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cellulose nanofibrous mats and their biosensing application
Applied Surface Science 349 (2015) 35–42
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Fabrication of polyaniline/carboxymethyl cellulose/cellulose nanofibrous mats and their biosensing application Jiapeng Fu, Zengyuan Pang, Jie Yang, Fenglin Huang, Yibing Cai, Qufu Wei ∗ Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China
a r t i c l e
i n f o
Article history: Received 12 March 2015 Received in revised form 26 April 2015 Accepted 29 April 2015 Available online 6 May 2015 Keyword: Electrospinning Cellulose Polyaniline Laccase Biosensor Catechol
a b s t r a c t We report a facile approach to synthesizing and immobilizing polyaniline nanorods onto carboxymethyl cellulose (CMC)-modified cellulose nanofibers for their biosensing application. Firstly, the hierarchical PANI/CMC/cellulose nanofibers were fabricated by in situ polymerization of aniline on the CMC-modified cellulose nanofiber. Subsequently, the PANI/CMC/cellulose nanofibrous mat modified with laccase (Lac) was used as biosensor substrate material for the detection of catechol. PANI/CMC/cellulose nanofibers with highly conductive and three dimensional nanostructure were characterized by scanning electron microscopy (SEM), transmission electron microscope (TEM), Fourier transform infrared spectra (FT-IR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under optimum conditions, the Lac/PANI/CMC/cellulose/glassy carbon electrode (GCE) exhibited a fast response time (within 8 s), a linear response range from 0.497 M to 2.27 mM with a high sensitivity and low detection limit of 0.374 M (3). The developed biosensor also displayed good repeatability, reproducibility as well as selectivity. The results indicated that the composite mat has potential application in enzyme biosensors. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, electrochemical biosensors have attracted most of the attention in the detection of different analytes such as glucose [1–3], H2 O2 [4,5] and catechol [6–8] due to their simplicity, fast response, high selectivity and sensitivity. Most effort has been focused on developing suitable matrices used for the fabrication of electrochemical biosensors. Those matrices include metals [9,10], metals oxides [11,12], carbon-based materials [13,14] and polymers [15,16]. The performance of electrochemical biosensors largely relies on the physical and chemical properties of the electrode materials. For this purpose polyaniline (PANI) has aroused much interest to construct novel and improved biosensors due to its good electrical conductivity, good environmental stability, ease of synthesis [17–19]. Nevertheless, the biosensing performance of PANI-based bisoensors is unsatisfactory with a small interfacial area between PANI and biomolecules. With the mass loading increases, PANI has
∗ Corresponding author at: Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Tel.: +86 510 8591 3653. E-mail addresses: fi[email protected] (J. Fu), [email protected] (Z. Pang), [email protected] (J. Yang), fl[email protected] (F. Huang), [email protected] (Y. Cai), [email protected] (Q. Wei). http://dx.doi.org/10.1016/j.apsusc.2015.04.215 0169-4332/© 2015 Elsevier B.V. All rights reserved.
the tendency of aggregation and the decreased accessible surface area, resulting in poor electrical conductivity and increasing mass transfer limitation [20]. Therefore, the development of nanostructured PANI with increased surface-to-volume ratio, high electrical conductivity and reduced distance for electron and mass transfer is of great importance to obtain excellent biosensing performance. Meanwhile, the lightweight and portable structures are considered necessary to broaden their applications in the surrounding environment in real time [21]. An effective strategy to achieve this goal is to deposit PANI on the surface of flexible fibrous mats, which could overcome the difficulty of electrospinning PANI solution. The three dimensional nanostructure of electrospun fibrous mats provides space for the deposition of PANI and improves their interfacial area with immobilized enzyme. Electrospinning is an effective technique that produces continuous nanoscaled polymeric fiber with several remarkable characteristics such as high surface area to volume ratio, high porosity and flexibility. Meanwhile, electrospun nanofibrous mats are easily handled and retrieved. It is well documented that biocompatible electrospun polymer nanofibers, like polylatic acid [22] and polyvinyl alcohol [16,23], serve as matrix or template for the application of biosensors. However, the relatively hydrophobic property of polylactic acid is unfavourable to maintain the enzyme activity. Electrospun PVA nanofibers have poor water stability and become a clear gelation like material in water, limiting their practical applications [24]. Nanocellulose materials could overcome
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these problems and recently show promising results in biomolecule immobilization and biosensors [25,26]. Cellulose is one of most abundant biopolymers and possesses desirable properties such as hydrophilicity, biocompatibility and biodegradation. Besides, cellulose could not be dissolved in water and most common organic solvents because of its high crystallinity, which makes it difficult to fabricate cellulose nanofibers via direct electrospinning [27]. Alternatively, cellulose nanofibers have been produced by electrospinning of cellulose acetate and subsequent deacetylation [28,29]. PANI/cellulose conductive composites have been fabricated in heterogeneous reaction [30,31]. However, the accessibility and reactivity of the –OH group are limited by numerous intermolecular and intramolecular hydrogen bonds. It is necessary to overcome the problem to obtain PANI-modified composites with desirable conductivity and good bonding. Carboxymethyl cellulose (CMC), a derivative of cellulose, can adsorb irreversibly on cellulose fibers under suitable condition and increase the negative charges of cellulose fibers [32,33]. The –COO− groups of CMC provide cationic exchangeable sites to capture electropositive molecules via electrostatic attraction in aqueous solutions, facilitating the in situ polymerization of aniline. In this present study, we fabricated hierarchical PANI/CMC/cellulose nanofibers by in situ polymerization of aniline on the CMC-modified cellulose nanofibers, which were employed as substrate materials to construct laccase (Lac) biosensors for the detection of catechol. The obtained fibrous mats with hierarchical structure combine the flexibility and high surface area of CMC-modified cellulose nanofibers with excellent conductivity of PANI, facilitating the immobilization of Lac as well as enhancing the mass and electron transport. Compared with previous biosensors based on other materials, the fabricated Lac/PANI/CMC/cellulose/glassy carbon electrode (GCE) exhibited a highly sensitive detection toward catechol with a broad linear range and low detection limit. This study demonstrated that PANI/CMC/cellulose nanofibrous mats are the promising material in the design of high-efficient biosensors.
2. Material and methods 2.1. Materials Cellulose acetate (CA, 39.8 wt% of acetyl content, Mn = 30,000), N,N-dimethylacetamide (DMAc), acetone, carboxymethyl cellulose (CMC), aniline monomer, ammonium persulfate (APS), hydrochloric acid (HCl, the concentration of 37%), NaCl, CH3 COOH, CH3 COONa, hydroquinone, resorcinol, vanillin, phenol and 3,5dinitrosalicylic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Laccase (from Trametes, activity ≥ 10 U/mg) and Nafion were purchased from Sigma–Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Catechol was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). All aqueous solutions were prepared with deionized water. All reagents were used as received without further purification.
