum in film viscosity when the molecules in the layer contain 75 base pairs. The experimental data during construction of a 3084 bp DNA concatemer layer were compared to predictions of the QCM-D response of a 1 μm thick film of rod-like polymers. A predicted nonmonotonous variation of dissipation with frequency (added mass) is in qualitative agreement with the experiments, but with a quantitative disagreement which likely reflects that the flexibility of such long DNA molecules is not included in the model.

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INTRODUCTION The behavior of DNA molecules when attached to macroscopic surfaces is important in applications such as chip technologies used for sequencing1 and diagnostics2,3 but also in microfluidic constructs such as DNA curtains.4 Tethered DNA molecules, where one end of the DNA molecule had been attached to a solid support, and the subsequent hybridization have been investigated by various techniques such as X-ray photoelectron spectroscopy (XPS),5 fluorescence microscopy,6,7 surface plasmon resonance (SPR),8−11 dual polarization interferometry (DPI),12 ellipsometry,13 and quartz crystal microbalance (QCM).14−18 In order to achieve an improved characterization of the immobilized DNA, a combination of these methods is often employed.15,16 For instance, the SPR optical method can be used to measure the number of surface-bound biomolecules (moptic) and the kinetics of their attachment, but little knowledge is gained regarding their conformation. QCM-D, on the other hand, is an acoustic technique which probes the structural properties of the layer in terms of its viscoelastic behavior, a key feature in the case of the soft DNA layers studied here. However, QCM-D alone cannot measure the amount of adsorbed biopolymer since it also detects water molecules that are associated with the attached biomolecules (macoustic). The effect of associated mass (hydration) is a major contribution in the case of the DNA films studied here, which tend to contain about 90% water.15 In order to derive the relative water content of the DNA film ((macoustic − moptic)/ moptic), a combination of QCM-D with an optical technique such as SPR or DPI is essential but also requires appropriate modeling of the acoustic mass (macoustic). © 2014 American Chemical Society

Investigations based on the combination of QCM-D and SPR have shown that the QCM-D response of biofilms can be quantified into layer thickness and viscoelastic properties by Voigt-based viscoelastic models based on fitting the QCM-D responses at different overtones,19,20 including DNA films,14 an improvement compared to the qualitative approaches to plotting the change in dissipation (ΔD) versus the change in resonance frequency (Δf)21 or using the acoustic ratio ΔD/Δf to evaluate the intrinsic viscosity of the DNA molecules.22,23 To our knowledge the viscoelastic modeling has been applied only to thin DNA films, containing molecules a few tens of base pairs long. Layers of longer DNA molecules attached to surfaces are used extensively, such as in DNA curtains for protein-interaction studies,4,24 mechanical experiments,25 and chromosome analysis,26 not only because longer DNA gives rise to a stronger signal. On the other hand, the base sequence is known to be important for the mechanical properties of DNA,27 and this parameter is difficult to vary systematically when the long DNA is of biological origin. Here we report on the use of QCM-D and SPR to monitor stepwise-assembled films of DNA concatemers on a streptavidin surface using synthetic oligonucleotides as building blocks to form molecules of up to 3084 base pairs long. We apply Voigt-based models28 to interpret QCM-D and SPR data in terms of the thickness and viscoelastic properties of these synthetic DNA films. The data strengthens the conclusion from a previous multilayer liposome study20 and theoretical Received: October 7, 2013 Revised: June 14, 2014 Published: June 27, 2014 8432

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calculations28,29 that Δf and ΔD in QCM-D do not necessarily exhibit a monotonous change during the formation of viscoelastic layers of increasing thickness and furthermore indicates that the viscoelastic properties of the tethered DNA molecules change drastically for molecules a few hundred base pairs long. Using DNA layers containing a molecule with wellknown structural and elastic properties allows us to evaluate different versions of Voigt-based viscoelastic models for polymer films.

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bases by alternatively adding oligonucleotides A′B′ and AB. The 25ss oligonucleotide A is added at the end of the assembly to form a capped DNA layer which is double-helical to the very end. DNA stock solutions of the oligonucleotides (25 μM strands) were prepared by dissolving the provided dry powder in deionized water (obtained from a Milli-Q unit, Millipore, France). QCM-D and SPR Measurements and Preparation of the Sensor Surfaces. QCM-D experiments were performed using a QSense E4 instrument (Q-sense E4, Sweden). AT-cut 5 MHz quartz crystals sputter-coated with a 100-nm-thick Au layer onto a 50 nm chromium adhesive layer were obtained from Q-Sense AB, Sweden. All changes in frequency are shown after dividing by overtone numbers (n = 3, 5, 7, 9, and 11). SPR experiments were performed using a BIAcore 2000 instrument (Biacore, Sweden) and a gold surface sensor SIA kit Au (GE Healthcare, Sweden). The gold surfaces on both QCM-D and SPR sensors were biotinylated by forming a mixed self-assembled monolayer (SAM) from an oligoethylene glycol (OEG) solution containing 1% biotin disulfides (−(S−C2H4−CO−NH−(CH2−O−CH2)9−NH−CO−C4H8−biotin)2 and 99% nonbiotin disulfides (−(S−CH2−(CH2−O−CH2)7−CH2−OH)2), both from Polypure, Norway following a published procedure.30 Real-Time Detection of Streptavidin and DNA Binding. In QCM-D experiments, changes in frequency (Δf) and dissipation (ΔD) were collected in real time while PBS buffer (5 mM NaH2PO4, 5 mM Na2HPO4, 27 mM KCl, 137 mM NaCl, pH 7.4) continuously flowed over the sensor surface at a rate of 50 μL/min. The measurement chamber was temperature stabilized to 22 ± 0.02 °C. Streptavidin (Sigma-Aldrich) was introduced onto the biotinylated gold surface at 25 μg/mL in PBS buffer. The streptavidin surface was exposed to biotinylated b-AB59 for 10 min. Hybridization of A′34, A′B, and AB took place until almost saturation after 50, 10, and 10 min, respectively, followed by the excess nonbound oligonucleotides being rinsed away from the surface by a flow of buffer for a minimum of 3 min. The same procedure was used for each alternating addition of semicomplementary oligonucleotides AB and A′B′. Oligonucleotide solutions were introduced into the QCM-D chamber and SPR flow cell at a concentration of 0.2 μM. When performing SPR experiments, each injection volume has a limitation of 340 μL. To optimize the use of the sample, we decided to use a slower flow rate of 5−20 μL/min to process the same procedures as in QCM-D but wait a longer time at each step to compensate for the slow flow. Experimental Assembly of a 3084 Base Pair Concatemer. QCM-D instrument Omega Auto (Q-sense, Sweden) was programmed to assemble a 3084 bp concatemer (124 steps in total) using the same protocol and conditions for DNA assembly as described above. Briefly, all hybridization steps were performed at 22 °C in PBS buffer (5 mM NaH2PO4, 5 mM Na2HPO4, 137 mM NaCl, 27 mM KCl at pH 7.4 if not otherwise stated) at a flow rate of 20 μL/ min and with an oligonucleotide concentration of 0.2 μM strands. The hybridization of A′34 and the first A′B′ (Figure 1) was programmed to last for 2 h and 30 min in order to ensure complete binding, and the subsequent hybridization steps of AB and A′B′ lasted 20 min each, considering that their hybridization takes about 10 min to complete under the present conditions. The protocol called for a 5 min rinse with PBS buffer between each injection of the oligonucleotides in order to remove any oligo from the previous step. The total assembly took 54 h. Gel electrophoresis control experiments of bulk samples showed that concatemers formed in the PBS buffer were stable at 22 °C for the 54 h used in the longest-lasting assembly experiments on surfaces. Modeling and Prediction of QCM-D Data. The two commonly used models in QCM-D are the Sauerbrey equation and the Voigtbased viscoelastic model. Both of the models assume that films have a homogeneous distribution. The former equation is used for thin and rigid films,31 while the latter one is often applied to the study of soft and viscous films.28 In the Sauerbrey equation, Δf is directly proportional to the change in mass, whereas in the Voigt-based model, Δf and ΔD are represented by viscoelastic components:

