Biomimetic Copper-Based InorganicâProtein Nanoflower Assembly

Subscriber access provided by University of Sussex Library

Article

Biomimetic Copper-based Inorganic-Protein Nanoflower Assembly Constructed on the Nanoscale Fibrous Membrane with Enhanced Stability and Durability Mufang Li, Mengying Luo, Fei Li, Wenwen Wang, Ke Liu, Qiongzhen Liu, Yuedan Wang, Zhentan Lu, and Dong Wang J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b03537 • Publication Date (Web): 13 Jul 2016 Downloaded from http://pubs.acs.org on July 14, 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.

The Journal of Physical Chemistry C 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

The Journal of Physical Chemistry

Biomimetic Copper-based Inorganic-Protein Nanoflower Assembly Constructed on the Nanoscale Fibrous Membrane with Enhanced Stability and Durability

Mufang Li, Mengying Luo, Fei Li, Wenwen Wang, Ke Liu, Qiongzhen Liu, Yuedan Wang, Zhentan Lu, Dong Wang*

College of Materials Science and Engineering, Wuhan Textile University, Wuhan, 430200

*Corresponding author: Dong Wang, Email: [email protected] Tel: +86 18627071668 Fax: +86 (27) 59367691

1

ACS Paragon Plus Environment


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

ABSTRACT: Copper-based inorganic-protein hybrid nanoflowers were constructed on the surface of a nanofiber membrane, creating a highly biocompatible, biomimetic and multilevel surface. The inorganic-enzyme nanoflowers exhibited enhanced durability and stability arising in part from the protection of the enzyme by the inorganic crystals. The high specific surface area and abundant functional groups of Cu2+ growth on the nanofiber membrane enables the dense and compact growth of Cu3(PO4)2·3H2O-protein nanoflowers. The structure, morphology, and crystallization of the copper-based inorganic-protein nanoflowers were analyzed, which led us to propose a formation mechanism of the hybrid nanoflower. The composition and structure of the copper-based inorganic-protein nanoflowers on the nanofiber membrane could be controlled by varying the structure of the nanofiber membrane, formulation of mineralizing solutions, concentraiton of protein, and growing time. Different structures of protein, such as BSA, papain, laccase and HRP show different morphologies. The construction of copper-based inorganic-protein nanoflowers on nanoscale fibrous membrane opens new possibilities for preparing biomimetic multilevel structured materials and makes them an ideal material for biosensor, biocatalysis, bioengineering and biodevices.

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


INTRODUCTION

The construction of biomimetic multilevel structured materials has drawn considerable interest potential applications of photonics, biocatalyst, biomedical implants, drug delivery, and biochemical separations.1-5 It has been demonstrated that the morphological and functional diversity of biomolecular architectures significantly depend on the contribution of inorganic materials in the formation process.6-7 Biomineralization is a typical process which reflects the remarkable level of organizing inorganic materials in living organisms.8-11 Accordingly, such natural biomineralization has inspired strategies for the synthesis of biomimetic multilevel structured materials. Although the variety of biomolecular architectures seems to be almost infinite, inorganic-organic materials based on calcium-, silicon-, and iron- are most common. 12, 13

Copper is the one of the most abundant inorganic components, while protein is the

most predominant constituent of living organisms.14 The coodination chemisty of copper protein has attracted a lot of interest.15, 16 However, there are few reports that focus on the copper-based inorganic-organic biomimetic materials. Recently, Zare et al. reported a method to synthesize hybrid organic-inorganic nanoflowers in solution using copper ions and various protein.17 Due to the high specific surface area and confinement of enzymes in the nanoflowers, the hybrid organic-inorganic nanoflowers exhibited enhanced enzymatic aciticity, durability and stability compared with the free enzyme.18-21 The biomimetic mineralization of copper and protein also provides a general method for encapsulating bioactive organics within a protective inorganic 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

framework.7 In contrast to previous reported free standing nanoflowers, this study fabricated enzyme-cupric phosphate hybrid nanoflowers on a controlled platform. Immobilization is an effective way to promote the stability, reusability and availability of the enzyme, thus making them an ideal material for a broad scope of applications, such as sensing, luminescence, diagnosis, tissue engineering and so on. 22, 23 For example, practical uses are

immobilization

of

horseradish

peroxidase

(HRP)

on

poly

(aniline-co-N-methylthionine) film to prepare hydrogen peroxide biosensor, immobilization of laccase on the electrode surface to design a biosensor for the detection of phenolic compound and immobilization of cysteine-modified azurin on nanopatterned surface for biodevices.24-26 Due to the very high aspect ratios, physical structures, and functional groups, nanofibers are recognized as very appropriate biomaterials which could be used in various fields including tissue engineering, drug delivery, separation and purification, wound dressing and sensor devices.27-29 In this study, we constructed copper-based inorganic-protein biomimetic nanoflowers on the surface of nanoscale fibrous membrane by a fast and simple method. The structure, morphology, crystallization and activity of the copper-based inorganic-protein nanoflowers were analyzed. Moreover, different kinds of proteins including BSA, HRP, papain and laccase were selected to test and verify the formation process. Accordingly, we proposed the formation mechanism of copper-based inorganic-protein nanoflowers. The storage stability and pH stablity of constructed copper inorganic-enzyme nanoflowers became better 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


compared with that of free enzyme. The immobilized copper-based inorganic-protein nanoflowers exhibit potential application in the fields of biodevices, biosensors, biocatalysis, and also provide new ideas for preparing biomimetic multilevel structured materials.

