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Controlled Fabrication of Silk Protein Sericin Mediated Hierarchical Hybrid Flowers and Their Excellent Adsorption Capability of Heavy Metal Ions of Pb(II), Cd(II) and Hg(II) Pradyot Koley, Makoto Sakurai, and Masakazu Aono ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11533 • Publication Date (Web): 06 Jan 2016 Downloaded from http://pubs.acs.org on January 13, 2016

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ACS Applied Materials & Interfaces

Controlled

Fabrication

of

Silk

Protein

Sericin

Mediated

Hierarchical Hybrid Flowers and Their Excellent Adsorption Capability of Heavy Metal Ions of Pb(II), Cd(II) and Hg(II)

Pradyot Koley,* Makoto Sakurai, and Masakazu Aono International Center for Materials Nanoarchitectonics (WPI MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, 305-0044, Japan

ABSTRACT Fabrication of protein-inorganic hybrid materials of innumerable hierarchical patterns plays a major role in the development of multi-functional advanced materials with their improved features in synergistic way. However effective fabrication and applications of the hybrid structures is limited due to the difficulty in control and production cost. Here, we report the controlled fabrication of complex hybrid flowers with hierarchical porosity through a green and facile co-precipitation method by using industrial waste natural silk protein sericin. The large surface areas and porosity of the micro-size hybrid flowers enable water purification through adsorption of different heavy metal ions. The high adsorption capacity depends on their morphology, which is changed largely by sericin concentration in their fabrication. Superior adsorption and greater selectivity of the Pb(II) ions have been confirmed by the characteristic growth of needle‒shaped nanowires on the hierarchical surface of the hybrid flowers. These hybrid flowers show excellent thermal stability even after complete evaporation of the protein molecules, significantly increasing the porosity of the flower petals. Simple, cost-effective, and

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environmental friendly fabrication method of the porous flowers will lead to new solution to water pollution required in the modern industrial society.

KEYWORDS: hybrid flowers, silk sericin, porosity, adsorption, nanowires

INTRODUCTION Structural complexity, manufacturing simplicity and the applications potentiality of innumerable natural patterns of functional hybrid structures through coupling between inorganic and organic building blocks have always fascinated researchers to understand and control their assembling processes.1-4 Laboratory-based synthetic approaches of hierarchical hybrid structures which consist of nano-scale components hierarchically while the total size is in the micro-scale, is still a major challenge to achieve similar levels of exquisite control in the fabrication of well-defined architectures with explicit functionality.5-12 Moreover, organic-inorganic hybrid ‘flowers’ derived from biomolecules have attracted increasing recent interest due to their broad applications in biocatalysis, drug delivery, biosensing etc.13-16 Mutual interactions between the biological molecules and the inorganic materials plays a key role in the enhancement of the added functionalities of these hybrid materials in a synergistic way. In comparison to the more compact structures such as solid spheres or polyhedra, a hierarchical flower-like structure has a much larger surface area, and the presence of the porosity extend their functionalities even further.13 Moreover, immobilization and entrapment of the biomolecules in the core of the nanoflowers, exhibit greater activity, durability and stability compared to free molecules in solution.13-16 Whereas active surface functional groups from confined biomolecules are highly required for the applications of hierarchical architectures in adsorption and separations.17 In addition if we can remove the confined biomolecules from the hybrid flowers without affecting


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their gross hierarchical morphology, it can have many applications due to the enhanced porosity and the surface area from the vacant space of the biomolecules.17-19 Considerable efforts have been devoted to fabricate bioinorganic hybrids not only due to the combined functional applications, but also their chemical diversity, composition flexibility and good biocompatibility. However, facile and low cost fabrication of bioinorganic porous architectures with specific functionalities is still difficult to achieve. Natural macromolecular silk, one of the highly promising biopolymers, have had a rich history in traditional use of textile industries and medical applications for thousands of years.20,21 The easy availability, good mechanical strength, optical properties, biocompatibility and controllable degradation makes it an outstanding candidate for multifunctional advanced materials.20,21 Substantial recent advances have revealed exciting new opportunities for silk proteins in photonics,22 resist for lithography,23 microstructure patterns,24 inkjet printing,25 biomedical devices,26 and drug delivery,27 necessitating the further development of innovative approaches to multi-scale fabrication of silk based materials for a host of applications. Silk derived from silkworms consists primarily of two proteins - a fibrous core protein, fibroin, and a glue protein called sericin, which is coated on the fibroin fiber with successive sticky layers forming cocoon, through cementing the silk fibers together. In contrast to insoluble fibroin, sericin has its unique characteristics including excellent hydrophilicity, biodegradation, and resistance to oxidation, bacteria and ultraviolet light.28-30 It also exhibits various biological activities and pharmacological functions, while its addition to the culture media enhances cells attachment, growth and proliferation.31 Despite plenty of beneficial properties, implementation of sericin for the fabrication of functional architectures is still limited in scope due to the difficulty in processing it into useful materials in comparison to the well-studied fibroin. However some



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recent studies have explored interesting applications of silk sericin opening up new prospects of its usefulness.32,33 Apart from these potentialities, there are two main reasons that strongly motivated our choice of sericin for the fabrication of hierarchical hybrid flowers. Firstly, most of the amino acids of sericin possess strongly polar functional groups such as hydroxyl, carboxyl, and amino groups, rendering it an excellent water-soluble and strong metal binding material.21,34 Secondly, sericin is very cheap and abundantly available material, and it has typically been discarded as byproduct during the degumming processes in raw silk production.20,21 In recent years, water pollution caused by heavy metal ions such as Pb(II), Cd(II), and Hg(II) have become one of the most serious problem in the modern industrial society, not only due to their high toxicity to human health and the ecosystem even at trace amount but also their resistance to degradation naturally.35-38 Various methods have been developed for the removal of toxic metals from an aqueous system, among them adsorption techniques have attracted significant attention due to their efficiency, inexpensiveness and simplicity of operation.39-43 However, the selective adsorption of very low concentration of heavy metal ions in presence of highly concentrated comparatively light-metal ions such as Ca(II), Mg(II), Na(I) etc. is a major concern for the drinking water treatment. At the same time fast and tight adsorption, structural integrity of the adsorbents and the recovery of the entrapped cations are highly required.44 Therefore, it is very crucial to design and synthesize high performance adsorbents in a costeffective and environmental friendly way. In this regard three dimensional flower-like bio-hybrid structures with porous nanoscale building blocks may become very effective adsorbent towards heavy metal ions due to their high surface area, facile mass transportation, abundant active adsorption sites and easy separation.


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Here, we report the controlled fabrication of complex organic-inorganic hybrid flowers with abundant surface porosity through a green and facile one-pot co-precipitation method by using silk proteins sericin as the organic component and the copper (II) phosphate as the inorganic counterpart without altering the inherent protein structure. They are thoroughly characterized with different microscopic and spectroscopic techniques. We have preciously optimized the growth conditions of biomolecules and metal ions for the formation of bio-hybrid flowers to meet manufacturing scalability. The morphological diversities of the as-synthesized hybrid flowers strongly depend on the concentration of the sericin. Moreover, the as-grown hybrid flowers show excellent thermal stability even after complete evaporation of the sericin molecules through calcinations, however, the process significantly increases the porosity of the flower petals. These flower-like porous architectures have a large surface area including different surface functional groups from sericin and show excellent adsorption properties for the heavy metal ions such as Pb(II), Cd(II) and Hg(II) from waste water. Further the experimental results exhibit the facile growth of uniformly distributed needle‒shaped nanowires on the hierarchical surface of the hybrid flowers, exploring the higher adsorption ability and the greater selectivity of Pb(II) ions. In order to demonstrate high performance drinking water purification, a continuous filtering device for the rapid and selective adsorption of heavy metals was also designed by using these synthetic hybrid materials.