2.2. Fabrication of regenerated cellulose nanofibers CA was dissolved in a mixed solution of acetone and DMAc (3:2, V/V). Electrospinning was carried out with the CA solution in which the content of CA polymer was 15%. The electrospinning conditions were a voltage of 18 kV, a flow rate of 1 mL/h and a needle tip-tocollector distance of 16 cm. Electrospun fibers were collected as a mat using a drum collector wrapped with aluminum foil. Then electrospun CA fibrous mat was deacetylated in a 0.05 M NaOH in ethanol for 12 h to regenerate into cellulose nanofibers. After
deacetylation, the fibers were rinsed with an excess of water to remove residual NaOH and ethanol. 2.3. Preparation of PANI/CMC/cellulose nanofibers Before the deposition of PANI on electrospun nanofibrous mats, the fabricated cellulose nanofibrous mats were firstly modified by irreversibly adsorbing 0.5 mg/mL CMC solution (50 mM NaCl) at pH 5.0 for 2 h, thus obtaining CMC-modified cellulose nanofibers. The pH of solution was adjusted to by 50 mM NaOAc buffer. PANI/CMC/cellulose composite nanofibers were prepared by in situ polymerization of aniline on the surface of CMC/cellulose nanofibers. The polymerization of aniline was carried out in an ice/water bath of aniline solution (1.007 g of aniline dissolved in 230 mL of 1.4 M HCl solution). First, CMC/cellulose nanofibers were soaked in aniline solution to adsorb the aniline monomer for 0.5 h. Then 20 mL of 1.4 M HCl solution containing APS was slowly added into the mentioned solution for initiating the polymerization reaction. The mole ratio of aniline to APS was 1:1. The successive polymerization lasted for 5 h. After PANI was formed on the surface of the CMC/cellulose nanofibers, PANI/CMC/cellulose nanofibrous mats were taken out and washed with HCl solution to remove oligometric product loosely bound on the fibers. Finally, the samples were washed with deionized water and dried at ambient temperature. The preparation procedure of PANI/CMC/cellulose composite nanofibers is schematically illustrated in Fig. 1. 2.4. Preparation of Lac/PANI/CMC/cellulose/GCE Prior to modification, the glassy carbon electrode (GCE) was successively polished to a mirror-like surface with 0.05 m alumina slurry, rinsed thoroughly by deionized water and then washed with ethanol for 5 min in an ultrasonic bath. The PANI/CMC/cellulose fibrous mat was glued on the pretreated GCE by 1 wt% Nafion aqueous solution containing 3 mg/mL of Lac and left to dry in refrigerator at 4 ◦ C. The modified electrode was denoted as Lac/PANI/CMC/cellulose/GCE. The control samples were fabricated using similar procedures for the preparation of Lac/PANI/CMC/cellulose/GCE. All the modified electrodes were stored at 4 ◦ C in a refrigerator before use. 2.5. Characterization SEM images of different electrospun composite nanofibers were taken on a Hitachi SU1510 scanning electron microscope at an acceleration voltage of 1 kV. Transmission electron microscope (TEM) images were taken with JEOL/JEM-2100 transmission electron microscope. FT-IR spectra were recorded using a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientific) at the wave-number range of 4000–500 cm−1 under ambient conditions. 2.6. Electrochemical measurement All electrochemical measurements were performed using a CHI 660d electrochemical workstation (CH Instruments, Shanghai, China). The modified electrode obtained via the above methods was used as the working electrode, a platinum wire (radius 0.5 mm) and an Ag/AgCl electrode were used as the auxiliary and reference electrode. The cyclic voltammetric measurements were taken in an unstirred electrochemical cell. The electrochemical impedance spectroscopy (EIS) measurements were carried out in acetate buffer solution containing 5 mM [Fe(CN)6 ]3−/4− . Time–current curves were obtained by consequently adding catechol solution into acetate buffer solution. The 0.1 M acetate buffer
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Fig. 1. Schematic of the preparation procedure of PANI/CMC/cellulose nanofibers.
Fig. 2. SEM images of CMC/cellulose nanofibers (a) and PANI/CMC/cellulose nanofibers (b).
solution was used as electrolyte and its volume was equal to 20 mL. All the experiments were performed at room temperature. 3. Results and discussion 3.1. Structure and morphology of PANI/CMC/cellulose nanofibers Fig. 2 represents SEM images of the CMC/cellulose nanofibers (a) and PANI/CMC/cellulose nanofibers (b). The CMC/cellulose nanofibers had an average diameter of 310 ± 8 nm with smooth surfaces, and were randomly oriented. After in situ polymerization of
aniline, highly dense PANI nanorods were grown onto the surface of CMC/cellulose nanofibers. The nanorods showed a diameter of 60 nm and a length of 180 nm. The CMC could improve the ability of electrospun nanofibers to capture aniline monomers in aqueous solutions through electrostatic attraction, contributing to the formation and uniform distribution of PANI. It is noticeable that the cylindrical fibrous structures of the individual fibers and the threedimensional structure of fibrous mats were preserved, which could be beneficial for the enzyme immobilization. Fig. 3 shows TEM images of PANI/CMC/cellulose nanofibers at different magnifications. It can be seen that PANI nanorods were
Fig. 3. TEM images of PANI/CMC/cellulose nanofibers at different magnifications.
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Fig. 4. FT-IR spectra of pure PANI, cellulose, CMC/cellulose and PANI/CMC/cellulose.
parallelly coated on the surface of CMC/cellulose nanofibers. Meanwhile, the dense of PANI nanorods was high and they could form an effective access for the electron transfer, which was expected to improve the sensitivity of biosensors based on PANI/CMC/cellulose nanofibers. The FT-IR spectra of pure PANI, cellulose nanofibers, CMC/cellulose nanofibers and PANI/CMC/cellulose nanofibers are shown in Fig. 4. FT-IR spectra of pure PANI is well known in literatures. The characteristic peaks at 1568 cm−1 and 1482 cm−1 attributed to the C–C stretching of the quinonoid and benzenoid rings of PANI were clearly observed [34]. The peaks around 809 cm−1 , 1125 cm−1 and 1294 cm−1 resulted from out of bending vibration of C–H band of p-disubstituted benzene ring, vibration of C–H in benzene ring and stretching of C–N [35], respectively. Cellulose nanofibers exhibited the adsorption bands at 3354 cm−1 and 2915 cm−1 , assigned to the stretching vibration of O–H and the asymmetrically stretching vibration of C–H [30]. After the adsorption of CMC to cellulose nanofibers, the band at 3348 cm−1 became broader. The spectrum of PANI/CMC/cellulose composite nanofibers showed overlapped adsorption bands of all components, illustrating the successful preparation of PANI/CMC/cellulose nanofibers. Compared with the spectrum of CMC/cellulose nanofibers, it was found that the peak at 3348 cm−1 in the spectrum of CMC/cellulose was shifted to 3293 cm−1 in the spectrum of PANI/CMC/cellulose, suggesting the existence of interaction between PANI and the –COO− groups. Furthermore, the absorption intensity of the band in the PANI/CMC/cellulose nanofibers became weaker, indicating that the intermolecular hydrogen bands were broken. Thus, the –COO− made the composite nanofibers accessible and benefited to form the uniform dispersion of PANI in the nanofibrous mats.
