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Soft, Elastic Macroporous Monolith by Templating High Internal Phase Emulsions with Aminoclay: Preparation, Pore Structure and Use for Enzyme Immobilization Anees Khan, and Guruswamy Kumaraswamy ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00622 • Publication Date (Web): 13 Jun 2018 Downloaded from http://pubs.acs.org on June 14, 2018

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ACS Applied Nano Materials

Soft, Elastic Macroporous Monolith by Templating High Internal Phase Emulsions with Aminoclay: Preparation, Pore Structure and Use for Enzyme Immobilization

Anees Y. Khan‡ and Guruswamy Kumaraswamy*

J-101, Polymers and Advanced Materials Laboratory, Complex Fluids and Polymer Engineering, Polymer Science and Engineering Division, CSIR-National Chemical Laboratory, Pune 411008, Maharashtra, India

‡

Present address

Department of Chemical Engineering, Faculty Block 3, 1 AB, Manipal University Jaipur, Off Jaipur-Ajmer Expressway, Post: Dehmi Kalan, Jaipur 303007, Rajasthan, India.

*

Corresponding author

Phone: +91 20 2590 2182 Fax: +91 20 2590 2618 E-mail: [email protected] 1

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Abstract We describe the preparation of macroporous monolithic structures by templating high internal phase emulsion with plate-like aminoclay nanoparticles. We demonstrate that choice of surfactant affords control over pore structure within the monolith. Scanning electron microscopy shows that using anionic surfactant (sodium dodecyl sulfate) leads to the formation of closed pores that template oil droplets in the emulsion, while cationic surfactant (cetyltrimethyl ammonium bromide) results in the formation of a network-like structure that does not directly replicate the oil droplets. Of particular interest are monoliths prepared using non-ionic surfactant (Pluronic F127) – these result in the formation of an interconnected open pore monolith, which is soft, and exhibit remarkable elasticity: they can recover from compressive strains as large as 80%. As a consequence of this, these monoliths are robust and do not crack on air drying at room temperature despite experiencing large (≈ 45%) volume shrinkage. We intercalate glucose oxidase enzyme in the aminoclay and use these constructs to prepare monoliths with an interconnected porous structure. We demonstrate monolith porosity can be tuned by increasing the oil volume fraction. Increasing the oil fraction in the emulsion from 74 to 82.6%, increased the monolith porosity from 72.5 to 84.4%, resulting in an increase in enzyme activity. Enzymes encapsulated in the monoliths are stable against chaotropic solvents and changes in pH.

Keywords: High internal phase emulsion, macroporous materials, enzyme immobilization, enzyme activity, aminoclay, monoliths.

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Introduction High-internal-phase emulsion (HIPE) refers to an emulsion with a high volume fraction (74% or higher) of the dispersed phase with respect to the dispersion medium.1,2 HIPEs are highly viscous, due to the dense packing of droplets at these high volume fractions. As the dispersed phase is surrounded by a thin film of the dispersion medium, these can be used as a template to synthesize porous materials.3 Both oil-in-water (o/w) and waterin-oil (w/o) HIPEs have been effectively used to prepare macroporous materials.3,4 The advantages of preparing macroporous monoliths by templating HIPEs include ease of fabrication and scalability, as well as the ability to access high porosity while independently tuning pore size across a wide range. 2,5,6,7 Due to their versatility, such materials find a wide range of applications.8 Commercially, HIPE based porous materials have found use in personal care hygiene products and as supports for chromatography.9,10 Such materials have also been explored as hosts for enzyme immobilization, typically using a multi-step preparation protocol where the macroporous materials are first prepared and then the enzyme is adsorbed on their surface.11 However, such materials suffer from the possibility of leaching of adsorbed enzyme. Therefore, covalent coupling of the enzyme onto macroporous monolith walls has been attempted to reduce leaching.12

Conventionally, polymerization in a HIPE template is used to prepare macroporous monoliths. The species required for the preparation of such monoliths, referred to as poly HIPEs, include monomer and co-monomers, an initiator (for the polymerization reaction) and a surfactant.3,4,5,13,14,15 Typically, a large amount of surfactant (5-50% concentration with respect to the continuous phase) is required to stabilize the HIPE.3 The resultant macroporous materials have either closed,16 partially open or interconnected pores.3 In HIPE based macroporous materials, control over pore architecture is important to tailor the monoliths for 3



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specific applications. For example, a closed pore structure is optimal for thermal insulating materials.17,18 On the other hand, interconnected porous structures are necessary for applications such as absorption and separation.19 Recently, solid particles have gained interest as emulsifiers to stabilize HIPEs.5,20,21,22 Both organic and inorganic particles have been used to stabilize emulsions.23,24 There are also reports of the use of surface active proteins or biopolymers that have been crosslinked to make a polyHIPE structure through emulsion templating.25,26 Emulsions stabilized using particles are referred to as Pickering emulsions. In some cases, functionalization of these particles is needed to modify their amphiphilicity to render them surface active (e.g. functionalized silica and surface-active polymer colloids).21,27 In most cases where particulate stabilizers were used, the resultant macroporous materials had closed pores.22,28,29 Interconnected macroporous materials could be prepared only when surfactant molecules were used in addition to solid particles in stabilization of the HIPE.28 Thus, it is not possible to control pore architecture by templating Pickering emulsions, stabilized purely using particles. Researchers have also demonstrated the synthesis of macroporous monoliths from particle stabilized emulsions, by sintering the particles30,31 and very recently with depletion attraction.32 Typically, poly HIPEs require the use of monomers and initiator as well as additional agents (such as salts, acid, etc) and high energy inputs (e.g. high speed homogenization), special conditions for reaction (for monolithic structure formation) and drying the scaffolds. Therefore, it is attractive to develop a simple route to HIPE-templated macroporous materials with tailorable pore morphologies and using less energy intensive synthesis.