EXPERIMENTAL SECTION Materials. Pure sericin (Wako Chemicals, Japan), anhydrous copper (II) sulfate (SigmaAldrich) and phosphate buffer saline (Lonza, USA) were used as precursor reagents for the coprecipitation reaction. Phosphate buffer without saline was purchased from Nacalai Tesque, Japan. The Milli-Q purified deionized water was used to prepare the stock solutions.



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Synthesis of Hybrid Flowers. In a typical experiment, calculated amount of aqueous CuSO 4 solution (100 mM) was added to 4 mL of 1X PBS (pH 7.4) containing different concentrations of sericin so that their optimum ratio (0.1 mg mL-1 sericin : 1 mM CuSO 4 ) was maintained in the mixture, followed by incubation at 25 °C for 12 h. A light blue precipitates of hybrid flowers were collected, washed repeatedly with deionized water, and dried at room temperature.

Characterizations. For SEM analysis, the suspension of the thoroughly washed as prepared hybrid flowers was deposited and dried on the glass substrates followed by platinum coating. SEM measurements were carried out with Hitachi S4800 and SU8000 field emission microscopes. Elemental analysis was accomplished with an energy dispersive X-ray (EDX) instrument in conjunction with the SEM. For TEM analysis, 2 µL dilute suspension of the hybrid flowers was added to the molybdenum TEM grids and dried at room temperature followed by imaging with a JEOL JEM-2100F high resolution electron microscope with operating voltage 200 kV. High angle annular dark field (HAADF) image and EDX mapping were carried out with scanning transmission electron microscopy (STEM) mode. LSM imaging of the hybrid flowers were carried with a KEYENCE VK-9710 (Violet Laser). XRD analysis of the dried hybrid flowers were carried out with Rigaku, Rint 2000 Ultima III Xray diffractometer using Cu Kα radiation (λ=1.5406Å, 40 kV / 40 mA). The 2 θ scanning range was from 1 to 80° at 0.02° intervals with a scanning speed of 1°/min. XPS analysis was carried out with a Thermo Fisher Scientific, Theta Probe system. For ATR-FTIR analysis, 10 µL of dense water suspension of the sample (different hybrid flowers) were deposited on a silicon wafer with a micropipette and dried in room temperature


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under vacuum forming a thin layer of hybrid flowers. ATR-FTIR measurements of this thin layer of hybrid flowers were carried out with a NICOLET 4700 FTIR instrument.

Thermal Analysis. For the thermal stability measurement, the suspension of as grown hybrid flowers were dried on a Si wafer, which was then heated under Ar atmosphere in an atmosphere controlled Muffle furnace (FUA112DB, ADVANTEC) at 400 °C for 2 h, followed by SEM analysis. TG analysis was carried out on a SII Exstar TG/DTA 6200 thermal analyzer in a dynamic atmosphere of dinitrogen (flow rate = 30 cm3 min−1). The sample was heated from 25 to 500 °C in an alumina crucible at a rate of 5 °C min−1. DSC measurement was done with Exstar X-DSC 7000 high sensitivity thermal analyzer.

Surface Area and Pore Size Measurements. The specific surface area was determined by the Brunauer −Emmett−Teller (BET) method from the nitrogen adsorption−desorption isotherm with a Quantachrome Autosorb iQ2 automated gas sorption analyzer after the samples were vacuum dried at 100 °C for 20 h. The pore-size distribution curves of the hybrid flowers were calculated based on the desorption branch of nitrogen isotherms using the Barrett−Joyner−Halenda (BJH) method.

Heavy Metal Ions Adsorption. Aqueous solution with different concentrations of Pb(II), Cd(II) and Hg(II) ions were prepared by dissolving Pb(NO 3 ) 2 , Cd(NO 3 ) 2 .4H 2 O and Hg(NO 3 ) 2 .H 2 O respectively, as the source of heavy metal ions. In general, 5 mg of hybrid flowers was added to 20 mL of above solutions of heavy metals with an initial concentration of 15 mg L-1 under stirring at room temperature for 2 h. After a specified time, the solid and liquid were separated immediately by centrifugation and analyzed by inductively coupled plasmaoptical emission spectroscopy (ICP-OES) to measure the concentration of metal ions in the



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remaining solution. The adsorption isotherm was obtained by varying the initial concentration of Pb(II) (2‒150 mg L-1) under stirring at constant time periods at room temperature. Similar adsorption technique was followed by using annealed hybrid flowers and copper phosphate powder (as-synthesized and commercial) as adsorbents. For the competitive experiments CaCl 2 solution was used as the source of competing Ca(II) ions.

RESULT AND DISCUSSION Synthesis and Characterization. The general procedure for the synthesis of silk protein sericin-inorganic hybrid flowers was illustrated in Scheme 1. In a typical experiment, an aqueous solution of CuSO 4 (1 mM) was added into the 10 mM phosphate buffered saline (1X PBS, pH 7.4) containing clear solution of silk protein sericin (0.1 mg mL-1). The resultant mixture was gently shaken for few minutes and allowed to incubation at room temperature (~25 °C). After 12 h, a large amount of bluish precipitate appeared with porous, uniformly grown flower-like structures. Whereas individually silk sericin self-assembled to form nanoparticles of different diameters and copper sulfate precipitated out forming large crystals of copper phosphate but no trace of flower-like morphology from 1X PBS (Supporting Information Figure S1). Figure 1a shows the scanning electron microscopy (SEM) image of a single hybrid flower (average size 5‒10 µm), which exhibits explicit hierarchical arrangements of flower petals having thickness in the range of 200‒600 nm (Figure 1b). A critical observation revealed that each petal was actually a combination of several tens of nanosheets (average thickness 15‒30 nm) sticking together as multilayer arrangements with many pores or rifts appearing on their surfaces (Figure 1c,d). Transmission electron microscopy (TEM) image and a laser scanning microscopy (LSM) image of the hybrid flowers are shown in Figures 1e, f and 1g respectively. Multilayer porous morphology of the nanosheet petals and the crystal lattice fringe with lattice distance 0.2 nm


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were clearly visible in the high-resolution TEM images (inset of Figures 1e and 1f). Energydispersive X-ray (EDX) analysis of the hybrid flowers manifested the presence of C, O, P, Cl and Cu (Supporting Information Figure S2). The X-ray diffraction (XRD) was employed to characterize the crystal structure of the hybrid flowers. The sharp, strong diffraction peaks confirmed the highly crystalline characteristics of the hybrid flowers in comparison to the amorphous silk sericin, and fitted well with that of Cu 3 (PO 4 ) 2 .3H 2 O (Supporting Information Figure S3). Moreover, X-ray photoelectron spectroscopy (XPS) was performed for additional characterization of the individual components along with their atomic concentration in the hybrid materials (Supporting Information Table S1 and Figures S4, S5). The characteristic binding energy peaks clearly indicate the presence of protein molecules and the copper phosphate in the hybrid structures. To optimize the growth condition for the formation of flower-like hierarchical hybrid structures, we had performed several systematic experiments with varying reaction conditions. The morphology of the as grown hybrid materials mainly depends on the concentration of the precursor molecules and the reaction time. Combination of a range of concentration ratio of sericin and Cu+2 in 1X PBS produced different types of flower-like architectures (Supporting Information Figure S6). On the contrary, concentrated PBS solution (20 mM) led to the comparatively quicker precipitations of the hybrid structures with some ambiguous morphology, whereas dilution of the PBS (5 mM) left the solution very few detectable microstructures rather some aggregated solid precipitates (Supporting Information Figure S7). Fabrication of hybrid flowers with 0.1 mg mL-1 sericin and 1 mM Cu+2 in 1X PBS had been chosen for further experimentation (Figure 1). At this stage we became interested to check the effect of sericin concentration on the formation of hybrid flowers maintaining the above optimum ratio of the



