Functionalized Titania Nanosheet Dispersions of Peroxidase Activity

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C: Surfaces, Interfaces, Porous Materials, and Catalysis

Functionalized Titania Nanosheet Dispersions of Peroxidase Activity Paul Rouster, Marko Pavlovic, Szilárd Sáringer, and Istvan Szilagyi J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b03271 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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The Journal of Physical Chemistry

Functionalized Titania Nanosheet Dispersions of Peroxidase Activity Paul Rouster,† Marko Pavlovic,‡ Szilárd Sáringer,§,║ Istvan Szilagyi*,§,║ †

Institute of Condensed Matter and Nanosciences - Bio and Soft Matter, Université Catholique

de Louvain, Louvain-la-neuve, Belgium ‡

§

Department of Inorganic and Analytical Chemistry, University of Geneva, Geneva, Switzerland MTA-SZTE Lendület Biocolloids Research Group, University of Szeged, Szeged, Hungary

║

Department of Physical Chemistry and Materials Science, University of Szeged, Szeged,

Hungary

ACS Paragon Plus Environment

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ABSTRACT: Nanocomposites of titania nanosheets (TNS), horseradish peroxidase (HRP) and poly(diallyldimethylammonium chloride) (PDADMAC) were prepared and their colloidal and functional stabilities were assessed. HRP quantitatively adsorbed on bare TNS and the adsorption process did not affect the charging and aggregation behavior of the colloidal system. The obtained TNS-HRP composite was functionalized by PDADMAC to stabilize the enzyme on the surface and to maintain good colloidal stability. Depending on the PDADMAC dose applied, its adsorption on TNS-HRP led to charge reversal of the particles from negative to positive. The formation of a saturated polyelectrolyte layer on the TNS-HRP (TNS-HRP-PDADMAC) gave rise to highly stable colloids, especially the resistance against salt-induced aggregation was excellent. The enzymatic activity of different systems was investigated as a function of the pH of the medium and over time. The results indicated that HRP remained enzymatically active upon immobilization, in addition, the pH range of application broadened compared to its native form. The developed TNS-HRP-PDADMAC system can thus be used in a wider pH range and possesses the advantages of a heterogeneous catalysts compared to the bare enzyme.


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INTRODUCTION Titania nanoparticles of spherical or elongated structures have been extensively studied in the past decades due to their advantageous chemical properties utilized in various applications.1-3 Among them, bio-applications, e.g. in medical devices4, as bio-sensors5 or biomimetic materials,6 to improve the biocompatibility of polymeric scaffolds7 and in drug delivery,8 became especially important. Indeed, these materials take the advantage of the biocompatibility of titania and also of the suitability of its surface for the deposition of various types of coating agents to obtain improved surface properties.9-11 In addition, preparation of 2-dimensional titania and other materials of sheet-like structure was in the focus of several research group.12-16 Enzymes, on the other hand, are sensitive biocatalysts that can partially or totally lose their enzymatic activity as a consequence of any changes occurring in their surrounding environment (e.g. pH, temperature, pressure or ionic strength).17,18 This major drawback on their environmental response can be overcome by immobilization on solid supports.17,19-21 However, depending on the enzymes and the supports used, contradictory results were reported.17,22-24 In some cases, immobilized enzymes performed better in harsher conditions compared to their native form in solution, whereas in some situations, immobilization resulted in denaturation over time. Among all the different classes of enzymes, oxidoreductase enzymes are of special importance for the removal of reactive oxygen species (ROS) like superoxide radical anions and hydrogen peroxide (H2O2).25-27 The superoxide dismutase enzyme is known for the efficient dismutation of superoxide radical anions by converting them to peroxide, which in turn is decomposed by catalase or horseradish peroxidase (HRP). When both types of enzymes are used together, they perform a cascade reaction leading to the complete decomposition of ROS. However, in some