MATERIALS AND METHODS

Design of DNA. DNA oligonucleotides were synthesized at the 1 μmol scale and purified by HPLC by Atdbio (Southampton, U.K.). Sequences of the oligonucleotides can be found in the Supporting Information, Table S1. Figure 1 schematically shows the sequential

Figure 1. (Top) Schematic representation of the first five steps for building DNA concatemer layers on a gold surface. (1) Formation of a streptavidin monolayer on a self-assembled layer of biotinylated OEG. (2) Binding of a biotinylated oligonucleotide (b-AB59) to the streptavidin layer. (3) Hybridization of oligo A′34 to the bottom half of b-AB59, which gives rise to a restriction site close to the surface. (4) Hybridization of semicomplementary A′B′ to the top of the DNA layer using b-AB59 as a seed. (5) Hybridization of AB after washing off excess A′B′. (Bottom) Real-time QCM-D measurements of frequency change Δf (black, left axis) and dissipation ΔD (red, right axis) during assembly steps 1−5 using the 7th overtone. Measurements at other overtones, 3rd, 5th, 9th, and 11th, exhibited similar trends. The zero level for Δf and ΔD corresponds to the biotinylated gold surface. assembly platform of DNA multilayers on a streptavidin-modified surface. The oligonucleotides are named b-AB59, A′34, A′B′, AB, and A, where the basic building blocks (AB and A′B′) are 50-mer semicomplementary oligonucleotides. The sequences of the A and A′ parts are designed to hybridize into 25 base pair (bp) duplexes and similarly for parts B and B′. The 59-mer oligonucleotide b-AB59 carries a biotin at the 5′ end which is used to immobilize the first (seed) DNA strand of the concatemers to the streptavidin-modified surface. For future use, compared to AB it also contains the extra four bases GATC that form a restriction site for the endonuclease Dpn II when b-AB59 is hybridized with the 34-mer oligonucleotide A′34. The restriction site is distanced from the surface using a 5T spacer in b-AB59 and a corresponding 5A part in A′34. The hybrid between b-AB59 and A′34 form a 34 ds +25 ss DNA layer which can be extended in steps of 25 8433

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Table 1. Characterization of the DNA Platform by QCM-D and SPR name of layera SA b-AB59 A′34 A′B′ AB

Δf 0 (Hz)b −22.0 −43.6 −17.7 −18.5 −19.5

± ± ± ± ±

0.45 2.86 2.05 0.96 1.58

ΔD0 (10−6)b 0.1 4.7 0.6 3.1 2.4

± ± ± ± ±

0.03 0.15 0.16 0.15 0.26

ΔmQCM (ng/cm2)c 389 798 285 352 366

± ± ± ± ±

7.94 31.65 25.07 53.90 27.28

ΔmSPR (ng/cm2)d 181.6 95.3 31.5 38.9 30.1

± ± ± ± ±

degree of hydration (%)e

6.15 2.42 0.64 0.71 1.32

53 88 89 88 92

± ± ± ± ±

1.62 0.01 1.01 1.60 0.48

Δdeff (nm)f 3.4 7.2 2.8 3.5 3.3

± ± ± ± ±

0.08 0.30 0.27 0.27 0.22

surface coverage (pmol/cm2)g 3.43 5.27 3.00 2.52 1.9

± ± ± ± ±

0.10 0.11 0.05 0.04 0.07

binding efficiency (%)h NA 153 ± 59 ± 84 ± 78 ±

2 2 2 3

See the top part of Figure 1. The base sequences of the oligonucleotides are given in Table S1. bFrequency and dissipation changes at the first overtone. cFor streptavidin from the Sauerbrey equation, ΔmQCM = C·(−Δf),27 where C = 17.7 ng·cm−2·Hz−1 for a 5 MHz quartz crystal. For the DNA multilayer, ΔmQCM is obtained from extended Voigt modeling using the same procedure as Larsson et al.15 dFrom SPR data (Figure 2B) assuming that a 1000 RU response corresponds to a change in surface concentration of 1 ng/mm2 for protein or DNA.35,36 eCalculated as (ΔmQCM − ΔmSPR)/ΔmQCM. fEffective thickness of the layer obtained from Voigt modeling, where the effective density was assumed to be ρ = 1.06 g·cm−3 for a water-rich DNA film (Materials and Methods).15 gSurface coverage is calculated from ΔmSPR as the number of bound target DNA molecules divided by the molecular weight. MWSA = 52.8 kDa, MWb‑AB = 18.1 kDa, MWA′34 = 10.5 kDa. MWA′B′ = 15.4 kDa, and MWAB = 15.4 kDa. h Hybridization efficiencies are calculated as the fraction of hybridized target coverage at saturation divided by the immobilized probe coverage. a

the first step of duplex formation (results not shown) were used as fixed numbers throughout the layer assembly. The theoretical concatemers were assembled by adding 25 bp duplex building blocks (the number of base pairs which in effect are added by each concatemer) rather than hybridizing two semicomplementary single strands. By this assumption, the DNA layers are still considered to be viscoelastic films but consist of stiff duplexes so that the effective thickness of the DNA layer is proportional to the added number of base pairs.

