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Thickness Dependence of Bovine Serum Albumin Adsorption on Thin Thermoresponsive Poly(diethylene glycol) Methyl ether Methacrylate Brushes by Surface Plasmon Resonance Measurements Ekram Wassel, Siyu Jiang, Qimeng Song, Stephan Vogt, Gilbert Nöll, Sergey I. Druzhinin, and Holger Schönherr Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02708 • Publication Date (Web): 16 Aug 2016 Downloaded from http://pubs.acs.org on August 22, 2016
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Thickness Dependence of Bovine Serum Albumin Adsorption on Thin Thermoresponsive Poly(diethylene glycol) Methyl ether Methacrylate Brushes by Surface Plasmon Resonance Measurements
Ekram Wassel,1 Siyu Jiang,1 Qimeng Song,1 Stephan Vogt,2 Gilbert Nöll,2 Sergey I. Druzhinin1 and Holger Schönherr1,* 1
Physical Chemistry I, Department of Chemistry and Biology & Research Center of Micro
and Nanochemistry and Engineering (Cµ), University of Siegen, Adolf-Reichwein-Strasse 2, 57076 Siegen, Germany 2
NRW Junior Research Group for Nanotechnology, Organic Chemistry, Department of
Chemistry and Biology & Research Center of Micro and Nanochemistry and Engineering (Cµ), University of Siegen, Adolf-Reichwein-Strasse 2, 57076 Siegen, Germany
Corresponding Author *Tel.: +49 271 740 2806. Fax: +49 271 740 2805. E-mail:
[email protected].
ABSTRACT: This study reports on the dependence of the temperature-induced changes in the properties of thin thermoresponsive poly(diethylene glycol) methylether methacrylate (PDEGMA) layers of end-tethered chains on polymer thickness and grafting density. PDEGMA layers with a dry ellipsometric thickness of 5 - 40 nm were synthesized by surface-initiated atom transfer radical polymerization on gold. To assess the temperature-induced changes, the adsorption of bovine serum albumin (BSA) was investigated systematically as a function of film thickness, temperature and grafting density by surface plasmon resonance (SPR), complemented by wettability and quartz crystal microbalance dissipation (QCM-D) measurements. BSA adsorption on PDEGMA brushes is shown to differ significantly above and below an apparent transition temperature. The surface transition temperature found to depend linearly on PDEGMA thicknesses and changed from 35°C at 5 nm thickness to 48°C at 23 nm. Similarly, a change of the grafting density enables the adjustment of this transition temperature presumably via a transition from the mushroom to the brush regime. Finally, BSA that adsorbed irreversibly on polymer brushes at temperatures above the transition temperature can be desorbed by reducing the temperature to 25°C, underlining the reversibly 1 ACS Paragon Plus Environment
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switchable properties of PDEGMA brushes in response to temperature changes.
KEYWORDS: Polymer brushes, stimulus response, lower critical solution temperature (LCST), protein adsorption
INTRODUCTION Polymer brushes play an important role in the area of surface modification and nano- and microfabrication of functional and also addressable (bio)interfaces.1,2 If the grafting density of endtethered polymer chains on a solid support is high enough, the chains extend and stretch away from the surface and thus generate a so-called "brush".3,4,5,6,7,8 Among the remarkable attributes of stimulus-responsive brushes are profound property changes in response to small changes in the environment.3,9 In general, stimulus-responsive materials possess several unique characteristics. For instance, they exhibit fast reversible changes in their structure, shape, surface characteristic, or solubility in response to environmental triggers, which are caused either by chemical or physical stimuli.10,11 While the chemical stimuli comprise changes in pH or ionic strength or the presence of certain metabolic biochemicals, physical stimuli include changes in electric or magnetic field, redox, light or much more importantly changes in temperature.2,10,11,12,13,14,15,16 Among the most intensively and widely
investigated
temperature-switchable
surfaces
are
poly(N-isopropyl
acrylamide)
(poly(NIPAAM))-based coatings, which change their properties at the lower critical solution temperature (LCST). Depending on the structure, the molar mass of the polymer and the solution conditions, the LCST of poly(NIPAAM), above which the polymer becomes insoluble and exhibits a coil-to-globule transition, has been reported to be located near 32°C.12,14,15,17 End-tethered polymer chains exhibit a very similar collapse, which can be tuned to occur close to physiological temperatures.15,18,19,20,21 Recently, there has been increasing evidence for a vertical 2 ACS Paragon Plus Environment