Fig. 5. Cyclic voltammograms of bare GCE, CMC/cellulose/GCE, PANI/CMC/cellulose/GCE and Lac/PANI/CMC/cellulose/GCE in 0.1 M acetate buffer containing 5 mM [Fe(CN)6 ]3−/4− redox indicator at a scan rate of 100 mV/s.
increase was assigned to the PANI nanorods which improved the electron communication between the electrode and the electrolyte as well as the surface area. After laccase was immobilized on the PANI/CMC/cellulose/GCE, the redox peaks in the cyclic voltammogram decreased from 278 A to 170 A, which was due to the cover of laccase to the fibrous mat. 3.3. Electrochemical impedance spectroscopy (EIS) studies Electrochemical impedance spectroscopy (EIS) is an effective method to study the interfacial electron-transfer resistance of electrode surface. Fig. 6 indicated the typical EIS of different electrodes in 0.1 M acetate buffer solution containing 5 mM [Fe(CN)6 ]3−/4− respectively. Nyquist plot (EIS curve) was recorded over the frequency range of 0.01 Hz to 100 kHz. In EIS spectrum, the diameter of the semicircle represents the electron transfer resistance (RCT ), which could be used to describe the interface properties. It can be seen that the RCT value of PANI/CMC/cellulose/GCE was about 2.2 × 103 , smaller than 1.39 × 104 of CMC/cellulose/GCE, implying that incorporation of PANI nanorods into CMC/cellulose nanofibers increased the electron transfer rate. Meanwhile, the large surface area of PANI/CMC/cellulose nanofibers facilitated the diffusion of [Fe(CN)6 ]3−/4− towards the electrode surface. The value of RCT was found to increase to 4.52 × 103 after the immobilization of Lac on the PANI/CMC/cellulose/GCE. The observed increase could be attributed to the increase of insulation area by
3.2. Cyclic voltammetric (CV) studies Fig. 5 compares the results of the cyclic voltammetric (CV) studies carried out in 0.1 M acetate buffer containing 5 mM [Fe(CN)6 ]3−/4− redox indicator. It can be seen that the redox peaks of all the fabricated electrodes were well-defined. Compared with GCE, the redox peaks of CMC/cellulose/GCE increased to 103 A, indicating the CMC/cellulose nanofibers broaden the surface area of electrode substantially. Meanwhile, a small change in peakto-peak separation voltage was attributed to the electrostatic interaction between the negatively charged carboxy group of CMC and [Fe(CN)6 ]3−/4− , resulting in reduced electron transfer kinetics. Further increase in oxidation current was observed after the deposition of PANI on electrospun nanofibrous mats. The obvious
Fig. 6. EIS spectra of CMC/cellulose/GCE, PANI/CMC/cellulose/GCE and Lac/PANI/CMC/cellulose/GCE taken in 0.1 M acetate buffer containing 5 mM [Fe(CN)6 ]3−/4− indicator.
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Fig. 7. Cyclic voltammograms of GCE, Lac/GCE, PANI/CMC/cellulose/GCE and Lac/PANI/CMC/cellulose/GCE in 0.1 M acetate buffer solution (pH 4.0).
the hydrophobic protein layers, confirming the effective immobilization of Lac. The results were in good agreement with that of cyclic voltammograms. 3.4. Direct electron transfer of Lac/PANI/CMC/cellulose/GCE Fig. 7 depicts typical CVs of different electrodes in 0.1 M acetate buffer solution at a scan rate of 100 mV/s. No redox peaks were observed for the GCE. The PANI/CMC/cellulose/GCE showed a pair of well-defined redox peaks with the anodic and cathodic peak potentials at 0.40 V and −0.03 V, suggesting that the composite mat could undergo the redox reaction. After Lac was immobilized onto PANI/CMC/cellulose/GCE, the redox peak current of Lac/PANI/CMC/cellulose/GCE decreased to 10.4 A, with potentials shifting to 0.3 V and 0 V. An obvious change of the peak-topeak separation was observed. The results confirmed that Lac was immobilized in the composite mat. The redox peak potentials of Lac/GCE attributed to the direct electron transfer of Lac were closed to that of Lac/PANI/CMC/cellulose/GCE. Meanwhile, the oxidation current of Lac/GCE (2.5 A) was smaller than that of Lac/PANI/CMC/cellulose/GCE (10.4 A). The results demonstrated that PANI/CMC/cellulose nanofibers could provide a suitable microenvironment for the immobilization of Lac and enhance the direct electron transfer of Lac. Fig. 8 shows the cyclic voltammograms of Lac/PANI/CMC/ cellulose/GCE at different scan rate (50–200 mV/s). The anodic peak
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Fig. 9. CVs of Lac/GCE, PANI/CMC/cellulose/GCE and Lac/PANI/CMC/cellulose/GCE in 0.1 M acetate buffer solution (pH 4.0) containing 4.97 × 10−5 M catechol at a scan rate of 100 mV/s.
potential at 0.273 V and cathodic peak potential at 0.03 V were observed at a scan rate of 50 mV/s. The peak potential separation (Ep ) of about 270 mV was larger than expected for a reversible one-electron reaction (Ep = 57 mV), indicating that Lac on the composite mat could display a quasi-reversible electrochemical reaction [36]. Besides, the redox peak currents showed linear behaviour with increasing scan rate in the range of 50–200 mV/s. The result revealed a typical surface-controlled redox process and a thin layer electrochemical behaviour. Also, it confirmed that the immobilized Lac was stable. The surface coverage of the modified electrode, = 5.76 × 10−10 mol cm−2 , by integrating the peak on the cyclic voltammogram was calculated at a low scan rate, v = 100 mV/s. It was higher than the theoretically calculated value of 1.3 × 10−11 mol cm−2 reported in a previous study [37], implying that more Lac has been immobilized onto the PANI/CMC/cellulose nanofibrous mat because of its large surface area. 3.5. Electrocatalysis of Lac/PANI/CMC/cellulose/GCE Fig. 9 exhibited cyclic voltammograms of different modified electrodes in 0.1 M acetate buffer solution containing 4.97 × 10−5 M catechol. In the presence of catechol, two pairs of CV peaks were obtained at the PANI/CMC/cellulose/GCE, (0.35 V and −0.08 V) and (0.73 V and 0.42 V), corresponding to the redox reaction
Fig. 8. CVs (a) of Lac/PANI/CMC/cellulose/GCE in acetate buffer solution at a scan rate from 50 mV/s to 200 mV/s and calibration plots (b) of anodic and cathodic peak current vs. scan rates.
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Fig. 10. The dependence of the response of Lac/PANI/CMC/cellulose/GCE toward 4.97 × 10−5 M catechol on the pH of acetate buffer solution (a), and the detection potential (b). Every point is an average value of three independent measurements.
of PANI and catechol, respectively. The redox peak current of Lac/GCE was about 6 A, with the potentials at 0.47 V and 0.15 V, attributed to the catalysis of Lac toward catechol. While for Lac/PANI/CMC/cellulose/GCE, the oxidation peak current (9.5 A) caused by the catalysis of Lac toward catechol increased significantly in accompany with a small peak potential change from 0.47 V to 0.53 V. The results indicated that the PANI/CMC/cellulose nanofibrous mat had a little effect on the physicochemical properties of the Lac. Besides, PANI/CMC/cellulose mats played a positive role in enhancing the response signal as well as the sensitivity of biosensors.