In the present work, we demonstrate for the first time, the facile synthesis of macroporous monoliths templated with solid aminoclay nanoparticles. We can adjust pore connectivity in such monoliths simply by varying the surfactant used for stabilization of the 4


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HIPE. These structures mimic poly HIPEs in terms of pore morphologies – however, in contrast to traditional poly HIPEs, preparation of these monoliths does not rely on conventional polymerization reactions or high temperature sintering. The use of functional aminoclay nanoparticles33,34 render the monoliths versatile. Monoliths prepared by crosslinking these nanoparticles are soft and demonstrate unprecedented mechanical elasticity. We demonstrate that they can recover from large compressive deformation and do not fail when air dried, despite experiencing volume shrinkage. Further, we note that a one pot protocol can be employed to prepare macroporous HIPE monoliths by crosslinking aminoclay stabilized enzymes, that render the enzyme robust to pH and chaotropic solvents. This route compares favourably with conventional two-step protocols11 for the preparation of enzyme immobilized monoliths. Our protocol affords leaching as low as ≈5%, as compared with up to 70% leaching observed35 when using currently practiced or popular strategies based on mesoporous inorganic materials.36 Finally, we note that process intensification efforts in the biotechnology industry have prompted a move from batch to continuous processes.37 Enzymes immobilized in mesoporous inorganic particles require a separation step and are incompatible with continuous processes. The macroporous nature of the monolith that we describe allows easy access of the substrate to the encapsulated enzyme, and offers the possibility of low pressure drops,38 important for continuous flow processing.

Experimental Materials and methods

3-Aminopropyltriethoxysilane (APTES, Sigma), magnesium chloride hexahydrate (Thomas Baker) and ethanol (Merck) were used for aminoclay (AC) synthesis. Toluene (Merck), Pluronic F127 (Sigma–Aldrich), sodium dodecyl sulphate (SDS, Sigma-Aldrich), 5



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cetyltrimethyl ammonium bromide (CTAB, Sigma-Aldrich) and isopropyl alcohol (IPA) were used as received. 1, 4 butanediol diglycidylether (CL, Sigma) was used as a crosslinker to crosslink the aminoclay particles. Glucose oxidase (GOx, EC number 1.1.3.4) from Aspergillus niger and horseradish peroxidase (HRP, EC number 1.11.1.7) were purchased from Sigma-Aldrich. Buffer solutions of pH 2, 4, 7 and 10 were purchased from SigmaAldrich. Phenol (Merck), dye 4-aminoantipyrine (AAP, Thomas Baker), and d-dextrose (Thomas Baker) were used for GOx enzyme activity assay.

Synthesis of aminoclay (AC) AC was prepared using protocols reported in literature.33,34 Briefly, 840 mg of magnesium chloride hexahydrate was dissolved under magnetic stirring in 20 g ethanol. A clear solution thus obtained becomes turbid when APTES is added (1.3 ml) drop-wise. The reaction is continued for 24 h. Thereafter, the reaction mixture is centrifuged at 8000 rpm for 3 min. A clear supernatant is separated. The bottom product contains AC particles and is washed two times with ethanol and dried at 40o C. Formation of layered aminoclay nanoparticles was confirmed using XRD (SI, Figure S1).

Enzyme adsorption on AC

To immobilize enzyme, aminoclay particles were exfoliated by sonication. Exfoliation relates to the delamination of the sheet-like aminoclay particles in aqueous dispersions. The as-prepared sample of aminoclay in dry powder form shows a stacked lamellar structure (SI, Figure S1). When dispersed in a polar solvent (like water), protonation of amine groups takes place, which results in repulsion between the charged clay sheets and delamination (or exfoliation) of the clay layers takes place, resulting in a clear dispersion.34 This property is essential for using aminoclay for immobilizing enzyme and making 6


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monoliths. On the other hand, the addition of a low polarity solvent (relative to water, like ethanol) to such aqueous dispersion of aminoclay, deprotonates the amine groups, which results in the stacking of clay layers. This helps in drying the aminoclay samples and its storage.

We prepare a stock solution of AC particles by dispersing 400 mg AC in 1 ml water. The AC was exfoliated by sonication.34 Then a measured aliquot of AC from its stock solution was taken in an eppendorf tube to which a known aliquot of GOx (5 mg GOx per ml water) was added. The contents were mixed by vortexing and stored for 1 h, to allow adsorption of the enzyme on the AC nanosheets. As the solution remains transparent, the unadsorbed enzyme could not be separated until the solution pH was changed to 6. This results in the suspension turning cloudy due to precipitation of aminoclay/enzyme complexes. The suspension is centrifuged at 8000 rpm for 3 min. to separate the AC/GOx from unadsorbed free enzyme. Supernatant was analyzed for enzyme activity and the amount of unadsorbed enzyme was estimated by using the reaction rate of free enzyme and enzyme concentration calibration plot (SI, Figure S2).

Emulsion preparation

Stock solutions of surfactants (100 mg/ml, SDS, F127, CTAB) and AC (400 mg/ml) were made in DI water, while that of GOx (5 mg/ml) was made in pH 7 phosphate buffer. Aliquots of AC and GOx were mixed together and kept on a shaker for 2 h at room temperature to allow adsorption of GOx on AC. Then an aliquot of oil was added followed by an aliquot of surfactant. The contents were vortexed for 1 min. in order to emulsify the oil and obtain an oil-in-water (o/w) emulsion. Finally, CL was added to the emulsion in order to crosslink the amine groups present on AC. No destabilization of the emulsion was observed 7



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for more than 24 h, with or without CL. In all experiments, viz. using different surfactants (F127 or CTAB or SDS) and at different oil loadings in the emulsions (corresponding to different porosities, details in SI, Table S3), we used the same mass ratio of aminoclay to crosslinker (butanediol diglycidylether) = 1.2:1. This was optimized so that it forms a selfstanding scaffold on crosslinking in the aqueous phase. Finally, the scaffolds were washed with water and 20% IPA to remove unreacted CL, surfactant and oil. We note that the aminoclays by themselves are not surface active and cannot stabilize the oil/water emulsion. Preparation of emulsions using only aminoclays without surfactant resulted in unstable emulsions where oil and water phases separated within about 5 minutes after vortexing (see details in section S5 of SI). In order to estimate the porosity of these scaffolds, we measured the dimensions and masses of dry scaffolds as well as scaffolds saturated with water. Since the monoliths are highly hydrophilic in character, they readily absorb water (and expand in volume) when soaked in water. Typically, the scaffold expands on soaking in water over about 10 minutes. To ensure complete expansion, we immersed the scaffolds in water for at least 2 hours before measuring their sizes. The volume of the absorbed water is a measure of the pore volume and is obtained by measuring the weight of the water required to saturate the scaffold. Normalizing by the measured volume of the dry scaffold yields the porosity.