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collapse of poly(NIPAAM) brushes, which starts upon approaching the LCST at the brush-substrate interface.22 In general poly(NIPAAM) and related polymer brushes afford thermoresponsive surfaces, which allow for instance to switch reversibly bio-interactions, and are highly relevant for applications in bioseparation, biosensors, bio-assays or cell engineering, among others.19,23,24 A striking application is the temperature-triggered cell sheet desorption,25,26 which is very mild in contrast to conventional cell removal methods, such as mechanical dissociation and enzymatic digestion. These established methods are known to be damaging to the cells and the extracellular matrix (ECM) beneath them.19,23,24 Despite the popularity of poly(NIPAAM), this material has been reported by some authors to be cytotoxic, 27,28 leading to activation of platelets on contact with blood,16 and has been considered a non-biocompatible polymer.29 Even though these findings are controversially discussed in the literature, 27,30,31,32,33,34,35,36 the design and synthesis of alternative polymers for efficient switchable surfaces has garnered increasing attention, also driven by the need to fabricate biointerfaces with improved resistance to protein adsorption and reduced toxicity. In this context poly(oligoethylene glycol) methacrylate (poly(OEGMA)) derived brushes have been in the focus. 37,38,39,40,41 As known stimulus-responsive polymer brushes, they also exhibit excellent protein resistance37,39,40,42 and are surpassed
only
by
poly[N-(2-hydroxypropyl)
methacrylamide]
(poly(HPMA))
and
poly(carboxybetaine acrylamide) (poly(CBAA)) brushes that show no protein adsorption even in blood.43 The temperature-induced collapse occurs for poly(OEGMA)475 in the bulk and for brushes at a LCST of 90 - 92°C,44,45,46 which is far away from physiological temperatures. Since it is well known that the LCST will be tuned by changes of different parameters, such as the polymer molar mass,
46,47
side chain length,46,48 main chain end group,46
molecular structure,46 and also
concentration, it is not surprising that strategies have been developed to reduce the LCST. This was achieved, e.g., by copolymerization of different oligo(ethylene glycol) monomers that differed in length of the OEGMA side chain.51 This approach afforded materials with tunable LCST. 3 ACS Paragon Plus Environment
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Interestingly, the LCST values for poly(diethylene glycol) methylether methacrylate (PDEGMA) and poly(triethylene glycol) ethyl ether methacrylate (poly(TEGMA)) brushes were reported to deviate significantly from the corresponding bulk LCST values.20,21,44,51 For instance, the LCST was found to increase for PDEGMA brushes from 26°C (for the bulk)52,53 to 32°C - 34°C (for brushes).20,54 Poly(TEGMA) brushes showed according to Dworak et al. LCST - like cell release behavior, when the solution temperature was dropped from 37°C to 17.5°C.25 However, the bulk LCST determined by these authors in cloud point measurements of 20°C in cell medium and 24°C in water is in stark contrast to other values of bulk LCST of 52°C reported in the literature.46,53,55 PDEGMA based brushes are also known to show a temperature-induced collapse, to be relatively resistant to adsorption of cells and proteins, from temperatures below the LCST to above.37 A recent report by the group of Jonas unveiled more subtle differences in the temperature response of these and related polymer brushes and helped to identify some experimental inconsistencies in the acquisition of contact angle data.54 In particular, they differentiate the transition in the bulk from that on the surface. In an earlier work, the difference in collapse temperature of PDEGMA “bulk brush” chains and the “brush surface” was reported by Jonas and co-workers,20 analyzing them with a combination of quartz crystal microbalance dissipation (QCM-D) measurements and water contact goniometry. These authors found that PDEGMA brushes start to collapse from the substrate interface at 22°C (from QCM data). The surface transition occurs in the vicinity of 32°C and is not accompanied by a drastic, step-wise change in contact angle.54 For applications, like the cell sheet engineering mentioned above, the temperature-induced property changes of thermo-responsive surface coatings are of paramount importance. We discovered while exploring alternatives for poly(NIPAAM) brushes that the thermo-responsive properties of relatively thin end-grafted layers of PDEGMA show a pronounced thickness dependence. This dependence was observed, as reported here, in protein adsorption (this study and in a companion cell release study),56 but also in AFM force displacement measurements.57 In order to exploit this thermo-responsive behavior of PDEGMA brushes, we addressed in detail, as 4 ACS Paragon Plus Environment
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reported here, the temperature dependence of the non-specific adsorption of the soft 66.0 kDa protein bovine serum albumin (BSA) by surface plasmon resonance (SPR) for systematically altered PDEGMA brush thicknesses and grafting densities (Scheme 1).58,59,60,61
(a)
(b)
Scheme 1. Schematic of the synthesis of PDEGMA brushes from (a) a neat SAM of the initiator thiol (ωmercaptoundecyl bromoisobutyrate, MuBiB) and (b) from a mixed SAM of MuBiB and a diluent thiol (16mercaptohexadecanoic, MHDA) on gold by surface-initiated atom transfer radical polymerization (SIATRP).
While transitions in PDEGMA brushes and concomitantly a chain collapse are known close to physiological temperatures,20 BSA does not exhibit any conformational changes in aqueous solution in the temperature interval of 15°C to 45°C.62,63 The altered BSA adsorption as a consequence of the temperature-induced chain collapse hence allows us to determine the apparent surface transition temperature of PDEGMA brushes by SPR and to unveil in detail, how this transition depends on brush thickness and grafting density.
