“Active Surfaces” Formed by Immobilization of Enzymes on Solid

Sep 10, 2014 - In various domains ranging from catalysis to medical and environmental sciences, there is currently much focus on the design of surface...
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“Active Surfaces” Formed by Immobilization of Enzymes on SolidSupported Polymer Membranes Camelia Draghici,†,‡,§ Justyna Kowal,†,§ Alina Darjan,‡ Wolfgang Meier,† and Cornelia G. Palivan*,† †

Chemistry Department, University of Basel, Klingelbergstrasse 80, 4056 Basel, Switzerland Department of Product Design, Mechatronics and Environment, Transilvania University of Brasov, 29 Eroilor Blv, 500036 Brasov, Romania

‡

S Supporting Information *

ABSTRACT: In various domains ranging from catalysis to medical and environmental sciences, there is currently much focus on the design of surfaces that present active compounds at the interface with their environments. Here, we describe the design of “active surfaces” based on solid-supported monolayers of asymmetric triblock copolymers, which serve as templates for the attachment of enzymes. A group of poly(ethylene glycol)-block-poly(γ-methyl-ε-caprolactone)-block-poly[(2-dimethylamino) ethyl methacrylate] amphiphilic copolymers, with different hydrophilic and hydrophobic domains (PEG45-b-PMCLx-b-PDMAEMAy) was selected to generate solid-supported polymer membranes. The behavior of the copolymers in terms of their molecular arrangements at the air−water interface was established by a combination of Langmuir isotherms and Brewster angle microscopy. Uniform thin layers of copolymers were obtained by transferring films onto silica solid supports at optimal surface pressure. These solid-supported polymer membranes were characterized by assessing various properties, such as monolayer thickness, hydrophilic/hydrophobic balance, topography, and roughness. Laccase, used as an enzyme model, was successfully attached to copolymer membranes by stable interactions as followed by quartz crystal microbalance with dissipation measurements, and its activity was preserved, as indicated by activity assays. The interaction between the amphiphilic triblock copolymer films and immobilized enzymes represents a straightforward approach to engineer “active surfaces”, with biomolecules playing the active role by their intrinsic bioactivity.

1. INTRODUCTION There is currently focus on the design of “active surfaces” for developing hybrid materials with specific properties and functionality at the interface with their environments for applications in domains, such as catalysis, medicine, and technology. A particularly appealing strategy is to design active surfaces by immobilization of biomolecules on solid supports, which will confer functionality by the intrinsic activity of the biomolecules. Various methods have been proposed for enzyme immobilization: (i) physical adsorption on a solid support, (ii) covalent binding to modified surfaces, (iii) cross-linking, and (iv) entrapment in matrices, such as polymer gels, channels, or capsules.1−3 In order to obtain high enzyme loading, immobilization is frequently performed on porous materials, for example, mesoporous silicates,4 nanoporous gold,5 or nanozeolites deposited on indium tin oxide surfaces.6 For example, α-chymotrypsin immobilized on mesoporous silica remained active for at least 100 recycles over 10 days.7 However, porous materials suffer diffusional limitations because of the large molecular weight substrates involved in the enzymatic reaction. While nonporous materials should overcome this limitation, their drawbacks are low enzyme loading and the risk of protein denaturation on contact with a hard support.8 Thus, to avoid protein denaturation, the solid support © 2014 American Chemical Society

has to be covered with soft layers, for example, lipids or polymers.9 There are numerous examples of successful enzyme immobilization on surfaces covered with lipid layers via physical adsorption. Examples include immobilization of rat osseous plate alkaline phosphatase on phospholipid films deposited on gold by Langmuir−Blodgett transfers (LB)10 and immobilization of tyrosinase by ionic interactions between the enzyme and a solid support.1 Various approaches have been reported for enzyme immobilization on lipid membranes, for example, by Langmuir−Schaefer transfer of a mixed tyrosinase−phospholipid (1,2-dipalmitoyl-S,N-glicero-3-phosphocholine) monolayer to a quartz support11 or by incubation of an enzyme solution with a monolayer of a mixture of linoleic acid, octadecyltrimethylammonium bromide, and poly[(N-nonylphenoxazine-3,7-diyl-alt-(1,2,3-benzothiadiazole)] prepared on glass via LB transfer.12 As stability in time and robustness are key factors for potential applications of active surfaces, an elegant approach is to use polymer instead of lipid membranes. Because of their greater stability, polymer membranes have advantages in fineReceived: July 18, 2014 Revised: September 10, 2014 Published: September 10, 2014 11660

