A green approach to the synthesis of novel “Desert rose stone”-like nanobiocatalytic system with excellent enzyme activity and stability

3D hierarchical layer double hydroxides (LDHs) have attracted extensive interest due to their unique electronic and catalytic properties. Unfortunately, the existing preparation methods require high temperature or toxic organic compounds, which limits the applications of the 3D hierarchical LDHs in biocatalysis and biomedicine. Herein, we present a green strategy to synthesize “Desert Rose Stone”-like Mg-Al-CO3 LDH nanoflowers in situ deposited on aluminum substrates via a coprecipitation method using atmospheric carbon dioxide. Using this method, we construct a novel “Desert Rose Stone”-like nanobiocatalytic system by using HRP as the model enzyme. Compared with the free HRP, the HRP/Mg-Al-LDH nanobiocatalytic system exhibits higher catalytic activity and stability. A smaller apparent Michaelis-Menten constant (0.16 mM) of this system suggests that the encapsulated HRP shows higher affinity towards H2O2.

O wing to their large surface area, high mechanical strength and good biocompatibility, nanomaterials (such as carbon materials, inorganic minerals, metal-organic frameworks, etc.) have emerged as a new class of promising candidates for applications inbiomedicine 1 and nanobiocatalysis [2][3][4][5] . Nanobiocatalytic systems could be constructed by incorporating enzymes into nanostructured materials by various strategies. Compared with the free enzymes, the enzymes in nanobiocatalytic systems usually exhibit the enhanced enzymatic stability. In addition, the incorporated enzymes could be easily separated from their substrates and products and thus present the improved reusability. However, it is worth noting that the catalytic performance of the incorporated enzymes is sensitive to the properties of nanomaterials and the immobilized methods 6,7 . Therefore, exploring the appropriate nanomaterials and immobilized methods is becoming a key and challenge for the applications of nanobiocatalytic systems in industrial biocatalysis.
Layer double hydroxides (LDHs) are known as an important class of anionic lamellar clays and could be generally described by the formula M 2z 1{x M 3z 21 and M 31 are divalent and trivalent metal cations, and A n2 is an anion intercalated in the gallery). Due to their tunable composition, anion exchange ability, excellent adsorption capacity, good biocompatibility and stability, LDHs have been employed in a wide range of potential applications in various areas [8][9][10][11][12] . Until now, LDHs have been prepared by various methods such as coprecipitation 13 , ion exchange 14 , calcination-rehydration 15 , hydrothermal 16 and electrochemistry 17 . The morphology and size of LDHs influencing their multifunctional properties and applications can be tailored by various environmental factors. However, LDHs prepared by conventional methods are usually stonelike or plate-like 18,19 and their unwelcome surface areas urgently need to be improved for their potential applications. Recently, 3D hierarchical LDHs have attracted extensive interest due to their unique optical, electronic, magnetic, catalytic and absorption properties [20][21][22][23][24][25][26][27] . Various 3D hierarchical M-Al-LDHs with tunable architecture have been fabricated by calcination-rehydration or hydrothermal methods [20][21][22] . Especially, a hierarchical tri-metal Mg/Ni/Al-LDH nanoflowers on Al-foam was synthesized by an in situ multistep crystallization technique at 70uC 23 . Unfortunately, these methods usually require high reaction temperature, long aging time, complicated operating or organic compounds (such as ethylene glycol 22 , CTAB 28 ), which greatly limits the application of 3D hierarchical LDHs in biocatalysis and biomedicine. Therefore, exploiting a novel and green strategy to fabricate the 3D hierarchical LDHs may shed lights on their potential applications in nanobiocatalytic systems with excellent performance. Herein, we demonstrate an environmentally friendly approach to the synthesis of ''Desert Rose Stone''-like Mg-Al-CO 3 layer double hydroxide (Mg-Al-LDH) nanoflowers via a coprecipitation method as shown in Figure 1. Atmospheric CO 2 in air is used as a source of CO 3 22 and Al 31 in the form of Al(OH) 3 originated from the dissolution of aluminum sheet under mild alkaline conditions. The in situgrown 3D hierarchical Mg-Al-LDH nanoflowers exhibit larger specific surface area than traditional plate-like LDHs. The proposed coprecipitaion method does not rely on high temperature or organic compounds and thus can be used for constructing nanobiocatalytic systems by one-step loading of biomolecules. As demonstration, a nanobiocatalytic system (HRP/Mg-Al-LDH nanoflowers) using horseradish peroxidase (HRP) as the model enzyme is constructed, showing excellent biocatalytic activity.