EXPERIMENTAL SECTION Materials. The following materials were purchased from the listed suppliers and were use as 5 ACS Paragon Plus Environment
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received unless otherwise noted: Di(ethylene glycol) methyl ether methacrylate (DEGMA), bovine serum albumin (BSA) 95.0% as well as phosphate saline buffer (PBS) tablets: Sigma Aldrich, Schott D263 borosilicate circle glass substrates (diameter: 26 mm, thickness: 1 mm), isopropanol: J.T. Baker, gold 99.99% (granules): Allgemeine Gold- und Silberscheideanstalt AG (Pforzheim), titanium 99.99% (pieces: 3 mm): Chempur, hydrogen peroxide (H2O2) 30%: Roth, sulfuric acid (H2SO4) 95–97% extra pure: Riedel de Häen, ethanol 97%, denaturated: Fischer Scientific, chloroform 99% p. A.: Roth, methanol 99.8%: J. T. Baker, aluminium oxide neutral, for column chromatography: Macherey-Nagel, bipyridine (2,2’-bipyridyl) 99%: Sigma Aldrich, 16mercaptohexadecanoic (MHDA): Sigma Aldrich, dichloromethan p.A.: Fisher Scientic, pyridine p.A.: Merck, 11-mercaptoundecanol 97%: Sigma Aldrich, bromoisobutyryl bromide 98%: Sigma Aldrich, 4-(dimethylamino)-pyridin 99%: Sigma Aldrich, ammonium chloride: Sigma Aldrich, magnesium sulfate (MgSO4): Sigma Aldrich, silica gel, neutral: Sigma Aldrich, hexane: Sigma Aldrich, triethylamine: Sigma Aldrich, copper (II) sulfate pentahydrate 98%: Sigma Aldich, sodium bromide, p.A.: Fluka, diethylether: Sigma Aldrich, toluene: Sigma Aldrich. Piranha solution was prepared as a 1:3 (v/v) mixture of 30% H2O2 and concentrated H2SO4. Caution: Piranha solution should be handled with extreme care. It has been reported to react violently with organic matter and has exploded unexpectedly. Milli-Q water from a Millipore Direct Q8 system (Millipore, Schwalbach, Germany) with a resistivity of 18.0 MΩ cm was used for preparation of all aqueous media
Preparation of surface plasmon resonance (SPR) gold substrates. For evaporation a thermal evaporation machine (Edwards Ltd. EA306 coating system, F.D. Edwards, Crawley, United Kingdom, 1982) was used that was equipped with a rotary vane pump (Edwards Ltd, Crawley, United Kingdom) and a diffusion pump (Edwards Ltd, Crawley, United Kingdom), which afford a final pressure of 2 × 10-7 mbar and including a liquid N2 cooled baffle. At a pressure of 10-6 mbar approximately 2 nm of titanium and 48 nm of gold were evaporated with an evaporation speed of 0.1 nm/s onto the pre-cleaned Schott D263 Borosilicate circle glass substrates (diameter: 26 mm, 6 ACS Paragon Plus Environment
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thickness: 1 mm). Prior to this the glass substrates were first cleaned in a dish washer with at 95°C, followed by isopropanol vapor and finally by UV light in the thermal evaporation machine. Evaporated gold substrates (Glass/Ti/Au) were cleaned with chloroform, followed by rinsing with ethanol and Milli-Q water. Afterwards the substrates were further cleaned by Piranha solution for 60s (for safety notice: see above). After Piranha cleaning, the substrates were immersed immediately in Milli-Q water and then rinsed thoroughly with Milli-Q water, ethanol and immersed for more than 10 hours into 1 mM ethanolic initiator (MuBiB, for details: see Supporting Information) solution for the later synthesis of polymer brushes and in ethanolic solutions of various fractions of inactive MHDA and MuBiB as initiator (total concentration: 1 mM) for the later synthesis of PDEGMA brushes with different grafting densities, respectively. After the formation of SAMs, the samples were thoroughly rinsed with methanol and Milli-Q water.
Synthesis of PDEGMA brushes. For the synthesis of PDEGMA brushes, the monomer (DEGMA, 3.08 mg, 16.7 mmol) purified by filtering through a column filled with aluminum oxide, bipyridine (312 mg, 2.0 mmol), Milli-Q water (3 mL) and methanol (12 mL) were added to a 100 mL round-bottom flask with a stir bar. The mixture was stirred under argon flow for 45 minutes, then CuBr (143 mg, 1.0 mmol, for details see Supporting Information) was added and the dark-red solution was bubbled with argon for further 15 min. The mixture was transferred via a metal cannula (compare Figure S-1, Supporting Information) to a flask containing the initiator-coated gold surfaces under inert argon atmosphere at 23°C. The polymerization was stopped by removing the substrates from the reaction mixture, then rinsing with copious amount of methanol and ethanol before drying in a nitrogen stream.
Ellipsometry. Film thickness measurements were performed in ambient air with an alpha-SE variable angle spectroscopic ellipsometer (J.A. Woollam Co., Lincoln, NE). The measurements were performed on two different points in the center area of each sample at two different incidence angles (65° and 70°)64 with wavelengths between 380 and 900 nm. The data were fitted using a 7 ACS Paragon Plus Environment
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three-layer model in EASE software. The constants of gold substrates were derived from ellipsometric measurements with a Cauchy layer model provided with the instrumental software conducted on 2 different spots on a bare gold substrate. The recorded substrate constants of the background were used to identify the correct thickness of the attached initiator and the grown polymer (we used a refractive index of 1.54 at a wavelength of 632.8 nm for the polymer). Thus the ellipsometric thickness for each sample was independently measured at two different locations and was reported as the average and its standard deviation. The ellipsometric thickness data were double-checked by Fourier transform infrared spectrometry (FTIR).
