Facile and Controlled Fabrication of Cu-Al Layered Double Hydroxide

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Facile and Controlled Fabrication of Cu-Al Layered Double Hydroxide Nanosheets/Laccase Hybrid Films: A Route to Efficient Biocatalytic Removal of Congo Red from Aqueous Solutions Yao Zhu, Jian Rong, Tao Zhang, Jicheng Xu, Yuting Dai, and Fengxian Qiu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00149 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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ACS Applied Nano Materials

Facile and Controlled Fabrication of Cu-Al Layered Double Hydroxide Nanosheets/Laccase Hybrid Films: A Route to Efficient Biocatalytic Removal of Congo Red from Aqueous Solutions Yao Zhu, † Jian Rong, † Tao Zhang, †,* Jicheng Xu, †, ‡ Yuting Dai, † Fengxian Qiu†,*

†

School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang

212013, China

‡

School of the Materials Science & Engineering, Jiangsu University, Zhenjiang

212013, China

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ACS Applied Nano Materials 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

ABSTRACT: The development of bio-based hybrid materials that can efficiently remove environmentally contaminants has broad practical applications for areas. In this work, a novel biomaterial-inorganic hybrid film was fabricated with Cu-Al layered double hydroxide (LDH) nanosheets as host and laccase as guest by using exfoliation/self-assembly monolayer technology. It included two processes: first, a nitric acid intercalation Cu-Al-NO3- LDHs was prepared with Cu-Al-CO32- LDHs through the ion exchange method, and then Cu-Al-NO3- LDHs was exfoliated into a single layer nano dispersion system with abundant Cu2+ ions on the surface of the positively charged layered double hydroxides to assembly the (LDHs/laccase)n hybrid multilayer film. The resulting (LDHs/laccase)n hybrid film was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), high resolution transmission electron microscopy (HRTEM), scanning probe microscope (SPM) and UV-visible absorption spectroscopy, which all supported the formation of the layer-by-layer (LBL) sandwich structures. The performance of the as-prepared film was evaluated for efficient biocatalytic removal of congo red from aqueous solutions. The loaded amounts of laccase of (LDHs/laccase)n were estimated to be 0.0626 mg/cm2 (n=10), 0.1731 mg/cm2 (n=30) and 0.2809 mg/cm2 (n=50) by Bradford protein assay method. Taking advantage of the combined benefits of LDH and protein, the as-prepared (LDHs/laccase)n hybrid film exhibited high uptake removal performance and high storage stability in contrast with free laccase. The superior removal performance of (LDHs/laccase)n hybrid film supports a potential strategy for decontamination of congo red dye from wastewater, and it may provide new insights 2


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for efficient removal of dye in environmental pollution cleanup.

KEYWORDS: Layer-by-layer assembly, exfoliated nanosheets, biocatalytic; hybrid, film, laccase

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INTRODUCTION

Hybrid assemblies that integrate both protein and inorganic substances, have wide applications in the fields of chemistry, biochemistry, medicine, pharmaceutical science, food and textile due to their high catalytic activity, high selectivity and low toxicity.1-5 Bio-inorganic hybrid nanomaterial6 can combine the properties of the inorganic, such as mechanical and chemical activity, thermal stability, and pressure resistivity, with the biodegradability, flexibility and process ability of biomaterials.7 Moreover, the hybrids may exhibit further enhanced property tunability and new synergistic properties that arise from the interactions between the biological molecules and inorganic materials. Ge and co-workers8 reported the first example of protein–inorganic hybrid nanoflowers using copper(II) ions as the inorganic component and laccase as the organic component, which exhibit enhanced enzymatic activity and stability after immobilization. It could be concluded that the high surface area of nanoflowers can reduce mass transfer limitations significantly and the cooperative effects of the enzyme molecules and inorganic may help to enhance the protein activity. Among numerous hybrid materials, hybrid film with open structure has arisen widely attention due to the diversification of raw materials and various novel properties.9 Layered double hydroxides (LDHs),10-13 known as a brand of inorganic materials, consisting of positively charged layers and exchangeable anions located in the interlayer for charge balance, have been extensively studied for their potential

