Sol-Gel Synthesis of Carbon Xerogel-ZnO

materials Article

Sol-Gel Synthesis of Carbon Xerogel-ZnO Composite for Detection of Catechol Dawei Li 1,† , Jun Zang 2,† , Jin Zhang 1 , Kelong Ao 1 , Qingqing Wang 1 , Quanfeng Dong 2 and Qufu Wei 1, * 1

2

* †

Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, Wuxi 214122, China; [email protected] (D.L.); [email protected] (J.Z.); [email protected] (K.A.); [email protected] (Q.W.) State Key Laboratory for Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, and Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China; [email protected] (J.Z.); [email protected] (Q.D.) Correspondence: [email protected]; Tel.: +86-510-8591-2007; Fax: +86-510-8591-3100 These authors contributed equally to this work.

Academic Editor: Javier Narciso Received: 19 February 2016; Accepted: 8 April 2016; Published: 12 April 2016

Abstract: Carbon xerogel-zinc oxide (CXZnO) composites were synthesized by a simple method of sol-gel condensation polymerization of formaldehyde and resorcinol solution containing zinc salt followed by drying and thermal treatment. ZnO nanoparticles were observed to be evenly dispersed on the surfaces of the carbon xerogel microspheres. The as-prepared CXZnO composites were mixed with laccase (Lac) and Nafion to obtain a mixture solution, which was further modified on an electrode surface to construct a novel biosensing platform. Finally, the prepared electrochemical biosensor was employed to detect the environmental pollutant, catechol. The analysis result was satisfactory, the sensor showed excellent electrocatalysis towards catechol with high sensitivity (31.2 µA¨ mM´1 ), a low detection limit (2.17 µM), and a wide linear range (6.91–453 µM). Moreover, the biosensor also displayed favorable repeatability, reproducibility, selectivity, and stability besides being successfully used in the trace detection of catechol existing in lake water environments. Keywords: carbon xerogel; ZnO; laccase; phenolic biosensor

1. Introduction A biosensor, as a type of analytical tool, can be employed to detect analyte existing in various environments. It consists of a biological recognition component and a physicochemical transduction device [1]. Since Leland C. Clark invented enzyme based electrodes in 1962, enzyme based biosensors have attracted a great deal of attention from scientists and researchers [2–4]. Due to the great advantages of enzyme biosensors over conventional analytical techniques, such as low price, high sensitive/selective, rapid response, and amenable miniaturization, etc., they have been applied in multifarious fields, such as clinical medicine, environment monitoring, and food safety, as well as homeland security [5,6]. Laccase (Lac) is a multicopper oxidase which can catalyze phenolic compounds to give their oxidation form accompanied by reduction of molecular oxygen [7]. As a consequence, numerous Lac based biosensors have been prepared to detect phenols in tea infusions and wines, as well as in watery environments [8–10]. In order to improve the sensitivity and selectivity of biosensors, a variety of conductive materials or conductive nanomaterials have been added to the biosensing system, which mainly contain metal nanoparticles, metal oxide nanoparticles, carbon materials, and conductive polymers. Among these materials, carbon materials, including carbon black, carbon nanotube, graphene, carbon nanofiber,

Materials 2016, 9, 282; doi:10.3390/ma9040282

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mesoporous carbon, etc., are commonly used due to their low cost, outstanding electron transfer ability, good chemical stability, and biocompatibility [11]. Specifically as a type of porous carbon material, carbon xerogel (CX) harbors many merits, e.g., low mass density, large surface area, and excellent electrical conductivity. On the basis of these advantages, CX has been widely applied in adsorbents, catalyst supports, and electrode materials for supercapacitors and rechargeable lithium-ion batteries. Only a few literature examples have reported biosensors utilizing CX which possesses a huge application potential in biosensors. Zinc oxide (ZnO), as an admirable semiconductor material, has attracted wide attention in various application fields, such as piezoelectric devices, sensors, transparent electronics, optics, optoelectronics, and actuators [12,13]. The satisfactory electron conduction ability, good biocompatibility, and chemical stability renders ZnO to be an outstanding modification material in biosensors [14]. Besides, the isoelectric point (IEP) of ZnO is about 9.5, which is very favorable for adsorption of proteins with low IEP [15]. However, direct modification of ZnO nanoparticles on electrodes usually leads to their aggregation, which considerably restricts their electrocatalysis and electrochemical performance. Combination of ZnO nanoparticles with conductive substrate materials can effectively solve this problem. In this work, we synthesized carbon xerogel-zinc oxide (CXZnO) composites through a simple method of sol-gel condensation polymerization of formaldehyde and resorcinol solution containing zinc salt followed by drying and thermal treatment. ZnO nanoparticles were evenly dispersed on the surfaces of the carbon xerogel microspheres in the final products. The as-prepared CXZnO composites were further employed to modify the electrode with Lac and Nafion to construct a novel biosensing platform. The obtained biosensor showed excellent bio-electrocatalysis towards the phenolic compound catechol, with high sensitivity, low detection limit and a wide linear range. Moreover, the sensor demonstrated its practical application potential by detecting catechol existing in real lake water with satisfactory recovery. Our study expands the application of carbon xerogel in the biosensing field and offers theoretical support for exploiting high-efficient enzyme based biosensors. 2. Materials and Methods 2.1. Chemicals and Reagents Laccase (Lac, enzyme activity ě10 U/mg) from Trametes versicolor was purchased from Sigma-Aldrich. Nafion (5% w/w) was obtained from Shanghai Branch, Du Pont China Holding Co., Ltd. (Shanghai, China). Catechol was purchased from Shanghai Aladdin Chemical Reagent Company (Shanghai, China). Zinc acetate dihydrate (C4 H6 O4 Zn¨ 2H2 O), formaldehyde, resorcinol, and other chemicals were purchased from the Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). All of the chemicals were of analytical grade and used without further purification. In addition, 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 TGA measurement was conducted using a Mettler Toledo analyzer in an air atmosphere, the temperature range was from ambient temperature to 800 ˝ C with a heating rate of 10 ˝ C/min. The chemical components of CXZnO composites were analyzed by a Powder D8 Advance X-ray diffraction (XRD, Bruker AXS D8, Coventry, UK). The nitrogen absorption and desorption isotherms of CXZnO composites at 77 K were measured by using a TriStar 3020 surface area and pore analyzer (Micromeritics, America). The morphologies of CXZnO composites were observed by using a field emission scanning electron microscope (FE-SEM, Hitachi S-4800, Tokyo, Japan) and a high-resolution transmission electron microscope (TEM, JEOL/JEM-2100, Tokyo, Japan). Prior to scanning under the FE-SEM, the samples were sputter coated with gold for 90 s to avoid charge accumulations. Electrochemical experiments were conducted at room temperature using a CHI 660E electrochemical workstation (CH Instruments, Inc., Shanghai, China). A three-electrode cell with a glass carbon

