A new nanosensor composed of laminated samarium borate and immobilized laccase for phenol determination

A new nanosensor composed of laminated samarium borate and immobilized laccase was developed for phenol determination. The laminated samarium borate was synthesized by a mild solid-state-hydrothermal (S-S-H) method without any surfactant or Template. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) were used to characterize the samples. The morphology of the as-prepared materials was characterized by SEM, which shows that laminated samarium borate are uniform nanosheets with a layer-by-layer self-assembled single-crystal structure. These laminated samarium borate have typical diameters of 3 ~ 5 μm and the thickness of each layer is in the range of 10 ~ 80 nm. And then, these SmBO3 multilayers were used to immobilize the laccase. The proposed nanosensor composed of laminated samarium borate and immobilized laccase was successfully developed for phenol determination. Cyclic voltammetry were used to study the nanosensor. The proposed nanosensor displayed high sensitivity toward phenolic compounds. The linearity of the nanosensor for the detection of hydroquinone was obtained from 1 to 50 μM with a detection limit of 3 × 10-7 M (based on the S/N = 3).


Background
With the development of the economy, more and more pollutants are eroding the human survival environment. Then the detection and treatment of environmental pollutions have aroused great attentions of scientists. Belonging to multicopper proteins, laccases are widely existed in nature especially fungi [1,2]. It is a phenol oxidase that can catalyze oxidation of many organic pollutants in water [3]. Wan and his group [4] had elaborated the progress on the research of laccases, namely the active center of copper ions, the three-dimensional structure of protein, and its catalytic mechanism. Substrate specificity of laccases was exploited to remove pollutants from the environment without creating the negative effects associated with many other methods [5,6]. It is well known that the enzyme is often easily inactivated in practical applications due to complex environment conditions, which limit its further industrial application [7,8]. Consequently, immobilized laccases have received much attention from researchers in recent years because of its substantial advantages over free laccases such as continuous reuse, easy separation of the product from the reaction media, easy recovery of the enzyme, and improvement in enzyme stability. Nowadays, many different types of methods have been employed in the immobilization of enzymes, such as adsorption, entrapment, cross-link, and covalent attachment. Recently, it is reported that laccase has been successfully immobilized [9][10][11] on many different types of supports, such as activated carbon [3], magnetic chitosan [12], alginate chitosan [13], porous glass [14], chitosan/poly(vinyl alcohol) composite nanofibrous membranes [15], cellulosepolyamine composite [16], alginate, kaolinite, polymer beads and membranes polystyrene microspheres, shortrange ordered aluminum hydroxide, and so on [17][18][19][20]. However, leakage, desorption, and the loss of enzyme activity were major problems in laccase immobilization, which was related to many factors involving the enzyme itself, polymer matrix, reaction reagents, and process conditions [9]. Therefore, it is of great interest in developing novel technologies on laccase immobilization to improve catalytic activity of laccase and increase its industrial application.
Among those laccase supports, inorganic materials are more attractive because of their regular structure, good mechanical, chemical, and thermal stabilities [21][22][23]. Nanomaterials have attracted increasing attention for their novel properties and potential applications with small dimensions [24,25]. Inorganic nanomaterials of rare-earth borate compounds show high vacuum ultraviolet (VUV) transparency and exceptional optical damage thresholds. Acentric lanthanide borate crystals are useful in a wide variety of photonic devices for unique optical, nonlinear optical, laser, electronic, and other physical properties [24,25]. In the past decades, the rareearth borates are widely used in many fields [26][27][28][29][30] and a number of synthetic methods have been employed to fabricate them. However, many routes suffer from the use of high temperature, tedious processes, and environmental pollution. Therefore, it is still an attractive and necessary topic for the development of environmentally friendly, facile, and reproducible methods to fabricate rare-earth borate nanometer materials.
In this paper, we choose a novel laminated SmBO 3 multilayer as support for the immobilization of laccase. The SmBO 3 multilayer samples were synthesized via the solid-state-hydrothermal (S-S-H) method, which exhibits many advantages, such as no side products, facile operation, and low cost. Then laccase was immobilized in SmBO 3 nanosheets for the fabrication of the nanosensor. The performance of the proposed nanosensor composed of the laminated samarium borate and immobilized laccase in the catalytic determination of phenolic compounds has been investigated in detail.
The morphology and structure of the samples were inspected by using a field emission scanning electron microscope (FE-SEM, Hitachi S4800, Tokyo, Japan) at an accelerating voltage of 5 KV. The phase purity and crystallinity of the samples were characterized by X-ray powder diffraction (XRD) performed on a D8 FOCUS diffractometer (Bruker, Madison, WI, USA) with CuKα radiation (λ = 0.154056 nm), employing a scanning rate of 0.02°· s −1 , in the 2θ ranges from 10°to 70°. Infrared spectra (4,000 to 400 cm −1 ) are recorded by Nicolet 5DX Fourier transform infrared spectroscopy (FTIR; Thermo Fisher Scientific, Waltham, MA, USA) equipped with a TGS/PE detector and a silicon beam splitter with 1 cm −1 resolution.
Electrochemical experiments were carried out with a CHI-660B electrochemical workstation (Shanghai, China). Measurements were performed at least three times on a glassy carbon electrode (GCE). A conventional threeelectrode system was employed, comprising a GCE (3-mm diameter) as the working electrode, a platinum wire as the auxiliary electrode, and an Ag/AgCl (saturated KCl) as the reference electrode. Voltammetric responses were recorded in 50 ml of substrate solutions prepared in PBS buffer solution. First, the modified electrode was activated by several successive voltammetric cycles from −0.20 to 0.80 V. Second, cycle voltammograms (CVs) at the rate of 50 mV · s −1 were carried out from −0.20 to 0.80 V after subtracting the background. Finally, the GCE was regenerated by 10 successive cyclic voltammetric sweeps in the blank solution. After several measurements, the GCE should be repolished. All the electrochemical measurements were carried out at room temperature.

