An Efficient, Recyclable, and Stable Immobilized Biocatalyst Based on

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

An Efficient, Recyclable and Stable Immobilized Biocatalyst Based on Bioinspired Microcapsules-in-Hydrogel Scaffolds Shaohua Zhang, Zhongyi Jiang, Jiafu Shi, Xueyan Wang, Pingping Han, and Weilun Qian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09483 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

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

ACS Applied Materials & Interfaces

An Efficient, Recyclable and Stable Immobilized Biocatalyst Based on Bioinspired Microcapsules-in-Hydrogel Scaffolds Shaohua Zhang†,§, Zhongyi Jiang†,§, Jiafu Shi*,‡,§, Xueyan Wang†,§, Pingping Han†,§, Weilun Qian†,§ †

Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical

Engineering and Technology, Tianjin University, Tianjin 300072, China; ‡

School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China;

§

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, China. ABSTRACT: Design and preparation of high-performance immobilized biocatalysts with exquisite structures and elucidation of their profound structure-performance relationship are highly desired for green and sustainable biotransformation processes. Learning from nature has been recognized as a shortcut to achieve such impressive goal. Loose connective tissue, which composes of hierarchically organized cells by extracellular matrix (ECM) and is recognized as an efficient catalytic system to ensure the ordered proceeding of metabolism, may offer an ideal prototype for preparing immobilized biocatalysts with high catalytic activity, recyclability and stability. Inspired by the hierarchical structure of loose connective tissue, we prepare an immobilized biocatalyst enabled by Microcapsules-in-Hydrogel (MCH) scaffolds via biomimetic mineralization in agarose hydrogel. In brief, the in situ synthesized hybrid microcapsules encapsulated with glucose oxidase (GOD) are hierarchically organized by the fibrous framework of agarose hydrogel, where the fibers are intercalated into the capsule wall. The as-prepared immobilized biocatalyst shows structure-dependent catalytic performance. The porous hydrogel

ACS Paragon Plus Environment

1


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 2 of 32

permits free diffusion of glucose molecules (diffusion coefficient: ~6×10-6 cm2 s-1, close to that in water) and retains the enzyme activity as much as possible after immobilization (initial reaction rate: 1.5×10-2 mM min-1). The monolithic macroscale of agarose hydrogel facilitates the easy recycling of the immobilized biocatalyst (only by using tweezers), which contributes to the non-activity decline during the recycling test. The fiber-intercalating structure elevates the mechanical stability of the in situ synthesized hybrid microcapsules, which inhibits the leaching and enhances the stability of the encapsulated GOD, achieving immobilization efficiency of ~95%. This study will, therefore, provide a generic method for the hierarchical organization of (bio)active materials and the rational design of novel (bio)catalysts. KEYWORDS: immobilized biocatalyst; loose connective tissue; hierarchical structure; microcapsules-in-hydrogel scaffolds; in situ synthesis 1. INTRODUCTION Biocatalysis holds great promise in biotransformation process with exceptional regio-, chemo-, and stereo-selectivity.1-3 Enzymes and cells (as two major biocatalysts) usually exhibit superior activity, whereas the difficult recycling and easy denaturation of these biocatalysts remarkably restrict their widespread applications.4-6 To address these two problems, tremendous efforts have been devoted to design and prepare immobilized biocatalysts with different structures.7-9 It seems that rational transfer of the structure-performance relationship of the catalytic systems in nature may provide an ingenious and feasible approach to obtain high-performance immobilized biocatalyst. In nature, hierarchical structures can be commonly found in both living and non-living systems.10 Particularly, in living systems, complex functions can be realized by the evolved hierarchical structures in an efficient and ordered manner. As the structure unit and building


2

Page 3 of 32

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


block of living systems, cells enable the effective implementation of biochemical reactions by cytoplasm, a relative stable micro-environment enclosed in lipid bilayer, and mediate the critical cellular functions.11 Rather than random stacking, cells are often hierarchically organized by extra-cellular matrix (ECM) to form tissues. This not only provides appropriate microenvironment and protection for the cells, but also enables the substrate transport in a controllable way.12,13 Specifically, ECM is a highly hydrated fibrous network gel with collagen fibers, assembled from collagen molecules, acting as the framework.14 Cells are bound to ECM (cell adhesion) through a transmembrane protein-integrin that bridges the actin filament of cytoskeleton in cells with collagen fibers.13 The porous network of ECM facilitates the transportation of nutrients and oxygen, thus ensures the efficient implementation of metabolic reactions in cells. The fibrous framework of ECM can provide mechanical protection to the neighboring cells. More importantly, the binding of cells to ECM avoids the metastasis of normal cells, preserving intact and stable tissue structure. As the cell analog, hybrid microcapsules have been extensively explored in recent years.15 Since favorable micro-environment and high mechanical stability can be achieved by regulating the organic and inorganic moieties of the capsule wall, hybrid microcapsules are well recognized as an excellent scaffold for the preparation of efficient immobilized biocatalysts.16,17 However, the recycling of hybrid microcapsules always requires energy/labor-intensive operations (centrifugation or filtration), which greatly restricts their practical applications. Meanwhile, the loss and breakage of hybrid microcapsules during recycling/reuse process usually result in decreased activity of immobilized biocatalysts. Organizing hybrid microcapsules into macroscopic assemblies based on the structure-performance relationship may offer a feasible


3


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 4 of 32

strategy to synergistically optimize the activity, recyclability and stability of immobilized biocatalysts.18,19 Inspired by the hierarchical structure of loose connective tissue, herein, we prepared an immobilized biocatalyst based on Microcapsules-in-Hydrogel (MCH) scaffolds. Agarose hydrogel with fibrous network was used to mimic the ECM of loose connective tissue.20,21 MCH scaffolds were prepared through biomimetic mineralization in agarose hydrogel. Specifically, poly(sodium 4-styrenesulfonate) (PSS)-doped CaCO3 particles derived from co-precipitation were firstly embedded into the agarose hydrogel to acquire CaCO3 Particles-embedded-Hydrogel (CPH).22 The obtained CPH was alternately immersed in the aqueous solutions of protamine and Na2SiO3 to implement biomimetic mineralization in agarose hydrogel. MCH scaffolds were obtained after removing CaCO3 particles by ethylenediaminetetraacetic acid (EDTA) treatment. The formation mechanism of MCH scaffolds and fiber-intercalating structure were elucidated. Based on this MCH scaffolds, glucose oxidase (GOD) as a commonly used enzyme was incorporated to acquire the immobilized biocatalysts (denoted as G-MCH scaffolds). The GMCH

scaffolds

displayed

remarkable

structure-dominated

performance,

which

was

systematically discussed. 2. EXPERIMENTAL SECTION 2.1. Materials Agarose was obtained from Biowest. Glucose oxidase from Aspergillus niger (GOD, 100000250000 U g-1, E.C. 1.1.3.4), poly(sodium 4-styrenesulfonate) (PSS, powder, average Mw ~70000), 3,3',5,5'-tetramethylbenzidine (TMB), and protamine sulfate salt from salmon were purchased from Sigma-Aldrich. Peroxidase (horseradish) (HRP, ≥300000 U g-1, E.C. 1.11.1.7) was obtained from Shanghai Yuanye Biotechnology Company Limited (Shanghai, China).


