Dopamine-Assisted Deposition of Dextran for Nonfouling Applications

Mar 3, 2014 - Materials. Dextran with an average molecular weight of 2000 kDa (Dextran T2000) was obtained from Pharmacosmos (Denmark). Dopamine ...
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Dopamine-Assisted Deposition of Dextran for Nonfouling Applications Yunxiao Liu,*,† Chia-Pin Chang,‡ and Tao Sun† †

Miniaturized Medical Device Program and ‡Bio-Electronic Program, Institute of Microelectronic, A* STAR (Agency for Science, Technology and Research), 11 Science Park Road, Science Park II, Singapore 117685 S Supporting Information *

ABSTRACT: Nonfouling surfaces are essential for many biomedical applications, such as diagnostic biosensors and blood- or tissue-contacting implants. In this study, we demonstrate a simple one-step method to introduce dextran onto various substrates based on dopamine polymerization. It has been shown for the first time that dextran molecules could be incorporated into a dopamine polymerization product via mixing dextran with dopamine in a slightly alkaline solution. The codeposited film was characterized by X-ray photoelectron spectroscopy (XPS), the water contact angle, ellipsometry, and atomic force microscopy (AFM). Results reveal that it is possible to control the thickness and surface roughness via the deposition time and deposition repeat cycles. Furthermore, quartz crystal microbalance (QCM) measurements show that the dextran-modified surface inhibits protein adhesion. In addition, cell attachment has been significantly inhibited on dextranmodified surfaces even after exposure to water for as long as 2 months. The described dopamine-assisted dextran modification represents a simple and universal method for nonfouling surface preparation and can be potentially applied to improve the performance of various medical devices and materials.

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INTRODUCTION The development of nonfouling surfaces is critical to the success of many biomedical devices, including biosensors, drug-delivery systems, and implantable devices. The nonspecific protein or cell adhesion on biomedical device surfaces often lead to adverse effects, such as foreign-body inflammation for implants and nonspecific responses for biosensors.1−4 As a result, the functions of biomedical devices can be severely impaired. A common strategy to limit nonspecific protein or cell adhesion is to introduce antifouling polymers, such as poly(ethylene glycol) (PEG), dextran, and polyacrylates,5−9 onto device surfaces. Physical adsorption and covalent immobilization methods have been applied to introduce antifouling polymers. For example, PEG was conjugated with poly(ethylene imine) (PEI) and then deposited onto a negatively charged surface via electrostatic interactions.10 Although physical adsorption is a simple method, the lack of long-term stability of the coating is a limitation. In comparison, covalent conjugation may provide long-term stability; however, specific functional groups such as amino and carboxyl groups need to be displayed by the substrate and/or polymer. Modification of the tethering polymers and substrates is often required, and conjugation approaches are to be developed case by case. For example, to modify a gold surface, PEG molecules need to be functionalized with thiol terminals,8,11 and to graft dextran onto a substrate surface, the modification of dextran to introduce aldehyde groups and the amination of glass are required.7 These methods are often time-consuming. Recently, a robust surface-coating strategy based on dopamine has been demonstrated.12−14 Dopamine, a simple catecholamine © 2014 American Chemical Society

better known as a neurotransmitter, was shown to selfpolymerize in a slightly basic solution in the presence of dissolved oxygen.15,16 The resultant material readily adhered to a wide array of substrates that were dipped into the solution, including metal, metal oxides, semiconductors, glass, and polymers.12 The chemical composition of the coating is complex, and recent findings showed that it was more likely to be a mixture of covalently bound and physically associated dopamine oxidation products.17,18 Nevertheless, the resultant materials are generally called polydopamine (PDA). The PDA coating has good stability in various environments except under strongly alkaline conditions (pH >13) or in the presence of a strong oxidant such as sodium hypochlorite (NaClO) or potassium periodate (KIO4).17,19−21 The PDA coating harbors latent reactivity toward nucleophiles, thus polymers with nucleophilic groups, most commonly amino and thiol groups, can be readily grafted onto a PDA coating to create diverse functional surfaces including nonfouling ones.22,23 For example, PDA coatings have been postmodified with amine- and thiol-containing PEG, leading to a dramatic reduction of nonspecific protein adsorption and fibroblast cell attachment.12 Polymers can also be preconjugated with dopamine, and the incorporated catechol moieties are utilized for polymer immobilization onto various substrates.2,24,25 For example, dextran was conjugated with dopamine via a two-step Received: January 6, 2014 Revised: February 24, 2014 Published: March 3, 2014 3118