Preparation of SmBO 3 nanocrystals
Precursor-laminated SmBO 3 multilayers were synthesized by solid-state-hydrothermal method. In a typical synthesis, 0.6 mmol Sm 2 O 3 , 0.72 mmol H 3 BO 3 , 14 ml deionized water are mixed in a 20-ml-capacity Teflonlined autoclave. The autoclave is sealed and maintained at 200°C constantly for 36 h and then cooled to room temperature naturally. The precipitation is centrifuged and washed with deionized water several times. Finally, as-obtained products are dried under vacuum at 60°C for 4 h. We propose that the formation processes of SmBO 3 in the solid-state-hydrothermal system at 200°C can be assigned to two stages: Sm 2 O 3 is first transformed into hydroxide, Sm(OH) 3 , then the hydroxide interacts with H 3 BO 3 to form products. The formation reactions of SmBO 3 are proposed and shown in Figure 1.

Immobilization of laccase on SmBO 3 nanocrystals
The SmBO 3 multilayers were employed as carriers for the immobilization of laccase, and the laccase was immobilized on these materials by the physical adsorption method. In a typical procedure, 100 mg of SmBO 3 support was suspended in 10 ml of phosphate buffer (pH = 7.0) containing a certain amount of laccase (about 20 mg). The mixture of the supports and laccase solution was slowly stirred at room temperature for 12 h. Subsequently, the laccase immobilized on SmBO 3 was separated by a centrifuge. Then the samples were washed with 10 ml of buffer solution by shaking for 5 min and separated quickly using a centrifuge. The washing procedure was repeated several times until no protein was detected in the supernatant. Finally, the laccase immobilized by SmBO 3 were stored at 4°C before using. The percentage of the immobilized laccase on the SmBO 3 samples is in the range of 10.7%~15.2%.

Preparation of the glassy carbon electrode
Ultrasonic agitation was used to disperse 1-mg SmBO 3immobilized laccase into 1-ml Nafion to give a suspension (1 mg · ml −1 ). Before an experiment, the GCE was polished successively with 0.1-μm γ-Al 2 O 3 powder, and then on a polishing cloth. Residual polishing material was removed from the electrode surface by ultrasonic agitation in concentrated HNO 3 , distilled water, and absolute ethanol. Then, the GCE was coated with 10 μl of laccase immobilized by SmBO 3 -Nafion suspension   (1 mg · ml −1 ) and the solvent evaporated under room temperature for 1 h. The modified electrode was cleaned with distilled water before use.

Results and discussion
SEM studies Figure 2a shows SEM micrographs of as-prepared SmBO 3 multilayer obtained via the additive-free S-S-H method at 200°C for 36 h. Figure 2b was the corresponding highmagnified images. The multilayer shapes consist of multilayer nanosheets. These nanosheets have typical diameters of 3~5 μm while the thickness of the single layer are in the range of 10~80 nm. These microparticles are nonaggregated with narrow size distribution. The pseudovaterite self-assembled SmBO 3 multilayers exhibit advantages in high-ratio surface area and analogy-graphite layer structure, which are favorable for potential application in enzyme immobilization. Figure 2c shows that the laccase was effectively filled among layers of SmBO 3 by physical absorption. Inspired by this, we inferred the multilayer structures of SmBO 3 suitable for immobilization of other enzymes.

