Direct electrochemistry of laccase and a hydroquinone biosensing application employing ZnO loaded carbon nanofibers

Abstract

ZnO loaded carbon nanofibers (ZnO/CNFs) were successfully fabricated by a combination of electrospinning, carbonization and a hydrothermal process. A novel biosensor was fabricated based on a composite of ZnO/CNFs, laccase (Lac), and Nafion. The addition of ZnO/CNFs apparently facilitated the direct electron transfer (DET) between the active center of Lac and the surface of the glassy carbon electrode (GCE). A pair of stable and well-defined redox peaks was observed on the Nafion–Lac–ZnO/CNF modified GCE. Meanwhile, square wave voltammetry (SWV) was employed to investigate the biosensor, and the sensor showed highly efficient electrocatalysis toward hydroquinone with a sensitivity of 28.50 μA μM⁻¹, a detection limit of 9.50 nM (S/N = 3), a linear range from 5.00 × 10⁻⁷ to 2.06 × 10⁻⁶ M, as well as good selectivity and stability. Furthermore, this novel biosensor was successfully used in detecting hydroquinone in real water samples.

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1. Introduction

Hydroquinone is a widely used chemical applied in the environment, cosmetics, human diet and medicines.¹ It is extremely harmful to animals and plants in an aquatic environment even at very low concentration; a high concentration of HQ can lead to headache, fatigue, tachycardia, even kidney damage and cancer in humans.² So it becomes an urgent issue that requires a simple and highly efficient detection method for hydroquinone. At present, various methods such as gas chromatography, liquid chromatography, electroanalytical techniques and enzyme immunoassays, have been employed for measuring phenols.^3–5 However the use of these methods in on-line or field monitoring is restricted because of the fixed space location, large size of the analytical equipment and complicated purification procedure for the samples. Compared with these traditional testing instruments, a biosensor is a fast, simple, facile and amenable to miniaturization analytical apparatus which can satisfy the requirements of multi component detection in situ.⁶

Laccase (Lac), a multi-copper oxidase, can catalyze the oxidation of phenols with the simultaneous reduction of molecular oxygen to water.⁷ Various biosensors employing Lac have been reported for the detection of phenols.^8–14 Nowadays, reagentless enzyme electrodes, which can achieve direct electron transfer (DET) by combining conductive materials with enzymes, are becoming a hotspot in biosensor research.^11,15–19 Carbon materials, such as carbon black,²⁰ carbon nanotubes,¹² graphene,⁸ mesoporous carbon²¹ and carbon nanofibers (CNF),²² have been widely used as enzyme immobilization materials in biosensors, because of their excellent conductivity, large specific surface area and satisfactory biocompatibility. It is noticeable that the functionalized surface area of CNFs is much larger than that of CNT and CNFs are a more suitable matrix for enzyme immobilization. Based on the research,²² CNFs are an excellent material for biosensor development, far superior to carbon nanotubes.

Many methods can be used for preparing CNFs, including chemical vapor deposition (CVD) methods,²³ laser ablation,²⁴ arc-discharge²⁵ and others. Electrospinning is a convenient and facile process technology for producing nanofibers or microfibers. CNFs can also be obtained through carbonizing electrospun polyacrylonitrile nanofibers.²⁶ Usually some impurities, e.g., graphite particles and metal catalyst, can be found in CNFs obtained from CVD, which needs a further complicated purification process. However, electrospun CNFs (ECNF) show higher purity and a simple fabrication process. Currently, some catalytic metal nanoparticle/ECNF composites and nonenzymatic sensors based on ECNF have been reported.^27–29 ECNF shows great potential in the biosensing field.