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cases, when dealing only with H2O2, only one type of enzyme is needed. By mixing HRP with H2O2, the heme group of the HRP reacts with H2O2 and this intermediate compound behaves as an oxidizing agent towards aromatic substances.28,29 This reaction has been used to develop biosensors in order to detect the presence of H2O2 in solutions and to monitor its concentration and/or to decompose aromatic compounds.30-33 Even though both HRP forms, native and immobilized, are highly efficient, the later one is more favorable due to its possibility to be recycled and reused.29 Adsorption of HRP or other enzymes on solid supports (e.g. titania nanostructures, clay platelets or silica nanoparticles) occurs through one or several types of interactions like covalent linkage, electrostatic attraction, hydrogen bonding and hydrophobic interaction.11,34-36 In general, these enzyme functionalized surfaces possess enzymatic activity as emphasized in the case of HRP by the reduction of the H2O2 content in solutions.22,37 Moreover, HRP enzymes deposited in multilayered films retain their enzymatic function. During the build-up of such thin films, one can play on the location and amount of deposited enzymes, which in turn will affect their efficiency in catalyzing the removal of H2O2.38,39 Similarly, core-shell structures or capsules were also investigated, where HRP enzymes were encapsulated either in their center or in their surrounding polyelectrolyte multilayers.40-42 These materials possessed the advantage of further protecting the enzymes towards the external medium due to the presence of the polyelectrolyte multilayer coating.43 Moreover, the enzymatic activity studies performed revealed that the enzymes were still efficient and possessed an improved lifespan compared to their free (nonimmobilized) form. In general, the structure of the enzyme-nanoparticle composites is characterized in detail, while little attention is paid to the colloidal stability of these systems. However, this is a critical issue,


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since many applications, especially the bio-related ones, occurs in dispersions. Indeed, aggregation of the nanocarriers leads to their unsuccessful use in delivery processes due to the formation of irregularly shaped clusters giving rise to a loss of their enzymatic activity.44 Despite the importance of aggregation and charging of these colloids, only a limited number of studies reported systematic investigations in this field. Here, we aim to design stable dispersions containing HRP immobilized on TNS acting as nanocarrier. The colloidal behavior of the nanocomposites was tuned by functionalization with poly(diallyldimethylammonium chloride) (PDADMAC) polyelectrolyte. The charging and aggregation features of the different systems upon their surface functionalization were investigated at each step of the preparation in order to determine the conditions, under which the colloids were stable or tend to aggregate. The enzymatic activity of the nanocomposites was determined in biochemical assays to assess the influence of HRP immobilization on the activity and the results were compared to its native form.

EXPERIMENTAL SECTION Materials. HRP (type VI, EC number 1.11.1.7), PDADMAC (20 wt.% in water, Mw=100-200 kg/mol), Coomassie Brillant Blue G, 2-methoxyphenol (guaiacol), phosphoric acid (85 v/v%) and analytical grade salts including sodium chloride (NaCl), sodium phosphate monobasic monohydrate (NaH2PO4·H2O) and sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) were purchased from Sigma Aldrich and used without further purification. Ethanol of reagent grade was acquired from Fisher Scientific. H2O2 (30%) was purchased from Reactolab SA. TNS were synthesized according to a procedure described elsewhere.45 Ultrapure water (Millipore) was


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used directly after production for all the sample preparations. The measurements were carried out at pH 7 and 25 °C. Prior to their use, the TNS were dispersed in ultrapure water. A pH 7 phosphate buffer, at a concentration of 1 M, was prepared by mixing appropriate amount of Na2HPO4 and NaH2PO4 solutions. Samples containing PDADMAC in a concentration range of 0.01-1000 mg/L were prepared by dissolving the polyelectrolyte in a 5 mM NaCl solution. HRP was dissolved in ultrapure water and solutions with a concentration range of 0.01-1000 mg/L were prepared. TNS-HRP-PDADMAC dispersions were prepared by mixing TNS-HRP with PDADMAC to obtain a solution, where the particle concentration was 1 mg/mL and the final NaCl concentration was 4.5 mM. After two hours of PDADMAC adsorption time, the solution was centrifuged at 10000 rpm for 20 min. The supernatant was removed and the slurry was redispersed in a 5 mM NaCl solution. More details concerning the sample preparation protocols are given later. Characterization Methods. Electrophoretic mobility (EM) measurements were performed on a Zetasizer Nano ZS (Malvern Instruments) device. For the EM experiments, 5 mL solutions were prepared, where the final particle concentration was 1 mg/L. In general, 0.5 mL of the particles was added to 4.5 mL of solution containing calculated amount of salt and/or coating material (HRP, PDADMAC). The sample was then allowed to rest overnight prior to the EM experiments. The reported results were the average of 5 independent measurements made under the same experimental conditions. Dynamic light scattering (DLS) experiments were carried out on a CGS-3 goniometer system (ALV) at a scattering angle of 90°. To follow the aggregation processes, time-resolved DLS experiments were performed, where the correlation function was collected for 20 seconds and the