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applications in the fields of adsorption/separation, catalysis, electrochemistry, biotechnology and biomedicine. The increasing interest on LDHs is mainly from their diversity of intrinsic properties in terms of chemical composition both of layer and interlayer ions, their high and tunable charge density, and anion exchange capacity. However, LDH with large size has strongly restricted its utilization. To date, much concern has been focused on delamination of LDHs into colloidal nanosheets.14,15 The exfoliated LDH nanosheets can serve as one promising material owing to its open structure with nanometer scale, which can be potentially applied in the field of supramolecular assembly via layer-by-layer (LBL) technique.16-18 In recent years, water pollution technology has shifted toward enzymatic treatment, of which a laccase mediator system is found to be highly efficient and eco-friendly. Conventional method by using free laccase as reactive substance, suffers from various problems, e.g. instability, low efficiency, difficult separation and recovery and high cost. Therefore, there is a strong interest in preparing a novel bio-based hybrid material19,20 to improve the above problems. Zhu et al.21 reported the fabrication of an enzyme–inorganic hybrid nanoflower of laccase with copper (II) ions as the inorganic component acting as a catalyst incorporated into a membrane. The growth of aggregates formed between protein molecules and Cu component is owing to coordination reactions between laccase and Cu2+, where the protein serves as a “glue” to bind together the petals. As we all know, laccase is full of histidine residues, which can form coordinate bonds with transition metal ions such as Cu2+, and thus to reduce the distortion and desorption of laccase to the most extent. 5



In this work, we describe an easy and convenient technique that takes advantage of the stability of the LDH plates and the biocatalytic property of laccase in fabrication of (LDHs/laccase)n ultrathin films (UTFs) via strong binding interaction provided by metal affinity adsorption. A general belief is that, covalent binding between laccase and exfoliated LDH nanosheets can lead to the improvement of enzyme activity and stability and hybrid film with open structure can cause the increase attachment between enzyme and carriers. In addition, LBL assembly provides a broad platform for the attachment between enzyme and support, which retains the enzyme activity due to the reduction in the conformational transition without compromising on the stability aspect. The main aim of the current study is to understand the assembly of the (LDHs/laccase)n hybrid film via a synergistic effect between Cu2+ and laccase toward removal of congo red. The Cu containing LDH nanosheets used for laccase immobilization served as a platform and copper source that have a positive effect on the removal performance. It is demonstrated that the (LDHs/laccase)n UTFs highly enhanced biocatalytic activities for removal of congo red compared with free laccase. To the best of our knowledge, this is the first time that the fabrication of bio-inorganic (LDHs/laccase)n ultrathin hybrid film is reported. Furthermore, the concept presented herein can be expanded to the preparation of other functional hybrid film materials for environmental governance. EXPERIMENTAL SECTION Materials. Copper nitrate (Cu(NO3)2·3H2O), aluminum nitrate (Al(NO3)3·9H2O), sodium hydroxide (NaOH), ethanol, formamide, nitric acid, sodium nitrate (NaNO3), sulphuric acid, hydrochloric acid, coomassie brilliant blue G250, bovine serum albumin (BSA), congo red (C32H22N6Na2O6S2; formula weight=696, Sigma) and anhydrous sodium carbonate of analytical grade were purchased from Sinopharm Chemical Regent Co., Ltd. (Shanghai, China). Polystyrene sulfonic acid (PSS) was

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supplied from Shanghai Macklin Biochemical Co., Ltd. Laccase having an activity ≥120 U/g was provided from Shanghai Yuanye Bio-Technology Co., Ltd. All the reagents above were used without further purification. Deionized and decarbonated water was used in all solutions. Synthesis of brucite-like Cu-Al-CO32- hydroxides. Highly crystalline particles of Cu-Al-CO32- LDHs were prepared by co-precipitation method.22 Generally, Cu (NO3)2·3H2O (0.025 mol) and Al (NO3)3·9H2O (0.0125 mol) were dissolved in 100 mL deionized water. Then a mixture solution of NaOH (0.6 M) and Na2CO3 (1.2 M) was added dropwise under vigorous agitation until the pH level reached 8.7. The obtained suspension was stirred at room temperature for a few hours and then treated at 75 °C for 12 h. The solid products, brucite-like Cu-Al- CO32- hydroxides containing anion CO32- (Cu-Al-CO32- LDHs), were separated by filtration, rinsed thoroughly with deionized water, and finally air-dried overnight at 70 °C. Anion-exchange and exfoliation of anion intercalated Cu-Al LDHs. A nitric acid intercalation Cu-Al-NO3- LDHs was prepared with Cu-Al-CO32- LDHs through the ion exchange method, and then Cu-Al-NO3- LDHs was exfoliated into a single layer nano dispersion system with abundant Cu2+ ions on the surface of the positively charged layered double hydroxides. Typically, the Cu-Al-CO32- LDHs (0.5 g) was dispersed into 500 mL of an aqueous solution containing 1.5 M sodium nitrate (NaNO3) and 4 mM HNO3. After purging with N2 gas, the reaction vessel was tightly capped. Then the mixture was homogenized by sonication and stirred vigorously for 1 day at room temperature. The sample prepared was filtered and washed with deionized water. 7