ZnO is an important functional material, and has been widely applied in photocatalysis,³⁰ biosensors³¹ and dye-sensitized solar cells³² due to its distinctive properties such as large exciton binding energy, wide direct band gap, good piezoelectric properties, and excellent chemical and thermal stability.³³ ZnO nanoparticles, as one of the promising metal oxides, has been extensively applied in biosensors due to its superior surface properties, electrochemical catalytic activity, nontoxicity and biocompatibility.³⁴

In this article, we prepared ZnO loaded carbon nanofibers (ZnO/CNFs) and fabricated a novel biosensor through dropping a certain amount of a mixture of ZnO/CNF, Lac and Nafion onto the surface of a polished glassy carbon electrode (GCE). These ZnO/CNFs facilitated the DET between the active center of Lac and the surface of the GCE. Furthermore, the biosensor showed highly efficient electrocatalysis toward hydroquinone with high sensitivity and a low detection limit. Meanwhile, the biosensor also possessed satisfactory selectivity, good reproducibility and stability. Finally, the biosensor was successfully employed to detect hydroquinone in a real water environment. This study demonstrates that ZnO/CNFs are a promising material for biosensing applications.

2. Materials and methods

2.1. Chemicals and reagents

Laccase (from Trametes, activity ≥10 U mg⁻¹) and polyacrylonitrile (PAN, average molecular weight = 79 100) powder were obtained from Sigma-Aldrich and utilized without further purification. N,N-Dimethylformamide (DMF) with a purity of 99.5%, zinc acetate dihydrate (C₄H₆O₄Zn·2H₂O), potassium ferricyanide (K₃[Fe(CN)₆]), potassium hexacyanoferrate (K₄[Fe(CN)₆]·3H₂O), NaOH and hydroquinone were purchased from the Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). Nafion (5% w/w) was obtained from E. I. Du Pont Company. Acetate buffer solution (0.1 M HAc–NaAc, pH = 5.0) was used as a supporting electrolyte. All aqueous solutions were prepared with deionized water (DIW).

2.2. Apparatus

A scanning electron microscope (SEM, Hitachi SU1510) and a high-resolution transmission electron microscope (TEM, JEOL/JEM-2100, Japan) were employed to observe the ZnO loaded carbon nanofibers (ZnO/CNFs). Prior to scanning under the SEM, the ZnO/CNFs were sputter coated with gold for 90 s to avoid charge accumulations. Powder D8 Advance X-ray diffraction (XRD, Bruker AXS D8) and energy dispersive X-ray spectroscopy (EDX, EDAX-TSL, AMETEK USA) were used to analyze the chemical components of ZnO/CNFs. Fourier transform infrared (FT-IR) spectra were recorded by a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientific) in the range of 500–4000 cm⁻¹. KBr pellets were used to prepare the samples for FT-IR measurements. A 3D Nanometer Scale Raman PL Microspectrometer (Tokyo Instruments, Inc., a 785 nm He–Ne laser) was used to analyze the graphite structure of CNFs at room temperature. Electrochemical experiments were carried out using a CHI 660E electrochemical workstation (CH Instruments, Shanghai, China) at room temperature. The electrochemical measurements were implemented by a three-electrode cell with a GCE (4.0 mm in diameter, purchased from Gaoss Union Technology Co., Ltd., Wuhan, China), a platinum wire auxiliary electrode and an Ag/AgCl reference electrode (saturated KCl).

2.3. Synthesis of CNFs

The CNFs were prepared by the following steps. First, the electrospinning solution was prepared by dissolving 10 wt% PAN powder with magnetic stirring for 12 h. Second, the prepared solution was added into a syringe for electrospinning. The experimental parameters were set with a voltage of 15 kV, a working distance of 15 cm, and a flow rate of 0.5 mL h⁻¹ respectively. Finally, a high temperature furnace was used to stabilize and carbonize the PAN nanofibers. The whole procedure was conducted in an N₂ atmosphere and could be divided into two steps: (1) heating up to 260 °C at a heating rate of 2 °C min⁻¹ in and keeping at this temperature for 1 h, this process was for stabilizing the shape of the nanofibers; (2) heating up to 900 °C at a heating rate of 5 °C min⁻¹ to carbonize the nanofibers, maintaining at the highest temperature for 1 h, and then cooling down to room temperature.

2.4. Fabrication of ZnO/CNFs

Firstly, the CNFs and zinc acetate dihydrate (with the mass ratio of 1 : 2) were put into 120 mL of DIW. Next, 0.5 mol L⁻¹ NaOH solution was added into the solution until the pH value reached 10 under continuous magnetic stirring. Finally, the mixture was dropped into a 200 mL Teflon-lined stainless autoclave, sealed and kept at 180 °C for 12 h, and cooled to room temperature. The fabricated products were gathered, washed several times with ethanol and DIW respectively, and dried at 60 °C.