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tuning properties, such as flexibility and responsiveness, due to their chain size and compositions. For example, when a polymer film is formed by Langmuir compression, various packing states are obtained compared with the discrete states available for lipids.13 Most polymer membranes are symmetric, being formed by self-assembly of AB or ABA amphiphilic block copolymers, for example, poly(butadiene)-block-poly(ethylene oxide) (PB-bPEO),14 polystyrene-block-poly(acrylic acid),15 and poly(2methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2methyloxazoline) (PMOXA-b-PDMS-b-PMOXA).16 They have been successfully used to host membrane proteins, either as vesicles in nanoreactors,17 or solid-supported membranes containing porins.18 In order to perform a directional membrane protein insertion/attachment or to generate membranes with a different specificity at each surface, asymmetric triblock copolymers, ABC, represent ideal candidates. For example, thin films of nonamphiphilic ABC triblock copolymers have been used to interact with bovine serum albumin.19 However, to the best of our knowledge, solidsupported asymmetric polymer membranes based on amphiphilic copolymers have not yet been used for immobilization of enzymes in order to engineer active surfaces. The asymmetry of a polymer membrane is a key factor favoring the functionality of active surfaces with the desired orientation (at the external membrane interface), while the lower polymer block serves for binding to the solid support. Here, we present a strategy for engineering active surfaces by immobilization of enzymes on a monolayer of asymmetric poly(ethylene glycol)-block-poly(γmethyl-ε-caprolactone)-block-poly[(2-dimethylamino) ethyl methacrylate] (PEG45-b-PMCLx-b-PDMAEMAy) amphiphilic copolymers that are solid supported. We selected these ABC block copolymers, because of their chemical nature and related properties: PEG is a hydrophilic biocompatible block, PMCL is a flexible hydrophobic block, and PDMAEMA is a second hydrophilic block with tertiary amine active groups.20 Polymer monolayers formed at the air−water interface were transferred to a solid support by the LB transfer technique, known as a “grafting to” deposition method, which results in defect-free monolayers on any kind of slides of unrestricted size.21 In addition, we selected the LB transfer technique because it involves no chemical reactions, as in the case of “grafting from” methods for development of solid-supported synthetic membranes.22 In addition, the LB transfer technique allows controlling of the density of the resulting monolayer and has a high reproducibility compared to other “grafting to” methods (i.e., direct polymer adsorption from the solution, spreading, and fusion of vesicles).23 We were interested in understanding the following: (i) how ABC copolymers are organized under compression at air−water interfaces, (ii) the orientation of the A, B, and C blocks at air− water interfaces, (iii) the structural properties, which determine film formation and availability for enzyme adsorption, and (iv) the stability and reactivity of the active surface on an enzyme− ABC polymer film. First, we investigated the behavior of PEG45-b-PMCLx-bPDMAEMAy block copolymers at the air−water interface in order to find the optimum conditions for their transfer to the solid support. Then, the polymer monolayers were transferred to silica slides by the LB deposition method, which resulted in well-organized solid-supported polymer films. Surface properties of the films were characterized in terms of thickness, wettability, topography, and roughness, in order to select the

best for enzyme immobilization. We chose to immobilize the enzyme by physical adsorption, which is both simple and straightforward. Solid-supported enzyme−ABC films were then characterized in terms of their morphology, stability, and enzyme activity. Since this is the first report of enzyme immobilization on a solid-supported membrane of asymmetric amphiphilic triblock copolymers, we have combined an indepth characterization of the behavior of the copolymers at the air−water interface, with determinations of the stability and activity of the immobilized enzymes on the LB films. Our intention was to generate active surfaces in a straightforward and biofriendly way and to allow further functionality by simply changing the type of enzyme.

2. MATERIALS AND METHODS 2.1. Materials. PEG45-b-PMCLX (AB) and PEG45-b-PMCLx-bPDMAEMAy (ABC) block copolymers (Figure S1, Supporting Information) were synthesized as described previously.20 In brief, ring-opening polymerization of γ-methyl-ε-caprolactone (MCL) was performed using PEG as a macroinitiator. The modified PEG-b-PMCL diblock copolymer containing an atom transfer radical polymerization (ATRP)-initiating group was then used for synthesis of the third, PDMAEMA block. Silica slides were obtained from Si-Mat Silicon Materials, Germany. Laccase from Trametes versicolor, 2,6-dimethoxy phenol (DMP), and solvents (of highest purity grade) were purchased from Sigma−Aldrich. 2.2. Preparation of Silica Slides. Silica slides were cut into pieces of about 1 cm2 area and then cleaned by ultrasonication in chloroform (three times for 15 min). Prior to use, the slides were placed in an UV/ozone chamber for 15 min. 2.3. Langmuir Monolayers at the Air−Water Interface. Langmuir isotherms were recorded with a KSV Inc. (Finland) Langmuir Teflon trough (area 420 cm2) equipped with two symmetrically moving hydrophilic Delrin barriers and a Wilhelmy plate (ashless filter paper) as a surface pressure sensor. All experiments were performed at a constant temperature of 20 °C, with water as the subphase, at pH 7.5. The compression procedure consisted of the following steps: (i) the trough was thoroughly cleaned with chloroform and ethanol and then filled with the aqueous subphase (double distilled water); (ii) the barriers were cleaned with ethanol and rinsed with water; (iii) the Wilhelmy plate was equilibrated in the subphase for 30 min; (iv) the water surface was cleaned through compression−aspiration−expansion cycles, and then, a certain volume of copolymer solution in chloroform (1 mg mL−1) was spread dropwise on the surface of the water subphase; (v) the solvent was allowed to evaporate for 10−15 min; and then, (vi) the monolayers were compressed to the desired surface pressure at a constant barriers rate of 10 mm min−1. 2.4. LB Transfer Technique. LB transfers of polymer monolayers onto solid supports were performed with a Mini-trough (KSV Instruments, Finland) with surface area of 242 cm2. In order to perform the transfer, the cleaned silica slides were placed in the water subphase, the polymer was added, and the film was formed at the air− water interface, following the same procedure as described for the Langmuir isotherms. The Mini-trough barriers were stopped at a surface pressure value lower than the one corresponding to film collapse, determined when recording the Langmuir isotherms. At that surface pressure, monolayers of ABC copolymers were transferred to the silica slides by lifting the dipper at a constant rate of 0.5 mm min−1. 2.5. Immobilization of Enzyme on Polymer Films. Immobilization of enzymes on polymer films was performed in two different ways: (i) before transfer of the films to the solid support and (ii) after film transfer to the solid support. For (i), the polymer films were compressed to a surface pressure of 20 mN m−1, and then, 25 or 100 μL of laccase solution (2 mg mL−1 in PBS or bidistilled water) was spread dropwise at the air−water interface. After 30 min of stabilization, ABC−enzyme films were compressed to 30 mN m−1 and then transferred to the silica substrate (further indicated as 11661

