Molecular and Thermodynamic Factors Explain the Passivation

Oct 6, 2015 - We finally report for the first time on the detailed thermodynamic values that show how adsorption results from a balance between large ...
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Molecular and Thermodynamic Factors Explain the Passivation Properties of Poly(ethylene glycol)-Coated Substrate Surfaces against Fluorophore-Labeled DNA Oligonucleotides Chun-lai Ren,† Robert Schlapak,‡ Roland Hager,‡ Igal Szleifer,*,§ and Stefan Howorka*,‡,∥ †

National Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China Center for Advanced Bioanalysis GmbH, Linz, Austria § Department of Biomedical Engineering, Department of Chemistry and Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States ∥ Department of Chemistry, Institute of Structural and Molecular Biology, University College London, London, United Kingdom ‡

S Supporting Information *

ABSTRACT: Poly(ethylene glycol) (PEG) nanofilms are used to avert the nonspecific binding of biomolecules on substrate surfaces in biomedicine and bioanalysis including modern fluorescence-based DNA sensing and sequencing chips. A fundamental and coherent understanding of the interactions between fluorophore-tagged DNA, PEG-films, and substrates in terms of molecular and energetic factors is, however, missing. Here we explore a large parameter space to elucidate how PEG layers passivate metal oxide surfaces against Cy3-labeled DNA probes. The driving force for probe adsorption is found to be the affinity of the fluorophore to the substrate, while the high-quality PEG films prevent adsorption to bare ITO surfaces. The amount of nonrepelled, surface-bound DNA strongly depends on oligonucleotide size, PEG chain length, and incubation temperature. To explain these observations, we develop an experimentally validated theory to provide a microscopic picture of the PEG layer and show that adsorbed DNA molecules reside within the film by end-tethering the fluorophore to the ITO surface. To compensate for the local accumulation of negatively charged DNA, counterions condense on the adsorbed probes within the layer. The model furthermore explains that surface passivation is governed by the interdependence of molecular size, conformation, charge, ion condensation, and environmental conditions. We finally report for the first time on the detailed thermodynamic values that show how adsorption results from a balance between large opposing energetic factors. The insight of our study can be applied to rationally engineer PEG nanolayers for improved functional performance in DNA analysis schemes and may be expanded to other polymeric thin films.

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formation in solution at high PEG concentrations,34−36 and simulations of DNA hybridization on hydrophilic surfaces.37 However, little is known about simple yet practically important questions such as how DNA length or differently sized PEG chains influence surface passivation or adsorption (Figure 1). At a more basic scientific level, there is a lack of understanding of how structural and thermodynamic factors influence the interaction of DNA with PEG-coated substrates. For example, the detailed entropic and enthalpic contributions are not known despite the central role of entropy in explaining the passivation mechanism of structurally fluctuating PEG chains.25,38 Furthermore, it is unclear how deep DNA molecules penetrate into the PEG layer upon adsorption. As several of these questions cannot be answered solely with experiments, a combined synergistic experimental and theoreti-

INTRODUCTION Thin films composed of poly(ethylene glycol) (PEG) are of scientific and technological interest as they avert the nonspecific adsorption of biomolecules on biologically relevant materials. PEG layers coat, for example, substrates used in bioanalysis, bioimaging, drug delivery, therapeutics,1−7 and research.8−12 As surface passivation is caused by the hydrophilic nature and structural flexibility of the PEG chains, studies have experimentally examined13−17 the polymer films with regard to chemical18,19 and structural factors20−23 such as chain length18 and chain density21,24 and developed molecular models.25−27 Reflecting their use in protein-rich samples or fluids, PEG films have been extensively explored to avoid protein binding.28 The interaction of PEG films with DNA is, however, less well understood, even though DNA microarrays,29−31 modern sequencing chips,1 and biophysical studies on nucleic acids30−32 use passivating PEG films. Existing reports have focused on PEG and oligo(ethylene glycol) as linkers for the surface immobilization of DNA capture strands,29,33 duplex © XXXX American Chemical Society

Received: July 20, 2015 Revised: October 2, 2015

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DOI: 10.1021/acs.langmuir.5b02674 Langmuir XXXX, XXX, XXX−XXX

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passivation is then used to validate a molecular theory which is developed to understand the structure and thermodynamics of the soft nanomaterial and biopolymer repulsion and adsorption. The molecular model is finally exploited to infer molecular and energetic properties of the layers and their interaction with DNA which can be generalized to cover a wider range of controlled variables. As overarching output, our study provides insight which could not have been obtained from experiments alone and achieves a comprehensive scientific understanding which can guide the rational design of functionally improved nanofilms on substrates of planar andwith further adaption curved geometry.

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RESULTS AND DISCUSSION Formation and Characterization of PEG Layers. We studied the passivation properties of polymer films composed of PEG with a molecular weight of 5000 Da (PEG-5000) and 500 Da (PEG-500). The chain length influences the nanoscale density and molecular structure of the PEG films. PEG-500 is more tightly packed than the molecularly looser PEG-5000, as illustrated in Figure 1. Both PEG films were formed on glass slides coated with a 17 nm thick indium−tin oxide (ITO) layer. The ITO layer was deliberately chosen as it is optically transparent yet electrically conductive and enables a wide range of analytical and cell biological applications.41 It is also used to fabricate nanoparticles.42 The polymeric film was coupled onto the inorganic substrate by forming covalent bonds between trialkoxysilane bearing PEG24,43 and the hydroxyl groups of ITO. Characterizing the films by X-ray photoelectron spectroscopy (XPS) confirmed the presence of the expected chemical signatures for PEG. Complementary analysis with atomic

Figure 1. Schematic drawing of the two PEG layers and fluorophorelabeled DNA molecules of different nucleotide length used in our study, and several unresolved questions addressed herein. The blue circles represent the Cy3 fluorophores. The PEG films and DNA probes are drawn to scale.