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individual components constant. It is very interesting to see that there was a great variation in morphology of the synthesized hybrid flowers by means of regulating the concentrations of sericin ranging from 0.01 to 1 mg mL-1. The results showed that the hybrid flowers synthesized from 1 mg mL-1 sericin were more compact and uniformly grown spherical objects (SP-hybrid) (Figure 2a,b). Gradually decreasing the concentration of sericin to 0.1 and 0.01 mg mL-1, the morphologies of the hybrid flowers changed significantly to less compact structures mimicking full bloom natural flowers shown at Figure 1 and Figure 2c,d respectively. At very low sericin concentration (0.01 mg mL-1) the width of the individual flower petals seems very thin like single layer of nanosheets petals (SL-hybrid), which combine themselves to form much thicker and compact one of multilayer hybrid flowers (ML-hybrid) as the concentration goes up to the 0.25 mg mL-1 (Supporting Information Figure S8). Further increase of concentration the morphology turns to the spherical shape where surface looks like network arrangements of the dense petals having crooked and wavy fringes with many void spaces in between (inset of Figure 2a,b). Thus, the results clearly indicate that the formation and the morphology of the hybrid flowers strongly depend on the concentrations of sericin. This is a unique example of hierarchical hybrid structures where the dramatic changes of their surface morphology were dictated by the fine tuning of protein concentrations. Since the peak positions in XRD patterns of the hybrid flowers are the same to those of copper phosphate crystals (Supporting Information Figure S3) and the large regularly arranged lattice structures are also observed in the HR-TEM image of the petal (Figure 1f), it could be concluded that the petals in the hybrid flowers were formed by regular arrangement of copper phosphate crystals. To understand the formation mechanism, time dependent growth of the hybrid flowers was carried out with two different sets of sericin concentrations. The gradual morphological


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changes of the petals at each incubation time are shown in Figure 3 and Supporting Information Figure S9, suggesting at least two stages in their growth. At the initial stage within 2 h of incubation, very thin (~10 nm) curling nanosheets are grown from the primary crystals of copper phosphate. At this stage, protein molecules formed complexes with the copper ions predominantly through the coordination of amino acid backbone and these complexes provided the nucleation of the primary crystals (Figure 3a,b and Supporting Information Figure S9a,b). In the second stage (6h), further nucleation and the subsequent growth of the copper phosphate nanocrystals originates on the surfaces of the thin nanosheet petals forming comparatively thicker and layered petals (Figure 3c and Supporting Information Figure S9c,d). In the last stage (12 h), the growth process continued to form full grown multi-layer flower structures (Figure 3d and Supporting Information Figure S9e,f). Surprisingly, kinetically controlled growth of hybrid flowers with thin bending petals had been observed at very early growth stage (Figure 3a,b). This is due to the ability of the inherent ‘glue-like’ protein sericin to form very strong complex with metal ions predominantly through the coordination facility of the different metal binding moieties in the silk sericin. Apart from the amide backbone, different polar side chains of sericin such as hydroxyl, carboxyl, and amino groups can also form complex with Cu+2 facilitating the formation of large folding nanosheets like petals of copper phosphate at the initial growth mode. The role of sericin is also consistent with the aforesaid results where the decreasing concentration of sericin led to the corresponding decrease in the number of nucleation sites and ‘glue-like’ binder, ensuing the flowers with thin petals, less compact and open up structures (Figure 2). Further, the time required (~12 h) for the formation of full bloom sericin hybrid flowers is strikingly lower than that of typical bio-hybrids flowers (~72 h).13 This may be attributed to the facile co-precipitation reaction due to the dynamic interactions between Cu+2



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ions and the binding sites of sericin protein, which is the main driving force for the formation of hierarchical hybrid structures. Overall, in this proposed growth process, the sericin induces the nucleation to form the large grain of copper phosphate crystals and serves as a ‘glue’ to bind the large grains together. We also remember that without sericin molecules, large crystals but no flowers were formed (Supporting Information Figure S1c). Moreover, controlled treatment of the hybrid flowers with glutaraldehyde, which was known as an excellent cross-linker for silk sericin by sequestering its binding site,45 led to the complete deformation of the flowery morphology and aggregation of the petals (Figure 4a and Supporting Information Figure S10a). Whereas addition of ethylenediaminetetraacetic acid (EDTA), a strong chelating agent for Cu+2 ions, converted the hybrid flowers to a collapsed structures and scattered petals (Figure 4b and Supporting Information Figure S10b). The findings further indicate the necessity of strong proteins-metal ions interactions which serves as pillars for stabilizing such hierarchical architectures. Moreover, it was observed that the chloride ions (Cl-) played an important role for the growth of hierarchical hybrid structures. EDX and XPS spectra also indicated its presence (Supporting Information Figure S2 and S4). No hybrid flowers had been formed from the phosphate buffer solution without Cl- ions, although the proportion of Cu+2 and sericin remained same as before (Supporting Information Figure S11). Additions of alkali metal salts of other halides (Br- or I-) of similar concentrations were also unable to produce any flower-like morphology rather than some solid precipitates from identical experimental conditions (Supporting Information Figure S12). Hence, Cl- ions assist the hierarchical growth of hybrid flowers by governing the crystallization process of copper phosphate and also favoring the preferred orientation of the protein-induced nucleation and the crystalline grains of copper phosphate.46


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Infrared spectroscopy is an important tool for the determination of secondary structures of proteins mainly through analyzing their characteristic amide bands.47 The study of attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy not only gives a direct proof for the formation of hybrid flowers but also provides valuable informations about the conformational constancy of the sericin protein. ATR-FTIR spectra showed no significant changes of the major amide bands of the sericin protein ‘before’ and ‘after’ the hybrid formation (amide I: 1636 and 1634 cm-1, amide II: 1515 and 1514 cm-1, and amide III: both 1238 cm-1) (Figure 5a and Supporting Information Figure S13).48 This indicates that the structural integrity of the protein remains intact after forming the hybrid flowers. Whereas the newly formed strong IR band at 1048 cm-1 was attributed to the phosphate group of the hybrid flowers. These as-synthesized hierarchical bio-hybrid flowers were expected to have large surface area, which was determined using the standard Brunauer–Emmett–Teller (BET) method. Figure 5b shows the typical nitrogen adsorption-desorption isotherm and BJH (Barrett–Joyner–Halenda) pore size distribution curves of the three different types of hybrid flowers and the synthesized copper phosphate. The BET surface area of different hybrid flowers was calculated to be 35.6 m2 g-1 (SL-hybrid), 43.9 m2 g-1 (ML-hybrid) and 54.9 m2 g-1 (SP-hybrid) respectively, which was much higher than the as-synthesized precursor copper phosphate (13.3 m2 g-1).49,50 This enhancement of specific surface areas is due to the interconnected stacked nanosheet petals with dominant surface porosity of the hybrid flowers. Whereas the increasing orders of BET surface area from SL to ML to SP-hybrid may be justified by their respective morphological signatures and the extent of porosity. Dense, compact and the network arrangement of the petals make the SP-hybrid with superior surface area than the others. The typical hysteresis loop at lower relative pressure range pointed out to the presence of mesopores in the flowery structures, having



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relatively narrow range of corresponding pore size distribution with average pore diameters 3.09, 3.67 and 3.85 nm and cumulative pore volume 0.085, 0.109 and 0.285 cm3 g-1, for SL, ML and SP-hybrid respectively from BJH desorption (Figure 5b (inset)). These preferentially grown smaller mesopores (3 – 10 nm) reflect pores within the nanosheets, whereas few larger ones (> 10 nm) can be correlated to the pores formed between interlace stacked nanosheets of the flower petals.