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“transfer technique”). For (ii), silica slides with transferred polymer monolayers were immersed in enzyme solutions for 30 min or 1 h (0.5 mg mL−1 in PBS pH = 4.25) and then rinsed with PBS buffer (further indicated as “immersion technique”). 2.6. Activity of Free and Immobilized Enzyme. Activities of free laccase and laccase immobilized on polymer films were investigated with DMP as substrate, with a final concentration of 0.06 mM in bidistilled water, at pH 7. The activity of free enzyme was measured after 12 h with a laccase solution with a final concentration of laccase of 500 ng mL−1. Slides with immobilized enzymes were immersed in DMP solutions, also for 12 h. The UV−visible (UV−vis) spectra were then recorded in the wavelength range 200−800 nm (with an accuracy of 1 nm) using a Thermo Scientific NanoDrop 2000c UV−vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a xenon flash lamp. 2.7. Brewster Angle Microscopy. Polymer film compression was monitored by an EP3SW system (Nanofilm Technologie GmbH, Göttingen, Germany) equipped with a Nd/YAG laser (λ = 532 nm), long distance objective (Nikon, 20×), and monochrome CCD camera. The size of the Brewster angle microscopy (BAM) image corresponds to 220 × 250 μm2, with a resolution of 1 μm. 2.8. Ellipsometry. The thickness of the polymer monolayers on the silica slide was measured with an EP3SW imaging ellipsometer (Nanofilm Technologie GmbH, Göttingen, Germany). Measurements were performed for 10 incident angles ranging from 55° to 75°. The thickness of the silicon dioxide layer (∼2 nm) was taken into account when estimating the polymer thickness. The refractive index value used for the polymers was 1.5. Each type of sample was measured at least 5 times on two different slides, and average values were calculated for values determined with a mean squared error less than 1. 2.9. Contact Angle. The wetting properties of polymer monolayers were investigated with a contact angle goniometer, CAM 100 (KSV Instruments, Finland) based on a CDD camera with 50 mm optics. Measurements were performed by placing droplets of ultrapure water with a microsyringe on the substrate, and automatic curve fitting was performed by the equipment software. The average value of the contact angle was calculated from at least five measurements with each silica slide for each type of sample. 2.10. Atomic Force Microscopy. The topography of the polymer monolayers transferred to the silica slides was investigated by atomic force microscopy (AFM) using an Agilent 5100 AFM/SPM microscope (PicoLe System, Molecular Imaging). All measurements were carried out in the tapping mode in air, using silicon cantilevers (PPP-NCHR, Nanosensors) with a nominal spring constant of 42 N m−1. The images were analyzed with the data analysis software Gwyddion (v. 2.37). 2.11. Quartz Crystal Microbalance with Dissipation. Adsorption of the enzymes onto polymer films was determined with a quartz crystal microbalance with a dissipation (QCM-D) system Q-Sense E1 (Biolin Scientific, Sweden). The polymer film was first transferred by the LB technique to the silicon dioxide QCM-sensor on the Minitrough and placed in the QCM chamber. After 1 h in PBS buffer for stabilization, the enzyme solution (0.5 mg mL−1) was introduced into the QCM chamber with a flow speed of 100 μL min−1 and then allowed to stabilize for approximately 15−30 min, before washing thoroughly with buffer. Measurements with laccase were performed at pH 4.25.

Table 1. Properties of the ABC Monolayers roughness (nm) ABC block copolymer

transfer ∏a (mN m−1)

A45B84C30 A45B84C85 A45B101C3 A45B101C3 A45B101C12 A45B101C17 A45B101C27 A45B101C27

30 30 32 30 30 30 30 26

film thicknessb (nm) 7.01 7.91 9.11 8.57 7.96 7.65 7.66 5.31

± ± ± ± ± ± ± ±

0.11 0.03 0.02 0.04 0.20 0.04 0.16 0.38

contact angleb (deg)

pH 3

pH 7

± ± ± ± ± ± ± ±

0.20 0.18 0.20 0.26 0.29 0.21 0.61 0.62

0.83 0.13 0.23 0.26 0.55 0.34 0.97 0.80

55.9 60.3 62.4 61.2 77.8 66.9 64.8 73.0

1.0 1.2 1.2 2.1 1.5 2.2 1.5 0.8

a Surface pressure at which the transfer was done. bAverage values calculated from measurements taken on two different plates, and on five different zones, with the related standard deviations.