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measurements lasted for typically 10-120 minutes depending on the speed of aggregation. A second order cumulant fit was used to determine the diffusion coefficient, which was then introduced in the Stokes-Einstein equation to calculate the hydrodynamic radius.46 The stability of the colloidal dispersions was expressed in terms of stability ratio.45-47 The sample preparation for DLS experiments was similar to the one applied for the EM measurements, but differed in the fact that the aggregation rate of the particles was probed immediately after sample preparation. Infrared (IR) spectra were recorded in the attenuated total reflectance mode (ATR) using a Spectrum 100 FT-IR spectrometer (PerkinElmer). The ATR crystal was made of diamond. Prior to the measurements, the internal reflectance unit was washed with ethanol and dried. The obtained spectrum is an average of 48 scans acquired at a resolution of 4 cm-1. Transmission electron microscopy (TEM, Tecnai G2 Sphera microscope, FEI) images of the bare and coated TNS were recorded at each stage of their surface functionalization. The device was equipped with a LaB6 cathode and operated at a voltage of 120 kV. The colloidal suspension was deposited on a plasma activated carbon hexagonal mesh (CF200H-CU-UL, Electron Microscopy Sciences). After 2 minutes adsorption time, the excess solution was removed with a filter paper. The mesh was then placed on the specimen and mounted in the microscope for imaging. Quantification of the HRP Amount in Solution. The amount of HRP that did not adsorb on the TNS, i.e. remained dissolved in the bulk, was determined by the Bradford test.48,49 A stock solution of Coomassie Brillant Blue dye was prepared as follows. 100 mg of the dye was dissolved in 50 mL of 95 v/v% ethanol and 100 mL of 85 v/v% phosphoric acid was added and the solution was completed to 1 L with ultrapure water. Thereafter, standard solutions of HRP with a concentration range of 0-100 mg/L were prepared by dilution. After mixing 400 µL of the


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HRP standard solution with 1.6 mL of the dye solution, the UV-Vis spectrum of the solution, after 5 minutes equilibration time, was recorded on a Lambda 35 spectrophotometer (Perkin Elmer). The change in the absorbance bands at 464 nm (free dye) and at 594 nm (enzyme-dye and free dye) was monitored. The ratio in the intensity between these two peaks indicated the amount of HRP that did not adsorb on the surface of the TNS. Enzymatic Assay. The determination of the HRP activity was performed by using the guaiacol assay.22,50,51 In brief, 240 µL of a HRP solution at a concentration of 5 mg/L was mixed with 1.872 mL phosphate buffer at a concentration of 12.9 mM followed by the addition of 240 µL guaiacol at a given concentration. The cuvette was then sealed and vortexed for a few seconds. Finally, 48 µL of H2O2 at 135 mM was added in the cuvette, which was vortexed again and immediately introduced into the UV-vis spectrophotometer to follow the formation of the guaiacol degradation products at 470 nm wavelength (see TOC Graphic for the color changes).52,53 The increase in the absorbance at 470 nm was monitored as a function of the reaction time, where the linear part of the curve was fitted in order to obtain the reaction rate of the system. The results were analyzed with the Michaelis-Menten model as54 v=

vmax [ S ] Km + [S ]

(1)

where v is the reaction rate, K m is the Michaelis-Menten constant, vmax is the maximum reaction rate for the investigated system and [ S ] refers to the substrate concentration. For the time-dependent measurements, the HRP containing samples were rested at room temperature. Appropriate amount of aliquots were taken each day and the enzymatic activities were tested in the above described probe reaction.