Thermal Stability. To the best of our knowledge, these sericin mediated hybrid flowers exhibit exceptional thermal stability even after complete evaporation of the sericin molecules at high temperature incubation. Calcination of the hybrid flowers at 400 °C for 2 h under Ar atmosphere decomposed all the protein molecules without affecting the gross flower-like pattern of the hybrid structures (Figure 6a-c). However the extent of thermal stability largely depends on the different hierarchical morphology of the hybrid flowers. The observed thermal stability decreases in the order SL>>ML>SP-hybrids. Where the SL-hybrid showed absolute stability under high temperature incubation, ML and SP-hybrids were vulnerable to form uniformly distributed highly porous structures (Figure 6a-c and Supporting Information Figure S14). EDX analysis and ATR-FTIR spectra confirmed the absence of any protein molecules in the hybrid flowers

after

thermal

treatment

(Supporting

Information

Figure

S15

and

S16).

Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) had also been performed to investigate their thermal behavior (Figure 6d and Supporting Information Figure S17). As can be seen from the TG curve, the initial weight loss ~ 6.2 % (SL-hybrid), ~ 6.6 % (ML-hybrid) and ~ 7.8 % (SP-hybrid) in the low temperature range at 25 ‒ 130 °C (DSC endothermic peak at 125 °C) was due to the loss of water. The second weight loss occurred in the range of 150 ‒ 400 °C (DSC endothermic broad peaks around 230 and 355 °C) and was


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attributed to the complete decomposition of the sericin molecules of the hybrid flowers.51 The amount of weight loss in this temperature range was approximately about 4.6 % (SL-hybrid), 7 % (ML-hybrid) and 10 % (SP-hybrid) which should be ascribed to the amount of sericin molecules in the hybrid materials, whereas the residual mass left after heat treatment corresponds to the inorganic building blocks of the respective structures. The phenomenon was further confirmed by the TG / DSC analysis of the precursor materials sericin and copper phosphate (Supporting Information Figure S18). Thermal analysis data provide direct evidence to explain the large difference in thermal behavior of the three different types of hybrid structures. Due to the presence of comparatively higher percentage of sericin in the SP-hybrids, it was the most susceptible to heat treatment through decomposition of the sericin molecules which may acts as a binder for the hierarchical growth. Thus the increased porosity of the ML and SP-hybrids was due to the vacant sites of the occupied sericin molecules. Though, the overall structural motif remained intact. Moreover, the enhanced porosity after thermal incubation was also confirmed by the increased in BET surface area (Supporting Information Figure S19). Whereas thermal decomposition of very small amount of sericin from SL-hybrid does not affect their morphological signature, because the higher percentage of inorganic building block may compensate the small loss of organic sericin from the single layer petals.

Study of Heavy Metal Ions Adsorption. On the basis of hierarchical 3D morphology, abundant surface functional groups from silk sericin, large surface areas and high porosity, the as-prepared hybrid flowers were expected to be very useful for the removal of toxic heavy metals from waste water. In addition, as the total size of the flowerlike nanostructured hybrid materials was several micrometers, the separation of heavy metals adsorbed spheres would be fairly easy. The typical heavy metal ions Pb(II), Cd(II) and Hg(II) were chosen for the investigation by both



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batch and continuous free flow filtering methods. They are among the major environmental hazardous and highly toxic pollutants in water resources, and their efficient removal is of great importance.35-43 In this study, different types of as-synthesized hybrid flowers (SL, ML and SPhybrids) were used to investigate their applicability in waste water treatment. Figure 7a shows the corresponding adsorption rates of Pb(II) ions with an initial concentration of 15 mg L-1 on the different hybrid flowers. The adsorption processes were very fast during the first 15 min, and the equilibrium were achieved within 1 h. All the three different hybrid flowers showed excellent adsorption capabilities; however, SP hybrid performed the best among them when the SL and ML hybrid showed almost identical capacities. To further investigate their adsorption property, the adsorption isotherm experiments of ML and SP hybrids (sample dose 5 mg per 20 mL) were obtained with a range of initial concentrations of Pb(II) ion from 2 to 150 mg L−1 at different time intervals (Supporting Information Figure S20). The maximum adsorption capacity of ML and SP-hybrid flowers for Pb(II) ions was 223 and 525 mg g−1 respectively, which is much higher than those of previously reported phosphate compounds at comparable experimental conditions.52,53 The very high adsorption capacity of SP-hybrid motivated us to try their adsorption with much lower dose (1 mg per 40 mL) of the flowers. Supporting Information Figure S21 shows their adsorption rate with a maximum adsorption capacity of 541 mg g−1 within 2 hrs of their addition. Moreover, we also investigated the captivity of commercial and assynthesized copper phosphate powder, but their adsorption rate was far below the flowerlike hybrid structures under similar experimental conditions (Supporting Information Figure S22). These hierarchical hybrid flowers were also proved to be efficient adsorbent for the removal of other hazardous heavy metals such as Cd(II) and Hg(II) from contaminated water (Figure 7b and Supporting Information Figure S23a). Here also the rapid adsorption processes were observed


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within the first 15 min, and thereafter it proceeded at a relatively slower rate achieving the equilibrium within 1 h. However, the adsorption efficiencies of Cd(II) and Hg(II) were much lower than that of Pb(II) at a particular time, having percentage of removal 75.3, 64.3 and 99.8%, respectively after 1 h. But, the adsorption power of SP-hybrid is still higher than the other types of hybrid flowers following the previous trends of Pb(II) adsorption (Supporting Information Figure S23b,c). Furthermore, we had examined the adsorption efficiency of thermally annealed hybrid flowers. Due to the enhanced surface area and porosity they were expected to show superior adsorption. But the observed adsorption rate of annealed hybrid flowers was much lower than that of without annealing one, and the difference is the most pronounced in case of SP-hybrids and least for SL-hybrid flowers (Supporting Information Figure S24).The results clearly indicate the beneficial role of various surface functional groups from silk sericin for the high adsorption of hybrid flowers. SP and ML-hybrid possess higher percentage of sericin molecules, so the difference in their adsorption rate between annealed and without annealed is more prominent, whereas this difference is negligible in case of SL-hybrids having comparatively less amount of sericin. Selectivity is one of the foremost criteria to be a good adsorbent for the removal of trace amount of heavy metals in presence of other competing metal ions. It is worthy to mention that there are many highly concentrated coexisting cations such as Ca(II), Mg(II), Na(I) etc. in commonly used drinking water, which may hinder the adsorption capacity of the adsorbent.44 Moreover presence of these comparatively light weight minerals in drinking water “mineral water” is very important for our health. So, it should be highly desired to fabricate such adsorbents which will selectively capture toxic heavy metal ions rather than coexisting beneficial light metal ions. In our experiment, Ca(II) was selected as a competitive cation to test its effect on the adsorption of