3.1. Formation of ABC Block Copolymer Films at the Air−Water Interface. First, the behavior at the air−water interface of ABC polymer films was investigated by compressing the copolymer monolayer and recording changes in the surface pressure at pH 7.5. Langmuir (surface pressure− area) isotherms indicated that A 45 B84 C x and A 45 B101 C x copolymers had similar behavior with a plateau zone where the polymer domains rearranged at the air−water interface and ending at surface pressures (Π) at which the polymer monolayer collapsed (Figure 1a, b).24 The length of the B and C blocks influenced the compression behavior: the plateau zone is more evident for block copolymers with a higher ratio between the hydrophobic B and hydrophilic C block lengths. Therefore, A45B84C85 compression resulted in an isotherm with almost no plateau (Figure 1a). As the overall shapes of the Langmuir isotherms for A45B101Cx polymers (Figure 1b) were similar, the film behavior was controlled mainly by the size of the hydrophobic B block, with very low contribution from the C block. Similar results were described previously for the behavior of AB amphiphilic block copolymers at the air−water interface.25 Langmuir isotherms also provide information regarding film transfer onto the solid support. Surface pressures close to film collapse ranged between 30 and 35 mN m−1 and were dependent on A, B, and C monomers units of the triblock copolymers. There was an increase in the mean molecular area at the collapse point with increasing number of C monomer units in the A45B101Cx copolymers. Thus, whereas A45B101C3 achieved the collapse surface pressure up to the mean molecular area of 200 Å2, a mean molecular area of about 350 Å2 was necessary for A45B101C27 (Figure 1c). This behavior is explained by higher volumes of ABCs with increased number of C monomers units, which required more space for molecular arrangement during film formation. Similar isotherm shapes were obtained for the A45B101Cx triblock copolymers and the corresponding AB diblock copolymers (Figure 1d−f). However, because of the absence of the C block, AB copolymers were characterized by both lower surface pressures for film rearrangement during the compression (plateau zone) and lower mean molecular areas at the surface pressure close to the collapse pressure. As expected, there was almost no difference in the isotherms of A45B101C3 and the corresponding AB copolymer (A45B101), because of the short hydrophilic C block of the triblock copolymer (Figure 1f).

3. RESULTS AND DISCUSSION Solid-supported membranes of asymmetric triblock copolymers on which an enzyme was immobilized were prepared to serve as stable active surfaces with functionality resulting from the intrinsic enzymatic reaction and stabilization by support by the polymer films. In this respect, a small library of ABC triblock copolymers with PEG as the A block, PMCL as the B block, and PDMAEMA as the C block (PEG45-b-PMCL101-bPDMAEMA27) was used to self-assemble and form solidsupported films (Table 1). 11662

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Figure 1. Surface pressure−area isotherms of the AB and ABC block copolymers: (a) A45B84C30 (black) and A45B84C85 (red); (b) A45B101C3 (pink), A45B101C12 (black), A45B101C17 (red), and A45B101C27 (green); (c) A45B101Cx block copolymers at surface pressure close to the collapse points; (d) A45B84 (red) and A45B84C85 (black); (e) A45B101 (red) and A45B101C27 (black); (f) A45B101 (red) and A45B101C3 (black).

Figure 2. BAM images recorded during A45B84Cx and A45B101Cx block copolymer compressions at the air−water interface, at different surfaces pressures (expressed in mN m−1): 10, 16, and 30, respectively.

In order to optimize film transfer to solid surfaces, we tested both the stability and elasticity of the polymer film. The stability of thin films was assessed by recording the surface pressure−time isotherms, at Π corresponding to the surface pressure of transfer, at constant mean molecular area, with a stop run of at least 30 min (Figure S2, Supporting Information). The very slow decrease in time of the surface pressure indicates that the polymer films are stable. The elasticity of ABC films was evaluated by recording three reversible compression−expansion cycles (Figure S3, Supporting Information). As no relevant hysteresis was observed, ABC block copolymers did not dissolve in water, and their monolayers were elastic. For the A45B84C85 block copolymer, slight shifts were observed to smaller mean molecular areas

corresponding to the expansion cycle. This indicates a slight loss of material in the aqueous subphase, because not the entire polymer was relaxing during the expansion. 3.2. ABC Block Copolymer Arrangements at the Air− Water Interface. Modifications of polymer arrangements during Langmuir compression, registered as surface pressure− area isotherms, were confirmed by the differences in BAM images (Figure 2), which showed different phase transitions. At the beginning of the compression process, at low surface pressures, the ABC block copolymers have a large space at their disposal, and large molecular areas, as in the gaseous phase.26 Therefore, they start with a “pancake” conformation, in which no interactions between polymer chains occur; then, they organize themselves by adopting a “mushroom” conformation, 11663

