Hairy Particles with Immobilized Enzymes: Impact of Particle Topology

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Hairy Particles with Immobilized Enzymes: Impact of Particle Topology on the Catalytic Activity Claudia Marschelke,†,‡ Martin Müller,† Dorina Köpke,§ Anke Matura,§ Marco Sallat,∥ and Alla Synytska*,†,‡ †

Leibniz Institute of Polymer Research Dresden e.V., Hohe Str. 6, 01069 Dresden, Germany Faculty of Mathematics and Science, Institute of Physical Chemistry and Polymer Physics, and §Faculty of Mathematics and Science, Institute of Biochemistry, Dresden University of Technology, 01062 Dresden, Germany ∥ Sächsisches Textilforschungsinstitut e.V., Annaberger Straße 240, 09125 Chemnitz, Germany Downloaded via UNIV OF SOUTH DAKOTA on December 21, 2018 at 12:12:04 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Enzymes are described as ideal green biocatalysts because they are highly specific and selective. However, their practical application is hampered because of the low stability and missing reusability of free enzymes. One method to overcome these problems is the immobilization of enzymes onto carriers. Although numerous publications discuss different immobilization strategies, optimization of these carriers for the highest enzyme activity and loading capacity, enzyme selectivity, reusability, and reactor system configuration still remains a challenging task. In this contribution, we aim to address the role of the core−shell particle design with respect to their geometry as well as the polymer shell thickness on the immobilization of biomolecules. We discovered that spherical particles with a core diameter of 200 nm and intermediate shell thickness as well as platelet-like particles exhibited excellent results with a maximum immobilization yield of laccase from Trametes versicolor of up to 92% and an activity on the carrier material of 5.722 U/(g particle). Especially, the platelet-like particles offered a scalable and convenient alternative for the immobilization of laccase. Circular dichroism measurements proved that the secondary structure of the enzyme is not impaired by immobilization onto all kinds of carrier particles. Moreover, the immobilized laccase was successfully used for the decolorization of Cibacron blue P-3R in up to 18 cycles. Finally, particle separation was achieved via citrate-induced flocculation within 10 min. This detailed study contributes to the understanding of rational design of catalytically active hybrid materials and their effective performance at interfaces for applications in textile industry and environmental technologies. KEYWORDS: stimuli-responsive particles, polymer interface, enzyme immobilization, laccase, particle size and shape, circular dichroism surface availability for enzyme immobilization,2,4,5,8,9,11−14 independently of the investigated size domain, because of higher particle surface to volume ratio, lower mass transfer resistance, lower diffusion resistance, and special surface effects offering better enzymatic performances such as higher activity retention and enzyme loading.1 Talbert and Goddard investigated the influence of nanoparticle size on activity retention of lactase covalently attached to carboxylic acid functionalized magnetic nanoparticles and demonstrated that reducing the particle size can increase the activity retention of conjugated lactase.3 The same trend was found for penicillin G acylase onto glyoxyl−agarose beads9 and glucose oxidase onto silica particles.5,12 Sisak et al. compared the activities of βgalactosidase immobilized onto macro-, micro- and submicrometer-sized chitosan supports.15 The best performance regarding activity retention was found for particles in the

1. INTRODUCTION The use and recycling of enzymes as versatile but highly specific and selective biocatalysts in industrial processes offer possible savings of chemicals, water, and energy consumption. Thus, enzyme-based procedures are more environmentally friendly, cost-effective, and sustainable than conventional catalytic methods. Immobilization of enzymes provides an excellent approach for the reduction of operating expenses by enhancing enzymatic stability in different environmental conditions, reducing product inhibition and facilitating recovery of the enzymes. Particle-based materials have attracted significant interest as supports for enzyme immobilization because of their wide diversity and large surface areas to obtain higher activity, recovery, and reusability.1 Particle size, which can be varied from nano- to micro-scale, impacts the performance of the immobilized enzyme and, thus, is an important parameter for the design of an efficient immobilization support. Several groups have introduced particles of a broad size spectrum, from nanoparticles2−6 over sub-micrometer particles7,8 up to microparticles.8−10 In general, they showed that smaller particle sizes offer better © XXXX American Chemical Society