The XRD pattern analysis of as-prepared SmBO 3 samples
To ascertain the structure of as-prepared SmBO 3 samples, corresponding XRD patterns of samples were investigated and shown in Figure 3. The pattern is inconsistent with aragonite-type, which are indexed in the standard pattern database listed in JCPDS. To make clear the crystal structure, the MDI Jade (5.0 Edition) software was applied to auto index the similar patterns in JCPDS. It was found that the peak positions are in accordance with the primitive-lattice hexagonal phase SmBO 3 (No. 13-0479).
FTIR spectra analysis  [31][32][33][34]. To investigate the binding between the laccase and the laminated SmBO 3 multilayers, FTIR spectra for the laminated SmBO 3 multilayers, lacasse, and laminated SmBO 3 multilayers with immobilized laccase were measured. Figure 4b,c shows the FTIR spectra laccase and SmBO 3immobilized lacasse. Compared to the typical absorption peaks of lacasse at 3,401, 2,923, and 1,649 cm −1 and the main absorption peaks of SmBO 3 at 1,110, 960, 894, and 827 cm −1 , the absorption of SmBO 3 -immobilized lacasse include all of the above peaks. So it is evident that the laccase was successfully immobilized on SmBO 3 nanosheets. Moreover, it can be seen from Figure 4 that the positions of lacasse and those immobilized in SmBO 3 are nearly at   the same place, suggesting that the lacasse retains its native structure in SmBO 3 -immobilized lacasse.

Electrochemical properties
The response of laccase-immobilized SmBO 3 nanosheets for phenolic compound detection is based on the mechanism in which a substrate (hydroquinone in this case), laccase, and oxygen are involved. The enzymatic mechanism involved in laccase-immobilized SmBO 3 for phenolic compound detection is the same as the bare laccase [4]. Laccase as one of the multicopper oxidases contains four copper atoms and catalyzes the four-electron reduction of O 2 to H 2 O at a trinuclear copper cluster. The catalytic process consists of the oxidation of hydroquinone by laccase followed with the reduction of O 2 by laccase ( Figure 5).
The electrochemical behaviors of laccase-immobilized SmBO 3 -modified GCE in various solutions were studied using cyclic voltammetry and the results are shown in Figure 6. The laccase-immobilized SmBO 3 -modified GCE remain its redox behaviors in pH 4.0 PBS at room temperature with the presence of 5 × 10 −5 mol · l −1 hydroquinone. The anodic peak currents of laccase-immobilized SmBO 3 -modified GCE are 3.0 μA. Compared to the anodic peak current of bare electrode which is 1.48 μA, the anodic peak current of modified GCE is at least two times greater. These demonstrate that the electrode of the SmBO 3 -immobilized laccase has a better sensitivity to the substrate. At the same time, we found that the ΔE of laccase-    immobilized SmBO 3 -modified GCE (0.51 V) is larger than bare electrode (0.47 V). According to the Gibbs-Helmholtz equation ΔG = −nFΔE, ΔG of the laccase-immobilized SmBO 3 -modified GCE is smaller than the bare electrode. These results suggest that the reaction occurs on the laccase-immobilized SmBO 3 electrode is much easier than the bare electrode.

Optimal parameters
We used 0.2 mol · l −1 Na 2 HPO 4 · 12H 2 O and 0.1 mol · l −1 C 6 H 8 O 7 · H 2 O solutions to adjust the pH of the buffer solutions from 3.0 to 8.0. Figures 7 and 8 show the relationship between the pH values and the anodic peak potentials, the anodic peak currents from CV, respectively. The potentials shifted negatively with increasing pH value. At the same time, the anodic peak currents increased slightly with increasing pH, and when the pH exceeded 4.0, the anodic peak currents decreased immediately. It may be due to the high oxidation potentials and the serious interference at low pH values. Therefore, pH 4.0 was chosen as the optimum pH in this work.
Cycle voltammograms were employed to investigate the influence of scan rate on hydroquinone oxidation at the laccase-immobilized SmBO 3 -modified electrode. The results are shown in Figure 9. At scan rates in the range of 0.01 to 0.1 V · s −1 , the oxidative peak currents of the laccase-immobilized SmBO 3 -modified electrode in hydroquinone solution increased linearly with the square root of the scan rate, which proved that the electro-oxidation of hydroquinone was a diffusion-controlled process.

Calibration graphs
The anodic peak currents (I p ) of laccase-immobilized SmBO 3 -modified electrode of the CV are proportional to the concentration of hydroquinone from 1 × 10 −6 to 5 × 10 −5 mol · l −1 . The picture is shown in Figure 10.
The calibration curve under optimal conditions is shown in Figure 11. The linear response range of laccaseimmobilized SmBO 3 -modified electrode to hydroquinone concentration is from 1 to 50 μM with a correlation coefficient of 0.998 (I = 4.13c +0.42, r = 0.998). The detection limits of the compounds are estimated to be 3 × 10 −7 mol · l −1 .

Conclusions
In summary, we have demonstrated a nanosensor composed of laminated samarium borate and immobilized laccase for phenol determination. These SmBO 3 nanosheets have been successfully prepared via a mild solidstate-hydrothermal method without any surfactant or template, and laccase was successfully immobilized on these multilayers through physical adsorption method. The uniform multilayer-intersected structure could play an important role in the adsorption of laccase. This novel laccase immobilization method based on SmBO 3 improved the performance of the laccase for phenol determination. The linear range and bioactivity of laccase-modified electrode can also satisfy the practical application. The present study has enlarged the family of support for laccase immobilization and may provide an efficient approach for phenol determination.