4

Page 5 of 32

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


Ethylenediaminetetraacetic acid (EDTA) and sodium silicate (Na2SiO3) were purchased from Shanghai Aladdin Biological Technology Company Limited (Shanghai, China). All of the other chemicals were analytical grade. The pH values of sodium silicate solution, PBS buffer and HAc-NaAc buffer were adjusted by adding 100 mM HCl and 100 mM NaOH solutions. Water utilized in the experiments was treated by Millipore water purification system. 2.2. Preparation of CPH PSS-doped CaCO3 particles with diameter of 4-6 µm were synthesized through coprecipitation method as reported previously.22 Hereafter, CaCO3 particles referred to PSS-doped CaCO3 particles without special illustration. 1% w/v agarose solution was prepared by adding agarose powder to corresponding deionized water and then heated to dissolve. CaCO3 particles (100 µL, 300 mg mL-1) were dispersed into agarose solution (2 mL) at around 40 oC. Then, the solution was left to cool until agarose hydrogel was formed. 2.3. Preparation of MCH scaffolds CPH was immersed in the solution of protamine (3 mg mL-1) for 15 min. After soaked by water for 5 min and three times, the hydrogel was immersed in the solution of Na2SiO3 (30 mM, pH=7~8) for another 15 min. The number of bilayers could be adjusted by repeating the above process. Finally, the hydrogel was immersed in the solution of EDTA (50 mM) to remove the CaCO3 particles. 2.4. Encapsulation of GOD in MCH scaffolds GOD was encapsulated in CaCO3 particles during the co-precipitation process. The GODencapsulated CaCO3 particles were embedded into the agarose hydrogel to prepare CPH. Then CPH was immersed in the solution of protamine and Na2SiO3, respectively. After removing the CaCO3 particles, G-MCH scaffolds were generated.


5


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 6 of 32

2.5. Activity assay of G-MCH scaffolds The activity of G-MCH scaffolds was determined by absorption intensity at 652 nm with UVvis spectrometer (Hitachi U-3010). Briefly, β-D-glucose would first react with oxygen catalyzed by the encapsulated GOD generating gluconic acid and H2O2. The concentration of H2O2 was monitored using TMB substrate through a second reaction catalyzed by HRP. 2.6. Recycling stability G-MCH scaffolds could be simply recycled after washing using a tweezer to hold the sample. Then, G-MCH scaffolds was washed by PBS buffer and reused. For comparison, the recycling stability of GOD-encapsulated PSi-W hybrid microcapsules were also evaluated, which was collected by centrifugation. The recycling stability was calculated according to equation (1), dividing the residual activity after nth cycle to their first cycle activity. Recycling stability =

enzyme activity of nth cycle × 100 enzyme activity of 1st cycle

(1)

2.7. pH, thermal and storage stability G-MCH scaffolds and free GOD were incubated in different temperatures (30-70 oC) or buffers with different pH values (3-9) for 2 hours. Then, the residual activities were measured. The pH and thermal stability of G-MCH scaffolds and free GOD were presented by the quotient of the residual activity and the highest residual activity. The storage stability of G-MCH scaffolds and free GOD was determined by measuring the residual activity after incubating for different time. 2.8. Enzyme distribution in G-MCH scaffolds To monitor the enzyme distribution in G-MCH scaffolds, FITC-labeled BSA was used as an enzyme analog to be encapsulated in MCH scaffolds as described above. Besides, to better


6

Page 7 of 32

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


visualize the spatial distribution of enzyme in G-MCH scaffolds, optical section and 3D reconstruction were conducted. 3. RESULTS AND DISCUSSION 3.1. Preparation and characterizations of Microcapsules-in-Hydrogel (MCH) scaffolds

Scheme 1. Schematic preparation process of MCH scaffolds inspired by loose connective tissue.

Figure 1. (a) SEM image of 1% w/v agarose hydrogel, (b) photograph of CPH that was casted into cubes (1 cm×1 cm×1 cm) using silicone mold during cooling process, (b1) optical micrograph and (b2, 3) SEM images of CPH (top view) and (c) FTIR spectra of (i) pristine


7


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 8 of 32

agarose powder, (ii) CaCO3 particles and (iii) CPH (after drying). The insets in (a) was the photograph of pristine agarose hydrogel.

Scheme 1 illustrated the preparation process of MCH scaffolds, where agarose hydrogel composed of agarose fibers was chosen as the ECM analog (Figure 1a) and hybrid microcapsules was in situ synthesized as the cell mimic. Initially, CPH was prepared by embedding CaCO3 particles into 1% w/v agarose hydrogel through sol-gel process. Specifically, 1% w/v agarose solution was prepared by adding agarose powder to deionized water and then heated to dissolve. CaCO3 particles were mixed with the agarose solution, which was cast into cubes (1 cm×1 cm×1 cm) using silicone mold, and cooled down to form cubic CPH. The successful embedding of CaCO3 particles in agarose hydrogel could be observed through the color change from transparent to milky white (insets in Figure 1a, Figure 1b). The CaCO3 particles were distributed in the porous hydrogel without aggregation (Figure 1b1). When amplifying the interfacial region, agarose fibers was observed to partially adhere on the surface of CaCO3 particles, showing a fiber-contacting structure (Figure 1b2 and b3). Considering the electrical neutrality of agarose, hydrogen bond interaction between the hydroxyl groups of agarose chains and the carbonate groups of CaCO3 particles may help agarose fibers to adhere onto the surface of CaCO3 particles. To verify the existence of the hydrogen bond interaction, we analyzed the FTIR spectra of pristine agarose powder, CaCO3 particles and CPH (Figure 1c). For CaCO3 particles, the bands at 743 and 876 cm-1 were assigned to the out-of-plane bending vibration and asymmetric stretching vibration of carbonate group, suggesting the vaterite form of CaCO3 particles.23,24 The band at 1083 cm-1 was assigned to symmetric stretch of CO32−. The bands at 1129 and 2925 cm-1 belonged to the characteristic absorption of PSS,25 which was pre-