Received: October 10, 2018 Accepted: December 7, 2018 Published: December 7, 2018 A

DOI: 10.1021/acsami.8b17703 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Representative SEM images of the synthesized PDMAEMA-modified core−shell particles with a core diameter of 100 nm (a), 200 nm (b), 400 nm (c), 800 nm (d), with platelet-like geometry (e), and 200 nm particles with varied polymer brush shell thickness of 1 nm (f), 4 nm (g), 7 nm (h), and 16 nm (i). Cryo-TEM image of a PDMAEMA-modified 200 nm particle (j). Cryo-TEM image of a PDMAEMA-modified plateletlike particle (k).

immobilized enzyme as well. Gopalan et al. presented polymer brush-based model systems composed of poly(acrylic acid) or poly(2-vinyl-4,4-dimethyl azlactone) and demonstrated, that the surface density of RNase increased linearly as the thickness of the brushes increased.20,21 The same tendency was found by Shang et al., who also confirmed that the amount of covalently immobilized glucose oxidase increased with the increase in thickness of the grafted poly(acrylic acid).22 Further examples for this correlation were shown by the groups of Mao23 and Gao,24 who immobilized trypsin and lysozyme onto poly(glycidyl methacrylate)-based brushes. To the best of our knowledge, there are no studies considering both the impact of particle core size and particle shell thickness of hybrid, “hairy” particle systems on the immobilization of enzymes. Herein, we report in detail how the design of hybrid polymer-modified particles will influence the effectiveness of immobilization, activity, and performance in dye decolorization of laccase from Trametes versicolor. Laccase is electrostatically adsorbed onto polyelectrolyte brushmodified silica- or kaolinite-based particles. The parameters that have been varied are the particle core size and shape (spherical and platelet-like), as well as the polymeric shell thickness. The immobilization capacity for laccase, the secondary structures of free and immobilized laccase via circular dichroism (CD), and Fourier-transform infrared spectroscopy (FTIR) studies are discussed. The best-performing laccase-carrier-system was finally used for the decolorization of Cibacron blue P 3R to demonstrate the convenient applicability of the system in textile industry and environmental technologies. Furthermore, we suggest the fast recovery of the laccase-loaded particles via directed aggregation and immediate sedimentation as a technological relevant separation method.

sub-micrometer range, which is also in focus of our studies presented herein. Particle size is of importance not only with respect to enzyme activity and loading capacity but also regarding enzyme selectivity, reusability, and reactor system configuration.16 Reduction of enantioselectivity or change of reaction selectivity can occur because of diffusion constraints in conjunction with the particle size.16 Thus, Hedenström et al. demonstrated that immobilization of Candida rugosa lipase onto smaller microscopic polypropylene carrier particles (Accurel) with smaller pore diameters gave higher enantiomeric ratios in esterification reactions.17 A decrease of the silica particle size may also cause higher maximum enzymatic reaction rates for immobilized lipase.18 Finally, the size of the silica support also affects the properties of the reaction product, as shown by He and co-workers for lipase-catalyzed ring-opening polymerization.10 Intermediate-sized particles offered highest activity and molecular weight of the obtained polymer. However, a too small particle size adjunctive with a too large surface area may not be a positive parameter. For instance, lipase molecules tend to maximize their contact area with the carrier surface which is hindered for very small particles with a high degree of curvature and which may lower the activity recovery of lipase.1 A similar tendency was found by Holmberg and co-workers who immobilized lipases onto mesoporous silica particles and showed that sub-micrometer-sized particles of 300 nm performed best with respect to the specific activity.7 Furthermore, separation and recovery of small carrier-enzyme systems are considerably hindered. Besides, a pressure drop in column reactors was reported for small particles.16 In general, smaller particles hamper their separation by filtration or centrifugation while large particles may cause loss of activity due to diffusion limitations. Thus, a compromise has to be found where particles are large enough to allow separation and small enough that diffusion limitations are prohibited.19 In the case of inorganic−organic (hybrid), “hairy” particles, the thickness of the polymer brush shell affects the amount of

2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of Hairy Hybrid Particles. In this work, we synthesized a series of hybrid hairy core−shell particles with various defined core diameters, B


