Facile fabrication of silk protein sericin-mediated hierarchical hydroxyapatite-based bio-hybrid architectures: excellent adsorption of toxic heavy metals and hazardous dye from wastewater

Abstract

Environmental pollutants, especially water pollutants, are a serious problem in modern industrial societies; therefore, there is a significant need for new approaches to water purification through the development of eco-friendly functional materials with cost-effective fabrication methods. Here, we report the inexpensive fabrication of hierarchical bio-hybrid architectures through a green and facile co-precipitation method by employing an industrial waste natural silk protein, sericin, and hydroxyapatite (HAP), the main component of bones and teeth. These hierarchical bio-hybrids show excellent adsorption of toxic heavy metal ions of Pb(II), Cd(II) and Hg(II), with adsorption efficiencies in the order of Hg(II) ≪ Cd(II) < Pb(II), and a hazardous dye, Congo red (CR), due to their large surface area and the chemical natures of sericin and HAP. The microscale size and structural integrity of the hybrids enables easy separation of the adsorbents from waste water. More than 99% adsorption of Pb(II) ions is observed within 15 min. Moreover, the simply stacked layers of hybrid flowers function as excellent filters for free-flowing polluted water, with a removal efficiency of 99.5%. This system can be used as a practical natural water treatment. Further, these hybrid adsorbents can be reused following recovery with dilute acetic acid treatment; the process also regenerates the adsorbed Pb(II) ions. The simple, economical and environmentally benign fabrication method of these hybrids and their excellent water-purifying ability will lead to a new solution to the worldwide issue of water pollution.

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Introduction

In recent years, water pollution caused by heavy metal ions such as Pb(II), Cd(II), and Hg(II) from various natural and anthropogenic sources has become one of the most serious problems in modern industrial society, not only due to their high toxicity even at trace amounts but also due to their natural resistance to degradation.^1–8 These ions have a tendency to accumulate in the body via the food chain, posing a long-term threat to human health and natural ecosystems. In addition, a large variety of colored effluents in the form of hazardous dye stuffs released into the environment from various unregulated industrial activities, such as textiles, leather, plastic, and paper, have also become major contributors to water pollution, giving rise to carcinogenicity, teratogenicity, and even mutagenicity.^9–12 Moreover, due to their complex molecular structures and large sizes, dyes are resistant to aerobic digestion and are stable to oxidizing agents; thus, their removal and degradation are very difficult to achieve by conventional physical and biological treatments.^9–12 Therefore, removal of these micro-pollutants from waste water is of utmost importance. A variety of methods have been developed to efficiently remove these heavy metals and hazardous dyes from aqueous systems; among these, adsorption techniques, one of the most promising strategies, have attracted significant attention due to their efficiency and low cost, the availability of different adsorbents, and their simplicity of operation.^13–17 A number of adsorbents, such as hierarchical metal oxides,^2,14 functionalized graphene or graphene oxide,^6,8,11,18 zeolites,¹⁹ poly-glutamic acid,^20,21 functionalized biomolecules,²² and agricultural residues,²³ have been used for the removal of heavy metal ions and dye stuffs from waste water. However, the major disadvantages of the available adsorbents are their low adsorption capacities, resulting in incomplete removal, their relatively weak interactions with adsorbates, and their difficulties of separation, high cost and slow capture kinetics, which greatly hamper their practical and affordable applications. At the same time, fast and tight adsorption, structural integrity of the adsorbents and recovery of the entrapped cations are greatly required.²⁴ Moreover, leaching of the adsorbent materials into purified water and their subsequent toxicity is a matter of concern for safe drinking water. Therefore, it is crucial to develop high performance adsorbents in a cost-effective and environmentally friendly way in order to rapidly and efficiently remove toxic heavy metals and hazardous dyes from wastewater. In this regard, hierarchical bio-hybrid architectures with porous nanoscale building blocks may function as very effective adsorbents towards heavy metal ions and dyes due to their high surface area, facile mass transportation, abundant active adsorption sites, easy separation and nontoxic biological origins.

Organic–inorganic hybrids derived from biomolecules have attracted increasing interest recently, due to their broad applications in biocatalysis, drug delivery, biosensing, etc.^7,25–29 These materials often present superior properties and synergistically enhanced functionalities of each of their components. Considerable efforts have been devoted to fabricate bioinorganic hybrids, not only due to their combined functional applications, but also to their chemical diversity, composition flexibility and good biocompatibility. However, facile and low cost fabrication of bioinorganic hybrid architectures with excellent adsorption capacities remains difficult to achieve.