Estimation of droplet diameter, pore diameter and interconnection pore diameter

Optical microscopic images of the emulsion stabilized by different surfactants were taken and droplet diameters were measured using open source ImageJ software. Similarly, pore diameter and interconnection pore diameter were measured from analysis of a large number of SEM images using ImageJ software. In all the cases, multiple images were analysed with more than 150 pores being measured. 8


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Characterization Compression/expansion experiments were performed using a strain-controlled rheometer, TA-ARES G2, equipped with a normal force transducer. We use a cylindrical sample and make measurements on the sample in the wet state. We use 8.4 mm roughened aluminium plates and place the sample in a pool of water, on the bottom fixture to prevent drying. A compression rate of 0.1 mm/s was employed and the sample was subjected to a normal force of 0.5 g at the beginning of the experiment. The morphologies of monoliths were imaged using a Quanta 200 3D scanning electron microscope. Wide-angle X-ray diffraction (WAXD) was performed on a Rigaku Micromax system. The data were collected using an image plate system. Optical microscope (Nikon Eclipse E600 POL, upright) was used to image emulsion stabilized by different surfactants. ImageJ software was used to analyse the size distribution of the emulsion droplets, pore sizes and interconnected pore sizes of the monoliths. UV-Visible spectrophotomter (Perkin Elmer, lambda 950) was used for enzymatic assay. A digital Vernier calliper (Mitutoyo Absolute AOS digimatic) was used to measure the dimensions of monoliths.

Enzyme activity and stability test GOx enzyme activity was assayed based on the protocol reported in literature.39,40 The stability of enzyme against pH was measured by dipping enzyme loaded scaffolds in 0.2 M glucose solutions with pH values of 2, 4, 7 and 10 and reacting for 10 min. Afterwards, an aliquot from the reaction mixture is taken and added to a dye solution which contains 4-AAP, Phenol and HRP, such that the colour changes to pink. The absorbance is then measured at 510 nm spectrophotometrically.

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The stability of enzyme against chaotropic solvent was measured by dipping enzyme loaded scaffolds in solvents such as 5 M urea and 4 M guanidine chloride for 2 h. Similarly, stability against ethanol was measured after dipping in with 100% ethanol for 15 min. Stability against pH change was measured by dipping these scaffolds in different pH solutions for 2 h. Thermal stability was measured by dipping the scaffolds in pH 7 buffer and keeping them in an oven for 2 h at various temperatures. In all cases, the scaffolds were washed with DI water before activity measurement. Free GOx was also exposed to such pH solutions, chaotropic solutions and temperature variations under the same conditions. The activity of the immobilized enzyme has been reported in terms of specific activity, relative activity and residual activity. Specific activity is defined as micromoles of glucose reacted per unit time per mg of enzyme. Relative activity is a ratio of specific activity of immobilized enzyme with that of free enzyme. Residual activity is a ratio of specific activity of immobilized enzyme at a particular condition to that of a reference condition. For example, residual activity of immobilized enzyme at pH 2 is normalized by that of at pH 7 (max. activity).

Results and discussion Aminoclay based macroporous monoliths with controlled pore microstructures

We start with synthesis of aminoclay, which is obtained by reaction between magnesium chloride hexahydrate and APTES in ethanol. This reaction is carried out at room temperature and results in the formation of a crystalline layered material that comprises a central dioctahedral magnesium oxide layer sandwiched between tetrahedral silicate layers (Figure 1a). WAXD confirms the stacking between these trilayer sheets (d001 = 1.33 nm) and provides evidence for the in-plane order in the individual sheets (SI, Figure S1). 10


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Figure 1. (a) Schematic of the aminoclay structure; (b) SEM image of exfoliated aminoclay particles; (c) schematic of emulsion preparation.

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These materials are organo-phyllosilicates,33,41 that are analogous to natural 2:1 magnesium silicate clays. In these, each silicon atom is bonded to a propyl amine group. Figure 1b shows an SEM image of the exfoliated aminoclay particles, which shows that they are irregular plate-like structures characterized by an average lateral dimension of ~ 60 nm. The AC particles thus obtained are washed and dried before dispersing them in water phase for emulsion. Next, we examine the effect of using different surfactant emulsifiers on the structure of HIPE-templated monoliths. We prepare monoliths in 2 ml eppendorf tubes. To dispersions of exfoliated AC particles in water, we add either SDS or CTAB or F127 surfactant (structural formula in Figure 1a). Oil was added drop-wise and the tube was vortexed continuously, till addition of the required amount of oil was complete. Thus, the exfoliated aminoclay dispersion in water forms a continuous phase, stabilizing the oil-in-water emulsion. All three surfactants gave stable emulsions as confirmed by optical microscopic images (SI, Figure S3 and S4). Surfactants are necessary to stabilize the emulsions. When we add oil to aqueous AC suspensions, stable emulsions cannot be formed, suggesting that the AC particles are not themselves surface active. Thus, the emulsion comprises oil droplets stabilized by surfactant, and surrounded by thin film of water containing AC particles with crosslinkable amine groups. Finally, crosslinker (CL, Figure 1c) was added to the emulsions while vortexing, to crosslink amine groups on the aminoclay particles, as shown schematically in Figure 1c. Crosslinking takes place over 24 hours at 40o C to afford a macroporous monolithic structure.

Figure 2a shows the SEM image of SDS stabilized emulsion templated macroporous monolith. We observe that the pores formed are polydisperse, and are several tens of microns in diameter. All the pores appear as closed pores, viz. there are no interconnections between 12


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the void spaces of neighbouring pores. SDS is an anionic surfactant and we anticipate that it localizes to the oil drop/water matrix interface in the emulsion. The surfactant tail is solvated in the oil while the head group dissociates in the aqueous phase to form negatively charged sulphonate group. It is likely that the presence of AC results in deposition on the negatively charged SDS head group to form a compact layer. This is the likely reason for the formation of the closed pore architecture observed when the AC moieties are subsequently crosslinked in the aqueous phase.

Figure 2. SEM images of the macroporous material obtained by surfactant such as (a) SDS (b) F127 and (c) CTAB. Scale bar is 50 µm for (a)-(c).