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tion. The thickness of the film with the PDMAEMA block externally oriented with respect to the film surface was about 24 nm, that is, ∼2-3 times thicker than our ABC monolayers with a similar number of total monomer units. 3.5. ABC Block Copolymer Film Wettability. In order to ensure a proper interaction of the ABC copolymer films with enzymes, their wettability, resulting from the hydrophilic-tohydrophobic balance, was determined by contact angle measurements using drops of bidistilled water. First, we assumed that, at the air−water interface, the ABC block copolymer adopts an orientation in which PEG, as hydrophilic block A, with constant length (45 units) is oriented toward water, while PDMAEMA, as the hydrophilic block C with variable lengths, is oriented toward the air phase. Such organization of polymer chains is also expected because PEG is more hydrophilic (PEG film has contact angle of approximately 30°)30 than PDMAEMA (contact angle of this polymer is approximately 50°).31 Second, when transferring the ABC film onto the silica slides, we assumed that the ABC block copolymers are preferentially adsorbed with PEG directly linked to the silica slides through hydrogen bonding with silanol groups available at the silica surface. PEG acts as an anchor block during adsorption onto the hydrophilic silica substrate, whereas PDMAEMA is expected to be externally oriented. As the PEG length is constant for all the ABC block copolymers, the orientation during compression at the air− water interface of the PDMAEMA, which has variable block length, is expected to induce differences in contact angle values, if our assumption regarding the orientation of blocks inside the polymer film is correct. The contact angle values for all ABCs monolayers were lower than 90° (Table 1), which indicates that the films transferred onto silica slides generated a rather hydrophilic surface. Indeed, the differences in contact angle values support our hypothesis that the PDMAEMA block is externally oriented. The preferential orientation of PDMAEMA block as an external block is expected to support the enzyme immobilization, compared to the PEG block, which is known to be protein repellent. The asymmetry of the membrane is the key factor, which favors a selective interaction of the enzyme with the desired interface of the membrane. 3.6. ABC Block Copolymer Films Topography. In order to increase the surface area available for interaction with enzymes, roughness is a key molecular property of membranes. The surface topography of the ABC films was evaluated by AFM, performed after immersion of the solid-supported monolayer in PBS buffer at different pH values (3, 7, and 10). AFM images offer both qualitative (Figure 3 and Figure S5, Supporting Information) and quantitative information regarding the roughness of polymer films, based on average roughness values (Table 1). The influence of pH on the roughness of the monolayer is mainly the result of pH sensitivity of the external PDMAEMA C block. All films formed by ABC block copolymers had high roughness at pH 10 that was difficult to determine. The roughness of the ABC triblock copolymer films increased with increasing buffer pH from acidic to neutral values, which is in agreement with previously reported results for diblock copolymer poly(2-vinylpyridine)-block-poly(dimethylaminoethyl methacrylate) (PVP-b-PDMAEMA).28 An exception to this behavior was obtained with A45B84C85, possibly due to the high number of the C block units inducing electrostatic interactions at the C block domain, which increased the polymer chain packaging within the film. For

in which small lateral interactions force the water-soluble chains (A blocks) to coil. At surface pressures corresponding to the plateau zones (16 mN m−1) BAM images of A45B101Cx copolymers indicate the formation of “brushlike” arrangements. When compression is increased, the copolymers adopt a more ordered “cigarlike” conformation, and the final conformation is a highly packed monolayer corresponding to the collapse point (Figure S4, Supporting Information).24,26 Only the A45B84C85 block copolymer did not show a mushroom or brushlike arrangement, in agreement with the isotherm shape with no plateau zone. Below the collapse surface pressure (30 mN m−1), both groups of ABC copolymers (with B84 and B101 blocks) formed uniform films. The combination of Langmuir isotherms and BAM clearly indicate that ABC block copolymers formed wellorganized, closely-packed, defect free, elastic, and stable films at the air−water interface. 3.3. LB Transfers of Copolymer Films to Silica Slides. ABC films were transferred from the air−water interface to silica plates at Π values below the collapse pressure, to obtain a densely packed polymer monolayer with stretched chains. For all transfers, the transfer ratio was about 1, indicating a successful and defect-free deposition, with yield close to 100%.27 In order to understand the availability of the polymer film for enzyme adsorption, various surface properties of the film were studied: (i) thickness (by ellipsometry), (ii) hydrophilic/hydrophobic balance (by contact angle), and (iii) topography and roughness (by AFM) (Table 1). 3.4. ABC Block Copolymer Monolayer Thickness. In order to obtain robust polymer monolayers, we studied the effect of the transfer surface pressure and lengths of the B and C blocks on the monolayer thickness. As expected, the thickness of the film increased with increase in the transfer surface pressure for the same ABC block copolymer (Table 1). This behavior is the result of the ABC copolymer arrangements adopted before collapse of the polymer film. At surface pressures below the plateau values, the block copolymers formed brushlike arrangements, whereas at higher surface pressure, they adopted cigarlike arrangements. In this case, both hydrophilic and hydrophobic domains are stretched due to space limitations.24 The difference in the ratio between the number of B hydrophobic and C hydrophilic monomer units also influenced monolayer arrangements at surface pressures below that of film collapse. As expected, for the B84 copolymers group, a larger film thickness was registered for a higher number of C monomer units. For the B101 copolymer group, the behavior was the opposite when transferred at the same surface pressure (Table 1). As the influence of the lengths of the B and C blocks on film thickness cannot be explained solely by their volume and conformation adopted during compression, we also took account of charge effects. Since the hydrophilic block C is positively charged at pH values less than 8,28 lateral electrostatic attractions at the C block domains favor the formation of more compact structures and as a consequence less thick films. Polymer film thickness also depends on the method used to prepare the film. As an example, an AB dihydrophilic block copolymer film was obtained by a “grafting from” method based on direct surface-initiated ATRP.29 The diblock copolymer poly(butyl methacrylate)-block-poly(2,2-dimethylaminoethyl methacrylate) (PBMA115-b-PDMAEMA58) had a folded conformation for the PBMA hydrophilic block, whereas the second PDMAEMA hydrophilic block adopted a stretched conforma11664

