Letter www.acsami.org
Enzymatic Inverse Opal Hydrogel Particles for Biocatalyst Huan Wang,† Hongcheng Gu,† Zhuoyue Chen, Luoran Shang, Ze Zhao, Zhongze Gu, and Yuanjin Zhao* State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China S Supporting Information *
ABSTRACT: Enzymatic carriers have a demonstrated value for chemical reactions and industrial applications. Here, we present a novel kind of inverse opal hydrogel particles as the enzymatic carriers. The particles were negatively replicated from spherical colloidal crystal templates by using magnetic nanoparticles tagged acrylamide hydrogel. Thus, they were endowed with the features of monodispersity, small volume, complete penetrating structure, and controllable motion, which are all beneficial for improving the efficiency of biocatalysis. In addition, due to the ordered porous nanostructure, the inverse opal hydrogel particles were imparted with unique photonic band gaps (PBGs) and vivid structural colors for encoding varieties of immobilized enzymes and for constructing a multienzymes biocatalysis system. These features of the inverse opal hydrogel particles indicate that they are ideal enzymatic carriers for biocatalysis. KEYWORDS: inverse opal, hydrogel, particles, biocatalysis, photonic crystal
1. INTRODUCTION Enzymes are powerful biological catalysts for a broad variety of chemical reactions. Benefiting from their impressive levels of stereospecificity, regioselectivity, and chemoselectivity,1,2 enzymes have found numerous industrial applications.3 During these processes, enzymes could be dispersed and used directly in solution for achieving a high efficiency of biocatalysts, although this usually reduces the recycle and reuse of the enzymes because of the difficulty of enzyme separation from products. To improve the recovery and reuse efficiency of the biocatalysis, researchers need to immobilize enzymes on or in specific carriers, as well as optimize their activity and stability.4−7 To date, a variety of carriers, such as porous materials8 and Pickering emulsion,9−11 have been developed for enzyme immobilization. However, most of the carriers can only provide a limited surface area for the enzymes immobilization, or provide irregular or nonconnected pore structure, which prevents the free flow of the substrates.12,13 This is exacerbated by the bulk volume and uncontrolled motion of these carrier materials,14 which restrict the biocatalysts efficiency and cause the catalysis mechanism to be more complex. In addition, because of the indistinguishability of the enzymatic carriers, the investigation of the multienzymes biocatalysis systems is lacking.15,16 Thus, the creation of novel enzymatic carriers is still anticipated. In this paper, we employed inverse opal hydrogel particles as the desired enzymatic carriers. Inverse opals are a kind of structural materials with three-dimensionally ordered macro© XXXX American Chemical Society
porous, which are negatively replicated from colloidal crystal templates.17−25 The homogeneous pore structure endows the inverse opal materials with huge specific surface areas for the enzymes immobilization and interconnected channels for the access of the substrates. Thus, the efficiency of biocatalysts can be improved to some extent by employing the inverse opal materials, which are usually based on film or its derivate morphology. However, the restrictions of incomplete penetrating structure, bulk volume, uncontrolled motion, and indistinguishable identity, still exist for these enzymatic inverse opal carriers. To overcome these bottlenecks, we herein used magnetic nanoparticles-tagged hydrogel to negatively replicate spherical colloidal crystal templates. The resultant inverse opal hydrogel particles are endowed with the features of complete penetrating structure, monodispersity, small volume and controlled motion, which are all beneficial for improving the efficiency of biocatalysts. More attractively, as the hydrogel particles had ordered porous structure, they were imparted with interesting optical properties and vivid structural colors, which could be employed for encoding different kinds of immobilized enzymes and constructing a multienzymes biocatalysis system.26−29 These features make the inverse opal hydrogel particles described here ideal for biocatalysis applications. Received: February 8, 2017 Accepted: April 4, 2017 Published: April 4, 2017 A
DOI: 10.1021/acsami.7b01866 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
2. RESULTS AND DISCUSSION In a typical experiment, the inverse opal hydrogel particles were fabricated by replicating spherical silica colloidal crystal templates, as indicated in Scheme 1. These colloidal crystal
enzymatic particles on the bottom of a vessel were generated with superb capability to response to the extra magnetic field (Figure 1a−c and Movie S1). Besides the simple two-
Scheme 1. Illustration of the Formation of the Enzymatic Inverse Opal Hydrogel Particles and the Enzymatic Catalysis Process: (a) Spherical Silica Colloidal Crystal; (b) Hybrid Particle; (c) Inverse Opal Particle; (d) Enzyme-Immobilized Inverse Opal Particle
Figure 1. Magnetic response of the magnetic nanoparticles-tagged inverse opal hydrogel particles (a−c) on the bottom of a vessel and (d−f) in a tube.
