Stable Bioactive Enzyme-Containing Multilayer Films Based on

Article pubs.acs.org/Langmuir

Stable Bioactive Enzyme-Containing Multilayer Films Based on Covalent Cross-Linking from Mussel-Inspired Adhesives Johan Longo,† Tony Garnier,† Mihaela Mateescu,‡,§ Florian Ponzio,‡,§ Pierre Schaaf,†,‡,§,∥,⊥,#,∇ Loïc Jierry,†,∥,⊥,# and Vincent Ball*,‡,§ †

Institut Charles Sadron, Centre National de la Recherche Scientifique, Université de Strasbourg, UPR 22, 23 rue du Loess, BP 84047, 67034, Strasbourg Cedex 2, France ‡ Biomatériaux et Bioingénierie, INSERM, UMR-S 1121, 11 rue Humann, 67085 Strasbourg Cedex, France § Faculté de Chirurgie Dentaire, Université de Strasbourg, 8 rue Sainte Elisabeth. 67000 Strasbourg Cedex, France ∥ Ecole de Chimie, Polymères et Matériaux, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France ⊥ Institut d’Etudes Avancées de l’Université de Strasbourg, 5 allée du Général Rouvillois, 67083 Strasbourg, France # International Center for Frontier Research in Chemistry, 8 allée Gaspard Monge, 67083 Strasbourg, France ∇ Institut Universitaire de France, 103 boulevard Saint-Michel, 75005 Paris, France S Supporting Information *

ABSTRACT: The use of immobilized enzymes is mandatory for the easy separation of the enzyme, the unreacted substrates, and the obtained products to allow repeated enzymatic assays without cumbersome purification steps. The immobilization procedure is however critical to obtain a high fraction of active enzyme. In this article, we present an enzyme immobilization strategy based on a catechol functionalized alginate. We demonstrate that alkaline phosphatase (ALP) remains active in multilayered films made with alginate modified with catechol moieties (AlgCat) for long duration, that is, up to 7 weeks, provided the multilayered architecture is crosslinked with sodium periodate. This cross-linking reaction allows to create covalent bonds between the amino groups of ALP and the quinone group carried by the modified alginate. In the absence of cross-linking, the enzymatic activity is rapidly lost and this reduction is mainly due to enzyme desorption. We also show that NaIO4 cross-linked (AlgCat-Alp)n films can be freeze-dried and reused at least 3 weeks later without lost in enzymatic activity.

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INTRODUCTION Confinment of enzymes on surfaces of lipid monolayers,1 in lipid vesicles,2,3 in porous materials such as inverse opals,4 in polyelectrolyte multilayer films5−7 or in gels8 offers many opportunities not only to confer biological activity to such materials but also to increase their enzymatic activity. This increase in enzymatic activity is manifested by an increase in the conversion rate of the enzyme substrate complex (in the framework of the Michaelis Menten model) to the product of the reaction.9 The same holds true when the enzymes are encapsulated in polyelectrolyte complexes10 or when they are immobilized at the surface of nanoparticles.11,12 To achieve the realization of either a controlled and sustained release of active enzymes or a long-term enzymatic activity without release, which is needed for the production of stable biosensors, it is mandatory to control the immobilization mechanism of the enzymes. In the case of stable enzyme immobilization, the active molecules should be bound preferentially by means of covalent bonds but without undergoing a significant conformational change. In addition, the immobilization matrix should be © 2015 American Chemical Society

porous enough to allow access of the substrate to all the immobilized enzymes. It has been found that in the case of polyelectrolyte multilayer films13−16 produced by the alternated layer-by-layer deposition of chitosan and glucose oxidase (GOX), the sensitivity for glucose sensing was maximal when only the last layer pair contained GOX17 implying that the enzyme embedded in the internal layers of the film remained either inaccessible to the substrate or underwent some severe denaturation. This last assumption seems however unlikely because it has been shown that many proteins acquire increased conformational stability when embedded in polyelectrolyte multilayered films.18,19 Other authors have demonstrated the importance of the accessibility of horse radish peroxidase (HRP) to its substrate when pH sensitive films made from the deposition of poly(allylamine) (PAH) and poly(acrylic acid) (PAA) are deposited on top of the enzyme; the enzymatic Received: September 7, 2015 Revised: October 13, 2015 Published: October 28, 2015 12447