2.5. Preparation of biosensors

Considering the optimal response and stability of the modified electrode, among control-experiments, the concentration and mass ratio of Nafion, ZnO/CNFs and Lac were optimized. Ultimately, the biosensor was prepared from a mixture containing 1.5 wt% Nafion, 0.4 mg mL⁻¹ ZnO/CNFs and 3 mg mL⁻¹ Lac.

The process for the preparation of the Nafion–Lac–ZnO/CNF modified GCE is as follows: first of all, adding 4 mg ZnO/CNFs into 10 mL pH 5.0 acetate buffer to prepare the ZnO/CNF suspension with the aid of ultrasonication and stirring. Then to a mixture containing the ZnO/CNF suspension, a certain volume of Nafion (5 wt%) and the appropriate mass amount of Lac were added and stirring was maintained for 1 h. Finally, the Nafion–Lac–ZnO/CNF modified GCE was fabricated by dropping 10 μL of the mixture on the surface of a freshly processed GCE. The GCE was processed as follows: first, it was polished with alumina; next, it was rinsed with DIW and ultrasonicated in DIW; finally, it was dried under a nitrogen atmosphere. The dried Nafion–Lac–ZnO/CNF modified GCE was stored at 4 °C.

For comparison, Nafion–CNF modified GCE, Nafion–ZnO/CNF modified GCE, Nafion–Lac modified GCE, Nafion–Lac–ZnO modified GCE and Nafion–Lac–CNF modified GCE were prepared. Herein, the mass amount of Lac in the different electrodes was kept equal. All the modified electrodes were dipped into pH 5.0 acetate buffer for 30 min to remove any unstable substances before electrochemical measurements. In addition, for the electrochemical impedance spectroscopy (EIS) test, the CNF modified GCE and ZnO/CNF modified GCE were fabricated by dropping 10 μL of CNFs or ZnO/CNF suspension with a concentration of 1 mg mL⁻¹ in DIW onto the surface of the GCE followed by a drying process at room temperature.

3. Results and discussions

3.1. Characterization

Fig. 1a depicts the SEM image of the prepared ZnO/CNFs. As shown in Fig. 1a, some nanoparticles can be observed on the surface of the randomly distributed CNFs. Although some nanoparticles were aggregated, which may be caused by the mutual attraction of the nanoparticles, most of the nanoparticles were relatively dispersed on the surfaces of the CNFs. This can be confirmed by the TEM image (Fig. 1b), the nanoparticles were successfully attached on the surfaces of CNFs with little aggregation. Furthermore, some triangle-shaped nanoparticles can be observed, which may be attributed to the formation of a special crystal structure of ZnO nanoparticles in the hydrothermal process. Meanwhile, as shown in the inset of Fig. 1b, the diameter distribution of ZnO nanoparticles mainly ranged between 80 and 180 nm, and the mean diameter was ca. 131.2 nm. The EDX result (see Fig. 1c) suggests that the main elements of the ZnO/CNFs were C and Zn. The Kα line of O can be ascribed to the oxygen in air or the ZnO nanoparticles. XRD analysis was employed to further study the composition of the ZnO/CNFs, the result is shown in Fig. 1d. It displays nine sharp characteristic diffraction peaks at ca. 31.8°, 34.4°, 36.3°, 47.5°, 56.6°, 62.9°, 66.3°, 67.9° and 69.1°, which correspond to the (100), (002), (101), (102), (110), (103), (200), (112) and (201) crystalline planes of hexagonal zinc oxide, respectively.³⁵ Meanwhile, the peak at around 25.6° was assigned to the (002) plane of carbon.³⁰ Due to the high peak intensity of ZnO, the peak of the CNFs is relatively indistinguishable. In summary, the EDX and XRD results fully demonstrated the successful preparation of ZnO nanoparticles in the ZnO/CNF heteroarchitectures.