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electrode (GCE) (3.0 mm in diameter, purchased from Gaoss Union Technology Co., Ltd., Wuhan, China), platinum wire (GCE) auxiliary electrode, and an Ag/AgCl reference electrode were used glass acarbon electrode (3.0 mm in diameter, purchased from Gaoss Union Technology Co., for Ltd., Wuhan, measurements. China), a platinum wire auxiliary solution electrode,was andbubbled an Ag/AgCl reference electrode were for electrochemical The electrolyte with highly pure nitrogen usedbefore for electrochemical measurements. solution was bubbled with highly pure 15 min electrochemical experimentsThe andelectrolyte a nitrogen atmosphere was kept over the solution nitrogen the for 15 min beforeexcept electrochemical experiments and a nitrogen atmosphere was kept over throughout experiments for the amperometric experiments. the solution throughout the experiments except for the amperometric experiments.

2.3. Synthesis of CXZnO Composites 2.3. Synthesis of CXZnO Composites

The synthesis procedure of CXZnO composites can be described as follows: 1.375 g of resorcinol procedure of CXZnO composites can be described as follows: 1.375 g of resorcinol and 2.23The g synthesis of C4 H6 O 4 Zn¨ 2H2 O were dissolved in 10 mL of DIW with the aid of stirring and and 2.23 g of C4H6O4Zn·2H2O were dissolved in 10 mL of DIW with the aid of stirring and ultrasonication followed by adding 2.375 mL of formaldehyde into the formed solution. After fast ultrasonication followed by adding 2.375 mL of formaldehyde into the formed solution. After fast ˝ stirring for a while, the solution was sealed by plastic wrap and placed in an oven at 76 C. stirring for a while, the solution was sealed by plastic wrap and placed in an oven at 76 °C. Through Through a sol-gel reaction for three days, the plastic wrap was poked to dry the composite gel. a sol-gel reaction for three days, the plastic wrap was poked to dry the composite gel. Eventually, the Eventually, gel awas into a high tube furnace it in a N2 dried gel the wasdried put into highput temperature tubetemperature furnace to carbonize it in atoNcarbonize 2 atmosphere. The ˝ C/min, maintaining 300 ˝ C and 800 ˝ C for 1 h and 2 h, respectively, atmosphere. The heating rate was 5 heating rate was 5 °C/min, maintaining 300 °C and 800 °C for 1 h and 2 h, respectively, and cooling anddown cooling downtemperature. to room temperature. The final products were solely CXZnOand composites, and the to room The final products were solely CXZnO composites, the composites composites were further ground to powders for the following experiments. For comparison, ZnO were further ground to powders for the following experiments. For comparison, ZnO powders were powders were by directly with thetreatment same thermal treatment process. prepared byprepared directly heating C4H6heating O4Zn·2HC24OHwith the 2H same process. 6 O4 Zn¨ 2 Othermal 2.4. 2.4. Preparation of Biosensors Preparation of Biosensors preparation procedures of biosensors described as follows: of CXZnO TheThe preparation procedures of biosensors areare described as follows: 1.5 1.5 mgmg of CXZnO waswas added added into actetate buffer of pH = 5.0 and with the aid of ultrasonication stirring, a CXZnO into actetate buffer of pH = 5.0 and with the aid of ultrasonication stirring, a CXZnO suspension was suspension was obtain. Subsequently, 15 µL mg of ofNafion Lac andsolution 75 µL of solution (5 wt%) were obtain. Subsequently, 15 mg of Lac and 75 (5Nafion wt %) were added to the above added to the above CXZnO suspension, whichwas the achieved. final mixture solution CXZnO suspension, by which the final mixtureby solution Eventually, 10 was µL ofachieved. the mixture 10 µL of the mixture wasglass dropped onto a freshly(GCE) polished glass to carbon electrode (GCE) wasEventually, dropped onto a freshly polished carbon electrode surface fabricate the biosensor, surface to fabricate the biosensor, and the dried modified GCE was named GCE/Lac-CXZnO-Nafion, ˝ and the dried modified GCE was named GCE/Lac-CXZnO-Nafion, which was stored at 4 C for use. which was stored at 4 °C for use. GCE/Lac-Nafion and GCE/Lac-ZnO-Nafion modified electrodes were fabricated by a similar GCE/Lac-Nafion and GCE/Lac-ZnO-Nafion modified electrodes were fabricated by a similar methods with the same amount of Lac for comparison experiments. All the modified electrodes were methods with the same amount of Lac for comparison experiments. All the modified electrodes were immersed into a buffer for 30 min to remove impurities before electrochemical measurements. immersed into a buffer for 30 min to remove impurities before electrochemical measurements.