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In contrast, Figure 2b shows that F127 stabilized emulsion results in an interconnected porous network, where neighbouring pores are connected through channels in the pore walls. Here, non-ionic surfactant F127 stabilizes the o/w emulsion, while AC is in the continuous water phase. The oil/water emulsion comprises densely packed oil droplets stabilized by F127. While there is a strong attractive interaction between exfoliated AC nanoplates with the SDS anionic head group, we do not expect similar interactions between the AC and F127. Therefore, when AC is crosslinked, we anticipate that there is no localization of AC at the thin boundaries between oil drops. During crosslinking, a thin crosslinked AC layer forms at oil drop/oil drop boundaries, that develops into pores to relieve mechanical stresses during drying of the monolith (Video of the monolith drying process, in the SI pore formation video VS1 shows the formation of connections between pores). Thus, the resulting monolith is characterized by an interconnected pore structure, with large macropores with a mean diameter of ≈ 38 ± 5.9 µm (SI, Figure S5), connected by holes in the pore walls. These interconnecting holes have an average size of 8.8 ± 1.1 µm (SI Section S4, Figure S5). We reiterate that the monoliths prepared are macroporous, and we observe no evidence for microporosity in these. Macropores result from templating oil droplets. The macropore sizes are very similar to the drop diameter in the emulsions. We measure a mean droplet diameter in the F127 stabilized emulsion = 28.7 ± 6.3 µm. The droplet distribution data from imaging the emulsions (SI, Figure S4 and Table S1) show the presence of smaller size oil drops. Corresponding small pores are not observed in the final monolith, suggesting that these smaller droplets might coarsen, possibly by Ostwald ripening or coalescence, during the process of pore formation while drying (also see pore formation video VS1 in SI). We note that even doubling the surfactant concentration does not significantly change the size distribution of emulsion droplets. Therefore, we believe that the droplet size distribution (and, therefore, the macropore size in the final monolith) is primarily determined by the 14


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vortexing protocol to prepare the emulsion rather than surfactant concentration. We attempted to prepare emulsions using a homogenizer (Ultraturrax, IKA). This resulted in the formation of emulsions with significantly smaller oil droplet size distribution. However, the amount of surfactant added was insufficient to stabilize the emulsion, for the added volume of oil. This influences the scaffold porosity and pore size. Since we were interested in highly porous monoliths for enzyme immobilization, the use of a homogenizer was unsuitable for this application (details given in section S11 of SI).

Finally, we examine monolith formation from templating emulsions stabilized by cationic surfactant (CTAB). We observe that here, emulsion templating results in the formation of a network like structure, viz. the emulsified oil droplet structure is not replicated in the final monolith. We note that in this case too, CTAB is able to stabilize the emulsion droplets, with the cetyl tail solvated in the oil droplets with the positively charged head group disposed to the aqueous phase. Positively charged droplet surfaces repel positively charged AC particles, and it is possible that the AC nanoplates are excluded from the thinnest regions separating the oil droplets. Therefore, on crosslinking, AC particles are unable to retain the emulsion structure and phase separate to form monoliths with network-like internal structure. Hence, the choice of surfactant used to stabilize the oil droplets in the emulsion determines connectivity in the porous structure of the monoliths. Details of the emulsion compositions used for obtaining macroporous scaffolds with different porous structures have been given in Table S2 of SI. In AC particles, each silicon atom is linked to a propylamine group. Therefore, when ACs exfoliate in water, their surface is characterized by a high density of positively charged protonated amine groups.33 AC particles exfoliated in water have been employed in the literature, to complex with negatively charged biomolecules (such as enzymes, DNA, lipid 15



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etc.) and stabilize them even in harsh environments (such as when pH and temperature are varied above/below physiological conditions).34 However, separating AC/biomolecule complexes from their aqueous suspensions is challenging. Thus, to reuse the stabilized biomolecules, one needs to employ processes that are difficult to scale, such as centrifugation. To avoid the use of centrifugation for separation and reuse of AC/biomolecule complexes, we explore the assembly of the AC/biomolecule hybrids into macroporous, monolithic structures. There are fewer transport limitations for the flow of substrates into, and products out of macroporous monoliths (relative to microporous materials). Further, we anticipate that assembling the stabilized biomolecules into a macroporous monolith, by crosslinking the amine groups on the AC particle surface will not adversely affect the activity of the encapsulated enzyme. Finally, using such a monolith will eliminate the need for complicated separation processes between cycles.

Enzyme immobilized macroporous monoliths Figure 3a shows a schematic of exfoliated AC particles exposed to aqueous solution of enzyme. As the isoelectric point of GOx is 4.2, it is negatively charged in the pH 7 phosphate buffer and gets electrostatically adsorbed onto positively charged AC particles. We estimate the enzyme immobilization efficiency by measuring the activity of free, unimmobilized enzyme in solution after separation of AC/GOx complex by centrifugation. Figure 3b shows that enzyme immobilization efficiency, measured in this manner, is ~99%. There is a very small amount of enzyme (< 5%) that is leached from the AC-GOx complexes after 24 h and no leaching thereafter observed till 48 h (SI, Figure S7).

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(a)

120

(b)

100 Immobilization efficiency (%)

80 0h 24 h 48 h

60 40 20 0 0.02

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Glucose oxidase (mg/ml) 30

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Relative activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20

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0 SDS

F127

C TAB

S u rfa c ta n t

Figure 3. Enzyme immobilization on AC particles and relative activity in macroporous monoliths (a) Enzyme immobilization (b) Enzyme immobilization efficiency. (c) Relative activity of GOx in scaffolds prepared with different surfactants.

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In order to test the activity of the immobilized enzyme, a control experiment was performed with the free enzyme alone. We found the specific activity of the free enzyme to be 43.9 ± 0.5 µmol min-1 (mg of enzyme)-1, which decreased to 35.7 ± 3.4 µmol min-1 (mg of enzyme)-1for the aminoclay-enzyme complex. Therefore, the activity of the immobilized enzyme relative to the free enzyme is 81.3%. This suggests that aminoclay does not cause any significant change to the native structure of the immobilized enzyme. However, when the enzyme immobilized aminoclay particles are incorporated into macroporous monoliths, the relative activity of the enzyme decreases to ~30-50% of the free enzyme value. Here, the enzyme activity is a function of pore wall thickness and porosity of the monoliths and will be discussed in detail later in the manuscript.

Now, for the case of immobilized enzyme in the monoliths, the preparation of macroporous scaffolds by crosslinking AC/GOx complexes in an oil-in-water emulsion proceeds as in the case of the neat AC suspensions discussed previously. We prepare scaffolds using AC/GOx complexes. Figure 3c shows that among these three porous structures, the interconnected porous network obtained by templating the F127 stabilized emulsion yields the highest relative activity of the immobilized enzyme. This is not unexpected and we attribute it to the higher access of the substrate to the immobilized enzyme that comes from the interconnected pore structure.