thickness (δ), density (ρ), shear viscosity (η), and shear elastic modulus (μ).28 The thickness and density cannot be determined independently, so we follow the procedure of Larsson et al.15 and assume a fixed value of ρ = 1.06 g·cm−3 for a water-rich DNA film. As discussed by Larsson et al., the assumed value of density in a range compatible with hydrated biomolecules has little effect on the fitted values of the other parameters in the case of thin films. The thicker and more flexible films studied here may potentially have a higher degree of hydration, but the hydration of thin films is already so high (reference 15 and Table 1 here) that the possible effect on the assumed density of the thicker layers studied here is neglected. The shear viscosity and the shear elastic modulus contribute to the complex-valued shear modulus G* of the film24,32,33

G* = G′ + jG″ = μ + j 2πfη

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RESULTS Platform for Building DNA Concatemers. The schematic graph in Figure 1 (top) shows the first five steps in the formation of surface-attached DNA concatemers and the Voigtbased model to represent the QCM-D response. The assembly is divided into layer 1, an essentially rigid lipid/streptavidin film, and layer 2, a viscoelastic film of end-on-immobilized DNA concatemers obtained by the sequential hybridization of A′34, A′B′, and AB to the platform (steps 2−5). Layer 1 is treated as a rigid layer with a given density (ρ1) and thickness (δ1) because ΔD is very low during step 1, while layer 2 is assumed to be a viscoelastic layer with a given density (ρ2), shear viscosity (η2), shear elastic modulus (μ2), and layer thickness (δ2). Utilizing this platform, we assembled DNA layers of controlled contour lengths in a stepwise fashion, where each concatemer step (A′B′ or AB) in effect adds 25 base pairs (bp) to the DNA layer. Figure 1 (bottom) shows the real-time QCM-D measurements during the first steps of the DNA layer assembly, where the frequency change (Δf) is inversely correlated to the addition of mass including water (i.e., for reasonably rigid layers, a negative Δf result corresponds to the accumulation of mass) and the corresponding change in dissipation (ΔD) depends on the viscoelastic properties of the layer (a softer layer will yield larger ΔD signals). During step 1, a monolayer of streptavidin (SA) is formed on a solid surface. During step 2, a biotinylated single-stranded oligonucleotide (b-AB59) is attached to the SA layer, while step 3 is to ensure that the DNA layer is double-stranded also in the part closest to the surface by hybridizing a 34 base oligonucleotide (A′34). Steps 4 and 5 correspond to the two first stages of subsequent concatemer assembly of oligonucleotides A′B′ and AB. Taken together, the observed QCM-D responses indicate that the platform in Figure 1 can be used to construct concatemers in a sequential fashion, as confirmed below by showing how A′B′ and AB can be sequentially hybridized in at least 20 steps using this platform

(1)

where G′ and G″ are the storage and loss moduli, respectively, and f is the shearing frequency. In the standard viscoelastic model, the shear viscosity and elastic modulus are assumed to be independent of frequency. In more advanced models they depend on frequency, and in what we will refer to as the extended viscoelastic model the shear viscosity follows the power law

ηn = η0(fn /f0 )α

η

(2a)

where n is the overtone number and f 0 = 5 MHz. The shear viscosity η0 at the fundamental frequency and the exponent αη (−2 ≤ αη ≤ 0) are used as fitting parameters. For the shear elastic modulus

μn = μ0 (fn /f0 )α

μ

(2b)

where μ0 (the shear elastic modulus at the fundamental frequency) and the exponent αμ (0 ≤ αμ ≤ 2) also are used as fitting parameters. The extended viscoelastic model thus contains five fitting parameters (δ, η0, αη, μ0, and αμ) since also in the extended model the density ρ is kept fixed. In this study, the streptavidin layer and DNA multilayer (Figure 1 top) are treated as rigid and viscous layers, respectively. The SA layer is well described as a rigid film, so here we assume that it acts as a part of the crystal. For the modeling of the DNA multilayer, the experimental QCM-D data were therefore set to zero after the formation of the SA layer (Figure 1, bottom). The QCM-D data were analyzed using the extended Voigt-based QTools modeling software (Q-sense, Sweden), using the combined data of Δf and ΔD at five harmonics (n = 3, 5, 7, 9, 11).24 Prediction of QCM Data for Thick DNA Films. In what we will refer to as the thick-film Voigt model we make the following simplifying assumptions to calculate the value of Δf and ΔD for effective film thicknesses of up to 1000 nm in steps of 8 nm for overtones 3, 7, and 11 using the standard Voigt model. The values of shear viscosity (η = 2.4 mPa·s) and shear elastic modulus (μ = 0.27 MPa) obtained from fitting the standard model (single-layer DNA film with frequency-independent parameters) to the experimental data in 8434

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Twenty-Step Assembly of a Double-Stranded 534 bp DNA Multilayer. Utilizing the platform in Figure 1, a 534 bp synthetic DNA concatemer multilayer was constructed by sequential hybridization of semicomplementary oligonucleotides A′B′ (10 times) and AB (9 times) in an alternating manner. In a final step, oligonucleotide A (Supporting Information) was hybridized to the top of the layer in order to make the DNA molecules fully double-stranded. Figure 2 shows the QCM-D and SPR measurement data during the whole assembly process.

whole assembly, albeit with decreasing magnitudes for all overtones as the number of concatemer steps increases. A similar pattern of negative−zero−positive changes in Δf combined with a monotonous but weakening increase in ΔD has been reported in a QCM-D study of layer-by-layer growth in the DNA-assisted assembly of lipid vesicles.20 Viscoelastic Modeling of the DNA Multilayer. Figure 3 shows the best fit of the experimental data (lines, from Figure

Figure 3. Experimental data (lines) and modeling results (symbols) for a 534 bp DNA film. QCM-D data on changes in frequency (Δf) and dissipation (ΔD) versus time at overtones n = 3 (solid line), 7 (dashed line), and 11 (dotted line) during the 20-step assembly in Figure 2. Best fit of the extended (frequency-dependent) Voigt model (symbols) to the experimental QCM-D data on Δf and ΔD at overtones n = 3 (squares), n = 7 (triangles), and n = 11 (diamonds).