Fig. 1 (a) SEM image of the ZnO/CNFs; (b) TEM image of the ZnO/CNFs; (c) EDX spectra of the ZnO/CNFs; (d) XRD pattern of the ZnO/CNFs. The inset is the size distribution of the ZnO nanoparticles.

Fig. 2a shows the FT-IR spectra of the CNFs and ZnO/CNFs. It can be easily seen that the two curves possess almost the same absorption peaks, which indicates that the CNFs maintained their intrinsic functional groups in the hydrothermal process. The peaks appearing at around 3443 cm⁻¹ and 1453 cm⁻¹ were related to the O–H band, and the adsorption peak at about 1636 cm⁻¹ indicated the presence of carboxyl functional groups (C=O).³⁶ Besides, the peak at around 2921 cm⁻¹ can be attributed to the C–H stretching band of the [double bond splayed left]CH₂ and the peak at around 1065 cm⁻¹ can be ascribed to the oxygen groups (C–O–C).³⁷ The large number of carboxyl groups existing on the surface of CNFs were expected to enhance the electrocatalytic property and biocompatibility of the CNFs.³⁸ Raman spectroscopy has been widely utilized as a surface analysis technique for carbon materials.³⁹ As shown in Fig. 2b, two distinct peaks at around 1330 cm⁻¹ and 1590 cm⁻¹, relating to the D-band and the G-band respectively, can be observed in the CNFs and ZnO/CNFs. The D band was ascribed to the defective carbon structure of the CNFs, and the G band is attributed to the in-plane carbon–carbon stretching vibrations of the graphite layers.⁴⁰ This suggested that the proposed CNFs possessed polycrystalline structures and massively disordered and defective graphite layers, which were favorable for the electrocatalysis of CNFs.²²

Fig. 2 (a) FT-IR spectra of the CNFs and ZnO/CNFs; (b) Raman spectra of the CNFs and ZnO/CNFs.

EIS was used to investigate the interface properties of different modified GCEs, and the results of AC impedance spectra of bare GCE, CNFs modified GCE and ZnO/CNFs modified GCE are illustrated in Fig. 3. The semicircle part at higher frequencies is related to the electron transfer process, and the linear part at lower frequencies is related to the Warburg diffusion process. The electron transfer resistance of the electrochemical reaction (R_et) corresponds to the semicircle diameter of the Nyquist plots, and the R_et controls the electron transfer kinetics of the redox electrochemical probe at the electrode interface. The electron transfer resistance (R_et) of bare GCE was 755 Ω. Apparently, the R_et for the CNF modified GCE decreased dramatically, and was only 229 Ω. This proved that the interface resistance of the CNF modified GCE was significantly diminished in comparison with that of bare GCE due to the excellent conductivity of CNFs. In addition, it can be easily seen that the R_et for the ZnO/CNF modified GCE was 677 Ω, which was larger than that for the CNF modified GCE. Herein, the enhanced interface resistance might be ascribed to the inferior conductivity of ZnO. Despite this, the R_et for the ZnO/CNF modified GCE was still lower than that for bare GCE, showing that the ZnO/CNFs are a potential candidate for a biosensor material.

Fig. 3 Nyquist plots for bare GCE, CNF modified GCE and ZnO/CNF modified GCE in a solution of 0.1 M KCl containing 5 mM Fe(CN)₆^3−/4− with a frequency range from 0.01 Hz to 100 000 Hz and signal amplitude of 5 mV. Inset: equivalent circuit used to model impedance data in the presence of redox couples.