cal strategy25,26,39 is the best route to obtain a coherent and deep scientific understanding. To settle the above questions, this report examines how PEG films passivate solid substrates against the adsorption of fluorophore-labeled, single-stranded DNA oligonucleotides. Fluorescence-labeled DNA strands are widely used in bioanalytical sensing schemes29−31,40 and additionally facilitate the fast scanning and quantification of the amount of adsorbed DNA. We first study how surface passivation is influenced by structural parameters including PEG chain length and nanoscale density, and length of the single-stranded DNA, as well as the biophysical parameters of DNA concentration and incubation temperature. The large experimental data set on

Figure 2. PEG films avert the adsorption of Cy3-DNA probes onto ITO surfaces in dependence of PEG length, DNA size and concentration, and temperature. (A) Fluorescence micrographs of (1, 2) bare ITO surfaces and substrates coated with a film composed of (3, 4) PEG-5000 and (5, 6) PEG-500 before (2) and after (2−6) incubation with a fluorescent probe. The probes of indicated length were incubated at a concentration of 2 μM and a temperature of 22 °C. The micrographs show an area of 78 × 78 μm. (B, C) Quantitative summary of experiments showing the surface densities of DNA adsorbed on (B) PEG-5000 films as a function of probe length, probe concentration, and temperature for (B-1) 7 °C, (B-2) 15 °C, (B-3) 22 °C, and (B-4) 37 °C. (C) Surface densities for binding onto PEG-500 films in dependence of DNA length and incubation temperature at a probe concentration of 2 μM. The data in (B) and (C) represent averages and standard deviations from at least three independent experiments. B

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contrast, binding to bare ITO strongly increased with DNA concentration (Figure S5). Quantitative information on the amount of material adsorbed on PEG-ITO interfaces was obtained by first correcting the fluorescence counts for the background signal of blank slides and then by normalizing to account for the different emission coefficients of the four DNA probes. Finally, reference standards carrying a radiolabel were applied to convert the fluorescence counts into surface densities of pmol/cm2.43 These experimental data featuring averages and standard deviations from at least three independent experiments are numerically tabulated in Table S1 and for PEG-5000 surfaces and in Table S2 for PEG-500 surfaces as a function of DNA length, DNA concentration, and temperature. Quantitative information for adsorption on bare ITO cannot be provided because particular amount of adsorbed DNA varied strongly depending on the age (up to 5 days) of purified surfaces. By contrast, PEG-coated slides were largely independent of the age of surface modification. Figure 2B graphically summarizes the amount of DNA-Cy3 adsorbed on PEG-5000 ITO surfaces as a function of nucleotide length and concentration and incubation temperature (Figure S6, semilogarithmic plot). Three trends are observed. (i) The 1 nt sample has the highest nonspecific adsorption while the amount of bound DNA decreases with higher probe length (e.g., Figure 2B-1) suggesting that the smallest DNA probes permeate readily into the porous meshwork of the PEG chains. Longer DNA strands did not adsorb likely due to size-exclusion effect, or the large entropic cost of binding larger nanoscale objects onto the otherwise structurally flexible PEG chains, or a combination of both factors. (ii) Higher incubation temperatures led to weaker adsorption (Figure 2B1−4; 7, 15, 22, and 37 °C, respectively), likely because higher temperatures render PEG chains structurally more flexible, thereby increasing the entropic cost associated with DNA binding or reducing in the van der Waals attraction between DNA and PEG. (iii) Higher DNA concentrations led to strong binding to the surfaces. In contrast to the PEG-5000 film, DNA binding onto substrates with shorter PEG-500 depends strongly on neither the incubation temperature nor the length of the probe (Figure 2C and Table S2). This could be due to the inability of DNA to permeate into the less porous and more densely packed PEG-500 film. Additional analysis clarified the molecular origin for the adsorption of Cy3-labeled DNA onto PEG-coated ITO surfaces. When disregarding the possibility of strong binding of Cy3-DNA to the PEG film, there are two main possible reasons. Affinity of either DNA or the fluorophore to the ITO substrate. The first can be discarded as a related study found poor DNA binding to negatively polarized ITO at a pH of 8.0 which was also used in our assay.42 To confirm the second possibility, we tested the adsorption of the Cy3 fluorophore to the ITO surfaces. The fluorescence microscopic results show strong binding of the fluorophore (Figure S7) in line with the known affinity of related aromatic structure.50,51 The affinity constant Kd was (1.2 ± 0.4) × 105 M−1, as inferred from the Langmuir isotherm for the concentration dependence of binding (Figure S7), corresponding to a Gibbs free energy of −28 ± 9 kJ mol−1. A similar and partly even stronger binding to the metal oxide surface was observed for fluorophores Cy5 and Alexa 647 and Rhodamine B (Figures S8 and S9) even though the structures had differences of up to three negative charges (compare Cy5 and Alexa 647). Binding largely independent of