RESULTS AND DISCUSSION


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TNS Aggregation in Phosphate Buffer. Bare titania, like other inorganic (e.g. clay, silica and gold) nano-objects, often possess a limited stability in the presence of monovalent salt solutions.45,55,56 Therefore a convenient way to increase the colloidal stability of such particles is to functionalize them with polyelectrolytes45,57 or dendrimers.58 In some cases, multivalent ions can be used to improve the stability of the bare particles.56,59 Indeed, in the presence of such ions, the particles first undergo fast aggregation above the first critical coagulation concentration (CCC) value, which marks the transition between stable and unstable dispersions. A further increase in the ionic strength above the first CCC value can lead to a restabilization of the particles marked by a second CCC value. This is due to further ion adsorption on the surfaces, which either leads to charge reversal or to an increase in the magnitude of the electrokinetic potential.56 Therefore, the influence of the phosphate buffer concentration at pH 7 on the charging and aggregation behavior of the bare TNS was investigated (Figure 1) in this study first in order to optimize the conditions for the further enzyme immobilization step. The particles are negatively charged in the phosphate buffer concentration range investigated (Figure 1a) due to the fact that their point of zero charge was reported to be 5.2 earlier.56 In this case, the phosphate ions are the coions, i.e. they possess the same sign of charge as the nanosheets. Furthermore, above a concentration of about 20 mM the magnitude of the EM decreases due to the increase of the surface charge screening by the sodium counterions. The colloidal stability was assessed in time-resolved DLS experiments and expressed in terms of stability ratio, which is calculated from the initial increase of the hydrodynamic radius in aggregating samples.34,46,56 Note that stability ratios close to unity indicate rapidly aggregating unstable colloids, while in the case of higher values, the aggregation slows down and the samples


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are more stable. In other words, a stability ratio of 10 means that 1/10 fraction of the particle collisions leads to dimer formation for instance. The trend in the stability ratios of the system shows that the samples are stable up to a phosphate buffer concentration of 78 mM (Figure 1b). Above this value, TNS undergo fast aggregation as indicated by stability ratios close to one. These results are in line with the theory developed by Derjaguin, Landau, Verwey and Overbeek (DLVO).60,61 Indeed, the DLVO theory states that the overall interparticle force is the sum of the repulsive electrical double layer force and the attractive van der Waals force. By increasing the ionic strength, the surface charges of the TNS are screened by the ions in the phosphate buffer leading to a thinning of the electrical double layer around the particles. Above the CCC, the repulsive electrical double layer forces no longer prevail and the attractive van der Waals forces predominate giving rise to rapid aggregation of the particles and thus, to unstable dispersions. It is obvious from these results that the particles are highly stable and negatively charged at a phosphate buffer concentration of 10 mM. Therefore, this condition was chosen in order to obtain stable TNS dispersions for further enzyme immobilization.

Immobilization of HRP. The isoelectric point of HRP ranges from 5 to 9 depending on its origin and purification process.28,33 In our case, however, the enzyme was positively charged at pH 7 and thus, it was expected to adsorb on the oppositely charged nanosheets by electrostatic attraction. It is also important to avoid the significant decrease in the magnitude of the surface charge upon HRP adsorption, because it may lead to unwanted particle aggregation. Therefore, EM was recorded at different HRP doses to follow the charging properties (Figure 2). It was found that for an enzyme dose less than 100 mg/g, the EM values were not affected by the amount of HRP in solution. Above this loading, the magnitude of the EM decreases. No stability