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Pb(II) by the hybrid flowers. Figure 7c shows the faster and superior adsorption of Pb(II) in comparison to Ca(II) from their mixtures, revealing the super selectivity of these hybrid flowers for the removal of Pb(II) ions. The selective adsorption of Pb(II) in presence of Ca(II) from their mixtures may be due to the comparable electronegativity value of Pb(II) with copper(II) phosphate based hierarchical porous structures, in comparison to much lower electronegativity value of Ca(II). In general, the greater the electronegativity and ionic size, the greater will be the affinity for sorption. The high electronegativity values of Pb(II) would induce the strong interaction, which, in turn, corresponds to the more adsorption sites with different adsorption energy levels, while the low electronegativity values will lead to less adsorption sites, due to the week interaction.54 Further, this high selectivity for Pb(II) may also be qualitatively interpreted using the hard and soft acid-base (HSAB) theory. In aqueous solution, Pb(II) ion is much softer than Ca(II) ion and acts as a borderline Lewis acid, so it possesses a higher priority to interact with the borderline Lewis base orthophosphate ion of the hybrid flowers. These combine effects may be responsible for the selective adsorption of Pb(II) by the hybrid flowers.55 The high adsorption rate of hybrid flowers inspires us to check their feasibility in continuous filtering process for water purification. Therefore, a continuous filtering adsorption device was designed to remove heavy metal ions from contaminated water. The schematic diagram is shown in Figure 7d, where the filter part was constructed by a few layer of SP hybrid flowers coated on a special support system (KIRIYAMA paper, pore diameter ~1µm). As the diameter of the microspheres is about 5–10 µm, there should be the gap between microspheres in contact with each other, which provides a path for filtering the polluted water. Upon addition of contaminated water from the top of the funnel, it travelled a long zig zag path through the porous hybrid flower ensuring the complete adsorption of heavy metal ions. The whole system was connected with a


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pumping device to control the free flow of the purified water. As a representative example, two different concentrations of Pb(II) ions were treated for continuous filtering process by using SPhybrid flowers. A very high adsorption efficiencies (>98%) was observed in this continuous filtering process (Supporting Information Table S2), suggesting its potentiality to eliminate the heavy metal ions from polluted water. Moreover, this arrangement is very practical for application to water treatment. Furthermore, the average concentrations of copper ions released into the purified water at different heavy metals treatment were in the range of 0.1–0.3 ppm, which are far below the limits of drinking water standards. Therefore, these flower-like hybrid structures are safe and reliable adsorbent for the removal of heavy metal ions from water. In general, the realization of adsorption mechanism of hierarchical architectures for the heavy metal ions are believed to involve electrostatic interaction, surface complexation and/or ion exchange.44,56,57 The superior performance of as prepared hybrid flowers could be attributed to their hierarchical porous morphology with high surface area as well as abundant surface functional groups from silk sericin, providing much more active sites for heavy metal ions removal. The higher adsorption capacity of SP-hybrid in comparison to SL and ML-hybrids is consistent with their respective surface area measurement (Figure 5b), as well as more compact and dense structures of SP-hybrid with greater mass fraction of sericin in comparison to that of SL & ML-hybrids which otherwise show relatively open up morphology. Whereas the observed difference in the efficiency of the adsorption process for different heavy metals depends on the combine effect of electronegativity, ionic radii and the mutual interactions between the adsorbent and adsorbate.53,58 As mentioned above, the higher relative adsorption of Pb(II) ions into the hybrid flowers in comparison to other heavy metals may be due to its suitable electronegativity and ionic radius which facilitates a better electrostatic interactions with sericin mediated



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hierarchical porous structures of copper phosphate. Therefore due to the superior interactions, a large amount of Pb(II) ions were adsorbed on the surface of the hybrid flowers very quickly and subsequently started to grow elongated needle–shaped nanowires structure. It is very interesting to see the unique growth of uniformly distributed needle‒shaped nanowires having diameter 20– 100 nm and the length 200–500 nm on the hierarchical surface of hybrid flowers (inset of Figure 7a, Figure 8 and Supporting Information Figure S25). Sequential elemental mapping and point analysis from EDX undoubtedly confirmed the presence of Pb in the newly grown nanowires whereas their crystal lattice fringes with lattice distance 0.3 nm were clearly observable from high‒resolution TEM images of single nanowire (Figure 8c-e and Supporting Information Figures S26, S27). Moreover, the extent of growth and the morphology of the nanowires were directly related to the accumulation of the Pb(II) ions on the hybrid flowers. At low initial concentrations of Pb(II) ion, we observed very few nanowires on the hybrid flowers, whereas at higher concentration comparatively thicker nanowires were grown due to the merging of individual thin nanowires (Figure 8f and Supporting Information Figure S28). These elongated nanowires were grown vertically as well as horizontally on the petals surfaces. Moreover, it should be noted that the no collapse structures or distortion of the hybrid flowers was observed after adsorption of heavy metals, demonstrating the stability of the as-prepared hybrid flowers to be used for water treatment (Supporting Information Figure S29). To the best of our knowledge this is the first and direct experimental evidence showing the tight immobilization of heavy metal ions on the adsorbent surface and their consequent growth to nanowire morphology. On the contrary due to the lower electrostatic interactions between the adsorbents and the Cd(II) or Hg(II) ions, very few amount of these metals were accumulated on the surface of the hybrid flowers leaving no such unique growth of nanowires of the corresponding heavy metals


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(Supporting Information Figure S30). Thus the higher adsorption capacity and greater selectivity of Pb(II) ions on the hierarchical surface was due to the large porous surface area and the electronic properties of the copper phosphate facilitating the growth of needlelike nanowires. This is of great significance for the complete removal of hazardous cations from water and their safe disposal to avoid leaching from the adsorbents. Further, the formation of uniformly distributed needle‒shaped nanowires may provide a way for the recovery and reusability of the entrapped heavy metals.

CONCLUSION In summary, silk protein sericin has been employed for the controlled fabrication of organicinorganic hybrid flowers through a green and facile co-precipitation method by exploiting the favorable material characteristics of silk sericin and copper phosphate. Representing only a few percent of the total mass, silk sericin facilitates the formation of well-ordered flowery structures with great precision and manufacturing scalability. Moreover, a large variation in the hierarchical morphology of SL, ML and SP-hybrids strongly depend on the of sericin concentration of their initial growth. The overall structural patterns of the hybrid flowers remains intact even after complete decomposition of the protein molecules at high temperature incubation; however, the process significantly increases the porosity of the flower petals, opening up a broad range of new and exciting applications including separation and detection, catalysis, and energy conversion and storage.17-19 These as-prepared hierarchical porous architectures possess a large BET surface area and show excellent adsorption properties for toxic heavy metal ions Pb(II), Cd(II) and Hg(II) from waste water by batch filtration as well as continuous free flow filtering method. Controlled experiments confirm the beneficial role of several surface functional groups from sericin for superior adsorption property of hybrid flowers. Moreover we have observed a post-



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adsorption growth of uniformly distributed needle-shaped nanowires on the hierarchical surface of the hybrid flowers, exploring the direct experimental evidence for the higher adsorption and greater selectivity of Pb(II) ions in comparison to other heavy metals. Due to the various advantages, such as low cost, large surface area, high adsorption capacity, structural stability and easy separation these flowerlike hybrid materials prove to be an attractive adsorbent for the water purification. Moreover, intrinsic structural integrity of the silk protein was preserved in the hybrid structures extending their possibility for various biological functions.59 Biocompatible silk sericin is well known for its applications in culture media,31 therefore these hierarchical porous structures would be very effective scaffolds for 3D cell cultures. Whereas, copper based hierarchical nanostructures materials may be useful as a promising antibacterial agents.60 Overall, the work may provide a good example for the fruitful utilization of industrial waste sericin in developing value-added materials offering great potential in environmental and economic benefits.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed synthesis of hybrid flowers, XPS characterization details, supporting data and supporting Figures S1 – S30.