force microscopy (AFM) and water contact angle goniometry films established that the films were very homogeneous.20,24 In addition, XPS, AFM, and ellipsometry determined the films thickness20,24 to be 2.6 nm for PEG-5000 and 1.1 nm for PEG500 in the dry state.20 These values correspond to an average molecular cross section of 3.2 nm2 for one PEG-5000 chain and 0.76 nm2 for PEG-500 showing that more short-length PEG chains are coupled per unit area. The equivalent surface densities are 0.32 and 1.3 chains per nm−2, or 52 and 218 pmol/cm2, respectively. The values reflect the larger entropic cost of confining longer chains and are also in line with the pore size of the PEG film meshwork which has been determined by probing the permeation properties with small electrochemical molecules.20 PEG Films Avert the Nonspecific Binding of Cy3Labeled DNA Probes onto ITO Surfaces. We experimentally determined to which degree Cy3-labeled DNA adsorbed onto PEG ITO interfaces as a function of DNA size, PEG length, and temperature. To clarify the influence of DNA size, probes with lengths of 1, 5, 8, and 14 nucleotides (nt) were chosen (Figure 1 and Supporting Information, Figure S1). The 1 nt sample was a deoxyuridine triphosphate whichalong with other fluorophore-labeled nucleotidesis used in DNA sequencing-by-synthesis strategies.1,44 The other DNA probes were synthetic oligonucleotides with a conventional phosphodiester backbone and sequences containing all bases (Figure 1 and Figure S1), as often employed in hybridization or primerinduced sequencing approaches.1,44,45 All four nucleic acids molecules were labeled with the cyanine Cy3 fluorophore which is widely used in the life sciences.46,47 The fluorophore was attached either at a base (1 nt) or terminal position (5, 8, and 14 nt) to facilitate their detection via fluorescence scanning (Figure S1). In the assay on surface passivation, PEG-coated and bare ITO surfaces were incubated with fluorescence-labeled DNA and, after washing, subjected to fluorescence microscopic scanning. The incubation was carried out using a buffered solution at pH 8.0 at a given DNA concentration and temperature. The incubation lasted for 3 h to ensure equilibration of the system which is required for the theoretical model. Incubation for 3 h is usually sufficient to achieve saturation in DNA oligonucleotide adsorption,48,49 and this was also found for our assay (Figure S2). The washing of the slides removed buffer components which would haveupon drying of the slidescaused salt crystals and undesired scattering during fluorescence scanning. The washing did not interfere with the equilibration of the system as rinsing was very short (less than 2 s) and only led to a 1.5% decrease in fluorescence (Figure S3A). The short washing step was essential as longer incubation with the buffer led to a marked decrease in the fluorescence signal (Figure S3B). The PEG film caused a strong reduction in the binding of DNA probes. For example, on bare ITO slides the 1 nt probe (at 2 μM and 22 ± 1 °C) adsorbed strongly (Figure 2A-1; compare to nonexposed ITO in Figure 2A-2). By contrast, nonspecific binding was reduced at least 10-fold by the presence of a PEG-5000 film (Figure 2A-3, based on comparing the fluorescence counts) and 31-fold by a PEG-500 layer (Figure 2A-5). When testing with 10 μM 1 nt probe, the PEG500 layer reduced adsorption 150-fold compared to bare ITO (not shown). Both films decreased adsorption also for the longer probes 5 nt (Figures 2A-4 and 2A-6) and 8 and 14 nt (Figure S4) and did not depend on DNA concentration. By C

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Figure 3. Comparing experiment and theory on the adsorption of DNA onto PEG-modified ITO surfaces. Top panels: the amount of adsorbed DNA as a function of DNA length for different DNA concentrations: (A) 10, (B) 2, and (C) 0.4 μM. Bottom panels: the amount of adsorbed DNA as a function of the DNA concentration for different DNA lengths: (D) 1, (E) 5, and (F) 8 nt. Four temperatures are compared in both cases. Filled circles with error bare correspond to the experimental data, and the lines are theoretical predictions.

from experiment and additionally predict properties for different PEG lengths parameters not experimentally covered in our study. Principles of the Theoretical Model. We used a molecular theory25 that explicitly considers the size, shape, charge, charge distribution, and conformations of every molecule in the system. As shown in previous studies,25,26 the model provides accurate information as confirmed by experimental observations for the structure and thermodynamics of tethered polymer layers26 as well as the amount of protein adsorption on surface-passivating PEG and peptoid layers.25,26 The model is based on previous studies25,26 but includes, newly, the effect of counterion condensation which is relevant for highly charged molecules such as DNA. The basic idea is to consider conformations of the immobilized PEG chains and the DNA molecules and write a free energy function in terms of the probabilities of those conformations and the density of the free species (ions and water) and include all the inter- and intramolecular interactions. The free energy per unit area is given by

a particular chemical structure or net charge demonstrates the wider relevance of our findings for adsorption of fluorophorelabeled DNA to ITO interfaces. The experimental results are of scientific and practical value. For example, our finding that PEG-500 films prevent adsorption of 1 nt DNA much better than PEG-5000 (Figure 2B,C) strongly favors the former PEG film in applications that use fluorescence-labeled trinucleotides such as primer extension and sequencing by synthesis.1,44 To further increase the scientific benefit of our study, we sought to develop a coherent theoretical model that is in agreement with the quantitative experimental data and additionally explains why PEG and DNA length influence surface adsorption. The model should also provide a molecular picture of the PEG films and their dimensions, clarify whether Cy3-DNA binds on the surface or inside the polymer film, and detail changes in the electrostatic distribution within the PEG layer upon DNA binding. Another question to be answered is the magnitude of energetic or steric contributions by which PEG avoids the adsorption of DNA. Hence, the model should provide insight that is not accessible βF = σ ∑ P(α) ln P(α) A α +

∫

ρDNA (z){∑ P(γz)[ln P(γz) + βU (0)] + ln ρDNA (z)vw − 1 − βμDNA } dz γz

+

∫

+

∑∫

0 ′ (z)⟩{fN (z)[ln fN (z) + βμ N0 ] + (1 − fN (z))[ln(1 − fN (z)) + βμDNA ⟨ρDNA ]} dz

ρi (z)[ln ρi (z)vw − 1 − βμi ] dz

i

+

χ vw

∫

+

∫

⎡ 1 2⎤ ⎢⎣⟨ρq (z)⟩ψ (z) − 2 ε(∇ψ (z)) ⎥⎦ dz

⟨ϕp(z)⟩⟨ϕDNA (z)⟩ dz (1)