8

Page 9 of 32

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


doped in the particles to stabilize the crystal form of CaCO3 particles.26 A broad band appeared at around 3448 cm-1 was assigned to the stretching vibration of -OH due to the presence of absorbed water, indicating the strong hydrophilicity of CaCO3 particles.27 The FTIR spectrum of pristine agarose powder exhibited bands at 931 and 1160 cm-1 which were assigned to the bending and stretching modes of C-O-C.28,29 The bands at 3575 cm-1 were attributed to the stretching modes of -OH.30 After embedding CaCO3 particles into the agarose hydrogel, the band assigned to -OH stretching mode shifted to a lower wavenumber from 3575 to 3426 cm-1. Moreover, the band of symmetric stretch of CO32− also shifted to a lower wavenumber from 1083 to 1075 cm-1. These blue shifts should be ascribed to the hydrogen bond interaction between agarose chains and CaCO3 particles,31 which was beneficial for adhering the agarose fibers onto the surface of CaCO3 particles. Therefore, the formation of CPH should include 1) the adsorption of agarose chains on CaCO3 particles through hydrogen bond interaction in solution and 2) the co-assembly of free agarose chains and CaCO3-adsorbed agarose chains through multiple physical interactions during sol-gel process.21,32

Figure 2. (a) Optical micrograph, (b) FTIR spectrum and (c) SEM image of MCH scaffolds.

Subsequently, CPH was alternately immersed in the solutions of protamine and Na2SiO3 to implement biomimetic mineralization in agarose hydrogel. Owing to the hierarchical self-


9


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 10 of 32

assembly of agarose chains, ~200 nm pore diameter could be obtained for 1% w/v agarose hydrogel determined by electrophoresis of DNA.33 Since the electrical neutral fibrous network of agarose hydrogel would hardly adsorb the positively charged protamine molecules, the spherical protamine molecules with a diameter of 2-4 nm could easily diffuse in agarose hydrogel driven by osmotic pressure.34,35 These protamine molecules would be adsorbed on the surface of the embedded, negatively charged CaCO3 particles through electrostatic interaction. Similarly, when immersed in Na2SiO3 solution, the silicate ions could also diffuse in agarose hydrogel and be enriched near the particle surface by the adsorbed protamine layer.36 The concentrated silicate ions would undergo hydrolysis/condensation, then forming a silica layer on the surface of the particles.16,37 The above process was repeated twice and capsule wall with two bilayers of Protamine/Silica (PSi) was obtained. Finally, MCH scaffolds were obtained by removing CaCO3 particles through EDTA treatment. As shown in Figure 2a, hybrid microcapsules with two bilayer of PSi (denoted as PSi2 hybrid microcapsules) were uniformly distributed in the agarose hydrogel inheriting from CaCO3 particles. Hereafter, MCH scaffolds with the in situ synthesized PSi2 hybrid microcapsules were denoted as PSi2-MCH scaffolds. The hollow structure of the in situ synthesized PSi2 hybrid microcapsules could be observed from the low contrast interior. The FTIR spectrum of PSi2-MCH scaffolds exhibited not only the characteristic absorption bands of agarose (1160 and 931 cm-1), but also the bands of protamine and silica at 1642 and 1078 cm-1,16 indicating the successful implementation of biomimetic mineralization in agarose hydrogel (Figure 2b).


10

Page 11 of 32

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


Figure 3. SEM images of (a, b) PSi2 hybrid microcapsules recovered from MCH scaffolds (denoted as PSi2-H hybrid microcapsules hereafter) and (c, d) PSi2 hybrid microcapsules prepared in water (denoted as PSi2-W hybrid microcapsules). The insets in (b, d) were TEM images of PSi2-H and PSi2-W hybrid microcapsules. Distribution of (e) the hole diameter on the surface of PSi2-H hybrid microcapsules and (f) the fiber diameter of frozen dried 1% w/v agarose hydrogel. The data of the hole diameter and fiber diameter was collected from the corresponding SEM images (Figure S1, Supporting Information) through "Nano Measure".

Considering the fiber-contacting structure of CPH, some agarose fibers may be intercalated into the wall of the in situ synthesized PSi2 hybrid microcapsules after biomimetic mineralization (Figure 2c), which was similar to the structure of collagen fibers of ECM


11


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 12 of 32

connecting with cell membranes.38,39 To demonstrate the fiber-intercalating structure of MCH scaffolds, agarose hydrogel was dissolved by heating and PSi2 hybrid microcapsules were recovered (denoted as PSi2-H hybrid microcapsules). For comparison, PSi2 hybrid microcapsules were also prepared in water (denoted as PSi2-W hybrid microcapsules). As shown in Figure 3a and c, both PSi2-H and PSi2-W hybrid microcapsules with a diameter of 4-6 µm kept standing after drying, suggesting the good mechanical stability of PSi2 hybrid microcapsules. Hollow structure of PSi2-H and PSi2-W hybrid microcapsules was verified by TEM images (insets in Figure 3b and d). Besides, the surface of both PSi2-H and PSi2-W hybrid microcapsules was composed of fused nanoparticles that should be generated through biomimetic mineralization (Figure 3b and d).40 Si element in EDS spectra for PSi2-H and PSi2W hybrid microcapsules further confirmed the successful implementation of biomimetic mineralization in agarose hydrogel and water (Figure S2, supporting information). N element originated from protamine was also observed for PSi2-H and PSi2-W hybrid microcapsules, indicating the hybridization of organic and inorganic moieties in the capsule wall. In addition, similar bands were observed in the FTIR spectra of PSi2-H and PSi2-W hybrid microcapsules. Briefly, the band at 1086 cm-1 was ascribed to the stretching vibration of Si-O-Si, whereas the bands at 3448, 1662 and 1545 cm-1 were ascribed to the absorption of primary amines belonging to protamine (Figure S3, supporting information). The above results evidenced the proposed mechanism of biomimetic mineralization in agarose hydrogel as depicted above. Interestingly, as shown in Figure 3b, numerous holes were observed on the wall of PSi2-H hybrid microcapsules, which was not found for PSi2-W hybrid microcapsules (Figure 3d). Upon this phenomenon, we conjectured that these holes should be generated through the dissolution of agarose fibers that intercalated into the wall of the in situ synthesized PSi2 hybrid microcapsules.35 To confirm this


12

Page 13 of 32

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


assumption, the diameter of these holes and agarose fibers were counted by "Nano Measure". As shown in Figure 3e, the diameter of the holes was distributed between 27 and 128 nm with an average value of ~68 nm. This value was in the range of the diameter of agarose fibers (50-100 nm for 1% w/v agarose hydrogel).41 However, the fiber diameter of frozen dried 1% w/v agarose hydrogel, obtained from the SEM image (Figure S1, supporting information), was distributed in 10-38 nm with a mean value of 21 nm (Figure 3f). The much decreased diameter should be attributed to the shrinkage of agarose fibers during lyophilization. Hence, we conjectured the dissolution of intercalated agarose fibers caused the generation of numerous holes (Figure 3b).

