Combining natural enzymes with synthetic membranes on solid support enables creation of functional surfaces able to serve for efficient biosensing. Enzymes (laccase and tyrosinase) integrated on soft copolymer mono- and bilayer membranes preserve their activity and specifically detect the presence of phenols. The straightforward approach to create these bio-hybrid membranes allows changing the enzyme type and thus producing functional surfaces for sensitive detection of desired molecules

Draghici, Camelia

Gunkel-Gabrol, Gesine

Kowal, Justyna

Meier, Wolfgang

Mikhalevich, Viktoria

Palivan, Cornelia G.

edoc

IntroductionCombinations of biomolecules and syn-thetic assemblies to generate bio-hybrid materials emerge in nanoscience as novel solutions for various domains, such as medicine, catalysis, technology. They benefit from the activity and functionality of the biomolecules (enzymes, proteins, DNA, mimics), while the assemblies rep-resent stable templates or compartments [1]. We created functional surfaces by combining enzymes (laccase and tyrosi-nase) with soft polymer membranes at-tached on solid support. These functional surfaces employ simple enzymatic reac-tions and aim sensitive detection of phe-nols (green spots in Fig. 1) as proof of concept, because phenols are known as harmful pollutants of drinking and natu-ral waters. The enzymes are attached to synthetic membranes composed of am-phiphilic asymmetric block copolymers that are immobilized on solid support in order to form functional surfaces (Fig. 1). Triblock copolymers of poly(ethylene-glycol)-block-poly(γ-methyl-ε-capro-lactone)-block-poly [(2-dimethylamino) ethylmethacrylate], named ABC copoly-mers, with different hydrophilic and hy-drophobic domains are selected to gener-ate solid supported membranes [2]. The advantage of our synthetic membranes is the possibility to use all the options of-fered by modern polymer chemistry to fine-tune and control the different molec-ular properties of the copolymers and re-sulting membranes. Notably, the ability of these amphiphilic copolymers to form uniform and stable films at the air-water interface serves to generate homoge-neous monolayers and bilayers upon transfer onto silica solid supports by us-ing the Langmuir–Blodgett (LB) tech-nique. The most promising membranes in terms of their properties (film thick-ness, wettability, topography and rough-ness) are used for enzyme attachment and production of functional surfaces. Af-ter successful attachment of the enzymes on copolymer membranes by stable non-covalent interactions, both enzymes pre-serve their activity. As the properties of the synthetic membranes affect the enzy-matic activity, they serve to distinguish the most promising bio-hybrid mem-branes in terms of overall functionality as biosensors for phenols: bilayer films pro-vide the best conditions in terms of over-all stability and enzymatic activity [3].Formation of MembranesABC triblock copolymers with poly(ethyleneglycol) as A block, poly(γ-methyl-ε-caprolactone) as B block and poly[(2-dimethylamino) ethylmethacry-late] as C block (PEG45-PMCLX-PDMAE-MAY) were used to self-assemble and form solid-supported polymer mem-branes. The behavior at the air-water in-terface of these ABC polymer films was investigated by using Langmuir iso-therms (surface pressure-area) and Brewster angle microscopy (BAM). At the optimal surface pressure, ABC films were transferred from the air-water interface to silica plates, resulting in densely packed copolymer monolayers [2]. As dif-ferences in the architecture of the copoly-mer membranes affect the enzymes im-mobilization and accessibility on the membrane surface, we first evaluated their properties (thickness, hydrophilic/hydrophobic balance, topography and roughness). As A45-B101-C27 block copoly-mer formed the best membranes, it was selected for further combination with the enzymes in order to obtain functional surfaces. To favor enzyme attachment, copoly-mer films resulting from different trans-fer directions (up lifting, arrow up, or down deeping, arrow down, as indicated in Fig. 2A), and sequences (single or two transfers) were prepared [3]. We intro-duced a new terminology for the polymer membranes formed by Langmuir–Blodgett transfers of ABC block copoly-mers: the resulting polymer films based on single transfers were labeled as “up” (arrow up) and “down” (arrow down), re-spectively, while the combination of two transfers were labeled accordingly “down-down”, “up-down”, “down-up” or “up-up”. This terminology is more ap-propriate to describe the membranes preparation procedure by using amphi-philic block copolymers, compared to the z- and y-type of membranes obtained Functional SurfacesBio-Hybrid Membranes for Biosensing ▪ Camelia Draghici1,2, Viktoria Mikhalevich1, Gesine Gunkel-Grabole1, Justyna Kowal1, Wolfgang Meier1, Cornelia G. Palivan1Combining natural enzymes with synthetic membranes on solid support enables cre-ation of functional surfaces able to serve for efficient biosensing. Enzymes (laccase and tyrosinase) integrated on soft copolymer mono- and bilayer membranes preserve their activity and specifically detect the presence of phenols. The straightforward approach to create these bio-hybrid membranes allows changing the enzyme type and thus pro-ducing functional surfaces for sensitive detection of desired molecules.Figure 1. Functional surfaces for phenol biosensing based on attachment of specific enzymes (a. lac-case and b. tyrosinase) on solid supported synthetic membranes. Reproduced by permission of The American Chemical Society [3].G.I.T. Laboratory Journal 5/2019, pp 36–38, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim     www.laboratory-journal.comfrom lipid films. The polymer films gen-erated by these transfer directions and sequences were evaluated in terms of thickness, wettability, topography