The resulting sample was used for delamination and the Cu-Al-NO3- LDHs containing anion NO3- was obtained. 0.1 g Cu-Al-NO3- LDHs was mixed with 100 mL of formamide23 in a three-necked flask, which was capped air-tight after purging with nitrogen gas. Then, the mixture was magnetic stirred, yielding a translucent colloidal suspension. After that, a resulting blue product was collected by sand filtration, washed with deionized and decarbonated water several times to obtain exfoliated Cu-Al LDH nanosheets. Layer-by-layer assembly of bio-inorganic (LDHs/laccase)n multilayer hybrid ultrathin films. Multilayer hybrid ultrathin films of LDHs/laccase were fabricated by using the layer-by-layer assembly procedure. Substrates (silicon wafer or quartz glass slide) were in turn immersed in the methanol-HCl (V : V = 1 : 1) and concentrated sulfuric acid for 30 min, followed by soaking in the PSS for 20 min. Deionized water washing was applied at the end of every steps for thoroughly rinsing, then dried by nitrogen. Treated silicon wafer or quartz glass slide with negative charge set aside was obtained. The freshly cleaned silicon substrate with negative charge was immersed in the colloidal solution of obtained Cu-Al LDH nanosheets for 15 min and washed with copious decarbonated deionized water, which was followed by drying at nitrogen atmosphere. Then, the substrate treated with LDH nanosheets was dipped into a laccase aqueous solution (0.2 g/L 50 mL) for another 15 min and treated as above. Subsequently, sequential deposition operations for LDH nanosheets and laccase were

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repeated n times to produce multilayer films of (LDHs/lacccase)n (Figure S1). The resulting films were finally dried under nitrogen gas flow at room temperature. The synthesis schematic is depicted in Scheme 1.

Scheme 1. Schematic illustration of ion- exchange and exfoliation of Cu-Al LDHs (A and B) and LBL assembly process (C) of the (LDHs/laccase)n hybrid multilayer film

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Determination of loaded amount of laccase. The loaded amount of laccase on the LDHs was determined by measuring the amount of protein lost in the solution after immobilization.20 In this work, the concentration of protein was measured using the Bradford protein assay method24 at 595 nm and BSA as the standard protein. The loaded amount of laccase was calculated as the following Equation (1):

⁄ =

(1)

where and are the initial and final laccase concentrations in the solution (mg/mL); V is the volume of the solution (mL); S is the total area of the quartz glass (cm2). Dye removal experiments. The performance test was carried out employing four pieces preformed (LDHs/laccase)n film with 40 mL aqueous solution of the congo red (10 mg/L) in a thermostat water bath at 35 °C. The pH of the dye liquor was adjusted to 4 and maintained constant during the process. 4 mL of the sample was withdrawn and separated centrifugally (10000 rpm/min) to remove hybrid before analysis with the UV–vis spectrophotometer at 499 nm at regular time intervals. The removal efficiency (RE%) was calculated as the following Equation (2):

RE% =

× 100

(2)

where is the initial concentration of congo red solution, is the concentration at time t (h) of congo red solution. Similarly, the removal efficiency of the same amount of free laccase was also evaluated.