Results
Synthesis and characterization of Mg-Al-LDH and HRP/Mg-Al-LDH nanoflowers. Mg-Al-LDH nanoflowers are synthesized in a solution of MgCl 2 via the proposed coprecipitation approach. Atmospheric CO 2 in air and aluminum sheet are used as the source of CO 3 22     occurrence of the characteristic reflections of a typical LDH with a series of (00l) peaks confirms the successful synthesis of LDHs. The peaks at 11.56u, 23.48u and 34.96u correspond to (003), (006) and (009) reflections, respectively. According to the Bragg formula nl 5 2dsinh, the interlayer spacing of the as-prepared LDHs is calculated to be 0.765 nm, consistent with the value of Mg-Al-CO 3 LDHs reported previously 22 . Compared with the Mg-Al-LDH nanoflowers, the intensity of diffraction peaks of HRP/Mg-Al-LDH nanoflowers decreases, which suggests a decreased crystallinity due to the introduction of HRP. In addition, no diffraction peaks are observed in the low-angle XRD pattern (0.5-10u) of HRP/Mg-Al-LDH nanoflowers ( Figure S1), which demonstrates that the intercalation of HRP into LDH layers cannot occur within the immobilization process. The possible structure model of HRP/Mg-Al-LDH hybrids may be similar to the one reported by Vial et al 29 .
The main interactions between HRP and LDHs are presumed to be hydrogen-bonding and/or electrostatic interactions since the HRP molecule (pI 5 8.9 30 ) carries positive charges at pH 10. The molar ratio of Mg and Al in LDHs obtained by ICP-AES is about 1.751 which is slightly lower than 251. The observed result may be ascribed to the contamination of aluminum compounds 31 .
The encapsulation of proteins into the layered nanostructures was also confirmed by infrared spectroscopic characterizations ( Figure 2b). In the case of Mg-Al-LDH nanoflowers, the adsorption peaks around 3500 cm 21 is attributed to the O-H stretching modes of hydroxyl groups in LDH octahedron layers and interlayer water molecules. The weak peak at 1647 cm 21 is associated with the deformation mode of interlayer water molecules. The strong peak at 1363 cm 21 belongs to the vibration mode of CO 3 22  The thermal stability of the as-prepared LDH nanoflowers was also studied by thermal gravimetric analysis. The TG-DTG curves of Mg-Al-LDH and HRP/Mg-Al-LDH nanoflowers are shown in Figures 2c-d. Two distinct weight loss steps are observed for Mg-Al-LDH nanomaterials. The first step (25-195uC) with a mass loss of 18.95% corresponds to the removal of the absorbed water on the surface of LDHs and interlayer water. The second step (200-550uC) with a mass loss of 26.77% is ascribed to the dehydroxylation of the Mg-Al hydroxide layers and the decomposition of intercalated CO 3 22 ions. Similar to Mg-Al-LDH, the HRP/Mg-Al-LDH nano-  flowers also exhibit two typical weight loss steps with mass losses of 11.92% in the first step and 51.34% in the second step. It is worth noting that the mass loss in the second step for HRP-containing LDHs is significantly larger than that for the pure LDHs, which can be explained by that the dehydroxylation and anion decomposition of LDHs are accompanied by the decomposition of HRP molecules. The results further confirm that HRP has been successfully incorporated into the nano-biohybrid. According to the HRP concentration in the supernatant tested by UV-vis spectroscopy, the encapsulation efficiency of HRP (defined as the ratio of the amount in LDHs and the added total amount) is calculated as 85.2%. The typical SEM and TEM images of the as-prepared Mg-Al-LDH nanoflowers at different magnifications are shown in Figures 3a-d. Lots of Mg-Al-LDH particles with a diameter about 300 nm, whose structure is similar to ''Desert Rose Stone'', could be observed from the low-magnification SEM image (Figure 3a). The high-magnification SEM images show that these LDH nanoparticles exhibit unique 3D hierarchical nanoflower structures constructed by plentiful intercalated round nanoplates with 300 nm in lateral size and 20 nm in thickness (Figures 3d-c). The TEM image indicates the presence of the nanoflowers with thin and smooth nanoplates (Figure 3d). The N 2 adsorption-desorption isotherm of Mg-Al-LDH nanoflowers is shown in Figure S2. The BET surface area of the LDH nanoflowers is 79.4 cm 2 /g, which is larger than those of the conventional plate-like LDHs 32,33 . In addition, the as-prepared HRP/Mg-Al-LDH nanoflowers display the similar 3D nanoflower structures constructed by round nanoplates with 500 nm in diameter (Figures 3e-f). The TEM image of HRP/Mg-Al-LDH nanoflowers (Inset in Figure 3e) further confirms the presence of intercalated nanostructures.