EXPERIMENTAL SECTION

Materials. Cellulose acetate butyrate (CAB, 381-20, butyryl content 37 wt%, acetyl content 13.5 wt% and hydroxyl content 1.8 wt%), was purchased from Acros Chemical (Pittsburg, PA). The melting point of CAB is 195-205 °C, and the molecular weight is 70,000 g/mol. Poly(vinyl alcohol-co-ethylene) copolymer (PE content 44%), was purchased from Sigma-Aldrich Milwaukee, WI. Acetone, sodium hydroxide, hydrogen peroxide (H2O2), copper sulfate, sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate and guaiacol were purchased from Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin (BSA), papain, laccase, cyanuric chloride, dioxane, iminodiacetic acid (IDA) were purchased from Aladdin (shanghai). Horseradish peroxidase (HRP) was provided by Biosharp. Preparation of Nanofiber Membrane. The PVA-co-PE nanofibers are prepared by previously invented methods in our group.30,

31

After dispersing the PVA-co-PE

nanofibers in the certain solution, the PVA-co-PE/PET composite membrane was prepared by coating the PVA-co-PE nanofibers suspension on the flat PET spunbond nonwoven substrate and subsequently peeling off the PET spunbond nonwoven 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

substrate from the PVA-co-PE/PET composite membrane. Surface Activation of PVA-co-PE Nanofiber Membranes. The PVA-co-PE nanofiber membranes were soaked into the 3 M sodium hydroxide solutions for 60 minutes at 30 °C. Then, the nanofiber membranes were immersed into the dioxane solution containing 10 wt% of cyanuric chloride for 2 hours at 30 °C. After removal from the solution, the cyanuric chloride activated nanofiber membranes were thoroughly rinsed with the solvent and deionized water. Adsorption of Copper Ions on the Surface of Nanofiber Membrane. 2 M Iminodiacetic acid (IDA) solution was prepared by adding IDA into the 4 M sodium hydroxide solutions. The cyanuric chloride activated nanofiber membranes were immersed into the 2 M iminodiacetic acid (IDA) solution for 2 hours at 50 °C. The copper ions (Cu2+) were adsorbed onto the surface of nanofiber membrane by immersing the IDA functionalized nanofiber membranes into 0.03 M CuSO4 solution for 12 hours at room temperature. After that, the nanofiber membranes were thoroughly rinsed with the deionized water to remove the free Cu2+. Construction of Copper-Based Inorganic-Protein Nanoflowers. Different kinds of proteins or enzyme including BSA, HRP, papain, laccase were added to the 0.01 M phosphate buffered saline (PBS, pH 7) respectively at a certain concentration. The Cu2+ functionalized nanofiber membranes were immersed into the pristine PBS and protein/PBS solution for different times at 3 °C to construct copper-based inorganic-protein nanoflowers on the surface. As control, the free-standing nanoflowers were prepared by adding 0.1 ml 120 mM CuSO4 solution into 15 ml 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


protein/PBS solutions at 37 °C. After 48 hours, the free standing nanoflowers were collected by filtration with the cellulose microfiltration membrane. Characterization. The chemical structures of pristine PVA-co-PE nanofiber membrane, cyanuric chloride activated, IDA functionalized nanofiber membranes were characterized by Fourier Transform Infrared-Attenuated Total Reflectance (FTIR-ATR, Tensor 27, Bruker). Afte rremoval from the PBS and protein/PBS solution, the surface morphology and structure of Cu2+ functionalized nanofiber membrane, nanofiber membrane in pristine PBS solution, and nanofiber membrane in protein/PBS solution were studied by the Scanning Electron Microscope (SEM, Hitachi X-650) and X-ray photoelectron spectrometer (XPS, MULT1LAB2000). The crystal strucure of the copper-based inorganic-protein nanoflowers were analyzed by the XRD (X′Pert PRO) and TEM (JEM2100F). The constructed Cu3(PO4)2·3H2O-HRP hybrid nanoflowers were used to catalyze the oxidation reaction of guaiacol by H2O2. To investigate activity and stability of the immobilized hybrid nanoflowers, 1 cm2 nanofiber membrane with hybrid nanoflowers prepared in the HRP concentration of 0.5 g/L, and the same amount of free HRP (same amount as the rough amount of HRP on the 1cm2 membrane) were incubated in the 3.0 ml PBS (0.1 M, pH=7) solution containing 2 ml guaiacol and a different concentration of H2O2 for 3 minutes. The guaiacol is an indicator in the chemical reactions that produce oxygen. When HRP is used to catalyze the oxidation reaction of guaiacol in the presence of H2O2, the complex turns yellowish brown. The activity was characterized by the UV–Vis spectrophotometer (UV-2550, Shimadzu). The stability was analyzed 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

by testing the activity of enzyme after 90 days’ storage, the membrane with the hybrid nanoflowers was stored in the air atmosphere at the room temperature of 25 °C, while the free HRP was stored in the PBS solution at 5 °C. The pH stability was analyzed by testing the activity of immobilized nanoflowers and free HRP after storage in the solutions with different pH value for different times.