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Characterization techniques. X-ray diffraction (XRD) analysis was employed to identify the crystallography of LDH and the periodic structure of the (LDHs/lacccase)n UTFs at room temperature using Shimadzu XRD-6100 instrument with Cu Kα radiation at 40 kV and 30 mA, a scanning rate of 4 °/min, and a 2 theta angle ranging from 5° to 70°. The morphologies of thin films were investigated using a scanning electron microscope (SEM Hitachi S-4800) equipped with an EDS attachment and the accelerating voltage of 20 kV. The SEM specimens were prepared by sputter coating a thin gold layer approximately 3 nm thick. The morphologies and dimensions of the particles were recorded on a Tecnai G2 F30 S-Twin high-resolution transmission electron microscope (HRTEM) with the accelerating voltage of 300 kV. The surface roughness and thickness data were obtained by using the Scanning Probe Microscope (SPM) (Shimadzu SPM-9600). The UV-vis absorption spectra were obtained in the range from 200 to 800 nm on a Shimadzu UV-2450 spectrophotometer, the width of the slit is 1.0 nm. RESULTS AND DISCUSSION Characterization of Cu-Al-CO32- LDHs. A series of intense and sharp diffraction peaks of (003), (006), (012), (015), (107) and (018) in Figure 1A(a) indicate the rhombohedral structure of the samples with the refined lattice parameters of a = 0.308 nm and c=2.285 nm. The peaks of the CO32--type CuAl-LDH sample can be indexed to Cu6Al2(OH)16(CO3)3·4H2O (JCPDS 37-0630)2, suggesting the high crystallinity for the synthesized CO32- intercalated products. The two major characteristic diffraction peaks corresponding to Cu-Al LDHs (003) and (006) 11



reflections can be found at 2θ=11.75 and 23.60o, respectively.25 The interlayer spacing of CO32- form was 0.76 nm. No XRD peaks of impurities appear, demonstrating the high purity of the as-prepared product. In addition, the sharp and symmetric features clearly indicate that the produced Cu-Al-CO32- LDHs were highly crystallized with a three-dimensional order, which is in line with the analysis of transmission electron microscope. As can be seen from Figure 1B and C, the Cu-Al-CO32- LDHs material is composed of many well-defined and thin mono-dispersed platelets stacked together. Evidently, the as-prepared Cu-Al-CO32- LDHs product was of high quality in terms of morphology and crystallinity (Figure 1D), which could be attributed to the slow and homogeneous nucleation process due to the slow drop of the solution containing intercalation ions.

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Figure 1. (A) XRD pattern of Cu-Al-CO32- LDHs (a) and the Cu-Al-NO3- LDHs (b); (B and C) HRTEM images of Cu-Al-CO32-LDHs under different magnification; (D) The magnified image of the circle in Figure 1(C)

Anion exchange and exfoliation behavior of Cu-Al LDHs. Due to the layered structure of LDHs, it is difficult to contact the inner surface of LDHs, thus limiting the application of LDHs in many aspects. Therefore, delamination of LDHs becomes an interesting research for producing positively charged single layer platelets. Due to their high charge density, interlayer CO2− anions show high affinity to adjacent LDH 3 laminates, resulting in the difficulty for LDH to peel off directly. Hence, for

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monodisperse nanosheets, the as-synthesized CO32- type Cu-Al LDHs required decarbonation and exchanged some anions that had weak affinity to the host layers, such as NO3-. In this work, a salt-acid mixed solution was developed to obtain Cu-Al-NO -3

LDHs sample according to the method of ion exchange reported

previous.26 The basal reflections ((003) and (006) reflections) for the CO32- form disappeared (Figure 1A(b)), replaced by a new series of intense basal reflections at lower 2θ angles, as the (003) and (006) diffraction peaks appeared at 2θ angles of 10.1° and 20.1°, suggesting that the ion-exchange was successful. After anion exchange, the interlayer spacing increased to 0.85 nm for NO3- owing to the low affinity between host layers and NO3-. A clear Tyndall light scattering was observed (Figure 2A), implying the exfoliated LDH nanosheets were well dispersed. Further treated by sand filtration, a blue, gel-like aggregate was recovered from the suspension. The obtained exfoliated LDH nanosheets showed a noticeable amorphous-like halo in the 2θ range of 20-30° (Figure 2B(a)), which can be arisen from the scattering of liquid formamide within the gel-like aggregate of LDHs. As seen from Figure 2B(a), all the sharp basal reflections of the pure LDH samples disappeared in this pattern, indicating that the lamellar structure had been destroyed, that is to say, the successful exfoliation of LDH. There also existed three sharp intense peaks in the 2θ range of 35-70°, which could be attributed to the filter membrane. As a contrast, XRD pattern (Figure 2B(b)) of the characteristic peaks (35-70°) of the filter membrane that was consistent with the above three sharp intense peaks. Moreover, no sediment was observed after long-term 14