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each sample, and the results were expressed as the mean ± standard deviation. XPS spectra were taken with a Thermo Scientific Theta Probe with a monochromatic Al Kα (1486.8 eV) X-ray source at a power of 100 W. A taken-off angle of 40° was used. The chamber was kept at a pressure of 8.8 × 10−10 mbar. High-resolution scans were performed to calculate the chemical compositions of the modified surfaces. The surface roughness and morphologies of the modified surfaces were obtained using atomic force microscopy (AFM, NanoWizard3 NanoOptics) in a tapping mode. For the thickness determination, samples were scratched with a clean syringe needle, and the height difference between the scratched and unscratched areas was measured for the thickness of the coating. For both XPS and AFM measurements, coatings were deposited on a silicon wafer. The coating thickness was also determined via a spectroscopic ellipsometry measurement (model HS-190, J. A. Woollam Co. Inc.). The measurements were performed at a continuous wavelength ranging from 300 to 800 nm and angles of incidence of 65, 70, and 75°. Six independent measurements were taken for each sample over an area of 2 × 2 cm2. The ellipsometric data were fitted to a three-layer model (Dex/ PDA layer, native silicon oxide layer, and silicon layer) using WVASE software. The thickness of the native silicon oxide layer was determined to be 2.3 nm using a Fresnel model by fixing the real part of the refractive index at 1.465. Then this value was fixed, and only parameters related to the Dex/PDA layer were fitted. The dielectric function of the Dex/PDA layer was modeled using the Lorentz oscillator model. Thickness and Lorentz parameters (amplitude An, center energy En, real part of the high-frequency dielectric constant ε1(∞), and broadening Brn,) of the Dex/PDA layer, were set as the fitting parameters. QCM measurements (Q-Sense E4) were used to analyze protein adhesion on modified surfaces. Gold-coated crystal sensors were precoated with PDA or Dex/PDA. The resulting polymer-coated sensors were mounted into the liquid-exchange chambers of the instrument. A stable baseline in PBS was achieved, after which a protein solution (RPMI-1640 plus 10% FBS) was introduced onto the crystals. When adsorption on the surface was saturated, the chambers were rinsed with PBS again. Normalized frequencies using the third overtone are presented. For the cell adhesion test, breast cancer cell line MCF-7 and lung cancer cell line H460 obtained from ATTC were used. Both cell lines were cultured at 37 °C in a humidified incubator containing 5% CO2. MCF-7 was cultured in DMEM (E15-810, from PAA) with 10% fetal bovine serum (FBS), and H460 was maintained in RPMI-1640 (E15842, from PAA) with 10% FBS. After growth to almost confluence, the cells were harvested using trypsin−EDTA solution. Cell suspensions were added to modified surfaces. After either 1 or 18 h of incubation, the wells were washed twice with phosphate-buffered saline (PBS) to rinse away the unattached cells. The attached cells were recorded using an Olympus microscope. Either bright-field (BF) images or differential interference contrast (DIC) images were taken. Cell attachment was also determined using a CyQUANT cell proliferation assay kit (Invitrogen). Briefly, cells were cultured on 96-well TCPS that had been premodified with PDA or Dex/PDA. After 18 h of culturing, the cell culture medium was discarded, and the plates were washed twice with PBS and then stored at −70 °C. Before measurement, cells were thawed at room temperature. The CyQUANT GR dye/cell-lysis buffer (200 μL) was added to each well of the microplates, and then the entire solution was transferred to a black microplate. A fluorescence measurement was made using a microplate reader (Enspire monochromator plate reader, PerkinElmer) with excitation at 485 nm and emission detection at 530 nm. A reference standard curve was generated with cell numbers ranging from 30 to 7000 cells per 200 μL of sample, and the attached cell numbers were calculated using this standard curve. Six measurements were read for each sample. The data are reported as the mean ± standard deviation. A student’s t test was performed between different groups. Probabilities of P ≤ 0.05 were considered to be significant differences.

