CoreâShell Magnetic Mesoporous Silica Microspheres with Large

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Surfaces, Interfaces, and Applications

Core-Shell Magnetic Mesoporous Silica Microspheres with Large-Mesopores for Enzyme Immobilization in Biocatalysis Yu Zhang, Qin Yue, Moustafa Zagho, Jiajie Zhang, Ahmed A. Elzatahry, Yongjian Jiang, and Yonghui Deng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18721 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 25, 2019

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ACS Applied Materials & Interfaces

Core-Shell Magnetic Mesoporous Silica Microspheres with LargeMesopores for Enzyme Immobilization in Biocatalysis Yu Zhanga, b Qin Yue*, a,c, Moustafa M. Zaghod, Jiajie Zhange, Ahmed A. Elzatahry*, d, Yongjian Jiang*, e, Yonghui Deng*, a a Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, and iChEM, Fudan University, Shanghai 200433, China b School of Electronic and Computer Engineering, Shenzhen Graduate School, Peking University, Shenzhen 518055, China Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, Chengdu 610051, China d Materials Science and Technology Program, College of Arts and Sciences, Qatar University, PO Box 2713, Doha, Qatar e Department of Pancreatic Surgery, Nephrology & Radiology, Huashan Hospital, Fudan University, Shanghai 200040, China c

ABSTRACT: Magnetic mesoporous silica microspheres with core-shell structure and large pores are highly desired in macromolecules delivery and biocatalysis, biospeparation and adsorption. In this work, a controllable solvent evaporation induced solution-phase interface co-assembly approach was developed to synthesize core-shell structural magnetic mesoporous silica microspheres with ultra large mesopore size (denoted as LP-MMS). The synthesis was achieved by employing large-molecular weight amphiphilic block copolymers poly(ethylene oxide) –block– Poly (methyl methacrylate) (PEO-b-PMMA) and small surfactant cetyltrimethylammonium bromide (CTAB) as co-templates which can co-assemble with silica source in THF/water solutions. The obtained LP-MMS microspheres possess uniform rasberry-like morphology with a diameter of 600 nm, large primary spherical mesopores (ca. 36 nm), large specific surface area (348 m2/g), high specific pore volume (0.59 cm3/g) and fast magnetic responsivity with a high magnetization (15.9 emu/g). The mesopore morphology can be transformed from spherical to cylindrical shape through introducing a shearing force during the interfacial co-assembly in the synthesis system. The designed LPMMS microspheres turn out to be good carriers for enzyme (trypsin) immobilization with a high loading capacity of 80 μg/mg and demonstrate excellent biocatalysis efficiency up to 99.1 % for protein digestion within 30 min and good recycling stability with negligible decay in digestion efficiency after reuse for 5 times. INTRODUCTION 1-11,

Among various functional mesoporous materials microspheres with a magnetic core and mesoporous shell have triggered extensive attention owing to their integrated properties of functional core including magnetic responsiveness and magnetocaloric, and highly porous shell such as large surface area, high pore volume, well connected uniform mesopores and easily modifiable pore walls 12, These advantages endow them with great potential for various applications in biomedicine 13, catalysis 14, 15, water treatment 16, etc. Mesoporous silica shell is usually constructed via an interface sol-gel co-assembly process using cationic surfactant cetyltrimethylammonium bromide (CTAB) as the soft template, generating mesopore size less than 3.0 nm and the pore arrangement is limited to two-dimensional hexagonal structures 17-19. Such small pore size restricts their applications, especially for adsorption, separation or immobilization of large guests like biomacromolecules (e.g. proteins and enzymes). Magnetic mesoporous silica microspheres coreshell structures with large mesopores are in urgent demand for applications. Recently, a micelle swelling approach has been developed in our group to synthesize magnetic mesoporous microspheres with cylindrical and radially arranged large mesopores (up to 9.0 nm) in the silica shell by introduction of a swelling agent (e.g. n-hexane) during the deposition of mesostructured silica shells using CTAB as the template 20,21. Later on, in order to directly create large mesopores in the shell, our group for the first time employed high molecular weights amphiphilic block copolymers (ABC) 22,23 as soft template and successfully