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used to calculate the mass that adsorbed to the sensor covered by polymer film, by using the Sauerbrey equation33 (Δm = −CΔf), where C is a proportionality constant, depending on the quartz properties (C = 18 ng cm−2 Hz−1). The transferred films formed by the A45B101Cx group of ABCs and A45B84C30 adsorbed laccase (Figure 4a and Figure S7, Supporting Information), and depending on the polymer, the amount of adsorbed enzyme ranged from 63 to 450 ng cm−2 (Table 2). Our values are in agreement with previous reports that indicated that gold surfaces covered with a zirconated layer of 11-mercaptoundecanol adsorbed 520 ng cm−2 of laccase34 and a self-assembled monolayer formed by a mixture of glutaraldehyde and cysteamine adsorbed 280 ng cm−2 of laccase.35 The immobilization of laccase was durable for at least 24 h at room temperature as measured for PEG45-b-PMCL101b-PDMAEMA27 film (Figure 4b). Ongoing experiments aim to establish the maximum stability of enzyme adsorption on solidsupported polymer films. Interestingly, the A45B84C85 copolymer monolayer possessed antifouling property by repelling laccase. On the basis of the molecular weight of the laccase unit of 68 kDa,5 the surface coverage was calculated as the Δm/Mw ratio (Table 2). We calculated the maximum number of laccase that can theoretically be attached in a completely packed mode on the silica slide surface (of about 1 cm2) by taking into account the laccase size (6.5 nm × 5.5 nm × 4.5 nm).36 The mostly packed orientation of the enzyme on the surface was considered, with an occupied area of 5.5 × 4.5 = 24.75 nm2. The immobilization yield was determined by reporting the experimental number of immobilized enzyme to the maximum number of laccase possible to be attached (Table 2). The immobilization yield exceeding 100% for the A45B101C27 monolayer is due to the fact that laccase was both adsorbed on the polymer surface and inserted between the chains of the polymer film. Adsorption of the enzyme on the polymer film is influenced by various factors, such as surface roughness and charge, with electrostatic attraction as the driving force for adsorption. Indeed, as the PDMAEMA (C) block is positively charged at pH values less than 8, due to protonation of the amino groups,28 and laccase possesses a negative charge at pH greater than 3.5,5 they interact through electrostatic attraction. We chose pH 4.25 to be optimal for enzyme immobilization due to the electrostatic character of both polymer and enzyme. We consider that the limited enzyme adsorption properties of A45B84C85 and A45B101C3 were due to the low roughness of monolayers formed on silica slides due to the internal

Figure 3. AFM two-dimensional topography of A45B101Cx block copolymers, at pHs 7 and 3, respectively. Scale bars are 2 μm.

both acidic and neutral pH values, low roughness characterized the films of A45B101C3 copolymer, whereas higher values were obtained for A45B101C27, because of the influence of the C block length on the orientation of the polymer chains within the film. In order to ensure high available surface for interaction with active biocompounds, assays of adsorbed enzymes were performed at neutral pH. In order to check the stability of the polymer films, we compared freshly prepared films with those transferred seven months earlier (Figure S6, Supporting Information). There were no appreciable differences in topography between fresh and old transferred monolayers for any of the polymers, thus indicating that the films have long-term stability in air. Note that these thin films were prepared by a “grafting to” method, which involves physical interactions of polymer film with the solid support. Therefore, they might be lower shear resistant than polymer brushes prepared by the “grafting from” method of surface-initiated polymerization, or polymer networks, which are cross-linked to increase the stability.32 A necessary balance between the resistance of the polymer films formed by a “grafting to” method and the requirement to preserve the integrity and functionality of enzymes is an essential criterion for development of active polymer surfaces by immobilization of enzymes. 3.7. Adsorption of Enzyme on ABC Block Copolymer Films. The ability of ABC block copolymer films to adsorb active biomolecules was investigated by QCM-D measurements. We selected an oxidase enzyme, laccase, as a protein model to generate active surfaces upon adsorption on the solidsupported monolayer. The change in frequency value (Δf) was