procedure.25 The resultant catechol-grafted dextran was found to bind strongly to titania and suppressed cell adhesion. A third option is that by simply mixing polymers into dopamine solution polymers can be codeposited during PDA formation.26−30 Initially, the codeposited polymers have been limited to carboxyl, amine, thiol, quaternary ammonium, and catechol groups, which are believed to form covalent bonds with dopamine. For example, poly(ethylene imine)-graf t-poly(ethylene glycol) (PEI-g-PEG) was successfully introduced onto different substrates using the codeposition method, and resultant coatings inhibited serum protein adsorption as well as fibroblast attachment.31 Later, poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) without any of the above-mentioned functional groups have also been incorporated onto surfaces via dopamine-assisted codeposition.32 It is believed that noncovalent interactions such as hydrogen bonding are responsible for PEG and PVA entrapment in the PDA coating. In fact, intermolecular hydrogen bonding between PVA and dopamine has been utilized to enhance the loading efficiency of dopamine in poly(D,L-lactic-co-glycolic acid) microspheres.33 Dextran (Dex) has been widely used as an antifouling coating layer on various substrates.7,25,34 However, to immobilize dextran onto a substrate, chemical modification of dextran is generally required.7,25 In this work, we aim to assess whether it is feasible to incorporate dextran via simple one-step dopamine-assisted assembly without any chemical modification. Because dextran has abundant hydroxyl groups that can act as both hydrogen acceptors and donors, intermolecular interactions are expected to form between dextran and dopamine. Using the water contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) characterizations, we have shown for the first time that a high-molecular-weight dextran (Mw = 2000 kDa) was successfully incorporated within the PDA coating via noncovalent interactions onto various surfaces, including silicon (Si), glass, polystyrene, and poly(dimethylsiloxane) (PDMS). Quartz crystal microbalance (QCM) analysis showed that the deposited coating reduced the protein adhesion. Furthermore, reduced cell adhesion has also been demonstrated.

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EXPERIMENTAL SECTION

Materials. Dextran with an average molecular weight of 2000 kDa (Dextran T2000) was obtained from Pharmacosmos (Denmark). Dopamine hydrochloride and tris(hydroxymethyl) aminomethane (Tris) were purchased from Sigma-Aldrich and used as received. Surface Coating. Dopamine solution was prepared in 10 mM Tris buffer (pH 8.5) at a concentration of 2 mg/mL. For codeposition, a mixture of dopamine (2 mg/mL) and dextran (10 mg/mL, unless otherwise specified) was prepared in Tris buffer, which is referred to as dextran/dopamine solution hereafter. Pieces of silicon wafer (2 × 2 cm2), glass slides (18 × 18 mm2), and PDMS (diameter around 11 mm, thickness around 200 μm) were cleaned by sonication in ethanol for 10 min, followed by 10 min in deionized water, and then blow-dried under a stream of nitrogen. Tissue culture polystyrene (TCPS) Petri dishes (FALCON), 96-well TCPS plates (Cellstar), and 8-well Lab-Tek Chamber slides (Thermo Scientific) were used as received. For surface coating, the samples were immersed in dopamine solution or dextran/ dopamine solution and kept for a certain time (either 2 or 18 h) on an orbital shaker. The coated samples were then rinsed with deionized water and dried in a stream of nitrogen. We will call a dopamine-treated surface a PDA surface and a dextran/dopamine-treated surface a Dex/ PDA surface. Characterization. The surface hydrophilicity of pristine and modified surfaces was characterized by water contact angle measurements (DSA 100, Krüss). At least six measurements were performed for 3119

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Figure 1. (A) XPS survey scans of unmodified, PDA-coated, and Dex/PDA-coated silicon substrates. Enlarged images on the right are high-resolution Si 2p scans. (B) High-resolution O 1s spectra of unmodified and modified Si substrates. (C) High-resolution C 1s spectra of PDA- and Dex/PDA-coated Si substrates.