Fourier transform infrared spectrometry (FTIR). Reflection FTIR spectra (spectral resolution 4 cm-1, 1000 scans) were obtained using an IFS 66V model FTIR spectrometer (Bruker) employing a liquid nitrogen-cooled cryogenic mercury cadmium telluride (MCT) detector. Background spectra were measured using Piranha-cleaned gold, as reported in the literature.65
Surface plasmon resonance (SPR). The surface plasmon resonance data were acquired with an imaging IBIS-iSPR (IBIS Technologies B.V., Hengelo, The Netherlands), which constitutes a flexible SPR imaging platform with an imaged area of about 7×7 mm² with a minimal spot-to-spot distance of 15 µm.66,67 In the IBIS-iSPR the light (840 nm) is reflected on an angle controlled mirror with a maximal scanning angle of 8° before passing through a hemispherical prism to enable fast scanning of the angle shift of the SPR-dip of the individual regions of interest (ROI's). 96 ROI's were defined in the center area of the sample. The SPR angle shift and the amount of adsorbed protein per each ROI are related approximately as follows: 1 m° ≈ 1 ng/cm².66,67 In the experiments the flow-cell (volume: 3 µL) was placed over the brush coated SPR sensor slide and was sealed with a rubber O-ring by fixing the screws in the sample rack. The temperature of the sample was precisely adjusted in the range of 19°C to 43°C by two independently controlled Peltier elements: one located in the sample rack and another high precision element (< 0.01°C) around the flow cell.66,67 The SPR experiments were performed sequentially by injection of PBS (800 µL) to record a baseline, injection of BSA dissolved in PBS (1 mg/mL, 800 µL) and finally the rinsing step by 8 ACS Paragon Plus Environment
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injection of 800 µL pure buffer solution, each with a speed of 30 µL/s and controlled by the microfluidic handling system of the iSPR. After the injection of 800 µL liquid in each sequence, the flow was stopped. The analysis software allows one to fit the minimum of the reflectivity curves obtained for each ROI (Figure S-2, Supporting Information) as a function of time and provides the shift in the SPR minimum ∆θ vs. time. The data were further corrected by a custom made software to correct for baseline drift. To determine the irreversible -, reversible - and total adsorption of BSA, the calculation of three angle shifts is necessary: between region I and region IV for the irreversible adsorption, between region II and region III for the reversible adsorption and between region I and region II for the total adsorption (compare Figure S-3, Supporting Information).
Contact angle measurements. Static contact angles of the PDEGMA brushes were measured with an OCA 15plus instrument (Data Physics Instruments GmbH, Germany) using the captive bubble method. The protocol was double checked by using well-defined SAMa, such as octadecane thiol, which possess known contact angles. During the measurements, the substrates were put upside down in a glass cell (GC20, Data Physics Instruments GmbH, Germany), which was filled with Milli-Q water. The temperature was controlled with model 350 temperature controller (Newport, USA) with a sensitivity to 0.1°C. A thermal sensor was fixed in the glass cell on the same depth as the sample position, to avoid any temperature difference between sensor and sample. For reaching a thermal equilibrium, the sample was kept at each temperature for at least 5 min. Afterwards, an air bubble of ≈ 15 uL volume was injected from below by a U-shape syringe needle. After 1 min equilibration, the left and right contact angle values were recorded. New air bubbles were injected for each measurement. The contact angle data are reported as the arithmetic mean (± standard deviation) of at least 3 independent experiments.
Quartz crystal microbalance (QCM) measurements. 14 mm diameter AT-cut quartz crystal sensors (QSX301, Q-Sense, Västra Frölunda, Sweden) with a thickness of 0.3 mm and 9 ACS Paragon Plus Environment
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correspondingly a resonance frequency of 4.95 MHz ± 50 kHz were used. Before modification, the sensors were cleaned in a UV/ozone cleaner (BioForce, Ames, USA) for 10 minutes and then placed in a heated (75°C) mixture of Milli-Q water, ammonia (25%) and hydrogen peroxide (30%) (v:v:v =5:1:1) for 5 minutes. Afterwards the sensors were rinsed with Milli-Q water and dried in a stream of nitrogen gas. Finally, they were treated again in the UV/ozone cleaner for 10 minutes. PDEGMA layers were synthesized on the QCM sensor chips as reported in the Supporting Information. QCM-D measurements were performed in Milli-Q water with a Q-sense E1 microbalance (Biolin Scientific / Q-sense, Göteborg, Sweden), which was controlled by the QSoft 401 acquisition software. After the modified quartz crystal was inserted into the flow chamber, Milli-Q water was injected. The temperature was ramped between 15°C and 60°C at a rate of 0.25°C/min, while the frequency and dissipation data were continuously acquired. The cooling rate in the subsequent cooling run was also 0.25°C/min.
RESULTS AND DISCUSSION Thickness determination of PDEGMA brushes. For the systematic study of BSA adsorption on PDEGMA layers with various thicknesses and grafting densities, PDEGMA brushes were synthesized via SI-ATRP starting from an initiator monolayer on gold. The polymerization was carried out at 23°C in an oxygen-free environment using CuBr/bipyridine as a catalyst in a Milli-Q water/methanol mixture, with DEGMA as the monomer. The polymerization afforded PDEGMA layers with a dry ellipsometric thickness between 5 ± 1 nm to 40 ± 1 nm. The dependence of the PDEGMA thickness on polymerization time (Figure 1a) agrees well with data in the literature.
65
The film thickness increases monotonically and for longer polymerization times
approaches asymptotically a maximum value. This slowing down has been attributed to termination reactions of the growing brush by radical recombination68 and chain pull out followed by chain transfer.69 10 ACS Paragon Plus Environment
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Figure 1. (a) Thicknesses of PDEGMA layers measured by ellipsometry. (b) Grazing incidence reflection FTIR spectra of several PDEGMA layers.