D

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Langmuir where the first line represents the conformational entropy of PEG layer. The second line represents the position-dependent conformational entropy of DNA, the attraction between Cy3 and ITO surface, translational entropy of the DNA molecules, and the chemical potential term to ensure thermodynamic equilibrium with the bulk solution. The third line contains the free energy arising from the counterion condensation on DNA. The fourth line is the position-dependent translational (mixing) entropy of the mobile species. The fifth line represents van der Waals interaction between PEG layer and DNA, where χ < 0 is the attractive strength. The last line is the electrostatic free energy. By minimization of the system’s free energy, the probability of each of those conformations and the density profile for all the species are determined as a function of the bulk solution conditions, i.e., ionic strength and DNA concentration. In this way, the theory enables the study of both structural and thermodynamic properties of the equilibrium state. For details on the theory, its formulation, numerical implementation, and parameter selection see Theoretical Methods in the Supporting Information and ref 52. The input necessary to solve the theory includes the amount of DNA and salt in the bulk solution, the surface coverage of PEG on the surface, the set of conformations of the PEG and the DNA molecules, and a molecular model for the ions from the salt (details in the Supporting Information). Furthermore, the theory requires the bare interaction between the Cy3 group and the surface. We take a value of βU(0) = −12, which translates after accounting for the temperature into free energy values of −28.0, −28.8, −29.4, and −30.9 kJ/mol for 7, 15, 22, and 37 °C, respectively. The range of these free energies is line with the experimental value of −28 kJ mol−1 obtained at 22 °C. The choice is also reasonable as it is of the order of a strong hydrogen bond. Using this affinity, we predict the DNA adsorption under all conditions and, as shown below, provide a comprehensive picture of the thermodynamic driving force for the passivating properties of the PEG layers as well as the structural properties of the combined PEG−DNA layer. Comparison between Theory and Experiment. For validation of the model, we compared the theoretical predictions with the experimentally found amount of adsorbed DNA, Γads, on PEG−ITO interfaces. Figure 3 shows the comparison for the different experimental conditions whereby the experimental data are shown as filled circles with error bars and theoretical predictions as lines. The agreement is very good as illustrated for plots of Γabs as a function of DNA length for various temperatures and DNA concentrations (Figure 3A−C) and of bulk concentration of DNA (Figure 3D−F). A discrepancy is found for the special case of the lowest concentration of 1 nt DNA at the highest temperature and when the amount of adsorbed DNA is very small and the relative errors in the measurements are large (Figure S10). Given the very good overall agreement, our theoretical model is validated and, consequently, allows to infer system properties not directly accessible from the experiments. Molecular Organization of PEG and DNA. To describe the molecular organization within the PEG−DNA film, we use the volume fraction, Φi(z). This quantity represents the fraction of volume occupied by molecules of type i in the region between z and z + dz, i.e., in the direction perpendicular to the surface. The plots of Φi(z) vs z for PEG-5000 and PEG-500 for the experimental surface coverages of 0.32 and 1.32 chains nm−2 are shown in Figures 4A and 4B, respectively. A comparison highlights that the PEG-5000 film at 12 nm is

Figure 4. Polymer volume fraction, Φi(z), and charge distribution within PEG layers as a function of distance from the surface, z. (A, B) Polymer volume fraction of (A) PEG-5000 and (B) PEG-500. The insets show the volume fraction for DNA. (C, D) Charge distribution within PEG-5000 films for (C) the total local charge and (D) charge contributed by DNA molecules with condensed counterions. The inset in (C) shows the charge distribution for the cations (solid lines) and anions (dashed lines), and the inset in (D) displays the fraction of ions condensed at DNA. All panels are for a temperature of 7 °C and a DNA concentration of 2 μM.

considerably thicker than the 2 nm thin PEG-500 layer, which is a direct reflection of the different polymer lengths and surface densities. Hence, the PEG-5000 film is less dense and more porous than PEG-500. Given the higher porosity of PEG-5000, one would expect that it is also more permeable for DNA. Indeed, this is shown in the DNA volume profiles, ΦDNA(z), which have higher values in PEG-5000 than in PEG-500 films (insets of Figure 4A and 4B, respectively). Furthermore, the more facile size-dependent permeation through the loose PEG5000 layer leads to higher volume fractions for the smaller DNA probes (inset of Figure 4A). The predictions also show that DNA partitions close to the ITO interface due to the short-range attraction between the aromatic Cy3 group of the nucleic acid probe and the inorganic ITO surface. In other words, DNA does not reside on top of the PEG brush but penetrates into the PEG layer and adsorbs close to the ITO surface. The penetration into the PEG-5000 layer is in contrast to the rejection of DNA by PEG-500, as can be gleaned from the different y-axis scales for insets of Figures 4A and 4B, respectively. We note that DNA binding does not affect the volume profiles of the polymer film because the amount of adsorbed DNA is too small (compare the scales of Figures 4A and 4B with its inset) to perturb the polymer layer significantly. Given the highly negatively charged nature of DNA, electrostatics is an important component within the interactions of the system. To obtain a more complete picture, we determined the charge distribution within the PEG layers. It can be assumed that the distribution is altered by different amounts of adsorbed DNA strands. This is indeed confirmed by plots of the total charge (Figure 4C) that show large negative charge in the vicinity of the surface due to the adsorbed DNA. This is followed by a net positive charge distribution at up to 5 nm due to more cations (acting as counterions to the DNA negative charge) in the inner layer E

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Figure 5. Total energetics and contributions for the interaction between DNA and PEG layers on ITO substrates. (A, B) The potential of mean force for (A) PEG-5000 and (B) PEG-500 as a function of the distance between the chromophore of Cy3−DNA and the solid surface for different DNA length at 7 °C (solid lines) and 22 °C (dashed lines in insets). (C) Different contributions to the potential of mean force for nt =14 at 22 °C for PEG-5000 as a function of distance to the surfaces. The total potential (magenta) results from a delicate balance between much larger attractive and repulsive contributions. (D) Gibbs free energy of adsorption for the interaction of DNA probes with PEG-5000 films at different temperatures. (E) Gibbs free energy, enthalpy, and entropic change for DNA adsorption as a function of the DNA length for 22 °C and PEG-5000.