3.6. Optimizing the parameters of the biosensing performance To improve the performance of the Lac/PANI/CMC/cellulose/GCE toward catechol, the effects of pH of the buffer solution and the detection potential on the current response were optimized. The amperometric response of the proposed biosensor was recorded in 0.1 M acetate buffer solution containing 4.97 × 10−5 M catechol. In the range of pH variant from 4.0 to 6.5, a maximum response was found at pH 4.5 (Fig. 10a). Besides, the response increased with the detection potential from 0.40 V to 0.50 V (Fig. 10b) and then showed a dramatic decrease. Therefore, pH value of 4.5 and a detection potential of 0.50 V were selected in the following experiments.
3.7. Amperometric characteristics of Lac/PANI/CMC/cellulose/GCE To further evaluate the biological sensing properties of the Lac/PANI/CMC/cellulose/GCE for catechol, amperometric measurement of catechol was carried out with the biosensor by successively adding of the analytes into the buffer solution at pH 4.5 (Fig. 11). When catechol was added into the buffer solution, the response current rose sharply and achieved 95% of steady-state currents within 8 s (Fig. 11a), indicating that the electrode responded rapidly to the change of catechol concentration. Fig. 11(b) shows the calibration plot for catechol detection. The response displayed a linear range from 0.497 M to 2277.8 M with a correlation coefficient of 0.9999. The limit of detection (LOD) of the constructed biosensor was determined according to the formula LOD = 3 × SD/slope (where SD is the estimated standard deviation for the points used to construct the calibration curve). From the slope of 0.0154 A/M, the detection limit was estimated to be at S/N = 3 with 0.374 M. The comparison of the recently reported laccase-modified biosensors on different electrode substrates was summarized in Table 1. As shown in Table 1, the prepared Lac/PANI/CMC/cellulose/GCE displayed improved biosensing performance in terms of the limit of detection. Moreover, the linear range of the biosensor was wider than other ones, which suggested that the biosensor was suitable for both low concentration detection of catechol and the extract samples containing high concentration without dilution.
Fig. 11. Typical steady-state current–time response (a) of Lac/PANI/CMC/cellulose/GCE by successively adding of catechol into acetate buffer solution at pH 4.5, and calibration curve (b) of the current vs. the concentration of catechol.
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Table 1 The comparison of the recently reported laccase-modified biosensors on different electrode substrates toward catechol. Electrode
Method
Linear range (M)
Detection limit (M)
References
MB-MCM-41/PVA/Lac Lac/Cu-OMC/CS/Au Lac-FSM7.0-GCE Lac/AP-rGOs/GCE Lac/(N-OMC)/PVA/Au PDA-Lac-NiCNFs/MGCE Lac/MWCNT/CB/Nf/OsO4/P4VP/GCE Lac/PANI/CMC/cellulose/GCE
i–t i–t i–t i–t i–t i–t DPV i–t
4–87.98 0.67–15.75 2–100 15–700 0.39–8.98 1–9100 3.98 × 10−3 –1.67 × 10−2 0.497–2277.8
0.331 0.67 2 7 0.31 0.69 2.82 × 10−3 0.374
[39] [40] [41] [14] [42] [38] [43] This work
According to the Michaelis–Menten equation, I = Imax × c/(KM + c), where I is the steady-state current after the addition of substrate, Imax is the maximum current response under the condition of saturated substrate, c is the concentration of substrate and KM is the apparent Michaelis–Menten constant); the values of KM was calculated to be 1.74 mM, smaller than that reported for Lac in PDA-Lac-NiCNFs/MGCE [38]. The lower KM value demonstrated that Lac immobilized onto PANI/CMC/cellulose/GCE had higher enzymatic activity and showed higher affinity for catechol. 3.8. Repeatability, reproducibility, storage stability and selectivity of Lac/PANI/CMC/cellulose/GCE Different aspects regarding the characteristics of the proposed biosensor were evaluated. The relative standard deviation (RSD) of Lac/PANI/CMC/cellulose/GCE to catechol was about 2.23% obtained from 5 successive measurements, revealing an acceptable repeatability. The electrode-to-electrode reproducibility was estimated from the determinations of five different and freshly prepared electrodes at a catechol concentration of 4.97 × 10−5 M. A RSD of 3.03% was obtained, indicating the good reproducibility in the construction of the electrode. The storage stability of the modified electrode was examined by carrying out response measurements at the interval of time and it was found that the response current retained about 98% of its initial value after 2 weeks when stored in acetate buffer (pH 4.5) at 4 ◦ C. There are some other phenolic compounds, for example, hydroquinone, resorcinol, vanillin, phenol and 3,5-dinitrosalicylic acid, etc. might affect the performance of the modified electrode. The selectivity and anti-interference advantage of the modified electrode was demonstrated (Fig. 12). The addition of hydroquinone resulted in 19.05% of the current response from catechol because of their similar oxidation potential, while the addition of other
interferences resulted in negligible signals. Hence, the modified electrode exhibited excellent selectivity for catechol. 4. Conclusion A hierarchical PANI/CMC/cellulose nanofibrous mat was fabricated by the combination of electrospinning and in situ polymerization process and it showed a potential application as biosensor substrate materials. CMC adsorbed on the surface of cellulose nanofiber to improve its accessibility and increase the ability to capture aniline monomers. The obtained PANI/CMC/cellulose nanofibers maintained the cylindrical fibrous structures of the individual fibers as well as the three-dimensional structure of fibrous mats to provide a high electroactive surface area for facile electron transfer and mass diffusion. Under their optimized condition, the prepared Lac/PANI/CMC/cellulose/GCE showed the excellent response characteristics with the detection limit of 0.374 M and the linear response range from 0.497 M to 2.27 mM, which were comparable and even superior to many laccase biosensors based on other matrices in the detection of catechol. Furthermore, the constructed biosensor displayed good repeatability, reproducibility and selectivity. These results indicated that the PANI/CMC/cellulose nanofiber would be a promising substrate material for the construction of enzyme bisoensors. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21201083), National High-tech R&D Program of China (2012AA030313), Changjiang Scholars and Innovative Research Team in University (IRT1135), 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), the Innovation Program for Graduate Education in Jiangsu Province (CXZZ13 07 and KYLX 1133), and Hubei Key Laboratory of Low Dimensional Optoelectronic Material and Devices (13XKL01002). References
Fig. 12. Amperometric response of the Lac/PANI/CMC/cellulose/GCE upon subsequent additions of (a) catechol, (b) hydroquinone, (c) resorcinol, (d) vanillin, (e) phenol, and (f) 3,5-dinitrosalicylic acid at 0.50 V, 0.1 M acetate buffer solution (pH 4.5).