RESULTS AND DISCUSSION

Figure 1. (a) Chemical modification of PVA-co-PE nanofiber membrane to adsorb Cu2+ on the surface; (b) FTIR spectra of pure PVA-co-PE nanofiber membrane, cyanuric chloride activated nanofiber membranes, and IDA functionalized nanofiber membrane; (c) XPS pattrerns of IDA-Cu functionalized nanofiber membrane. To immobilize the protein on the surface of PVA-co-PE nanofiber membrane, Cu2+ is adsorbed onto the surface of the membrane. The chemical modification of PVA-co-PE 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


nanofiber membrane is shown in Figure 1a. The PVA-co-PE nanofiber membrane was activated by cyanuric chloride first, and then the iminodiacetic acid (IDA) was adsorbed on the surface of the membrane. Due to the good affinity between IDA and metal ions, the Cu2+ was adsorbed on the surface of membrane by formation of stable metal-IDA chelates. Figure 1b shows the FTIR spectra of PVA-co-PE nanofiber membrane, cyanuric chloride activated PVA-co-PE nanofiber membrane, and IDA functionalized PVA-co-PE nanofiber membrane. Compared with the pure PVA-co-PE nanofiber membrane, the new peaks of cyanuric chloride activated PVA-co-PE nanofiber membrane located at 1505 and 1540 cm-1 correspond to the planar triazine rings in the cyanuric chloride.32 After grafting the IDA, the two peak which appeared at 1565 and 1393 cm-1 corresponded to the COO- antisymmetrical vibration and COOsymmetric vibration, indicating the carboxyl groups in IDA.33, 34 To demonstrate the adsorption of Cu2+ on the surface of nanofiber membrane, Figure 1c gives XPS spectra of Cu2+ functionalized PVA-co-PE nanofiber membrane. The peak at 935 eV correspoding to the binding energy of Cu2p, indicated the presence of Cu2+ on the surface of nanofiber membrane.

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

Figure 2. Formation mechanism of copper-based inorganic-protein hybrid crystals

Figure 3. SEM images of (a) pure PVA-co-PE nanofiber membrane, (b) Cu2+ functioned nanofiber membrane, (c) IDA functioned nanofiber membrane immersed in the BSA/PBS solution (without Cu2+), (d) Cu2+ functioned nanofiber membrane immersed in the BSA/water solution (without PBS), (e) Cu2+ functioned nanofiber membrane immersed in the PBS solution (without BSA), the growing time is 6 hours.

In this study, the copper-based protein-inorganic nanoflowers are constructed on 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


the surface of nanofiber membrane by immersing the Cu2+ functioned nanofiber membrane into the BSA/PBS solution. In this process, Cu2+, PO43- and BSA are three key elements to make the construction successful, the formation mechanism can be seen in Figure 2. To demonstrate the above mentioned mechanism, Figue 3 shows some pictures about the surface morphology of membranes prepared by different methods. As shown in Figure 3a,b, after several stages of chemical modification, the nanofibers still maintain their original morphology and structure. In Figure 3c, no protein is adsorbed on the surface of nanofiber membrane if the IDA functioned nanofiber membrane is immersed into the BSA/PBS solution without Cu2+. Meanwhile, if the Cu2+ functioned nanofiber membare is put into the BSA/water solution without PBS, the BSA is adsorbed onto the surface of nanofiber membrane with irregular bulk morphology because the protein molecules are able to coordinate with Cu2+ through their amide groups, as shown in Figure 3d. In Figure 3e, the Cu2+ funciotned nanofiber membrane was immersed into the PBS solution. The flower-like Cu3(PO4)2·3H2O crystals appeared due to the reaction of Cu2+ and PO43-.35 However, due to there is no BSA in the PBS solution, the Cu3(PO4)2·3H2O nanoflowers are incompact, just like a large number of petals grow and aggregate together on the surface of nanofiber membrane.

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

Figure 4. Effect of surface morphology on the construction of the inorganic-organic nanoflowers (a) PVA-co-PE membrane, (b) cellulose filter paper, (c) PVA-co-PE nanofiber membrane, the concentration of BSA is 0.1g/L, the growing time is 6 hours. To construct the copper-based inorganic-protein nanoflowers on the surface of nanofiber membrane, the Cu2+ functioned nanofiber membrane is immersed into the BSA/PBS solution. Because both the BSA molecules and PO43- can combine with Cu2+, as shown in Figure 2, the BSA embed into the Cu3(PO4)2·3H2O crystals formed by the reaction of Cu2+ and PO43-.36 The Cu3(PO4)2·3H2O-protein hybrid nanoflowers are constructed by the oriented growth and aggregation of the Cu3(PO4)2·3H2O-protein hybrid crystal. It should be noted that the submicro-/nano-scale surface morphology is also important for the construction of inorganic-protein nanoflowers. As shown in Figure 4, the Cu2+ functioned PVA-co-PE membrane, cellulose filter paper and PVA-co-PE nanofiber membrane are selected to immobilize the inorganic-organic nanoflowers in the same condition. Due to possessing high specific surface areas and abundant functional groups, compact Cu3(PO4)2·3H2O-protein hybrid nanoflowers are constructed on the surface of nanofiber membrane. However, only a few nanoflowers formed on the surface of PVA-co-PE membrane and cellulose filter paper.

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


Figure 5. SEM morphology of Cu3(PO4)2·3H2O-protein hybrid nanoflowers formed on the surface of nanofiber membrane with the time evolution, the concentration of BSA is 0.05 g/L.