In case of the enzyme activity in the scaffolds prepared by using CTAB and SDS surfactants,we note the following observations. Stabilized oil droplets in the emulsion are separated by thin layer of water. Since CTAB is cationic, it repels the cationic enzyme aminoclay complex in this thin water layer surrounding oil droplets (This water layer contains enzyme immobilized aminoclay, crosslinker and surfactant head groups at the oilwater interface). Thus, the monolith that forms on crosslinking does not have a well-defined 18


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pore structure. The enzyme immobilized aminoclay composites are crosslinked in an illdefined structure. Therefore, when these scaffolds are exposed to the substrate for enzyme catalysis, it is possible that the enzyme is not readily accessible to the substrate. It appears that there are significant constraints on substrate diffusion to the enzyme active site, thus leading to low relative activity. On the other hand, the cationic enzyme-aminoclay complex interacts with anionic SDS at the oil-water interface to form thin walls that bound a closed pore structure. The enzyme-aminoclay complex is crosslinked to form a self-standing macroporous material that replicates the emulsion structure. In the case of enzyme catalysis, the diffusional resistance of substrate through the pore wall is governed by the pore wall thickness. It appears that the diffusional resistance due to the walls that enclose the pores is less than that to access the enzyme buried in the ill-defined structure that forms in the presence of SDS. Therefore, we observe a higher relative activity of the enzyme in the monolith prepared using SDS surfactant compared to CTAB.

Thus, in monoliths with interconnected pores obtained using F127 surfactant, access to the immobilized enzyme by glucose substrate appears to be better than in the case of monoliths obtained using anionic or cationic surfactants. Therefore, all further experiments were done with the monolith with interconnected porous structure. We note that complexing the aminoclay with enzyme alters the amphiphilicity of the aminoclay particles. At the concentrations used in this work (0.06% by weight of enzyme relative to the continuous phase), there is no change in the structure of monoliths prepared using neat aminoclay when compared with aminoclay/GOx composites. However, significantly increasing the GOx content relative to the aminoclay results in a qualitative change in the pore structure of the scaffold (details given in section S8 of SI).

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We observe that, in addition to control over pore connectivity, the volume porosity of these scaffolds can also be easily controlled simply by changing the oil content of the emulsion (Composition details given in Table S3 of SI). Figure 4a is an image of a stable o/w emulsion with increasing oil volume fraction from 74 to 82.6% (left to right). In these, only the oil content is varied. Figure 4b shows the scaffolds obtained after crosslinking amine groups of the AC/GOx complexes. SEM images show the porous structure obtained after emulsion templating (Figures 4c-e). Furthermore, one can see from Figure 4b that the length of the scaffold increases with increase in the amount of oil (porogen). Therefore, the porosity can be increased simply with increase in oil volume fraction in the emulsion (Figure 4f). We observe no systematic change in the pore size distribution over this range of oil fractions used in the emulsion templating process (SI, Figure S9). BET data indicates that monoliths prepared using aminoclay capped enzymes are not characterized by micro/mesoporosity. In summary, the present work shows that two variables i.e., surfactant type and volume fraction of the oil in o/w emulsion, can be used to control the porous structure and porosity of the scaffolds.

Monoliths with an interconnected pore structure, obtained by emulsion templating oil droplets stabilized with F127, using AC/GOx complexes exhibit remarkable softness, even though their walls are comprised of particles. Typically, monoliths prepared by consolidation of particles are brittle and fail on being subjected to even strains as small as 5%. As a consequence, such monoliths are unable to tolerate the capillary stresses experienced during drying and require to be lyophilized or vacuum dried.42,43 In contrast, enzyme immobilized emulsion templated monoliths can be obtained by simple air drying following our preparation protocol. This is a consequence of the mechanical robustness of the obtained scaffolds. We observe that compression/expansion studies (Figure 5a) of water-saturated enzyme 20


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immobilized monolith demonstrate their ability to elastically recover from up to 80% compressive strain.

On compressing the monolith, there is an initial linear region in the stress-strain curve (Figure 5b), followed by a non-linear increase in stress, characteristic of soft open pore foams.44 The monolith is soft, and is characterized by a Young’s modulus of 57.2 kPa. After compression to a strain of 80%, the monolith is allowed to expand. On expansion, the stress decreases with some hysteresis between compression and expansion steps. However, we observe complete recovery to the initial monolith size, indicating the remarkable elasticity of these materials (SI, compression/expansion test video VS2). Another consequence of the mechanical resilience of these scaffolds is that it is capable of being air dried at room temperature without cracking, despite a volume shrinkage of ≈ 45% (SI, Section S12, Figure S11). Thus, lyophilization is not required to prepare these porous monoliths. Further, we observe that the dry monolith recovers to ≈90% of its original volume on rehydrating.

Therefore, unusually, these emulsion templated monoliths are elastic despite being comprised largely of crosslinked AC/GOx complex particles. The key advantage of this is that it allows the preparation of macroporous monoliths without needing to resort to expensive lyophilization. The monoliths are sufficiently robust that they can be simply air dried.

21



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Figure 4. Effect of oil volume fraction in the o/w emulsion (with enzyme present in water phase) on porosity of the macroporous scaffolds. (a) Images of w/o emulsions with different oil volume fractions; (b) scaffolds obtained after emulsion templating; (c)-(e) SEM images of the scaffolds shown in (b); (f) plot showing variation of porosity with oil volume fraction. Scale bar is 50 µm for (c)-(e).

22


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Stress (Pa)

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180000 160000 140000 120000 100000 80000 60000 40000 20000 0

(b)

0

0.2

0.4 0.6 Strain (mm/mm)

0.8

Figure 5. (a) The scaffold showing elastic recovery from 80% compressive strain, (b) Stressstrain data obtained from compression/expansion test performed on the scaffold.