In order to investigate the formation process of 3D hierarchical nanoflowers, the morphology of aluminum substrate at different reaction intervals was characterized ( Figure 4). The aluminum sheet exhibits a relatively smooth surface (Figure 4a) before the coprecipitation reaction. After reacted for 30 min, lots of nanoparticles can be observed at the surface of aluminum sheets (Figure 4b), which should be ascribed to the formation of the crystal nucleus of Mg-Al-CO 3 LDHs. At this nucleation stage, Al(OH) 3 is released continuously from the aluminum sheet and further reacted with Mg 21 , OH 2 and CO 3 22 to form the LDH nucleus. The proposed reaction equations relating to the formation of LDH nanoflowers are shown in Equations (1-3). When the reaction time is prolonged, the small flakes appear (Figure 4c) and grow in the direction of the solution. Therefore, 3D hierarchical nanomaterials consisted of intercalated irregular nanoplates at the surface of aluminum sheet are formed (Figure 4d). At a reaction time of 5 h, the physical structure of the asprepared nanomaterials tends to be the perfect one that the 3D hierarchical nanoflowers consist of plentiful regular round nanoplates (Figure 4e). After the gradual growth of nanoflowers with a certain height, the in situ-grown nanoflowers will fall off the aluminum sheet into solution, which makes the aluminum sheet to the nucleation stage (Figure 4f). Many literatures reported that 3D hierarchical LDHs could be prepared in the presence of organic compounds 22,28 . Fortunately, both organic compounds and high temperature are not required in the present approach. It is speculated that the possible reason of the formation of 3D nanoflowers is as follows: CO 3 22 ions are derived from the atmospheric CO 2 at pH 10 and react with Mg 21 and Al(OH) 3 near the aluminum substrate surface. The distance-dependent concentration gradient of these ions at the surface of aluminum substrate may avoid the fast growth of LDHs 34 . Therefore, the Mg-Al-LDHs can grow in a certain orientation and self-assembled into the flower-like nanostructures.
(1-x)Mg 2z zxAl(OH) 3 zx=2CO 2-3 z(2-3x)OH -zmH 2 O? : In addition, the effect of MgCl 2 concentration on the formation of Mg-Al-LDH was further studied. Figures 5a-b show the XRD patterns of the precipitations obtained from 1 mM and 100 mM MgCl 2 precursors, respectively. Compared with the standard card of Al(OH) 3 (ICCD PDF no. 20-0011), the sample from 1 mM MgCl 2 can be indexed to be the Al(OH) 3 crystal (Figure 4a). This phenomenon is explained by that, at low concentration of MgCl 2 , the Mg 21 ions are limited and hardly coprecipitated with Al(OH) 3 to generate Mg-Al-LDHs. As shown in Figures 5c-d, the Al(OH) 3 precipitation displays the rod-shaped or triangular nanostructures with 600-800 nm in lateral size. At high MgCl 2 concentration (100 mM), the obtained sample exhibits the characteristic reflections of a LDH phase, which indicates the successful synthesis of Mg-Al-CO 3 LDHs ( Figure 4b). As shown in Figures 5e-f, the as-prepared LDHs exhibit the similar 3D hierarchical nanostructures with larger nanoplates in lateral size and thickness compared with the LDHs formed from 20 mM MgCl 2 . These results demonstrate that a higher MgCl 2 concentration will result in a larger concentration gradient of Mg 21 ions at the surface of aluminum substrate, which accelerates the growth of LDH nanoflowers. As a result, the larger LDH nanoflowers are obtained.