2) to an extended Voigt model (symbols) where the shear viscosity and elastic modulus of the DNA layer (layer 2 in Figure 1) depend on frequency (overtone) according to eq 2. The underlying experimental data for the change Δf in frequency (black) and ΔD in dissipation (red) at three overtones (n = 3, 7, 11) when semicomplementary oligonucleotides A′B′ and AB were hybridized alternatingly to the platform are found in Figure 2. The modeling also included the data at overtones n = 5 and 9, which exhibit similar trends (fitting results not shown). Figure 3 clearly shows the good agreement between the fit and the measured data at all overtones, where the overall χ2 is 7.5 × 106. However, it is important to point out that even though Voigt-based model can reproduce the measured data it is not a true representation of the real system since the model assumes that the molecular film can be regarded as a continuous material. The obtained viscoelastic parameters should therefore be considered to be effective parameters. Figure 4 shows how the effective thickness (δ) and effective shear viscosity (η) obtained from the modeling vary with time during the assembly of the DNA layer. It is seen that the modeled layer thickness increases in a monotonous fashion with each added DNA concatemer, whereas the effective shear viscosity exhibits a marked maximum at the third step as the DNA layer is built thicker. The shear elastic modulus (Supporting Information, Figure S1) has a similar maximum at the fourth step. The inset in Figure 4 eliminates the time factor by plotting the effective shear viscosity versus the layer thickness. The viscosity is seen to exhibit three distinct phases as the DNA layer becomes thicker: (1) a strong increase in viscosity when the single-stranded b-AB59 is anchored to the rigid SA surface and a continued increase in viscosity by the hybridization of A′ at the bottom of b-AB59; (2) a slower

Figure 2. (A) QCM-D measurements and (B) SPR sensorgram of the stepwise assembly of a 534 bp synthetic double-stranded DNA multilayer. (A) Changes in frequency (Δf) and dissipation (ΔD) versus time at indicated overtones during the 20-step assembly when semicomplementary oligonucleotides A′B′ (10 times) and AB (9 times) are hybridized to the platform in Figure 1. The zero levels for Δf and ΔD correspond to a fully formed streptavidin layer. (B) Separate SPR measurement during the assembly of the same 534 bp layer. In both QCM-D and SPR, the first step corresponds to the biotin-DNA b-AB59 being immobilized on the streptavidin surface.

As can be seen, in the beginning of the assembly, each hybridization step of A′B′ or AB causes a decrease in Δf at all harmonics as expected from the added mass, and also the magnitude of the change is slightly smaller with each step. As the DNA layers are grown further, the change in frequency corresponding to a certain hybridization step becomes nondetectable and finally is positive. Overtone 11 (Δf n=11) is the first to show a slight increase in frequency during the 10th step, and then Δf n=9, Δf n=7, and Δf n=5 display similar weak increases after the 11th, 14th, and 16th steps, respectively. In Figure 2B the SPR response per step clearly decreases as the film grows longer due to the limited sensing depth, but importantly the SPR data show that the oligonucleotides continue to hybridize sequentially on the surface also when there is a positive change in Δf according to QCM-D. In contrast to the pattern of negative−zero−positive changes in frequency, the dissipation ΔD is seen to increase during the 8435

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Figure 4. Derived changes in effective thickness (black, left axis) and effective shear viscosity (blue, right axis) versus time during the stepwise assembly of a 534 bp DNA film as obtained from the Voigtbased modeling shown in Figure 3. (Inset) Plot of effective viscosity versus effective thickness.

increase as the first two concatemers are formed; (3) a slow decrease in the layer viscosity as the concatemers are grown even longer. Experiments and Prediction for Layers of Even Longer DNA. On the basis of theoretical considerations, QCM-D responses of thick biomolecular films are expected to be complex, as supported experimentally by the nonmonotonous pattern of negative−zero−positive changes in Δf while ΔD increases monotonously (Figure 2), first observed in multilayers of 100 nm liposomes.20 It should be noted that the DNA experiment in Figure 2 reaches a similar final film thickness as the vesicle study but in steps which are 10 times shorter than the liposome sizes and so provide a much denser data set which is better suited for comparison with theory. In order to exploit this advantage of DNA-based layers to better understand the nonlinear viscoelastic behavior of thick polymer films, we performed a prediction of Δf and ΔD for a 1-μm-thick DNA layer using a Voigt type of model (Figure 5A) and compared it to QCM-D experiments on thick concatemeric layers containing DNA molecules of up to 3084 bp in size (Figure 5B). The predicted values in Figure 5A were obtained using the thick-film Voigt model (Material and Methods) which makes the simplifying assumption of constant viscosity and shear elasticity throughout the assembly, with the aim of capturing the general QCM-D response of a thick DNA-based film while keeping in mind the simplicity of the model. The predicted Δf and ΔD follow a linear decrease and increase, respectively, at low thicknesses. However, as the film thickness increases further, both parameters exhibit a turning point after which Δf instead increases and (for even thicker films) ΔD increases. Similar oscillatory shapes in theoretical QCM-D data can be found in other studies with different assumed viscosity and shear elasticity values.28,34 Figure 5A also shows that the predicted responses enter the nonlinear region at a thinner thickness the higher the overtone number, which reflects how the penetration depth in QCM-D varies with frequency.28 To test these theoretical predictions, a 3084 bp DNA concatemer layer was assembled in 25 bp steps (Figure 5B) using the automated Omega-Auto QCM-D instrument because of the long assembly time (54 h). The frequency shift Δf initially decreases with increasing hybridization time and then exhibits an upward turning point starting with the highest overtone, in qualitative agreement with the predictions in Figure 5A albeit with considerably less marked oscillations.