constructed a monolayer of mesoporous shell on magnetic core through an sol-gel interface co-assembly process using polystyrene-block-poly(4-vinylpyridine) (PS-b-P4VP) with protonatable P4VP segment as soft template, and unique radially aligned mesopores with large pore size (20–32 nm) in outer aluminosilicate shell were formed. However, the hydrophilic segment P4VP can be easily protonated, and in typical synthesis solutions containing inorganic acids like HCl, these copolymers suffer flocculation and precipitation in the THF-dominant solution, which is unfavorable for the interface co-assembly coating process. Thus, the synthesis condition for interface co-assembly using PS-b-P4VP as surfactant needs to be precisely controlled. In our previous reports, amphiphilic block copolymers with PEO segments as hydrophilic chain (e.g. PEO-b-PS, PEO-b-PMMA) have been successfully used to synthesize large-pore mesoporous silica materials with various distinct morphologies, including spheres (or polyhedrons) and nanofibers (or nanotubes) via solvent evaporation induced aggregating assembly (EIAA) 24-29. However, it is difficult to construct core-shell mesoporous structure by simply employing these ABC surfactants because of the weak interface interaction between the large polymer and the functional magnetic colloids; instead, it usually leads to the phase-separation of co-assembled ABC surfactant-silica composite and magnetic colloids. Till now, it still remains a great challenge to conveniently fabricate core-shell mesoporous structures with large mesopores using high molecular weights amphiphilic block copolymers. In this study, a novel, facile and general cationic surfactant assisted interface co-assembly method was developed to

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controllable synthesize core-shell mesoporous silica structures with large mesopores by employing the high molecular weights amphiphilic block copolymers, poly(ethylene oxide)– block–Poly(methyl methacrylate) (PEO-b-PMMA) as soft templates and CTAB as a co-surfactant. The synthesis was achieved through a controllable solvent evaporation induced interface co-assembly by introducing a small amount of cationic surfactant CTAB as bridging molecules to enhance the interface interaction among large PEO-b-PMMA micelles, silica oligomer and nonporous silica-coated magnetite colloids (i.e. Fe3O4@nSiO2) via the electrostatic force and hydrogen binding in THF/H2O solutions. The obtained Fe3O4@nSiO2@LP-mSiO2 (denoted as LP-MMS) has uniform spherical morphology and a mean diameter ~600 nm, large primary spherical mesopores (36 nm), high specific surface area (348 m2/g), large specific pore volume (0.59 cm3/g) and high magnetization (15.9 emu/g). It is worth mentioning that the mesopore structures can be tuned from spherical to cylindrical morphology by introducing a shearing force during the co-assembly process in the solution under dynamic stirring. Because their surface is formed by closely packed spherical mesopores, the microspheres exhibit rough pollen grain-like external morphology with enhanced surface hydrophobic property. Enzyme immobilization and enzymolysis results demonstrate that these microspheres have a high loading capacity for trypsin (80 μg/mg) and superior biocatalysis performance for digestion of bovine serum albumin (BSA) due to the enhanced affinity of their rough surface toward BSA molecules and high density of immobilized trypsin catalytic centers in the porous shell, and through convenient magnetic separation, the biocatalyst can be readily recycled for reuse with well-retained catalysis performance. MATERIALS AND METHODS Chemicals. tetraethyl orthosilicate (TEOS), hydrochloric acid (HCl), concentrated ammonia solution (28 wt%), Ethanol, tetrahydrofuran (THF) (>99%), 3glycidyloxypropyltrimethoxysilane (GLYMO) and cetyltrimethylammonium bromide (CTAB) were purchased from Shanghai Chemical Corp . For enzymolysis, cytochrome C (Cyt-C), bovine serum albumin (BSA) and tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin were purchased from Sigma-Aldrich Chemical. Millipore water was used in all experiments. Any other reagents were used as received. Synthesis of Fe3O4@nSiO2 Microspheres. Colloidal Fe3O4 magnetic particles with a size of ~200 nm were synthesized according to our previous report 30. An aqueous dispersion of magnetite particles (4 mL, 0.02 g/ml) was added to a glass flask containing 300 mL of absolute ethanol and 4.0 mL of concentrated ammonia solution under the mechanical stirring (150 rpm) at 35 oC. After stirring for 30 min, TEOS (5.0 ml) was added dropwise within 5 min, and the solution was kept continuous mechanical stirring for12 h. After that, the Fe3O4@nSiO2 core-shell microspheres were separated using a magnet, followed by washing with ethanol and water three times, respectively. Synthesis of core-shell magnetic mesoporous silica microspheres with large mesopores. The amphiphilic block copolymer with a composition of PEO114-b-PMMA130 (Mw: 18000 g/mol) was synthesized via atom transfers radical polymerization (ATRP) technique according to our previous