Enzymatic activity of the HRP/Mg-Al-LDH nanobiocatalytic system. Nanobiocatalytic systems based on nanostructured materials  have attracted increasing attention due to their potential applications in biofuel cells, antifouling and proteomic analysis 35,36 . In the present work, HRP, as a model enzyme, has been incorporated into the LDH nanoflowers by the proposed coprecipitation method to construct a ''desert rose stone''-like HRP/Mg-Al-LDH nanobiocatalytic system. H 2 O 2 and TMB are chosen as the model substrates for HRP. In the presence of H 2 O 2 , HRP can catalyze the oxidation of TMB to generate TMB ox which exhibits two absorption bands centered at 370 nm and 652 nm. Therefore, the catalytic activity of HRP can be easily evaluated by monitoring the absorbance at 652 nm in time-scan mode. Figure 6a shows the typical kinetic curves for free HRP and HRP/Mg-Al-LDH nanobiocatalytic systems. The final concentration of HRP is 1 ng/mL for each biocatalytic system. Compared with the free HRP system, the HRP/Mg-Al-LDH nanobiocatalytic system exhibits a much higher catalytic reaction velocity, which demonstrates that the catalytic activity of HRP incorporated into the Mg-Al-LDH nanoflowers is greatly enhanced. It is speculated that the enhanced enzyme activity may be ascribed to the conformation change of the immobilized HRP, which further results in an increase in the proportion of the exposed active sites.
In general, the amide I band in FTIR spectrum is sensitive to the changes of secondary structure in proteins and thus is usually used to monitor the conformation change of proteins. The contents of secondary structure can be calculated by integrating the distinct Gaussian peaks [37][38][39] . The FTIR spectrum of free HRP in 4000-400 cm 21 is shown in Figure S3. In order to prove the above speculation, the amide I bands for free HRP and the immobilized HRP are deconvoluted ( Figure S4). In the amide I band, the bands at 1622, 1632 and 1695 cm 21 are assigned to b-sheet components; the bands at 1642, 1657 and 1678 cm 21 are attributed to random coil, a-helix and b-turn structures, respectively (Table S1) 40 . The contents of each secondary structure for free HRP and HRP immobilized in Mg-Al-LDH nanoflowers are shown in Table 1. After incorporated into Mg-Al-LDH nanoflowers, the content of a-helix structure of HRP molecules increases from 34.62% to 37.34%, which is accompanied by an decrease of the amount of b-structure (b-sheet and b-turn, from 55.12% to 47.38%) 41 . The change of secondary structure compositions reflects the change of protein conformation. These phenomena indicate that the conformation of HRP is changed due to the hydrogen-bonding and/or electrostatic interactions with LDH nanoflowers after HRP is incorporated into the nano-biohybrid.
Affinity of enzymes toward the substrates can be evaluated by Michaelis-Menten constant K m . The smaller Michaelis-Menten constant indicates the higher affinity. The Michaelis-Menten constant K m and the maximum reaction velocity u max can be estimated from the Lineweaver-Burke equation 41 : Michaelis-Menten plot (Figure 6b) and Lineweaver-Burke plot (Inset in Figure 6b) of the HRP/Mg-Al-LDH nanobiocatalytic system are  obtained by monitoring the absorbance change at 652 nm in the presence of different H 2 O 2 concentration ( Figure S5). Based on the slope and intercept of the Lineweaver-Burke plot, the apparent K m and u max are calculated to be 0.16 mM and 1.02 3 10 27 M/s, respectively. It is obvious that the apparent K m of the proposed nanobiocatalytic system is smaller than that of free HRP (3.7 mM) and other reported systems such as HRP/Titania Sol-Gel Matrix (1.89 mM) 42 , HRP/CMCS-Au NPs (0.57 mM) 43 , HRP/ZnO-GNPs-Nafion (1.76 mM) 44 . These results demonstrate that the ''Desert Rose Stone''-like HRP/Mg-Al-LDH nanobiocatalytic system shows higher affinity toward H 2 O 2 .
Detection of H 2 O 2 by the HRP/Mg-Al-LDH nanobiocatalytic system. In the present work, a simple and sensitive H 2 O 2 biosensor was constructed based on the high affinity of HRP/Mg-Al-LDH nanoflowers to H 2 O 2 . Figure 6c shows the UV-vis spectra of TMB ox in the presence of different H 2 O 2 concentration. As the H 2 O 2 concentration increases, the absorbance at 652 nm increases accordingly (Figure 6d). The proposed biosensor shows a linear response to H 2 O 2 in the concentration range from 1 to 20 mM with a detection limit of 0.34 mM, which is better than those of the HRPbased biosensors 42,45 . In addition, the stability of the proposed biosensor was also studied. After the storage for 72 h at room temperature, the HRP/Mg-Al-LDH nanobiocatalytic system retains 80.3% of the initial activity, while the free HRP system retains only 4.4% of its initial activity. The results suggest that the HRP/Mg-Al-LDH nanobiocatalytic system exhibits higher stability than the free HRP system.