In the protocol described in the present work, aminoclay particles are localized in the aqueous regions of the emulsion and the amine groups on these are crosslinked to form a monolith. This strategy is achieved using conditions that are sufficiently benign that they do not affect the activity of enzymes encapsulated by the aminoclay. Particle based monoliths have been typically prepared using sintering protocols.30,31 It is clear that this strategy cannot be used to prepare monoliths from enzyme immobilized aminoclay particles. Further, thermally sintered monoliths are likely to be rigid and will not exhibit such remarkable elasticity. 23



Activity of immobilized enzyme inside interconnected macroporous scaffolds

60

(a)

Relative activity(%)

50 40 30 20

84.4% 81.50%

10

72.50%

0 0

1

2

3 Cycle

4

5

6

120 Residual activity(%)

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Page 24 of 34

(b)

100 80 60 40

84.40% 81.50%

20

72.50%

0 0

1

2

3 Cycle

4

5

6

Figure 6. (a) Effect of porosity and (b) recyclability on the activity of the immobilized enzyme.

GOx stabilized by complexing with AC has been shown to demonstrate robustness against thermal and pH changes.34 Despite these advantages, GOx/AC complexes are not widely used due to difficulties in separation of the enzyme from the reaction mixture. 24


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We now explore the activity of GOx/AC complexes assembled into a monolith by emulsion templating.

Figure 6a shows that the specific activity of the immobilized enzyme in the macroporous scaffold increases with increase in porosity. The porosity in the monolith is determined by the volume fraction of oil in the emulsion. With increase in oil fraction, there is an increase in the number of pores and in the volume porosity of the monolith. Thus, for a given enzyme loading, as the monolith porosity increases, there is a reduction in the thickness of the pore walls. We anticipate that this results in a decrease in diffusional resistance of the substrate to the enzyme, leading to increased access of the substrate and hence higher enzyme activity.

The volume fraction of the oil in o/w emulsion determines the porosity and therefore, directly influences encapsulated enzyme activity. Figure 6b shows that the residual activity of the enzyme does not show an appreciable decrease up to 5 cycles of reuse. The ability to reuse the enzyme over multiple cycles of catalysis addresses one of the key challenges outlined in previous reports.45,46,47 Previously, difficulty in separation of the encapsulated enzyme from the reaction mixture precluded the reuse of enzymes.34 In contrast, here, encapsulation of the GOx in a monolith allows us to manually remove the monolith that can be washed and reused multiple times, without any loss of activity.

Stability of the immobilized enzyme

Figure 7a shows that GOx immobilized in interconnected pore scaffolds with varying porosities, has improved pH stability compared to free GOx. This is consistent with the literature reports34 that show that free enzyme loses 60% activity at lower pH while the enzyme-aminoclay complex loses only 10% of its activity at similar conditions. This 25



stability against pH has been attributed to the protective environment provided by the aminoclay to the enzyme. In the present work, the free enzyme loses almost 90% of its activity at low pH, while the enzyme in the scaffold loses only 20-30% of its activity. We reiterate that the free enzyme in the control experiments was subjected to the harsh conditions experienced by the immobilized enzyme. This includes exposing free enzyme to oil, vortexing, etc. Our data suggest that the immobilized enzyme embedded in the macroporous scaffold is stable against these harsh conditions. As the activity of both free and immobilized GOx is highest at pH 7, activity at pH 7 was used to calculate the residual activity at all other pH values.

(a)

100

Free GOx 72.5% 81.5% 84.4%

(b)

100

80

Residual activity (%)

80

Residual activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 34

60

40 Free GOx 72.5% 81.5% 84.4%

20

60

40

20

0

2

4

6

pH

8

10

0

Buffer

Urea

GdCl

Ethanol

Solvent

Figure 7. Stability of GOx immobilized in the macroporous scaffolds with varying porosities in the range of 72.5% to 84.4% against (a) pH and (b) chaotropic solvents.

We also tested the stability of GOx encapsulated in monoliths against chaotropic solvents. This was performed by incubating the scaffolds with several widely used denaturants, such as urea, guanidinium chloride and ethanol. These solvents disrupt hydrogen bonding between water and enzyme and thereby may induce unfolding of enzyme. Figure 7b shows that GOx immobilized in the monolithic scaffolds show improved stability against 26


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chaotropic solvents such as urea (5 M), guanidinium chloride (4 M) and ethanol (100%), when compared with free GOx. We have also examined the thermal stability of the GOx encapsulated in the monoliths and find that the thermal stability is similar to that of free GOx (SI, Figure S12). Our results suggest that complexing with plate-like aminoclay protects the enzyme21 so that it can be used even under harsh conditions of varying pH and on exposure to chaotropic solvents. Assembling the GOx/AC into macroporous monoliths additionally allows transport of substrate to and products from the enzyme, while allowing for facile recovery of the monolith from the reaction mixture and its reuse.

Conclusions In the present work, we demonstrate for the first time that aminoclay nanoparticles can be crosslinked to make a macroporous scaffolds by templating a high internal phase emulsion. Aminoclay nanoparticles and aminoclay/glucose oxidase enzyme complexes were used to prepare macroporous scaffolds. This is achieved by using a surfactant stabilized o/w emulsion where the aminoclay is present in the continuous aqueous layer surrounding the oil droplets. Amine groups on aminoclay nanoplatelet surfaces are crosslinked by a hydrophilic crosslinker to prepare the macroporous monolith. The porous structure and the volume porosity can simply be controlled by varying surfactant type and the oil volume fraction of the o/w emulsion, respectively. Use of CTAB, results in the formation of a network like structure that does not directly template the emulsion structure. The oil drop structure in the emulsion is templated when SDS or F127 is used to stabilize the emulsion. Use of SDS results in a closed pore structure, while a monolith with interconnected pores is formed using F127. Remarkably, the interconnected open pore networks prepared by templating emulsions 27



Page 28 of 34

stabilized with F127, using AC/GOx complexes yields a monolith that can recover elastically after mechanical compression to a strain of 80%. The mechanical resiliency of these monoliths render it possible to air dry them at room temperature without cracking. We further describe that these enzyme immobilized aminoclay particles serve as building blocks and can be converted into a macroporous monolithic structure. This offers minimum leaching of enzyme, and ease in suspending them in the substrate solution and their manual removal from the reaction medium without any energy intensive steps such as centrifugation. The enzyme in the monolith is stabilized by the aminoclay against the effect of chaotropic solvents and variation of pH. Such monolithic structures are likely to find many useful applications for continuous flow operations.

Conflict of interest There is no conflict of interest to declare.

Acknowledgements Authors gratefully acknowledge funding from DST project no. GAP309326. AYK gratefully acknowledges Ms. Kathika Suresh and Mr. Saurabh Usgaonkar working in the group of GK, for their helps in compression/expansion test of the monolith and emulsion preparation by a homogenizer experiments, respectively.