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Fabrication of polyaniline/carboxymethyl cellulose/cellulose nanofibrous mats and their biosensing application Jiapeng Fu, Zengyuan Pang, Jie Yang, Fenglin Huang, Yibing Cai, Qufu Wei ∗ Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China
a r t i c l e
i n f o
Article history: Received 12 March 2015 Received in revised form 26 April 2015 Accepted 29 April 2015 Available online 6 May 2015 Keyword: Electrospinning Cellulose Polyaniline Laccase Biosensor Catechol
a b s t r a c t We report a facile approach to synthesizing and immobilizing polyaniline nanorods onto carboxymethyl cellulose (CMC)-modified cellulose nanofibers for their biosensing application. Firstly, the hierarchical PANI/CMC/cellulose nanofibers were fabricated by in situ polymerization of aniline on the CMC-modified cellulose nanofiber. Subsequently, the PANI/CMC/cellulose nanofibrous mat modified with laccase (Lac) was used as biosensor substrate material for the detection of catechol. PANI/CMC/cellulose nanofibers with highly conductive and three dimensional nanostructure were characterized by scanning electron microscopy (SEM), transmission electron microscope (TEM), Fourier transform infrared spectra (FT-IR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under optimum conditions, the Lac/PANI/CMC/cellulose/glassy carbon electrode (GCE) exhibited a fast response time (within 8 s), a linear response range from 0.497 M to 2.27 mM with a high sensitivity and low detection limit of 0.374 M (3). The developed biosensor also displayed good repeatability, reproducibility as well as selectivity. The results indicated that the composite mat has potential application in enzyme biosensors. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, electrochemical biosensors have attracted most of the attention in the detection of different analytes such as glucose [1–3], H2 O2 [4,5] and catechol [6–8] due to their simplicity, fast response, high selectivity and sensitivity. Most effort has been focused on developing suitable matrices used for the fabrication of electrochemical biosensors. Those matrices include metals [9,10], metals oxides [11,12], carbon-based materials [13,14] and polymers [15,16]. The performance of electrochemical biosensors largely relies on the physical and chemical properties of the electrode materials. For this purpose polyaniline (PANI) has aroused much interest to construct novel and improved biosensors due to its good electrical conductivity, good environmental stability, ease of synthesis [17–19]. Nevertheless, the biosensing performance of PANI-based bisoensors is unsatisfactory with a small interfacial area between PANI and biomolecules. With the mass loading increases, PANI has
∗ Corresponding author at: Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Tel.: +86 510 8591 3653. E-mail addresses: fi[email protected] (J. Fu), [email protected] (Z. Pang), [email protected] (J. Yang), fl[email protected] (F. Huang), [email protected] (Y. Cai), [email protected] (Q. Wei). http://dx.doi.org/10.1016/j.apsusc.2015.04.215 0169-4332/© 2015 Elsevier B.V. All rights reserved.
the tendency of aggregation and the decreased accessible surface area, resulting in poor electrical conductivity and increasing mass transfer limitation [20]. Therefore, the development of nanostructured PANI with increased surface-to-volume ratio, high electrical conductivity and reduced distance for electron and mass transfer is of great importance to obtain excellent biosensing performance. Meanwhile, the lightweight and portable structures are considered necessary to broaden their applications in the surrounding environment in real time [21]. An effective strategy to achieve this goal is to deposit PANI on the surface of flexible fibrous mats, which could overcome the difficulty of electrospinning PANI solution. The three dimensional nanostructure of electrospun fibrous mats provides space for the deposition of PANI and improves their interfacial area with immobilized enzyme. Electrospinning is an effective technique that produces continuous nanoscaled polymeric fiber with several remarkable characteristics such as high surface area to volume ratio, high porosity and flexibility. Meanwhile, electrospun nanofibrous mats are easily handled and retrieved. It is well documented that biocompatible electrospun polymer nanofibers, like polylatic acid [22] and polyvinyl alcohol [16,23], serve as matrix or template for the application of biosensors. However, the relatively hydrophobic property of polylactic acid is unfavourable to maintain the enzyme activity. Electrospun PVA nanofibers have poor water stability and become a clear gelation like material in water, limiting their practical applications [24]. Nanocellulose materials could overcome
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these problems and recently show promising results in biomolecule immobilization and biosensors [25,26]. Cellulose is one of most abundant biopolymers and possesses desirable properties such as hydrophilicity, biocompatibility and biodegradation. Besides, cellulose could not be dissolved in water and most common organic solvents because of its high crystallinity, which makes it difficult to fabricate cellulose nanofibers via direct electrospinning [27]. Alternatively, cellulose nanofibers have been produced by electrospinning of cellulose acetate and subsequent deacetylation [28,29]. PANI/cellulose conductive composites have been fabricated in heterogeneous reaction [30,31]. However, the accessibility and reactivity of the –OH group are limited by numerous intermolecular and intramolecular hydrogen bonds. It is necessary to overcome the problem to obtain PANI-modified composites with desirable conductivity and good bonding. Carboxymethyl cellulose (CMC), a derivative of cellulose, can adsorb irreversibly on cellulose fibers under suitable condition and increase the negative charges of cellulose fibers [32,33]. The –COO− groups of CMC provide cationic exchangeable sites to capture electropositive molecules via electrostatic attraction in aqueous solutions, facilitating the in situ polymerization of aniline. In this present study, we fabricated hierarchical PANI/CMC/cellulose nanofibers by in situ polymerization of aniline on the CMC-modified cellulose nanofibers, which were employed as substrate materials to construct laccase (Lac) biosensors for the detection of catechol. The obtained fibrous mats with hierarchical structure combine the flexibility and high surface area of CMC-modified cellulose nanofibers with excellent conductivity of PANI, facilitating the immobilization of Lac as well as enhancing the mass and electron transport. Compared with previous biosensors based on other materials, the fabricated Lac/PANI/CMC/cellulose/glassy carbon electrode (GCE) exhibited a highly sensitive detection toward catechol with a broad linear range and low detection limit. This study demonstrated that PANI/CMC/cellulose nanofibrous mats are the promising material in the design of high-efficient biosensors.
2. Material and methods 2.1. Materials Cellulose acetate (CA, 39.8 wt% of acetyl content, Mn = 30,000), N,N-dimethylacetamide (DMAc), acetone, carboxymethyl cellulose (CMC), aniline monomer, ammonium persulfate (APS), hydrochloric acid (HCl, the concentration of 37%), NaCl, CH3 COOH, CH3 COONa, hydroquinone, resorcinol, vanillin, phenol and 3,5dinitrosalicylic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Laccase (from Trametes, activity ≥ 10 U/mg) and Nafion were purchased from Sigma–Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Catechol was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). All aqueous solutions were prepared with deionized water. All reagents were used as received without further purification.