3.2. Direct electrochemistry of the proposed biosensor

As shown in Fig. 4, seven electrodes were prepared to investigate the direct electrochemistry of Lac existing in the composites. It is apparent that the bare GCE (Fig. 4, curve a), Nafion–CNF modified GCE (Fig. 4, curve b) and Nafion–ZnO/CNF modified GCE (Fig. 4, curve c) showed no redox peaks, indicating the electrochemical inertness of CNFs and ZnO within the potential window. However, two pairs of distinct redox peaks can be observed on the Nafion–Lac modified GCE (Fig. 4, curve d), which may be attributed to the existence of electrical communication between two copper based redox centers in the Lac with the surface of GCE. Herein, the Nafion may offer a favorable microenvironment for the immobilization of Lac. Notably, Lac did not show direct electrochemistry in the Nafion–Lac–ZnO modified GCE (Fig. 4, curve e), which can be ascribed to the poor conductivity of the aggregated ZnO nanoparticles, and this was consistent with the result reported in the literature.⁴¹ A pair of distinct redox peaks can be observed for the Nafion–Lac–CNF modified GCE (Fig. 4, curve f) and the right redox peaks almost disappeared, it is believed that the addition of CNFs facilitated the electron transfer in the T₁ area of Lac to the electrode surface, leading to the decrease of electron number in the T₂ area of Lac. Compared with the left redox peaks observed on Nafion–Lac modified GCE, although the cathodic peak current decreased, the anodic peak current increased, and the peak-to-peak distance decreased, which indicated faster electron transfer. Besides, the reversibility of the redox reaction of the Nafion–Lac–CNF modified GCE also became better than that of the Nafion–Lac modified GCE. These results suggested that the addition of CNFs improved the DET between the surface of the GCE and the electroactive centre of the immobilized Lac to some extent. Remarkably, the Nafion–Lac–ZnO/CNF modified GCE (Fig. 4, curve g) showed higher redox peak currents in comparison with those of the Nafion–Lac–CNF modified GCE. This indicated that the ZnO/CNFs can further facilitate the DET between the surface of GCE and the electroactive centre of immobilized Lac, which may be attributed to the larger electroactive surface area of ZnO/CNFs for the immobilization of Lac. It can be easily observed that the anodic (E_pa) and cathodic (E_pc) peak potentials were located at 0.214 and 0.086 V (vs. Ag/AgCl), respectively. The formal potential (E⁰′) was ca. 0.150 V, and the peak-to-peak separation (ΔE_p) was 128 mV at a scan rate of 100 mV s⁻¹, indicating a quick electron transfer.

Fig. 4 Cyclic voltammograms of (a) bare GCE, (b) Nafion–CNF modified GCE, (c) Nafion–ZnO/CNF modified GCE, (d) Nafion–Lac modified GCE, (e) Nafion–Lac–ZnO modified GCE, (f) Nafion–Lac–CNF modified GCE, and (g) Nafion–Lac–ZnO/CNF modified GCE in 0.1 M acetate buffer solution (pH 5.0) at a scan rate of 100 mV s⁻¹.

It can be seen from Fig. 5a, that as the scan rate increased, the anodic and cathodic peak currents increased simultaneously. Moreover, the peak currents improved linearly with the scan rate from 50 to 400 mV s⁻¹ (see Fig. 5b), indicating that the whole electrochemical process was a surface-controlled redox process. Notably, the E_pa and E_pc peak potentials moved to more positive and more negative values respectively, which suggested that the electrochemical reaction process was a quasi-reversible process. Based on the Laviron equation,⁴² the apparent electron-transfer rate constant (k_s) and the charge-transfer coefficient of the Lac at the Nafion–Lac–ZnO/CNF modified GCE were estimated to be 1.64 s⁻¹ and 0.5 at a scan rate of 100 mV s⁻¹. This k_s value was larger than the value of 1.17 s⁻¹ observed for the Lac immobilized on an AP-rGO/chit modified GCE,⁸ that of 0.50 s⁻¹ observed for the Lac immobilized on a gold nanoparticle modified GCE,¹³ and that of 1.28 s⁻¹ reported for the Lac immobilized on AuNP encapsulated-dendrimer bonded conducting polymer.⁴³ The high k_s value may be attributed to the excellent conductivity of ZnO/CNFs and the favorable biocompatibility of the immobilization matrix. According to the formula Q = nFAΓ, where n is the number of transferred electrons, F is the Faraday constant, A is the surface area of electrode (cm²), Γ is the surface coverage of electroactive species (mol cm⁻²), Q is the consumed charge (C), the surface coverage for the electroactive Lac was calculated to be 1.67 × 10⁻¹⁰ mol cm⁻², which was higher than the value theoretically calculated in a previous report (1.30 × 10⁻¹¹ mol cm⁻²).⁴⁴ This result indicated that the great specific surface area of ZnO/CNFs can offer more immobilization sites for the Lac.