3. Results and Discussion

3. Results and Discussion

3.1. Characterization of CXZnO Composites

3.1. Characterization of CXZnO Composites

Thermogravimetric analysis wasemployed employedtoto determine contents Thermogravimetric analysis(TGA) (TGA)characterization characterization was determine thethe contents of of carbon xerogel in CXZnO composites. The result is displayed in Figure 1, which shows the weight carbon xerogel in CXZnO composites. The result is displayed in Figure 1, which shows the weightloss of the composites terminated at around 640 ˝ C.640 The°C. weight loss below 640 ˝ C640 can°Cbecan ascribed lossCXZnO of the CXZnO composites terminated at around The weight loss below be solely to carbon, the hence, contentthe ofcontent ZnO inofthe composites can be assessed in Figure 1, which ascribed solelyhence, to carbon, ZnO in the composites can be assessed in Figure 1, is about 40%. which is about 40%.

Figure Thermogravimetric analysis analysis (TGA) Figure 1. 1. Thermogravimetric (TGA)curve curveofofCXZnO CXZnOcomposites. composites.

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X-ray diffraction (XRD) analysis was used to investigate the chemical ingredients and crystal X-ray diffraction (XRD) analysis was used to investigate the chemical ingredients and crystal structures of the CXZnO composites, and the result is shown in Figure 2A. The diffraction peak structures of the CXZnO composites, and the result is shown in Figure 2A. The diffraction peak occurring at about 24° is attributed to the {0 0 2} plane of graphite carbon [16]. In addition, the peaks occurring at about 24˝ is attributed to the {0 0 2} plane of graphite carbon [16]. In addition, the peaks at ca. 31.8°, 34.4°, 36.3°, 47.5°, 56.6°, 62.9°, 66.3°, 67.9°, and 69.1° correspond to the {1 0 0}, {0 0 2}, {1 0 1}, at ca. 31.8˝ , 34.4˝ , 36.3˝ , 47.5˝ , 56.6˝ , 62.9˝ , 66.3˝ , 67.9˝ , and 69.1˝ correspond to the {1 0 0}, {0 0 2}, {1 0 2}, {1 1 0}, {1 0 3}, {2 0 0}, {1 1 2} and {2 0 1} crystalline planes of zinc oxide, respectively [17]. The {1 0 1}, {1 0 2}, {1 1 0}, {1 0 3}, {2 0 0}, {1 1 2} and {2 0 1} crystalline planes of zinc oxide, respectively [17]. XRD result demonstrates that the CXZnO composite was successfully synthesized. The XRD result demonstrates that the CXZnO composite was successfully synthesized. Figure 2B displays the nitrogen adsorption-desorption isotherm of CXZnO composites. Figure 2B displays the nitrogen adsorption-desorption isotherm of CXZnO composites. According to the BDDT classification [18], the isotherm belongs to a combination of type I and type II According to the BDDT classification [18], the isotherm belongs to a combination of type I and of micro-mesoporous materials. Meanwhile, the specific surface area of CXZnO composites was type II of micro-mesoporous materials. Meanwhile, the specific surface area of CXZnO composites tested to be 340.5 m2/g with a pore volume of 0.161 cm3/g, and the average pore size was about 2.2 nm was tested to be 340.5 m2 /g with a pore volume of 0.161 cm3 /g, and the average pore size was about through the Barrett-Joyner-Halenda (BJH) model (Figure 2C). The large specific surface area and 2.2 nm through the Barrett-Joyner-Halenda (BJH) model (Figure 2C). The large specific surface area and special micro-mesoporous structure were both favorable for the immobilization of biological special micro-mesoporous structure were both favorable for the immobilization of biological enzyme enzyme protein, leading to high bioelectrocatalytic properties. protein, leading to high bioelectrocatalytic properties.

Figure Figure2.2.X-ray X-raydiffraction diffraction(XRD) (XRD)pattern pattern(A); (A);nitrogen nitrogenadsorption-desorption adsorption-desorptionisotherm isotherm(B); (B);and andpore pore size sizedistribution distributioncurve curve(C) (C)ofofCXZnO CXZnOcomposites. composites.

Figure 3A 3Ashows showsthe theSEM SEMmorphology morphologyimage imageofofCXZnO CXZnOcomposites. composites. A A large large number number ofof Figure microspheres were gathered together, some of which formed a chain-like structure. The average microspheres were gathered together, some of which formed a chain-like structure. The average diameter of these microspheres was about 1.8 µm. It can be seen from the enlarged SEM image diameter of these microspheres was about 1.8 µm. It can be seen from the enlarged SEM image ofof CXZnOcomposites composites(Figure (Figure3B) 3B)that thatmost mostmicrospheres microspherespossessed possessedsmooth smoothsurfaces surfaceswhile whilethere therewere were CXZnO also some nanoparticle loaded microspheres, which may be the CXZnO composite microspheres. also some nanoparticle loaded microspheres, which may be the CXZnO composite microspheres. Figure3C,D 3C and Figurethe 3DTEM display theofTEM image of CXZnOit composites, it observed can be clearly observed Figure displays image CXZnO composites, can be clearly that there are that there are two kinds of morphologies, one shows smooth surfaces with no nanoparticles, and the two kinds of morphologies, one shows smooth surfaces with no nanoparticles, and the other has rough other has rough surfaces with evenly distributed nanoparticles. The average diameter of these surfaces with evenly distributed nanoparticles. The average diameter of these nanoparticles is around nanoparticles is around 103uniform nm. Sodistribution the smallofsizes andnanoparticles uniform distribution of the ZnO 103 nm. So the small sizes and the ZnO were both favorable to nanoparticles were both favorable to perform their electrocatalytic activities. Meanwhile, the perform their electrocatalytic activities. Meanwhile, the nanoparticles also offered abundant active nanoparticles also offered abundant active sites for Lac immobilization. sites for Lac immobilization.