Figure 5 shows the morphology of Cu3(PO4)2·3H2O-protein hybrid nanoflowers formed on the surface of nanofiber membrane with the time evolution. Interestingly, due to the incorporation of protein molecule into the Cu3(PO4)2·3H2O crystal, the morphology of Cu3(PO4)2·3H2O-protein hybrid nanoflowers changed comparing with the pure Cu3(PO4)2·3H2O nanoflowers in Figure 3e. The Cu3(PO4)2·3H2O-protein hybrid nanoflowers appeared in 10 minutes after the Cu2+ functioned nanofiber membrane was immersed into the BSA/PBS solution, and the hybrid nanoflowers covered the full membrane after only 3 hours. The Cu3(PO4)2·3H2O nanoflowers appeared in 30 minutes and grew completely in 3 hours after the Cu2+ functioned nanofiber membrane was immersed into the PBS solution. The initial growing speed of Cu3(PO4)2·3H2O-protein hybrid nanoflowers was much faster than that of Cu3(PO4)2·3H2O nanoflowers. It indicated the BSA accelerated the growth of 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

Cu3(PO4)2·3H2O crystal. Besides, the crystal plates in the hybrid nanoflowers, or we can call it the petals, became thicker and smaller compared with the crystal plates in the Cu3(PO4)2·3H2O nanoflowers. This is mainly because the incorporation of BSA molecules with the Cu3(PO4)2·3H2O plates, which accelerate the nucleation of Cu3(PO4)2·3H2O crystals and limit the growth of Cu3(PO4)2·3H2O plates. Third, more and more hybrid nanoflowers produced and the morphology of hybrid nanoflowers became complete and compact with the time evolution. It is because that more and more petals produce and aggregate together with the time evolution. The requirement of lower surface energy make the petals reorganize to form the complete and compact nanoflowers.

Figure 6. SEM morphology of Cu3(PO4)2·3H2O-protein hybrid nanoflower formed in different BSA concentration, the growing time is 6 hours.

Moreover, the morphology of Cu3(PO4)2·3H2O-protein hybrid nanoflowers significantly depends on the BSA concentration. As shown in Figure 6, the BSA concentration changed from 0.02 to 1 g/L, and the hybrid nanoflowers shows very 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


different morphologies, just like different flowers in nature. In the BSA concentration of 0.02 g/L, 0.1 g/L, 0.5 g/L and 1 g/L, the average size of these nanoflowers were approximately 20 um, 17 um, 7 um and 5 um, respectively. This is explained by the competition between reaction of Cu2+-PO43- and Cu2+-BSA. When the BSA concentration is 0.02 g/L, the hybrid nanoflowers compose of abundant thin and small petals because the single petal contains fewer BSA on it. It also indicates the former result of protein can accelerate the nucleation of Cu3(PO4)2·3H2O crystal and limit the growth of Cu3(PO4)2·3H2O plates, so the quantity of the petals increases but the size decreases. With the increase of BSA concentration, the petals become thicker and the size of hybrid nanoflowers become smaller. When the concentration of BSA increase to 0.5 g/L, the petals become much thicker than before because plenty of BSA molecules are incorporated into the petals and some petals even stick together, as shown in Figure 6c. In the concentration of 1 g/L, nearly all the petals stick together and the hybrid nanoflowers become much smaller. With the concentration of protein increases from 0 to 1 g/L, the weight percentage of encapsulated proteins in the hybrid nanoflowers increases from 0 to 19.8%.

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

Figure 7. TEM characterization of (a, b) Cu3(PO4)2·3H2O nanoflowers (c, d) Cu3(PO4)2·3H2O-protein nanoflowers on the surface of nanofiber membrane with different magnification.

Figure 8. XRD patterns of (a) PVA-co-PE nanofiber substrate, Cu3(PO4)2·3H2O nanoflower and Cu3(PO4)2·3H2O-protein nanoflower on the surface of nanofiber membrane (b) XRD patterns of free Cu3(PO4)2·3H2O nanoflower and free-standing Cu3(PO4)2·3H2O-protein nanoflowers prepared in the solution.

TEM images of inorganic nanoflowers and inorganic-protein hybrid nanoflowers 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


are reflected in Figure 7. The planes with the interlayer distance of 0.215 nm are observed in Figure 7b and Figure 7d, which correspond to the Cu3PO4·3H2O crystal structure. The corresponding XRD patterns are displayed in Figure 8a. The broad diffraction peak around 2θ = 20° of PVA-co-PE nanofiber substrate conceals the real diffraction peaks of inorganic nanoflower and inorganic-protein nanoflower in this area. To avoid the influence of the PVA-co-PE nanofiber substrate, free-standing Cu3(PO4)2·3H2O nanoflower and Cu3(PO4)2·3H2O-protein nanoflowers were prepared in solution for contrast. As shown in Figure 8b, the XRD pattern of the free-standing inorganic nanoflowers are nearly the same as the free-standing hybrid nanoflowers, fitting those obtained from the JCPDS card (00-022-0548). It indicates that the Cu3(PO4)2·3H2O-protein hybrid crystals are mainly composed of Cu3(PO4)2·3H2O crystals.17 Moreover, although a broaden diffraction peak of PVA-co-PE nanofiber substrate conceals some information, several small peaks can still be observed. Those small peaks in accordance with the corresponding peaks in Figure 8b, indicating the same crystal structure of immobilized nanoflower with the free-standing one.