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acid) (ABTS) as substrate. The immobilization process is strongly contingent on the pH of the medium, in which particles and enzyme are dispersed in, as recently shown in our own work.25 The maximum immobilization yield was achieved at pH 4.0, where PDMAEMA brushes are protonated and positively charged (ZP ≥ +30 mV, Figures S2 and S5) and laccase carries a negative net charge (ZP = −18 mV, Figure S8). To optimize the particle-based carrier system with respect to immobilization yield and activity found on the carrier, we varied the particle core size (from 100 to 800 nm in diameter) and the particle shape (spherical and platelet-like). In general, core−shell particles of all sizes and shapes could successfully adsorb laccase in considerable amount. As control study, we performed immobilization experiments of laccase onto unmodified (“native”) spherical and platelet-like particles. The activity yield in this control study was 6.1% for native spherical silica particles, and 2.9% for native platelet-like particles. However, the immobilization yield strongly depends on the initially employed enzyme concentration (Figure 2). The yield is comparatively low for very low enzyme concentrations. Increase of the enzyme concentration to 20−40 U/mL resulted in remarkably higher yields (up to 92% for 200PDMAEMA). A further increase of the enzyme concentration reduced the immobilization yield. This effect could be explained by two scenarios: first, the brush is completely saturated with laccase molecules and, thus, unable to adsorb even more; secondly, a very dense loading of the brush layer with enzyme molecules lowers their activity, for example, lack of sufficient substrate. Notably, the core diameter and geometry of the particles also influenced the yield of immobilization of laccase. The yield increased with decreasing particle size in the diameter range of 200−800 nm because of their increasing specific surface area (Figure 2). However, a further decrease of the particle diameter down to 100 nm did not further improve the yield (Figure 2, black curve). The best performance with a maximum yield up to 92% and also high yield for high initially employed enzyme concentrations was found for particles with a core diameter of 200 nm (Figure 2, red curve, polymer shell thickness: 11 nm). The platelet-like kaolinite-based particles (Figure 2, orange curve) showed almost as high yields as for 200-PDMAEMA spherical particles (91% vs 92%). This offers a good opportunity for a large-scale production of kaolinitebased particles in future because these are low-cost materials. Connected to the above-mentioned tendencies, activity found on particle and polymer also depends on the particle size and geometry. In analogy to the yield of immobilization, spherical 200-PDMAEMA particles showed the highest maximum activity on the carrier material of 5.7 kU/(g particle), whereas neither smaller nor larger particles could further improve this parameter (Figure 3, dark blue columns). Activity found on the polymer is also determined by the degree of curvature (Figure 3, light blue columns). A smaller particle size caused an increase of the maximum activity (Figure 3, light blue columns). This effect is presumably because of the stronger curvature of the particle surface which leads to a better accessibility of the polymer chain for the enzyme molecules. 2.3. Impact of Polymer Shell Thickness on Immobilization of Laccase. The properties of the hairy core−shell particles are also contingent upon the thickness of the polymeric shell which can be adjusted by the chain length or

shapes, and thicknesses of the polymer brush shell. To vary the core diameter of the particles, we prepared spherical silica particles with a diameter of 100−800 nm. Moreover, the core shape was variegated by using platelet-like, kaolinite-based particles. All kinds of particles were premodified with 3aminopropyltriethoxysilane (APTES) and afterward with 50 mol % of an ATRP initiator. Subsequently, the particles were modified with the polyelectrolyte poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) via ATRP obtaining core− shell particles with an inorganic core and a polymer brush shell with defined thickness. The grafting density of the polymer brushes was reduced down to 0.2 chains/nm2. Our previous studies showed that intermediate grafting densities (0.1−0.34 nm−2) provide best swelling and switching behavior and facilitate the diffusion of the enzyme into the polymeric brush layer.25 After grafting of the polymer, the hairy hybrid particles were characterized by scanning electron microscopy (SEM) (Figures 1 and 2), (cryo-)transmission electron microscopy

Figure 2. Immobilization yield of laccase from TvL onto spherical and platelet-like PDMAEMA-modified core−shell particles of varied sizes as a function of the employed enzyme concentration. The particles have a core diameter of 100 nm (black circles), 200 nm (red circles), 400 nm (green circles), 800 nm (blue circles), or are of platelet-like shape (orange circles). The core−shell particles were loaded with different concentrations of laccase. Immobilization yield was calculated according to eq 2 (see Experimental Section) employing the ABTS assay.