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Figure 3. Scanning electon microscopy (SEM) topographies of CXZnO composites (A); partial

Figure 3. Scanning electon microscopy (SEM) topographies of CXZnO composites (A); partial enlarged enlarged drawing of SEM image of CXZnO composites (B); transmission electron microscopy (TEM) drawingimages of SEM imagexerogel of CXZnO composites (B); transmission electron microscopy (TEM) images of of carbon (C) and CXZnO composites (D). carbon xerogel (C) and CXZnO composites (D). 3.2. Electrochemical Studies of Modified Electrodes

3.2. Electrochemical Studies Impedance of ModifiedSpectroscopy Electrodes (EIS) characterization was utilized to compare the Electrochemical interface resistances of different modified electrodes, the result of the Nyquist plot of impedance is

Electrochemical Impedance Spectroscopy (EIS) characterization was utilized to compare the shown in Figure 4A. The values of charge transfer resistance (Rct) of modified electrodes are interface resistances of electrodes, the result of the Nyquist plotalmost of impedance proportional to thedifferent diametersmodified of semicircles. Apparently, bare electrode (GCE) shows a is shown in Figure 4A. Thethe values of charge transfer resistance (Rct) of modified are straight line, indicating negligible Rct value. However, an obvious semicircle occurredelectrodes on the curve for GCE/Lac-Nafion, Rct value isApparently, about 227 Ω, suggesting that the interface resistance was proportional to the diameters ofthe semicircles. bare electrode (GCE) shows almost a straight increased by immobilization process of Lac. In addition, the Rct semicircle value of GCE/Lac-ZnO-Nafion line, indicating thethe negligible Rct value. However, an obvious occurred on the curve was almost equal to that of GCE/Lac-Nafion, implying that the addition of ZnO did not decrease the for GCE/Lac-Nafion, the Rct value is about 227 Ω, suggesting that the interface resistance was interface resistance of the modified electrode. This may be explained by the fact that ZnO is a type increased by the immobilization process of Lac. similar In addition, Rct value of GCE/Lac-ZnO-Nafion of semiconductor material, which may possess electronthe conductive ability to Lac. While, the was almost equaldiameter to that of implying that the addition of ZnO not of decrease semicircle of GCE/Lac-Nafion, the curve for GCE/Lac-CXZnO-Nafion was smaller thandid those GCE/Lac-Nafion the RctThis valuemay was be also decreased by to the 145 Ω. the interface resistanceand of GCE/Lac-ZnO-Nafion, the modified electrode. explained factThis that ZnO demonstrated that the CXZnO composites accelerated the electron transfer therebyconductive weakening the is a type of semiconductor material, which may possess similar electron ability to interface resistance of the modified electrode hence revealing the excellent electron conductivity of Lac. While, the semicircle diameter of the curve for GCE/Lac-CXZnO-Nafion was smaller than carbon xerogel. those of GCE/Lac-Nafion and GCE/Lac-ZnO-Nafion, the Rct value was also decreased to 145 Ω. The electrocatalytic properties of four modified electrodes, including bare GCE, This demonstrated thatGCE/Lac-ZnO-Nafion, the CXZnO composites accelerated the electron transfer thereby GCE/Lac-Nafion, and GCE/Lac-CXZnO-Nafion, were compared usingweakening the the interface of theofmodified electrode hence the excellent electron conductivity of cyclic resistance voltammograms these electrodes in pH = 5.0revealing acetate buffer solution containing 100 µM catechol, the result is shown in Figure 4B. These electrodes all show a pair of distinct redox peaks, carbon xerogel. can be ascribed to the redox electrochemical reaction of catechol occurring on the electrode Thewhich electrocatalytic properties of four modified electrodes, including bare GCE, GCE/Lac-Nafion, surface. It was observed that bare GCE possessed the smallest oxidation and reduction peak current GCE/Lac-ZnO-Nafion, and GCE/Lac-CXZnO-Nafion, were compared using the cyclic voltammograms values, indicating the poor electrocatalytic activity of bare GCE. GCE/Lac-Nafion presented higher of thesepeak electrodes in pH = 5.0 acetate buffer solution containing 100 µM catechol, the result is shown in current values as compared to bare GCE, the oxidation peak current value reached to 12 µA, Figure 4B. electrodes showvalue a pair ofca. distinct redox cantobetheascribed to the redox andThese the reduction peakall current was 11.67 µA. Thispeaks, can be which attributed high-efficient electrochemical reaction of catechol on thevalues electrode surface. It was observed that bare GCE catalysis of Lac toward catechol. occurring The peak current for GCE/Lac-ZnO-Nafion were decreased to some extent, which suggested the aggregated ZnO particles impair the possessed the smallest oxidation and that reduction peak current values,may indicating theelectrocatalytic poor electrocatalytic activity of the modified electrode. It is noticeable that GCE/Lac-CXZnO-Nafion shows the largest to bare activity of bare GCE. GCE/Lac-Nafion presented higher peak current values as compared redox peak current values, which were increased to 14.68 µA and 14.31 µA, respectively. This GCE, the oxidation peak current value reached to 12 µA, and the reduction peak current value was demonstrated that the CXZnO composites can enhance the electrocatalytic properties of the ca. 11.67modified µA. This can be maybe attributed to thetohigh-efficient catalysisofofCXZnO Lac toward catechol. The peak electrode, attributing the good conductivity composites and the current values for GCE/Lac-ZnO-Nafion were decreased to some extent, which suggested that the aggregated ZnO particles may impair the electrocatalytic activity of the modified electrode. It is noticeable that GCE/Lac-CXZnO-Nafion shows the largest redox peak current values, which were increased to 14.68 µA and 14.31 µA, respectively. This demonstrated that the CXZnO composites can enhance the electrocatalytic properties of the modified electrode, maybe attributing to the good conductivity of CXZnO composites and the synergetic catalysis of ZnO nanoparticles. The whole