The synthesized polymer brushes were also characterized by FTIR spectroscopy (Figure 1b) and Xray photoelectron spectroscopy (XPS, data not shown). The FTIR spectra the PDEGMA films show, in accordance with the literature The FTIR spectra (Figure 1b) revealed all expected bands associated to the vibrations of PDEGMA. The peak positions for the symmetric and asymmetric CH stretching vibrations of the methyl groups appear at 2883 cm-1 and 2983 cm-1, and those of the methylene groups are located at 2821 cm-1 and 2927 cm-1. The peak at 1732 cm-1 can be attributed to the symmetric vibrations of the C=O double bond and the peak at 1160 cm-1 to the C-O-C bonds, which is in agreement with the literature.
70
The linear correlation of the integrated absorbance
under the peak at 1732 cm-1 with the brush thickness corroborates the ellipsometric thickness measurements (see Figure S-4, Supporting Information).
Adsorption of BSA on PDEGMA as a function of film thickness. The adsorption of BSA from PBS was followed in real time by imaging SPR on a series of PDEGMA layers with systematically varied thickness, as seen in Figure 2a. After recording a baseline in PBS for 70 min (Figure 2a, region I), 1 mg/mL BSA solution in PBS was injected into the sample cell (region II) and allowed to interact under “no flow” conditions with the brush for 60 min (Figure 2a, region III). Immediately after injection of the BSA solution, a monotonic increase in the SPR signal (reported 11 ACS Paragon Plus Environment
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as SPR minimum angle shift in m°) was observed, which is caused by a minor change in bulk refractive index due to replacement of the buffer with protein in buffer, as well as by BSA molecules that adsorb on the brushes. After 60 min, the surface was rinsed with PBS (Figure 2a, region IV). The difference in the finally obtained ∆θ between baseline and the final steady-state signal is directly proportional to the mass of the remaining adsorbed protein (1 m° ≈ 1 ng/cm²).67 We consider the protein that remains bound to the surface after rinsing with PBS as “irreversibly adsorbed”.
Figure 2. (a) Mean curves of 96 simultaneously acquired SPR curves for BSA adsorption from 1 mg/mL PBS solution on PDEGMA layers with different thicknesses (0 nm thickness: MuBiB on gold) at 25°C. (b) PDEGMA thicknesses dependence of irreversible BSA adsorption at 25°C.
The SPR angle shifts observed in experiments carried out at 25°C were plotted as a function of brush thickness in Figure 2b. The amount of irreversibly adsorbed protein at 25°C decreased from ≈ 130 ng/cm² to ≈ 1.3 ng/cm² with increasing PDEGMA film thickness from 0 nm (i.e. a SAM of the initiator) to 14 nm. The adsorption of the proteins, fibronectin (1 mg/mL), 100% fetal bovine serum (FBS) and 10% FBS, on poly(OEGMA) brushes with a thickness of 15 nm (at an unspecified temperature) was reported before to be below 1 ng/cm²,37 while the BSA adsorption on PDEGMA brush with a thickness of 14 nm studied here was to within the uncertainty virtually identical (≈ 1.3 12 ACS Paragon Plus Environment
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± 1.0 ng/cm2). In a companion study we observed ≈ 8 ng/cm² irreversibly adsorbed fibronectin and ≈ 68 ng/cm² proteins etc. from cell culture medium on 5 nm thick brushes under comparable conditions.56 Hence thicker PDEGMA brushes are not fouled significantly by BSA at 25°C. Such non-fouling properties of thick brushes have been attributed primarily to the steric hindrance effect of the highly hydrated DEGMA side chains.71,72
Temperature response of PDEGMA brushes. The temperature-induced changes of the PDEGMA brushes were then first investigated by contact angle measurements and QCM-D. The contact angle of PDEGMA brush films were measured by the captive bubble approach using separate bubbles for each temperature. It was observed that the static contact angles are practically independent of brush thickness and remain constant to within the experimental error between 25°C and approximately 32 - 35°C and then increase slightly with further increasing temperature (Figure 3). The change in static contact angles does not exceed 5° and occurs continuously. Hence a surface transition the vicinity of the reported brush LCST of 32°C can be confirmed also for these thin brushes,54 however, the surface hydrophobicity changes only to a small extent and not stepwise. While the qualitative trend and temperature dependence are fully in agreement compared to the work by Jonas and co-workers, the absolute values are off-set by 15° to higher values. This may be attributed to the much lower thickness in our case.
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5 nm 25 nm
55
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θ [°]
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Temperature [°C]
Figure 3. Contact angle for PDEGMA brushes of 5 and 25 nm thickness, determined by the captive bubble method as a function of temperature.
The collapse of PDEGMA brushes by QCM-D is evident from the change in slope of the QCM-D data that was acquired during a linear temperature increase with a rate of 0.25°C/min. In Figure 4 the change in slope of the frequency as well as the dissipation of the 3rd overtone is plotted for PDEGMA brushes with 3 nm and 21 nm dry thickness as well as for a QCM sensor covered with an initiator monolayer in water are depicted. The corresponding raw data is shown in Figures S-6 and S-7 in the Supporting Information. While the initiator layer shows no pronounced changes, the frequency and the dissipation of the brushes show maxima and minima, respectively, at 21°C 22°C. The dissipation of the 3 nm thick brush is naturally smaller. Very similar results were obtained for other overtones.