report 31. The large mesoporous core-shell LP-MMS was synthesized as follows. A THF solution with dissolved PEO-bPMMA (1.3 wt %, 9.0 g) was mixed with CTAB (0.03 g), H2O (4.0 mL) and HCl aqueous solution (2 M, 0.5 mL). The obtained solution was further kept stirring at 120 rpm for 1 h before TEOS (0.45 g) was added. After stirring for 0.5 h, the generated solution was let to stand for 4 h in a hood for THF evaporation slowly in air at 25 ºC When the transparent solution turned into pale blue, 0.2 mL of THF solution containing Fe3O4@nSiO2 microspheres (10 mg) was added into the reaction medium with stirring. After a gentle stirring for 5 min, the solution was let to stand for further evaporation of THF for 48 h, the yellow precipitate was separated by a magnet, followed by washing with ethanol four times. After vacuum drying at 50 ºC, the as-made samples undergo calcination at 450 oC in a muffle for 6 h to remove the organic templates by decomposition, generating core-shell magnetic microspheres with large spherical mesopores (LP-MMS). TEOS is much excess in the synthesis, the efficient yield of silica in this step is calculated about 13 %. Similar synthesis was carried out to construct cylindrical mesoporous silica shell except that, after adding THF solution containing Fe3O4@nSiO2 seeds, the solution was kept gentle stirring (200 rpm) for 48 h to allow a complete evaporation of THF, followed by similar post treatment including ethanol washing and thermal treatment. Enzyme Immobilization and Protein Digestion. Trypsin was immobilized into LP-MMS microspheres via two steps of surface reactions as follows. Firstly, 0.2 g of LP-MMS microspheres were dispersed in 50 mL of toluene containing 0.5 g of GLYMO. The mixture was stirred at 35 oC for 8 h. The resultant microspheres were separated and washed with ethanol three times and vacuum dried at 40 oC for further use. Secondly, the GLYMO modified microspheres (5.0 mg) was dispersed in 500 μL NH3HCO3 (25 mM) buffer solution with dissolved trypsin (1.0 g/L) in an Eppendorf tube and then incubated at 37 oC for 2 h in a shaker. Then, the magnetic microspheres were obtained through magnetic separation and washed with water four times. Finally, the enzymeimmobilized microspheres (denoted as LP-MMS-trypsin) were redispersed in NH3HCO3 solution (500 μL, 25 mM) for further use. For magnetic mesoporous silica core-shell microspheres carriers with rasberry-like morphology pore size of 5.7 nm (MMS), the procedure of trypsin immobilization is the same except to replace LP-MMS with MMS microspheres. To assess the biological activity of immobilized enzyme, bovine serum albumin (BSA, MW = 66 400 Da, 5.0 × 7.0 × 7.0 nm3) was selected as a model protein to be digested. Typically, appropriate amount of BSA was dissolved in NH4HCO3 (25 mM, pH=8.0) buffer solution. The suspension containing 0.01 mg/mL of LP-MMS-trypsin (25 mM NH4HCO3 buffer) was added into the BSA solution (0.01 nmol/mL, 25 mM NH4HCO3). After that, the mixture solution was agitated in a shaker at 37 oC for 30 min. LP-MMS-trypsin catalysts were collected from the mixture with a magnet, and the supernatant was analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Measurements and Characterization. Scanning electron microscopy (SEM) analysis was applied to observe the morphology of samples at 20 kV using Philips XL30 electron