Silk, a highly promising biopolymer derived from silkworm cocoons, consists primarily of two proteins – a fibrous core protein, fibroin, and a glue protein called sericin.^30–35 Apart from the various beneficial properties of sericin in comparison to the well-studied fibroin,^36–41 there are two main reasons that strongly motivated our choice of sericin for the fabrication of hierarchical bio-hybrid structures. Firstly, most of the amino acids of sericin possess high contents of polar side chains containing hydroxyl, carboxyl, and amino groups; thus, sericin is a highly water-soluble, hydrogen bond-forming and strongly metal-binding material.^31,42 Secondly, sericin is comparatively inexpensive and abundantly available material which is typically discarded as a byproduct during degumming processes in raw silk production.^30,31 Contrastingly, hydroxyapatite (HAP), the main inorganic component of vertebrate hard tissues such as bones and teeth, plays an important role in many biomedical fields, including bone repair,⁴³ tissue engineering,⁴⁴ and drug and gene delivery,^45,46 owing to its biocompatibility, bioactivity, non-toxicity and high stability. Moreover, various HAP nanostructured materials are of great significance in many other fields, such as catalysis,⁴⁷ gas sensing⁴⁸ and water treatment.⁴⁹ Thus, we believed it would be worthwhile to fabricate sericin-mediated hierarchical HAP hybrids for possible multimodal applications. Herein, we report the facile fabrication and characterization of hierarchical bio-hybrid architectures using the silk protein sericin as the organic component and hydroxyapatite as an inorganic counterpart of biological origin. These synthetic flower-like hybrid architectures have large surface areas with different surface functional groups from sericin; they act as excellent environmental pollutant scavengers through very fast adsorption of heavy metal ions, such as Pb(II), Cd(II) and Hg(II), and a carcinogenic textile dye, Congo red (CR), from waste water. Moreover, the hybrid flowers can be recovered and the corresponding heavy metal ions can be regenerated by dilute acetic acid treatment. The simple fabrication method, biocompatible nature and good water purifying abilities of these hybrids can be expanded to large-scale applications.

Experimental

Materials

Pure sericin (Wako Chemicals, Japan), calcium chloride dihydrate (Sigma-Aldrich) and phosphate buffer saline (Lonza, USA) were used as precursor reagents for the co-precipitation reaction. Milli-Q purified deionized water was used to prepare the stock solutions.

Synthesis of hybrid flowers

In a typical experiment, 200 μL of aqueous CaCl₂ solution (100 mM) was added to 4 mL of 1× PBS (pH 7.4) containing 0.5 mg mL⁻¹ sericin; the resulting mixture was gently shaken for a few minutes, followed by incubation at 25 °C for 24 h. A white precipitate of hybrid flowers was collected, washed repeatedly with deionized water, and dried at room temperature.

Characterizations

For SEM analysis, a suspension of the thoroughly washed as-prepared hybrid flowers was deposited and dried on a glass substrate, followed by platinum coating. SEM measurements were carried out with Hitachi S4800 and SU8000 field emission microscopes.

For TEM analysis, 2 μL dilute suspension of the hybrid flowers was added to molybdenum and copper TEM grids and dried at room temperature, followed by imaging with a JEOL JEM-2100F high resolution electron microscope with an operating voltage of 200 kV. High angle annular dark field (HAADF) imaging and EDX mapping were carried out in scanning transmission electron microscopy (STEM) mode.

XRD analysis of the dried hybrid flowers was carried out with a Rigaku, Rint 2000 Ultima III X-ray diffractometer using Cu Kα radiation (λ = 1.5406 Å, 40 kV/40 mA). The 2θ scanning range was from 1° to 80° at 0.02° intervals with a scanning speed of 1° min⁻¹.

XPS analysis was carried out with a Thermo Fisher Scientific Theta Probe system.

Thermogravimetric 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 °C to 500 °C in an alumina crucible at a rate of 5 °C min⁻¹.

Surface area and pore size measurements

The specific surface area was determined by the Brunauer–Emmett–Teller (BET) method from nitrogen adsorption–desorption isotherms 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 branches of the nitrogen isotherms using the Barrett–Joyner–Halenda (BJH) method.