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synergetic catalysis of ZnO nanoparticles. The whole electrochemical reaction was a quasi-reversible cyclic process and the sensing mechanism of Lac on GCE/Lac-CXZnO-Nafion is illustrated in Materials 2016, 9, 282 6 of 11 Scheme 1. Under the presence of molecular oxygen, the catechol was oxidized to 1,2-benzoquinone by Lac, electrochemical coupled withreaction the electrocatalytic reduction oxygen the surface was a quasi-reversible cyclic of process and to the water sensingon mechanism of LacofonGCE. The reactionGCE/Lac-CXZnO-Nafion process can be described as follows: is illustrated in Scheme 1. Under the presence of molecular oxygen, the catechol was oxidized to 1,2-benzoquinone by Lac, coupled with the electrocatalytic reduction of + + 2e− Catechol Lac(oxy) → The 1,2-Benzoquinone + Lac(deoxy) + 2H oxygen to water on the +surface of GCE. reaction process can be described as follows:

Lac(deoxy) + O2 + 4H+ → Lac(oxy) + 2H2O Catechol ` Lacpoxyq Ñ 1, 2-Benzoquinone ` Lacpdeoxyq ` 2H+ ` 2e´ Lacpdeoxyq ` O2 ` 4H+ Ñ Lacpoxyq ` 2H2 O

(1)

(1)

Figure 4. (A) Electrochemical impedance spectroscopy (EIS) of modified electrodes: bare GCE,

Figure GCE/Lac-Nafion, 4. (A) Electrochemical impedance (EIS) of inmodified electrodes: bare GCE, GCE/Lac-ZnO-Nafion, andspectroscopy GCE/Lac-CXZnO-Nafion 0.1 M KCl containing 5 mM 3´/4´ GCE/Lac-Nafion, GCE/Lac-ZnO-Nafion, and GCE/Lac-CXZnO-Nafion 0.1 voltammograms M KCl containing 5 mM Fe(CN)6 . Frequency range: 0.01 Hz–100,000 Hz. Amplitude: 5 mV; (B) in Cyclic of 3−/4− (a) bare GCE; (b) GCE/Lac-Nafion; (c) GCE/Lac-ZnO-Nafion; and (d) GCE/Lac-CXZnO-Nafion in Fe(CN)6 . Frequency range: 0.01 Hz–100,000 Hz. Amplitude: 5 mV; (B) Cyclic voltammograms of pH 5.0 acetate buffer solution containing 100 µM catechol at 100 mV/s; (C) Cyclic voltammograms of (a) bare GCE; (b) GCE/Lac-Nafion; (c) GCE/Lac-ZnO-Nafion; and (d) GCE/Lac-CXZnO-Nafion in pH GCE/Lac-CXZnO-Nafion in pH = 5.0 acetate buffer solution containing 100 µM catechol at scan rates 5.0 acetate buffer solution containing 100 µM catechol at 100 mV/s; (C) Cyclic voltammograms of of 50, 100, 120, 150, 200, 250, and 300 mV/s (a–f), respectively. Inset: Plots of the corresponding anodic GCE/Lac-CXZnO-Nafion in pHvs. = 5.0 solution of containing 100 µM catechol at scan rates and cathodic peak currents scanacetate rate; (D)buffer Electrocatalysis GCE/Lac-CXZnO-Nafion towards catechol in pH = 5.0 acetate buffer solution with scan rate 100 mV/s. Catechol concentrations (µM): of 50, 100, 120, 150, 200, 250, and 300 mV/s (a–f), respectively. Inset: Plots of the corresponding anodic (a) 100; (b) 200;currents (c) 300. vs. scan rate; (D) Electrocatalysis of GCE/Lac-CXZnO-Nafion towards and cathodic peak catechol in pH = 5.0 acetate buffer solution with scan rate 100 mV/s. Catechol concentrations (µM): Figure 4C shows the influence of scan rates on the cyclic voltammograms of (a) 100; (b) 200; (c) 300.

GCE/Lac-CXZnO-Nafion. As the scan rates grew from 50 mV/s to 300 mV/s, both the anodic peak and cathodic peak current values increased. It can be seen from the inset of Figure 4C, the peak current Figure 4C shows the rates on to the cyclic voltammograms of values enhanced linearly withinfluence the scan ratesofandscan were proportional the scan rates. This indicated GCE/Lac-CXZnO-Nafion. Asconduction the scan rates grew 50 mV/s to 300 mV/s, both the anodic peak that the electrochemical occurring on from the electrode surface was a surface-controlled electrochemical reaction process. Figure 4D displays the electrocatalysis of GCE/Lac-CXZnO-Nafion and cathodic peak current values increased. It can be seen from the inset of Figure 4C, the peak with different concentrations. Obviously, the current values of redox peaks increased current towards values catechol enhanced linearly with the scan rates and were proportional to the scan rates. This with the increment of substrate (catechol) concentration, implying that GCE/Lac-CXZnO-Nafion indicated that the electrochemical conduction occurring on the electrode surface was a possessed exceptionally good electrocatalytic properties toward catechol and can be applied in a surface-controlled electrochemical catechol enzyme-based biosensor. reaction process. Figure 4D displays the electrocatalysis of

GCE/Lac-CXZnO-Nafion towards catechol with different concentrations. Obviously, the current values of redox peaks increased with the increment of substrate (catechol) concentration, implying that GCE/Lac-CXZnO-Nafion possessed exceptionally good electrocatalytic properties toward catechol and can be applied in a catechol enzyme-based biosensor.