AUTHOR INFORMATION Corresponding Author *P. Koley. E-mail: [email protected] [email protected]


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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported in part by the World Premier International Center (WPI) Initiative on Materials Nanoarchitectonics, MEXT,

Japan, and in part by JPSP

KAKENHI (24241047). We thank Dr. A. Tanaka and Dr. H. Iwai for measurement of the XPS and Dr. K. Kurashima and Dr. T. Takei for measurement of the TEM.

REFERENCES (1) Ball, P. In The Self-Made Tapestry, Oxford Univ. Press, Oxford, UK 1999. (2) Lowenstam, H. A.; Weiner, S. In On Biomineralization, Oxford Univ. Press, New York, USA 1989. (3) Veis, A. A Window on Biomineralization. Science 2005, 307, 1419-1420. (4) Mann, S. Self-Assembly and Transformation of Hybrid Nano-Objects and Nanostructures under Equilibrium and Non-Equilibrium Conditions. Nat. Mater. 2009, 8, 781-792. (5) Glotzer, S. C.; Solomon, M. J. Anisotropy of Building Blocks and Their Assembly into Complex Structures. Nat. Mater. 2007, 6, 557–562. (6) Sanchez, C.; Arribart, H.; Guille, M. M. G. Biomimetism and Bioinspiration as Tools for the Design of Innovative Materials and Systems. Nat. Mater. 2005, 4, 277-288. (7) Sanchez, C.; Soler-Illia, G. J. A. A.; Ribot, F.; Lalot, T.; Mayer, C. R.; Cabuil, V. Designed Hybrid Organic-Inorganic Nanocomposites from Functional Nanobuilding Blocks. Chem. Mater. 2001, 13, 3061‒3083.



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) Descalzo, A. B.; Máñez, R. M.; Sancenón, F.; Hoffmann, K.; Rurack, K. The Supramolecular Chemistry of Organic–Inorganic Hybrid Materials. Angew. Chem. Int. Ed. 2006, 45, 5924 – 5948. (9) Jia, L.; Zhao, G.; Shi, W.; Coombs, N.; Gourevich, I.; Walker, G. C. A Design Strategy for the Hierarchical Fabrication of Colloidal Hybrid Mesostructures. Nat. Commun. 2014, 5 (3882), 1‒8. (10) Shi, J.; Jiang, Y.; Wang, X.; Wu, H.; Yang, D.; Pan, F.; Suad, Y.; Jiang, Z. Design and Synthesis of Organic–Inorganic Hybrid Capsules for Biotechnological Applications. Chem. Soc. Rev. 2014, 43, 5192‒5210. (11) Kaushik, A.; Kumar, R.; Arya, S. K.; Nair, M.; Malhotra, B. D.; Bhansali, S. OrganicInorganic Hybrid Nanocomposite-Based Gas Sensors for Environmental Monitoring. Chem. Rev. 2015, 115, 4571-4606. (12) Chaudhari, A. K.; Han. I.; Tan, J.-C. Multifunctional Supramolecular Hybrid Materials Constructed from Hierarchical Self-Ordering of In Situ Generated Metal-Organic Framework (MOF) Nanoparticles. Adv. Mater. 2015, 27, 4438-4446. (13) Ge, J.; Lei, J.; Zare, R. N. Protein–Inorganic Hybrid Nanoflowers. Nat. Nanotechnol. 2012, 7, 428‒432. (14) Wang, R.; Zhang, Y.; Lu, D.; Ge, J.; Liu, Z.; Zare, R. N. Functional Protein– Organic/Inorganic Hybrid Nanomaterials WIREs Nanomed. Nanobiotechnol. 2013, 5, 320-328. (15) Wang, L.-B.; Wang, Y.-C.; He, R.; Zhuang, A.; Wang, X.; 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. (16) Lyu, F.; Zhang, Y.; Zare, R. N.; Ge, J.; Liu, Z. One-Pot Synthesis of Protein-Embedded Metal−Organic Frameworks with Enhanced Biological Activities. Nano Lett. 2014, 14, 5761‒5765.


Page 24 of 38

Page 25 of 38

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


(17) Du, X.; Li, X.; Huang, H.; He, J.; Zhang, X. Dendrimer-Like Hybrid Particles with Tunable Hierarchical Pores. Nanoscale 2015, 7, 6173-6184. (18) Tan, K. W.; Jung, B.; Werner, J. G.; Rhoades, E. R.; Thompson, M. O.; Wiesner, U. Transient Laser Heating Induced Hierarchical Porous Structures from Block Copolymer– Directed Self-Assembly. Science 2015, 349, 54-58. (19) Parlett, C. M. A.; Wilson, K.; Lee, A. F. Hierarchical Porous Materials: Catalytic Applications. Chem. Soc. Rev. 2013, 42, 3876‒3893. (20) Omenetto, F. G.; Kaplan, D. L. New Opportunities for an Ancient Material, Science 2010, 329, 528‒531. (21) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Silk-Based Biomaterials. Biomaterials 2003, 24, 401‒416. (22) Kim, S.; Mitropoulos, A. N.; Spitzberg, J. D.; Tao, H.; Kaplan, D. L.; Omenetto, F. G. Silk Inverse Opals. Nat. Photon. 2012, 6, 818‒823. (23) Kim, S.; Marelli, B.; Brenckle, M. A.; Mitropoulos, A. N.; Gil, E.-S.; Tsioris, K.; Tao, H.; Kaplan, D. L.; Omenetto, F. G. All-Water-Based Electron-Beam Lithography using Silk as a Resist. Nat. Nanotechnol. 2014, 9, 306‒310. (24) Kurland, N. E.; Dey, T.; Kundu, S. C.; Yadavalli, V. K. Precise Patterning of Silk Microstructures Using Photolithography. Adv. Mater. 2013, 25, 6207–6212. (25) Tao, H.; Marelli, B.; Yang, M.; An, B.; Onses, M. S.; Rogers, J. A.; Kaplan, D. A.; Omenetto, F. G. Inkjet Printing of Regenerated Silk Fibroin: From Printable Forms to Printable Functions. Adv. Mater. 2015, 27, 4273-4279. (26) Mandal, B. B.; Grinberg, A.; Gil, E. S.; Panilaitis, B.; Kaplan, D. L. High-Strength Silk Protein Scaffolds for Bone Repair. Proc. Natl. Acad. Sci. 2012, 109, 7699–7704. (27) Tsioris, K.; Raja, W. K.; Pritchard, E. M.; Panilaitis, B.; Kaplan, D. L.; Omenetto, F. G. Fabrication of Silk Microneedles for Controlled-Release Drug Delivery. Adv. Funct. Mater. 2012, 22, 330–335.