Figure 5. (A) Predicted data of changes in Δf and ΔD versus the theroretical thickness of the DNA layer up to 1 μm. The calculated data were obtained by assuming constant viscoelastic properties of the DNA layers (thick-film Voigt model in Materials and Methods) with viscosity η set to 2.45 ms·Pa and the shear elastic modulus μ set to 0.27 MPa. (B) QCM-D measurements during the stepwise assembly of a 3084 bp synthetic double-stranded DNA layer. (Main figure) Experimental data of changes in Δf and ΔD versus time during the assembly in steps of 25 bp using the same semicomplementary oligonucleotides and conditions as in Figure 2. (Inset) Magnification of QCM-D data during 4 of the hybridization steps (out of 125 steps in total) approximately 20 h into the assembly. (C) Effective thickness and viscosity plotted as a function of time, using the extended Voigt model to fit the data in panel B.

After 15 h (approximately 850 bp), the Δf response is essentially constant even though the sequential hybridization process continues as seen in the dissipation (inset in Figure 5B). By contrast, ΔD shows a monotonous increase during the assembly and the predicted turning point in ΔD (Figure 5A) is not observed in the experiments. The experimental data in Figure 5B were fitted to the extended (frequency-dependent) Voigt model, and Figure 5C shows that the thickness increases linearly up to 15 h, followed by a much slower growth rate in the later stages of the assembly which reaches an effective thickness of about 90 nm at the end. The viscosity displays the same maximum seen with the thinner DNA layers in Figure 4, and beyond 15 h it increases only very slowly as the concatemer layer is further extended.

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DISCUSSION Characterization of the Platform for Concatemer Formation. Table 1 summarizes measured QCM-D and SPR data as well as some calculated layer properties during the first five steps in the assembly (Figure 1). 8436

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nm (Table 1 and Figure 4), in agreement with a QCM-D study15 of the hybridization of 30-mer oligonucleotides to a surface-tethered complementary strand where the effective thickness increased by 2.3 nm. The good agreement with our measured thickness indicates that the duplexes formed by the A′34 hybridization to the bottom half of b-AB59 stiffen and extend the DNA layer. Figure 4 shows that the subsequent hybridization of oligonucleotides A′B′ or AB (steps 4 and 5 in Figure 1) initially causes an increase in thickness of ∼3.4 nm each. The thickness increase in each step is about 59% lower than the known contour length of DNA (25 bp × 0.34 nm/bp = 8.5 nm), even though the DNA molecule on this length scale is supposed to be rigid. The low effective thickness is likely due to the tilting of DNA helices, allowed by the low surface coverage and a flexible linker between the biotin and the oligonucleotides, and would correspond to a tilting angle of about 24° from the surface. The tilting is not likely to affect the hybridization efficiency in subsequent steps, however, because the surface coverage of DNA is too low (1 ds DNA/SA) to hinder the hybridization: the immobilized streptavidin molecules provide each DNA a 8 × 8 nm2 space to the closest neighbor molecule,40 which is large enough to allow for the hybridization of a 25 bp DNA molecule (8.5 nm long). This explains why the hybridization does take place as indicated by the QCM-D and SPR responses (Figure 2). Figure 4 also shows that the increase in thickness per concatemer decreases for every step after the eighth hybridization step. This observation rules out the DNA layer consisting of helices standing up as rigid rods, consistent with that 200-bp-long DNA molecules are considerably longer than the persistence length of double-helical DNA (150 bp at the present ionic strength), so their flexibility and tendency to form coils have to be taken into account. The coiling up of DNA molecules could hide the protruding single strand from the DNA layer surface inside the DNA layer. Such an effect may explain the lower hybridization efficiency as the DNA concatemers increase in length as seen in SPR (Figure 2B). Shear Viscosity. The contrast between the maximum in effective shear viscosity (η) of the DNA/water film (Figure 4 blue) and the monotonous increase in the effective layer thickness (Figure 4 black) shows that the viscoelastic properties of the DNA film are different for short and long concatemers. Starting with the binding of b-AB59 (step 2 in Figure 1), the effective viscosity (1 mPa·s) at low coverage (time zero) is close to the dynamic viscosity of water. When b-AB59 reaches saturated binding, the effective viscosity almost doubles to 1.94 mPa·s, in good agreement with a previous study using 30-base single-stranded DNA (1.8 mPa·s)15 considering that b-AB59 is longer. These observations lend credibility to the modeling that at low DNA coverage the effective viscosity should be close to that of the solvent but as more DNA molecules are attached their retarding effect on the motion of the associated water or their own viscoelastic behavior is expected to increase the effective viscosity of the ad-layer. When A′34 is hybridized to the b-AB59 platform (step 3 in Figure 1), the effective viscosity increases further (to 2.38 mPa·s) as expected if the DNA layer extends further from the surface as concluded from the thickness data. Figure 4 shows that when A′B′ is hybridized to the platform (step 4 in Figure 1), the effective viscosity increases a little further (to 2.45 mPa·s) but less than expected considering that A′B′ is comparable in size to b-AB59. It is therefore interesting that the subsequent hybridization of AB