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RESULTS AND DISCUSSION Film Assembly. To verify whether dextran can be codeposited during dopamine polymerization, we first choose silicon as the substrate for deposition. XPS analysis (Figure 1)

was then performed on coated as well as pristine Si. Figure 1 shows the XPS survey scans as well as high-resolution O 1s and S 1s scans. Compared to pristine silicon, the appearance of the N 1s peak at a BE of ∼400.2 eV (Figure 1A), the decrease in Si 2p 3120

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deposition onto the substrate surface. We have observed brownblack precipitation in the solution during the PDA coating process (Supporting Information Figure S1 left), which has been suggested to be supramolecular aggregates of dopamine oxidative products (such as 5,6-dihydroxyindole (DHI), 5,6-indolequinone (IDQ), and DHI oligomers) held together via noncovalent interactions such as hydrogen bonding, π stacking, and chargetransfer interactions.9,12 In the presence of dextran, the color change of the dopamine solution to black has been observed, which is characteristic of PDA formation.21 However, no aggregate was observed for dextran/dopamine solution even after centrifugation (Supporting Information Figure S1 right). We also noticed a difference in the color of the deposited films on PDMS, glass, and TCPS: the deposited PDA is darker than the codeposited Dex/PDA. All of these indicate that dextran has inhibited the noncovalent assembly of dopamine oxidative products. Two aspects of the inhibition effect may be explained: (i) dextran itself forms hydrogen bonds with oxidative products such as DHI and (ii) high-molecular-weight dextran causes a steric hindrance effect for the aggregation process. We also increased the dextran concentration and found that PDA could be formed at a dextran concentration of up to 100 mg/mL. Note that the prevention of PDA formation, in which case the darkening of the solution is no longer visible, has been observed in the presence of poly(N-vinylpyrrolidone).32 The rinse stability of the Dex/PDA coating was tested via measuring the optical thickness changes, which were obtained by fitting the ellipsometry data with the Lorentz oscillator model  after immersion in various solutions for 6 h. For all of the tested buffers except pH 3 DI water, there was no significant decrease in thickness (Supporting Information Figure S2), indicating that the Dex/PDA coating has good rinse stability. For DI water pH 3, a slight decrease in thickness was observed, which may be caused by the disruption of hydrogen bonding. It is interesting that we have found when immersing dopamine and Dex/PDA coatings into reductive NaBH4 solution (10 mg/ mL in 0.1 M NaHCO3) overnight that the black color on the surface of the substrates (glass, PDMS, and PS) disappeared and the reductant solution became green. We suspect that the coating was degraded by the reductant, and this is confirmed by the water contact angle measurement (Supporting Information Figure S3). For hydrophobic PDMS and PS substrates, after NaBH4 treatment, both the previous PDA- and Dex/PDA-coated substrates become hydrophobic again with the water contact angles almost returning to the values of pristine substrates. However, for hydrophilic Si and glass substrates after NaBH4 treatment, the substrates become even more hydrophilic with water contact angles similar to that of piranha solution-treated substrates. The degradation of PDA in the presence of NaBH4 has also been observed by others. It is probably triggered by a decrease in the oxidized dione content. This decrease may cause the disappearance of hydrogen bonding, which has been reported to be responsible for PDA formation. The same may happen for the Dex/PDA film. Note that in the presence of a strong oxidant, such as NaClO, the degradation of PDA has also been observed.9,20 All of these suggest that the balance of diol and its oxidized carbonyl structures in dopamine solution is important for PDA formation. However, this has to be investigated further. The surface morphologies of modified substrates were investigated using an AFM. The deposition of PDA increased the Si surface roughness, consistent with previous reports in the literature.16 However, to our surprise, the Dex/PDA-coated