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

(28) Zhang, Y.-Q. Applications of Natural Silk Protein Sericin in Biomaterials. Biotechnol. Adv. 2002, 20, 91−100. (29) Nishida, A.; Yamada, M.; Kanazawa, T.; Takashima, Y.; Ouchi, K.; Okada, H. Sustained-Release of Protein from Biodegradable Sericin Film, Gel and Sponge. Int. J. Pharm. 2011, 407, 44−52. (30) Kaur, J.; Rajkhowa, R.; Tsuzuki, T.; Millington, K.; Zhang, J.; Wang, X.-G. PhotoProtection by Silk Cocoons. Biomacromolecules 2013, 14, 3660−3667. (31) Kundu, S. C.; Dash, B. C.; Dash, R.; Kaplan, D. L. Natural Protective Glue Protein, Sericin Bioengineered by Silkworms: Potential for Biomedical and Biotechnological Applications. Prog. Polym. Sci. 2008, 33, 998−1012. (32) Wang, H.; Meng, F.; Cai, Y.; Zheng, L.; Li, Y.; Liu, Y.; Jiang, Y.; Wang, X.; Chen, X. Sericin for Resistance Switching Device with Multilevel Nonvolatile Memory. Adv. Mater. 2013, 25, 5498–5503. (33) Kurland, N. E.; Dey, T.; Wang, C.; Kundu, S. C.; Yadavalli, V. K. Silk Protein Lithography as a Route to Fabricate Sericin Microarchitectures. Adv. Mater. 2014, 26, 4431–4437. (34) Dudev, T.; Lim, C. Competition among Metal Ions for Protein Binding Sites: Determinants of Metal Ion Selectivity in Proteins. Chem. Rev. 2014, 114, 538–556. (35) Hu, J. S.; Zhong, L. S.; Song, W. G.; Wan, L. J. Synthesis of Hierarchically Structured Metal Oxides and their Application in Heavy Metal Ion Removal. Adv. Mater. 2008, 20, 2977-2982. (36) Fu, F.; Wang, Q. Removal of Heavy Metal Ions from Wastewaters: A Review. J. Environ. Manage. 2011, 92, 407-418. (37) Liu, A. M.; Hidajat, K.; Kawi, S.; Zhao, D. Y. A New Class of Hybrid Mesoporous Materials with Functionalized Organic Monolayers for Selective Adsorption of Heavy Metal Ions. Chem. Commun. 2000, 1145-1146.


Page 26 of 38

Page 27 of 38

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


(38) Chen, B.; Ma, Q.; Tan, C.; Lim, T. T.; Huang, L.; Zhang, H. Carbon-Based Sorbents with Three-Dimensional Architectures for Water Remediation. Small 2015, 11, 33193336. (39) Zhong, L. S.; Hu, J. S.; Cao, A. M.; Liu, Q.; Song, W. G.; Wan, L. J. 3D Flowerlike Ceria Micro/Nanocomposite Structure and Its Application for Water Treatment and CO Removal. Chem. Mater. 2007, 19, 1648-1655. (40) Wu, Z. X.; Zhao, D. Y. Ordered Mesoporous Materials as Adsorbents. Chem. Commun. 2011, 47, 3332-3338. (41) Ide, Y.; Ochi, N.; Ogawa, M. Effective and Selective Adsorption of Zn2+ from Seawater on a Layered Silicate. Angew. Chem. Int. Ed. 2011, 50, 654-656. (42) Liang, H. W.; Cao, X.; Zhang, W. J.; Lin, H. T.; Zhou, F.; Chen, L. F.; Yu, S. H. Robust and Highly Efficient Free-Standing Carbonaceous Nanofiber Membranes for Water Purification. Adv. Funct. Mater. 2011, 21, 3851-3858. (43) Chen, B.; Zhu, Z.; Liu, S.; Hong, J.; Ma, J.; Qiu, Y.; Chen, J. Facile Hydrothermal Synthesis of Nanostructured Hollow Iron-Cerium Alkoxides and Their Superior Arsenic Adsorption Performance. ACS Appl. Mater. Interfaces 2014, 6, 14016-14025. (44) Li, N.; Zhang, L.; Chen, Y.; Fang, M.; Zhang, J.; Wang, H. Highly Efficient, Irreversible and Selective Ion Exchange Property of Layered Titanate Nanostructures. Adv. Funct. Mater. 2012, 22, 835-841. (45) Wang, Z.; Zhang, Y.; Zhang, J.; Huang, L.; Liu, J.; Li, Y.; Zhang, G.; Kundu, S. C.; Wang, L. Exploring Natural Silk Protein Sericin for Regenerative Medicine: an Injectable, Photoluminescent, Cell-Adhesive 3D Hydrogel. Sci. Rep. 2014, 4, 7064. (46) Dar, M. I.; Arora, N.; Gao, P.; Ahmad, S.; Grätzel, M.; Nazeeruddin, M. K. Investigation Regarding the Role of Chloride in Organic ‒Inorganic

Halide Perovskites

Obtained from Chloride Containing Precursors. Nano Lett. 2014, 14, 6991–6996. (47) Barth, A. Infrared Spectroscopy of Proteins. Biochim. Biophys. Acta 2007, 1767, 1073‒1101.



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Page 28 of 38

(48) Teramoto, H.; Miyazawa, M. Molecular Orientation Behavior of Silk Sericin Film as Revealed by ATR Infrared Spectroscopy. Biomacromolecules 2005, 6, 2049‒2057. (49) Onoda, H.; Okumoto, K.; Nakahira, A.; Tanaka, I. Mechanochemical Effects on the Synthesis of Copper Orthophosphate and cyclo-Tetraphosphate Bulks by the Hydrothermal Hot Pressing Method. Materials 2009, 2, 1-9. (50) Onoda, H.; Okumoto, K. Synthesis, Acid and Base Resistance of Various Copper Phosphate Pigments by the Substitution with Lanthanum. Mater. Sci. Appl. 2011, 2, 209214. (51) Mazzi, S.; Zulker, E.; Buchicchio, J.; Anderson, B.; Hu, X. Comparative Thermal Analysis of Eri, Mori, Muga, and Tussar Silk Cocoons and Fibroin Fibers. J Therm Anal Calorim 2014, 116, 1337–1343. (52) Jia, K.; Pan, B.; Zhang, Q.; Zhang, W.; Jiang, P.; Hong, C.; Pan, B.; Zhang, Q. Adsorption of Pb2+, Zn2+, and Cd2+ from Waters by Amorphous Titanium Phosphate. J. Colloid Interface Sci. 2008, 318, 160-166. (53) Zhao, X. Y.; Zhu, Y. J.; Zhao, J.; Lu, B. Q.; Chen, F.; Qi, C.; Wu, J. Hydroxyapatite Nanosheet-Assembled Microspheres: Hemoglobin-Templated Synthesis and Adsorption for Heavy Metal Ions. J. Colloid Interface Sci. 2014, 416, 11-18. (54) Cai, W.; Duan, G.; Li, Y. In Hierarchical Micro/Nanostructured Materials: Fabrication, Properties, and Applications, CRC Press, 2014. (55) Wang, X.; Yang, X.; Cai, J.; Miao,T.; Li, L.; Li, G.; Deng, D.; Jiang, L.; Wang, C. Novel flower-like titanium phosphate microstructures and their application in lead ion removal from drinking water. J. Mater. Chem. A 2014, 2, 6718–6722. (56) Zhong, L. S.; Hu, J. S.; Liang, H. P.; Cao, A. M.; Song, W. G.; Wan, L. J. SelfAssembled 3D Flowerlike Iron Oxide Nanostructures and Their Application in Water Treatment. Adv. Mater. 2006, 18, 2426-2431.