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ratio values could be determined in the HRP concentration regime investigated due to the high stability, i.e. the lack of aggregation processes, of the colloidal suspension. However, applying a high amount of HRP dose to functionalize the particles does not necessarily result in a quantitative adsorption of the enzyme on the TNS. As a matter of fact, it is primordial to determine a HRP dose, which does not affect the colloidal behavior of the TNS and on the other hand, also results in quantitative adsorption on the TNS, i.e. no HRP partitioning between the surface and the bulk. The quantification of the amount of adsorbed HRP on the TNS was performed by the Bradford test,48 which determines the amount of HRP present in the bulk solution after enzyme adsorption. For HRP doses lower than 10 mg/g, it was found that after two hours of adsorption time, 99.9% of the enzyme adsorbed on the TNS. Such a strong adsorption is most likely due to the combined effects of electrostatic attraction, hydrogen bonding and hydrophobic interaction between the enzyme and the nanosheet surface. Accordingly, 10 mg/g HRP dose was applied in the further studies, since no HRP is present apart from the one on the surface and the obtained TNS-HRP hybrid is of high colloidal stability. Moreover, the adsorption of the enzyme on the TNS was also confirmed by IR spectroscopy. In Figure 3, the IR spectra of the native enzyme and TNS are shown along with the TNS-HRP hybrid. In the wavenumber region 800-1800 cm-1, the TNS possess no characteristic peaks, whereas several vibrational bands can be assigned to the HRP in this regime. The peaks at 1645 cm-1 and 1525 cm-1 are attributed to the amide I and amide II functions, respectively.62-64 Upon immobilization of HRP on the TNS, the IR spectrum of TNS-HRP shows the appearance of the amide I function meaning that the enzyme was successfully adsorbed on the TNS. Thus, the combination of the Bradford test results with the IR spectrum of the TNS-HRP system unambiguously confirms that the HRP is quantitatively adsorbed on the TNS, once a dose of 10


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mg/g is applied. Note that the TNS-HRP abbreviation refers to a 10 mg/g HRP coating of one gram of TNS in the followings.

Surface Functionalization by PDADMAC Adsorption. Although HRP was successfully immobilized on TNS, the leakage of the enzyme from the nanoparticulate support may occur over time or during the operational use.65,66 Therefore, the surface of TNS-HRP was functionalized by PDADMAC polyelectrolyte in order to prevent or minimize any leakage of the enzyme and also to reverse the surface charge of the nanocarrier from negative to positive in order to match the positive net charge of the native enzyme. PDADMAC is a strong polyelectrolyte, whose charge density is pH independent meaning that any variation of the pH in the solution does not affect the surface charge of the functionalized TNS. To optimize the polyelectrolyte dose for the functionalization, the charging and aggregation behavior of the particles were initially investigated. The effect of HRP adsorbed on the TNS on the surface charge properties and colloidal stability was also probed and the EM values of both TNS and TNS-HRP measured at different PDADMAC doses were determined and compared. Let us first discuss the influence of the PDADMAC dose on the EM values in both TNS and TNS-HRP systems (Figure 4a). It was reported several times earlier that polyelectrolytes tend to strongly absorb on oppositely charged surfaces.45,55,57,67,68 For both systems at low PDADMAC dose, the EM values are similar to the ones of the bare particles. By increasing the amount of PDADMAC adsorbing on the surface, the EM values increase until reaching the charge neutralization point. Further addition of PDADMAC leads to charge reversal of the system and to the appearance of an adsorption saturation plateau. Beyond the dose corresponding to the onset of this plateau, no more polyelectrolyte can adsorb on the surface of the particles and the remaining PDADMAC stays dissolved in the bulk. Such a trend in the EM data for oppositely


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charged polyelectrolyte-particle systems is typical and was reported previously for different types of dispersions.34,45,47,58,67 It was observed that the charging behavior of both systems was very similar giving rise to similar charge neutralization points and their surface saturates more or less at the same PDADMAC loadings. Note that TNS-HRP-PDADMAC refers to PDADMAC coating of the TNS-HRP particles at the onset value for the saturation plateau (144 mg/g). The colloidal stability of the TNS and TNS-HRP was also investigated (Figure 4b) under exactly the same experimental conditions as applied in the EM study. At low and high PDADMAC doses, the systems are highly stable, whereas aggregation occurs around the neutralization point, as indicated by stability ratio values close to unity at the minima of the plots. As for the electrophoresis measurements for both types of particles, similar aggregation results were obtained meaning that the applied dose of 10 mg/g of HRP used to functionalize the TNS did not affect the overall colloidal behavior of the particles. In addition, the TNS-HRPPDADMAC hybrid material of saturated polyelectrolyte layer on the surface forms stable dispersion under the experimental conditions investigated. Furthermore, the tendency of the stability ratios upon the PDADMAC coating of the particles is in line with the DLVO theory. Indeed, at low PDADMAC dose, the repulsive electrical double layer forces of the particles overcome the attractive van der Waals forces leading to stable dispersions. Increasing the PDADMAC dose, the electrical double layer forces weaken due to the surface charge compensation of the nanoparticles by the PDADMAC adsorption. Around the charge neutralization point, the particles are close to an electrostatically neutral state and thus, aggregation occurs due to the predominance of the attractive van der Waals forces. Further adsorption of PDADMAC above the neutralization point leads to a restabilization of the systems, where aggregation is hindered by the generation of a positive electrical double layer around the