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 9. XPS spectras of (a) Cu3(PO4)2·3H2O nanoflowers and (b) Cu3(PO4)2·3H2O-protein hybrid nanoflowers on the nanofiber membrane. Table 1. Elemental Compositions (%) of (a) Cu3(PO4)2·3H2O and (b) Cu3(PO4)2·3H2O-Protein Hybrid Nanoflowers on the Nanofiber Membrane Sample C O Cu N P S Cl Na Cu3(PO4)2·3H2O

20.83

45.09

10.52

1.12

10.99

0

2.95

8.51

Cu3(PO4)2·3H2O-protein

51.61

26.52

2.76

9.25

4.03

0.42

1.87

3.54

The

elemental

compositions

of

Cu3(PO4)2·3H2O

nanoflowers

and

Cu3(PO4)2·3H2O-protein nanoflowers were analyzed by XPS. As shown in Figure 9 and in Table 1, the pure Cu3(PO4)2·3H2O nanoflowers contain little N element on the surface. After incorporation with the BSA molecule, a new peak at 400 V is observed, which is ascribed to the binding energy of N. The element compositions of O, Cu, and P decreased from 45.09%, 10.52%, and 10.99% to 26.52%, 2.76%, and 4.03%, while the element compositions of N and C increased from 1.12% and 20.83% to 9.25% and 51.61%. The BSA molecules are embedded into the Cu3(PO4)2·3H2O crystal to form the Cu3(PO4)2·3H2O-protein nanoflowers and the elements of N and C are the essential 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


components of protein. The XPS results demonstrate the combination of BSA molecules with the Cu3(PO4)2·3H2O crystal. The FTIR spectra of the hybrid nanoflower is given in Figure 1S. Compared with the functional PVA-co-PE nanofiber membrane, two new obvious peaks appeared at 1074 cm-1 and 990 cm-1, which corresponded to the symmetric stretching vibration absorption peak and the asymmetric stretching vibration absorption peak of the phosphate respectively. The spectra from 1600 to 1700 cm-1 corresponding to the amides I of BSA, indicating the presence of the protein in the Cu3(PO4)2·3H2O-BSA hybrid nanoflower.

Figure 10. Growing mechanism of Cu3(PO4)2·3H2O-protein nanoflowers.

We propose the growing mechanism of inorganic-protein hybrid nanoflowers on the surface of nanofiber membrane, as shown in Figure 10. Initially, when the Cu2+ 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

functioned nanofiber membrane is immersed into the BSA-PBS solution, Cu3(PO4)2·3H2O seed crystals form due to the reaction between Cu2+ and PO43-. The nucleation then occurs with the increase and aggregation of the seed crystals.37 Due to the chelation between Cu2+ and IDA, the Cu3(PO4)2·3H2O nuclei would construct on the surface of nanofiber membrane. The formation of Cu3(PO4)2·3H2O crystal has been demonstrated in the pure PBS solution even without BSA. However, the BSA molecules could combine with Cu3(PO4)2·3H2O crystal and facilitate the nucleation of Cu3(PO4)2·3H2O crystal as the coordination between Cu2+ and protein molecules. The growth process of copper-based protein-inorganic hybrid nanoflowers could be explained by the Oswald’s ripening and oriented attachment growing mechanism.38 First, the aggregated Cu3(PO4)2·3H2O nuclei forms geminate crystals, the active sites generated at the middle interface of geminate crystals. The existence of active sites stimulate the growth of Cu3(PO4)2·3H2O plates along the certain direction.39 Meanwhile, BSA molecules are successfully embedded into the crystals in the whole growing process to form the Cu3(PO4)2·3H2O-protein crystal plates. The oriented growth and self-organization of the Cu3(PO4)2·3H2O-protein crystal plates result in the formation of flower-like Cu3(PO4)2·3H2O-protein hybrid structures on the surface of nanofiber membrane.40

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


Figure 11. Cu3(PO4)2·3H2O-enzyme hybrid nanoflowers prepared by (a) papain (b) laccase (c) HRP, the concentration of papain is 0.01 g/L, laccase and HRP is 0.1 g/L, the growth time is 6 hours.

To confirm the formation mechanism of copper-based inorganic-protein hybrid nanoflowers, different enzymes including papain, laccase and HRP were chosen. As shown in Figure 11, different copper-based inorganic-enzyme hybrid nanoflowers were prepared and constructed on the surface of nanofiber membrane by successfully incorporated the papain, laccase and HRP molecule into Cu3(PO4)2·3H2O crystal. The morphological diversity caused by the types of enzymes mainly come from the different structures of enzymes. The Cu2+-protein binding sites are dominated by the histidine, cysteine, and methionine residues in the protein molecules. The coordination between enzyme and Cu2+ relates to the composition and geometries of hybrid nanoflowers. Due to the different structures of papain, laccase and HRP, the corresponding

Cu3(PO4)2·3H2O-protein

hybrid

nanoflowers

show

different

morphologies.2 Moreover, the effect of protein concentration on the morphology of hybrid nanoflowers is also distinguishing, that is why we prepared the hybrid nanoflowers in different protein concentrations.

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

Figure 12. (a) Immobilized Cu3(PO4)2·3H2O-HRP hybrid nanoflowers and free HRP were incubated in the PBS solution containing guaiacol and different concentration of H2O2 for 3 minutes, (1#) 200 µM (2#) 500 µM (3#) 1 mM (4#) 5mM (5#) 10 mM (b) the catalytic activity of immobilized Cu3(PO4)2·3H2O-HRP hybrid nanoflowers in different concentrations of H2O2 (c) storage stability of immobilized Cu3(PO4)2·3H2O-HRP hybrid nanoflowers and free HRP (d) pH stability of immobilized Cu3(PO4)2·3H2O-HRP hybrid nanoflowers and free HRP.

