Reversible Light-Switching of Enzymatic Activity on Orthogonally

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Reversible Light-Switching of Enzymatic Activity on Orthogonally Functionalized Polymer Brushes Matthias Dübner, Victor J. Cadarso, Tugce Nihal Gevrek, Amitav Sanyal, Nicholas D Spencer, and Celestino Padeste ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01154 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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ACS Applied Materials & Interfaces

Reversible Light-Switching of Enzymatic Activity on Orthogonally Functionalized Polymer Brushes Matthias Dübner†, ‡, Victor J. Cadarso†, Tugce N. Gevrek§, Amitav Sanyal§, Nicholas D. Spencer‡, and Celestino Padeste*, † †

Laboratory for Micro- and Nanotechnology, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland. Laboratory for Surface Science and Technology, Department of Materials, ETH Zürich, 8093 Zurich, Switzerland. § Department of Chemistry, Bogazici University, 34342 Bebek (Istanbul), Turkey. ‡

Supporting Information

ABSTRACT: Copolymer brushes, composed of glycidyl methacrylate and a furan-protected maleimide-containing monomer, were grafted from radical initiators at the surface of irradiation-activated fluoropolymer foils. After post-polymerization modification with enzymatically active microperoxidase-11 and photochromic spiropyran moieties, the polymer brushes catalyzed the oxidation of 3,3’5,5’-tetramethylbenzidine. Exposure to either UV or visible light allowed switching the turnover by more than one order of magnitude, as consequence of the reversible, light-induced spiropyran-merocyanine transition. The modified samples were integrated into an optofluidic device that allowed the reversible switching of enzymatic activity for several cycles under flow, validating the potential for application in smart lab-on-a-chip systems. KEYWORDS: smart materials, orthogonal post-polymerization modification, light-responsive polymer brushes, lab-on-achip devices, switchable enzyme activity Smart surfaces allow the stimulus-induced switching of properties and functionalities on demand, based on their responsiveness to a certain trigger, such as temperature, pH or light.1-7 Combining bioconjugated systems with stimulus-responsiveness is very desirable, as it provides a handle to control functionality by modulation of the properties such as polarity, solvency and chemical structure of the environment in the near vicinity of the biomolecule. Thermoresponsive copolymer systems have been described to control the release of fouling8, hemoglobin9 and cells10, 11. Light as an external stimulus, on the other hand, benefits from a spatial and temporal control without suffering from diffusion effects and allows tuning of the response via its wavelength and intensity.12 Light-controlled electron-transfer reactions at photoisomerizable monolayer electrodes have been described by Willner and Katz13-15. Spiropyran-terminated self-assembled monolayers (SAMs) formed on Au electrodes were used to control the communication of redox proteins with the electrodes upon photostimulation. More recently, incorporation of photochromic azobenzenes into biomolecules has been used for photocontrolled bioprocesses, relying on the reversible deformation of active centers in enzymes16, 17 or pore sizes in transmembrane proteins18 caused by the cis/trans transformation of the molecular photoswitches upon light exposure. Photoresponsiveness has also been used to tailor cell adhesion19 and indirectly to trigger capturing of proteins in thermoresponsive polymer brushes via light-to-heat-transfer20. Poloni et al.21 reported the photocontrolled deactivation of lipase immobilized on a quartz surface modified with an azobenzene-containing SAM.

However, the lack of flexibility of the SAM and the direct connection of the chromophore to the enzyme resulted in irreversible deactivation of the enzyme. Here we report on a new approach to the reversible, light-induced switching of enzymatic activity on stimuliresponsive polymer brushes. Polymer brushes consist of long and flexible chains, which are mechanically and chemically stable due to their covalent attachment to the substrate, and act as an extension of the surface into a third dimension, providing a high and tunable density of functional groups for conjugation of functional moieties. We took advantage of the highly chemoselective orthogonal post-polymerization modification (PPM) of copolymer brushes, in which nucleophilic amines and thiols were attached specifically to epoxides and maleimides, respectively. The light-induced change in the local environment caused a reversible switch in enzymatic turn-over, as demonstrated by the oxidation of 3,3’5,5’tetramethylbenzidine (TMB). Furthermore, implementation of multifunctional brushes into a lab-on-a-chip device was demonstrated. Extreme-ultraviolet (EUV) lithography was used for activating the surface of foils of ETFE (poly(ethylene-cotetrafluoroethylene)). Exposures using 13.5 nm (92.5 eV) EUV caused bond breaking and radical formation at the polymer surfaces, which allowed subsequent grafting of copolymer brushes by means of free-radical polymerization. In recent years, we have established this approach as a robust method for obtaining polymer brushes on polymeric surfaces with structuring capabilities in the sub-µm range.6, 22-25

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values (Figure 1b). The experimentally obtained stoichiometry of the two building blocks in the copolymer-brush structures showed an incorporation of FuMaMA almost equal to its mole fraction in solution (Figure 1c). The deprotection of the maleimide groups was achieved via heat treatment under vacuum, to remove the furan moiety via the retro Diels-Alder (rDA) reaction (Figure 1a). This reaction step is expected to be nearly quantitative32 and is not affecting the epoxide groups, as confirmed using IR and contact angle measurements (SI).