Heavy metal ions adsorption

Aqueous solutions with different concentrations of Pb(II), Cd(II) and Hg(II) ions were prepared by dissolving Pb(NO₃)₂, Cd(NO₃)₂·4H₂O and Hg(NO₃)₂·H₂O, respectively, as the sources of the heavy metal ions. In general, 5 mg of hybrid flowers was added to 20 mL of the 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 plasma-optical emission spectroscopy (ICP-OES) to measure the concentration of metal ions remaining in the solution. The adsorption isotherms were obtained by varying the initial concentration of Pb(II) (2 to 60 mg L⁻¹) under stirring at constant time periods at room temperature. The desorptions of the heavy metals and the corresponding recoveries of the hybrid materials were carried out using 20 mL of a 1% acetic acid solution. The hybrid materials used in the adsorption kinetics experiments were mixed with the 1% acetic acid solution and stirred for 12 h. The amount of heavy metals in the supernatant was tested to calculate the desorption efficiency of the adsorbents and the recovery of the hybrid materials for repeated use.

Congo red dye adsorption

The dye adsorption properties of the hybrid flowers were examined by time-dependent UV adsorption study of the dye molecules. In a typical experiment, 6 mg of hybrid flowers was added to 3 mL of aqueous Congo red solution (0.05 mg mL⁻¹). UV measurements were performed on a UV-VIS-NIR spectrophotometer (V-570, JASCO) at different incubation times.

Results and discussion

Synthesis and characterization

The hierarchical hybrid structures were prepared through simple mixing of two components by adding an aqueous solution of CaCl₂ to a clear phosphate buffered saline (PBS) solution of the silk protein sericin. The resulting mixture was then gently shaken for a few minutes and allowed to incubate at room temperature (∼25 °C). After 24 h, a large amount of whitish precipitate appeared with a porous, uniform flower-like morphology. Fig. 1a and b show the general morphologies of the hybrid flowers (average size 2 to 5 μm) imaged by scanning electron microscopy (SEM). The high resolution SEM image exhibits explicit hierarchical arrangements of the flower petals with thicknesses in the range of 10 to 15 nm (Fig. 1c). Meanwhile, individually, silk sericin self-assembled to form nanoparticles of different diameters, and calcium chloride precipitated from 1× PBS, forming large crystals of aggregated calcium phosphate but no trace of flower-like morphology (Fig. S1, ESI†). Transmission electron microscope (TEM) images of the as-synthesized hybrid flowers are shown in Fig. 1d–f. The hierarchical morphology of the nanosheet petals consists of crooked and wavy shapes with many void spaces in between them, and the crystal lattice fringes in the high-resolution TEM images correspond to the periodicity of the structures (Fig. 1e and f). Energy-dispersive X-ray (EDX) analysis of the hybrid flowers manifested the presence of C, N, O, P and Ca elements (Fig. S2, ESI†). Meanwhile, EDX elemental mapping demonstrated that the Ca, P and O elements, which are the main components of HAP materials, were predominantly distributed in the nanosheet petals of the hybrid flowers in comparison to other elements (Fig. 2). Moreover, it was also found that the C and N elements from the sericin protein were distributed uniformly on the petals (Fig. 2). X-ray diffraction (XRD) was employed to characterize the crystal structure of the hybrid flowers. The diffraction peaks confirmed the highly crystalline characteristics of the hybrid flowers in comparison to the broad features of amorphous silk sericin; the peaks also fitted well with those of natural HAP materials (JCPDS no. 09-0432) (Fig. 3a). Moreover, the surface properties of the hybrid flowers deposited on a Si substrate were further characterized by X-ray photoelectron spectroscopy (XPS) (Fig. 3b and Table S1, Fig. S3, ESI†). Fig. 3b shows the XPS profiles of the samples scanned in the range of 0 to 1400 eV, indicating the presence of C, N, O, P, and Ca elements. Table S1 in the ESI† represents the binding energies of the C 1s, N 1s, O 1s, P 2p, and Ca 2p peaks along with their atomic concentrations. The high-resolution XPS spectrum of C 1s and N 1s clearly indicates the presence of characteristic functional groups from the silk protein sericin. Meanwhile, the binding energy data of Ca confirms the presence of calcium phosphate (Ca 2p: 347, 350.5 eV), whose two main components, the O 1s peak (531.5 eV) and the P 2p peak (133.3 eV), are attributed to the PO₄³⁻ groups (Fig. S3, ESI†). Thus, the characteristic binding energy peaks clearly indicate the presence of protein molecules and calcium phosphate in the hybrid structures. Thermogravimetric analysis (TGA) was performed to investigate the percentage of different components in the hybrid flowers by analyzing their different weight loss steps (Fig. S4a, ESI†). As can be seen from the TG curve, the initial weight loss of ∼5.6% in the temperature range of 25 to 200 °C was due to the removal of physically adsorbed and bound water molecules. The second weight loss step occurred in the range of 200 to 450 °C and was attributed to the complete decomposition of the sericin molecules of the hybrid flowers.⁵⁰ The amount of weight loss in this temperature range was approximately 5%, which should be ascribed to the total weight percentage of the sericin molecules in the hybrid material, whereas the residual mass remaining after heat treatment corresponds to the inorganic components of the hybrid structures and was calculated to be ∼89.4%. The phenomenon was further confirmed by TG analysis of the precursor material, sericin (Fig. S4b, ESI†).