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deposing, indicating the resulting colloidal suspension was stable enough. Figure 2C shows a great many of thin sheetlike particles. It is generally known that LDHs have been found for more than 100 years since the discovery, and now the preparation process and exfoliation technology of LDH are both mature technology. Previous research results show the ultrathin nature and uniform thickness of LDH nanosheets with the thickness is generally less than 1 nm.27-30 Similarly, the Cu-Al LDH nanosheets (Figure 2D) presented very faint but homogeneous contrast, reflecting the ultrathin and transparent nature like other LDH nanosheets. In comparison with the LDHs crystallites obtained in Figure 2, the nanosheets were dimensionally diminished and morphologically irregular, implying the fracture of the nanosheets during the delamination process. The HRTEM image (Figure S2) of exfoliated nanosheets shows the clear crystal lattice, and the spacing of the lattice fringe is about 0.23 nm that corresponding to the (012) plane of the Cu-Al LDH. On the basis of these results, it can be concluded that the exfoliation of LDHs follows two separate processes: CO32intercalated Cu-Al LDHs were first prepared, followed by the Cu-Al-NO3- LDHs via ion exchange, while the laminates of LDHs didn’t change during the process of anion exchange. After that, the Cu-Al-NO3- LDHs crystal was swelling in the formamide for exfoliation under the external force, thus obtaining the well-dispersed exfoliated nanosheets.

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Figure 2. (A) Photo of colloidal suspension of Cu-Al LDHs in formamide; (B) XRD patterns of the exfoliated nanosheets (a) and the filter membrane (b); (C and D) TEM images of the exfoliated single-layer nanosheets under different magnification

Assembly of the (LDHs/laccase)n UTFs and characterization. The surface of blank silicon wafer (Figure S3) is quite smooth. The surface of the (LDHs/laccase)10 film (Figure 3A) was rougher than the blank silicon wafer, demonstrating that the protein and Cu-Al LDH nanosheets were successfully assembled to the silicon wafer surface. While the assembly times increased, the surface trended toward uniformity and smoothness. The film surface (Figure 3B) was covered with plate-like nanosheets while some partially over-lapped with others and no excess sediment was observed. Further increasing the depositing cycles, when n reached 50, a top-view of SEM image for the (LDHs/laccase)50 film (Figure 3C) showed that an increasing number of sediments were deposited on the surface, indicating the surface roughness increased. 16


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Due to the fracture of the nanosheets during the delamination process, the nanosheets could hardly arrange orderly on the surface or fully cover the substrate without any defect in the first depositing cycle. However, as shown in the SEM image, with the increase of the depositing cycles, the surface deficiency decreased. This can be interpreted that the interfacial deficiencies were filled up by the nanosheets progressing then leading to the decrease of the surface roughness. While increasing the number of the depositing cycles continuously (50 assembly times), there existed more sediment on the surface of the silicon wafer, presenting that the roughness was increased. Consequently, after 30 depositing cycles, a smooth and uniform surface morphology of the assembly multilayer film was obtained. The multilayer film (Figure 3D) was composed of C, N, O, Cu and Al elements, indicating the co-existence of Cu-Al LDHs and laccase in the product.

Figure 3. SEM images of different multilayer films of (LDHs/laccase)n (n = 10(A), 30(B) and 50(C)); EDS pattern of LBL assembly (LDHs/laccase)n films (D) 17



UV absorption intensity (275 nm) increased gradually with the increasing in deposition cycles (Figure 4A). The nearly linear increment of absorbance distinctly proved a stepwise and regular assembly between LDH nanosheets and laccases. As the number of the depositing cycles increases, the intensity (Figure 4B) of the reflection showed the same trend, indicating that the (LDHs/laccase)n UTFs possessed an ordered periodic structure in the direction perpendicular to the substrate. From the small-angle XRD patterns (Figure S4) of the multilayer films (n= 30 and 50), the evolution of a Bragg peak at 2θ=1.8° was attributed to a so-called superlattice reflection of the inorganic/organic repeating nanostructure. The peak intensity increased progressively with the increase of the number of layer pairs, thus supporting the successful formation of an ordered hybrid structure via regular arrangement of the layers despite the fact that many defects could be present in the film.