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(a) 0.10
(b)
Initiator 3nm PDEGMA 21nm PDEGMA
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Temperature [oC]
Temperature [ C]
Figure 4. QCM-D data acquired at different temperatures: Variation of the slope of (a) frequency and (b) dissipation versus temperature for a SAM of the initiator and 3 nm as well as 21 nm thick PDEGMA brushes (heating rate 0.25°C/min).
One can conclude at this stage that the thin PDEGMA brushes show that in agreement with previous work20 the collapse starts inside the brush at around 22°C according to QCM and that the surface hydrophilicity changes continuously starting at ca. 35°C according to the contact angle measurements. No obvious thickness dependence of the thermo-responsiveness of the layers was observed.
Irreversible BSA adsorption as a function of temperature. Subsequently, it was analyzed, how the thermo-responsive behavior detected in accordance with the literature by contact angle and QCM-D measurements may affect the adsorption of BSA. The mean SPR kinetics for PDEGMA brushes with a thickness of 5 ± 1 nm at different temperatures are shown in Figure 5a. The amount of reversibly and irreversibly adsorbed BSA increased markedly with increasing temperature. As shown in the Supporting Information (Figure S-8) reversibly adsorbed BSA desorbs rapidly upon changing the medium to PBS. In contrast to the PDEGMA brushes, the irreversible adsorption of BSA on polystyrene as well as the irreversible adsorption of proteins from cell culture 15 ACS Paragon Plus Environment
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medium on the initiator monolayer both decreased with increasing temperature (Figure S-8, Supporting Information). For temperatures > 25°C, a transient maximum (overshoot) was observed in the initial phase of BSA adsorption on PDEGMA (Figure 5a). In Figure 5b the SPR angle shift for irreversible BSA adsorption is plotted vs. temperature (see also Figure S-8, Supporting Information). After a near constant low angle shift (≈ 12 m°) for temperatures below 31°C, the values increase to (≈ 40 m°), showing an overall sigmoid temperature dependence.
Figure 5. (a) Mean curves of 96 SPR curves each, measured for the adsorption of BSA (1 mg/mL in PBS) on PDEGMA layers with a thickness of 5 ± 1 nm at different temperatures. (b) Each point represents resulting arithmetic mean values and standard deviations from 96 SPR curves for irreversible BSA adsorption on a sample covered with poly (DEGMA) layers at chosen temperature. The arithmetic average values of SPR angle shifts increase with increasing temperature.
The protein resistant properties of swollen PDEGMA brushes in water at lower temperatures are known to be strongly correlated with a hydration layer near the surface.71,72 This tightly bound water layer prevents the protein adsorption on the surface by formation of physical and energetic barriers. However, the change in hydration of these brushes as a result of increasing temperature leads to breaking of hydrogen bounds between water and the ether oxygens of the DEGMA side groups, so that the surface becomes less protein resistant. Thus, the transition of PDEGMA brushes from a hydrated, highly swollen and extended to a collapsed state at higher temperature leads to the 16 ACS Paragon Plus Environment
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apparent transition of the 5 nm thick PDEGMA layers in BSA adsorption at approx. 35°C. Due to the collapse of the layer that is at 25°C swollen to > twice the dry thickness, which has been verified in independent AFM experiments,57 BSA molecules may interact partially with the underlying substrate. The approximate thickness of the irreversibly adsorbed BSA layer is with ≈ 0.4 nm (estimated from an angle shift of 40 × 10-3 ° ≈ 40 ng/cm2 and a density of BSA of ≈1000 g/L)73,74 much smaller than the thickness of a monolayer of BSA, which is ≈ 4 nm in side on orientation (BSA is known to possess approximate dimensions of 4 nm x 4 nm x 14 nm in aqueous solution.75 In addition, the maximum absorbed mass coverage of 40 ng/cm2 is more than 3 times smaller than that reported for weakly interacting monolayers (0.143±0.005 µg/cm2 mass coverage for BSA adsorption on a hydroxy-terminated SAM).76 Hence the collapse may enable access of BSA to interact with defects in the underlying PDEGMA layer. From the data presented in Figures 2b and 5b one can see that BSA irreversible adsorption on the initiator of ≈ 130 ng/cm2 at 25°C is an order of magnitude larger than that on PDEGMA layer at 25 °C and 3 times at 40 °C. The overshoot observed in the closer analysis of the SPR kinetic scans in Figure 5a, which was observed at all temperatures > 25°C, was not observed for poly(styrene) coated SPR sensors (no data shown). This overshoot could in principle represent a transient maximum of BSA surface coverage before the lower saturation coverage with adsorbed BSA is reached. It may be attributed to orientational changes and hence change in area requirement per molecule of some of the initially adsorbed BSA molecules and a concomitant reorganization within the adsorbed protein layer that results in desorption of a small fraction of the initially adsorbed BSA molecules. Such behavior has been reported for adsorption of the settlement-inducing protein complex on positively charged SAMs.77 Wertz and Santore78 and Daly et al.79 reported an overshooting effect for the adsorption of fluorescently labelled lysozyme on hydrophilic and hydrophobic surfaces at pH 7.4, caused by the orientational change of adsorbed molecules from an initial end-on to a final side-on orientation with respect to the surface. However, since the absolute amount of adsorbed protein on the hydrophobic surfaces is (much) higher than on the PDEGMA layers discussed here, it cannot be excluded that 17 ACS Paragon Plus Environment
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the sharp increase in SPR minimum angle observed for the hydrophobic reference surfaces (Figure S8) masks the overshoot that may be related to a transiently reduced temperature after injection of BSA solution and hence a transient refractive index increase.