Figure 1. (a) Synthetic strategy for grafting of P(GMA-coFuMaMA) and the subsequent retro Diels-Alder (rDA) reaction used to activate the maleimide groups. (b) ATR-IR spectra of P(GMA-co-FuMaMA) brushes grafted from ETFE and (c) the mole fraction of FuMaMA in GMA copolymer-brushes grafted from ETFE in dependence of the mole fraction of FuMaMA in the comonomer solution. The data were obtained from the relative areas of the carbonyl peaks of the ester and imide groups in the ATR-IR spectra.

Although relatively simple and very efficient multicomponent reactions26-28 for post-polymerization modifications have been recently described, we chose orthogonally functionalizable repeating units. This allowed us to chemospecifically control the concentration of the reactive centers and as a consequence the concentration and relative ratio of bound functional moieties on the flexible surface-bound macromolecules, using highly efficient PPM reactions. To fabricate bi-functional polymer brushes, two monomers were copolymerized that allowed chemoselective PPM of the brush structures via orthogonal reactions. As the monomers, we chose glycidyl methacrylate (GMA), and a furan-protected maleimide methacrylate monomer (FuMaMA)29 (Figure 1a). The grafting level in dependence of the monomer ratios of GMA to FuMaMA in the grafting solution was assessed by means of atomic force microscopy (AFM) (Figure S1). The dry thickness of the grafted brushes of all investigated compositions roughly showed a square-root dependence on EUV dose. For P(GMA) the thickness at highest dose exceeded 2 µm and reduced with increasing concentration of FuMaMA in the monomer feed, resulting in only 100 nm thickness for P(FuMaMA) homopolymer brushes. Hence, the grafting behavior was dominated by the grafting kinetics of the two different monomers, in good agreement with our previous findings for (poly)ethylene-methacrylate-containing copolymer brush structures22. The molecular weights of the grafted copolymers range up to several hundred kDa, as estimated from the dry thickness30, 31 of the brushes (Table S1) allowing us to conjugate functional moieties at very high densities. For quantification of the copolymer compositions, the ratio of areas under the carbonyl vibrations for the ester and imide groups, appearing in the IR spectra at 1728 and 1704 cm-1, respectively, were analyzed, using the relative areas of P(GMA) and P(FuMaMA) homopolymers as reference

Figure 2. (a) Synthetic strategy for chemoselective PPM of orthogonally functionalizable P(GMA-co-MaMA) brushes to covalently attach microperoxidase-11 (MP-11) via epoxide-amine and photochromic spiropyran (SP) via thiol-ene reactions. MP-11 catalyzed TMB oxidation (b) from the colorless diamine to the deep-blue colored diimine configuration causing (c) a color change in a droplet of TMB solution on non-exposed P(GMAMP-11-co-MaMA-SP) polymer brushes grafted from ETFE. MP11-free P(GMA-co-MaMA-SP) was used as a negative control and did not show any blue color appearing (data not shown). UVand visible light-induced switching (d) between uncharged colorless spiropyran (SP) and zwitterionic deep-purple colored merocyanine (MC) causing (e) reversible switching of the static contact angle on P(GMA-MP-11-co-MaMA-SP) brush surfaces.

The lysine residue in the peptide chain of MP-11 was used in an initial PPM step as a flexible linker for chemoselective binding to epoxides of the copolymers (Figure 2a). The very high efficiency of this reaction was indicated by the disappearance of the C-O-C vibration for ether-like epoxides and the appearance of strong vibrations for OH, NH and C=O in the IR spectrum corresponding to the ringopened epoxides and the amides in surface-bound MP-11, respectively (Figure S2b, Table S2). High chemoselectivity for the amine-epoxide vs amine-maleimide reaction was achieved by short reaction times, moderate reaction temperatures and low concentration of the amine, to reduce the chance of side reactions of MP-11 with the activated MaMA. In the second chemoselective PPM step, a thiolmodified spiropyran was bound to the unreacted maleimides on MP-11-containing P(GMA-MP-11-co-MaMA) brushes using the nucleophilic thiol-ene reaction which is highly efficient under ambient conditions22. MP-11 is a