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Scheme 1. Schematic illustration of the catalyzed oxidation of catechol by laccase on the electrode surface.

3.3. Analytical Performance for Detecting Catechol

Scheme 1.Scheme Schematic illustration catalyzed oxidation catechol byelectrode laccase on the 1. Schematic illustration ofofthethe catalyzed oxidation of catechol of by laccase on the electrode surface. surface.

Chrono-amperometry was employed to investigate the analytical performance of the 3.3. Analytical Performance for Detecting CatecholTo acquire the optimal current response and the as-prepared3.3. biosensor for detecting catechol. Analytical Performance for Detecting Catechol Chrono-amperometry was employed to investigate the analytical performance of the highest sensitivity of the biosensor, before the amperometric tests,performance some parameters like solution pH Chrono-amperometry was to investigate thethe analytical thethe as-prepared as-prepared biosensor foremployed detecting catechol. To acquire optimal current responseofand and appliedbiosensor work highest voltage were Asamperometric shown in some Figure 5, optimal pH and applied for detecting catechol. To acquire the optimal current response andthe thesolution highest sensitivity of theoptimized. biosensor, before the tests, parameters like pHsensitivity and applied work voltage were optimized. As shown in Figure 5,like the solution optimal pH and applied of the biosensor, before the amperometric tests, some parameters pH and applied potential were pH 5.0 and 0.5 V, respectively, which were fixed in work the following potential were pH 5.0 and 0.5 V, respectively, which were fixed in the following voltage were optimized. As shown in Figure 5, the optimal pH and applied potential were pH 5.0 and chrono-amperometry tests. chrono-amperometry tests. 0.5 V, respectively, which were fixed in the following chrono-amperometry tests.

Figure 5. (A) The pH effects; and (B) applied potentials of GCE/Lac-CXZnO-Nafion on the catalytic currents of catechol in 0.1 M acetate buffer solution containing 100 µM catechol.

Figure 6A depicts the typical current-time response curve of the GCE/Lac-CXZnO-Nafion upon successive additions of catechol into pH = 5.0 acetate buffer solution with 0.5 V of applied potential. Herein, two concentrations of catechol solution (2 mM and 20 mM) were successively added into the acetate buffer solution. It can be clearly seen that once the 100 µM of catechol was added, the response current increased instantly, the time for the current value reaching 95% of the next Figure 5.pH (A) The pH effects; and (B) applied potentials of of GCE/Lac-CXZnO-Nafion on the catalytic Figure 5. (A) The effects; (B) applied maximum responseand current value was only 3potentials s, indicating a fast GCE/Lac-CXZnO-Nafion response of biosensor, which may be on the catalytic currents of catechol in 0.1 M acetate buffer solution containing 100 µM catechol. film. Figure 6B attributed to the easy diffusion of catechol in the Lac-CXZnO-Nafion currents of catechol in 0.1 M acetate buffer solution containing 100composite µM catechol. shows the calibration curve of response currents vs. catechol concentrations. The current values increased linearly with the ascent of catechol response concentration. The of linear was from 6.91 µM to Figure 6A depicts the typical current-time curve therange GCE/Lac-CXZnO-Nafion upon 453 µM with a correlation coefficient (R2) of 0.983 (n = 13). The sensitivity was 31.2 µA/mM and the successive additions of catechol into pH = 5.0 acetate buffer solution with 0.5 V of applied potential. detection limit was estimated to be 2.17 µM at a signal-to-noise of 3 (S/N = 3). Table 1 compares the Herein, two concentrations of catechol (2 mM and 20 Our mM) were successively added into the biosensing performance of differentsolution laccase based biosensors. biosensor showed satisfactory detection resultsIttoward catechol with lowthat detection high µM sensitivity and wide linear range.the response acetate buffer solution. can be clearly seen oncelimit, the 100 of catechol was added,

Figure 6A depicts the typical current-time response curve of the GCE/Lac-CXZnO-Nafion upon successive additions of catechol into pH = 5.0 acetate buffer solution with 0.5 V of applied potential. Herein, two concentrations of catechol solution (2 mM and 20 mM) were successively added into the current increased instantly, the time for the current value reaching 95% of the next maximum response acetate buffer solution. can clearly seen thatofonce thewhich 100 may µMbeof catechol was current value wasItonly 3 s,be indicating a fast response biosensor, attributed to the easy added, the diffusionincreased of catechol in instantly, the Lac-CXZnO-Nafion Figure 6Bvalue shows the calibration curveof the next response current the timecomposite for thefilm. current reaching 95% of response currents vs. catechol concentrations. The current values increased linearly with the ascent maximum response current value was only 3 s, indicating a fast response of biosensor, which may be of catechol concentration. The linear range was from 6.91 µM to 453 µM with a correlation coefficient attributed to easy(n diffusion of catechol in µA/mM the Lac-CXZnO-Nafion composite (R2the ) of 0.983 = 13). The sensitivity was 31.2 and the detection limit was estimated film. to be Figure 6B shows the calibration curve of response currents vs. catechol concentrations. The current values 2.17 µM at a signal-to-noise of 3 (S/N = 3). Table 1 compares the biosensing performance of different laccase based biosensors. Our biosensor showed satisfactory detection results toward catechol with increased linearly with the ascent of catechol concentration. The linear range was from 6.91 µM to low detection limit, high sensitivity and wide linear range. 453 µM with a correlation coefficient (R2) of 0.983 (n = 13). The sensitivity was 31.2 µA/mM and the detection limit was estimated to be 2.17 µM at a signal-to-noise of 3 (S/N = 3). Table 1 compares the biosensing performance of different laccase based biosensors. Our biosensor showed satisfactory detection results toward catechol with low detection limit, high sensitivity and wide linear range.