than anions (Figure 4C, inset), and a net negative charge at the end of the PEG brush because of the larger number of anions gathering at the brush interface (Figure 4C, inset). The charge distribution shows that the layer has net local charge. Therefore, while the overall system is electroneutral it is not locally electroneutral. The local charge includes all contributions from DNA, bound ions, and free ions. The effective charge of DNA is dramatically reduced due to the condensed couterions (Figure 4D). A plot for the fraction of bound counterions (Figure 4D, inset) reflects the amount of neutralized nucleotides. The fraction of condensed counterions is similar for all DNAs except 1 nt with a much higher value. This is explained by the relatively low charge density of −1e per phosphate bearing nucleotide for DNA strands 5 to 14 nt in length, while the single nucleotide carries −5e condensed within the trinucleotide group. DNA Adsorption and Surface Passivation Result from an Interplay of Large and Opposing Interactions. In order to explore the mechanism of DNA adsorption, it is important to consider all the interactions together as the changes in molecular organization lead to a lack of additivity of the individual factors.53 The best measure of effective interactions between DNA and PEG-modified surface is the potential of mean force βUmf(z). This is the free energy that a single DNA molecule feels averaged over the degrees of freedom of all the other molecules in the system. The potential of mean force represents the work required to bring a DNA from the bulk solution to the position z. The chemical potential of a DNA molecule, μDNA, is given by βμDNA (z) =

δβF = ln ρDNA (z) + βUmf (z) δρDNA (z)

βUmf (z) = −ln q(z)

(3)

with q(z) =

∑ exp[−βU (0) − ∫

n(γz , z′) ln(1 − fN (z′)) dz′

γz

−

χ vw

∫

ϕp(z′)vDNA(γz , z′) dz′ −

vDNA(γz , z′) dz′ −

∫

∫

βπ (z′)

β Ψ(z′)qDNA (γz , z′) dz′]

(4)

where the sum runs over all the conformations of the DNA molecule, γz. The first term in eq 4 represents the attractions between DNA and the surface, which arise from the presence of the Cy3. The second term represents entropic contributions associated with counterion binding to DNA molecules. The third term accounts for the intermolecular (van der Waals) attractions between DNA and the PEG layer, where χ is a measure of the strength of the DNA−PEG attractions. The forth term accounts for the steric repulsions felt by the DNA. π(z′) represents the lateral (osmotic) pressures acting on the DNA at z′. The last term in eq 4 arises from the electrostatic interactions within the layer. It is clear from the expression that the potential of mean force depends on the molecular organization of the PEG layer, through the attractive, repulsive, and electrostatic fields, and on the specific conformations of the DNA. Figure 5A shows the potential of mean force for PEG-5000 for different DNA lengths at two temperatures. All the curves display a similar trend. At z values close to 0, the shape of the potential reflects the attraction of Cy3 to the ITO surface (negative potentials at contact). With increasing distance, the potential grows due to the repulsive nature of the PEG layer. The repulsive barrier is strengthened by increasing DNA length and temperature due to enhanced steric repulsion and decreased van der Waals attraction within the layer. Note that at low temperature the DNA feels a weak attraction after a

(2)

where βF is the total free energy per unit area of the system and ρDNA(z) is the DNA number density at distance z. The potential of mean force of DNA is given by F

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Langmuir small repulsive barrier for PEG-5000 films, which arises from the PEG-DNA van der Waals attraction. Furthermore, the influence of polymer chain length and film thickness is captured as the potential for thicker PEG-5000 (Figure 5A) has a longer distance range than the thinner PEG-500 layer (Figure 5B). The magnitude of the energetic barrier is bigger for the latter film due to the larger role of steric repulsions caused by the higher surface coverage of PEG-500. Because steric repulsions dominate in PEG-500 layer, the influence on the potential of mean force resulting from DNA length is smaller. In order to quantify the different contributions to the potential of mean force, Figure 5C details the components exemplarily for PEG-5000 and DNA of 14 nt. According to eq 4, there are five contributions to the potential of mean force.53 The major repulsive contribution to the overall potential arises from the steric factors due to molecular crowding, which is a combination of the local osmotic pressure and the entropic loss of PEG chains (Figure 5C, black line). The second repulsive contribution is due to counterion confinement via entropic factors (Figure 5C, blue line). The van der Waals forces and, to a lesser extent, the electrostatic interactions are two important attractions (Figure 5C, green and red lines, respectively). The other attraction at the surface is that of the fluorescent label (Figure 5C, orange). The distance dependence of each contribution is similar to each other and reflects the shape of the PEG layer. The overall potential of mean force results, hence, from the combination53 of strong repulsions between the DNA and the PEG arising from steric and entropic factors, which are partly offset by vdW and electrostatic attractions except at the surface for the strong Cy3−interface attraction (Figure 5C, magenta). The interaction between ITO and DNA (without Cy3) is net repulsive, in line with a previous study.42 Thermodynamics of the Interaction of FluorescentLabeled DNA Adsorption on PEG Film-ITO Interfaces. The potentials of mean force contain important quantitative thermodynamic information, namely, the value of Umf at z = 0. This represents the Gibbs free energy associated with the adsorption of a DNA molecule onto the ITO surface covered with the polymer layer. The free energies for PEG-5000 (Figure 5D) are all negative and increase with DNA length, reflecting the passivation of PEG-5000. However, the free energies for PEG-500 (Figure S11) are negative for only 1 nt DNA case, implying almost no adsorption for PEG-500. The thermodynamic values could not be experimentally derived from a Langmuir isotherm. The simple assumptions underpinning the Langmuir isotherm could not capture the complexity of our system which describe the interplay between different molecular factors. From the temperature variation of the Gibbs free energy we obtained the enthalpy and the entropy for the adsorption via ⎛∂ ⎜ ⎜⎜ ⎝ ∂