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fragment of ferric heme enzyme cytochrome c which catalyzes the oxidation of a variety of substrates33 – including TMB – in a buffered hydrogen peroxide (H2O2) solution (Figure 2b). The relatively small size of MP-11 (1.9 kDa) allowed binding a high number of active centers onto grafted brushes. The enzymatic activity of the MP-11 modified brushes was evident from the appearance of the characteristic blue color (Figure 2c) developing upon pipetting a droplet of TMB/H2O2 solution onto a modified surface. The light-responsiveness of brushes modified with SP was demonstrated via UV/vis-spectroscopy (Figure S3) and CA measurements (Figure 2d), illustrating a reversible switch between the colorless, non-charged spiropyran (SP) and the purple, zwitterionic merocyanine (MC) form upon alternating visible and UV-light exposure (30 s, λ = 366 nm, 40 W) (Figure 2e).

Figure 3. Sequences of absorbance spectra acquired at 90 s time intervals of TMB analyte solution in the presence of (a) P(GMAMP-11-co-MaMA-SP) and (b) P(GMA-MP-11-co-MaMA-MC), obtained by an initial 30 s activation time with UV-light. The absorption bands are characteristic for the diimine oxidation product of TMB. (c) Absorbance at 655 nm of TMB analyte solution. Measurements in the presence of P(GMA-MP-11-coMaMA-SP) (orange) and P(GMA-MP-11-co-MaMA-MC) (purple) obtained by 30 s activation with UV-light. (d) The reaction was started in the presence of P(GMA-MP-11-co-MaMA-SP). After 15 min the sample was activated for 30 s in situ with UVlight. This cycle was repeated after a washing step in between. The exposure changed the enzymatic turn-over drastically and was reversible after a relaxation after 1 h under visible light. In both cases MP-11-free P(GMA-co-MaMA-SP) (grey) was used as a negative control.

For determination of the influence of the local environment change evoked by the SP-MC-transition on the enzymatically activity of MP-11-containing polymer brushes, surface-modified ETFE samples were immersed into a TMB reaction solution and the absorbance spectrum of the solution was monitored using UV/vis-spectroscopy in the dark at room temperature. Two absorption bands at 655 and 370 nm, indicating the formation of TMB diimine evolved much faster for samples initially exposed to UV-light to form the P(GMA-MP-11-co-MaMA-MC), indicating a dramatically increased enzymatic turn-over of TMB upon

UV-exposure (Figures 3a-c). The overall enhanced TMB oxidation after UV exposure is interpreted as a consequence of the increased wettability and swelling behavior of the zwitterionic MC-containing P(GMA-MP-11-coMaMA-MC) brushes, causing a preferable environment for the MP-11 and a better accessibility of the reagents. While wetting effects are detected immediately, swelling of polymer brushes usually occurs on a timescale of minutes7. Repeated switching and in-situ activation were demonstrated in an analogous experiment. The enzymatic activity of an unexposed P(GMA-MP-11-co-MaMA-SP) sample leveled off after 15 min, but could be significantly amplified by means of UV-light exposure for 30 s, leading to an increase in absorbance by a factor of 2 within 15 min. After an extensive washing in buffer under ambient light to reconstitute the SP moieties, the procedure was repeated starting with a new TMB solution. The second cycle after a 1-h relaxation showed similar reaction kinetics, but with reduced intensity. A third cycle showed only very weak initial activity, indicating deactivation of the MP-11 by the peroxide solution in the top-most layers, but the enzymatic turn-over could still be activated by UV-light exposure. The strongest switch in enzymatic activity upon light irradiation was found for a ratio of MP-11/SP of 2:1 conjugated to the grafted brushes, in which the MP-11/SP ratio was determined by a GMA/FuMaMA ratio with an assumed quantitative PPM.

Figure 4. (a) Schematic illustration of the fabricated optofluidic device integrated with the modified ETFE substrate. (b) Measurement of absorbance at 635 nm of TMB analyte solution in the presence of P(GMA-MP-11-co-MaMA-SP) (orange): Three cycles of alternating exposure to either visible (orange) or UVlight exposure to grafted P(GMA-MP-11-co-MaMA-SP) brushes demonstrate a reversible switch in enzymatic activity in a modified microchannel under continuous flow. The spikes in the curves are due to air bubbles passing through the channels when switching solutions.