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ACS Applied Materials & Interfaces microscopy (Holland). Transmission electron microscopy (TEM) observation was performed at 200 kV on a JEOL 2011 microscope operated. Field-emission scanning electron microscopy (FESEM) images were acquired on the Hitachi (Japan) Model S-4800 field emission scanning electron microscope. Powder X-ray diffraction (XRD) patterns were applied to characterize the composition and crystallization of samples at 40 kV and 40 mA using a Bruker (Germany) D8 Advanced X-ray diffractometer. It is operated with Ni-filtered Cu K α radiation. Nitrogen sorption-desorption isotherms were recorded using a Micromeritics 3000 analyzer at 77 K. the samples were submitted to degas treatment in a vacuum at 180 oC for 6 h before measurements. The specific surface areas were calculated according to the Brunauer-Emmett-Teller (BET) method by adopting adsorption data recorded at a relative pressure (P/P0) from 0.05 to 0.25. According to the Barrett-Joyner-Halenda (BJH) model, total pore volumes were calculated from the adsorbed amount at a relative pressure P/P0 of 0.995 and pore size distributions were originated from the adsorption data. MALDI-TOF MS analysis were applied to monitor the enzymolysis of proteins on a 4700 Proteomics Analyzer in positive ion mode with the Nd-YAG laser at the wavelength of 355 nm, an acceleration voltage of 20 kV and a repetition rate of 200 Hz (Applied Biosystems, USA).

RESULTS AND DISCUSSION The preparation procedure of LP-MMS microspheres is schematically illustrated in Figure 1. Firstly, the citrate capped hydrophilic magnetite (Fe3O4) particles were prepared via a solvothermal method according to the previous report30. Then, a shell of nonporous dense silica (nSiO2) was coated onto it by a modified Stöber synthesis through the interface hydrolysis and condensation of TEOS catalyzed by alkaline. The dense silica shell served as a protective layer to avoid the corrosion of magnetic particles in subsequent acid media, improving the stability of magnetic core. Thirdly, amphiphilic block copolymers PEO114-b-PMMA130 macromolecular surfactants, together with CTAB, were used as structure-directing agent. A layer of surfactant/SiO2 nanocomposites were deposited onto the Fe3O4@nSiO2 seeds through a controllable solvent evaporation induced interface co-assembly of PEO-b-PMMA, CTAB micelles with silica oligomers by electrostatic interaction and hydrogen bond. After the sandwich structures were fixed by the further cross-linking of silica oligomers, the microspheres were calcined in air to remove organic surfactants, resulting in magnetic mesoporous silica core-shell microspheres (LP-MMS) with large mesopore size.

Figure 1. Synthetic route for magnetic mesoporous silica core-shell microspheres with rasberry-like morphology and ultra large mesopore size (LP-MMS) involving deposition of nonporous silica shell on colloidal magnetite particles, further interface co-assembly of PEO-bPMMA/CTAB/silica on silica shell, and finally removal of CTAB and PEO-b-PMMA/silica via combustion in air.



Figure 2. SEM images of (A) Fe3O4 particles, (B) Fe3O4@nSiO2 microspheres and FESEM images of (C) LP-MMS microspheres; The inset in panel A is the optical photo of Fe3O4 particles dispersed in alcohol. (B inset) is the TEM image of Fe3O4@nSiO2 microspheres. The inset in panel C is the high-magnification SEM (HRSEM) image of LP-MMS on polydimethylsiloxane (PDMS) layer for individual contact angle measurement using the gel trapping (GTT) strategy. (D–F) TEM images of LP-MMS microspheres with different magnifications. The inset in panel F is the enlarged TEM image of LP-MMS microspheres.