Supporting information Supporting information contains details of aminoclay characterization, calibration of enzyme quantity, droplet size distribution of emulsion and scaffold, composition details of scaffolds, 28


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interconnection pore diameter of the scaffold, enzyme isotherm, tuning pore structure of the scaffolds with enzyme content, pore size distribution for different oil volume fractions, composition details of scaffolds with enzyme, shrinkage and expansion of the scaffold, and thermal stability of the immobilized enzyme.

Supporting video VS1: This video shows the monolith drying process, revealing the formation of connections between pores.

Supporting video VS2: This video shows the compression/expansion test performed on the wet monolith.

References (1)

Lissant, K. J. Emulsions and Emulsion Technology Part 1; Lissant, K. ., Ed.; Mercel Dekker Inc.: New York, 1974.

(2)

Cameron, N. R. High Internal Phase Emulsion Templating as a Route to Well-Defined Porous Polymers. Polymer 2005, 46, 1439–1449.

(3)

Silverstein, M. S. Progress in Polymer Science PolyHIPEs : Recent Advances in Emulsion-Templated Porous Polymers. Prog. Polym. Sci. 2014, 39, 199–234.

(4)

Cameron, N. R.; Sherrington, D. C.; Albiston, L.; Gregory, D. P. Study of the Formation of the Open-Cellular Morphology of Poly(styrene/divinylbenzene) polyHIPE Materials by Cryo-SEM. Colloid. Polym. Sci. 1996, 274, 592–595.

(5)

Ikem, V. O.; Menner, A.; Bismarck, A. High Internal Phase Emulsions Stabilized Solely by Functionalized Silica Particles. Angew. Chem. Int. Ed. 2008, 47, 8277–8279.

(6)

Wu, D.; Xu, F.; Sun, B.; Fu, R.; He, H.; Matyjaszewski, K. Design and Preparation of Porous Polymers. Chem. Rev. 2012, 112, 3595–4015.

(7)

Brun, N.; Ungureanu, S.; Deleaze, H.; Backov, R. Hybrid Foams, Colloids and 29



Page 30 of 34

beyond: From Design to Applications. Chem. Soc. Rev. 2011, 40, 771–788. (8)

Gokmen, M. T.; Prez, F. E. Du. Porous Polymer Particles — A Comprehensive Guide to Synthesis , Characterization , Functionalization and Applications. Prog. Polym. Sci. 2012, 37, 365–405.

(9)

Krajnc, P.; Leber, N.; Štefanec, D.; Kontrec, S.; Podgornik, A. Preparation and Characterisation of Poly(high Internal Phase Emulsion) Methacrylate Monoliths and Their Application as Separation Media. J. Chromatogr. A 2005, 1065, 69–73.

(10)

Kovačič, S.; Silverstein, M. S. Superabsorbent, High Porosity, PAMPS-Based Hydrogels through Emulsion Templating. Macromol. Rapid Commun. 2016, 37, 1814– 1819.

(11)

Dizge, N.; Keskinler, B.; Tanriseven, A. Biodiesel Production from Canola Oil by Using Lipase Immobilized onto Hydrophobic Microporous Styrene – Divinylbenzene Copolymer. Biochem. Engg. J. 2009, 44, 220–225.

(12)

Pierre, S. J.; Thies, J. C.; Dureault, A.; Cameron, N. R.; Van Hest, J. C. M.; Carette, N.;

Michon,

T.;

Weberskirch,

R.

Covalent

Enzyme

Immobilization

onto

Photopolymerized Highly Porous Monoliths. Adv. Mater. 2006, 18, 1822–1826. (13)

Ikem, B. V. O.; Menner, A.; Horozov, T. S.; Bismarck, A. Highly Permeable Macroporous Polymers Synthesized from Pickering Medium and High Internal Phase Emulsion Templates. Adv. Mater. 2010, 22, 3588–3592.

(14)

Manley, S. S.; Graeber, N.; Grof, Z.; Menner, A.; Hewitt, G. F. New Insights into the Relationship between Internal Phase Level of Emulsion Templates and Gas – Liquid Permeability of Interconnected Macroporous Polymers. Soft Matter 2009, 5, 4780– 4787.

(15)

Haibach, K.; Menner, A.; Powell, R.; Bismarck, A. Tailoring Mechanical Properties of Highly Porous Polymer Foams : Silica Particle Reinforced Polymer Foams via Emulsion Templating. Polymer (Guildf). 2006, 47, 4513–4519.

(16)

Gurevitch, I.; Silverstein, M. S. Polymerized Pickering HIPEs : Effects of Synthesis 30


Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Parameters on Porous Structure. J. Polym. Sci. A 2010, 48, 1516–1525. (17)

Sumirat, I.; Shimamura, Y. A. S. Theoretical Consideration of the Effect of Porosity on Thermal Conductivity of Porous Materials. J. Porous Mater. 2006, 13, 439–443.

(18)

Sandra, F.; Depardieu, M.; Mouline, Z.; Vignoles, G. L.; Iwamoto, Y.; Miele, P.; Backov, R.; Bernard, S. Polymer-Derived Silicoboron Carbonitride Foams for CO2 Capture: From Design to Application as Scaffolds for the in Situ Growth of MetalOrganic Frameworks. Chem. A Eur. J. 2016, 22, 8346–8357.

(19)

Chatterjee, S.; Gupta, S. Sen; Kumaraswamy, G. Omniphilic Polymeric Sponges by Ice Templating. Chem. Mater 2016, 28, 1823–1831.

(20)

Ikem, V. O.; Menner, A.; Bismarck, A. High-Porosity Macroporous Polymers Sythesized from Titania-Particle-Stabilized Medium and High Internal Phase Emulsions. Langmuir 2010, 26, 8836–8841.

(21)

Xu, H.; Zheng, X.; Huang, Y.; Wang, H.; Du, Q. Interconnected Porous Polymers with Tunable Pore Throat Size Prepared via Pickering High Internal Phase Emulsions.

Langmuir 2016, 32, 38–45. (22)

Zheng, X.; Zhang, Y.; Wang, H.; Du, Q. Interconnected Macroporous Polymers Synthesized from Silica Particle Stabilized High Internal Phase Emulsions.

Macromolecules 2014, 47, 6847–6855. (23)

Zhang, S.; Chen, J. PMMA Based Foams Made via Surfactant-Free High Internal Phase Emulsion Templates. Chem. Commun. 2009, 2217.