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(57) Cao, C.-Y.; Qu, J.; Yan, W.-S.; Zhu, J.-F.; Wu, Z.-Y.; Song, W.-G. Low-Cost Synthesis of Flowerlike α-Fe2O3 Nanostructures for Heavy Metal Ion Removal: Adsorption Property and Mechanism. Langmuir 2012, 28, 4573-4579. (58) Vila, M.; Sánchez-Salcedo, S.; Cicuéndezb, M.; Izquierdo-Barba, I. Novel BiopolymerCoated Hydroxyapatite Foams for Removing Heavy-Metals from Polluted Water. J. Hazard. Mater. 2011, 192, 71-77. (59) 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 MetalOrganic Frameworks as Protective Coatings for Biomacromolecules. Nat. Commun., 2015, 6 (7240), 1. (60) Gao, F.; Pang, H.; Xuc, S.; Lu, Q. Copper-Based Nanostructures: Promising Antibacterial Agents and Photocatalysts. Chem. Commun. 2009, 3571–3573.



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Purification

PBS (1X)

(Degumming procedures)

(Gently shaking)

Silkworm cocoons

Sericin powder (Typically discarded as by product during fibroin extraction)

Clear sericin soln. (Required concentration) 12 hours incubation

5 µm

Aq. CuSO4 soln. (Calculated amount)

Hybrid flowers (Precipitate)

Scheme 1. Schematic representation of the synthesis of silk protein sericin mediated organicinorganic hybrid flowers.


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c

b

50 nm

d

2 µm

e

500 nm

50 nm

g

f 2 nm 50 nm

2 µm

5 nm

5 µm

Figure 1. SEM images of (a) single hybrid flower (inset: natural flower to make analogy), and (b) corresponding hierarchical arrangement of porous petals. High-resolution SEM images showing (c) multilayer stacked nanosheets and (d) surface porosity of a single petal. (e) TEM images of hybrid flowers (inset: edge of the flower petal showing surface porosity and multilayer arrangements) and (f) corresponding high-resolution image showing the crystal lattice structure of the petal (inset: magnified image shows lateral arrangement of the crystal lattice fringes). (g) LSM image of the hybrid flowers. Sericin concentration 0.1 mg L-1 (ML-hybrid).



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a

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b 200 nm

1 µm

10 µm

2 µm

50 nm

c

d 200 nm

1 µm

10 µm

50 nm

1 µm

Figure 2. SEM images showing the morphological diversity of the hybrid flowers with varying concentrations of sericin: (a), (b) 1 mg mL-1 (SP-hybrid) and (c), (d) 0.01 mg mL-1 (SL-hybrid). Inset of (b) indicates the network arrangement of dense petals with many void spaces in between and (d) shows single layer thin nanosheet petals. Inset of (a) and (c) indicate the TEM images of SP-hybrid and SL-hybrid with their corresponding magnified images showing the crooked and wavy fringe of network petals and thin nanosheet petals respectively.


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a

b 200 nm

2 µm

10 µm

c

d 200 nm

200 nm

2 µm

2 µm

Figure 3. SEM images showing the time dependent growth of hybrid flowers (0.1 mg mL-1 sericin): (a) 10 mins, (b) 2 h, (c) 6 h and (d) 12 h of incubation time. Insets: corresponding magnified images.



a

b

500 nm

5 µm

Figure 4. SEM images of hybrid flowers after treated with (a) glutaraldehyde and (b) EDTA.

b

104

102

100 Amide I (1634)

98

Amide III (1238)

Amide II (1514) Phosphate (1048)

96 3500

3000

1500

Wavenumber (cm-1)

1000

SL-Hybrid ML-Hybrid SP-Hybrid Synthesized Copper Phosphate

0.03

120

80

dV(r) (cm3/g/nm)

Quantity Adsorbed (cm3/g)

a Transmittance (%T)

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 34 of 38

0.02

0.01

0.00 5

10

15

20

25

Pore Diameter (nm)

40

0 0.0

0.2

0.4

0.6

0.8

1.0

Relative Pressure (P/P0)

Figure 5. (a) ATR-FTIR spectra of hybrid flowers, and (b) nitrogen adsorption-desorption isotherms (inset: pore size distribution) of different hybrid flowers and copper phosphate.


Page 35 of 38

a

b 200 nm

200 nm

2 µm

2 µm

d

c 200 nm

Weight (wt %)

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


100

SL-Hybrid ML-Hybrid SP-Hybrid

95 90 85 80

2 µm

75 100

200

300

400

500

o

Temperature ( C) Figure 6. SEM images showing the thermal stability of hybrid flowers after calcination at 400 °C for 2 h under Ar atmosphere: (a) SL-hybrid showing absolute stability, (b) ML-hybrid and (c) SP-hybrid with increased porosity on the flower petals. (inset: magnified image of the respective structures). (d) TG analysis curve of the different hybrid flowers.



a SL-hybrid

ML-hybrid

SP-hybrid

C/Co

0.8 0.6 Pb

adsorption

0.4 0.2 0.0 0

20

40

60

80

100

Heavy metal adsorbed (%)

b

1.0

100 80 60 Pb(II) Cd(II) Hg(II)

40 20 0 0

120

20

40

60

80

100

120

Time (min)

Time (min)

c

d 1.0 0.8

C/Co

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 36 of 38

Pb (II) Ca (II)

0.6 0.4 0.2 0.0 0

20

40

60

80

100

120

Time (min) Figure 7. (a) The adsorption rate curve for Pb(II) by different types of hybrid flowers (inset: SEM images before and after Pb(II) adsorption). (b) Comparative study of adsorption rate (%) of different heavy metals by SP-hybrid flowers. (c) Adsorption rate showing the selective adsorption of Pb(II) in presence of Ca(II) ions. (Initial concentrations of different heavy metal ions: 15 mg L-1, and the dose of different hybrid flowers used as an adsorbents: 5 mg per 20 mL). (d) Schematic diagram showing the designed continuous filtering adsorption devices.


Page 37 of 38

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


a

c

b

100 nm

200 nm

d

2 µm

500 nm

e

200 nm

f

1 nm

5 nm

50 nm

50 nm

Figure 8. (a), (b) SEM images showing the unique growth of uniformly distributed needle‒shaped nanowires with initial Pb(II) concentration 15 mg L-1 (inset: magnified image of nanowires). (c) HAADF-STEM image and (d) STEM-EDX mapping clearly indicates the formation of Pb nanowires (Green: Cu and Red: Pb). (e) HR-TEM images of single nanowire (inset: magnified image showing lateral arrangement of crystal lattice fringes). (f) TEM image showing the merging of nanowires at the edge with initial Pb(II) concentration 25 mg L-1 (inset: corresponding SEM image of the nanowires).



Table of Contents Graphic

Aq. CuSO4 soln.

Silkworm Cocoons

Heavy metals left (C/Co)

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

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1.0

PBS

Sericin Powder

Hybrid Flowers

Pb

0.5

adsorption

0.0 0

40

80

Time (min)


120

Controlled Fabrication of Silk Protein Sericin ... - ACS Publications

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