2.2. Fabrication of regenerated cellulose nanofibers CA was dissolved in a mixed solution of acetone and DMAc (3:2, V/V). Electrospinning was carried out with the CA solution in which the content of CA polymer was 15%. The electrospinning conditions were a voltage of 18 kV, a flow rate of 1 mL/h and a needle tip-tocollector distance of 16 cm. Electrospun fibers were collected as a mat using a drum collector wrapped with aluminum foil. Then electrospun CA fibrous mat was deacetylated in a 0.05 M NaOH in ethanol for 12 h to regenerate into cellulose nanofibers. After
deacetylation, the fibers were rinsed with an excess of water to remove residual NaOH and ethanol. 2.3. Preparation of PANI/CMC/cellulose nanofibers Before the deposition of PANI on electrospun nanofibrous mats, the fabricated cellulose nanofibrous mats were firstly modified by irreversibly adsorbing 0.5 mg/mL CMC solution (50 mM NaCl) at pH 5.0 for 2 h, thus obtaining CMC-modified cellulose nanofibers. The pH of solution was adjusted to by 50 mM NaOAc buffer. PANI/CMC/cellulose composite nanofibers were prepared by in situ polymerization of aniline on the surface of CMC/cellulose nanofibers. The polymerization of aniline was carried out in an ice/water bath of aniline solution (1.007 g of aniline dissolved in 230 mL of 1.4 M HCl solution). First, CMC/cellulose nanofibers were soaked in aniline solution to adsorb the aniline monomer for 0.5 h. Then 20 mL of 1.4 M HCl solution containing APS was slowly added into the mentioned solution for initiating the polymerization reaction. The mole ratio of aniline to APS was 1:1. The successive polymerization lasted for 5 h. After PANI was formed on the surface of the CMC/cellulose nanofibers, PANI/CMC/cellulose nanofibrous mats were taken out and washed with HCl solution to remove oligometric product loosely bound on the fibers. Finally, the samples were washed with deionized water and dried at ambient temperature. The preparation procedure of PANI/CMC/cellulose composite nanofibers is schematically illustrated in Fig. 1. 2.4. Preparation of Lac/PANI/CMC/cellulose/GCE Prior to modification, the glassy carbon electrode (GCE) was successively polished to a mirror-like surface with 0.05 m alumina slurry, rinsed thoroughly by deionized water and then washed with ethanol for 5 min in an ultrasonic bath. The PANI/CMC/cellulose fibrous mat was glued on the pretreated GCE by 1 wt% Nafion aqueous solution containing 3 mg/mL of Lac and left to dry in refrigerator at 4 ◦ C. The modified electrode was denoted as Lac/PANI/CMC/cellulose/GCE. The control samples were fabricated using similar procedures for the preparation of Lac/PANI/CMC/cellulose/GCE. All the modified electrodes were stored at 4 ◦ C in a refrigerator before use. 2.5. Characterization SEM images of different electrospun composite nanofibers were taken on a Hitachi SU1510 scanning electron microscope at an acceleration voltage of 1 kV. Transmission electron microscope (TEM) images were taken with JEOL/JEM-2100 transmission electron microscope. FT-IR spectra were recorded using a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientific) at the wave-number range of 4000–500 cm−1 under ambient conditions. 2.6. Electrochemical measurement All electrochemical measurements were performed using a CHI 660d electrochemical workstation (CH Instruments, Shanghai, China). The modified electrode obtained via the above methods was used as the working electrode, a platinum wire (radius 0.5 mm) and an Ag/AgCl electrode were used as the auxiliary and reference electrode. The cyclic voltammetric measurements were taken in an unstirred electrochemical cell. The electrochemical impedance spectroscopy (EIS) measurements were carried out in acetate buffer solution containing 5 mM [Fe(CN)6 ]3−/4− . Time–current curves were obtained by consequently adding catechol solution into acetate buffer solution. The 0.1 M acetate buffer
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Fig. 1. Schematic of the preparation procedure of PANI/CMC/cellulose nanofibers.
Fig. 2. SEM images of CMC/cellulose nanofibers (a) and PANI/CMC/cellulose nanofibers (b).
solution was used as electrolyte and its volume was equal to 20 mL. All the experiments were performed at room temperature. 3. Results and discussion 3.1. Structure and morphology of PANI/CMC/cellulose nanofibers Fig. 2 represents SEM images of the CMC/cellulose nanofibers (a) and PANI/CMC/cellulose nanofibers (b). The CMC/cellulose nanofibers had an average diameter of 310 ± 8 nm with smooth surfaces, and were randomly oriented. After in situ polymerization of
aniline, highly dense PANI nanorods were grown onto the surface of CMC/cellulose nanofibers. The nanorods showed a diameter of 60 nm and a length of 180 nm. The CMC could improve the ability of electrospun nanofibers to capture aniline monomers in aqueous solutions through electrostatic attraction, contributing to the formation and uniform distribution of PANI. It is noticeable that the cylindrical fibrous structures of the individual fibers and the threedimensional structure of fibrous mats were preserved, which could be beneficial for the enzyme immobilization. Fig. 3 shows TEM images of PANI/CMC/cellulose nanofibers at different magnifications. It can be seen that PANI nanorods were
Fig. 3. TEM images of PANI/CMC/cellulose nanofibers at different magnifications.
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Fig. 4. FT-IR spectra of pure PANI, cellulose, CMC/cellulose and PANI/CMC/cellulose.
parallelly coated on the surface of CMC/cellulose nanofibers. Meanwhile, the dense of PANI nanorods was high and they could form an effective access for the electron transfer, which was expected to improve the sensitivity of biosensors based on PANI/CMC/cellulose nanofibers. The FT-IR spectra of pure PANI, cellulose nanofibers, CMC/cellulose nanofibers and PANI/CMC/cellulose nanofibers are shown in Fig. 4. FT-IR spectra of pure PANI is well known in literatures. The characteristic peaks at 1568 cm−1 and 1482 cm−1 attributed to the C–C stretching of the quinonoid and benzenoid rings of PANI were clearly observed [34]. The peaks around 809 cm−1 , 1125 cm−1 and 1294 cm−1 resulted from out of bending vibration of C–H band of p-disubstituted benzene ring, vibration of C–H in benzene ring and stretching of C–N [35], respectively. Cellulose nanofibers exhibited the adsorption bands at 3354 cm−1 and 2915 cm−1 , assigned to the stretching vibration of O–H and the asymmetrically stretching vibration of C–H [30]. After the adsorption of CMC to cellulose nanofibers, the band at 3348 cm−1 became broader. The spectrum of PANI/CMC/cellulose composite nanofibers showed overlapped adsorption bands of all components, illustrating the successful preparation of PANI/CMC/cellulose nanofibers. Compared with the spectrum of CMC/cellulose nanofibers, it was found that the peak at 3348 cm−1 in the spectrum of CMC/cellulose was shifted to 3293 cm−1 in the spectrum of PANI/CMC/cellulose, suggesting the existence of interaction between PANI and the –COO− groups. Furthermore, the absorption intensity of the band in the PANI/CMC/cellulose nanofibers became weaker, indicating that the intermolecular hydrogen bands were broken. Thus, the –COO− made the composite nanofibers accessible and benefited to form the uniform dispersion of PANI in the nanofibrous mats.