Figure 4. Optical micrographs of PSi2-W hybrid microcapsules (a) before and (b) after treated in 40 w/v% PSS solution for 4 h. (c) Optical micrograph of PSi2-MCH scaffolds after treated in 40 w/v% PSS solution for 4 h. (d) Percentage of deformed hybrid microcapsules in PSi2-MCH scaffolds and PSi2-W hybrid microcapsules as a function of PSS concentration.


13


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 14 of 32

As shown in Figure 2c, a typical fiber-intercalating structure was observed. Multiple adhesion forces, such as hydrogen bonding interaction between residual Si-OH and C-OH of fiber, frictional force and bite force arisen from the rough interface by fused silica nanoparticles, coexisted between the capsule wall and the intercalated agarose fibers. These multiple adhesion forces could help the agarose fibers to tightly associate with the capsule wall. This structure may, therefore, elevate the resistance to compression and expansion of the in situ synthesized hybrid microcapsules and help to fix the hybrid microcapsules tightly in the agarose hydrogel.42-44 To demonstrate the mechanical property of the in situ synthesized hybrid microcapsules in MCH scaffolds, compression test was conducted using the osmotic pressure created by differential PSS (average Mw ~70000) concentration between bulk solution and interior of the hybrid microcapsules.45 When the elastic restoring force of the hybrid microcapsules could not compensate the hydrostatic pressure difference, the hybrid microcapsules would get deformed. For comparison, PSi2-W hybrid microcapsules were also treated in PSS solutions. As shown in Figure 4d, the percentage of deformed microcapsules increased with the increase of PSS concentration (2-40 w/v%). In detail, only ~10% of the in situ synthesized PSi2 hybrid microcapsules in MCH scaffolds got deformed after treated in 40 w/v% PSS (Figure 4c), while ~44% of PSi2-W hybrid microcapsules got deformed (Figure 4b). This indicated that fiberintercalating structure did elevate the mechanical stability of the in situ synthesized hybrid microcapsules in MCH scaffolds. 3.2. Encapsulation of GOD in MCH scaffolds


14

Page 15 of 32

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


Figure 5. (a) Z-stack and (b) 3D reconstruction confocal microscopy images of FITC-labeled BSA-encapsulated PSi2-MCH scaffolds. The image under (b) was the side view reconstruction of y-z plane as pointed by the white arrow. 3D reconstruction was obtained from the z-stack confocal images using "ZEN 2009 Light Edition" image analysis software. Herein, FITC-labeled BSA was used as the substitute of GOD.

GOD was a homodimeric glycoenzyme (E.C. 1.1.3.4) that can catalyze the oxidation of β-Dglucose to H2O2 and gluconic acid with oxygen, which was usually coupled with peroxidase to determine the concentration of glucose by detecting the as-generated H2O2 through chromogenic reaction. GOD was widely used in bioconversion, diagnostics, food industry and biotechnologies and so on. Herein, GOD was chosen as a model enzyme to prepare and evaluate the immobilized biocatalyst. Specifically, GOD was encapsulated in CaCO3 particles during the co-precipitation process, which was then used to prepare PSi2-MCH scaffolds as illustrated in Scheme 1. FITClabeled BSA, substitute of GOD with similar pI value (4.2 for GOD vs 4.6 for BSA),46,47 was encapsulated in PSi2-MCH scaffolds to confirm the successful encapsulation and distribution of GOD. To better visualize the enzyme distribution in PSi2-MCH scaffolds, z-stack confocal microscopy image were obtained by collecting images along z-axis every 1 µm across a thickness of 14 µm (Figure S4, supporting information). Figure 5a and S5 showed that the


15


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 16 of 32

green fluorescence was primarily emitted from the interior and wall of hybrid microcapsules, indicating the completely encapsulation of FITC-labeled BSA in the in situ synthesized PSi2 hybrid microcapsules. Moreover, partial enrichment of FITC-labeled BSA around the capsule wall was observed (Figure 5a), which could be attributed to the attraction of FITC-labeled BSA by the positive charged protamine in the capsule wall. Similar phenomenon was also observed for FITC-labeled BSA-encapsulated PSi2-W hybrid microcapsules (Figure S6, supporting information). Enzyme distribution visualization was further performed by 3D reconstruction confocal microscopy image of FITC-labeled BSA-encapsulated PSi2-MCH scaffolds. Obviously, FITC-labeled BSA was encapsulated in the evenly distributed hybrid microcapsules of PSi2MCH scaffolds (Figure 5b and Video S1, Supporting Information). 3.3. Catalytic activity, recyclability and recycling stability of GOD-encapsulated MCH (GMCH) scaffolds

Figure 6. (a) Schematic catalytic conversion of glucose by G-MCH scaffolds and detection of H2O2 using TMB substrate through a second reaction catalyzed by HRP. (b) Colorimetric change of HAc-NaAc buffer containing TMB and HRP by adding different concentration of H2O2 and


16

Page 17 of 32

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


(c) the corresponding plot of UV-Vis absorption at 652 nm (A652) against the concentration of H2O2.

Figure 6a illustrated the schematic catalytic conversion of glucose by G-MCH scaffolds. To trigger the reaction, glucose and oxygen need to pass through the agarose hydrogel and capsule wall, and contact with the active sites of encapsulated GOD. For 1% w/v agarose hydrogel, the diffusion coefficient of glucose was ca. 6×10-6 cm2 s-1, which was only a bit lower than that of glucose in water (6.76×10-6 cm2 s-1).48,49 Hence, glucose with a diameter of 0.86 nm could diffuse freely in the agarose hydrogel.50 Moreover, pore diameter of the capsule wall of the in situ synthesized hybrid microcapsules was determined to be 3.49 nm (Figure S7), which was consistent with previous literature.16 Thus, glucose could diffuse into the interior of the in situ synthesized hybrid microcapsules through mesoporous capsule wall driven by concentration gradient. Similarly, the dissolved O2 molecules with much smaller diameter could also reach the active site of enzyme through diffusion. The glucose would react with O2 catalyzed by GOD, thus generating gluconic acid and H2O2. The generated H2O2 was detected through a colorimetric method with TMB as the substrate.51 As shown in Figure 6b, blue color was observed after adding H2O2 into HAc-NaAc buffer solution containing TMB and HRP, which could be assigned to the generation of oxTMB. The blue color got deeper gradually with the increase of H2O2 concentration. To quantitatively calculate the amount of generated H2O2, UV-Vis absorption values at 652 nm (A652) of the blue-colored solution were detected. Figure 6c showed the standard curve of A652 versus the concentration of H2O2. A good linear relationship with R2=0.998 was observed. The specific amount of the generated H2O2 could be obtained by converting A652 with the standard curve.