Figure 4. (a) Representative QCM-D data for laccase adsorption on the PEG45-b-PMCL101-b-PDMAEMA27 monolayer; (b) long-term stability of the PEG45-b-PMCL101-b-PDMAEMA27 film with adsorbed laccase. 11665

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Table 2. Enzyme Adsorption on ABC Triblock Copolymer Films ABC block copolymer

protein adsorptiona

Δm (ng cm−2)

surface coverage (nmol cm−2)

no. of enzymes per cm2 × 10−12

immobilization yieldb (%)

A45B101C3 A45B101C12 A45B101C17 A45B101C27 A45B84C30 A45B84C85

Y Y Y Y Y N

63 324 270 450 216 0

0.001 0.005 0.004 0.007 0.003 −

0.6 3.01 2.41 4.21 1.81 −

14.85 74.50 59.65 104.21 44.80 −

a Y: yes, protein adsorbs to the polymer film; N: no, protein does not adsorb. bCalculated for 4.5 × 5.5 = 24.75 nm2 occupied by one laccase molecule.

polymer film and the enzyme, as described in the adsorption investigation performed by QCM-D. Immersion of polymer films in enzyme solutions was performed for a longer period of time (30 min to 1 h) than that used for assessing the capability of polymer films for enzyme adsorption by QCM-D (approximately 15−30 min) to improve the diffusion and stabilization of the enzyme. The presence of laccase after immersion influenced the topography of the A45B101C27 film. As indicated by AFM images, an increase in film roughness from 0.62 to 1.43 nm after laccase adsorption by immersion was observed (Figure 6a−c). Since the size of laccase is 6.5 nm × 5.5 nm × 4.5 nm,36 the bright points, having the approximate height of 5 nm and width of 200 nm, correspond to the adsorbed enzyme agglomerates (Figure 6c, d). In order to achieve enzyme immobilization by transferring the mixed polymer−laccase film onto a solid surface, we first evaluated the aspect of the Langmuir isotherms. Laccase spread on an A45B101C27 polymer monolayer at the air−water interface influenced the shape of the Langmuir isotherm, by a slight decrease in the surface pressure (Figure S8, Supporting Information). Hydrogen bonding and/or electrostatic interaction between amino groups from the C block of the ABC polymers and the reactive groups of the enzyme might have created a more closely packed structure, which reduced the surface pressure and the mean molecular areas. A similar effect was observed when laccase dissolved in PBS buffer was spread on the ABC block copolymer monolayer. During the mixed ABC−enzyme film compression, the uniformity of the A45B101C27 was evaluated by BAM. Less uniform films were formed at surface pressures close to that, which resulted in collapse, compared to the films formed in the absence of laccase. The mixed A45B101C27−enzyme films transferred to a solid support had a different roughness, depending on the quantity of the enzyme, which was spread on the air−water interface during monolayer formation. Spreading low volumes of enzyme solution (25 μL of 2 mg mL−1) induced a decrease in the monolayer roughness to 0.75 nm, compared to the laccase free A45B101C27 monolayer with Ra of 0.97 nm (Figure 6a, e). In contrast, a higher volume of enzyme solution (100 μL of 2 mg mL−1) spread on the surface induced an increase in roughness of the ABC−enzyme film to 1.79 nm (Figure 6f). When a low amount of laccase was spread, the enzyme occupied the free space available between the chains of ABC polymer film and induced the formation of a more compact film, thus decreasing Ra in agreement with the values of the immobilization yield. In contrast, spreading a high amount of laccase resulted in the free intra-ABC film space being exceeded and induced an increase of Ra. The different behavior of surface roughness as a function of the amount of the enzyme will allow further modulation of active surface properties.

rearrangement of copolymer chains during film formation (Figure 3). Roughness strongly influenced adsorption by providing a larger surface for enzyme attachment. Indeed, the highest enzyme adsorption was with A45B101C27 polymer films, which had the highest roughness. In addition, adsorption was improved by the presence of small gaps (depth of about 3 nm) in the films, which partly exposed the hydrophobic block. This slight increase in hydrophobicity enhanced the interactions between the enzyme and the polymer film and therefore improved laccase adsorption, in agreement with the results of Deere et al., who showed that hydrophobic interactions decreased protein desorption from the surface.37 The effect of roughness on enzyme adsorption is clearly seen for copolymer films with close compositions. Indeed, A45B101C12 adsorbed more laccase (324 ng cm−2) than A45B101C17 (270 ng cm−2), because of its greater roughness that resulted in the formation of more gaps than the A45B101C17 polymer film. As A45B101C27 block copolymer formed films with the highest monolayer roughness and was able to adsorb the highest amount of laccase, this one was used for further investigations of active surface. 3.8. Generation of Active Surfaces by Immobilization of Enzyme. There are various methods for protein immobilization on solid surfaces, including binding to a support (physical adsorption or chemical binding), cross-linking or entrapment.38 After demonstrating the ability of ABC block copolymer monolayers to adsorb enzymes, we used the A45B101C27 triblock copolymer to generate active surfaces by employing two different techniques, based on laccase immobilization. Both techniques are based on physical adsorption: (i) immersion of the ABC solid-supported monolayers in an enzyme solution, and (ii) spreading the enzyme solution on ABC polymer at the air−water interface, followed by the transfer of the mixed polymer−enzyme films to silica slides (Figure 5). Laccase immobilization by immersion of the ABC solidsupported monolayer in an enzyme solution was selected because of the electrostatic interactions that occur between the