The hydrophilic magnetic Fe3O4 microspheres obtained through the solvothermal approach demonstrate uniform spherical morphology with a size of ~200 nm (Figure 2A). Through the sol-gel process of TEOS, Fe3O4 microspheres were uniformly coated by a dense layer of silica with a shell thickness of ~100 nm (Figure 2B), and the resultant coreshell Fe3O4@nSiO2 have a diameter of about 400 nm. The nonporous silica layer plays an important role in the subsequent constructing large-pore mesoporous silica outer shell. Firstly, it protects Fe3O4 core from being etched or oxidized under harsh conditions. Secondly, it provides the microspheres with a large number of ionizable silanol groups in the surface, enabling the interaction between CTAB, PEOb-PMMA and silicate species at the solid-solution interface around the Fe3O4@nSiO2 colloids. After further interface coassembly and deposition of PEO-b-PMMA/CTAB/SiO2 nanocomposite layer onto Fe3O4@nSiO2 through cationic surfactant assisted approach and subsequent thermal decomposition treatment in air at 450 oC, the generated LPMMS microspheres remain uniform spherical morphology and possess a diameter of ~600 nm (Figure 2C). Compared with Fe3O4@nSiO2 microspheres, LP-MMS microspheres show a rough surface due to the fact that the porous shell are constructed by the packing of large spherical PEO-bPMMA/CTAB/SiO2 composite micelles and further thermolysis of PEO-b-PMMA spherical micelles and CTAB. By using the well-established gel trapping technique (GTT) 32,

the static water contact angle of a single LP-MMS microsphere (Figure 2C inset) was measured to be 117 ± 5°, implying a hydrophobic surface property mainly owing to the rough surface of microspheres 33. TEM observation further confirms the core-shell-shell structure of the obtained uniform LP-MMS microspheres (Figure 2D), and the large ellipsoidal pores with a mean pore size of 40 nm can be clearly observed in the shell (Figure 2E). The ellipsoidal pores were probably due to the spherical micelle distortion during the high temperature calcination. In the pore walls, some tiny worm-like pores templated by CTAB molecules can be observed (Figure 2F inset). According to the TEM image, the porous shell is about 50 nm in thickness (Figure 2F). It implies that the porous shell was derivate from packing of about two layers of CTAB-silicate-PEO-b-PMMA composite micelles. Nitrogen sorption isotherm of LP-MMS microspheres (Figure 3A) reveals representative type IV curves. In the P/P0 range of 0.85 –1.0, a sharp capillary condensation step was appeared, suggesting a uniform and large mesopore size. The isotherms exhibit a large H2-type hysteresis loop, indicating that LP-MMS possesses large spherical mesopores. The total pore volume and BET specific surface area of LP-MMS microspheres were calculated to be ~0.59 cm3/g and 348 m2/g. Based on the Barrett-Joyner-Halenda (BJH) method, the pore size distribution (PSD) curve (Figure 3B) originated from the adsorption data shows a primary mesopore centered at 36 nm,


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ACS Applied Materials & Interfaces in agreement with the mesopore size estimated from the TEM images. Such a large pore size is highly desired for various host-guest chemistry applications that involve large functional guest molecules or nanoparticles. Besides, another peak around 3 nm can be observed in the pore size distribution profile (Figure 3B, inset). These smaller mesopores are template by CTAB molecules, consistent with the previous report.31 The peak of smaller mesopore is relatively weak due to the fact that the small mesopores in the thin layer of silica shell has a small N2 absorption capacity. If Fe3O4@nSiO2 cores are absent in the synthesis system, pure mesoporous silica powders with clear dual mesopores centered at 2.9 templated by CTAB and 27.4 nm templated by block copolymers were obtained (Figure S1), further confirming the co-assembly behavior of CTAB and PEO-b-PMMA templates. Notably, the unique dual-mesopores structure is beneficial for the mass diffusion and guest delivery, making the functional particles ideal carriers and nanoreactors.

Figure 3. (A) Nitrogen sorption isotherms and (B) pore size distribution of LP-MMS microspheres. (C) The magnetic hysteresis loops of Fe3O4 microspheres and LP-MMS microspheres. (D) The magnetic separation-redispersion process of LP-MMS microspheres in their aqueous dispersion.