Fig. 5 (a) Cyclic voltammograms of the Nafion–Lac–ZnO/CNF modified GCE in 0.1 M acetate buffer solution (pH 5.0) at scan rates of 50, 100, 150, 200, 250, 300, 350 and 400 mV s⁻¹ (from inner to outer), respectively; (b) plots of the corresponding anodic and cathodic peak currents vs. scan rate; (c) cyclic voltammograms of the Nafion–Lac–ZnO/CNF modified GCE in 0.1 M acetate buffer solution with different pH values of 3.0, 4.0, 5.0, 6.0 and 7.0 (from right to left) at a scan rate of 100 mV s⁻¹; (d) plots of E_pa, E_pc and E⁰′ vs. pH.

Fig. 5c presents the influence of pH on the CVs of the Nafion–Lac–ZnO/CNF modified GCE in 0.1 M acetate buffer solution. It can be clearly seen that as the pH value altered from 3.0 to 7.0, the redox peaks gradually moved left. As the pH of the solution increased, the values of E_pa, E_pc and E⁰′ moved negatively and linearly (see Fig. 5d), suggesting that proton transfer happened in the electrochemical procedure. The linear slopes of the plots of E_pa, E_pc and E⁰′ vs. pH were −50.0 mV pH⁻¹ (r² = 0.994), −54.4 mV pH⁻¹ (r² = 0.989) and −52.2 mV pH⁻¹ (r² = 0.994), respectively. The expected value was −59.2 mV pH⁻¹ (25 °C), which was close to the above values. This indicated that a direct electrochemical reaction containing equal numbers of electrons (e⁻) and protons (H⁺) occurred in the Lac immobilized on the Nafion–Lac–ZnO/CNF modified GCE.¹³

3.3. Electrochemical response of the biosensor to hydroquinone

As shown in Fig. 6, to investigate the effect of different materials on the response of modified electrodes to hydroquinone, square wave voltammetry (SWV) was employed using bare GCE (curve a), Nafion–Lac modified GCE (curve b), Nafion–Lac–CNF modified GCE (curve c) and Nafion–Lac–ZnO/CNF modified GCE (curve d) in 0.1 M acetate buffer solution (pH 5.0) with 1.1 μM of hydroquinone at a frequency 60 Hz, pulse amplitude 100 mV and scan increment 5 mV. It can be easily seen from the inset, that the current value of the bare GCE (bar a) was the lowest one, at around 50.7 μA. Compared with the current value of bare GCE, that of the Nafion–Lac modified GCE (bar b) ascended significantly, indicating the high electrocatalysis of Lac toward hydroquinone. It is noticeable that the value of Nafion–Lac–CN modified GCE (bar c) rose by 10.0 μA in comparison with that of the Nafion–Lac modified GCE. This fully demonstrated that the CNFs strongly enhanced the electrocatalysis of modified GCE toward hydroquinone, which may be caused by the DET. Furthermore, compared with that of the Nafion–Lac–CNF modified GCE, the current value of the Nafion–Lac–ZnO/CNF modified GCE increased again, indicating that the addition of ZnO further enhanced the sensitivity of the modified GCE. Herein, the ZnO nanoparticles offered a larger electroactive surface area for the immobilization of Lac, leading to enhanced electrocatalytic ability of the Nafion–Lac–ZnO/CNF modified GCE towards substrate. Notably, the blank “d” was obtained in 0.1 M acetate buffer solution (pH 5.0) without hydroquinone by the Nafion–Lac-G/CNFs modified GCE, and the peak current of ca. 74 μA can be regarded as a blank signal, which may be ascribed to the DET. The reaction mechanism is illustrated in Scheme 1. First, the hydroquinone on contact with the Lac was oxidized to 1,4-benzoquinone in the presence of molecular oxygen. Subsequently, the 1,4-benzoquinone was reduced electrochemically on the surface of the GCE.