Fig. 1 SEM images of (a) a group of hybrid flowers, (b) a single hybrid flower and (c) the corresponding high resolution image showing the hierarchical arrangement of the nanosheet petals. TEM images of (d) a single hybrid flower, (e) thin nanosheet petals and (f) the corresponding high-resolution image showing the crystal lattice structure of the petal (inset: magnified image showing the lateral arrangement of the crystal lattice fringes).

Fig. 2 HAADF-TEM image and EDX elemental mapping of the hybrid flowers, showing different concentrations but uniform distribution of the corresponding elements.

Fig. 3 (a) XRD patterns of crystalline hybrid structures and the amorphous silk protein sericin. The bottom panel shows the XRD pattern of standard hydroxyapatite (JCPDS 09-0432). The same peak positions in the patterns of the hybrid structures as those of hydroxyapatite crystals suggest that the hybrid flowers are mainly formed from hydroxyapatite. (b) XPS spectral data of the HAP-hybrid structures and silk sericin.

The formation mechanism of these hierarchical hybrid structures is similar to those described in published reports of protein based copper phosphate hybrid flowers.^7,25–28 Since the peak positions in the XRD patterns of the hybrid structures are the same as those of HAP crystals (Fig. 3a), and large, regularly arranged lattice structures are also observed in the HR-TEM image of the nanosheet petals (Fig. 1f), it can be concluded that the petals in the hybrid flowers were formed by a regular arrangement of HAP. The uniform distribution of the C and N elements of sericin protein indicates that the HAP frames of the petals are covered by sericin layers. The inherently hydrophilic protein sericin formed complexes with the HAP surfaces, predominantly through the coordination ability of the amino acid backbone, and served as a glue to form the nanosheet petals. These nanosheets further self-assembled to form hierarchical porous architectures with a flower-like morphology. Since the HAP materials do not form hierarchical architectures without sericin, the protein sericin must play an important role in the self-assembled growth of the architectures.

The specific surface area, average pore size distribution and pore volume of the hierarchical architectures play significant roles in enhancing the adsorption performance of the materials. These synthetic flower-like hierarchical hybrid materials were expected to have a large surface area, which was measured using the standard Brunauer–Emmett–Teller (BET) method. Fig. 4a shows a typical nitrogen adsorption-desorption isotherm of the hybrid structures and the as-synthesized HAP. According to the investigative results, it is clear that the as-prepared 3D HAP hybrid flowers possess a very high BET surface area value of 373.2 m² g⁻¹, which is not only much higher than that of the as-synthesized precursor HAP materials (153.9 m² g⁻¹), but also shows a significant increase over previous reports of hierarchical HAP architectures.^51–53 The sorption results represent a type IV isotherm with a distinct hysteresis loop, indicating the presence of mesopores in the microstructures^53,54 with a relatively narrow pore size distribution, mainly centered in the range of 3 to 20 nm, and a cumulative pore volume of 1.239 cm³ g⁻¹ according to the Barrett–Joyner–Halenda (BJH) desorption method (Fig. 4b).

Fig. 4 (a) Nitrogen adsorption–desorption isotherms and (b) pore size distribution curves of the HAP hybrid flowers and as-synthesized HAP.