The wide-angle XRD pattern (Figure S2) of LP-MMS microspheres displays the typical specific diffraction peaks of magnetite crystalline phase, and the peak intensities are relatively lower than magnetite particles before coating, owing to the presence of amorphous silica shell. The magnetic property was analyzed using superconducting quantum interference device (SQUID) magnetometer at 300 K. Fe3O4 particles and LP-MMS microspheres have magnetization saturation values of 68.8 and 15.9 emu/g, respectively (Figure 3C) and they show superparamagnetic feature with no remanence because that magnetic Fe3O4 particles consisting of nanosized magnetite crystals of less than 15 nm 17. As a result, the obtained LP-MMS microspheres show a fast

separation-redispersion performance in the existence-absence of magnet (Figure 3D), respectively, which favors the separation and recycling in practical applications. It is worth mention that, the mesopore structure in the shell can be altered by changing the solvent evaporation process from static into dynamic stirring conditions. After introducing Fe3O4@nSiO2 microspheres into acidic THF-aqueous mixture in the presence of PEO-b-PMMA, CTAB and TEOS under the mild stirring and evaporation of THF, the obtained microspheres show a similar uniform spherical core-shell-shell magnetic mesoporous structures (Figure 4). However, on the surface of microspheres, regularly arranged tubular mesopore channels surround the Fe3O4@nSiO2 cores were distinctly observed, which is different from the ellipsoidal mesopores for LP-MMS microspheres. This kind of mesoporous magnetic silica microspheres with cylindrical pores in the shell was denoted as C-MMS microspheres. Nitrogen adsorptiondesorption isotherm (Figure S3A) confirms that it possesses cylindrical mesopore structures, and the mesopore size derived from the adsorption branch was about 33 nm (Figure S3B), close to that of LP-MMS microspheres due to the same molecular weight of PEO-b-PMMA surfactants. The total pore volume and BET surface area for C-MMS microspheres were calculated to be ~ 0.58 cm3/g and 361 m2/g. In addition to the Fe3O4@nSiO2 colloids, the cationic surfactant assisted interface co-assembly strategy can be extended to creation of mesoporous shell on other colloids. Taking Fe3O4@RF (resorcinol-formaldehyde resin) core-shell microspheres (Figure S4) as an example, by using Fe3O4@RF to replace Fe3O4@nSiO2 seeds following the same synthesis as LP-MMS, Fe3O4@RF@LP-mSiO2 microspheres with a mean diameter of ~600 nm can be generated (Figure S5a, b), and the microspheres show uniform rasberry-like morphology because the large spherical mesopores were formed through thermal decomposition of spherical PEO-b-PMMA/CTAB/silica composite micelles on the Fe3O4@RF core. TEM image (Figure S5c, d) further clearly reveals multilayer bubble-like mesopores with a size of about 25 nm in the shell. The successful deposition of mesoporous silica shell is mainly due to the large amount of phenolic hydroxyl groups (-OH) present on the surface of Fe3O4@RF colloids which can provide electrostatic attraction and hydrogen bond interaction between PEO-b-PMMA, CTAB, silica to ensure a controllable coassembly at the RF-liquid interface. Therefore, the PEO-bPMMA/CTAB/silica composite micelles tend to aggregate and assemble selectively on the different hydrophilic surfaces that have ionizable hydroxyl groups, because they can form both electrostatic attraction and hydrogen bond interactions with precursors and surfactants. It is thus reasonable to expect that this method can be generally applied to prepare mesoporous core-shell microspheres with large mesopores by adopting PEO-b-PMMA and CTAB as co-templates.



Scheme 1. Formation mechanism diagrams for LP-MMS (route i) and C-MMS (route ii) microspheres.

Based on the above results, the LP-MMAS microspheres formation mechanism was proposed as solvent evaporation induced interface aggregates and co-assembly of silica oligomers, PEO-b-PMMA, CTAB in the presence of colloidal Fe3O4@nSiO2 microspheres, as depicted in Scheme 1. Firstly, when the transparent acidic THF-H2O solution with completely dissolved PEO-b-PMMA, CTAB and TEOS was allowed to stand for slow evaporation of THF at room temperature (25 oC), the solvency intensity of the mixture for PMMA segments decreases and PEO-b-PMMA spherical micelles with hydrophobic PMMA segment core and hydrophilic PEO shell were formed. Meanwhile, as the THF evaporates, more and more silicate oligomers were generated in the solution because the solution becomes more acidic and catalyzed the hydrolysis and condensation of TEOS. These oligomers associate with the hydrophilic PEO moieties of the copolymers through hydrogen bonding, and they also coassemble with CTAB molecules via electrostatic attraction into rod-like composite micelles. As a result, the small rodlike and large spherical micelles dynamically co-exist in the solution, giving rise to light blue emulsion. When Fe3O4@nSiO2 seeds were introduced into the emulsions, cationic surfactant CTAB can adsorb on the surface to stabilize the cores. On the other hand, surface absorbed CTAB also served as molecular bridge to interact with the negative charged spherical PEO-b-PMMA/SiO2 composite micelles as well as rodlike CTAB-silica composite micelles, thus enhancing the interface interaction between composite micelles and core materials. It was found CTAB molecules play a key role to promote the interface deposition of PEO-bPMMA/SiO2 composites onto Fe3O4@nSiO2 particles. In the absence of CTAB, PEO-b-PMMA copolymers tend to coassemble with silicate oligomers homogenously in solution, leading to a macroscopic phase separation between Fe3O4@nSiO2 microspheres and mesostrutured PEO-bPMMA/silica composites, and thus no distinct core-shell LPMMS microspheres can be obtained (Figure S6). As THF