(TEM) (Figures 1 and S1), thermogravimetric analysis (TGA) (Figures S4 and S7), electrokinetics (electrophoresis, Figures S2, S5, S6, and S8), and dynamic light scattering (DLS) (Figures S3, S5, and S8). The synthesized core−shell particles exhibit a slightly rough surface morphology because of the successful modification with polymer brushes (Figure 1). The core−shell character with polymer brush morphology as well as the pHresponsiveness of the particle shell can be proved by cryoTEM measurements (Figures 1j and S1). The sizes of the final core−shell particles in aqueous medium (acetate buffer, pH 4.0, 10 mM) determined with DLS, as well as the polymer fractions, the corresponding shell thicknesses, and isoelectric points (IEPs) are summarized in Tables S1 and S2. 2.2. Impact of Particle Size and Geometry on Immobilization of Laccase. Next, laccase from T. versicolor (TvL) was adsorbed via electrostatic interactions onto the polyelectrolyte (PDMAEMA) brush layer of the hybrid particles. The activity of laccase was determined spectrophotometrically with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic C


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activity on particles compared with the values of the section we showed before result from the different polymer shell thicknesses of the particles used in the experiments: 11 nm in the particle size experiment versus 7 nm in the experiment analyzing the polymer shell thickness, see Tables S1 and S2). In contrast to that, particles with a 4 nm PDMAEMA shell provided the maximum activity found on polymer of 76.8 ± 9.7 kU/(g polymer) (Figure 4b, blue curve). This is probably due to complete swelling and stretching of these rather short polymer chains, which enables facilitated diffusion and adsorption of the laccase molecules down to deeper polymer regions close to the particle core. Thus, the polymer chain can be ideally loaded. Very low and very high polymer layer thicknesses did not improve yield and activity on the carrier. On the one hand, a thin polymer shell of 1 nm did not provide enough positively charged amino groups to sufficiently adsorb the laccase. On the other hand, the dispersibility of particles with a very thick polymer shell of 16 nm was very complicated. Because of entanglements of the long polymer chains, the distribution of the particles in the dispersion was insufficient. This led to hindered diffusion of laccase and adsorption to the particles and, thus, to a lower immobilization yield and activities on the carrier. Therefore, 200 nm large PDMAEMA-modified particles with intermediate polymer shell thicknesses of 4−7 nm were selected for continuing applications, that is, decolorization and separation experiments. 2.4. Identification of Laccase Uptake. In Figure S10a (see Supporting Information), full-range (4000−700 cm−1) attenuated total reflection (ATR)−FTIR spectra of unloaded and laccase-loaded PDMAEMA-modified silica particles are given for the particle diameters 100, 200, and 800 nm. Prominent IR bands at 1750 cm−1 prove the presence of PDMAEMA because of the ν(CO) mode (ester bond), those at 1650 and 1540 cm−1 indicate successful loading with laccase because of the amide I and amide II modes, and those at 1000−1200 cm−1 represent the silica core. In Figure S10b, difference attenuated total reflection fourier-transform infrared spectroscopy (ATR−FTIR) spectra in the amide band region (1800−1300 cm−1) between laccase-loaded and unloaded PDMAEMA-modified particles representing immobilized laccase are given. As shown in Figure S10b, the amide I band integrals were approximately similar for all laccase-loaded

Figure 3. Maximum activities on carrier (dark blue columns) and on polymer (light blue columns) loaded with laccase from TvL onto spherical and platelet-like PDMAEMA-modified core−shell particles. The particles have a core diameter of 100, 200, 400, and 800 nm, or are of platelet-like shape. Maximum activities on carrier material are defined as the maximum activity of laccase (determined with ABTS assay) per gram of particle or polymer (see eq 3 in the Experimental Section).