Materials 282 Materials 2016, 2016, 9, 9, 282 Materials 2016, 9, 282

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Figure 6. 6. (A) (A) Typical Typicalcurrent-time current-timeresponse responsecurve curveofofthe theGCE/Lac-CXZnO-Nafion GCE/Lac-CXZnO-Nafion upon upon successive successive Figure 6. (A) Typical current-time response curve of the GCE/Lac-CXZnO-Nafion upon successive catechol into pH pH == 5.0 acetate acetate buffer buffer solution, solution, applied applied potential: potential: 0.5 V; (B) (B) Calibration Calibration additions of catechol additions of catechol into pH = 5.0 acetate buffer solution, applied potential: 0.5 V; (B) Calibration curve of steady-state steady-state currents currents vs. vs. catechol catechol concentrations. concentrations. curve of steady-state currents vs. catechol concentrations. Table Table 1. 1. Biosensing Biosensing performance performance comparison comparison of of different different laccase laccasebased basedbiosensors biosensorstoward towardcatechol catecholaa.. Table 1. Biosensing performance comparison of different laccase based biosensors toward catechol a.

Detection Linear Range Sensitivity Sensitivity Detection Limit Limit Linear Range Detection Limit Linear Range Sensitivity Ref. Ref. (µM) (µM) (µA/mM)Ref. (µM) (µM) (µA/mM) (µM) (µM) (µA/mM) MB-MCM-41/PVA/lac 0.331 4–87.98 [19] MB-MCM-41/PVA/lac 0.331 4–87.98 –– [19] MB-MCM-41/PVA/lac 0.331 4–87.98 – [19] GCE/MCN/Tyr 0.01 0.05–12.5 – [20] GCE/MCN/Tyr 0.01 0.05–12.5 – [20] GCE/MCN/Tyr 0.01 0.05–12.5 – [20] Lac/APrGOs/Chit/GCE 7 15–700 15.79 [21] Lac/APrGOs/Chit/GCE 7 15–700 15.79 [21] Lac/AP- rGOs/Chit/GCE 7 15–700 15.79 [21] Lac-FSM7.0-GC 2–100 – [22] Lac-FSM7.0-GC 222 2–100 [22] Lac-FSM7.0-GC 2–100 –– [22] Cu-OMC/Lac/CS/Au 0.67 0.67–13.8 104 [23] Cu-OMC/Lac/CS/Au 0.67 0.67–13.8 104 [23] Cu-OMC/Lac/CS/Au 0.67 0.67–13.8 104 [23] GCE/Lac-CXZnO-Nafion 2.17 6.91–453 31.2 This work GCE/Lac-CXZnO-Nafion 2.17 6.91–453 31.2 This work GCE/Lac-CXZnO-Nafion 2.17 6.91–453 31.2 This work a The dashes in the table represent values that were not reported in the references. a The dashes in the table represent a The dashes in the table represent values that that were werenot notreported reportedininthe thereferences. references. Electrodes Electrodes Electrodes

The fabrication reproducibility of the GCE/Lac-CXZnO-Nafion was investigated by successive The fabricationreproducibility reproducibility of of the the GCE/Lac-CXZnO-Nafion GCE/Lac-CXZnO-Nafion was byby successive The fabrication wasinvestigated investigated successive detection of 100 µMµM catechol by six modified electrodes prepared in thein same way. The relative standard detection of 100 catechol by six modified electrodes prepared the same way. The relative detection of 100 µM catechol by six modified electrodes prepared in the same way. The relative deviation (RSD) was 3.2%, implying the acceptable reproducibility of the GCE/Lac-CXZnO-Nafion. standard deviation deviation (RSD) (RSD) was was 3.2%, 3.2%, implying implying the standard the acceptable acceptable reproducibility reproducibilityof ofthethe The RSD of the GCE/Lac-CXZnO-Nafion for 20 times of successive detection of 100 µM catechol was GCE/Lac-CXZnO-Nafion. The RSD RSD of of the the GCE/Lac-CXZnO-Nafion for GCE/Lac-CXZnO-Nafion. The GCE/Lac-CXZnO-Nafion for2020times timesof ofsuccessive successive 1.7%, indicating repeatability the GCE/Lac-CXZnO-Nafion. The repeatability selectivity experimental detection of excellent 100 µM catechol of was 1.7%, indicating excellent detection of 100 µM catechol was 1.7%, indicating excellent repeatability of ofthethe result is shown in Figure 7. The response of the GCE/Lac-CXZnO-Nafion for7.100 µMcurrent catechol GCE/Lac-CXZnO-Nafion. Thecurrent selectivity experimental result is shown in Figure The GCE/Lac-CXZnO-Nafion. The selectivity experimental result is shown in Figure 7. The current solution andof100 catechol solution containing 100 catechol µM interferents catechin, gallic response theµM GCE/Lac-CXZnO-Nafion for 100 µM solution (hydroquinone, and 100 µM catechol solution response of the GCE/Lac-CXZnO-Nafion for 100 µM catechol solution and 100 µM catechol solution acid, phenol, 100 andµM aminophenol) was measured,catechin, respectively. interferents almost containing interferents (hydroquinone, gallic Apparently, acid, phenol, these and aminophenol) was containing 100 µM interferents (hydroquinone, catechin, gallic acid, phenol, and aminophenol) was measured, these interferents almost indicating produced no on the current produced no respectively. effects on theApparently, current response of the biosensor, theeffects good selectivity of the measured, respectively. Apparently, these interferents almost produced no effects on the current response of the biosensor, indicating the good selectivity of the GCE/Lac-CXZnO-Nafion. GCE/Lac-CXZnO-Nafion. response of the biosensor, indicating the good selectivity of the GCE/Lac-CXZnO-Nafion.