DOI: 10.1021/acs.langmuir.5b03329 Langmuir 2015, 31, 12447−12454

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Scheme 1. (a) Schematic Representation of the Catalytically Active Enzyme-Confined Multilayer, Covalently Crosslinked by Using (b) Catechol-Modified Alginate (AlgCat)

pluronic F-127 triblock copolymers to yield thermosensitive hydrogels that gelate spontaneously at the body temperature. These gels are biocompatible and useful as hemostatic materials.25,26 In addition polyelectrolytes modified with catechol moieties have already been used to build up multilayer polyelectrolyte films in combination with clays allowing to produce ultrastrong coatings after oxidation of the catechol moieties to create covalent bonds in the composite architecture.27 Catechol-modified polymers allow to deposit polyelectrolyte multilayers on substrates that do not carry charged groups 28 owing to the strong and substrate independent adhesion strength provided by catechols in a manner reminiscent to the mechanism used by mussels to strongly adhere on solid substrates in a marine environment and under strong shear stresses.29,30 However, to our knowledge catechol-modified polyelectrolytes were never used to build up self-assembled films containing enzymes. The main topic of this article is to show the proof of concept of such architectures made by using the layer-by-layer (LBL) deposition method. The main result of this research is to show that the NaIO4 triggered cross-linking of the alginate catechol (AlgCat) based films not only impedes the desorption of alkaline phosphatase (ALP) from the PEI(AlgCat-ALP)n multilayer films, which occurs in the absence of cross-linking, but also allows for a long-term preservation, up to at least 7 weeks, of the enzymatic activity. Furthermore, the LBL films can be freeze-dried without damage and reused without reduction in the enzymatic activity. ALP was chosen as a model enzyme because its activity can be measured easily by means of UV−vis spectroscopy by tracking the appearance of paranitrophenol during the hydrolysis of the enzyme substrate, paranitrophenyl-phosphate (PNP) (see Scheme 1).

activity increases when pores appear in the (PAH/PAA)n capping layers.20 It has also been demonstrated that active enzymes can be encapsulated in gels21 and in polyelectrolyte complexes10 with the marked advantage that the preparation of such a material can be performed in one step whereas the deposition of films using the layer-by-layer (LBL) deposition method can be very long and cumbersome. If the growth regime of polyelectrolyte multilayer films obtained in an LBL manner is linear with the number of deposition cycles (one cycle corresponds to the deposition of one polycation and one polyanion in the case of polyelectrolyte multilayer films), the time required to prepare a film is proportional to its thickness. It is the aim of this article to investigate the influence of chemical cross-linking on the activity of a typical enzyme, alkaline phosphatase, deposited in an LBL manner with a polyanion carrying chemical cross-linkable moieties, namely catechol groups. The catechol groups carried by sodium alginate can undergo oxidation in the presence of an oxidant like sodium periodate. The obtained quinone groups are extremely reactive with nucleophiles like amino groups carried by the protein and should allow to stabilize the whole architecture of the film. In addition, we selected a modified alginate that is a polyanion and in addition is able to undergo gelation and hence a physical cross-linking process that should also contribute to the stabilization of the whole architecture. The swellability of alginate also offers the possibility to get highly porous and hydrated films in order to allow for a fast diffusion process of the enzyme’s substrate in the whole film. Indeed, catechol-modified alginate can be used to produce gels of tunable swelling ratios and elastic moduli.22 Gels made from L-DOPA containing proteins can easily be cross-linked using Fe3+ as a complexing agent.23 Subsequently the metallic cations undergo reduction whereas the catechol groups of L-DOPA are oxidized in quinone groups. Instead of metallic cations, sodium periodate can also be used as an oxidant to cross-link mussel inspired adhesive hydrogels.24 As an example, Chitosan modified with catechols was blended with thiol terminated