Immobilization is an effective way to promote the stability, reusability and availability of the enzyme. After immobilization on the surface of the nanofiber membrane, hybrid nanoflowers become easy to operate and collect. To analyze the activity and stability, the immoblized Cu3(PO4)2·3H2O-HRP hybrid nanoflowers were used to catalyze the oxidation reaction of guaiacol in the presence of H2O2. As shown in 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


Figure 12a, the color of the solution gradually deepens with the increase of H2O2 concentration. Figure 12b shows the UV absorption spectra of reaction solution containing constructed Cu3(PO4)2·3H2O-HRP hybrid nanoflowers. The increase in the adsorption peak at 410 and 475 nm exhibited the activity of the immobilized Cu3(PO4)2·3H2O-HRP hybrid nanoflowers. Figure 12c,d shows the storage and pH stability of the immobilized hybrid nanoflowers and free HRP. After 90 days, the relative activity of constructed Cu3(PO4)2·3H2O-HRP hybrid nanoflowers is above 80%, which is much higher than the free HRP of 5%. The hybrid nanoflowers can maintain higher relative activity from pH 5 to pH 7. However, the free HRP can only be used at pH 7. The combination of confinement effect with the encapsulation of enzyme in the matrix is effective way to improve enzyme stability. The immobilized HPR-inorganic nanoflowers enabled the entrapment of enzymes at any convenient pH that is suitable for their structural integrity and functionality. Besides, the multipoint immobilization could reduce any conformational change involved in enzyme inactivation and greatly increase the enzyme stability.41-43 Therefore, the immobilized HPR-inorganic nanoflowers have a high retention of activity over a wider pH range compared with the free HRP.

CONCLUSION

Biomimetic copper-based inorganic-protein nanoflowers were constructed on the surface of nanoscale fibrous membrane by a fast and simple method. The 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

Cu3(PO4)2·3H2O-protein hybrid nanoflowers appeared in 10 mins after the Cu2+ functioned nanofiber membrane was immersed into the BSA/PBS solution, and the hybrid nanoflowers covered the full membrane after only 3 hours. The morphology of inorganic-protein nanoflowers became more complete and compact with the time evolution, while the petals in the nanoflowers became thicker and smaller with the increase of protein concentration. The formation of the copper-based inorganic-organic nanoflowers on nanofiber membrane follows the process of nucleation, growth and self-organization. The protein molecules incorporate into the inorganic crystals in the growing process to form the hybrid crystal plates. Different structures of protein, such as papain, laccase and HRP result in the different morphology of inorganic-protein hybrid nanoflowers. After immobilization on the surface of nanofiber membrane, the hybrid nanoflowers exhibited enhanced durability, pH stability compared with the free enzyme, indicating their potential applications for biodevice, biosensor and biocatalysis.

ACKNOWLEDGEMENT

The authors are thankful for the financial support of National Nature Science Foundation (51403166, 51473129 and 51273152), National Science and Technology support program (2015BAE01B00), Nature Science Foundation of Hubei Province (2014CFB759), Excellent Innovative Team of Young Researchers from Hubei provincial department of education (T201408), and Creative research group of Hubei 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


province (2015CFA028).

REFERENCES

(1) Lee, T.; Yagati, A. K.; Min, J.; Choi, J. W. Bioprocessing Device Composed of Protein/DNA/Inorganic Material Hybrid. Adv. Funct. Mater. 2014, 24, 1781-1789. (2) Zhang, Z. P.; Shao, X. Q.; Yu, H. D.; Wang, Y. B.; Han, M. Y. Morphosynthesis and Ornamentation of 3D Dendritic Nanoarchitectures. Chem. Mater. 2005, 17, 332-336. (3) Tan, L.; Huang, C.; Peng, R. F.; Tang, Y. W.; Li, W. M. Development of Hybrid Organic-Inorganic Surface Imprinted Mn-doped ZnS QDs and Their Application as a Sensing Material for Target Proteins. Biosens. Bioelectron. 2014, 61, 506-511. (4) Cao, H. M.; Yang, D. P.; Ye, D. X.; Zhang, X. X.; Fang, X. E.; Zhang, S.; Liu, B. H.; Kong, J. L. Protein-Inorganic Hybrid Nanoflowers as Ultrasensitive Electrochemical Cytosensing Interfaces for Evaluation of Cell Surface Sialic Acid. Biosens. Bioelectron. 2015, 68, 329-335. (5) Niide, T.; Ozawa, K.; Nakazawa, H.; Oliveira, D.; Kasai, H.; Onodera, M.; Asano, R.; Kumagai, I.; Umetsu, M. Organic Crystal-binding Peptides: Morphology Control and One-pot Formation of Protein-displaying Organic Crystals. Nanoscale 2015, 7, 20155-20163. (6) Wang, Z. Y.; Wang, J. J.; Li, M. Y.; Sun, K. H.; Liu, C. J. Three-Dimensional Printed Acrylonitrile Butadiene Styrene Framework Coated with Cu-BTC Metal-organic Frameworks for the Removal of Methylene Blue. Sci. Rep. 2014, 4. 5939. 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