molecular weight of the polymer chains. To realize the variation of the polymer chain length, we altered the polymerization time between 5 and 25 min. Because of the linear correlation between polymerization time and polymer shell thickness, we were able to create polymer shells of 1, 4, 7, and 16 nm onto silica particles with a diameter of 200 nm (Figure 1f−i, Table S2). These values refer to the thickness of the grafted polymer layer in dry state and were determined from TGA results (see the Experimental Section). We assumed that the thickness of the polymer shell impacts the immobilization of enzymes with regard to immobilization yield and activity on the carrier material. Indeed, we found that immobilization yield (Figure 4a), maximum activities on particle, and polymer (Figure 4b) were strongly affected by the thickness of the polymer shell. The highest immobilization yield of 83% (Figure 4a) and activity found on carrier material of 3.4 kU/(g particle) (Figure 4b, black curve) could be achieved for particles with 7 nm thick shells. This polymer shell thickness exhibited the best ability to efficiently immobilize laccase from T. versicolor, which also led to high activity on particles (the lower absolute values of immobilization yield and

Figure 4. (a) Immobilization yield and (b) maximum activity on particle and polymer loaded with laccase from TvL onto 200 nm large PDMAEMA-modified core−shell particles. Immobilization procedure was carried out with an initial enzymatic activity of 40 U/mL. The particles have a polymer shell thickness of 1, 4, 7, and 16 nm. Maximum activities on carrier material are defined as the maximum activity of laccase (determined with ABTS assay) per gram of particle or polymer (see eq 3 in the Experimental Section). D


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convenient benchmark system for the implementation in the textile industry. In a typical procedure, 5 mg of PDMAEMAmodified particles (core diameter: 200 nm, shell thickness: 7 nm) loaded with laccase (2 U/[mg particles]) were applied to 1 mL of 0.02% Cibacron blue solution in presence of violuric acid (20 mM) as mediator. In preliminary experiments, adsorption of the dye onto the carrier particles could be observed which requested the determination of a blank value. For this, three types of particles unable to decolorize Cibacron blue were used for adsorption experiments: particles without any enzymes; particles with immobilized but inactive laccase (denaturation of laccase at 95 °C for 10 min); and particles with immobilized amylase. All three kinds of particles, which were used to determine the blank value, exhibited significant adsorption of the dye Cibacron blue up to the 8th cycle. Beginning from the 9th cycle, PDMAEMA-modified particles are saturated with dye assuming that subsequent decolorization will now be exclusively caused by laccase-induced oxidation of Cibacron blue. The use of 200 nm-PDMAEMA particles with immobilized, active laccase from T. versicolor enabled 13-fold decolorization of Cibacron blue P-3R adopting 10 min reaction time for each cycle. According to the pre-experiments, decolorization of the solution is partly determined by adsorption to the particles up to the 8th time and only by laccase-induced oxidation beginning from the 9th up to the 13th cycle. Figure 6 shows that laccase is involved to some extent in decolorization of Cibacron blue from the beginning because of observed changes in the absorption spectra. Furthermore, extension of the reaction time from 10 to 30 min led to improved results with 18-fold decolorization of Cibacron-blue (including eight cycles of decolorization caused by adsorption of the dye to the particles). 2.7. Separation via Aggregation. Finally, the carrier materials should be easily and fast separable from the reaction solution for technical feasibility. In our approach, we propose the targeted flocculation of the core−shell particles for separation induced by the addition of citric acid buffer at a defined pH. The addition of citric acid buffer at pH values between 3 and 5 did not lead to any observable effect on the particle suspensions (reference sample in Figure 7). However, the particles aggregated and sedimented rapidly in citrate buffer at pH 6.4 (Figure 7), which correlates exactly with the acid constant pKa,3 of citric acid. At this pH, half of the citric acid is completely dissociated into three-valent citrate anions, while PDMAEMA is still strongly positively charged. Because of electrostatic interactions, the citrate ions are adsorbed onto the PDMAEMA brushes leading to a loss of their positive charge. Caused by this neutralization process, the PDMAEMA brushes collapse, which triggers cohesion between the particles and their flocculation within several minutes. This way, the fast recovery of the laccase-loaded particles via directed aggregation and immediate sedimentation as a technological relevant separation method was enabled. To ensure that laccase retains its activity and is not dissociated from the particles due to the conditions used for flocculation, we determined the activity of laccase immobilized onto the particles after incubation in citrate buffer at pH 6.4. Indeed, we found that 87% of the initial enzymatic activity could be preserved.