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MATERIALS AND METHODS

All solutions were prepared from double distilled and deionized water (Milli QPlus, ρ = 18.2 MΩ.cm). The used buffers were 50 mM sodium acetate at pH = 5.0 (Sigma-Aldrich) and 50 mM tris(hydroxyethyl) aminomethane at pH = 8.5 (Euromedex, Schiltigheim, France). ALP 12448


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Scheme 2. Buildup Strategy to Prepare the PEI-(AlgCat-ALP)n Films through a Sequential Approach Based on a First Layer-byLayer Electrostatic Adsorption Followed by a Post-Construction Crosslinking in the Presence of Sodium Periodate

(EC. 3.1.3.1) was purchased from Sigma-Aldrich (ref P7640) and used without further purification. Some control experiments were performed by replacing ALP by Hen egg white lysozyme (SigmaAldrich ref L6876) during the deposition of the films. The substrate of the enzyme was PNP in the form of sodium salt (ref N7653) also from Sigma-Aldrich. AlgCat was synthesized as described elsewhere using dopamine, see Supporting Information.31 The grafting ratio was determined to be equal to 15% as determined by means of 1H NMR spectroscopy. The polyelectrolyte multilayer films were deposited on silica coated quartz crystals (QSX 303 from Q Sense, Sweden), on quartz plates (4 × 1 × 0.1 cm, Thuet, Blodelsheim, France), and on silicon wafers (Siltronix, Archamps, France) for their characterization by means of quartz crystal microbalance with dissipation monitoring (QCM-D), UV−vis spectroscopy, and atomic force microscopy (AFM), respectively. The UV−vis spectra were acquired on a mc2 double beam spectrophotometer (Safas, Monaco) and AFM imaging was performed on a Nanoscope IV (Veeco, Santa Barbara) in the dry state and in the contact mode using cantilevers of the MLCT type with a nominal spring constant of 0.1 mN·m−1. Surface plasmon resonance spectroscopy was perfomed with a Multiskop apparatus from Optrel (Germany). The architectures deposited were hence denoted PEI-(AlgCatALP)n, and the number of corresponding layer pairs of such a film were equal to n + 0.5 owing to the presence of the anchoring PEI layer. The optimal adsorption time to reach completion of an adsorbed layer was determined by means of QCM-D. An adsorption kinetics will be considered to be finished when the reduced frequency change ΔF ν/ν (where ν is the overtone number, ν = 3,5,7, 9, and 11 herein) is lower than 0.1 Hz during 1 min. The QCM-D experiments were performed with a E1 instrument from Q-Sense (Göteborg, Sweden) and all the solutions were injected with a flow rate of 0.25 mL.min−1 using a peristaltic pump. The enzymatic activity of the supernatant in contact with the PEI(AlgCat-ALP)n films was measured for its enzymatic activity in order to estimate the desorption of active enzyme from the films. To that aim, the freshly prepared films, cross-linked or not in the presence of 1 mM NaIO4 (dissolved in the 50 mM Tris buffer), were put in 20 mL of buffer solution. At regular time intervals, 1 mL of this buffer solution was mixed with 1 mL of PNP solution (Sigma-Aldrich, ref N7653). The whole experimental strategy is depicted in Scheme 2. This mixture was put in a quartz cuvette to follow the increase in absorbance at 405 nm reflecting the appearance of paranitrophenol

and hence the presence of active ALP in the supernatant. The absorbance was measured using a double beam UV-mc2 spectrophotometer from Safas (Monaco) using a mixture of Tris buffer (1 mL) and PNP (1 mL) in the reference cuvette to take the spontaneous hydrolysis of PNP into account. When the activity of the supernatant became equal to zero, the PEI-(AlgCat-ALP)n films were stored in the presence of 20 mL Tris buffer at 4 °C. At regular time intervals, the coated quartz slides were immersed in 2 mL of a 20-fold diluted PNP solution in a quartz cuvette. The absorbance of the solution was followed every 5 s during 2 h to monitor the kinetics of the enzymatic activity of the films. After the enzymatic assay, the functionalized quartz slide was immersed in 20 mL of fresh Tris buffer and stored again at 4 °C before the next enzymatic assay. The absorbance of the PNP solution after 2 h of contact with the polyelectrolyte multilayer films measured after t days of storage was compared to the activity of the same film at the time where the release of enzyme in the buffer solution was zero (defined as t = 0) to calculate the relative activity of ALP present in the polyelectrolyte multilayer film. In all those experiments, the quartz slides were immersed in identical volumes of diluted PNP solutions to ensure that the same surface area of the film was exposed to the PNP substrate.