dimensional plane surface, the particles could also be regulated in three-dimensional (3D) space. In a simple demonstration process, the inverse opal hydrogel particles were placed on the bottom of bottle at first (Figure 1d). When the magnet was positioned on the side of the bottle, the inverse opal hydrogel particles rose from the bottom and moved to the side near the magnet (Figure 1e), whereas the particles sank down to the bottom when the magnet was removed (Figure 1f). These results indicated that the inverse opal hydrogel particles had the capability to be separated and enriched from the solution by magnet easily, which benefited the recovery and reuse for reducing the cost. The microstructures of the inverse opal hydrogel particles and their spherical silica colloidal crystal templates (Figure S1a) were investigated by using a scanning electron microscope (SEM). It was observed that the monodispersed silica nanoparticles on the surface of the templates mainly formed an ordered hexagonal alignment (Figure 2a), and the ordered arrangement also extended to the inside of the spherical silica colloidal crystal templates (Figure 2b). Thus, the hydrogel particles replicated from the templates should have a similar highly ordered 3D inverse porous structure. However, as the hydrogel scaffolds of the inverse opal particles used for the enzyme immobilization were generated with relatively low concentrations and cross-linking degree, they tended to shrink and collapse during the drying process (Figures S1b and S2). Thus, to further investigate the actual nanostructure of the replaced particles, a high concentration of acrylamide hydrogel was employed to maintain the nanostructure during drying. It can be observed that the replicated hydrogel particles had a hexagonal symmetrical porous surface (Figure 2c), and the pores were also interconnected and extended inside the particles (Figure 2d). This complete penetrating inverse opal nanostructure can not only provide a high surface for the enzyme immobilization but also offer an easier access for the biocatalysis molecules. To conduct the biocatalysis research, we employed the inverse opal hydrogel particles with different sizes of nanopores and overall diameters as the enzymatic carriers. Usually, the catalytic efficiency of the porous enzymes carriers was restricted
templates with high monodispersity were prepared by drying of silica nanoparticles in microfluidic droplets. The silica nanoparticles became closely packed and formed an ordered structure in the bead templates during the evaporation of water. This ordered packing of the nanoparticles forms connected nanochannels throughout the templates for infiltration of the pregel solution. During the replicating process, the hydrophilically modified spherical colloidal crystal templates were immersed in the pregel solution, which could penetrate into the voids between the nanoparticles of the templates by capillary force. Then, the mixture was exposed to ultraviolet light to polymerize the pregel solution in and out of the templates. After removing the hydrogel outside the colloidal crystal templates and etching the silica nanoparticles in the templates, inverse opal hydrogel particles were obtained. The scaffold of the inverse opal hydrogel particles was mainly composed of acrylamide (AAm) and N,N′-methylenebis(acrylamide) (Bis). AAm is a kind of functional monomer with amide group, which can keep free after polymerization and hydrolyzed by NaOH and N,N,N′,N′-tetramethylethylenediamine (TEMED) to generate carboxyl group.30 The carboxyl group can form covalent bond with the molecules with amino group, such as proteinaceous enzymes, after excited by N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride crystalline (EDC) and N-hydroxysuccinimide (NHS). Thus, the concentration of the AAm in the hydrogel was closely related to the amount of the immobilized enzymes. It was found that although the hydrogel particles with low ratio of AAm and Bis could provide more inner pores for enzymes immobilization and substrate solution diffusion, the mechanical strength of the particles was decreased and the inverse opal structure became difficult to maintain. Therefore, an optimized concentration of AAm and Bis at 30 wt % and ratio of 29:1 were selected to balance the demand between the mechanical strength and porosity of the enzymatic carriers. To impart the functionality of controllable movement to the enzymatic inverse opal hydrogel particles, we tagged magnetic nanoparticles in the net of the hydrogel scaffold. It was found that with the addition of the magnetic nanoparticles, the B