Fig. 6 Square-wave voltammograms obtained using different electrodes: (a) bare GCE, (b) Nafion–Lac modified GCE, (c) Nafion–Lac–CNF modified GCE and (d) Nafion–Lac–ZnO/CNF modified GCE in 0.1 M acetate buffer solution (pH 5.0) with 1.1 μM of hydroquinone at a frequency 60 Hz, pulse amplitude 100 mV and scan increment 5 mV. Blank “d” was obtained in 0.1 M acetate buffer solution (pH 5.0) without hydroquinone. Inset: current values for hydroquinone obtained using each of the electrodes studied.

Scheme 1 Schematic representation of laccase catalyzed oxidation of hydroquinone with its subsequent electrochemical reduction on the GCE.

3.4. Optimization of the biosensor construction and experimental conditions

In order to obtain the optimal analysis performance, the biosensor construction and the experimental conditions of SWV were compared and optimized. Different volumes of Nafion–Lac–ZnO/CNF solution were dropped on the GCE, and the best response of the modified GCE in 0.1 M acetate buffer solution (pH 5.0) with 1.1 μM of hydroquinone was obtained using 10 μL of Nafion–Lac–ZnO/CNF solution. So, in the subsequent experiments, this volume was employed. Afterward, the effects of different pH values on the response of the biosensor were also investigated. The strongest current response was found at pH 5.0 from pH 3.0 to pH 7.0 using SWV. Finally, the parameters of SWV were optimized under the above experimental conditions. Scan increments from 0.5 to 10 mV, pulse amplitudes between 10 and 100 mV and frequency values in the range of 10–100 Hz were evaluated and the optimized parameters were as follows: scan increment 5 mV, amplitude 100 mV and frequency 60 Hz. So, these experimental conditions were selected for all following analytical determinations of hydroquinone.

3.5. Analytical performance of biosensor in hydroquinone determination

Fig. 7 shows the square-wave voltammograms and analytical calibration curve for hydroquinone concentration employing the Nafion–Lac–ZnO/CNF modified GCE. It can be seen that with the increment of hydroquinone concentration, the peak current value increased simultaneously. The linear range was from 5.00 × 10⁻⁷ to 2.06 × 10⁻⁶ M (ΔI = 61.28 (±0.6890) + 28.50 (±0.4990)C; r² = 0.9976); where ΔI is the peak current in μA and C is the concentration in μM. The sensitivity was as high as 28.50 μA μM⁻¹ and the detection limit (S/N = 3) was 9.50 nM. The sensing performances of several sensors toward hydroquinone are compared in Table 1. It can be seen that our sensor showed a low detection limit and high sensitivity. The analytical performance was satisfactory and so the low detection limit emphasized the advantage of Nafion–Lac–ZnO/CNF modified GCE for hydroquinone sensing over other modified electrodes.

Fig. 7 Square-wave voltammograms obtained using Nafion–Lac–ZnO/CNF modified GCE for (a) blank in 0.1 M acetate buffer solution (pH 5.0) and hydroquinone solutions at the following concentrations: (b) 0.50; (c) 0.70; (d) 0.90; (e) 1.10; (f) 1.29; (g) 1.49; (h) 1.69; (i) 1.86; (j) 2.06 μM at frequency 60 Hz, pulse amplitude 100 mV and scan increment 5 mV. Inset: calibration curve for hydroquinone.

Table 1 Sensing performance comparison of different sensors toward hydroquinone by SWV^a

Electrode description	Detection limit (μM)	Linear range (μM)	Sensitivity (μA μM^−1)	Reference
a The dash in the table represent values that were not reported in the reference.
GCE–PEI–AuNP–LAC	0.2100	2.90–22.00	1.12	12
[Cu(μ2-hep)(hep-H)]2·2PF6(1)–CPE	0.0150	0.05–1.81	0.51	45
GC/MWCNT/CoPc electrode	0.1600	0.99–8.30	36.10	46
Pd/ImS3-14/(Fe^IIICu^II)/GCE	0.0850	0.50–18.50	—	47
Nafion–Lac–ZnO/CNF modified GCE	0.0095	0.50–2.06	28.50	This work

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3.6. Stability and reproducibility of the biosensor