(24)

Hua, Y.; Chu, Y.; Zhang, S.; Zhu, Y.; Chen, J. Macroporous Materials from Water-inOil High Internal Phase Emulsion Stabilized Solely by Water-Dispersible Copolymer Particles. Polymer 2013, 54, 5852–5857.

(25)

Li, Z.; Xiao, M.; Wang, J.; Ngai, T. Pure Protein Scaffolds from Pickering High Internal Phase Emulsion Template. Macromol. Rapid Commun. 2013, 34, 169–174.

(26)

Oh, B. H. L.; Bismarck, A.; Chan-park, M. B. High Internal Phase Emulsion 31



Page 32 of 34

Templating with Self-Emulsifying and Thermoresponsive Chitosan- Graf T -PNIPAMGraft -Oligoproline. Biomacromolecules 2014, 15, 1777–1787. (27)

Li, Z.; Ming, T.; Wang, J.; Ngai, T. High Internal Phase Emulsions Stabilized Solely by Microgel Particles. Angew. Chem. Int. Ed. 2009, 48, 8490–8493.

(28)

Yin, D.; Guan, Y.; Li, B.; Zhang, B. Antagonistic Effect of Particles and Surfactant on Pore Structure of Macroporous Materials Based on High Internal Phase Emulsion.

Colloid. Surf. A 2016, 506, 550–556. (29)

Zhu, H.; Zhang, Q.; Zhu, S. Assembly of a Metal–Organic Framework into 3 D Hierarchical Porous Monoliths Using a Pickering High Internal Phase Emulsion Template. Chem. A Eur. J. 2016, 22, 8751–8755.

(30)

Studart, R.; Tervoort, E.; Gauckler, L. J.; Akartuna, B. I. Macroporous Ceramics from Particle-Stabilized Emulsions. Adv. Mater. 2008, 20, 4714–4718.

(31)

Akartuna, I.; Tervoort, E.; Wong, J. C. H. Macroporous Polymers from ParticleStabilized Emulsions. Polymer 2009, 50 (15), 3645–3651.

(32)

Kim, K.; Kim, S.; Ryu, J.; Jeon, J.; Jang, S. G.; Kim, H.; Gweon, D. G.; Im, W. Bin; Han, Y.; Kim, H.; Choi S. Q. Processable High Internal Phase Pickering Emulsions Using Depletion Attraction. Nat. Commun. 2017, 8, 1–8.

(33)

Burkett, S. L.; Press, A.; Mann, S. Synthesis, Characterization and Reactivity of Layered Inorganic - Organic Nanocomposites Based on 2 : 1 Trioctahedral Phyllosilicates. Chem. Mater. 1997, 9, 1071–1073.

(34)

Patil, A. J.; Muthusamy, E.; Mann, S. Fabrication of Functional Protein- Organoclay Lamellar Nanocomposites by Biomolecule-Induced

Assembly of Exfoliated

Aminopropyl-Functionalized Magnesium Phyllosilicates. J. Mater. Chem. 2005, 15, 3838–3843. (35)

Wang, Y.; Caruso, F. Mesoporous Silica Spheres as Supports for Enzyme Immobilization and Encapsulation. Chem. Mater. 2005, 17, 953–961.

32


Page 33 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36)

Jesionowski, T.; Zdarta, J.; Krajewska, B. Enzyme Immobilization by Adsorption: A Review. Adsorption 2014, 20, 801–821.

(37)

Sheldon, R. A. Enzyme Immobilization : The Quest for Optimum Performance. Adv.

Synth. Catal. 2007, 349, 1289–1307. (38)

Kumari, S.; Kulkarni, A.; Kumaraswamy, G.; Sen Gupta, S. Large Centimeter-Sized Macroporous Ferritin Gels as Versatile Nanoreactors. Chem. Mater. 2013, 25, 4813– 4819.

(39)

Khan, A. Y.; Noronha, S. B.; Bandyopadhyaya, R. Glucose Oxidase Enzyme Immobilized Porous Silica for Improved Performance of a Glucose Biosensor.

Biochem. Engg. J. 2014, 91, 78–85. (40)

Khan, A. Y.; Noronha, S. B.; Bandyopadhyaya, R. Impact of Structural Features of SBA-15 Host Particles on Activity of Immobilized Glucose Oxidase Enzyme and Sensitivity of a Glucose Sensor. J. Porous Mater. 2015, 22, 369–378.

(41)

Kumaraswamy, G.; Deshmukh, Y. S.; Agrawal, V. V.; Nisal, A. A. Composites of Polypropylene with Layered Mg-Silsesquioxanes Show an Unusual Combination of Properties. Ind. Eng. Chem. Res. 2008, 47, 3891–3899.

(42)

Naranda, J.; Sušec, M.; Maver, U.; Gradišnik, L.; Gorenjak, M.; Vukasović, A.; Ivković, A.; Rupnik, M. S.; Vogrin, M.; Krajnc, P. Polyester Type polyHIPE Scaffolds with an Interconnected Porous Structure for Cartilage Regeneration. Sci. Rep. 2016, 6, 1–11.

(43)

Costantini, M.; Colosi, C.; Guzowski, J.; Barbetta, A.; Jaroszewicz, J.; Święszkowski, W.; Dentini, M.; Garstecki, P. Highly Ordered and Tunable polyHIPEs by Using Microfluidics. J. Mater. Chem. B 2014, 2, 2290–2300.

(44)

Gibson, L. J.; Ashby, M. F. Cellular Solids: Structure and Properties, 2nd ed.; Cambridge Solid State Science Series; Cambridge University Press: New York, 1999.

(45)

Hudson, S. P.; White, S.; Goradia, D.; Essa, H.; Liu, B.; Qiao, L.; Liu, Y.; Cooney, J. C.; Hodnett, B. K.; Magner, E. Proteins in Mesoporous Silicates. Angew. Chem. Int. 33



Page 34 of 34

Ed. 2008, 986, 49–60. (46)

Ispas, C.; Sokolov, I.; Andreescu, S. Enzyme-Functionalized Mesoporous Silica for Bioanalytical Applications. Anal. Bioanal. Chem. 2009, 393, 543–554.

(47)

Forsyth, C.; Patwardhan, S. V. Controlling Performance of Lipase Immobilised on Bioinspired Silica. J. Mater. Chem. B 2013, 1, 1164.

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