17


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 18 of 32

Figure 7. (a) Reaction kinetics of GOD-encapsulated PSi1-MCH scaffolds (cube: 1 cm×1 cm×1 cm), PSi2-MCH scaffolds (cube: 1 cm×1 cm×1 cm, cuboid: 1 cm×2 cm×2 cm, cube: 2 cm×2 cm×2 cm), PSi1-W hybrid microcapsules, PSi2-W hybrid microcapsules and 1% w/v agarose hydrogel (cube: 1 cm×1 cm×1 cm). Notably, the initially added GOD was identical for these seven immobilized biocatalysts. (b) Recycling stability of GOD-encapsulated PSi1-MCH scaffolds (cube: 1 cm×1 cm×1 cm), PSi2-MCH scaffolds (cube: 1 cm×1 cm×1 cm), PSi1-W hybrid microcapsules and PSi2-W hybrid microcapsules. Note: PSi1- and PSi2-MCH scaffolds were recycled after washing by tweezers as shown in the inset of (b) after each reaction, while PSi1- and PSi2-W hybrid microcapsules were recycled by centrifugation.

To elucidate the structure-performance relationship of the immobilized biocatalyst, we prepared GOD-encapsulated PSi2-MCH scaffolds, PSi2-W hybrid microcapsules, PSi1-MCH scaffolds and PSi1-W hybrid microcapsules for comparison. Besides, GOD-encapsulated 1% w/v agarose hydrogel was also prepared by simply embedding the GOD-encapsulated CaCO3 particles into agarose hydrogel followed by the removal of CaCO3 through EDTA treatment. The amount of initially added GOD was identical for these five immobilized biocatalysts and all of the agarose hydrogel was casted into cubes (1 cm×1 cm×1 cm). As expected, for 1% w/v agarose


18

Page 19 of 32

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


hydrogel, most of the added GOD (8.6 nm in diameter) got leached during the removing process of CaCO3 particles primary as a result of the rather large pore diameter of the agarose hydrogel (~200 nm).52 For PSi2-MCH scaffolds and PSi2-W hybrid microcapsules, ~95% and 94% of the initially added GOD was immobilized, indicating that PSi2 hybrid microcapsules prepared in both agarose hydrogel and water could well inhibit the GOD leaching. Because of the low enzyme leaching, both GOD-encapsulated PSi2-MCH scaffolds and PSi2-W hybrid microcapsules exhibited enhanced activity in comparison to GOD-encapsulated 1% w/v agarose hydrogel (Figure 7a). Additionally, the activity of GOD-encapsulated PSi2-W hybrid microcapsules was higher than GOD-encapsulated PSi2-MCH scaffolds (activity of initial 40 min: 5.2×10-2 vs 1.5×10-2 mM min-1). This may be arisen from the stagnation flow in agarose hydrogel, which increased the external diffusion resistance and lowered the catalytic activity of G-MCH scaffolds. To verify this hypothesis, PSi2-MCH scaffolds were also casted into cuboids (1 cm×2 cm×2 cm) and cubes (2 cm×2 cm×2 cm). Decreased activity along with the increase of hydrogel size confirmed the negative influence of external diffusion resistance caused by stagnation flow in hydrogel scaffolds. Interestingly, although suffering from higher diffusion resistance, the affinity to the substrate got increased after encapsulation (Km: 4.1 vs 16.8 mM for G-MCH scaffolds and free GOD, Table S1). Moreover, GOD-encapsulated PSi2-MCH scaffolds exhibited superiority in recyclability and recycling stability in comparison to GOD-encapsulated PSi2-W hybrid microcapsules. The inset of Figure 7b revealed that GOD-encapsulated PSi2-MCH scaffolds can be simply recycled after washing by tweezers because of the monolithic macroscale of agarose hydrogel, while centrifugation was required to collect GOD-encapsulated PSi2-W hybrid microcapsules. The loss and break of the hybrid microcapsules would then occur during the recycling process of GOD-


19


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 20 of 32

encapsulated PSi2-W hybrid microcapsules (Figure S8, supporting information). As a result, only 17% of the initial activity was retained for GOD-encapsulated PSi2-W hybrid microcapsules, while no activity decline was observed for GOD-encapsulated PSi2-MCH scaffolds after 7 cycles. Such enhanced recycling stability for GOD-encapsulated PSi2-MCH scaffolds should be ascribed to their easy recyclability and unique structure. In detail, agarose hydrogel could protect the in situ synthesized hybrid microcapsules, whereas fiber-intercalating structure could improve the mechanical stability, both of which prevented the breakage of microcapsules. The fiber-intercalating structure also fixed the in situ synthesized hybrid microcapsules in agarose hydrogel tightly, avoiding the loss of the hybrid microcapsules. To further illustrate the feasibility of acquiring immobilized biocatalyst from MCH scaffolds, the catalytic performance of GOD-encapsulated PSi1-MCH scaffolds and PSi1-W hybrid microcapsules were also evaluated (Figure 7 and S9, supporting information). With the decrease of the layer number, ultralow immobilization efficiency (less than 30%) was achieved for PSi1W hybrid microcapsules. This should be as a result of the poor mechanical stability of PSi1-W hybrid microcapsules that could be easily destroyed by the generated CO2 during the removal of CaCO3 particles. Surprisingly, ~93% of GOD was immobilized for PSi1-MCH scaffolds, which should be owing to the fact of the fiber-intercalating structure of PSi1-MCH scaffolds elevating the mechanical stability of PSi1 hybrid microcapsules and inhibiting the GOD leaching. Thus, GOD-encapsulated PSi1-MCH scaffolds exhibited higher activity compared to GODencapsulated PSi1-W hybrid microcapsules (activity of initial 40 min: 1.2×10-2 vs 3.6×10-3 mM min-1). Besides, GOD-encapsulated PSi1-MCH scaffolds also showed much enhanced recycling stability. Specifically, ca. 67% of the initial activity was retained after 7 cycles for GOD-


20

Page 21 of 32

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


encapsulated PSi1-MCH scaffolds, whereas no activity could be detected only after 3 cycles for GOD-encapsulated PSi1-W hybrid microcapsules. 3.4. pH, thermal and storage stability of G-MCH scaffolds

Figure 8. (a) pH, (b) thermal and (c) storage stability of G-MCH scaffolds and free GOD.