intensity (Figure 1A), the increase in C 1s intensity (Figure 1A), and the shift in O 1s (Figure 1B) from ∼532.5 eV (Si−O) to ∼533.2 eV (C−O) and ∼531.5 eV (CO) indicate the spontaneous deposition of PDA on silicon after immersion in dopamine solution. Similar changes are observed for the dextran/ dopamine-treated silicon substrate, indicating that PDA was also deposited on Si in the presence of dextran. However, compared to PDA-coated silicon, dextran/dopamine-treated silicon has a lower N 1s intensity (Supporting Information Table S1) and an increased intensity of the oxygen- or nitrogen-substituted carbon (C−O/C−N) component at a BE of ∼286.6 eV in the highresolution C 1s scan (Figure 1C). Because dextran has no N element and a higher ratio of the ester carbon (C−O) component than dopamine, the mentioned changes above confirm that dextran was incorporated into the coating layer during PDA deposition. It is possible that dextran is physically entrapped in or bound to the PDA coating layer via noncovalent interactions such as hydrogen bonding. Note that when the dextran concentration varies from 2 to 100 mg/mL the content of dextran incorporated into the deposited film changes (Supporting Information Table S1). Because the PDA coating can be deposited onto almost any type of material surface,12 we expect the Dex/PDA coating to be applied to substrates other than Si. We immersed glass, PDMS, and PS substrates in dextran/dopamine solution and then measured the wettability changes using the water contact angle measurement. Figure 2 shows the result. Glass, PDMS, and PS

Figure 2. Water contact angles of pristine, PDA-coated, and Dex/PDAcoated substrates. Substrates investigated include glass, silicon (Si), PS, and PDMS.

substrates show exactly the same trend in wettability change as for the Si substrate: water contact angles decreased after immersion in dopamine solution for 18 h, and a larger decrease is observed for dextran/dopamine-treated substrates than for dopamine-treated substrates. This result confirms the codeposition of dextran onto the tested inorganic and polymeric substrates. It has been assumed that PDA deposition on a substrate surface is trigged by the absorption of dopamine oxidative monomers and oligomers and in a similar manner as to polymerization in the solution.5,13 Thus, investigation into the reaction solution helps us to understand the mechanism of 3121

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Figure 3. AFM phase images of pristine Si, PDA-coated Si, and Dex/PDA-coated Si.

the thickness of the PDA layer on the Si surface reaches ∼9 nm. For the Dex/PDA coating, the thickness is about 11 nm. We also evaluated the thickness of the coating layers based on the attenuation of the Si 2p signal in the XPS measurement. The following equation is used for calculation:

surface was much rougher than the PDA-coated surface (Figures 3 and S4). Aggregates with size ranging from tens to hundreds of nanometers were seen on the Dex/PDA-coated surface. Although we have observed precipitation in dopamine solution, we did not see many aggregates on the PDA-coated surface probably because the particles were so large that they were washed away from the surface by DI water.35 Note that particles from dopamine solution have been reported to be several micrometers in diameter.21,36,37 In the presence of dextran, only small aggregates (not large enough to precipitate) formed, which could adhere firmly to the surface. The measured average roughnesses (Ra) of Si, PDA-coated Si, and Dex/PDA-coated Si are 0.15, 0.85, and 1.98 nm, respectively, and the root-meansquared (rms) roughnesses of the above surfaces are 0.43, 1.11, and 4.89 nm, respectively. The morphology changes of the modified surfaces verify the formation of coatings on the substrates. The thickness of the coating layers was determined by AFM using scratched surfaces (Figure S5). After 18 h of immersion,