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RESULTS AND DISCUSSION The first film deposition experiments were performed by dissolving AlgCat (1 mg·mL−1) and ALP (1 mg·mL−1) in 50 mM Tris buffer at pH 8.5. These experiments were done in such conditions owing to the known pH sensitivity of the enzymatic activity of ALP, even if both ALP and AlgCat are negatively charged in these conditions, which is a priori not favorable for the deposition of polyelectrolyte multilayer films relying only on electrostatic interactions. Indeed, no noticeable film deposition was measured by means of QCM-D when both entities were dissolved at pH 8.5 (data not shown). For this reason, the deposition experiments were performed in the presence of 50 mM sodium acetate buffer at pH 5.0. In these conditions AlgCat is negatively charged whereas ALP is dissolved at a pH value below its isolelectric point (pHi = 7) and is hence positively charged. The film deposition was followed by means of QCM-D (Figure 1), by means of SPR (Figure 1 in the Supporting Information), as well as by means of UV−vis spectroscopy (Figure 2 of the Supporting 12449


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Figure 1. Follow-up of the deposition of PEI-(AlgCat-ALP)n films by means of QCM-D up to n = 11 layer pairs as followed at the third (black line), the fifth (blue line), and the seventh (red line) overtone of the quartz crystal. The injections of AlgCat and of ALP are labeled with a blue and green vertical line, respectively. The beginning of the buffer rinse steps are omitted for clarity. The beginning of the injection of CaCl2 (3.3 mM in the presence of Tris buffer) and of NaI04 (10 mM in the presence of Tris buffer) are also indicated.

Figure 2. PNP hydrolysis rate in the supernatant put in contact of PEI(AlgCat-ALP)10 film after different contact times with 50 mM Tris buffer for an non-cross-linked film (black line after 10 min, red line after 30 min, and blue line after 2 h of contact with Tris buffer, curves A), for a film put in the presence of 1 mM NaIO4 during 10 min (black circle 10 min, red circle 30 min, blue circle 2 h, curves B) and for a film put in the presence of 1 mM NaIO4 during 2 h (green circle after 24 h of contact with Tris buffer, curve C).

with a 1 mM NaIO4 solution during different contact times (Figure 2, curve B). After the exposure of the PEI-(AlgCat-ALP)8 films to 1 mM NaIO4 during 2 h; no significant enzymatic activity was found in solution even after 24 h of contact between the film and Tris buffer (Figure 2, curve C). This result is in agreement with the known covalent cross-linking mechanism between quinone groups formed upon the oxidation of catechols and amino groups present on proteins (Scheme 3).34 This cross-linking process occurs through two successive steps: first of all, the catechol units on the modified-alginate (AlgCat) need to be oxidized into quinone groups. This step is effective and easily observable thanks to the appearance of a strong yellow color of the so-prepared film, typical of quinone formation. Then, under this highly reactive chemical form a nucleophile, such as the amino groups from alkaline phosphatase (AP), react spontaneously with quinones in mainly two distinct pathways: the 1,4addition (called Mickael addition) between the amine on the enone system (quinone) leading to covalent N−C bonds and the Schiff base formation from the condensation between amine and carbonyl (oxidized catechol) leading to NC (imine) bonds. It must be taken into account that the modification degree of AlgCat is low (15%) and we can expect that maybe not all catechol groups contributed to the cross-linking process with AP. Thus, the detection and the quantification of this low proportion of new chemical bonds formed into a nanometersized film is a hard task, maybe impossible to reach using the current spectroscopic tools (UV, IR, XPS) applied on a surface. Furthermore, the new chemical bonds created (secondary amines and imines) into the film do not have significantly different features through UV or IR than all others bonds present in AP. The cross-linking is nevertheless efficient because it precludes the desorption of AP from the film At this point, it should be noted that a prolonged treatment with the strong oxidant IO4− ions could denature a part of the enzymes and at this stage one cannot exclude that enzymes