DOI: 10.1021/acsami.7b01866 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
concentration of enzyme (Figure S5). It was found that in all cases the catalytic efficiencies of proposed hydrogel particles were amplified by about 500% than the same structured films (Figure S6). The maximum enzyme activities of HRP and urease immobilized particles were up to 934 and 575 U/g, which means the minimum mass of the immobilized enzymes were about 4.67 and 14.2 mg/g, respectively. These results indicated the importance of the complete penetrating structure, small volume and controlled motion of the enzymatic carriers during working. On the basis of the results in Figure 3a, b, it was also found that the larger nanopores structure resulted in a higher catalysis activity of HRP, while causing a lower catalysis activity of urease. Theoretically, at a fixed volume of inverse opal materials, smaller nanopores represents a larger total surface for the enzyme immobilization, thus the catalysis activity should be improved, as was the case of urease. However, because of the fast reaction of the HRP catalysis, the products might aggregate and precipitate in the nanopores, which might even block the nanochannels (Figure S7). Thus, the enzymatic hydrogel carriers with smaller inverse nanopores could show their advantages of larger surface, while the carriers with larger nanopores could provide more stable nanochannels during the biocatalysis. As the enzyme carriers suffered from the precipitate of products and the loss of enzyme activities after repeatedly washing, the enzyme activities of the particles decreased every cycle gradually.14 The effects of the diameters of the enzymatic inverse opal hydrogel particles on the activities of biocatalyst were also investigated, as shown in Figure 3c, d. During this process, four kinds of inverse opal hydrogel particles with same nanopores but with different particle diameters were employed for the HRP and urease biocatalysis. It was found that in both cases the catalytic efficiency improved along with the decrease of the
Figure 2. (a, b) SEM images of a spherical silica colloidal crystal template: (a) high-magnification surface, (b) low-magnification crosssection. (c, d) SEM images of an inverse opal hydrogel particle: (c) high-magnification surface, (d) low-magnification cross-section. Scale bars are 500 nm in a, c and 2 μm in b, d.
by the incomplete penetrating nanostructure and the bulk volumes, both of which hindered the free flow of the substrate and products. But in our experiment, the inverse opal hydrogel particles were generated with connected nanopores and controllable overall diameters, thus they should exhibit high catalytic efficiency. To demonstrate this hypothesis, four kinds of uniform inverse opal hydrogel particles with different size of nanopores were immobilized with HRP and urease for the biocatalysis (Figure 3a, b and Figure S3). The immobilized enzymes were distributed uniformly in the particles (Figure S4) and their biocatalysis efficiency is closely related to the
Figure 3. (a, b) Effects of the sizes of the inverse pores on the activities of (a) HRP-immobilized and (b) urease-immobilized inverse opal hydrogel particles. (c, d) Effects of the diameters of the enzymatic carriers on the activities of (c) HRP-immobilized and (d) urease-immobilized inverse opal hydrogel particles. The number of cycles was 5. C
DOI: 10.1021/acsami.7b01866 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 4. (a) Schematic illustration and (b) the relative result of an encoded multienzymes biocatalysis system.
carriers not only had good interference tolerance for the impurities mixed with the corresponding substrate, but also kept a high specificity to their substrates. These features implied that the encoded inverse opal hydrogel particles were ideal carriers of multienzymes biocatalysis system for qualitative processing of complex samples, especially for those that need cascade biocatalysis processes.