Fig. 5. Cyclic voltammograms of bare GCE, CMC/cellulose/GCE, PANI/CMC/cellulose/GCE and Lac/PANI/CMC/cellulose/GCE in 0.1 M acetate buffer containing 5 mM [Fe(CN)6 ]3−/4− redox indicator at a scan rate of 100 mV/s.
increase was assigned to the PANI nanorods which improved the electron communication between the electrode and the electrolyte as well as the surface area. After laccase was immobilized on the PANI/CMC/cellulose/GCE, the redox peaks in the cyclic voltammogram decreased from 278 A to 170 A, which was due to the cover of laccase to the fibrous mat. 3.3. Electrochemical impedance spectroscopy (EIS) studies Electrochemical impedance spectroscopy (EIS) is an effective method to study the interfacial electron-transfer resistance of electrode surface. Fig. 6 indicated the typical EIS of different electrodes in 0.1 M acetate buffer solution containing 5 mM [Fe(CN)6 ]3−/4− respectively. Nyquist plot (EIS curve) was recorded over the frequency range of 0.01 Hz to 100 kHz. In EIS spectrum, the diameter of the semicircle represents the electron transfer resistance (RCT ), which could be used to describe the interface properties. It can be seen that the RCT value of PANI/CMC/cellulose/GCE was about 2.2 × 103 , smaller than 1.39 × 104 of CMC/cellulose/GCE, implying that incorporation of PANI nanorods into CMC/cellulose nanofibers increased the electron transfer rate. Meanwhile, the large surface area of PANI/CMC/cellulose nanofibers facilitated the diffusion of [Fe(CN)6 ]3−/4− towards the electrode surface. The value of RCT was found to increase to 4.52 × 103 after the immobilization of Lac on the PANI/CMC/cellulose/GCE. The observed increase could be attributed to the increase of insulation area by
3.2. Cyclic voltammetric (CV) studies Fig. 5 compares the results of the cyclic voltammetric (CV) studies carried out in 0.1 M acetate buffer containing 5 mM [Fe(CN)6 ]3−/4− redox indicator. It can be seen that the redox peaks of all the fabricated electrodes were well-defined. Compared with GCE, the redox peaks of CMC/cellulose/GCE increased to 103 A, indicating the CMC/cellulose nanofibers broaden the surface area of electrode substantially. Meanwhile, a small change in peakto-peak separation voltage was attributed to the electrostatic interaction between the negatively charged carboxy group of CMC and [Fe(CN)6 ]3−/4− , resulting in reduced electron transfer kinetics. Further increase in oxidation current was observed after the deposition of PANI on electrospun nanofibrous mats. The obvious
Fig. 6. EIS spectra of CMC/cellulose/GCE, PANI/CMC/cellulose/GCE and Lac/PANI/CMC/cellulose/GCE taken in 0.1 M acetate buffer containing 5 mM [Fe(CN)6 ]3−/4− indicator.
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Fig. 7. Cyclic voltammograms of GCE, Lac/GCE, PANI/CMC/cellulose/GCE and Lac/PANI/CMC/cellulose/GCE in 0.1 M acetate buffer solution (pH 4.0).
the hydrophobic protein layers, confirming the effective immobilization of Lac. The results were in good agreement with that of cyclic voltammograms. 3.4. Direct electron transfer of Lac/PANI/CMC/cellulose/GCE Fig. 7 depicts typical CVs of different electrodes in 0.1 M acetate buffer solution at a scan rate of 100 mV/s. No redox peaks were observed for the GCE. The PANI/CMC/cellulose/GCE showed a pair of well-defined redox peaks with the anodic and cathodic peak potentials at 0.40 V and −0.03 V, suggesting that the composite mat could undergo the redox reaction. After Lac was immobilized onto PANI/CMC/cellulose/GCE, the redox peak current of Lac/PANI/CMC/cellulose/GCE decreased to 10.4 A, with potentials shifting to 0.3 V and 0 V. An obvious change of the peak-topeak separation was observed. The results confirmed that Lac was immobilized in the composite mat. The redox peak potentials of Lac/GCE attributed to the direct electron transfer of Lac were closed to that of Lac/PANI/CMC/cellulose/GCE. Meanwhile, the oxidation current of Lac/GCE (2.5 A) was smaller than that of Lac/PANI/CMC/cellulose/GCE (10.4 A). The results demonstrated that PANI/CMC/cellulose nanofibers could provide a suitable microenvironment for the immobilization of Lac and enhance the direct electron transfer of Lac. Fig. 8 shows the cyclic voltammograms of Lac/PANI/CMC/ cellulose/GCE at different scan rate (50–200 mV/s). The anodic peak
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Fig. 9. CVs of Lac/GCE, PANI/CMC/cellulose/GCE and Lac/PANI/CMC/cellulose/GCE in 0.1 M acetate buffer solution (pH 4.0) containing 4.97 × 10−5 M catechol at a scan rate of 100 mV/s.
potential at 0.273 V and cathodic peak potential at 0.03 V were observed at a scan rate of 50 mV/s. The peak potential separation (Ep ) of about 270 mV was larger than expected for a reversible one-electron reaction (Ep = 57 mV), indicating that Lac on the composite mat could display a quasi-reversible electrochemical reaction [36]. Besides, the redox peak currents showed linear behaviour with increasing scan rate in the range of 50–200 mV/s. The result revealed a typical surface-controlled redox process and a thin layer electrochemical behaviour. Also, it confirmed that the immobilized Lac was stable. The surface coverage of the modified electrode, = 5.76 × 10−10 mol cm−2 , by integrating the peak on the cyclic voltammogram was calculated at a low scan rate, v = 100 mV/s. It was higher than the theoretically calculated value of 1.3 × 10−11 mol cm−2 reported in a previous study [37], implying that more Lac has been immobilized onto the PANI/CMC/cellulose nanofibrous mat because of its large surface area. 3.5. Electrocatalysis of Lac/PANI/CMC/cellulose/GCE Fig. 9 exhibited cyclic voltammograms of different modified electrodes in 0.1 M acetate buffer solution containing 4.97 × 10−5 M catechol. In the presence of catechol, two pairs of CV peaks were obtained at the PANI/CMC/cellulose/GCE, (0.35 V and −0.08 V) and (0.73 V and 0.42 V), corresponding to the redox reaction
Fig. 8. CVs (a) of Lac/PANI/CMC/cellulose/GCE in acetate buffer solution at a scan rate from 50 mV/s to 200 mV/s and calibration plots (b) of anodic and cathodic peak current vs. scan rates.
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Fig. 10. The dependence of the response of Lac/PANI/CMC/cellulose/GCE toward 4.97 × 10−5 M catechol on the pH of acetate buffer solution (a), and the detection potential (b). Every point is an average value of three independent measurements.
of PANI and catechol, respectively. The redox peak current of Lac/GCE was about 6 A, with the potentials at 0.47 V and 0.15 V, attributed to the catalysis of Lac toward catechol. While for Lac/PANI/CMC/cellulose/GCE, the oxidation peak current (9.5 A) caused by the catalysis of Lac toward catechol increased significantly in accompany with a small peak potential change from 0.47 V to 0.53 V. The results indicated that the PANI/CMC/cellulose nanofibrous mat had a little effect on the physicochemical properties of the Lac. Besides, PANI/CMC/cellulose mats played a positive role in enhancing the response signal as well as the sensitivity of biosensors.