and roughness. Only the “up-up” transfers formed bilayers films (Fig. 2C), while the “up”, “up-down” and “down-up” trans-fers resulted in formation of monolayer films (Fig. 2B).Enzyme AttachmentThe ability of the enzymes to adsorb on the most promising solid supported poly-mer membranes (monolayer, up-down monolayer and bilayer) was studied by quartz crystal microbalance with dissipa-tion (QCM-D) method and bicinchoninic acid assay (BCA). The enzymes adsorbed on polymer membranes remained stable for at least 18 h at room temperature, ac-cording to the QCM-D experiments. Less amounts of each enzyme were absorbed onto the monolayers than onto the bilay-er films. Higher amount of both enzymes adsorbed on bilayers is favoured by a higher roughness of these synthetic membranes, with larger surface area available for the enzyme adsorption. Combination of QCM-D and BCA indicat-ed that both enzymes adsorbed on the polymer films in different degrees, de-pending on the intrinsic architecture of the synthetic membranes. Stable, non-co-valent attachment of the enzymes to form bio-hybrid membranes on solid support represents a prerequisite for develop-ment of biosensors. Enzymes adsorption, Figure 2. Langmuir–Blodgett transfers to generate the polymer films. A) Polymer transfer onto a hydro-philic solid support (a – up transfer, up lifting; b – down transfer, down dipping), B) Schematic repre-sentation of a monolayer film formed by LB transfers and C) Schematic representation of a bilayer film formed by LB transfers. incubation and desorption served to ob-tain the conditions for production of effi-cient functional surfaces to sense and convert phenols [3]. Activity of the Attached EnzymesThe activity of biomolecules upon combi-nation with synthetic membranes repre-sents a crucial step in development of functional membranes [4-6]. We studied the activities of the attached enzymes on polymer monolayer and bilayer mem-branes and compared them with the ac-tivity of the free enzymes by using as substrates dimethoxyphenol (DMP) for laccase and 4-methoxyphenol (4-MP) ty-rosinase. Both enzymes preserved their activity upon attachment on synthetic membranes with respect to their sub-strates. The enzymatic activity was tested for different time periods: for laccase 24 h (with maximum activity at 19-24 h) and for tyrosinase 6 h (with maximum activi-ty at 2-3 h). The enzymes activity was dif-ferent depending on the type of polymer membrane: for monolayer membranes it increased from that corresponding to en-zyme attachment on “monolayer up” to that corresponding to enzyme attach-ment on “monolayer up-down”. The high-est enzyme activity was obtained when the enzymes were attached on bilayer membranes (Fig. 3A, B).Both enzymes immobilized on bilayers were tested in terms of their ability to contribute to phenols autooxidation at concentrations 10-fold lower (Fig. 3C). Successful phenols autooxidation dem-onstrates that lower concentrations of phenol derivatives can be efficiently de-tected by our functional surfaces. To-gether, the stability of the solid support-ed copolymer membranes and the preserved bioactivity of the enzymes in-dicate that these functional surfaces have high potential upon optimization, for efficient and sensitive phenols bio-sensing.ConclusionsIn this study we highlighted the poten-tial of functional surfaces produced by taking advantage on the interaction be-tween soft amphiphilic asymmetric co-polymer membranes and enzymes (lac-case and tyrosinase) to efficiently sense phenols. Triblock copolymer films were prepared by Langmuir–Blodgett transfer technique, resulting in stable, solid-sup-ported membranes for the enzymes ab-sorption. Mono- and bilayers with differ-ent properties (film thickness, wettability and roughness) were investigated and the three most promising polymer films (the monolayer, the up-down monolayer and the bilayer) were selected for devel-opment of functional surfaces. Both lac-case and tyrosinase preserved their en-zymatic activity upon adsorption on these polymer films, the best candidate in terms of the overall bio-activity being the bilayer films. Our approach to gener-ate functional surfaces is straightfor-ward and therefore allows extending the sensing of other desired molecules by a simple change of the enzyme types. In the future, these functional surfaces will not only allow to sense one type of mol-ecules (e.g. phenols), but also one day may offer an option for complex detec-tion by using multiple biomolecules si-multaneously present in the bio-hybrid membranes. AcknowledgementsThe authors would like to thank the sup-port from the Swiss National Foundation (SNF), the National Centre of Competence Molecular System Engineering (NCCR MSE). CD acknowledges the financial sup-port from the Swiss National Science Foundation, International Short Visit (IZ-K0Z2_150884).Affiliations1 Chemistry Department, University of Ba-sel, Basel, Switzerland2 Product Design, Mechatronics and Envi-ronment Department, Transilvania Uni-versity of Brasov, Brasov, Romania  ContactProf. Dr. Cornelia G. PalivanUniversity of BaselChemistry DepartmentBasel, Switzerlandcornelia.palivan@unibas.chFigure 3. Activity of enzymes immobilized on the A45-B101-C27 copolymer films as determined by oxida-tive product formation: A) laccase with DMP as substrate and B) tyrosinase with 4-MP as substrate. Ac-tivity of laccase and tyrosinase attached on the “up-up” bilayer for two different concentrations of the enzyme substrates C): 0.1 mM and 0.01 mM DMP, and 0.2 mM and 0.02 mM 4-MP, respectively. Re-produced by permission of The American Chemical Society [3].

Functional Surfaces: Bio-Hybrid Membranes for Biosensing

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Functional Surfaces: Bio-Hybrid Membranes for Biosensing

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