( ΔTG ) ⎞⎟ = ΔH , ( T1 ) ⎟⎟⎠P

⎛ ∂ΔG ⎞ ⎜ ⎟ = −ΔS ⎝ ∂T ⎠ P

terms, the total free energy, is a very small value compared to both contributions. Extending the Model To Predict Adsorption Isotherms of PEG Layers with Different Surface Coverage. All the calculations presented above were carried out at the fixed PEG surface coverage given by the experimental conditions, i.e., at a surface density, σPEG = 0.32 nm−2 for PEG-5000 and 1.32 nm−2 for PEG-500. To extend our findings to different PEG surface densities, Figure 6 shows the

Figure 6. Amount of adsorbed DNA of various lengths as a function of the surface coverage of PEG. The plots are for (A) PEG-5000 and (B) PEG-500. Solid lines are for 7 °C and dashed lines for 22 °C.

adsorption isotherms as a function of PEG surface coverage, as predicted by the experimentally validated molecular theory. The adsorption decreases monotonically with higher PEG surface coverage, reflecting stronger steric repulsions. Similarly, longer PEG chains reduce adsorption as do, to a larger extent, elevated temperatures. Figure 6 can be used as a design tool to select ideal PEG lengths and densities for a given application. Furthermore, it gives a complete picture of the adsorption behavior something that is hard, if not impossible, to achieve directly from the experimental observations.

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CONCLUSIONS In this report, we have examined the passivating properties of PEG films against fluorophore-labeled, single-stranded DNA probes. While PEG films are increasingly used in chip-based DNA analysis schemes, the knowledge of ideal film parameters and detailed scientific understanding of the interaction between DNA and PEG−substrate interfaces have been missing. Our report has, first, explored a large parameter space to identify those conditions which successfully prevent nonspecific binding. For example, under the experimental conditions PEG-500 films were identified to be better than PEG-5000 in preventing adsorption of a fluorophore-labeled nucleotide (Figure 7) and are hence ideal for sequencing-by-synthesis applications that use these reagents. The source for the better passivation is the higher surface coverage of the PEG-500-coated surfaces. Our report has, second, established that Cy3 and other fluorophores strongly adsorb to the metal oxide surface while PEG film repel DNA and prevent binding. This can in future work inform the choice of substrate and fluorophores to avoid non-specific binding. Our study, third, led to the development of an experimentally validated theoretical approach that provides detailed scientific insight. For example, we found that adsorbed DNA resides close to the substrates surfaces but not on top of the PEG brushes (Figure 7), which reflects the strong affinity of the fluorophore to the ITO surface. The theory also determines the previously unknown Gibbs free energy of adsorption and establishes that binding results from very large but opposing, entropic versus energetic, contributions. Our study, forth,

(5)

The results (Figure 5E) are a manifestation of the competition between the previously discussed opposing forces but are apparent in a more dramatic fashion here. Namely, the change in free energy for the adsorption of a short DNA oligomer to a PEG-5000 ITO surface results from the difference between two much larger quantities that correspond to the entropy (multiplied by the temperature) and enthalpy changes on adsorption. However, the combination of the two opposing G

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the slides in a 1% solution of PEG-5000 or in anhydrous toluene supplemented with 1% triethylamine as catalyst for 18 h at 60 °C under argon. Afterward, loosely bound PEG was removed from the slides by sonication in toluene and acetone for 5 min each, followed by rinsing with deionized water and drying with a nitrogen stream. The PEG-coated substrates were subjected to surface analytical characterization to confirm the chemical identity of the polymeric films and to determine their thickness and homogeneity, as described.20,24,43 Assays on the Surface-Interaction of PEG-Films with DNA Oligonucleotides. The ability of the grafted PEG films to passivate ITO surfaces against nonspecific adsorption of short DNA strands was tested with three fluorescence-labeled oligonucleotides and the mononucleotide dUTP-Cy3. The sequences of the three Cy3-labeled oligonucleotides of 14, 8, and 5 nt length were 5′-Cy3-AGG TGC GTG TTT GT-3′, 5′-Cy3-GTC AGA TT-3′, and 5′-Cy3-CTA GC-3′. The substrates were covered with a commercial 3 × 2 array of round incubation chambers each with a volume of 100 μL and covered by on the top side with a transparent lid featuring two holes for pipetting. The nucleic acids were diluted in buffer A containing 10 mM Tris HCl pH 8.0 and 100 mM NaCl. Bare ITO and PEGylated slides were incubated by pipetting the DNA solution into the chambers and incubating for 180 min at 7.0 ± 0.5, 15.0 ± 0.8, 22 ± 1, and 37 ± 1 °C. Unbound DNA and buffer were removed under vacuum suction, and briefly (2 s) washing with a total volume of 250 μL of deionized water under vacuum suction, followed by drying in a stream of nitrogen. For the 5- and 8-mer oligonucleotides, the assays were repeated three times, and for 1 and 14 nt probes five times. Fluorescence Microscopic Read-Out of Interaction Assays. Fluorescence images of all slides were obtained with an in-house developed fluorescence scanning device,54,55 which is based on an inverted epifluorescence microscope (Axiovert 200, Zeiss, Oberkochen, Germany). For our measurements, a 100× objective (Zeiss, αPlan-FLUAR 100×/1.45) was used. Samples were mounted on a scanning stage (Märzhäuser, Wetzlar-Steindorf, Germany) and illuminated with a diode-pumped solid-state laser with an emission line of 532 nm (Millennia Iis, Spectra-Physics, Irvine, CA) using a dichroic filter 570DCLP (AHF Analysentechnik, Tübingen, Germany) and an emission filter HQ610/75m (AHF Analysentechnik). For detection of Cy5 and Alexa647 fluorophores, a compact diode laser with an emission line at 642 nm (iBeam Smart, Toptica Photonics AG, Gräfelfing, Germany) was used in combination with dichroic filter Q660LP (Chroma Technology Coop, Olching Germany) and emission filter HQ700/75 M (AHF Analysentechnik). Images were taken with a Photometrics CoolSnap HQ digital camera (Roper Scientific, Trenton, NJ) (1392 × 1040 element CCD; pixel pitch 6.45 × 6.45 μm; 12-bit; QE 0.6) using a time-delayed integration mode.54,55 Slide were optionally scanned with a filter of the optical density 1 for bright fluorescence spots exceeding the maximum detection limit of the CCD camera. Image processing and analysis of the images were performed with V++ (Roper Scientific, Tucson, AZ) and Matlab (The MathWorks, Natick, MA). After obtaining average fluorescence counts, the values were corrected for background signal of blank slides and then corrected for the different emission coefficients of four Cy3-DNA conjugates which were determined using UV−vis absorption measurements at a solution of 100 nM and then normalized to the signal of the 14-mer. The fluorescence counts were converted into surface densities using reference DNA standards carrying a radiolabel.43