Figure 4. (A) UV-visible absorption spectra of the multilayer films from n=10 to 50; (B) XRD patterns of the multilayer films (n=10, 30 and 50)

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The morphology of the film can be accessed by SPM images (Figure 5). The roughness is increased for thicker films, as observed of (LDHs/laccase)10, (LDHs/laccase)30 and (LDHs/laccase)50, of which the average height profile was found to be approximately 8, 16 and 32 nm, respectively. Through the SPM tapping mode images, the multilayer films of well dispersed LDH nanosheets and laccase lying parallel to the substrate surface were observed, indicating the successful assembly of the LDHs and laccase. However, some nanosheets edges are rough and irregular in shape, which may be attributed to the breakage or fracture of the sheets in the process of the exfoliation.

Figure 5.

Tapping-mode SPM images: the thicknesses and roughness of

(LDHs/laccase)n (n = 10(A), 30(B) and 50(C))

Application of (LDHs/laccase)n hybrid multilayer films for removal of congo red. The biocatalytic removal activity of the as-prepared multilayer films was measured against a 10 mg/L congo red solution in order to evaluate their application potential. According to the results of Bradford protein assay method, the loaded amount of laccase (q) was 0.0626 mg/cm2 (n=10), 0.1731 mg/cm2 (n=30) and 0.2809 19



mg/cm2 (n=50), respectively. The hybrid film had relatively high removal efficiency in a wide pH range, with maximal activity at pH 4.0 (Figure 6A). There is, to emphasize, significant increase in the removal efficiency than that of free laccase. Additionally, when the solution is too acidic or too alkaline, the removal efficiency of both hybrid film and laccase was lower owing to enzyme inactivation. This is mainly because the binding interaction between Cu-Al LDH nanosheets and laccase has a reduction on the conformational transition under the change of pH. While the optimum pH of hybrid film was shifted to less acidic region, which may be due to the different concentration of H+ and OH- in the environment of dye solutions degraded by hybrid film and free laccase, respectively. From the beginning of 15 °C, the removal efficiency of both hybrid film and laccase presented an upward trend, and the relatively high removal efficiency of hybrid film was achieved range from 30 ~ 35 °C, the optimum temperature for free laccase was 30 °C (Figure 6B). Further increasing temperature, the removal efficiency of the two samples was downwards. Compared with the free laccase, the hybrid film presented a broader temperature profile with the activity maintained more than 70% in the range from 20 ~ 40 °C. In addition, the effect of layer numbers of hybrid film on the dye removal performance was systematically investigated under the optimum conditions. As shown in Figure 6C, (LDHs/laccase)10 showed a slight decrease on the removal of congo red in the aqueous solution. And (LDHs/laccase)50 presented a same trend as (LDHs/laccase)30 on the removal of dye within 1.5 h, while the ultimate removal 20


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efficiency is lower than that of the latter. One explanation could be that the assembly of nanosheets and laccase molecules was in equilibrium when the layer number of hybrid film reached 30, of which the surface tends to smooth. Going a step further in increasing the depositing cycles, the surface sediments increased, however, there has little impact on the dye removal performance. By comparison, (LDHs/laccase)30 hybrid would be the best in testing the dye removal performance.

Figure 6. Effects of pH (A) and temperature (B) on the removal efficiency of free laccase and hybrid film; Contrast curves of different layer numbers on removal performance (C)

Stated thus, at optimum conditions (pH 4.0 and temperature 35 ℃), the reaction of the dye removal was also monitored with a UV-Vis spectrophotometer on the basis of the change in the absorbance of the peak at 499 nm (Figure 7A). Evidently, the absorption intensity suffers a gradual decrease, but no changes in the shape and position of the peaks have occurred. The color of the dye solution gradually vanished with time increasing (Figure 7B). A fine relationship between the -ln(I0/I) and time confirmed that the degradation of the congo red by the hybrid film follows an overall first-order kinetics (I0 is the initial absorption intensity of congo red at 499 nm, I