3.6. Optimizing the parameters of the biosensing performance To improve the performance of the Lac/PANI/CMC/cellulose/GCE toward catechol, the effects of pH of the buffer solution and the detection potential on the current response were optimized. The amperometric response of the proposed biosensor was recorded in 0.1 M acetate buffer solution containing 4.97 × 10−5 M catechol. In the range of pH variant from 4.0 to 6.5, a maximum response was found at pH 4.5 (Fig. 10a). Besides, the response increased with the detection potential from 0.40 V to 0.50 V (Fig. 10b) and then showed a dramatic decrease. Therefore, pH value of 4.5 and a detection potential of 0.50 V were selected in the following experiments.
3.7. Amperometric characteristics of Lac/PANI/CMC/cellulose/GCE To further evaluate the biological sensing properties of the Lac/PANI/CMC/cellulose/GCE for catechol, amperometric measurement of catechol was carried out with the biosensor by successively adding of the analytes into the buffer solution at pH 4.5 (Fig. 11). When catechol was added into the buffer solution, the response current rose sharply and achieved 95% of steady-state currents within 8 s (Fig. 11a), indicating that the electrode responded rapidly to the change of catechol concentration. Fig. 11(b) shows the calibration plot for catechol detection. The response displayed a linear range from 0.497 M to 2277.8 M with a correlation coefficient of 0.9999. The limit of detection (LOD) of the constructed biosensor was determined according to the formula LOD = 3 × SD/slope (where SD is the estimated standard deviation for the points used to construct the calibration curve). From the slope of 0.0154 A/M, the detection limit was estimated to be at S/N = 3 with 0.374 M. The comparison of the recently reported laccase-modified biosensors on different electrode substrates was summarized in Table 1. As shown in Table 1, the prepared Lac/PANI/CMC/cellulose/GCE displayed improved biosensing performance in terms of the limit of detection. Moreover, the linear range of the biosensor was wider than other ones, which suggested that the biosensor was suitable for both low concentration detection of catechol and the extract samples containing high concentration without dilution.
Fig. 11. Typical steady-state current–time response (a) of Lac/PANI/CMC/cellulose/GCE by successively adding of catechol into acetate buffer solution at pH 4.5, and calibration curve (b) of the current vs. the concentration of catechol.
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Table 1 The comparison of the recently reported laccase-modified biosensors on different electrode substrates toward catechol. Electrode
Method
Linear range (M)
Detection limit (M)
References
MB-MCM-41/PVA/Lac Lac/Cu-OMC/CS/Au Lac-FSM7.0-GCE Lac/AP-rGOs/GCE Lac/(N-OMC)/PVA/Au PDA-Lac-NiCNFs/MGCE Lac/MWCNT/CB/Nf/OsO4/P4VP/GCE Lac/PANI/CMC/cellulose/GCE
i–t i–t i–t i–t i–t i–t DPV i–t
4–87.98 0.67–15.75 2–100 15–700 0.39–8.98 1–9100 3.98 × 10−3 –1.67 × 10−2 0.497–2277.8
0.331 0.67 2 7 0.31 0.69 2.82 × 10−3 0.374
[39] [40] [41] [14] [42] [38] [43] This work
According to the Michaelis–Menten equation, I = Imax × c/(KM + c), where I is the steady-state current after the addition of substrate, Imax is the maximum current response under the condition of saturated substrate, c is the concentration of substrate and KM is the apparent Michaelis–Menten constant); the values of KM was calculated to be 1.74 mM, smaller than that reported for Lac in PDA-Lac-NiCNFs/MGCE [38]. The lower KM value demonstrated that Lac immobilized onto PANI/CMC/cellulose/GCE had higher enzymatic activity and showed higher affinity for catechol. 3.8. Repeatability, reproducibility, storage stability and selectivity of Lac/PANI/CMC/cellulose/GCE Different aspects regarding the characteristics of the proposed biosensor were evaluated. The relative standard deviation (RSD) of Lac/PANI/CMC/cellulose/GCE to catechol was about 2.23% obtained from 5 successive measurements, revealing an acceptable repeatability. The electrode-to-electrode reproducibility was estimated from the determinations of five different and freshly prepared electrodes at a catechol concentration of 4.97 × 10−5 M. A RSD of 3.03% was obtained, indicating the good reproducibility in the construction of the electrode. The storage stability of the modified electrode was examined by carrying out response measurements at the interval of time and it was found that the response current retained about 98% of its initial value after 2 weeks when stored in acetate buffer (pH 4.5) at 4 ◦ C. There are some other phenolic compounds, for example, hydroquinone, resorcinol, vanillin, phenol and 3,5-dinitrosalicylic acid, etc. might affect the performance of the modified electrode. The selectivity and anti-interference advantage of the modified electrode was demonstrated (Fig. 12). The addition of hydroquinone resulted in 19.05% of the current response from catechol because of their similar oxidation potential, while the addition of other
interferences resulted in negligible signals. Hence, the modified electrode exhibited excellent selectivity for catechol. 4. Conclusion A hierarchical PANI/CMC/cellulose nanofibrous mat was fabricated by the combination of electrospinning and in situ polymerization process and it showed a potential application as biosensor substrate materials. CMC adsorbed on the surface of cellulose nanofiber to improve its accessibility and increase the ability to capture aniline monomers. The obtained PANI/CMC/cellulose nanofibers maintained the cylindrical fibrous structures of the individual fibers as well as the three-dimensional structure of fibrous mats to provide a high electroactive surface area for facile electron transfer and mass diffusion. Under their optimized condition, the prepared Lac/PANI/CMC/cellulose/GCE showed the excellent response characteristics with the detection limit of 0.374 M and the linear response range from 0.497 M to 2.27 mM, which were comparable and even superior to many laccase biosensors based on other matrices in the detection of catechol. Furthermore, the constructed biosensor displayed good repeatability, reproducibility and selectivity. These results indicated that the PANI/CMC/cellulose nanofiber would be a promising substrate material for the construction of enzyme bisoensors. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21201083), National High-tech R&D Program of China (2012AA030313), Changjiang Scholars and Innovative Research Team in University (IRT1135), 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), the Innovation Program for Graduate Education in Jiangsu Province (CXZZ13 07 and KYLX 1133), and Hubei Key Laboratory of Low Dimensional Optoelectronic Material and Devices (13XKL01002). References
Fig. 12. Amperometric response of the Lac/PANI/CMC/cellulose/GCE upon subsequent additions of (a) catechol, (b) hydroquinone, (c) resorcinol, (d) vanillin, (e) phenol, and (f) 3,5-dinitrosalicylic acid at 0.50 V, 0.1 M acetate buffer solution (pH 4.5).
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