The 53% degree of hydration of the streptavidin layer (Table 1) is in good agreement with previous studies,15 and the 3.4 nm thickness of the streptavidin layer is also expected.37 The 88% degree of hydration for the b-AB59 oligonucleotide layer confirms that surface-attached DNA tends to bind large amounts of water.15 Third, the 150% binding efficiency of bAB59 (Table 1) is in agreement with a previous study that showed that biotinylated single-stranded DNA tends to bind a streptavidin layer with 1.5 oligos per protein16 even though there are two biotin sites available per streptavidin. Finally, the binding of 34-mer oligonucleotide A′34 to the bottom part of the b-AB59 layer (step 3 in Figure 1) leads to a smaller decrease in frequency, as expected because its mass is only about half as large as that of b-AB59, and to a further increase in dissipation probably due to a stiffening and extension of the DNA film.15 The hybridization efficiency of this step (step 3 in Figure 1) is only 59 ± 2%, but a similarly low value of 61% has been reported in a previous study based on the same streptavidinimmobilized DNA layer.16 The DNA hybridization efficiency of 25 bp SH-DNA has been investigated by Peterson et al.38 It was observed that by increasing the coverage of the first strand in the range of (2−12) × 1012 molecule·cm−2 (3.3−19.9 pmol· cm−2) the hybridization efficiency dropped from 100 to 10%. Our system with a 5.27 pmol·cm−2 probe density (calculated from ΔmSPR) is thus in a range where a hybridization efficiency of about 50% can be expected. When oligonucleotides A′B′ and AB are hybridized (steps 4 and 5 in Figure 1) the result is essentially the same for the two regarding the layer thickness and degree of hydration (Table 1), as expected because they have similar molecular properties. Furthermore, the hybridization efficiency after step 3 (Figure 1) decreased the subsequent probe density to about 2 pmol·cm−2, so the increase in hybridization efficiency in the subsequent steps is expected. However, the SPR results (Figure 2B) suggest that the coupled amount decreases after the eighth hybridization step, which will be discussed below. In summary, the results in Table 1 show that the platform built from streptavidin, b-AB59 and A′34 (Figure 1), behaves in agreement with previous studies of thin oligonucleotide layers and can be used for the present purpose to build increasingly thicker DNA films through concatemerization. Layer Thickness. The immobilization of biotinylated oligonucleotide b-AB59 (step 2 in Figure 1) results in a 7.2nm-thick DNA layer according to the fits to the extended model (Figure 4). This value is higher than the value of 5.8 nm which can be expected if the DNA molecules are described by the wormlike chain (WC) model39 according to the following calculation. The equilibrium radius of gyration Rg of a WC polymer coil is given by Rg =

PLc /3

(3)

if the contour length Lc is assumed to be much longer than the persistence length P. Assuming a rise of 0.43 nm per base and a persistence length of 1 nm for single-stranded DNA,39 the single-stranded b-AB59 (Lc = 25 nm) should give a layer thickness (2Rg) of 5.8 nm. One possible explanation for the larger measured thickness is a certain degree of steric extension of the coils since the calculated coil dimension is somewhat larger than the available space given the coverage of 1.5 ssDNA per streptavidin SA. The hybridization of A′34 to the bottom half of b-AB59 (step 3 in Figure 1) increases the effective thickness from 7.2 to 10 8437

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Figure S4) may be ascribed to adding two more free parameters, but this explanation is unlikely since adding three parameters in the two-layer model did not improve the fit to the same degree (Figure S2). It is therefore interesting that existing bulk data on the dynamics of short DNA molecules are consistent with a frequency-dependent response in the MHz region used in QCM. When free in 200 mM NaCl aqueous solution, the DNA sizes used here (50−534 bp) have rotational correlation times of between 4 and 35 μs,45 indicating that the concatemers are short enough to respond to the MHz shearing motion in a frequency-dependent manner as they grow longer. We obtain a linear frequency dependence for shear elastic modulus G′′ (eq 1 combined with the fitted value αη = 0 ± 0.001 in eq 2a) as commonly observed for polymers in the MHz region, but to our knowledge there are no direct measurements of the viscoelastic response of such short DNA molecules in bulk solution. Mason and coworkers46 have performed measurements on 13 kbp DNA in 100 mM monovalent salt and have observed a frequency dependence in both G′ and G′′ (cf. eq 2) but in a comparatively low frequency range (100 Hz or less) consistent with the slower rotation of such large DNA molecules. In conclusion, the QCM-D response of the concatemer layers in Figure 1 is expected to be frequency-dependent because the DNA molecules can reorient in the MHz range, but additional bulk measurements on short DNA is needed for quantitative confirmation. Predicted and Measured QCM-D Data for Concatemers up to 1000 nm Long. Figure 6A (curves without

decreases the viscosity to 2.30 mPa·s, in effect giving rise to the observed maximum. One possibility is that the maximum in effective viscosity (Figure 4) occurs when the DNA concatemers become long enough to be flexible rather than behaving as rigid rods. A similar interpretation has been made in a study of viscoelastic properties of adsorbing polyelectrolytes.41 The maximum is observed during the third step, where the formed concatemer is 59ds + 25ss long and shorter than the persistence length of ds DNA under 150 mM conditions (150 bp),42 but recent studies indicate that DNA molecules as short as 67−100 bp may be intrinsically bendable.43 Notably, the presence of a nick every 25 base pairs in our present protocol is unlikely to affect the duplex stiffness since Hagerman and coworkers have shown that a nick (as opposed to a deleted base) has little effect on the persistence length of short oligonucleotide duplexes as used here.44 In order to test this possibility we attempted to fit the experimental data in Figure 2 to a two-layer model by assuming that the DNA film consists of two sublayers with different properties (Supporting Information, Figure S2). However, the two-layer model did not give a better fit of the experimental data than the one-layer model (Figure 3) even though the latter contains fewer fitting parameters. These observations indicate that the maximum in effective viscosity (Figure 4) is not mainly due to a change in the DNA bendability as the layer grows thicker. This conclusion is also supported by the observation (Supporting Information, Figure S3) that the viscosity maximum occurs at a similar concatemer length at higher ionic strength (10 mM PBS buffer with 537 mM NaCl and 127 KCl) even though the persistence length is known to decrease at higher salt content. An alternative explanation for the decrease in effective viscosity after the third concatemer step is the contribution from the single strands which are present at the top of the DNA layer in our assembly protocol, because single-stranded DNA is known to contribute considerably more to the effective shear viscosity in a DNA film than the corresponding doublestranded form. This effect was reported in a previous study,15 where a single-stranded layer increased the viscosity from 1.0 to 1.8 mPa·s while the fully hybridized ds DNA increased the viscosity only by 0.2 mPa·s, and the strong viscosity effect of ssDNA is confirmed by our results with b-AB59 (blue in Figure 4). In the beginning of the concatemer assembly, the singlestranded DNA layer on top of the film (Figure 1) contributes substantially to the QCM-detected viscosity, but it plays less and less of a role in the viscosity of the whole DNA layer as the duplex part grows thicker. In summary, the results in Figure 4 indicate that the DNA platform in Figure 1 is highly viscous but thin due to the flexibility of the single-stranded b-AB59 seed, until A′34 hybridization stiffens the layer and makes it thicker. As the concatemers grow longer, the film grows thicker, but less than expected for rigid DNA rods and with a decreasing effective viscosity because the contribution from the single-stranded DNA top layer becomes less important. Frequency Dependence of Voigt Parameters. The standard Voigt-based model was also applied to fit the QCM-D measurement data (Supporting Information, Figure S4), but the extended Voigt-based model described the measured data considerably better than the standard model. That the Voigtmodel fits become better when the viscoelastic parameters are allowed to be frequency-dependent (Figure 3 compared to