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particles upon PDADMAC adsorption. Similar tendency in the stability ratios were previously published in particle-oppositely charged polyelectrolyte systems.34,47,55 Under the applied coating conditions, all the different systems (TNS, TNS-HRP and TNSHRP-PDADMAC) appear to be stable as indicated by the aggregation studies. This statement was further investigated by recording TEM images of the samples. When dispersed in a 10 mM phosphate buffer at pH 7, the TNS are stable and low order of aggregates can be seen due to the drying process during the sample preparation for TEM imaging (Figure 5a). Similar observations were made, when recording TNS-HRP (Figure 5b) dispersions. Thus, the 10 mg/g dose of HRP used to functionalize the TNS does not affect the colloidal behavior of the system as already observed previously. The TEM images of the TNS-HRP-PDADMAC sample (Figure 5c) showed that upon charge reversal of the TNS-HRP by PDADMAC coating at the onset of the saturation plateau does not lead to any destabilization of the dispersion. Moreover, these TEM images clearly indicate that the morphology of the bare, enzyme functionalized and polyelectrolyte coated nanosheets is the same.

Resistance against Salt-Induced Aggregation. In the next step, the charging and aggregation processes of TNS, TNS-HRP and TNS-HRP-PDADMAC materials were studied at different ionic strengths. The main idea was to investigate the influence of the successive coatings on the colloidal stability of the particles and whether it has been improved or not. For comparison, note that the CCC of the bare TNS dispersed in ultrapure water (without phosphate buffer) was found to be 19 mM.45 Figure 6 shows the effect of the NaCl concentration on the EM and stability ratio values for the different systems. From the charging behavior of the different materials (Figure 6a), it was observed that the TNS and TNS-HRP are negatively charged, whereas the TNS-HRP-PDADMAC is of positive charge


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in almost the entire salt concentration regime studied. This is in line with the results of the mobility studies discussed in the earlier sections. Moreover, from the salt dependent electrophoresis experiments, the surface charge density of the materials was determined by converting the EM values at different salt concentrations to electrokinetic potentials by using the Smoluchowski equation followed by the fitting of the potentials at different ionic strength by the Debye-Hückel model.69 The calculated surface charge densities were -15 mC/m2, -16 mC/m2 and +7.5 mC/m2 for TNS, TNS-HRP and TNS-HRP-PDADMAC, respectively. Accordingly, the 10 mg/g of HRP deposited on the TNS did not affect the surface charge density of the material, whereas the PDADMAC coating led to a decrease in the magnitude of the surface charge density. Stability ratios were measured in the same systems to assess the colloidal stability (Figure 6b). The first observation was that the presence of 10 mM phosphate buffer increased the CCC from 19 mM to 78 mM compared to the bare TNS dispersed in water45 meaning that the stability of TNS raised 4 times. This is due to the specific phosphate adsorption and subsequent increase of the negatively charged group on the TNS surface.56,70 The influence of the 10 mg/g HRP adsorbed on the TNS followed by the PDADMAC coating of the TNS was also studied. The calculated CCCs were found to be 82.4 mM and 78.6 mM in NaCl salt solutions for the TNSHRP and TNS-HRP-PDADMAC, respectively. These results indicate that upon the successive adsorption of the enzyme and the polyelectrolyte, no destabilization of the system occurred and that no extra stabilization of the TNS could be observed. However, the PDADMAC coating of the nanoparticulate support led to a positively charged nanocarrier of similar charge compared to the utilized native enzyme.