The biocompatible porous network, in situ synthesized PSi2 hybrid microcapsules and fiberintercalating structure of G-MCH scaffolds may provide an appropriate and stable microenvironment for the encapsulated GOD. Such micro-environment would confer the immobilized GOD with higher pH, thermal and storage stabilities than GOD in free form. As shown in Figure 8a, after incubating at pH 9 and 4, GOD-encapsulated PSi2-MCH scaffolds exhibited 88% and 97% of the initial activity, while only 49% and 62% of the initial activity were retained for free GOD. When incubating at pH 3, G-MCH scaffolds still exhibited higher relative activity than free GOD (62% vs 45%). The enhanced pH stability of G-MCH scaffolds should be resulted from the buffering effect of the in situ synthesized PSi2 hybrid microcapsules. Specifically, amine groups (=NH and –NH2) of protamine and residual Si-OH of silica in the capsule wall could go through protonation and deprotonation, and neutralize the invasive H+ and OH-.53,54 Besides, enhanced thermal stability was also observed for G-MCH scaffolds (Figure 8b). Particularly, after incubating at 60 oC, GOD-encapsulated PSi2-MCH scaffolds and free GOD


21


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 22 of 32

retained 56% and 19% of the initial activity, respectively. For free GOD, the thermal inactivation was mainly due to the dissociation of flavin adenosinedinucleotide (FAD) from GOD and conformational changes and nonspecific aggregation of enzymes. After encapsulation, the electronic interaction between negative charged GOD and positive charged protamine moiety of the capsule wall could stabilize the active conformation of the enzyme. In addition, the sulfate groups of PSS could strengthen the hydrophobic interactions and make GOD a more compact structure, which would inhibit the dissociation of FAD from GOD. The confined volume created by microcapsules might reduce the water activity and increase the thermal stability of encapsulated GOD. Moreover, enhanced storage stability was observed for G-MCH scaffolds (Figure 8c), which retained 85% of the initial activity after incubating at -4 oC for 22 days while only 38% was retained for free GOD. Collectively, G-MCH scaffolds exhibited enhanced recycling, pH, thermal and storage stabilities due to the hierarchical structure of MCH scaffolds. Nevertheless, the activity of G-MCH scaffolds was indeed lower than the nano/micro-sized immobilized GOD.55-57 Further enhancement of the activity without sacrificing the stability was still under research. 4. CONCLUSIONS In this study, an immobilized biocatalyst with high catalytic activity, recyclability and stability was designed and prepared based on Microcapsules-in-Hydrogel (MCH) scaffolds. Specifically, GOD was encapsulated in the in situ synthesized hybrid microcapsules of MCH scaffolds by biomimetic mineralization in agarose hydrogel. The immobilized biocatalyst showed structure similarity to loose connective tissue in the hierarchically organized hybrid microcapsules by fibrous framework of agarose hydrogel with fibers intercalating into the capsule wall. The GMCH scaffolds provided an appropriate and stable micro-environment for the encapsulated


22

Page 23 of 32

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


GOD. Briefly, the porous network of agarose hydrogel allowed the free diffusion of glucose molecules, retaining the enzyme activity as much as possible after immobilization. The monolithic macroscale of agarose hydrogel facilitated the easy recycling of G-MCH scaffolds, contributing to the zero activity decline during the recycling test. The fiber-intercalating structure elevated the mechanical stability of the in situ synthesized hybrid microcapsules, inhibiting the leaching and enhancing the stability of the encapsulated GOD. Hopefully, this study could shed light on the rational design of high-performance immobilized biocatalyst through the bioinspiration strategies. ASSOCIATED CONTENT Supporting Information. SEM images of PSi2-H hybrid microcapsules, 1% w/v agarose hydrogel, PSi2-W hybrid microcapsules before and after recycling, PSi1-H and PSi1-W hybrid microcapsules; EDS and FTIR spectra of PSi2-H and PSi2-W hybrid microcapsules; bright field and confocal microscopy images of FITC-labeled BSA-encapsulated PSi2-MCH scaffolds and PSi2-W hybrid microcapsules; N2 adsorption-desorption isotherm and pore size distribution of MCH scaffolds and 1% w/v agarose hydrogel; surface zeta-potential as a function of layer number for PSSdoped CaCO3 particles coated with (protamine/silica)n layers; relative activity GODencapsulated CPH, PSi2-CPH and PSi2-MCH scaffolds; kinetic parameter of G-MCH scaffolds and free GOD. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Fax: +86-22-27890566; Tel: +86-22-27890566 (Jiafu Shi)


23


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 24 of 32

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT The authors thank the National Natural Science Funds of China (21406163, 91534126) and the Program of Introducing Talents of Discipline to Universities (B06006) for financial support. REFERENCES (1) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Industrial Biocatalysis Today and Tomorrow. Nature 2001, 409, 258-268. (2) Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K. Engineering the Third Wave of Biocatalysis. Nature 2012, 485, 185-194. (3) Straathof, A. J. J. Transformation of Biomass into Commodity Chemicals Using Enzymes or Cells. Chem. Rev. 2014, 114, 1871-1908. (4) Magner, E. Immobilisation of Enzymes on Mesoporous Silicate Materials. Chem. Soc. Rev. 2013, 42, 6213-6222. (5) Feng, W.; Ji, P. Enzymes Immobilized on Carbon Nanotubes. Biotechnol. Adv. 2011, 29, 889-895. (6) Ariga, K.; Ji, Q.; Mori, T.; Naito, M.; Yamauchi, Y.; Abe, H.; Hill, J. P. Enzyme Nanoarchitectonics: Organization and Device Application. Chem. Soc. Rev. 2013, 42, 63226345. (7) Sheldon, R. A.; van Pelt, S. Enzyme Immobilisation in Biocatalysis: Why, What and How. Chem. Soc. Rev. 2013, 42, 6223-6235.