further evaporates under static condition, the spherical PEO-bPMMA/SiO2 composite micelles with ever-increasing concentration can aggregate around the Fe3O4@nSiO2 seeds to decrease interface energy, give rising to core-shell spheres with rasberry-like morphology (Route 1). However, when a gentle stirring is applied during the THF evaporation, a shearing stress generated by the mild stirring can be produced in the solution, which causes a head-to-head assembly of the preformed spherical inorganic-organic PEO-b-PMMA/CTABsilicate composite micelles to fuse to spheroidal, and to final tubular micelles that deposit on the colloidal magnetic Fe3O4@nSiO2 particles. The shearing stress regulated assembly from spherical to cylindrical micelles was also found in previous reports 34. After the regular arranged structures were fixed through further condensation of silicate oligomers, the microspheres were separated and calcined in air to remove organic surfactants, thus generating LP-mSiO2 and C-MMS microspheres.


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ACS Applied Materials & Interfaces Figure 4. SEM (A, B) and TEM (C, D) images of C-MMS microspheres, the inset is the model of C-MMS microspheres.

Proteolysis, as an important biocatalysis process, was widely used to acquire information about proteins in proteome analysis 35, 36. As is well-know, peptide mapping is an important tool for protein identification and analysis 37. However, the free enzymes easily lose activity and are difficult to recycle due to unavoidable protein selfenzymolysis, unfolding and aggregation which limit the practical applications. To overcome the problems, enzyme immobilization has been developed to improve the activity and stability of enzyme 38-40. In this study, the obtained LP-MMS microspheres possess rough morphology, large mesopores, high surface area, plenty of silanol groups in the surface and fast magnetic responsivity, which is believed to be the ideal carriers for macromolecules like enzyme and proteins. Herein, a typical enzyme trypsin (3.8 × 3.8 × 3.8 nm3) was selected to be immobilized into the mesopores of LP-MMS microspheres. Before trypsin immobilization, GLYMO was served as a bridge agent for trypsin and carriers owing to the strong interaction between epoxy group from GLYMO and amino acid residues of trypsin. For comparison, similar rough coreshell magnetic mesoporous silica microspheres with smaller pore size of 5.7 nm synthesized through our previous reports 41 (Figure S7) were also applied for trypsin immobilization. Through the simple aqueous-phase reaction, trypsin could be immobilized in the mesoporous channel of LP-MMS microspheres within 1 h. The UV spectrophotometer was employed to measure the immobilization capability of LPMMS and MMS microspheres for trypsin, which can reach 80 and 71 μg/mg (enzyme/supports), respectively. Meanwhile, Fourier transform infrared spectra (FTIR) of trypsinimmobilized LP-MMS microspheres exhibit characteristic in the range of 1200 ~ 1600 cm-1, which is attributed to typical absorption peak of trypsin, further confirming the trypsin was immobilized successfully (Figure S8).

Figure 5. MALDI-TOF mass spectra obtained from BSA (4 mg/mL, 100 μL) digested with LP-MMS-trypsin microspheres at 37 oC for 30 min.