Figure 5. Strategies for enzyme immobilization: (a) immersion of the ABC solid-supported monolayer in the enzyme solution; (b) transfer of mixed polymer−enzyme film to silica substrate. Enzyme: green dots. 11666

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Figure 6. AFM images of A45B101C27 monolayers: (a) without laccase and (b) a corresponding profile; (c) after immersion in laccase solution and (d) a corresponding profile; after transfer of the polymer−enzyme mixed film from the air−water interface, with (e) low and (f) high enzyme concentrations. Scale bars: 2 μm.

3.9. Activity of Free and Immobilized Laccase. The activity values of free enzyme and of laccase immobilized by the immersion technique were determined in similar conditions of laccase concentration as calculated from QCM-D (Table 2). We assumed the amount of laccase immobilized in a static regime (by immersion) to be, in the first approximation, the same as in the dynamic regime (by QCM-D) in similar conditions (pH 7 and 30 min). Laccase activity was investigated by monitoring the oxidation product of the DMP substrate by absorbance measurements at λ = 470 nm (Figure 7). The enzymatic activity of laccase was preserved when adsorbed on A45B101C27 polymer films. The immobilization techniques did not influence the overall enzymatic activity; that is, the polymer monolayer with laccase immobilized by the immersion technique resulted in an average absorbance of 0.19, while the polymer monolayer with laccase immobilized by the transfer technique resulted in an absorbance of 0.21. The higher absorbance value obtained with the active surfaces compared to free laccase is presumably due to (i) a slightly higher amount of immobilized enzyme than that estimated by QCM-D and (ii) the immobilization process, which stabilizes the enzyme and increases its substrate accessibility.

Figure 7. Spectroscopic evaluation of laccase activity based on formation of the DMP oxidation product (λ = 470 nm) for a free laccase (black), a polymer monolayer with laccase adsorbed by immersion (red), and transfer of mixed ABC−laccase film (blue); a polymer monolayer without laccase (green).

4. CONCLUSIONS We have presented a strategy for engineering active surfaces by immobilization of enzymes on solid-supported polymer monolayers with asymmetric architecture that benefits from the stability and flexibility of the polymer films, and the functionality of the intrinsic biological activity of the enzyme. A group of six PEG45-b-PMCLx-b-PDMAEMAy asymmetric amphiphilic triblock copolymers were investigated in terms of their behavior at the air−water interface, formation of films on a solid support, and ability to adsorb laccase, which was used as a

model enzyme. We selected the LB technique for preparation of ABC block copolymer monolayers on solid supports, because it serves to obtain uniform films, favoring a reproducible enzyme immobilization. During the Langmuir isotherm compression, the ABC block copolymers adopted different arrangements, organized from pancakelike aggregates to fully packed monolayers. At the air− water interface, films were oriented with PEG in the water subphase and PDMAEMA facing toward air, that is, externally oriented. The properties of LB monolayers of ABC triblock 11667

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copolymers on silica slides, such as thickness, wettability, topography, and roughness were established by AFM, ellipsometry, and contact angle. These properties varied, depending on the surface pressure of transfer, ABC chemistry (hydrophilic/hydrophobic blocks length and active groups), and pH. We selected the ABC copolymer, which formed the most homogeneous and flexible films for enzyme immobilization investigations. We used two methods to prepare active surfaces by laccase immobilization: (i) immersion of solid-supported polymer films in enzyme solutions and (ii) transfer of mixed ABC−enzyme films on silica slides. Both methods successfully immobilized the enzyme, and the resulting solid-supported laccase−polymer films were both stable and active, as measured by QCM-D and activity assays, respectively. This study represents a new strategy for the design of active surfaces that are engineered by immobilization of enzymes on a soft asymmetric membrane attached to a solid support. Our simple and fast method to obtain well-organized uniform polymer membranes combined with straightforward techniques for enzyme immobilization indicates our strategy appealing for applications in the medical or ecological domains where the enzyme activity plays a key role.

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ASSOCIATED CONTENT

S Supporting Information *

Structure of ABC triblock copolymer; plots of stability, compression−expansion cycles, A45B101C3 film organization during compression at the air−water interface; AFM images; QCM data; and influence of laccase on the Langmuir isotherms. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions §

C.D. and J.K. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was provided by the National Centre of Competence in Nanoscience and the University of Basel, and this is gratefully acknowledged. C.D. acknowledges the financial support from the Swiss National Science Foundation, International Short Visit (IZK0Z2_150884). We thank Dr. Yves Matter for synthesizing the ABC block copolymer used in this study and Dr. B.A. Goodman for editing the manuscript.

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REFERENCES

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