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In addition, the Bradford test48 was performed on the supernatant of the TNS-HRPPDADMAC sample in order to check whether the HRP remained immobilized on the TNS surface upon the PDADMAC functionalization or desorbed from it. The results indicated that 99.7% of the enzyme was adsorbed on the TNS surface, i.e. no desorption of the enzyme took place during the coating process. Therefore, two types of stable nanocarriers (TNS-HRP and TNS-HRP-PDADMAC) were successfully developed.

Enzymatic Activity Tests. Although stable nanocarriers were obtained, their efficiency in the decomposition of H2O2 had to be verified. The enzymatic activity of the native and immobilized enzymes was determined by using the guaiacol assay.22,50,51 This biochemical test reaction consists of measuring the formation of the degradation products of guaiacol under the combined effect of HRP and H2O2. In brief, the HRP reacts with H2O2 to form an oxidizing agent towards aromatic compounds,28,29 like guaiacol for example. The formation of the guaiacol degradation products can then be monitored by following the color change of the solution over time.22,51 Prior to the determination of the enzymatic activity of the native and immobilized form of HRP, the influence of the bare TNS and TNS-PDADMAC (without any HRP added) were tested by the guaiacol assay. Both type of materials exhibited no enzymatic activity, i.e. these materials alone were unable to degrade guaiacol. Hence, if an enzyme-like function is observed during the further assays, it originates solely from the enzymes in the samples and not from the nanoparticulate supports. As mentioned in the experimental part, the Michaelis-Menten model54 was used to analyze the results of the assays. Accordingly, the Michaelis constants (Km) and the maximum reaction rate (vmax) were calculated from the reaction rate versus substrate concentration plots (equation 1). The Km value corresponds to the affinity of the enzyme towards the substrate. For example, a


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decrease in the Km value refers to a higher affinity of the enzyme to guaiacol. The vmax is the maximum reaction rate that can be achieved by the system once the active site of the enzymes is completely saturated by the substrate. The activities of the native (HRP), immobilized (TNS-HRP) and embedded (TNS-HRPPDADMAC) forms of the enzyme were then probed as a function of the pH. The relative Km (Figure 7a) and vmax (Figure 7b) data are plotted and correspond to the normalized Km and vmax values for each sample at the optimum pH conditions, which is pH 7.3 in these systems. One can conclude from Figure 7a that all the systems possess an optimum pH range of application around 7.3-7.8, where the relative Km is the lowest. Interestingly, the optimum relative vmax (Figure 7b) appears to be slightly different for all three systems. Indeed, the optimum pH range for HRP, TNS-HRP and TNS-HRP-PDADMAC is around pH 5.9-7.3, 5.9-7.8 and 4.8-7.8, respectively. Interpreting the relative Km and vmax values together, it can be observed that the immobilized and embedded form of the HRP exhibit a broader pH range of application, where their relative enzymatic activity is higher compared to the native enzyme. These systems appear particularly interesting, whenever HRP needs to be used in a broad pH range or in slightly acidic conditions. Knowing the optimum pH of application of these materials, the time dependent activity was also probed. Indeed, upon immobilization, it is known that the enzymes may change their conformation resulting in a loss of activity due to denaturation.17 Here, we probed the relative enzymatic activities for all three systems (HRP, TNS-HRP and TNS-HRP-PDADMAC) at pH 7.3 over several days. In Figure 8, only the relative vmax values are given as no significant information from the relative Km values could be obtained. Compared to the native HRP, which appears to be stable over time by maintaining its initial activity, the immobilized and embedded form of the HRP both losses their activity over time.