(7) Liang, K.; Ricco, R.; Doherty, C. M.; Styles, M. J.; Bell, S.; Kirby, N.; Mudie, S.; Haylock, D.; Hill, A. J.; Doonan, C. J.; Falcaro, P. Biomimetic Mineralization of Metal-Organic Frameworks as Protective Coatings for Biomacromolecules. Nat. Commun. 2015, 6, 7240. (8) Munro, N. H.; Green, D. W.; McGrath, K. M. In Situ Continuous Growth Formation of Synthetic Biominerals. Chem. Commun. 2013, 49, 3407-3409. (9) Bergström, L.; Sturm, E. V.; Salazar-Alvarez, G. Cölfen, H. Mesocrystals in Biominerals and Colloidal Arrays. Acc. Chem. Res. 2015, 48, 1391-1402. (10) Revilla-López, G.; Casanovas, J.; Bertran, O.; Turon, P.; Puiggalí, J.; Alemán, C. Modeling Biominerals Formed by Apatites and DNA, Modeling Biominerals Formed by Apatites and DNA. Biointerphases 2013, 8, 10-24. (11) Sato, S.; Ikemi, M.; Kikuchi, T.; Matsumura, S.; Shiba, K.; Fujita, M. Bridging Adhesion of a Protein onto an Inorganic Surface Using Self Assembled Dual-Functionalized Spheres. J. Am. Chem. Soc. 2015, 137, 12890-12896. (12) Sun, B. B.; Shen, H. Correlation of the Composition of Biominerals with Their Ability of Stimulating Intracellular DNA Sensors and Inflammatory Cytokines. Biomaterials 2015, 54, 106-115. (13) Kawata, M.; Azuma, K.; Izawa, H.; Morimotoa, M.; Saimoto, H.; Ifuku, S. Biomineralization of Calcium Phosphate Crystals on Chitin Nanofiber Hydrogel for Bone Regeneration Material. Carbohyd. Polym. 2016, 136, 964-969. (14) Lichtenegger, H. C.; Schöberl, T.; Bartl, M. H.; Waite, H.; Stucky, G. D. High Abrasion Resistance with Sparse Mineralization: Copper Biomineral in Worm Jaws. 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


Science 2002, 298, 389-392. (15) Rubino, J. T.;; Franz, K. J.. Coordination Chemistry of Copper Proteins: How Nature Handles a Toxic Cargo for Essential Function. J. Inorg. Biochem. 2012, 107, 129-143. (16) Hashimoto, A.; Kambe, T. Mg, Zn and Cu Transport Proteins: A Brief Overview from Physiological and Molecular Perspectives. J. Nutr. Sci. Vitaminol. 2015, 61, 116-118. (17) Ge, J.; Lei, J. D.; Zare, R. N. Nat. Protein-Inorganic Hybrid Nanoflowers. Nat. Nanotechnol. 2012, 7, 428-432. (18) Sun, J. Y.; Ge, J. C.; Liu, W. M.; Lan, M. H.; Zhang, H. Y.; Wang, P. F.; Wang, Y. M.; Niu, Z. W. Multi-enzyme Co-embedded Organic-inorganic Hybrid Nanoflowers: Synthesis and Application as a Colorimetric Sensor. Nanoscale 2014, 6, 255-262. (19) Lin Z.; Lu G.; Zhang, Y. F.; Wang, R.; Ge, J.; Liu, Z.; Zare, R. N. Rapid Detection of Phenol Using a Membrane Containing Laccase Nanoflowers. Chem. Asian J. 2013, 8, 2358-2360. (20) Huang, Y. Y.; Ran, X.; Lin, Y. H.; Ren, J. S.; Qu, X. G. Self-Assembly of an Organic-Inorganic Hybrid Nanoflower as an Efficient Biomimetic Catalyst for Self-Activated Tandem Reactions. Chem. Commun. 2015, 51, 4386-4389. (21) Lin, Z.; Xiao, Y.; Yin, Y. Q.; Hu, W. L.; Liu, W.; Yang, H. H. Facile Synthesis of Enzyme-Inorganic Hybrid Nanoflowers and Its Application as a Colorimetric Platform for Visual Detection of Hydrogen Peroxide and Phenol. ACS Appl. Mater. Inter. 2014, 6, 10775-10782. 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

(22) Junga, M.; Kim, S. U.; Oh, B. K.; Choi J. W. Immobilization of biomaterials on nanopatterned surface using nanoporous alumina for biodevices. Curr. Appl. Phys. 2009, 9, e111-e114. (23) Liang, K.; Carbonell, C.; Styles, M. J.; Ricco, R.; Cui, J. W.; Richardson, J. J.; Maspoch, D.; Caruso, F.; Falcaro, P. Biomimetic Replication of Microscopic Metal-Organic Framework Patterns Using Printed Protein Patterns. Adv. Mater. 2015, 27, 7293-7298. (24) Wu, Y. L.; Yan, M.; Cui, J. Y.; Yan, Y. S.; Li, C. X. A Multiple-Functional Ag/SiO2/Organic Based Biomimetic Nanocomposite Membrane for High-Stability Protein Recognition and Cell Adhesion/Detachment. Adv. Funct. Mater. 2015, 25, 5823-5832. (25) Chen, C. X.; Hong, X. Z.; Xu, T. T.; Chen, A. K.; Lu, L.; Gao, Y. H. Hydrogen Peroxide Biosensor Based on the Immobilization of Horseradish Peroxidase onto a Poly(Aniline-Co-N-Methylthionine) Film. Synthetic Met. 2016, 212, 123-130. (26) Nazaria, M.; Kashanian, S.; Rafipour, R. Laccase Immobilization on the Electrode Surface to Design a Biosensor for the Detection of Phenolic Compound such as Catechol. Spectrochim. Acta A 2015, 145, 130-138. (27) Garg, T.; Rath, G.; Goyal, A. K. Biomaterials-Based Nanofiber Scaffold: Targeted and Controlled Carrier for Cell and Drug Delivery. J. Drug Target 2015, 23, 202-221. (28) Jaiswal, D.; James, R.; Shelke, N. B.; Harmon, M. D.; Brown, J. L.; Hussain, F.; Kumbar, S. G. Gelatin Nanofiber Matrices Derived from Schiff Base Derivative for Tissue Engineering Applications. J. Biomed. Nanotechnol. 2015, 11, 2067-2080. 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