The Nafion–Lac–ZnO/CNF modified GCE displayed good reproducibility, repeatability and stability. Three biosensors were prepared independently under the same conditions to investigate the electrode-to-electrode reproducibility and the relative standard deviation (RSD) of the three modified electrodes was 2.6%, indicating the good reproducibility of the biosensor. The RSD of the biosensor response to hydroquinone was within 1.0% for 6 successive measurements, indicating the good repeatability of the biosensor. As shown in Fig. 8, through a week of storage in 0.1 M pH 5.0 acetate buffer solution at 4 °C, the response current of biosensor remained almost stable. Even after 30 days, the response current retained 93.1% of the initial value, indicating the stable Lac activity in the composite of the Nafion–Lac–ZnO/CNF. Thus the biosensor demonstrated its good stability.

Fig. 8 Storage stability of the Nafion–Lac–ZnO/CNF modified GCE in 0.1 M acetate buffer solution (pH 5.0) at 4 °C.

3.7. Selectivity study and real sample analysis

Selectivity is an important characteristic of biosensors. Herein, several sorts of phenolic compounds, including vanillin, guaiacol, 3,5-dinitrosalicylic acid and phenol, were used to study the selectivity of the biosensor via SWV. The current response was separately examined by the biosensor in 1.1 μM hydroquinone solution with the interferents in a 1 : 1 ratio. By recording the current values for hydroquinone before and after adding each interferent respectively, it was found that the contribution of all these phenolic compounds toward the biosensor response for hydroquinone was ≤3% (as shown in Fig. 9), indicating negligible interference, which may be attributed to the known anti-interference effect of Nafion.⁴⁸ So the biosensor exhibited satisfactory selectivity for hydroquinone.

Fig. 9 Selectivity of the Nafion–Lac–ZnO/CNFs modified GCE in 0.1 M acetate buffer solution (pH 5.0) containing different phenolic compounds.

To investigate the practical application of the biosensor, the response of the biosensor in real water samples was investigated. The water sample was Taihu Lake water from Taihu Lake without any treatment. Here, a recovery experiment was adopted and the experiment was repeated five times. The analytical results are illustrated in Table 2. The recoveries looked satisfactory, confirming that the new biosensor can be applied in trace detection of hydroquinone existing in a real water environment.

Table 2 Determination of hydroquinone in real water samples (n = 5)

Sample^a	Cadded (μM)	Cfound (μM)	Recovery (%)	RSD (%)
a 1: Taihu lake water.
1	1.10	1.04	94.50	3.20
		1.08	98.20
		1.13	102.70
		1.07	97.30
		1.11	100.90

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4. Conclusions

In summary, the ZnO/CNFs were prepared by a combination of electrospinning, carbonization and a hydrothermal process. After that, a novel biosensor was fabricated using the ZnO/CNFs, Lac, Nafion and GCE. The electrochemical experiments indicated that the ZnO/CNFs facilitated the DET between the active center of Lac and the surface of the GCE. The Nafion–Lac–ZnO/CNF modified GCE showed highly efficient catalysis towards hydroquinone. The novel biosensor displayed high sensitivity, a low detection limit as well as good reproducibility, repeatability and stability. Finally, hydroquinone existing in real water samples was successfully detected using the biosensor. This study demonstrates that ZnO/CNFs are good candidates for constructing a highly sensitive hydroquinone biosensor.

Acknowledgements

This research was financially supported by the National High-tech R&D Program of China (2012AA030313), Changjiang Scholars and Innovative Research Team in University (IRT1135), National Natural Science Foundation of China (51203064, 21201083 and 51163014), the Priority Academic Program Development of Jiangsu Higher Education Institutions, Industry-Academia-Research Joint Innovation Fund of Jiangsu Province (BY2012068), Science and Technology Support Program of Jiangsu Province (SBE201201094), Industry-Academia-Research Prospective Joint Research Program of Jiangsu Province (SBY201220335), the Innovation Program for Graduate Education in Jiangsu Province (CXZZ13_07) and Scientific Research Foundation Program for PhD in Jiangnan University (JUDCF13022). In addition, the State Scholarship Fund from China Scholarship Council is cheerfully acknowledged.

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