⎛ −d ⎞ ⎟ It = I0 exp⎜ ⎝ λ sin θ ⎠

(1)

Io and It are the Si 2p peak intensity before and after coating, θ is the taken-off angle (40° in our case), d is the thickness of the coating layer, and λ is the inelastic mean free path of Si 2p photoelectrons in the organic coating film, which depends on both the kinetic energy of the photoelectron and the specific materials that the photoelectron passes through. λ can be estimated on the basis of the structural formula of the coating molecules.38 For the PDA film, we estimate λ to be 3.7 nm, and for the Dex/PDA film, we estimate λ to be 3.6 nm. The detailed calculation of λ is shown in the Supporting Information. 3122

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disappears for the six layers. The calculated thickness using eq 1 for one layer is 2.5 nm, and that for three layers is 6.3 nm. The thickness of the three layers based on XPS data is slightly less than that of the 18 h-deposited film (7.8 nm), consistent with the optical thickness result (18.5 ± 0.6 nm for three layers vs 21.5 ± 0.5 nm for 18 h of deposition). High-resolution C 1s spectra can be peak fitted into three components: C−C (285 eV), C−O/C−N (286.6 eV), and CO (288.3 eV). We noticed that the ratio of the C 1s intensity at 286.6 eV to that at 285 eV decreased with the number of deposition cycles (1.21, 1, and 0.96 for one, three, and six layers, respectively). It is known that dextran has a higher content of ester carbon than dopamine, and thus a decrease in a C−O/C−N to C−C ratio indicates a decreased ratio of dextran to dopamine in the deposited film with the deposition cycle. Also, the 2 hdeposited film has a higher ratio of C−O/C−N to C−C than does the 18 h-deposited film (1.21 vs 1.05), indicating that the deposition of dextran was particularly pronounced during the initial process. This could be due to the adsorption of dextran on the Si surface. However, as the deposition time or the repeat cycle increases, the dextran content in the deposited film becomes stabilized, as indicated by the slight change in the C−O/C−N to C−C ratio. The LBL-assembled film shows morphology similar to that of the 18 h one-cycle-deposited film (Figure S6). Aggregates are seen on the surface. However, the size is smaller on the LBLassembled film than on the one-circle-deposited film (18 h). The LBL-deposited surfaces are smoother. The measured Ra and rms for three layers are 1.26 and 2.88 nm, respectively. For six layers, Ra and RMS are 1.45 and 3.23 nm, respectively. The above values are smaller than that of the 18 h one-cycle-deposited film (1.98 and 4.89 nm). Thus, it is possible to obtain a thick yet smooth Dex surface, which is expected to be more efficient in resisting protein or cell adhesion than a thin and rough surface is, via repeated but short-time immersion in fresh dextran/dopamine solution. Protein and Cell Adhesion. We evaluated whether codeposited dextran exhibits protein-repellent properties. Films were deposited on QCM gold sensors (1 layer, 18 h). A color change in the gold sensors has been observed after PDA and Dex/PDA deposition, indicating the successful coating of the gold surface. When proteins were introduced onto the sensors, fast drops in the resonance frequency were observed (Figure 6A), indicating the adsorption of protein onto the sensor surface. For bare and Dex/PDA-coated gold sensors, the resonance frequencies reach steady state quickly (about 6 min after protein injection) whereas for the PDA-coated sensor the frequency drop lasts for a longer time. This is due to the fact that PDA can react with amine or thiol groups containing proteins, and the reaction takes time to finish. In the presence of dextran, the reactivity of PDA toward protein has been inhibited, thus the resonance frequency quickly drops to a steady state. The bare gold sensor shows the highest Δf, followed by the PDA coating and then the Dex/PDA coating, indicating that protein adsorption has been inhibited. When considering the dissipation changes in the absorbed protein layers, a greater dissipation change is observed for the PDA surface than for the Dex/PDA surface (Figure 6B), indicating that a soft protein layer (water-rich) has been formed on the PDA surface whereas a rigid film (less water) has been formed on the Dex surface. The difference in viscoelasticity of the adsorbed protein layer may be due to the different conformational arrangement of protein molecules on the surface.39