Figure Selectivityofofthe theGCE/Lac-CXZnO-Nafion GCE/Lac-CXZnO-Nafion in containing Figure 7. 7. Selectivity in pH pH==5.0 5.0acetate acetatebuffer buffersolution solution containing different phenolic compounds. different phenolic compounds. Figure 7. Selectivity of the GCE/Lac-CXZnO-Nafion in pH = 5.0 acetate buffer solution containing different phenolic compounds.


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It that the the storage storage stability stability of of the the GCE/Lac-CXZnO-Nafion GCE/Lac-CXZnO-Nafion in in pH pH = = 5.0 It can can be be seen seen from from Figure Figure 88 that 5.0 ˝ acetate buffersolution solutionatat satisfactory. Furthermore, over one of month of the storage, the acetate buffer 4 4C °C waswas satisfactory. Furthermore, over one month storage, response response current value could retain of value, the original value,thesuggesting the excellent storage current value could retain 93.6% of the93.6% original suggesting excellent storage stability of the stability of the GCE/Lac-CXZnO-Nafion. GCE/Lac-CXZnO-Nafion.

˝ C. Figure 8. 8. Storage stability of the GCE/Lac-CXZnO-Nafion GCE/Lac-CXZnO-Nafion in Figure in pH pH == 5.0 5.0 acetate acetate buffer buffer solution solution at at 44 °C.

3.4. 3.4. Real Real Sample Sample Analysis Analysis To test the the practical To test practical application application of of the the biosensor, biosensor, we we conducted conducted recovery recovery experiments experiments using using real real lake water from Taihu Lake, Wuxi, China. An amount of 100 µM of catechol was added into the lake lake water from Taihu Lake, Wuxi, China. An amount of 100 µM of catechol was added into the lake water samples, which which was was named namedCCadded.. The determined content was denominated Cfound and the water samples, added The determined content was denominated Cfound and the result 2. It result is is shown shown in in Table Table 2. It is is seen seen that that the the recovery recovery was was very very close close to to 100%, 100%, and and the the RSD RSD was was only only 2.52%. This real sample analysis test demonstrated that the as-prepared biosensor can be 2.52%. This real sample analysis test demonstrated that the as-prepared biosensor can be successfully successfully applied the trace detectioninofa catechol in aenvironment. lake water environment. applied for the trace for detection of catechol lake water Table 2. Determination of catechol catechol content content in in real real water water samples samples (n (n = = 5). Table 2. Determination of 5). Sample Cadded (µM) Cfound (µM) Recovery (%) RSD (%) Sample 100.00 Cadded (µM) Cfound (µM) Recovery (%) RSD (%) 2.52 a 103.26 103.26 a 100.00 103.26 103.26 2.52 – 100.00 98.93 98.93 – – 100.00 98.93 98.9397.31 – – 100.00 97.31 – – 100.00 97.31 97.31 – – 100.00 102.47 102.47 – – 100.00 102.47 102.47 – – 100.00 101.95 101.95 – – 100.00 101.95 101.95 – a: Taihu Lake water. a: Taihu Lake water.

4. Conclusions 4. Conclusions In summary, a sol-gel condensation polymerization of formaldehyde and resorcinol solution In summary, sol-gel condensation polymerization formaldehyde andThe resorcinol solution containing zinc salta was used to prepare the precursors of of CXZnO composites. precursors were containing zinc salt was used to to obtain prepare the precursors of CXZnO composites. Thewas precursors were further dried and carbonized the CXZnO composites. A novel biosensor fabricated by further dried and carbonized to CXZnO obtain the CXZnO composites. A novel biosensor was fabricated modifying a mixture containing composites, Lac, and Nafion on the electrode surface. The by modifyingbiosensor a mixtureshowed containing composites, Lac, and Nafion on the electrode surface. as-prepared veryCXZnO good biological electrocatalysis towards catechol with high The as-prepared biosensorlimit, showed biological electrocatalysis towards catechol with high sensitivity, low detection andvery widegood linear range. Besides, the sensor was successfully used in sensitivity, lowof detection andinwide linear range. the sensor successfully in trace detection catechollimit, existing the real lake waterBesides, environment. Thiswas novel composite used further trace detection of catechol in the real lake water environment. This novel expands the application of existing carbon xerogel materials in the field of biosensing, andcomposite paves the further way to develop highly sensitive phenolic biosensors.


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expands the application of carbon xerogel materials in the field of biosensing, and paves the way to develop highly sensitive phenolic biosensors. Acknowledgments: This research was financially supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Six talent peaks project in Jiangsu Province (2014-XCL001), the Fundamental Research Funds for the Central Universities (JUSRP51505 and JUSRP51621A), and Scientific Research Foundation Program for PhD in Jiangnan University (JUDCF13022). Author Contributions: Dawei Li and Jun Zang conceived and designed the experiments; Dawei Li and Jun Zang performed the experiments; Jin Zhang and Kelong Ao analyzed the data; Qingqing Wang contributed materials and tools; Quanfeng Dong and Qufu Wei wrote the paper. Conflicts of Interest: The authors declare no conflicts of interest.

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