Information) as a function of time and hence as a function of the number of deposition steps. Each deposition step led to a maximal amount of deposited molecules in less than 5 min (Figure 1). The QCM-D experiment shows that the frequency changes measured at the different overtones of the resonance frequency of the quartz crystal do not overlap, meaning that the deposit does not behave as a rigid coating. This finding impedes to use the Sauerbrey equation to calculate the surface coverage in deposited molecules. The amount of deposited macromolecules (i.e., AlgCat and ALP) increases linearly with the number of deposition steps as shown by means of QCM-D and UV−visible spectroscopy (Figure 2 of the Supporting Information). But a closer look at Figures 1 and Figure 1 in the Supporting Information shows that the injection of the enzyme containing solution produces a net deposition of molecules (the surface plasmon angle increases and the frequency of the quartz crystal decreases) whereas the injection of AlgCat produces some desorption, most probably in the form of AlgCat-ALP polyelectrolyte complexes. Such an adsorption/desorption behavior is not uncommon during the deposition of polyelectrolyte multilayer films.32,33 The morphology of the obtained films was investigated by atomic force microscopy (Figure 3 of the Supporting Information). These deposits covered the whole surface of the substrate and appear as island-like and very thin with a thickness of about 7 nm after 8 deposition cycles. After their deposition from sodium acetate buffer, the films were exposed to Tris buffer at pH = 8.5. In these conditions, the obtained PEI-(AlgCat-ALP)n films display a fast release of ALP as evaluated by measurement of the enzymatic activity of the supernatant in contact with the quartz slide on which the multilayer film was deposited (Figure 2, curve A). This enzyme release from the film could be reduced by cross-linking the film in two steps: first upon contact with 3.3 mM CaCl2 during 10 min (known to induce gelation of alginate) and upon contact 12450


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Scheme 3. Schematic Representation of the Covalent Crosslinking That Occurs between ALP and AlgCat in the Presence of Sodium Periodatea

a

We include the possibility of either Mickael addition of Schiff base formation. Only the Mickael addition leads to an irreversible bond. Indeed, after the addition of the amine onto the quinone, a re-aromatization step occurs and the reversibility of this reaction becomes impossible. Schiff bases are reversible bonds by essence through a hydrolysis reaction that occurs overtime but can be accelerated (catalyzed) in the presence of protons or metallic Lewis acids.

8.5 and stored at 4 °C before measurement of the hydrolysis of PNP after different storage times. It appears that the activity of the NaIO4 cross-linked PEI(AlgCat-ALP)10 films did remain constant over 7 weeks (Figure 3). In addition, the kinetics of paranitrophenol production displayed an initial lag phase in the experiments performed in the first days of storage and progressively disappears after longer storage (Figure 3). Such phenomena have already been observed in the case of metal nanoparticles immobilized in polymer brushes during the catalytic reduction of 4-nitrophenol into 4-aminophenol.35 The lag phase was attributed to a slow diffusion of the substrate to the active catalytic sites immobilized through the polymer brush. We believe that the lag phase progressively disappears for increased storage times of the PEI-(AlgCat-ALP)10 films in Tris buffer due to a progressive swelling of the film with the consequence of an increased accessibility of the enzyme for its substrate. This finding allows one to exclude the previously formulated assumption that prolonged contact with IO4− anions may induce some denaturation of ALP. After 11 days, the PEI(AlgCat-ALP)8 uncross-linked films displayed no enzymatic activity. In a control experiment, we checked that the catalysis of the PNP hydrolysis was indeed due to the enzyme and not to the catechol-quinone moieties present on the AlgCat polymer. For this aim, we deposited a PEI-(AlgCat-Lysozyme)8 film on a quartz slide, performed the two cross-linking steps as for the films containing ALP and measured the hydrolysis rate of PNP. The hydrolysis rate of PNP was found to be an order of magnitude smaller in the case of PEI-(AlgCat-Lysozyme)10 films than for their ALP containing counterparts (Figure 5 of the Supporting Information). Unfortunately, we were not able to determine the fraction of ALP present in the film that remains active in the hydrolysis of PNP after the cross-linking steps. To that aim we tried to dissolve the as-prepared and cross-linked films upon an increase in ionic strength (2 M in NaCl, which is sufficient to completely suppress the interactions between AlgCat and ALP) to dialyse the obtained AlgCat- and ALP-containing solution against water and then to determine the enzyme concentration using the Bradford method (after calibration with