24

Page 25 of 32

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


(8) Pfruender, H.; Amidjojo, M.; Kragl, U.; Weuster-Botz, D. Efficient Whole-Cell Biotransformation in a Biphasic Ionic Liquid/Water System. Angew. Chem. Int. Ed. 2004, 43, 4529-4531. (9) Chen, Z.; Ji, H.; Zhao, C.; Ju, E.; Ren, J.; Qu, X. Individual Surface-Engineered Microorganisms as Robust Pickering Interfacial Biocatalysts for Resistance-Minimized Phase-Transfer Bioconversion. Angew. Chem. Int. Ed. 2015, 54, 4904-4908. (10) Fratzl, P.; Weinkamer, R. Nature’s Hierarchical Materials. Prog. Mater. Sci. 2007, 52, 1263-1334. (11) Ingber, D. E. Tensegrity I. Cell Structure and Hierarchical Systems Biology. J. Cell Sci. 2003, 116, 1157-1173. (12) West, J. L. Protein-Patterned Hydrogels: Customized Cell Microenvironments. Nat. Mater. 2011, 10, 727-729. (13) Humphrey, J. D.; Dufresne, E. R.; Schwartz, M. A. Mechanotransduction and Extracellular Matrix Homeostasis. Nat. Rev. Mol. Cell Biol. 2014, 15, 802-812. (14) Kubow, K. E.; Vukmirovic, R.; Zhe, L.; Klotzsch, E.; Smith, M. L.; Gourdon, D.; Luna, S.; Vogel, V. Mechanical Forces Regulate the Interactions of Fibronectin and Collagen I in Extracellular Matrix. Nat. Commun. 2015, 6, 8026. (15) Shi, J.; Jiang, Y.; Wang, X.; Wu, H.; Yang, D.; Pan, F.; Su, Y.; Jiang, Z. Design and Synthesis of Organic-Inorganic Hybrid Capsules for Biotechnological Applications. Chem. Soc. Rev. 2014, 43, 5192-5210. (16) Zhang, Y.; Wu, H.; Li, J.; Li, L.; Jiang, Y.; Jiang, Y.; Jiang, Z. Protamine-Templated Biomimetic Hybrid Capsules: Efficient and Stable Carrier for Enzyme Encapsulation. Chem. Mater. 2008, 20, 1041-1048.


25


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 32

(17) Jiang, Y.; Yang, D.; Zhang, L.; Sun, Q.; Sun, X.; Li, J.; Jiang, Z. Preparation of ProtamineTitania Microcapsules Through Synergy Between Layer-by-Layer Assembly and Biomimetic Mineralization. Adv. Funct. Mater. 2009, 19, 150-156. (18) Jiang, Y.; Sun, Q.; Zhang, L.; Jiang, Z. Capsules-in-Bead Scaffold: a Rational Architecture for Spatially Separated Multienzyme Cascade System. J. Mater. Chem. 2009, 19, 90689074. (19) Chen, K.; Zhou, S.; Yang, S.; Wu, L. Fabrication of All-Water-Based Self-Repairing Superhydrophobic Coatings Based on UV-Responsive Microcapsules. Adv. Funct. Mater. 2015, 25, 1035-1041. (20) Wylie, R. G.; Ahsan, S.; Aizawa, Y.; Maxwell, K. L.; Morshead, C. M.; Shoichet, M. S. Spatially Controlled Simultaneous Patterning of Multiple Growth Factors in ThreeDimensional Hydrogels. Nat. Mater. 2011, 10, 799-806. (21) Asenath-Smith, E.; Li, H. Y.; Keene, E. C.; Seh, Z. W.; Estroff, L. A. Crystal Growth of Calcium Carbonate in Hydrogels as a Model of Biomineralization. Adv. Funct. Mater. 2012, 22, 2891-2914. (22) Wang, F.; Feng, J.; Tong, W.; Gao, C. A Facile Pathway to Fabricate Microcapsules by in situ Polyelectrolyte Coacervation on Poly(styrene sulfonate)-Doped CaCO3 Particles. J. Mater. Chem. 2007, 17, 670-676. (23) Xu, A. W.; Dong, W. F.; Antonietti, M.; Colfen, H. Polymorph Switching of Calcium Carbonate Crystals by Polymer-Controlled Crystallization. Adv. Funct. Mater. 2008, 18, 1307-1313.


26

Page 27 of 32

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


(24) Xie, L.; Song, X.; Tong, W.; Gao, C. Preparation and Structure Evolution of Bowknot-Like Calcium Carbonate Particles in the Presence of Poly(sodium 4-styrene sulfate). J. Colloid Interface Sci. 2012, 385, 274-281. (25) Jang, J.; Ha, J.; Cho, J. Fabrication of Water-Dispersible Polyaniline-Poly(4styrenesulfonate) Nanoparticles for Inkjet-Printed Chemical-Sensor Applications. Adv. Mater. 2007, 19, 1772-1775. (26) Yu, J. G.; Guo, H.; Davis, S. A.; Mann, S. Fabrication of Hollow Inorganic Microspheres by Chemically Induced Self-Transformation. Adv. Funct. Mater. 2006, 16, 2035-2041. (27) Michel, F. M.; MacDonald, J.; Feng, J.; Phillips, B. L.; Ehm, L.; Tarabrella, C.; Parise, J. B.; Reeder, R. J. Structural Characteristics of Synthetic Amorphous Calcium Carbonate. Chem. Mater. 2008, 20, 4720-4728. (28) Bassett, D. C.; Marelli, B.; Nazhat, S. N.; Barralet, J. E. Stabilization of Amorphous Calcium Carbonate with Nanofibrillar Biopolymers. Adv. Funct. Mater. 2012, 22, 34603469. (29) Singh, T.; Trivedi, T. J.; Kumar, A. Dissolution, Regeneration and Ion-Gel Formation of Agarose in Room-Temperature Ionic Liquids. Green Chem. 2010, 12, 1029-1035. (30) Li, J. X.; Guo, Z. Q.; Zhang, S. W.; Wang, X. K. Enrich and Seal Radionuclides in Magnetic Agarose Microspheres. Chem. Eng. J. 2011, 172, 892-897. (31) Ye, C.; Malak, S. T.; Hu, K.; Wu, W.; Tsukruk, V. V. Cellulose Nanocrystal Microcapsules as Tunable Cages for Nano-and Microparticles. ACS Nano, 2015, 9, 10887-10895. (32) Arnott, S.; Fulmer, A.; Scott, W. E.; Dea, I. C. M.; Moorhouse, R.; Rees, D. A. The Agarose Double Helix and its Function in Agarose Gel Structure. J. Mol. Biol. 1974, 90, 269-284.