The thickness determined by XPS for the PDA coating layer is 9.0 nm, and that for the Dex/PDA coating is 7.1 nm. Note that the thickness determined by XPS for the PDA film is consistent with that determined by AFM. However, for the Dex/PDA film, the thickness value calculated on the basis of XPS data is smaller than that determined by AFM. The difference is not surprising considering that AFM was measured under conditions of ambient humidity whereas XPS was performed in an ultrahigh vacuum chamber. Dehydration of the coating layers is expected in the chamber of an XPS analyzer. Not much dehydration was observed for the PDA coating in a vacuum chamber. For the Dex/PDA film, a smaller thickness in a vacuum chamber than under ambient conditions suggests the dehydration of the film. Because dextran is a very hydrophilic polymer, this dehydration is expected for the dextran-containing coating. Layer-by-Layer Deposition. Because PDA is able to grow on virtually any kind of substrate surface, it might also grow on a surface already covered with PDA. This has been proven to be true: the deposition of thick films is possible, provided that unpolymerized dopamine is refreshed regularly.12,19 Here, we also tested whether a thick Dex/PDA coating layer can be obtained by providing freshly prepared dextran/dopamine solution regularly. Si wafers were dipped into dextran/dopamine solution. After 2 h, wafers were taken out, washed, and dried in a steam of nitrogen. This process was repeated for several cycles. Note that it is a similar process to layer-by-layer (LBL) assembly. We consider one deposition cycle to be one layer of coating. Figure 4 shows the ellipsometric thickness changes obtained by fitting the measured data to the Lorentz oscillator model as a

Figure 4. Ellipsometric thickness changes in Dex/PDA deposits obtained by immersing Si substrates into dextran/dopamine solutions as a function of repeat immersion cycles. For each cycle, the Si substrate was immersed in dextran/dopamine solution for 2 h.

function of deposition repeat cycles. It clearly shows that the thickness of the deposited Dex/PDA layer increases linearly with the number of deposition repeat cycles, indicating the LBL deposition when fresh solution is provided. Note that the 18 hdeposited film has an ellipsometric thickness of 21.5 ± 0.5 nm, which is slightly greater than that of the three layers (18.5 ± 0.6 nm). Considering that the optical thickness may contain errors because the light-scattering loss caused by the presence of surface inhomogeneities is not accounted for by the Lorentz oscillator model, we also performed XPS analysis. The result confirms the feasibility of using the LBL process to deposit thick layers. The Si 2p intensity decreases with the increase in the number of layers (Figure 5A and Supporting Information Table S2) and totally 3123

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Figure 5. (A) XPS survey spectra and (B) high-resolution C 1s spectra of Si after the first layer of assembly in dextran/dopamine solution, after three layers of assembly, and after six layers.

and most of them exhibited a round morphology (Figure S7). When a piece of TCPS was partially modified by Dex/PDA, cells were found to attach only to the unmodified part. Quantitative analysis also confirms the cell-repellent effect of the Dex/PDA coating (Figure 7B). Cell attachment to Dex/PDA surfaces decreased by more than 90% compared to that of unmodified TCPS, whereas no significant difference is shown between the PDA surface and bare TCPS. It is worth noting that the coating is quite stable. After being stored in water for about 2 months, the Dex/PDA-coated surface suppressed cell adhesion to the same degree as the freshly prepared one did (old samples in Figure 7B). However, for the PDA-coated surface, significantly more cells attached to old samples than to fresh samples. This may be caused by PDA oxidation resulting in increased affinity for proteins during long-time storage. The Dex/PDA coating has also been shown to inhibit H460 cell adhesion (Figure S8). All of these indicate that the dextran codeposited surface inhibits cell adhesion effectively. Because PDA is able to bind proteins, the fact that the codeposited dextran surface inhibits protein and cell adhesion suggests that dextran molecules have completely covered the