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After one day, the relative vmax decreased by 91% and 65% for TNS-HRP and TNS-HRPPDADMAC, respectively. After three days, TNS-HRP exhibited no more activity, whereas TNSHRP-PDADMAC only retained 9% of its initial activity. To explain the decrease in the enzymatic activity of the HRP coated nanocarriers, two assumptions were made. The first one being that the enzyme was desorbing over time from the nanocarrier and the second one that the HRP was being denaturated leading to a decrease in its activity. To check the first assumption, Bradford tests on the supernatant were performed to determine if any enzymes were present in it. The results indicated that no HRP leaked from the surface of the nanocarriers and that they were still immobilized or embedded. Thus, we could rule out the first assumption. Hence, the loss of enzymatic activity of the HRP functionalized nanocarriers should be due to the denaturation of the enzyme over time. This process appeared to occur faster for TNS-HRP compared to TNSHRP-PDADMAC. Indeed, for TNS-HRP-PDADMAC, the HRP is trapped under the PDADMAC layer and its denaturation was slower due to a blocking effect of the PDADMAC on the enzyme denaturation. For TNS-HRP, the enzyme is not trapped under a polyelectrolyte layer and the loss in activity due to denaturation of HRP cannot be slowed down or hindered. Although, the enzymatic activity of the nanocomposites decreases over time, the TNS-HRP-PDADMAC maintains considerable activity for one day. In addition, it possesses a broader pH range of application compared to the native enzyme. Considering these facts and also the advantages gained by the immobilization (e.g. better separation from the reaction mixture) the obtained TNS-HRPPDADMAC hybrid is a promising approach for future applications in manufacturing processes for the removal of H2O2 or oxidation of organic contaminants.


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CONCLUSIONS We report here on the preparation and colloidal behavior of TNS and its HRP modified and PDADMAC coated derivatives. HRP quantitatively adsorbed on the TNS through electrostatic forces, hydrogen bonding and hydrophobic interactions. PDADMAC adsorption on the TNSHRP particles resulted in charge neutralization at lower and charge reversal at higher polyelectrolyte doses. Formation of a saturated polyelectrolyte layer on the nanosheets prevented enzyme leakage and aggregation of the particles. The TNS, TNS-HRP and TNS-HRPPDADMAC materials possessed similar behavior once dispersed in salt solutions of different concentrations. The relative enzymatic activity of HRP under its native, immobilized (TNSHRP) and embedded (TNS-HRP-PDADMAC) forms was also probed. The results showed that upon adsorption of the enzyme, the particles exhibited a broader pH range of application, where the relative enzymatic activity of HRP appeared to be higher compared to the native HRP in solution. However, the time dependent study pointed out that the immobilized enzyme was being denaturated, especially in the case of TNS-HRP. Nevertheless, the obtained TNS-HRPPDADMAC hybrid material can be used in a broader pH range and possess the advantages of a heterogeneous catalyst such as an easier separation from the reaction mixture.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This research was financially supported by the Lendület program of the Hungarian Academy of Sciences (96130) and the Swiss National Science Foundation (150162).


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Figure 1. Influence of the phosphate buffer concentration on (a) the EM values and (b) stability ratios of the bare TNS at pH 7.


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Figure 2. Evolution of the EM data of TNS as a function of the applied dose of HRP. The mg/g unit refers to mg of enzyme per gram of TNS.


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Figure 3. IR spectra of TNS, HRP and TNS-HRP hybrid at an enzyme dose of 10 mg/g.


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Figure 4. (a) EM data and (b) stability ratios of TNS and TNS-HRP as a function of the applied PDADMAC dose used to functionalize the materials.


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Figure 5. TEM images of (a) TNS, (b) TNS-HRP and (c) TNS-HRP-PDADMAC. The scale bars are equal to 100 nm.


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Figure 6. (a) EM and (b) stability ratio measurements of TNS, TNS-HRP and TNS-HRPPDADMAC as a function of the NaCl concentration.


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Figure 7. Relative (a) Km and (b) vmax values of HRP in native (HRP), immobilized (TNS-HRP) and embedded (TNS-HRP-PDADMAC) forms as a function of the pH in a 10 mM phosphate buffer. The lines are just to guide the eyes.


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Figure 8. Time dependence of the relative vmax values of the native, immobilized and embedded form of HRP in a 10 mM phosphate buffer at pH 7.3. The lines serve as eye guides.


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Functionalized Titania Nanosheet Dispersions of Peroxidase Activity

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