In contrast to the measurements under static conditions discussed above, in which the reaction turn-over is determined by kinetics superimposed upon the depletion of reagents and formation of products, measurements in flow systems allow a constant feed of fresh reagents of known concentration, enabling a more direct insight into the reaction kinetics. For the implementation of polymer brushes into an optofluidic device, a modified polymeric film was attached to a microfluidic channel fabricated from polydimethylsiloxane (PDMS) which was attached to a syringe pump system for precise control over the flow, and for fast switching between different solutions. A schematic illustration of the microchannel device that includes air mirrors for signal enhancement (for details see SI) is shown in Figure


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4a.34, 35 The measurement was started with a constant flow of buffer. Analogously to the static measurements, the absorbance in the modified microchannel was immediately enhanced due to the formation of the TMB oxidation product when changing the flow to a (initially colorless) TMB solution (orange part in Figure 4b). Under a flow of 0.5 µL min-1 the absorbance almost disappeared after 10 min. As PDMS is almost transparent to UV-light, an in-situ exposure (60 s) was possible, which increased the enzymatic turn-over drastically, indicated by the drastically enhanced absorption. This increase in enzymatic activity was reversible after a relaxation of 60 min under visible light and constant buffer flow. In the following, for the first time we demonstrated reversible switching of the enzymatic activity on functionalized microchannels in two additional cycles. The results were in good agreement with the findings under static conditions. However, in contrast to the static measurements, where the absorbance represents an accumulation of formed blue TMB diimine, the optofluidic device allowed us to determine the dynamic enzymatic turn-over under different levels of light exposure. Further, samples could simply be washed with buffered solution, resulting in an absorbance almost as low as that of the initial buffer solution. This implies that the oxidation products formed were not trapped in the modified thin film, highlighting the beneficial properties of flexible polymer brushes. Furthermore, the activity reduction observed in static measurements was significantly reduced in the dynamic case, since the exposure of the MP-11 to the peroxide contained in the TMB solution was decreased. Hence, these dynamic measurements demonstrate the continuous enzymatic activity of a MP-11 bioconjugated microchannel over an overall reaction time of several hours in the analyte solution opening the possibility of more than three cycles in the future. In summary, a polymeric brush biointerface that enables light-switching of an enzymatic reaction was realized by means of orthogonal functionalization of polymeric substrates with amine- and thiol-reactive copolymer brushes. Facile and effective amine-epoxide and thiol-ene conjugation were employed as a toolbox for sequential and chemospecific binding of enzymatically active MP-11 and lightresponsive SP to grafted brushes. The combination of both functionalities in a single brush allowed us to reversibly switch the enzymatic activity using light as an external stimulus. In addition, integration of multifunctional polymer brushes grafted from ETFE into an optofluidic device allowed reversible switching of the enzymatic turn-over of TMB under flow. This enabled the dynamic enzymatic activity of the bioconjugated microchannel to be controlled in an all-polymeric photonic lab-on-a-chip system. The results represent a major milestone in responsive bioconjugated polymeric surfaces, as they combine chemospecific highly efficient PPM in an orthogonal fashion to implement multifunctionality on a single brush. This new type of synthetic platform demonstrates the proof of concept with the chosen enzyme MP-11, being an exemplary biocatalyst. The well-controlled brush structures open up a broad variety of applications for responsive bioconjugated polymer brushes in, for example, all-polymeric smart diagnostic

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systems in which metabolic events can be in investigated in well-defined areas of interest.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Experimental procedures, step-height measurements by means of AFM, estimation of the molecular weight (Mw) of grafted brushes, ATR-IR spectroscopy, ultraviolet/visible (UV/vis) spectroscopy and CA measurements (PDF).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Celestino Padeste: 0000-0001-7577-5781 Matthias Dübner: 0000-0001-5167-3553 Author Contributions All authors have given approval to the final version of the manuscript. Funding Swiss National Science Foundation SNF, 200020_159758/1, SNF Ambizione project n° PZ00P2_142511, and Scientific and Technological Research Council of Turkey Graduate Scholarship TUBITAK 2211-D. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT EUV exposures were performed at the Swiss Light Source, Paul Scherrer Institute, Villigen. Thanks are due to Michaela Vockenhuber, Stefan Stutz, Yves Erdin and Camelia Nicoleta for assistance with EUV exposures, and UV/vis and ATR-IR microscopy. The authors gratefully acknowledge the financial support from the Swiss National Science Foundation (SNF, 200020_159758/1) and SNF Ambizione project (n° PZ00P2_142511; granted to V.J. Cadarso). T.N.G. acknowledges TUBITAK 2211-D Graduate Scholarship from the Scientific and Technological Research Council of Turkey.

ABBREVIATIONS AFM, atomic force microscopy EUV, Extreme ultraviolet FuMaMA, furan-protected maleimide methacrylate GMA, glycidyl methacrylate MC, merocyanine MP-11, microperoxidase-11 Mw, molecular weight PDMS, polydimethylsiloxane PPM, post-polymerization modification rDA, retro-Diels Alder SAM, self-assembled monolayer SP, spiropyran TMB, 3,3’5,5’-tetramethylbenzidine


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Reversible Light-Switching of Enzymatic Activity on Orthogonally

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