diameter of the inverse opal hydrogel particles. This should be explained that the larger inverse opal hydrogel particles were harder for the substrate and products getting in and out, and thus the immobilized enzymes could not exhibit entire capacity. The loss of enzyme activities after every cycle should be ascribed to the activities decrease of the immobilized enzymes after repeated washing and the accumulation of the reaction products in the nanopores, which hindered the substrate reaching the active site. It was worth noting that the interconnected inverse nanopores could not only provide nanochannels for biocatalysis, but also form a photonic band gap (PBG) in the inverse opal hydrogel particles. Thus, the enzymatic carriers were imparted with vivid structural colors and corresponding characteristic reflection peaks (Figure S8). Under normal incidence, the reflection peak positions λ of the hydrogel particles could be estimated by Bragg’s law
λ = 1.633dnaverage
3. CONCLUSION In summary, we have developed a new kind of enzymatic inverse opal carriers by using magnetic nanoparticles-tagged acrylamide hydrogel to negatively replicate spherical colloidal crystal templates. These particles were with high monodispersity, small volume, complete penetrating structure, and controllable motion, which were all beneficial for improving the efficiency of biocatalysis. As the inverse opal hydrogel particles were with unique PBGs and vivid structural colors, they were employed for encoding varieties of immobilized enzymes for constructing multienzymes biocatalysis systems. These features of the enzymatic inverse opal hydrogel particles indicated their potential values for chemical reactions and industrial applications.
(1)
where d is the center-to-center distance of two neighboring nanopores, and naverage is the average refractive index of the inverse opal hydrogel particles. Therefore, by using the spherical colloidal crystal templates with different silica nanoparticle compositions, a series of inverse opal hydrogel particles with different diffraction-peak positions and colors could be obtained (Figure S9), which can be derived for encoding different enzymes and constructing multienzymes biocatalysis system. To implement this concept, we immobilized three kinds of red, green, and blue structural-color encoded inverse opal hydrogel particles with HRP, urease, and amylase, respectively. These encoded enzymatic carriers were then mixed together and used for multienzymes biocatalysis, as schemed in Figure 4a. It was found that when the three kinds of encoded enzymatic carriers were incubated in the mixture of H2O2, phenol, urea, and starch, all three kinds of the enzymes exhibited high enzyme catalytic activities as their correspondingly catalyzed products or the reduce of the substrate could be detected (Figure 4b). Besides, the different combination of two kinds of encoded enzymatic carriers were also mixed and incubated in the mixture of urea, starch, H2O2 and phenol for the specificity investigation, as shown in Figure S10. It was found that the substrates could be degraded with high enzyme activity only when their corresponding enzyme carriers existed, whereas the substrates had little change and the products were undetectable when the corresponding enzyme carriers were absent. These results indicated that the encoded enzymatic
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
S Supporting Information *
The following files are available free of charge. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01866. Materials and experimental details, the SEM images of whole silica colloidal crystal bead and the collapsed inverse opal hydrogel; comparison of the activities of the inverse opal film and the inverse opal particles; the metalloscope images of the silica colloidal crystal bead, the inverse opal hydrogel particle, and the inverse opal hydrogel particle with enzyme; the reflection images of three kind of inverse opal hydrogel particles and the corresponding spectrum; the multiple biocatalysis results and the movie of magnetic response (PDF) Movie S1 (AVI)
Corresponding Author
*E-mail:
[email protected]. ORCID
Zhongze Gu: 0000-0001-8926-7710 Yuanjin Zhao: 0000-0001-9242-4000 D
DOI: 10.1021/acsami.7b01866 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces Author Contributions
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†
H.W. and H.G. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (Grants 21473029 and 51522302), the NSAF Foundation of China (Grant U1530260), the National Science Foundation of Jiangsu (Grant BK20140028), the Program for New Century Excellent Talents in University, the Scientific Research Foundation of Southeast University, the Graduate Innovation Program of Jiangsu (Grant KYLX16_0288), and the Scientific Research Foundation of Graduate School of Southeast University. Hongcheng Gu also thanks the Postdoctoral Science Foundation of China and Jiangsu.
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