(29) Squires, A. H.; Hersey, J. S.; Grinstaff, M. W.; Meller, A. A NanoporeNanofiber Mesh Biosensor to Control DNA Translocation. J. Am. Chem. Soc. 2013, 135, 16304-16307. (30) Wang, D.; Sun, G.; Chiou, B. S. A High-Throughput, Controllable, and Environmentally Benign Fabrication Process of Thermoplastic Nanofibers. Macromol. Mater. Eng. 2007, 292, 407-414. (31) Wang, D.; Liu, N.; Xu, W. L.; Sun, G. Layer-by-Layer Structured Nanofiber Membranes with Photoinduced Self-Cleaning Functions. J. Phys. Chem. C 2011, 115, 6825-6832. (32) Lu, Y.; Wu, Z. H.; Li, M. F.; Liu, Q. Z.; Wang, D. Hydrophilic PVA-co-PE Nanofiber Membrane Functionalized with Iminodiacetic Acid by Solid-Phase Synthesis for Heavy Metal Ions Removal. React. Funct. Polym. 2014, 82, 98-102. (33) Zhang, M.; He, X. W.; Chen, L. X.; Zhang, Y. K. Preparation of IDA-Cu Functionalized Core-Satellite Fe3O4/Polydopamine/Au Magnetic Nanocomposites and Their Application for Depletion of Abundant Protein in Bovine Blood. J. Mater. Chem. 2010, 20, 10696-10704. (34) Deng, S.; Zhang, G. S.; Wang, X.; Zheng, T.; Wang, P. Preparation and Performance of Polyacrylonitrile Fiber Functionalized with Iminodiacetic Acid under Microwave Irradiation for Adsorption of Cu(II) and Hg(II). Chem. Eng. J. 2015, 276, 349-357. (35) Aghjani, A.; Mirzaie, P. Synthesis and Characterisation of Novel Nano-Flowers Cu3(PO4)2·3H2O by Solid State Reaction at Room Temperature. Int. J. Nanopart. 2010, 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

3, 326-334. (36) Wang, L. B.; Wang, Y. C.; He, R.; Zhuang, A.; Wang, X. P.; Zeng, J.; Hou, J. G. A New Nanobiocatalytic System Based on Allosteric Effect with Dramatically Enhanced Enzymatic Performance. J. Am. Chem. Soc. 2013, 135, 1272-1275. (37) Ahsanulhaq, Q.; Kim, S. H.; Kim, J. H.; Hahn, Y. B. Structural Properties and Growth Mechanism of Flower-like ZnO Structures Obtained by Simple Solution Method. Mater. Res. Bull. 2008, 43, 3483-3489. (38) Zhang, Q.; Liu, S. J.; Yu, S. H. Recent Advances in Oriented Attachment Growth and Synthesis of Functional Materials: Concept, Evidence, Mechanism, and Future. J. Mater. Chem. 2009, 19, 191-207. (39) Liu, J. P.; Huang, X. T.; Li, Y. Y.; Duan, J. X.; Ai, H. H. Large-Scale Synthesis of Flower-like ZnO Structures by a Surfactant-Free and Low-Temperature Process. Mater. Chem. Phys. 2006, 98, 523-527. (40) Chao, C. Y.; Ren, Z. H.; Liu, Z. Y.; Xiao, Z.; Xu, G.; Wei, X.; Shen, G. Crystal Growth of Ferroelectric PbTiO3 Nanocrystals on Silicon Substrate via Intermediates. Mater. Res. Bull. 2012, 47, 912-916. (41) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of Enzyme Activity, Stability and Selectivity via Immobilization Techniques. Enzyme Microb Tech. 2007, 40, 1451-1463. (42) Cao, L. Q. Immobilised Enzymes: Science or Art? Curr. Opin. Chem. Biol. 2005, 9, 217-226. (43) Sheldon, R. A.; Pelt, S. V. Enzyme Immobilisation in Biocatalysis: Why, What and How. Chem. Soc. Rev. 2013, 42, 6223-6235. 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


- Figures 3-7, 10, 11: Remove all extraneous text from these figures, using instead simple scale bars referenced within the captions (i.e., scale bar = 20 µm). - Figures 8, 9: Left axes labels are missing unit of measurement (ex. a.u. or arb. unit).

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 IMAGE

32


Biomimetic Copper-Based InorganicâProtein Nanoflower Assembly

Recommend Documents

Biomimetic Copper-Based InorganicâProtein Nanoflower Assembly