Figure 6. ΔD versus Δf for experimental data (lines without symbols, from Figure 2) and as predicted by the thick-film Voigt model (lines with symbols, from Figure 5A) at overtones n = 3 (black), n = 7 (red), and n = 11 (blue). (A) Magnified plot in the range of the experimental data. (B) Plots of predicted data up to a theoretical layer thickness of 1000 nm. 8438

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postsynthesis analysis of the length distribution by gel electrophoresis. Second, in the present setup the DNA molecules contain nicks, and in our experience ligation is hampered in the DNA films, perhaps due to crowding. Attempts are underway to overcome this limitation by decreasing the surface coverage, which may also improve the hybridization efficiency. Third, during assembly the films contain a 25-base single-stranded top layer, which is the price to be paid in our approach to studying films of different lengths on a given chip instead of synthesizing films of different lengths on different chips. In the latter case and in applications of a film of a given length, the layers can be made fully double-stranded by the hybridization of the 25-base oligonucleotide A that we always perform here as a final step. Four different Voigt-type models were evaluated by comparison with the experimental data on the concatemeric DNA films. In the case of the three models with fitting parameters, the single-layer standard model, the two-layer model, and the single-layer frequency-dependent model, the latter gives the best fits to the data in spite of having fewer fitting parameters than the two-layer model. The good fits are used to extract important information about the DNA films, in spite of the limitations listed above. For the very thin films (75 bp in Figure 1), the fitted thicknesses are in agreement with previous results and indicate essentially rigid rods tilted with respect to the surface. For moderately thick films (up to 534 bp, Figure 4), the fitted thickness indicates the coiling up of the DNA due to the inherent helix flexibility, with potential effects on the hybridization efficiency. The derived effective shear viscosity is consistent with available data on the viscoelastic properties of such flexible but moderately sized DNA, and the need for frequency-dependent parameters to obtain good fits points to the importance of segment rotation for the viscoelastic properties of polymer films. For very thick films (up to 3084 bp, Figure 5C), the derived shear viscosity is nearly independent of DNA length, in agreement with the fact that such long DNA polymers add normal rotational modes with characteristic time constants in the millisecond range or longer39 and thus are probably too slow to respond to the MHz frequencies used in QCM-D. That the effective thickness increases very slowly in view of a 10-fold increase in DNA contour length points to possible limitations in the QCM-D technique due to a nonmonotonic response for thick films. The fourth model, the thick-film Voigt model, contains no fitting parameters and is the first attempt with a very simple molecularly based model (stiff DNA helices perpendicular to the surface and 0.34 nm contour length per base pair, constant layer viscosity, and elastic modulus of very short DNA). Not surprisingly, the comparison with experiment (Figure 6) shows that this molecular model needs to be improved, probably first by taking DNA flexibility into account by using the wormlike chain model instead of rigid rods, a task outside the scope of this study.

symbols) shows the experimental data in Figure 2 plotted as ΔD versus Δf, as commonly used to present the viscoelastic properties of a nonrigid layer as a function of the added mass. It is seen that the experimental relation between ΔD and Δf is linear for the first few steps but then the curve turns upward for all three overtones as the concatemers grow longer. A similar trend was observed in a study of liposome layers assembled by DNA linkers20 but in a more stepwise fashion because the liposome building blocks are 10-fold larger than the 25 bp DNA steps of our layers. Hence, in our case the experimental curve is essentially continuous even at the magnified scale of Figure 6A which allows for an improved comparison with theory. Figure 6A (curves with symbols) also includes the data predicted by the thick-layer Voigt model (Figure 5A). It is seen that the model reproduces the experimentally observed upward curvature, but the experimental data bends more strongly (i.e., the experimental dissipation increases faster than predicted for a given bound mass) and the deviation between theory and experiment is stronger the higher the overtone number. Plotting the predicted data up to a contour length of 1000 nm (Figure 6B) shows that if the viscoelastic properties were assumed to be constant then the plot of ΔD versus Δf exhibits a spiral trend with different radii at different overtones. This observation indicates that only the initial linear part of the spiral curve gives reliable information on the viscoelastic properties of the DNA film as monitored by QCM-D. Although the experimental data also shows the initial curvature (Figure 6A), the data on Δf and ΔD during the experimental assembly of the 3084 bp concatemers (Figure 5B) did not exhibit the strong undulations in Δf and ΔD (Figure 5A) that underlie the predicted spiral behavior in Figure 6B, indicating that the layer thickness is not reached where ΔD exhibit its turning point. One possibility is that DNA coiling prevents the layer from increasing linearly in thickness with DNA contour length or (as discussed in the section on thickness) that the coiling leads to a decrease in hybridization efficiency when the DNA molecules are much longer than the persistence length. Taken together, the results in Figure 5 indicate that films containing a large number of DNA concatemers cannot be modeled as consisting of rigid and aligned polymers as we assume in the thick-film Voigt model. From Figure 5C it is also seen that the assumption of constant viscosity (and elastic modulus) in this model is particularly unrealistic up to about 850 bp and is actually better satisfied for films containing longer DNA molecules, at least according to the effective viscosity obtained from fitting to the extended Voigt model. DNA Concatemer Layers as an Experimental System for Studying Viscoelastic Polymer Films. DNA films are useful experimental model systems for investigating the viscoelastic properties of polymer films because the molecular properties of DNA in the bulk are well understood, such as the structure and mechanical properties. An added advantage of the concatemeric films used here is the possibility of controlling the base sequence also in long molecules that give thick films and the potential for varying the DNA length on the surface by varying the number of concatemerization steps. The films used here have disadvantages as well that need to be taken into account or remedied. Even a small deviation from 100% hybridization efficiency will accumulate exponentially and result in length distributions after a given concatemeric step, especially in thick films. The purpose of the designed restriction enzyme site close to the surface (Figure 1) is to perform a

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CONCLUSIONS The QCM-D and SPR responses upon sequential hybridization of a DNA concatemer layer on an immobilized streptavidin layer was used to demonstrate how Voigt-based modeling can be applied to evaluate the thickness and viscolelastic properties of polymer films using DNA films as a model experimental system. The extended frequency-dependent version of the Voigt-based model shows considerably better fits to the measured data than the standard and two-layer models. 8439

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Compared to previous studies of DNA15 and other polymers using20,34,41 QCM-D, our modeled results show a good description of the polymer films for short DNA, whereas thicker DNA films did not behave as predicted by a simple rigid rod molecular model probably because helix coiling leads to a nonlinear growth in thickness.