27


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 28 of 32

(33) Stellwagen, N. C. Electrophoresis of DNA in Agarose Gels, Polyacrylamide Gels and in Free Solution. Electrophoresis 2009, 30, S188-S195. (34) Jiang, Y.; Yang, D.; Zhang, L.; Li, L.; Sun, Q.; Zhang, Y.; Li, J.; Jiang, Z. Biomimetic Synthesis of Titania Nanoparticles Induced by Protamine. Dalton Trans. 2008, 4165-4171. (35) Richardson, J. J.; Liang, K.; Kempe, K.; Ejima, H.; Cui, J.; Caruso, F. Immersive Polymer Assembly on Immobilized Particles for Automated Capsule Preparation. Adv. Mater. 2013, 25, 6874-6878. (36) Shi, J.; Zhang, X.; Zhang, S.; Wang, X.; Jiang, Z. Incorporating Mobile Nanospheres in the Lumen of Hybrid Microcapsules for Enhanced Enzymatic Activity. ACS Appl. Mater. Interfaces 2013, 5, 10433-10436. (37) Haase, N. R.; Shian, S.; Sandhage, K. H.; Kröger, N. Biocatalytic Nanoscale Coatings Through Biomimetic Layer-by-Layer Mineralization. Adv. Funct. Mater. 2011, 21, 42434251. (38) Li, H.; Xin, H. L.; Muller, D. A.; Estroff, L. A. Visualizing the 3D Internal Structure of Calcite Single Crystals Grown in Agarose Hydrogels. Science 2009, 326, 1244-1247. (39) Li, H.; Estroff, L. A. Calcite Growth in Hydrogels: Assessing the Mechanism of PolymerNetwork Incorporation into Single Crystals. Adv. Mater. 2009, 21, 470-473. (40) Sumper, M.; Brunner, E. Silica Biomineralisation in Diatoms: the Model Organism Thalassiosira Pseudonana. ChemBioChem 2008, 9, 1187-1194. (41) Tietz, D.; Chrambach, A. Analysis of Convex Ferguson Plots in Agarose Gel Electrophoresis by Empirical Computer Modeling. Electrophoresis 1986, 7, 241-250. (42) Liu, R.; Shi, Y.; Wan, Y.; Meng, Y.; Zhang, F.; Gu, D.; Chen, Z.; Tu, B.; Zhao, D. Triconstituent Co-Assembly to Ordered Mesostructured Polymer-Silica and Carbon-Silica


28

Page 29 of 32

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


Nanocomposites and Large-Pore Mesoporous Carbons with High Surface Areas. J. Am. Chem. Soc. 2006, 128, 11652-11662. (43) Yashchenok, A. M.; Bratashov, D. N.; Gorin, D. A.; Lomova, M. V.; Pavlov, A. M.; Sapelkin, A. V.; Shim, B. S.; Khomutov, G. B.; Kotov, N. A.; Sukhorukov, G. B.; Möhwald, H.; Skirtach, A. G. Carbon Nanotubes on Polymeric Microcapsules: Free-Standing Structures and Point-Wise Laser Openings. Adv. Funct. Mater. 2010, 20, 3136-3142. (44) Sagis, L. M. C.; de Ruiter, R.; Miranda, F. J. R.; de Ruiter, J.; Schroën, K.; van Aelst, A. C.; Kieft, H.; Boom, R.; van der Linden, E. Polymer Microcapsules with a Fiber-Reinforced Nanocomposite Shell. Langmuir 2008, 24, 1608-1612. (45) Wang, X.; Shi, J.; Jiang, Z.; Li, Z.; Zhang, W; Song, X.; Ai, Q.; Wu, H. Preparation of Ultrathin, Robust Protein Microcapsules through Template-Mediated Interfacial Reaction between Amine and Catechol Groups. Biomacromolecules 2013, 14, 3861-3869. (46) Davis, S. C.; Sheppard, V. C.; Begum, G.; Cai, Y.; Fang, Y.; Berrigan, J. D.; Kröger, N.; Sandhage, K. H. Rapid Flow-Through Biocatalysis with High Surface Area, EnzymeLoaded Carbon and Gold-Bearing Diatom Frustule Replicas. Adv. Funct. Mater. 2013, 23, 4611-4620. (47) Strozyk, M. S.; Chanana, M.; Pastoriza-Santos, I.; Pérez-Juste, J.; Liz-Marzán, L. M. Protein/Polymer-Based Dual-Responsive Gold Nanoparticles with pH-Dependent Thermal Sensitivity. Adv. Funct. Mater. 2012, 22, 1436-1444. (48) Weng, L.; Liang, S.; Zhang, L.; Zhang, X.; Xu, J. Transport of Glucose and Poly(ethylene glycol)s in Agarose Gels Studied by the Refractive Index Method. Macromolecules 2005, 38, 5236-5242.


29


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 30 of 32

(49) Mogi, N.; Sugai, E.; Fuse, Y.; Funazukuri, T. Infinite Dilution Binary Diffusion Coefficients for Six Sugars at 0.1 MPa and Temperatures from (273.2 to 353.2) K. J. Chem. Eng. Data 2007, 52, 40-43. (50) Cheema, U.; Rong, Z.; Kirresh, O.; MacRobert, A. J.; Vadgama, P.; Brown, R. A. Oxygen Diffusion through Collagen Scaffolds at Defined Densities: Implications for Cell Survival in Tissue Models. J. Tissue Eng. Regener. Med. 2012, 6, 77-84. (51) Zhang, S.; Jiang, Z.; Wang, X.; Yang, C.; Shi, J. Facile Method to Prepare Microcapsules Inspired by Polyphenol Chemistry for Efficient Enzyme Immobilization. ACS Appl. Mater. Interfaces 2015, 7, 19570-19578. (52) Wei, W.; Song, Y.; Shi, W.; Lin, N.; Jiang, T.; Cai, X. A High Sensitivity MEA Probe for Measuring Real Time Rat Brain Glucose Flux. Biosens. Bioelectron. 2014, 55, 66-71. (53) Li, J.; Jiang, Z.; Wu, H.; Zhang, L.; Long, L.; Jiang, Y. Constructing Inorganic Shell onto LBL Microcapsule through Biomimetic Mineralization: a Novel and Facile Method for Fabrication of Microbioreactors. Soft Matter 2010, 6, 542-550. (54) Milligan, A. J.; Morel, F. M. M. A Proton Buffering Role for Silica in Diatoms. Science 2002, 297, 1848-1850. (55) Wang. J. Carbon-Nanotube Based Electrochemical Biosensors: A Review. Electroanalysis 2005, 17, 7-14. (56) Novak, M. J.; Pattammattel, A.; Koshmerl, B.; Puglia, M.; Williams, C.; Kumar C. V. “Stable-on-the-Table” Enzymes: Engineering the Enzyme-Graphene Oxide Interface for Unprecedented Kinetic Stability of the Biocatalyst. ACS Catal. 2016, 6, 339-347.


30

Page 31 of 32

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


(57) Scodeller, P.; Flexer, V.; Szamocki, R.; Calvo, E. J.; Tognalli, N.; Troiani, H.; Fainstein A. Wired-Enzyme Core-Shell Au Nanoparticle Biosensor. J. Am. Chem. Soc. 2008, 130, 12690-12697.


31


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 32 of 32

TOC graphic


32

An Efficient, Recyclable, and Stable Immobilized Biocatalyst Based on

Recommend Documents