Transition temperature of PDEGMA brushes as a function of film thickness. The SPR angle shifts for irreversible BSA adsorption were found to depend markedly on the thickness of the PDEGMA layer. As shown in Figures 6a and 6b, the inflection point of the sigmoid curve shifts from 35 ± 1°C for a layer thickness of 5 nm to 39 ± 2°C for a thicker layer with 9 nm thickness. For the determination of the inflection points the data were fitted by a sigmoidal function:
∆θ = θ 2 +
(θ1 − θ 2 ) 1+ exp(T − T0 ) / d
(1)
in which θ1 is the initial value of the angle shift, θ2 the final value of angle shift (value for the saturation), T0 the inflection point of the curve, d the width of the curve, ∆θ is the value of SPR angle shift and T is the temperature. This equation can be linearized in the form
θ −θ T − T0 ln 1 2 − 1 = d θ −θ2
(2)
See eq S-1d in Supporting Information. The blank experiments shown in Figure 6c confirm that the observed changes in the SPR minimum only occur for varied temperature, if BSA adsorption takes place. The monotonic dependence of the SPR angle shifts observed in the absence of BSA on all different samples (neat gold sample, 5 nm thick PDEGMA brush on gold and spin coated polystyrene film on gold) agree to within the experimental error with the changes of the refractive index of the aqueous medium (and to a
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negligible extent of the glass) due to the increase or decrease in temperature, respectively (see also Figure S-5, Supporting Information). It is worthwhile to point out that the evanescent field in our experiments carried out with a wavelength of 840 nm decays over much longer distance from the surface compared to conventional experiments carried out at 632 nm. The penetration depth into the dielectric is with 400 nm more than twice that of the 162 nm for conventional red lasers.67 The absence of a pronounced change in the SPR angle shift is attributed to the fact that the PDEGMA layers are very thin compared to this rather long decay length of the evanescent field. As mentioned, from AFM data a swelling ratio of ≈ 2 was estimated.57
Figure 6. SPR angle shifts for irreversible BSA adsorption on PDEGMA coatings with a thickness of (a) 5 nm and (b) 9 nm vs. temperature. The solid lines are fits to a sigmoid function (eq1)). (c) SPR angle shift as a function of temperature for PDEGMA layer, PS film in PBS and neat Au in pure PBS.
For even thicker PDEGMA brushes (17 nm and 23 nm), the curves are effectively shifted to higher temperatures and a possibly present plateau at higher temperatures could not be observed because of the limited temperature range accessible by the temperature control unit and the SPR instrument (for the data see Figure S-8, Supporting Information). Here the transition temperatures were determined by the linearization of the sigmoidal function (see also Supporting Information, Figure 19 ACS Paragon Plus Environment
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S-9) and a linear fit to the measured data. For this linear fit, the intersection points are the sought inflection points. These fits afforded a monotonic dependence of the transition temperature on brush thicknesses (Figure 7). For brushes with increasing thickness from 5 nm to 23 nm, the transition temperature was found to increase from a 35°C to 48°C (see also Supporting Information, Figure S10).
Figure 7. Inflection points for BSA adsorption on PDEGMA brushes determined from the SPR data shown in Figures 4 and S-12 vs. PDEGMA film thickness. The solid line represents a linear fit, which shows a shift of inflection point to higher temperatures with increasing film thickness.
Temperature-triggered desorption of BSA. Next, we investigated the temperaturetriggered adsorption/desorption of BSA on PDEGMA brushes by switching of temperature between 25°C and 40°C. Figure 8 shows SPR angle shifts for BSA adsorption from PBS on a PDEGMA brush with a thickness of 17 nm at 25°C and subsequently at 40°C. While the data in Figure 8a shows the experimental data corrected for the baseline drift, the changes in refractive index due to the change in temperature of the aqueous buffer have been corrected in Figure 8b. The amount of adsorbed protein on swollen PDEGMA brushes was very small at 25°C (4 ± 2 ng/cm²). A subsequent adsorption on the same sensor at 40°C resulted in an increase of irreversibly adsorbed BSA on the now collapsed brushes up to (31 ± 6 ng/cm²). After reducing the temperature from 40°C 20 ACS Paragon Plus Environment
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back to 25°C, which results as shown independently by AFM in rehydrated, swollen brushes at least 80% of the adsorbed protein molecules are released from the surface (Figure 8c).57 A residual coverage of 6 ± 5 ng/cm² still remains on the surface. Additional protein adsorption on this sensor surface at 25°C increased the amount of adsorbed proteins on the surface slightly to (11 ± 3 ng/cm²).
Figure 8. (a) SPR angle shift for the switching of protein adsorption/desorption on thermoresponsive PDEGMA brush with a thickness of 17 nm by switching of temperature from 25°C to 40°C and again back to 25°C, (b) Correction of the changes in refractive index due to the change in temperature of the aqueous buffer (c) Mass coverage of irreversibly adsorbed BSA after each step determined from the data shown in panel (a).