particle samples (100, 200, 800 nm) evidencing similar protein contents. The peak maximum of the amide I band, whose position and shape is related to protein conformation,26 was around 1643 cm−1 for all three samples which is a qualitative hint for no significant differences in the secondary structure of laccase. Moreover, the amide band region is commonly used for the quantitative determination of secondary structure fractions (α-helix, β-sheet, turn, random coil) applying dedicated quantitative techniques based on Fourier-selfdeconvolution, band fitting by multiple (Gaussian/Lorentzian) components, and/or second derivative processing, which is compiled therein.26 However, in our case, caution should be taken because a spectral shoulder at around 1610 cm−1 showed up in the ATR−FTIR spectra (Figure S10a) for both laccaseloaded and unloaded particles. This effect could not be sufficiently compensated in the difference spectrum and therefore could be misleading. Hence, CD spectroscopy was used, which is given in the following. 2.5. Secondary Structure of Free and Immobilized Laccase. To get deeper insights into conformational states of laccase, CD spectra of free laccase and laccase immobilized onto PDMAEMA-modified particles with core diameters of 100, 200, and 800 nm were recorded (Figure S11), which were analyzed for their secondary structure composition as described in the Experimental Section. Generally, free laccase and immobilized laccase resulted in very similar secondary structure compositions of around 5/40/25/30% with respect to α-helix/β-sheet/turn/random coil (Figure 5), considering

Figure 5. Fractions of α-helix, β-sheet, turn and random protein structures in free and immobilized laccase from T. versicolor determined with CD measurements.

errors of single secondary structure portions lower than 5%. In detail, the α-helical content of pure and immobilized laccase was quite low with around 5%. Low α-helical contents of 11% for laccase from T. versicolor are also known from ref 27, as well as contents of 5% for Rhus vernicifera laccase by Filippi et al.28 For the β-sheet content, conflicting data are available. While Filippi found β-sheet contents up to 21% for laccase from T. versicolor,28 ref 27 and Cambria29 report on β-sheet contents of 37 and 45%, respectively, which both support our results. Conclusively, the CD signature of free and immobilized laccase onto PDMAEMA-modified particles is similar and no significant changes on the secondary structure level of laccase were obtained. 2.6. Decolorization of Dyes. To investigate the applicability of the immobilized laccase, the decolorization of Cibacron blue P-3R (Reactive Blue 49) was applied as a E


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Figure 6. Optical absorption spectrum of Cibacron blue P-3R before and after decolorization cycle 1−18 with (a) inactive, immobilized laccase from T. versicolor; and (b) active, immobilized laccase from T. versicolor. Insets show photographs of Cibacron blue solutions before (i) and after (ii) decolorization.

Figure 7. (a) Schematic illustration of the citrate-induced flocculation of PDMAEMA-particles at pH 6.4. (b) Representative photographs of the flocculation process of PDMAEMA-modified core−shell particles. After adjusting the citric acid buffer to pH 6.4, the particles aggregate and settle within 10 min.

3. CONCLUSIONS Herein, we demonstrated the important role of core−shell particle design with respect to particle size, shape, and polymer shell thickness for the efficient immobilization of laccase from T. versicolor, as well as the reusability of the system. For this, hybrid PDMAEMA-decorated particles with spherical geometries (core diameters between 100 and 800 nm) as well as platelet-like geometries were synthesized and thoroughly tested according the immobilization yield and activity found on the carrier. We have discovered that spherical particles with a core diameter of 200 nm showed the best performance with an outstanding immobilization yield of 92% and activity of 5.7 kU/(g particle). The platelet-like particles also offer a feasible, competitive system for large-scale applications because of the cost-efficient core material and good immobilization results. Moreover, we demonstrated that intermediate shell thicknesses ranging between 4 and 7 nm provide highest immobilization yields and activities on the carrier particles. The detailed CD measurements proved that the secondary structure of the enzyme is not impaired by immobilization onto all kinds of carrier particles. Finally, the immobilized laccase was used for decolorization of Cibacron blue P-3R and

reused up to 13 times; 18 cycles are realizable when extending the reaction time for decolorization. The particles were easily separated via citrate-induced flocculation in technologically relevant spaces of time. This comprehensive study contributes to the understanding of catalytically active hybrid materials as rational design tools and their performance in textile industry and environmental technologies such as water purification.