In this study, the immobilized trypsin in the mesopores served as the catalytic active sites. Since the biocatalysis mainly occured at the interface of solid LP-MMS-trypsin microsphere and protein solutions, the adsorption ability of the LP-MMS carriers can greatly influence the proteolysis efficiency. The adsorption behavior of LP-MMS-trypsin and MMS-trypsin microspheres for proteins with different size were investigated. Firstly, smaller Cyt-C (MW = 12 384 Da, 2.6 × 3.2 × 3.3 nm3) was employed to test the adsorption

ability of LP-MMS-trypsin and MMS-trypsin microspheres. As shown in Figure S9A, both LP-MMS-trypsin and MMStrypsin microspheres show a distinct adsorption performance in 4 h for Cyt-C with a saturated adsorption capacity of 120 and 100 µg/mg, respectively. Due to the small pore size and hydration layer in mesopores for MMS-trypsin, the adsorption capacity is lower than that of LP-MMS-trypsin microspheres, confirming the large mesopores are beneficial for protein absorption. Furthermore, if larger protein BSA which possesses size of 5.0 × 7.0 × 7.0 nm3 instead of smaller Cyt-C was used for adsorption study (Figure S9B), MMS exhibits negligible adsorption capacity because that BSA proteins (7.0 nm) cannot enter into such small mesopores (5.7 nm). However, LP-MMS-trypsin still maintains a faster adsorption rate and high adsorption capacity of 135 µg/mg owing to their ultra large dual-mesopores with large entrance size. After BSA digestion by LP-MMS-trypsin microspheres at 37 oC for 30 min, the reaction solution was subjected to MALDI-TOF MS analysis. As shown in Figure 5, various characteristic peaks of the peptide (Mw 1001.7, Mw 1193.7, Mw 1439.9, Mw 1479.9, Mw 1567.9, Mw 1640.1) derived from BSA were detected, confirming the successful BSA digestion by LP-MMS-trypsin microspheres. The high proteolysis efficiency is mainly ascribed to the large mesopores and high surface areas for high immobilization capacity of enzyme with easy accessibility, and the hydrophobic rough morphology of LP-MMS provides enhanced adhesiveness for fast enriching the substrate BSA molecules, achieving protein adsorption kinetics and digestion 42. Besides, after reaction, the microspheres can be recycled by a magnet and exhibit stable enzymolysis performance after reused for 5 times (Figure S10). 

CONCLUSIONS

In summary, a facile and general cationic surfactant assisted interface co-assembly method was demonstrated to synthesize magnetic mesoporous silica core-shell microspheres with rasberry-like morphology and ultra large mesopore size by employing ultrahigh molecular weight amphiphilic block copolymers PEO-b-PMMA and CTAB as templates. These obtained microspheres possess rough surface morphology, large ellipsoidal mesopores (ca. 36 nm), uniform diameter size (600 nm), large surface area (348 m2/g), high pore volume (0.59 cm3/g) and fast magnetic responsivity with high magnetization of 15.9 emu/g. Additionally, if mild stirring was added in the solvent evaporation induce interface assembly process, core-shell magnetic mesoporous silica microspheres with cylindrical mesopore channels in the shell were obtained. By virtue of their ultralarge mesopores and rough morphology, LP-MMS microspheres were demonstrated to be ideal carriers for trypsin immobilization with a large loading capacity of 80 μg/mg and reveal high digestion efficiency with good recycling stability. ASSOCIATED CONTENT Supporting Information. The wide-angle XRD pattern of LP-MMS microspheres. Fourier transform infrared spectra (FITR) of LP-MMS-trypsin microspheres. Magnetic separation and recycling of LP-MMS-trypsin microspheres. Adsorption behavior of the LP-MMSs and magnetic mesoporous silica core-shell microspheres with smaller pore size of 5.7 nm for different proteins. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION Corresponding Author Yonghui Deng, Email: [email protected] Qin Yue, Email: [email protected], Yongjian Jiang, Email: [email protected] Ahmed A. Elzatahry, Email: [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. 

ACKNOWLEDGMENT

This work was supported by the NSF of China (21673048, 21701153 and 21875044), Key Basic Research Program of Science and Technology Commission of Shanghai Municipality (17JC1400100), Qatar University under GCC Co-Fund Program Grant GCC-2017-001, and Youth Top-notch Talent Support Program of China.

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REFERENCES

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