Adsorption of heavy metal ions and organic dyes

In this study, the efficiency of the as-prepared hybrid flowers was investigated for waste water treatment. The typical heavy metal ions Pb(II), Cd(II) and Hg(II) were chosen for analysis by varying the contact time and the concentration of the adsorbate. These ions are among the major environmentally hazardous and highly toxic pollutants in water resources, and their efficient removal is of great importance for safe drinking water.^1–8 Fig. 5a shows the adsorption rate of Pb(II) ions with an initial concentration of 15 mg L⁻¹ by the hierarchical adsorbent. It was observed that the adsorption process proceeded very quickly, and equilibrium was achieved within 15 min. Thus, complete removal of the Pb(II) ions was achieved within 15 min after the separation of the microscale-size adsorbent through centrifugation. Rapid adsorption rates were also observed when the concentration of the heavy metal ion was varied (Fig. S5a, ESI†). To further investigate the adsorption properties, adsorption isotherm experiments of the HAP-hybrid flowers (sample dose 5 mg per 20 mL) were performed with a range of initial concentrations of Pb(II) ion from 2 to 60 mg L⁻¹ at different time intervals (inset of Fig. 5a and S5b, ESI†). The maximum adsorption capacity of the HAP-hybrid flowers for Pb(II) ions was calculated to be 248 mg g⁻¹, which is much higher and faster than those of previously reported HAP architectures under comparable experimental conditions.^49,52,53 Moreover, we investigated the capacity of as-synthesized bulk HAP as a control under similar experimental conditions; however, its adsorption rate was far below that of the HAP-hybrid flowers, even after 2 h of incubation. This further confirms the necessity of sericin-mediated flower-like hybrid architectures with a large surface area for superior adsorption efficiency. Further, these hierarchical hybrid flowers were proved to be an efficient adsorbent for the removal of other hazardous heavy metals, such as Cd(II) and Hg(II), from contaminated water (Fig. 5b and S6, ESI†). Here also, rapid adsorption processes were observed within the first 15 min; thereafter, adsorption proceeded at a relatively slower rate until equilibrium was achieved. However, a substantial difference in the adsorption rates was observed in the order Hg(II) ≪ Cd(II) < Pb(II), with respective percentages of removal of 42.5%, 89.5% and 99.9% after 15 min of adsorption (Fig. 5b). These results clearly indicate that the adsorption efficiency of Pb(II) was fairly higher than that of Cd(II), whereas the adsorption of Hg(II) showed much lower efficiency in comparison to the other two metal ions at a particular time. The high adsorption efficiency of Pb(II) may be qualitatively interpreted using the hard and soft acid–base (HSAB) theory.^55,56 In aqueous solution, Pb(II) ion is harder than Cd(II) and Hg(II) ions and acts as a borderline Lewis acid; therefore, it possesses a greater ability to interact with the borderline Lewis base orthophosphate ions of the hybrid flowers. Moreover, it should be noted that the adsorption mechanisms of hierarchical architectures for heavy metal ions are believed to involve electrostatic interactions, surface complexation, softness and/or ion exchange phenomena.^24,57,58 The superior performance of the as-prepared HAP-hybrid could be attributed to its hierarchical morphology with a large surface area as well as the abundant surface functional groups from silk sericin, which provide many more active sites for the removal of heavy metal ions. In general, the binding of metal onto the HAP surface can be described by an ion exchange mechanism which involves the replacement of Ca(II) ions with M²⁺ ions, i.e., Pb(II), Cd(II), or Hg(II).^59–61 Alkaline earth metals such as Ca(II) are easily replaced by divalent metal ions due to their labile nature by the ion exchange phenomenon.^59–61 However, the observed differences in the efficiency of the adsorption process for different heavy metals depend on the combined effects of electronegativity and ionic radii, which facilitate the ion exchange mechanism. Moreover, the very fast adsorption process (<15 min) also indicates an ion exchange mechanism.^59,62 As mentioned above, Pb(II) has strong affinity towards the HAP hybrid, which is in good agreement with previous reports.⁶³ The higher relative adsorption of Pb(II) ions onto the hybrid flowers in comparison to other heavy metals may be due to its suitable electronegativity and ionic radius, which facilitates a better cation exchange reaction between Pb(II) ions in aqueous solution and the Ca(II) ions of HAP in the sericin-mediated hierarchical porous structures. Therefore, a large amount of Pb(II) ions accumulated on the surface of the hybrid flowers very quickly and subsequently started to grow groups of elongated nanorods with hexagonal facets. It is very interesting to see this unique growth of densely packed nanorod structures with diameters of 20 to 200 nm on the hierarchical surface of the HAP-hybrid flowers (Fig. 6 and 7). EDX analysis and sequential elemental mapping from scanning transmission electron microscopy (STEM) undeniably confirmed the presence of Pb in the morphology of the newly grown nanorods (Fig. 7b–d and S7, ESI†). At low initial concentrations of Pb(II) ions, we did not observe any detectable nanorod morphologies, whereas at higher concentrations, a large number of dense nanorods were grown on the hybrid flowers (Fig. S8, ESI†). However, crystalline lead nitrate forms hexagonal microstructures from aqueous solution (Fig. S8f, ESI†). The extent of growth and the morphological differences of the nanorods were due to the different concentrations of adsorbed Pb(II) ions on the surface and near the surface for their crystal growth.⁶⁴ These results provide direct experimental evidence for the very fast and tight immobilization of heavy metal ions on the adsorbent surface and their consequent growth into a nanorod morphology. Contrastingly, because of unfavorable electronegativity and ionic radii in addition to less facile cation exchange phenomena between the adsorbents and Cd(II) or Hg(II) ions, very small amounts of these heavy metals accumulated on the surface of the hybrid flowers, with no unique growth of nanorods of the corresponding heavy metals under identical experimental conditions (Fig. S9, ESI†). This is of great significance for the complete and rapid removal of hazardous cations from water and their safe disposal to avoid leaching from the adsorbents. Further, the formation of uniformly distributed dense nanorod structures may provide a way to recover and reuse the entrapped heavy metals.