Irreversible BSA adsorption as a function of grafting density. Finally, the dependence of the transition temperature of PDEGMA layers on the grafting density was analyzed. PDEGMA layers with a thickness of 5 nm and 9 nm were prepared with different grafting densities. These samples were fabricated by immersion of cleaned gold substrates in various mixtures of 16mercaptohexadecanoic acid (MHDA) as "dummy" initiator and MuBiB with different ratios for more than 10 hours.70 Figure 9 shows in analogy to the experiments discussed above the dependence of the irreversible BSA adsorption for various temperatures on polymer layers with a constant thickness of 5 nm (Figure 9a, b) and 9 nm (Figures 9c, d) and different grafting density of 21 ACS Paragon Plus Environment
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6% and 43% initiator coverage and 20% and 60% initiator coverage, respectively. The curves resemble those shown in Figures 5 and 6, i.e. ∆θ increases sigmoidally with increasing temperature until a plateau is reached at temperatures of 38°C to 42°C. Fits to equation (1) afforded the corresponding inflection points and transition temperatures, which were found to increase with increasing grafting density.
Figure 9. SPR angle shift for the irreversible BSA adsorption on PDEGMA layers of 5-6 nm with different grafting densities of (a) 6% MuBiB initiator and 94%MHDA, (b) 43% MuBiB initiator and 57% MHDA at different temperatures and the fit of these data by the sigmoidal function (1), (c) (d) SPR angle shift for the irreversible BSA adsorption on PDEGMA brushes of 9-10 nm with different grafting densities at different temperatures and the fit of these data by the sigmoidal function (1), (e) Dependence of the inflection points on grafting density of polymer films with different thicknesses. The dotted lines serve as a guide for the eyes. The inflection points are indicated in red in panels (a)-(d).
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These SPR data show that the transition temperature values of PDEGMA brushes increase with an increasing fraction of MuBiB on the surface. For an initiator coverage > 40% a plateau is observed, as it is shown in Figure 9e. This coverage coincides with the transition from the mushroom regime to the brush regime, which has been reported previously.70 Hence on PDEGMA in mixed SAMS on gold the transition temperature decreases, if the polymer is in the mushroom regime (coverage > 20%) compared to the brush regime. As shown in companion papers,56,57 the brush collapse, apparent mechanical properties and the change in work of adhesion of a hydrophobized colloidal force probe show the same dependence on temperature as the irreversible adsorption of BSA.57 Cells that adhere and spread at temperatures above the transition temperature round up upon cooling to below this transition temperature and can be easily detached by a mild rinsing step.56 Likewise the changes in grafting density yield comparable effects in cell release experiments. Thus by adjusting carefully the thickness and grafting density of thermoresponsive PDEGMA layers, the thickness of irreversibly adsorbed protein can be controlled and efficiently modulated by temperature, which apparently controls cellsurface interactions. The associated apparent transition temperatures can hence be controlled and allow fine-tuning of the desired property changes of the thermoresponsive layers. Since the reported LCST of much thicker PDEGMA brushes is 32.5°C and our contact angle and QCM-D data agree well with the literature data, we attribute the observed changes in very thin PDEGMA layers to a thickness and grafting density dependent modulation of the interactions of BSA with the underlying substrate that is caused by the thermally triggered PDEGMA collapse.
CONCLUSIONS Stimulus responsive poly(diethylene) glycol methacrylate (PDEGMA) brushes with a dry ellipsometric thickness between 5 and 40 nm synthesized by surface-initiated atom transfer radical polymerization show a temperature dependence of the irreversible adsorption of the soft protein 23 ACS Paragon Plus Environment
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BSA. This behavior was unraveled by imaging surface plasmon resonance (iSPR) measurements as a function of polymer thickness and grafting density. BSA adsorption was found to be significantly reduced below an apparent surface transition temperature. This transition temperature was found to increase in a first approximation linearly from 35°C to 48°C with increasing film thickness from 5 nm to 23 nm and matches the layer collapse and work of adhesion determined in an independent AFM study.57 Similarly, an increase in brush grafting density also leads to an increase in the transition temperature of the PDEGMA layers due to perhaps a change of the brush morphology from the mushroom to the brush regime. The results discussed here, which agree quantitatively with temperature dependence of AFM force - displacement measurements57 and cell release experiments56 discussed elsewhere, highlight the need for the determination of the apparent surface transition temperatures in thermoresponsive interfacial architectures, which may differ from the established LCST values for bulk polymers and very thick polymer brushes.
ASSOCIATED CONTENT Experimental Details for the SPR analysis and brush synthesis on QCM sensor surface, scheme of the experimental setup for synthesis of PDEGMA brushes, data for correction and analysis of SPR curves, FTIR spectra of PDEGMA brushes with different thicknesses and their relations to measured ellipsometric thicknesses, SPR sensorgram of a PDEGMA layer in PBS measured at different temperatures, QCM-D raw data, mean values of SPR data for BSA adsorption on PDEGMA brushes with thicknesses of 17 nm and 23 nm at different temperatures and blanks of polystyrene, linearization of the sigmoidal function, 3D representation of BSA adsorption on PDEGMA layers with different thicknesses and different temperatures and linearization of equation (1). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION Corresponding Author *Tel: +49 271 740 2806, Fax: +49 271 740 2805, E-mail:
[email protected].
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT The authors gratefully acknowledge Dr. Elena Sperotto and Dipl.-Ing. Gregor Schulte (Physical Chemistry I, Department of Chemistry and Biology, University of Siegen) for advice and excellent technical support, M.Sc. Marc Steuber for help with the synthesis of the initiator and Dr. Thomas Paululat for the access to the FTIR spectrometer. Financial support by the European Research Council (ERC grant no. 279202) and the University of Siegen is gratefully acknowledged.
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ToC Graphic
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