could be desorbed in an inactive state after cross-linking, which could also explain the absence of enzyme activity in the supernatant after 2 h of interaction with the NaIO4 containing solution. However, if this would be the case the ability of the film to hydrolyze PNP would also be severely affected. The influence of cross-linking by CaCl2 and NaIO4 was also investigated by means of SPR spectroscopy and QCM-D (Figure 1). CaCl2 induced some desorption of the films as seen from a reduction of the resonance angle of the surface plasmons and a slight increase in the frequency of the quartz crystal. The contact with the NaIO4 solution produced a sudden decrease in the resonance angle of the surface plasmon spectrum and a sudden decrease in the resonance frequency of the quartz crystal (Figure 1) but no further change with prolonged contact time between the film and the NaIO4 containing solution. The sudden increase in the resonance frequency of the quartz crystal after the injection of NaIO4 could either be due to a mass decrease of the film or due to a slight increase in the mass density of the solution. The mass decrease could result from a desorption of the polymers, AlgCat or ALP, or release of water or a combination of both processes. Because the frequency changes in Figure 1 display an overshoot and a pretty slow relaxation we discard the influence of a the increase in the solution’s mass density and assume that the film undergoes a relaxation, most probably a deswelling process upon contact with the NaIO4 containing solution. The sodium periodate also produced a marked change in the optical properties of the alginate catechol solution with the appearance of a strong absorption band at 290 nm, which is characteristic for quinone groups, whereas the catechol groups where characterized by a strong absorption at λ = 275 nm (Figure 5 in the Supporting Information). These changes in the optical and in the gravimetric response sensed by SPR and QCM-D, respectively, could be attributed respectively to a change in refractive index (in the SPR experiment) and in the mass density (in the QCM-D experiment) of the solution upon injection of the NaIO4 containing solution. Hence the films were systematically cross-linked successively in the presence of 3.3 mM CaCl2 (in acetate buffer) and 1 mM NaIO4 (also in acetate buffer), rinsed with Tris buffer at pH = 12451


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about 7 nm in thickness (Figure 3 of the Supporting Information) and covering the two faces of a quartz slide, hence about 4 cm2 of surface area. The hydrolysis of PNP was also measured for PEI-(AlgCatALP)n films deposited by changing the number of deposition cycles, n, and hence the film thickness (Figure 1). All these films were cross-linked with 3.3 mM CaCl2 during 10 min and with 1 mM NaIO4 during 2 h, conditions that appeared to be optimal for the stability of the architecture since no enzyme is desorbed from the films in these conditions (Figure 2). These films were then stored at 4 °C in the presence of Tris buffer before following the hydrolysis rate of PNP (Figure 4). The absorbance obtained after 2000 s of PNP hydrolysis is proportional to the number of deposition cycles (inset of Figure 4). Because the film thickness and hence the amount of deposited enzyme per unit area is a linear function of n (Figure 1 and Figure 2 of the Supporting Information), the enzymatic activity of the PEI-(AlgCat-ALP)n films can be precisely controlled by the number of deposition cycles and that even the enzyme located far away from the film solution-interface is accessible to its substrate, PNP. This result is in marked contrast with the findings of Oliveira et al., who showed that in (chitosan-GOX)n films only the enzyme deposited in the last layer remains accessible to glucose.17 Finally, the PEI-(AlgCat-ALP)8 films deposited on quartz slides and cross-linked during 2 h in the presence of 1 mM NaIO4 were freeze-dried and stored at 4° during 3 weeks before the first measurement was performed. The obtained enzymatic activity was indistinguishable from that of the same film but estimated directly after its preparation and cross-linking with 1 mM NaIO4 (Figure 5). This shows that PEI-(AlgCat-ALP)n films can be used in a totally preserved state after freeze-drying. This result opens an avenue for the preparation of biosensors by layering AlgCat with enzymes presenting a real sensing ability like glucose oxidase, β-galactosidase, and so forth. Indeed the preparation of such films using the layer-by-layer deposition method can be realized in an automatized manner using dipping robots and the