Discussion
We propose a green coprecipitation method for the facile synthesis of ''desert rose stone''-like 3D hierarchical Mg-Al-CO 3 LDH nanoflowers. The growth process of the as-prepared3D hierarchical LDH nanoflowers has been investigated. The slow diffusion of CO 2 from air is considered to be a key process for tuning the morphology of LDH nanoflowers, which avoids the fast growth of LDHs. The proposed coprecipitaion method does not require high temperature or organic compounds, and can be used for preparing bio-nanomaterials. The prepared ''Desert Rose Stone''-like HRP/Mg-Al-LDH nanoflowers exhibit higher catalytic activity and stability than the free HRP, which suggests the LDH nanoflowers show good biocompatibility. The encapsulation of biomolecules is not limited to HRP. Biomolecules including individual and multiple enzymes, proteins and microRNA can also be incorporated to prepare nanobiofunctional systems for biocatalysis and biomedicine.

Methods
Synthesis of the Mg-Al-LDH and HRP/Mg-Al-LDH nanoflowers. The Mg-Al-LDH nanoflowers were synthesized in moderate conditions. Briefly, the pH of MgCl 2 (20 mM) aqueous solution was adjusted to ca. 10 using aqueous NaOH (0.5 M) solution. Then, a small piece of Al sheet was put into the solution. The reaction vessel was covered with perforated parafilm to allow CO 2 from air to diffuse into the system. After 18 h at room temperature, the obtained white precipitation (Mg-Al-LDH nanoflowers) was centrifuged and thoroughly washed with ultrapure water. The HRP/Mg-Al-LDH nanoflowers were synthesized by the same procedure with the addition of HRP (the final concentration 1.25 mg/mL) in MgCl 2 aqueous solution.
The fresh suspension was used for the analysis of enzyme activity and the dried power was used for characterizations. The HRP concentration in the supernatant was determined by the absorbance at 403 nm (e 403 59.1 3 10 4 L?mol 21 ?cm 21 ) 46 , which allows us to calculate the encapsulation yield of HRP in nanoflowers. The encapsulation efficiency of HRP in Mg-Al-LDH nanoflowers is defined as the ratio of encapsulation yield to the initial weight of HRP.
Kinetic Analysis and bioassay. Kinetic measurements were carried out at room temperature with 5 mL HRP/Mg-Al-LDH suspension or HRP solution in 500 mL PBS (25 mM, pH 5.0) containing 0.1 mM TMB and 20 mM H 2 O 2 . The absorbance at 652 nm in time-scan mode was monitored. The final concentration of HRP was 1 ng/ mL for each biocatalytic system. Detection of H 2 O 2 was finished as follows. Briefly, 5 mL HRP/Mg-Al-LDH suspension was added into 500 mL PBS (25 mM, pH 5.0) containing 0.1 mM TMB and different concentration of H 2 O 2 , followed by the reaction at 30uC for 20 min. The absorbance of the resulted solution at 652 nm was measured.
Characterization. UV-vis absorption spectra were obtained on a UV 3600 spectrophotometer (Shimazu, Japan).Fourier transform infrared (FTIR) spectra were collected on a Nicolet 6700 FTIR spectrometer (Nicolet, USA).X-ray diffraction (XRD) measurement was performed on a XRD-6000 diffractometer (Shimadzu, Japan), using Cu Ka radiation (l 5 1.5418 Å ) with a scan rate 2 deg/min. Scanning electron microscopy (SEM) images were obtained on a Hitachi S-4800 field-emission scanning electron microscope (Hitachi, Japan).Transmission electron microscopy (TEM) images were collected with a JEM-2100 high-resolution transmission electron microscope (JEOL, Japan). The molar ratio of Mg 21 /Al 31 in Mg-Al-LDH was determined by an optima 5300DV inductively coupled plasma atomic emission spectroscopy (ICP-AES, Perkin Elmer, USA). Thermogravimetry-differential scanning calorimetry (TG-DSC) analysis was performed on a STA 449C thermal analyzer (Netzsch, Germany) under the air flow with a heating rate of 10 K/min. The N 2 adsorption-desorption experiment was carried out by Micromeritics ASAP 2010 (Micromeritics, USA) at 77 K. Specific surface area of Mg-Al-LDH nanoflowers was calculated according to the Brunnauer-Emmett-Teller (BET) method.