Fig. 5 (a) Adsorption rate curve for Pb(II) ions by HAP-hybrid flowers (inset: adsorption isotherm for Pb(II) ions with initial concentrations from 2 to 60 mg L⁻¹ after 15 min of adsorption). (b) Comparative study of the adsorption rates (%) of different heavy metals, Pb(II), Cd(II) and Hg(II), by HAP-hybrid flowers (initial concentrations of different heavy metal ions: 15 mg L⁻¹; dose of HAP-hybrid flowers used as adsorbent: 5 mg per 20 mL).

Fig. 6 SEM images showing (a, b) HAP-hybrid flowers covered with a unique growth of uniformly distributed nanorods and (c) high magnification image of the nanorods. Pb(II) concentration: 15 mg L⁻¹.

Fig. 7 (a) TEM image showing the many as-grown nanorods on the HAP-hybrids. (b) STEM-EDX elemental analysis, (c) HAADF-TEM image and (d) STEM-EDX elemental mapping clearly indicate the formation of Pb nanorods (green, Pb; red, Ca).

However, stirring of the adsorbent materials and their separation from liquid by centrifugation after adsorption of heavy metal ions may be difficult for practical applications in natural water purification. The high adsorption rate of the hybrid flowers inspired us to check their feasibility in the continuous filtering process for water purification. Therefore, a continuous filtering adsorption device was designed to remove heavy metal ions from wastewater. Fig. 8 shows the schematic of this device, where the filter part was constructed from a few layers of hybrid flowers coated on a special support system, KIRIYAMA paper (pore diameter < 1 μm). As the size of the hybrid flowers is about 2 to 5 μm, gaps should be created between adjacent hybrid structures, providing a path for filtering polluted water. Upon addition of contaminated water from the top of the funnel containing layers of hybrid flowers, it travelled a long zig zag path through the hybrid flowers, ensuring complete adsorption of the heavy metal ions. The whole system was connected with a pumping device to control the free flowing rate of the purified water. As a representative example, particular concentrations of Pb(II) ions were treated by this continuous filtering process using the hierarchical hybrid flowers. The heavy metal ions were completely adsorbed by the hybrid flowers (adsorption efficiencies > 99.5%) in this continuous filtering process (Table S2, ESI†), suggesting its potential to eliminate heavy metal ions from polluted water. Considering that the arrangement is very practical, it can be used for the effective treatment of wastewater.