Because less protein was adsorbed on the Dex/PDA surface, an elongated fat arrangement of protein molecules may be the case. However, considering that the PDA surface adsorbed more proteins, a standing-up arrangement of the protein molecules with more water entrapped is more likely the case. A slow increase in dissipation is observed for the PDA coating, consistent with the slow decrease in the resonance frequency. We next evaluated whether codeposited dextran inhibited cell adhesion. Dex/PDA coatings were prepared on glass, TCPS, and hydrophobic PDMS (1 layer, 18 h). Two types of cells have been seeded on the modified surfaces: MCF-7 and H460. Figure 7A shows MCF-7 attachment after 1 h of culturing on glass and TCPS. Significantly more cells were attached to the PDA surface than to the bare substrate surface, whereas after Dex/PDA coating the number of attached cells decreased significantly. A more pronounced inhibition effect is observed on the Dex/PDAcoated glass surface: barely any cells are seen (Figure 7A). Longterm cell culturing was also performed (Figure S7), and the results show that after being cultured for 18 h, MCF-7 on bare TCPS and PDA-coated TCPS exhibits a spreading morphology. However, on Dex/PDA-coated TCPS, fewer cells were found 3124

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Figure 7. (A) DIC images of MCF-7 adhesion on bare, PDA-coated, and Dex/PDA-coated glasses and TCPS. Cells were cultured for 1 h before imaging. (B) MCF-7 attachment measured using a CyQUANT cell proliferation assay kit. (*) P ≤ 0.05 and (**) P ≤ 0.0001. Old samples have been stored in water for 2 months before cell culturing.

the interface reaction between device and biological media is required.

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Figure 6. QCM-D signals obtained for protein adsorption on bare and modified Au sensors. (A) Frequency change vs time. (B) Dissipation factor change vs time.

ASSOCIATED CONTENT

S Supporting Information *

Photographs of dopamine solution and dextran/dopamine solution, thickness changes in Dex/PDA films after 6 h of immersion in various buffer solutions, water contact angles of a modified surface after treatment in NaBH4 solution, AFM 3D images of pristine and coated surfaces, AFM height profiles of scratched surfaces, AFM phase and 3D images of LBL-deposited surfaces, MCF-7 attachment after 18 h of culturing, H460 attachment after 1 h of culturing, calculation of the inelastic mean free path λ of Si 2p photoelectrons in PDA and Dex/PDA films, and tables of XPS analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

surface and shielded the effect of PDA. Note that PEG-NH2 (Mw = 5000) has also been deposited on the surface via a similar dopamine-assisted one-step deposition.27 However, the cell resistance of such a PEG coating was not as encouraging as that of our Dex/PDA coating. One possible reason is that the PEG graft density is too low. However, a high coverage of PEG could be obtained via two-step deposition: first the PDA layer is deposited and then PEG is deposited on this PDA anchoring layer, but the process is time-consuming.37 Here, by using a high-molecularweight nonfouling dextran, we create a high density of polymer surface coverage that effectively inhibits protein and cell adhesion.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +65 67705438. E-mail: [email protected].

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Notes

CONCLUSIONS Herein, we have demonstrated dextran to be a nonfouling polymer that can be incorporated onto various substrates via simple immersion into dextran/dopamine solution. The deposited film has been confirmed to be mixture of PDA and dextran via XPS analysis. Interestingly, the thickness of the deposited film can be controlled by providing freshly prepared dextran/dopamine solution. The presence of dextran in the mixed film effectively inhibits protein adsorption and cell adhesion. Moreover, the coating is quite stable under wet conditions. We believe that this facile, one-step method of nonfouling surface preparation can be potentially applied to therapeutic and diagnostic biomedical devices, where control of

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Science and Engineering Research Council of the Agency for Science, Technology and Research (A*STAR) under grant 1021710159.

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REFERENCES

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