Figure 7. Experimentally found nonspecific binding or the repulsion of fluorophore-labeled single-stranded DNA molecules on PEG film− substrate interfaces, as shown, is explained by a model that encompasses structural, energetic, electrostatic, and thermodynamic factors. The PEG films and DNA probes are drawn to scale.

provides for the first time the thermodynamic quantities of DNA adsorption onto PEG interfaces which is important to accurately describe the interactions. The model, finally, shows that surface coverage is the most important determining factor in passivating the surface. At a fixed surface coverage, longer PEG chains lead to lower adsorption. The molecular and thermodynamic analysis has practical implications as the calculations can become an integral component in the design of surfaces to prevent the nonspecific adsorption DNA and other macromolecules in modern bioanalytical platforms as well as diagnostic and therapeutic nanoparticles. For these applications, the key to success isas shown by our studyto consider the multitude and interdependence of all factors which include the conformational degrees of freedom of the molecular components and the energetic interactions between the components. Rather than using simplistic models, adsorption behavior can only be understood as a result of the intrinsic interplay between all those factors.

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EXPERIMENTAL PROCEDURES

Reagents. All reagents were obtained from Sigma-Aldrich and used as received unless stated otherwise. Indium tin oxide (ITO)-coated glass slides (50 × 24 × 0.1 mm) with a film thickness of 17 ± 2 nm and a sheet resistance of 1200 ± 200 Ω/sq were obtained from Hans Tafelmaier Dünnschicht-Technik GmbH (Rosenheim, Germany). αMethoxy-ω-{N-[3-(triethoxysilyl)propyl]ureido}poly(ethylene glycol) (MeO-PEG-NH-CO-NH-(CH2)3-Si(OEt)3) with a molecular weight (MW) of 5 kDa (PEG-5000) (PDI = 1.27) was purchased from Rapp Polymere (Tü bingen, Germany). 2-[Methoxy(polyethylenoxy)propyl]trimethoxysilane (MeO-PEG-(CH2)3-Si(OMe)3) with a MW of 500D (PEG-500) was obtained from ABCR (Karlsruhe, Germany). DNA oligonucleotides were ordered from Integrated DNA Technologies (Leuven, Belgium), and 5-amino-propargyl-2′-deoxyuridine 5′-triphosphate coupled to Cy3 fluorescent dye (dUTP-Cy3) was procured from GE Healthcare Biosciences (Vienna, Austria). Formation of PEG Films. Following a published procedure,20,24 ITO slides were cleaned by sequentially sonication in methanol/ chloroform with a mixing ratio of 10:90, 50:50, and 90:10 for 15 min each followed by washing in deionized water and drying in a stream of nitrogen. The substrates were incubated twice for 20 min in a mixture of DI water/ammonia/hydrogen peroxide 30% (v/v/v 100/25/20) at 70 °C, rinsed thoroughly with DI water, and dried in a stream of nitrogen gas. PEG films were coupled onto ITO surfaces by incubating

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02674. Chemical diagrams of Cy3-labeled DNA probes; data on the equilibration of dUTP-Cy3 binding on PEG 5000coated surfaces; details on washing conditions to maintain the equilibrated state for dUTP-Cy3 adsorption; fluorescence micrographs on the binding of Cy3H