Fig. 8 (a) Schematic showing the designed continuous filtering adsorption device and (b) the corresponding experimental set-up.

Moreover, the hybrid flowers can easily be recovered and the adsorbed heavy metal ions can be regenerated by treatment with 1% acetic acid. ICP analysis showed that about 75% of the Pb(II) ions were regenerated by the dilute acetic acid treatment (Table S3, ESI†). Thus, the hybrid flowers can be recovered and reused through consecutive adsorption–desorption experiments, although their efficiency will diminish after every adsorption cycle.

Further, we also attempted to extend the use of sericin-embedded HAP hybrid for the removal of a hazardous dye from contaminated water. Congo red (CR), a benzidine-based anionic dye mainly generated from the textiles industry, was chosen as the model dye in this study.^9,10 It can cause acute toxicity to aquatic living organisms and is a human carcinogen.⁶⁵ Therefore, it is very important to remove residual CR from water sources before discharge into the environment. Fig. 9 shows the adsorption of CR dye with an initial concentration of 50 mg L⁻¹ on the hierarchical HAP hybrid structures. The results indicate that most of the dye molecules were transferred from water to the adsorbent, leaving a faint red solution, after 12 h. Almost 50% of the dye molecules were adsorbed within the first 4 h of the adsorption process; after that, the adsorption rate decreased (Fig. 9b). In the initial stage, the high adsorption rate is probably due to rapid contact of CR molecules with the active sites on the external surface of the adsorbent. The hierarchical HAP hybrids are covered with different surface activating groups, such as hydroxyl, carboxyl, and amine, from the amino acid backbone of silk sericin. Thus, the amine- and sulfonate-functionalized CR dye may undergo strong interactions with the sericin-mediated HAP hybrid through electrostatic interactions or hydrogen bonding, resulting in the apparent removal of this hazardous dye.⁹

Fig. 9 (a) UV-vis spectra showing the adsorption of Congo red dye by HAP-hybrid flowers at different incubation periods (inset: photographs of Congo red adsorption by HAP-hybrid flowers). (b) Adsorption rate curve of Congo red adsorption by HAP-hybrid flowers (concentration of Congo red: 0.05 mg mL⁻¹. Concentration of HAP-hybrid flowers: 2 mg mL⁻¹).

Conclusion

In summary, hierarchical HAP based bio-hybrid structures have been prepared through a green and facile co-precipitation method by exploiting the favorable material characteristics of a water-soluble biopolymer silk protein, sericin, and hydroxyapatite. Representing only a small percentage of the total mass, sericin plays a significant role in the formation of flower-like bio-hybrid materials. These synthetic hierarchical architectures possess a large BET surface area and show excellent adsorption activity for the toxic heavy metal ions Pb(II), Cd(II) and Hg(II) from wastewater. Moreover, these hybrid flowers were shown to be efficient in the continuous free flow filtering method, which is more practical for natural water treatment. One of the most important qualities of this bio-hybrid adsorbent is the short time (<15 min) required for more than 99% adsorption of Pb(II) ions. This is in accordance with our observation of the post-adsorption growth of uniformly distributed Pb nanorods on the hierarchical surface of the hybrid flowers, confirming their higher adsorption efficiency of Pb(II) in comparison to other heavy metals. Moreover, this hybrid adsorbent can be reused by recovering it through dilute acetic acid treatment, accompanied by regeneration of the corresponding heavy metal ions. Further, this HAP hybrid also exhibits promise as an adsorbent for the removal of a carcinogenic dye, CR, from contaminated water. Because of its various advantages, such as facile and low-cost fabrication, large surface area, very fast and high adsorption capacity, and easy separation, this flower-like hybrid material has been proved to be an attractive adsorbent for water purification. Moreover, biocompatible silk sericin is well known for its applications in culture media, whereas HAP based materials have wide applications in the biomedical field; therefore, the hierarchical porous structures formed by combining these materials may have synergistic effects and could be a very effective scaffold for a host of potential biomedical applications. Overall, the fabrication of HAP-based bio-hybrids may provide a good example of the fruitful utilization of industrial waste sericin in developing value-added materials, offering great potential for environmental and economic benefits.

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 H. Sugaya for the XPS measurements and Dr Kawata and Dr Ishitoya for the ICP-OES measurements. We also thank Mr S. Sarkar for drawing a schematic model.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra12818a

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