4. EXPERIMENTAL SECTION 4.1. Materials. Kaolinite (Sigma-Aldrich, natural, 22.3 ± 0.1 m2/ g), ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich, 99+%), sodium citrate dihydrate (Aldrich, 99%), sodium bicarbonate (SigmaAldrich, 99.7%), sodium dithionite (Sigma-Aldrich, 85%), tetraethylorthosilicate (TEOS, Fluka, 99%), ammonia solution (NH4OH, Acros, 28−30% solution), hydrogen peroxide (H2O2, VWR, 30%), ethanol abs. (EtOH, VWR, 99.9%), APTES (ABCR, 97%), αbromoisobutyryl bromide (BrIn, Aldrich, 98%), propionyl bromide (Aldrich, 97%), anhydrous dichloromethane (Fluka), triethylamine (Fluka), copper(II) bromide (CuBr2, Aldrich, 99.999%), tin(II) 2ethylhexanoate (Aldrich, 95%), tris(2-pyridylmethyl)amine (TPMA, Aldrich, 98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (Aldrich, 99%), ethyl α-bromoisobutyrate (EBiB, Aldrich, 98%), dichloromethane (Acros, 99.99%), 2,2′-azino-bis(3-ethylbenzthiazoF


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secondary structure portions were based on standard deviations of analysis results on three independent measurements. 4.9. Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy. ATR−FTIR spectroscopy was used to characterize unloaded and laccase-loaded PDMAEMA-modified silica particles and to identify enzyme loading similarly to earlier analytical work on unloaded and laccase-loaded poly(ethyleneimine)/poly(maleic acidco-propylene) complex nanoparticles.36 ATR−FTIR spectra of unmodified and laccase-modified hybrid PDMAEMA-silica particles were measured on a Tensor 27 FTIR spectrometer (Bruker Optics GmbH, Ettlingen, Germany) equipped with a dedicated ATR attachment (Optispec, Neerach, Switzerland). A spectral resolution of 2 cm−1 coadding and averaging 100 scans per sample was applied. Reference single channel intensity spectra IR were recorded from a germanium internal reflection element (Ge-IRE, 50 × 20 × 2 mm) cleaned by a low pressure Plasma Cleaner (Harrick, Ossining USA) and sample single channel intensity spectra IS were recorded from the Ge IRE coated by unloaded and laccase-loaded Si/PDMAEMA nanoparticles, which were casted from their original dispersions and dried (vacuum oven, 37 °C). ATR−FTIR spectra were obtained according to A = −log(IS/IR). FTIR measurements and data were processed using OPUS Software (OPUS 7.0, Bruker GmbH, Ettlingen, Germany). 4.10. Synthesis and Modification of Monodisperse SiO2 Particles. Silica particles (100−800 nm) were synthesized using a multistep hydrolysis−condensation procedure of TEOS in ammonium hydroxide−ethanol solution based on the Stöber approach and described in refs 30 and 31. TEOS was added sequentially into a mixture of ethanol and ammonia solution. The particles produced within one step were used as seeds for the next step. Each reaction was carried out by stirring the mixture at 500 rpm overnight at room temperature (starting from the last addition of TEOS). Subsequently, the particles of the desired size were separated from the solvent by centrifugation yielding monodisperse silica particles. The purified particles were dried in a vacuum oven under reduced pressure at 60 °C. The specific surface area for particles with a diameter of 200 nm was determined to be 18.7 ± 0.3 m2/g. Kaolinite particles were separated by sedimentation in deionized (DI) water for 2 days. Large particles (100 nm to 2 μm) are separated using grain size separation (Atterberg method). After the sample was poured into a sedimentation cylinder, DI water was added up to the desired settling height. The closed cylinder was shaken until the suspension was homogeneous. When the necessary settling time for a given equivalent diameter (e.g., 2 μm) was reached (calculated according to Stokes law), the supernatant suspension (e.g., only material

Hairy Particles with Immobilized Enzymes: Impact of Particle Topology

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