Figure 3. PNP hydrolysis rate of a 20-fold diluted PNP solution put in the presence of a PEI-(AlgCat-ALP)10 film after different times of storage of the film at 4 °C and in the presence of 50 mM Tris buffer at pH = 8.5. The films were cross-linked in the presence of 3.3 mM CaCl2 during 10 min and then in the presence of 1 mM NaI04 during 2 h before the first evaluation of the enzymatic activity at time t = 0. The storage time was of 0 (●), 5 (black line), 6 (red line), 8 (green line), 13 (magenta line), 20 (black dashed line), and 49 (red dashed line) days. The inset represents the evolution of the relative enzymatic activity as a function of the storage time, that is, the activity measured after 2 h of reaction at day t divided by the corresponding activity at day t = 0. The different symbols correspond to two films prepared in an independent manner and the open circles correspond to the data plotted in the main part of the figure.

ALP itself).36 Unfortunately the amount of ALP was below the detection limit of the assay, which is not surprising for a film

Figure 4. Influence of the number of deposition cycles of PEI-(AlgCat-ALP)n films on their enzymatic activity, in the case where n = 0 (blue circle, PEI alone), n = 7 (black circle), n = 11 (red inverted triangle) and n = 15 (black triangle). All these films were cross-linked with 3.3 mM CaCl2 and with 1 mM NaIO4 during 2 h before measurement of the enzymatic activity. The inset displays the evolution of the absorbance at 405 nm after 2000 s of exposure to the PNP solution as a function of the number of layer pairs. 12452


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solution (0.2 mg.mL−1) in the presence of 50 mM Tris buffer at pH = 8.5 before and after addition addition of NaIO4, and kinetics of the hydrolysis rate of a 20-fold diluted PNP solution in the presence of a PEI-(AlgCatALP)10 and a PEI-(AlgCat-Lysozyme)10 film.(PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Christian Ringwald is acknowledged for his technical help. Figure 5. Comparison of the activity of a PEI-(AlgCat-ALP)4 film cross-linked with 1 mM NaIO4 during 2 h, freeze-dried and stored at 4 °C during 20 days before measurement (red line) with the activity of a film prepared in the same conditions, cross-linked and immediately investigated for its enzymatic activity (blue line).