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(2) Caro, A.; Humblot, V.; Methivier, C.; Minier, M.; Salmain, M.; Pradier, C. M. Grafting of Lysozyme and/or Poly(Ethylene Glycol) to Prevent Biofilm Growth on Stainless Steel Surfaces. J. Phys. Chem. B 2009, 113, 2101−2109. (3) Aslan, S.; Deneufchatel, M.; Hashmi, S.; Li, N.; Pfefferle, L. D.; Elimelech, M.; Pauthe, E.; Van Tassel, P. R. Carbon Nanotube-Based Antimicrobial Biomaterials Formed Via Layer-by-Layer Assembly with Polypeptides. J. Colloid Interface Sci. 2012, 388, 268−273. (4) Trzcinska, R.; Balin, K.; Kubacki, J.; Marzec, M. E.; Pedrys, R.; Szade, J.; Silberring, J.; Dworak, A.; Trzebicka, B. Relevance of the Poly(Ethylene Glycol) Linkers in Peptide Surfaces for Proteases Assays. Langmuir 2014, 30, 5015−5025. (5) Xia, X.; Yang, M.; Wang, Y.; Zheng, Y.; Li, Q.; Chen, J.; Xia, Y. Quantifying the Coverage Density of Poly(Ethylene Glycol) Chains on the Surface of Gold Nanostructures. ACS Nano 2012, 6, 512−522. (6) Moczko, E.; Guerreiro, A.; Piletska, E.; Piletsky, S. Peg-Stabilized Core-Shell Surface-Imprinted Nanoparticles. Langmuir 2013, 29, 9891−9896. (7) Larsson, A.; Liedberg, B. Poly(Ethylene Glycol) Gradient for Biochip Development. Langmuir 2007, 23, 11319−11325. (8) Schlapak, R.; Danzberger, J.; Haselgrubler, T.; Hinterdorfer, P.; Schaffler, F.; Howorka, S. Painting with Biomolecules at the Nanoscale: Biofunctionalization with Tunable Surface Densities. Nano Lett. 2012, 12, 1983−1989. (9) Yu, C. Q.; Guan, J.; Chen, K. J.; Bae, S. C.; Granick, S. SingleMolecule Observation of Long Jumps in Polymer Adsorption. ACS Nano 2013, 7, 9735−9742. (10) Onses, M. S. Fabrication of Nanopatterned Poly(Ethylene Glycol) Brushes by Molecular Transfer Printing from Poly(StyreneBlock-Methyl Methacrylate) Films to Generate Arrays of Au Nanoparticles. Langmuir 2015, 31, 1225−1230. (11) Ghezzi, M.; Thickett, S. C.; Telford, A. M.; Easton, C. D.; Meagher, L.; Neto, C. Protein Micropatterns by Peg Grafting on Dewetted Plga Films. Langmuir 2014, 30, 11714−11722. (12) Schenk, F. C.; Boehm, H.; Spatz, J. P.; Wegner, S. V. DualFunctionalized Nanostructured Biointerfaces by Click Chemistry. Langmuir 2014, 30, 6897−6905. (13) Jeppesen, C.; Wong, J. Y.; Kuhl, T. L.; Israelachvili, J. N.; Mullah, N.; Zalipsky, S.; Marques, C. M. Impact of Polymer Tether Length on Multiple Ligand-Receptor Bond Formation. Science 2001, 293, 465−468. (14) Wong, J. Y.; Kuhl, T. L.; Israelachvili, J. N.; Mullah, N.; Zalipsky, S. Direct Measurement of a Tethered Ligand-Receptor Interaction Potential. Science 1997, 275, 820−822. (15) Aray, Y.; Marquez, M.; Rodriguez, J.; Vega, D.; Simon-Manso, Y.; Coll, S.; Gonzales, C.; Weitz, D. A. Electrostatics for Exploring the Nature of the Hydrogen Bonding in Polyethylene Oxide Hydration. J. Phys. Chem. B 2004, 108, 2418−2424. (16) Smart, T. P.; Mykhaylyk, O. O.; Ryan, A. J.; Battaglia, G. Polymersomes Hydrophilic Brush Scaling Relations. Soft Matter 2009, 5, 3607−3610. (17) Lee, C. L.; Muthukumar, M. Phase Behaviour of Polyelectrolyte Solutions with Salt. J. Chem. Phys. 2009, 130, 024904. (18) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. Factors That Determine the Protein Resistance of Oligoether Self-Assembled Monolayers - Internal Hydrophilicity, Terminal Hydrophilicity, and Lateral Packing Density. J. Am. Chem. Soc. 2003, 125, 9359−9366. (19) Balamurugan, S.; Ista, L. K.; Yan, J.; Lopez, G. P.; Fick, J.; Himmelhaus, M.; Grunze, M. Reversible Protein Adsorption and Bioadhesion on Monolayers Terminated with Mixtures of Oligo(Ethylene Glycol) and Methyl Groups. J. Am. Chem. Soc. 2005, 127, 14548−14549. (20) Schlapak, R.; Caruana, D.; Armitage, D.; Howorka, S. Semipermeable Poly(Ethylene Glycol) Films: The Relationship between Permeability and Molecular Structure of Polymer Chains. Soft Matter 2009, 5, 4104−4112. (21) Feldman, K.; Hahner, G.; Spencer, N. D.; Harder, P.; Grunze, M. Probing Resistance to Protein Adsorption of Oligo(Ethylene

labeled DNA probes on bare and PEG-coated ITO surfaces; surface densities for bound Cy3-labeled probes as a function of DNA length, DNA concentration, temperature, and PEG type, presented tabulated and in graphical format; concentration dependence of Cy3 binding onto bare ITO surfaces; chemical diagrams of four fluorophores rhodamine, Cy3, Alexa-C2-maleimide, and Cy5; fluorescence count data for the adsorption of the four fluorophores onto bare ITO surfaces; a comparison of experimental adsorption data and theoretically inferred binding of Cy3-DNA probes onto PEG-5000 surfaces; the dependence of the Gibbs free energy for the adsorption of Cy3-DNA probes onto PEG-500 surfaces; and a description of the theoretical methods including the formulation of the theory, minimization of the free energy function, the potential of mean force, the chain model, and parameter selection (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected]; Tel 0044 20 7679 4702 (S.H.). *E-mail [email protected]; Tel (847) 467-0674 (I.S.). Author Contributions

C.R. carried out the theoretical calculations, R.S. and R.H conducted the experiments, and S.H. supervised the project with I.S. and wrote the manuscript with I.S. using contributions from all authors. All authors have given approval to the final version of the manuscript. C.R. and R.S. contributed equally to the manuscript. Funding

C.R. acknowledges support by the National Natural Science Foundation of China under Grant 21274062. I.S. acknowledges support from the National Science Foundation of the USA under Grant CBET-1403058. S.H. acknowledges support by the Austrian Nano Initiative of the Austrian Research Promotion Agency (NSI project 819703 VO104-08-BI), the Austrian Science Foundation (P 25730), the European Regional Development Fund (EFRE), the state of Upper Austria, and the EPSRC Access to Research Equipment Initiative (Grant EP/F019823/1). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Jan Hesse for assistance with the fluorescence microscopic scanning of slides, Ha Phuong Nguyen for drawing chemical diagrams, and Jim Anderson and Giuseppe Battaglia from UCL Chemistry for critically reading the manuscript.

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