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(11) Yang, Y.; Wang, Q.; Guo, D. A Novel Strategy for Analyzing Rna-Protein Interactions by Surface Plasmon Resonance Biosensor. Mol. Biotechnol. 2008, 40, 87−93. (12) Lillis, B.; Manning, M.; Berney, H.; Hurley, E.; Mathewson, A.; Sheehan, M. M. Dual Polarisation Interferometry Characterisation of DNA Immobilisation and Hybridisation Detection on a Silanised Support. Biosens. Bioelectron. 2006, 21, 1459−1467. (13) Cardenas, M.; Campos-Teran, J.; Nylander, T.; Lindman, B. DNA and Cationic Surfactant Complexes at Hydrophilic Surfaces. An Ellipsometry and Surface Force Study. Langmuir 2004, 20, 8597− 8603. (14) Caruso, F.; Rodda, E.; Furlong, D. N.; Niikura, K.; Okahata, Y. Quartz Crystal Microbalance Study of DNA Immobilization and Hybridization for Nucleic Acid Sensor Development. Anal. Chem. 1997, 69, 2043−2049. (15) Larsson, C.; Rodahl, M.; Höök, F. Characterization of DNA Immobilization and Subsequent Hybridization on a 2D Arrangement of Streptavidin on a Biotin-Modified Lipid Bilayer Supported on SiO2. Anal. Chem. 2003, 75, 5080−5087. (16) Su, X.; Wu, Y.-J.; Robelek, R.; Knoll, W. Surface Plasmon Resonance Spectroscopy and Quartz Crystal Microbalance Study of Streptavidin Film Structure Effects on Biotinylated DNA Assembly and Target DNA Hybridization. Langmuir 2005, 21, 348−353. (17) Cho, Y.-K.; Kim, S.; Kim, Y. A.; Lim, H. K.; Lee, K.; Yoon, D.; Lim, G.; Pak, Y. E.; Ha, T. H.; Kim, K. Characterization of DNA Immobilization and Subsequent Hybridization Using in Situ Quartz Crystal Microbalance, Fluorescence Spectroscopy, and Surface Plasmon Resonance. J. Colloid Interface Sci. 2004, 278, 44−52. (18) Höök, F.; Ray, A.; Norden, B.; Kasemo, B. Characterization of Pna and DNA Immobilization and Subsequent Hybridization with DNA Using Acoustic-Shear-Wave Attenuation Measurements. Langmuir 2001, 17, 8305−8312. (19) Höök, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Variations in Coupled Water, Viscoelastic Properties, and Film Thickness of a Mefp-1 Protein Film During Adsorption and CrossLinking: A Quartz Crystal Microbalance with Dissipation Monitoring, Ellipsometry, and Surface Plasmon Resonance Study. Anal. Chem. 2001, 73, 5796−5804. (20) Graneli, A.; Edvardsson, M.; Höök, F. DNA-Based Formation of a Supported, Three-Dimensional Lipid Vesicle Matrix Probed by QCM-D and SPR. ChemPhysChem 2004, 5, 729−733. (21) Johnston, A. P. R.; Mitomo, H.; Read, E. S.; Caruso, F. Compositional and Structural Engineering of DNA Multilayer Films. Langmuir 2006, 22, 3251−3258. (22) Tsortos, A.; Papadakis, G.; Gizeli, E. Shear Acoustic Wave Biosensor for Detecting DNA Intrinsic Viscosity and Conformation: A Study with QCM-D. Biosens. Bioelectron. 2008, 24, 836−841. (23) Papadakis, G.; Tsortos, A.; Bender, F.; Ferapontova, E. E.; Gizeli, E. Direct Detection of DNA Conformation in Hybridization Processes. Anal. Chem. 2012, 84, 1854−1861. (24) Kim, S.; Blainey, P. C.; Schroeder, C. M.; Xie, X. S. Multiplexed Single-Molecule Assay for Enzymatic Activity on Flow-Stretched DNA. Nat. Methods 2007, 4, 397−399. (25) De Vlaminck, I.; Henighan, T.; van Loenhout, M. T. J.; Burnham, D. R.; Dekker, C. Magnetic Forces and DNA Mechanics in Multiplexed Magnetic Tweezers. PLoS One 2012, 7, e41432. (26) Herrick, J.; Bensimon, A. Imaging of Single DNA Molecule: Applications to High-Resolution Genomic Studies. Chromosome Res. 1999, 7, 409−423. (27) Bosaeus, N.; El-Sagheer, A. H.; Brown, T.; Smith, S. B.; Åkerman, B.; Bustamante, C.; Norden, B. Tension Induces a BasePaired Overstretched DNA Conformation. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 15179−15184. (28) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Viscoelastic Acoustic Response of Layered Polymer Films at Fluid-Solid Interfaces: Continuum Mechanics Approach. Phys. Scr. 1999, 59, 391−396. (29) Lucklum, R.; Behling, C.; Hauptmann, P. Role of Mass Accumulation and Viscoelastic Film Properties for the Response of

ASSOCIATED CONTENT

S Supporting Information *

Voigt-modeled effective shear elastic modulus, fit to a two-layer Voigt model, QCM-D data and Voigt modeling of a 534 bp concatemer in 10 mM PBS buffer, fit to standard Voigt model, and fitting comparison of frequency-dependent and frequencyindependent Voigt models. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +46 31 772 3052. Fax: +46 31 772 3858. E-mail: baa@ chalmers.se. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Jennie Wikström and Gabriel Ohlsson are gratefully acknowledged for discussions regarding QCM-D measurements and modeling. This research is funded by the Swedish research council (project 349-2007-8680) as a part of the Linnaeus Centre SUPRA for Bioinspired Supramolecular Function and Design.

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REFERENCES

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