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REFERENCES

(1) Caseli, L.; Oliveira, R. G.; Masui, D. C.; Furriel, R. P. M.; Leone, F. A.; Maggio, B.; Zaniquelli, M. E. D. Effect of Molecular Surface Packing on the Enzymatic Activity Modulation of an Anchored Protein on Phospholipid Langmuir Monolayers. Langmuir 2005, 21, 4090− 4095. (2) Walde, P.; Ichikawa, S. Enzymes Inside Lipid Vesicles: Preparation, Reactivity and Applications. Biomol. Eng. 2001, 18, 143−177. (3) Grotzky, A.; Atamura, E.; Adamcik, J.; Carrara, P.; Stano, P.; Mavelli, F.; Nauser, T.; Mezzenga, R.; Schlüter, A. D.; Walde, P. Structure and Enzymatic Properties of Molecular Dendronized Polymer-Enzyme Conjugates and Their Entrapment inside Giant Vesicles. Langmuir 2013, 29, 10831−10840. (4) Gornowich, D. B.; Blanchard, G. J. Enhancement of Enzyme Activity by Confinement in an Inverse Opal Structure. J. Phys. Chem. C 2012, 116, 12165−12171. (5) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Sequential Actions of Glucose Oxidase and Peroxidase in Molecular Films Assembled by Layer-by-Layer Alternate Adsorption. Biotechnol. Bioeng. 1996, 51, 163−167. (6) Derbal, L.; Lesot, H.; Voegel, J.-C.; Ball, V. Incorporation of Alkaline Phosphatase into Layer-by-Layer Polyelectrolyte Films on the Surface of Affi-gel Heparin Beads: Physicochemical Characterization and Evaluation of the Enzyme Stability. Biomacromolecules 2003, 4, 1255−1263. (7) Yao, H.; Hu, N. pH-Controllable On-Off Bioelectrocatalysis of Bienzyme Layer-by-Layer Films Assembled by Concanavalin A and Glucoenzymes with an Electroactive Mediator. J. Phys. Chem. B 2010, 114, 9926−9933. (8) Huang, C.; Bai, H.; Li, C.; Shi, G. A Grapheme Oxide/ Hemoglobin Composite Hydrogel for Enzymatic Catalysis in Organic Solvents. Chem. Commun. 2011, 47, 4962−4964. (9) Mahler, H. R.; Cordes, E. H. Biological Chemistry; Harper & Row Publishers: New York, 1966; Chapter 6. (10) Tirado, P.; Reisch, A.; Roger, E.; Boulmedais, F.; Jierry, L.; Lavalle, Ph.; Voegel, J.-C.; Schaaf, P.; Schlenoff, J. B.; Frisch, B. Catalytic Saloplastics: Alkaline Phosphatase Immobilized and Stabilized in Compacted Polyelectrolyte Complexes. Adv. Funct. Mater. 2013, 23, 4785−4792. (11) Ciaurriz, P.; Bravo, E.; Hamad-Schifferli, K. Effect of Architecture on the Activity of Glucose Oxidase/Horseradish Peroxidase/Carbon Nanoparticle Conjugates. J. Colloid Interface Sci. 2014, 414, 73−81. (12) Johnson, B. J.; Algar, W. R.; Malanoski, A. P.; Ancona, M. G.; Medintz, I. L. Understanding Enzymatic Acceleration at Nanoparticle

obtained films can be stored safely before their use as a biosensor. In addition, each sample can be used several times over a time duration of 1 or 2 weeks as shown in Figure 3 without significant loss of enzymatic activity. The possibility of producing a film with sensing abilities in which the amount of active enzyme can be controlled through the number of deposition steps (Figure 4) with a prolonged storage of activity up to 7 weeks in the present case (Figure 3) is the major originality of this work in comparison with other enzyme immobilization techniques.37

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CONCLUSIONS Stable, enzymatically active PEI-(AlgCat-ALP)n films can be deposited on solid surfaces using alginate modified with catechol moieties that can be oxidized in the presence of a strong oxidant like sodium periodate. The obtained quinone groups are able to react with uncharged amino groups present on the surface of the protein impeding its desorption in the buffer solution and allowing to maintain the enzymatic activity at the same level for at least 7 weeks. The cross-linked PEI(AlgCat-ALP)n films can also be freeze-dried and reused after at least 3 weeks of storage without loss in enzymatic activity in comparison to a freshly prepared film of the same composition. In the future, we aim to apply a similar strategy to more conformationally unstable enzymes like glucose oxidase to generalize the layer-by-layer deposition followed by oxidative cross-linking from a catechol-modified polymeric template.

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ABBREVIATIONS AlgCat: alginate modified with catechol groups ALP: alkaline phosphatase

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03329. Description of the synthesis of AlgCat and supplementary figures: deposition of a PEI-(AlgCat-ALP)5 coating as followed by means of SPR, deposition of a PEI(AlgCat-ALP)12 coating as followed by means of UV− visible spectroscopy, surface topographical image acquired by AFM in the dry state and in contact mode of a PEI-(AlgCat-ALP)8 film, absorption spectra of an AlgCat 12453


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Stable Bioactive Enzyme-Containing Multilayer Films Based on

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