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represents the actual absorption intensity of congo red at 499 nm at time t). A possible efficient removal pathway of congo red was proposed (Figure S5).31,32 An electron was transferred from the organic substrate to one of the copper containing site of laccase first, causing the formation of electron deficient reaction center. Owing to its high reactivity, asymmetric cleavage of azo bond was occurred, in other words, the chromophore in congo red was disappeared, leading to the vanish of the color of dye solution. The product obtained from the cleavage of azo bond was proved to be p-dihydroxybiphenyl and postulated diazine intermediate according to the earlier study. The latter was presumably unstable in a molecular oxygen atmosphere to lose an electron, which was obtained by oxygen molecule. Then the electron loss product further lost molecular nitrogen, followed by amine oxidation and desulfonation, confirming the removal of congo red. While the Cu containing LDH nanosheets used for laccase immobilization, it could provide a synergistic effect between Cu2+ and laccase, resulting in the enhancement of the removal performance. On account of the adsorption characteristic of LDH nanosheets, there is a possibility that nanosheets alone may also play a part in the removal of congo red. To verify this, an identical removal process was carried out over free laccase and exfoliated LDH nanosheets in contrast. A quite slow rate of removal was observed in the absence of laccase (Figure 7C). While the removal capacity of free laccase showed better than that of exfoliated nanosheets, it is still not meet the requirements. Nevertheless, the (LDH/laccase)30 hybrid multilayer film exhibited an efficient performance. It could be seen that the hybrid film retained above 90% after storage at 22


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4 °C for 5 days (Figure 7D), however, the removal efficiency of free laccase decreased from 44.6% to 31.1%. Further studies on the effect of layer numbers on the storage stability of hybrid film were carried out at optimum conditions (Figure 7E and F). It could be seen that the stability of either (LDHs/laccase)10 or (LDHs/laccase)50 was basically unchanged, namely, the hybrid film showed a good stability, indicating that the impact of layer numbers on the stability of hybrid film can be neglected. The improvement of storage stability was also probably due to the restricted conformational mobility of laccase after immobilization. This result confirm that (LDH/laccase)n hybrid multilayer films retained excellent stability after low temperature storage.

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Figure 7. The UV spectra of the removal process (A) and kinetic study (B) of congo red with (LDHs/laccase)30 hybrid films (the inset presents the color disappeared with time increasing); Contrast curves of removal on LDH nanosheets, free laccase and hybrid film (C); Storage stabilities of different layer numbers of hybrid film and free laccase (D~F).

CONCLUSIONS

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In summary, a convenient and reproducible procedure for the preparation of the bio-inorganic hybrid films has been developed in the manner of layer-by-layer assembly. Monolayer Cu-Al LDH nanosheets were obtained by delaminating the anion-exchanged Cu-Al-NO3- LDHs. By utilizing metal affinity interaction between Cu2+ and laccase, multilayer hybrid films were obtained by layer-by-layer technique. SEM, UV spectroscopy and SPM were employed to characterize the as-prepared hybrid films. The hybrid film showed high removal efficiency of congo red (more than 95%) under optimal reaction conditions, which was significantly improved compared with free laccase. And the (LDHs/laccase)n hybrid films show broader pH, temperature optimal and good storage stability. The (LDH/laccase)30 hybrid multilayer film exhibited an efficient performance and could retain above 90% after storage at 4 °C for 5 days, however, the removal efficiency of free laccase decreased from 44.6% to 31.1%. This facile method not only provides a novel idea for the treatment of dye containing wastewater but also opens up fascinating possibilities for the design of multi-functional homogeneous organic-inorganic hybrid films. ASSOCIATED CONTENT Supporting Information. Photo of hybrid film assembled on the quartz glass slide (Figure S1); HRTEM images of the exfoliated LDH nanosheets (Figure S2); SEM image of blank silicon wafer (Figure S3); Small-angle XRD patterns of the (LDHs/laccase)30 and (LDHs/laccase)50 films (Figure S4); A possible removal pathway of congo red via (LDH/laccase)n hybrid film (Figure S5). This material is available free of charge via the Inter-net at http://pubs.acs.org. 25



AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This project was supported by the National Natural Science Foundation of China (U1507115), the Natural Science of Jiangsu Province (BK20160500 and BK20161362), 333 High-Level Personnel Training Project of Jiangsu Province (BRA2016142) and China Postdoctoral Science Foundation (2016M600373, 1701067C and 1601016A). REFERENCES (1) Alcantara, A. C. S.; Aranda, P.; Darder, M.; Ruiz-Hitzky, E., Bionanocomposites Based on Alginate-Zein/Layered Double Hydroxide Materials as Drug Delivery Systems. J. Mater. Chem. 2010, 20 (42), 9495–9504.

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Table of Contents

A novel biomaterial-inorganic (LDHs/laccase)n hybrid film was fabricated for efficient removal of congo red.

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Facile and Controlled Fabrication of Cu-Al Layered Double Hydroxide

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