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polyaniline modified gold electrode for determination of phenolic content in fruit juices
Biochemical Engineering Journal 68 (2012) 76–84
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An amperometric biosensor based on laccase immobilized onto nickel nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode for determination of phenolic content in fruit juices Sheetal Chawla, Rachna Rawal, Swati Sharma, Chandra Shekhar Pundir ∗ Department of Biochemistry, M.D. University, Rohtak 124001, Haryana, India
a r t i c l e
i n f o
Article history: Received 16 January 2012 Received in revised form 23 May 2012 Accepted 11 July 2012 Available online 20 July 2012 Keywords: Biosensors Enzymes Immobilisation Optimisation NiNPs cMWCNT
a b s t r a c t A method is described for construction of an enzyme electrode for detection of phenolic compounds based on covalent immobilization of laccase onto nickel nanoparticles (NiNPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto gold (Au) electrode. The modified electrode was characterized at different stages of its construction by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammograms and electrochemical impedance spectroscopy (EIS). An amperometric biosensor for phenolic compounds was fabricated by connecting enzyme electrode (Lac/NiNPs/cMWCNTs/PANI/AuE) as working electrode, with Ag/AgCl as reference electrode and Pt wire as auxiliary electrode through potentiostat. The biosensor showed optimum response at pH 5.5 (0.1 M acetate buffer) and 35 ◦ C, when operated at a scan rate of 20 mV s−1 . Linear range, response time, detection limit and sensitivity of biosensor were 0.1–10 M (lower concentration range) and 10–500 M (higher concentration range), 8 s, 0.05 M and 0.694 A M−1 cm−2 respectively. The biosensor measured total phenolic content in fruit juices. The enzyme electrode was used 200 times over a period of four months, when stored at 4 ◦ C. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Polyphenols are the most abundant antioxidants in our diet. Diets containing an abundance of antioxidants are protective against a variety of diseases, particularly cardiovascular diseases and cancer. A number of analytical techniques are available for determination of phenolic content such as spectrophotometry, gas chromatography, liquid chromatography and capillary electrophoresis [1]. However, some of these techniques (e.g. spectrophotometry) are not sensitive and specific, while others (e.g. gas chromatography, liquid chromatography and capillary electrophoresis) are cumbersome and require time consuming sample pre-treatment, expensive equipments and skilled persons to operate. Therefore, there is an interest in developing a simple, sensitive, rapid, cost-effective and portable system such as biosensor for determination of phenolic compounds in important areas such as clinical, biomedical, environmental contamination, industrial and pharmaceutical analysis [2–4]. A number of biosensors for detection of phenolic compounds have been reported based on immobilization of laccase onto various supports such as graphite electrode [5,6], silane-modified
∗ Corresponding author. Tel.: +91 9416492413. E-mail address: [email protected] (C.S. Pundir). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.07.008
platinum surface [7], titania gel matrix [8] through adsorption; carbon nanotubes–chitosan composite [9] by crosslinking; hydrophilic matrix [10] by covalent coupling. All these biosensors suffer from leakage of enzyme except that based on covalent coupling and had a drawback of limited electron flow and storage stability. In recent years, the use of nanomaterials has led to improvement of the analytic performance of enzyme electrodes. Carbon nanotubes (CNTs) are known for their good electrocatalytic properties, unique structure, high surface-to-volume ratio, metallic, semiconducting and superconducting electron transport and high capacity for storing guest molecules [11–14], which make them extremely attractive nanomaterial for electrochemical biosensors. Composite materials based on integration of CNT with some conducting polymer have been exploited further for improvement in the analytic performance of electrochemical biosensors. Polyaniline (PANI) is unique among conducting polymers, primarily due to its high conductivity and chemical durability, good environmental stability and reversible control of conductivity both by protonation and by charge-transfer doping [15,16]. Thus, PANI/CNT composite film can be a useful platform for immobilizing biomolecules to improve analytic performance of biosensor [17]. Such a composite-modified electrode combines the ability of CNTs and conductive polymer to promote electron transfer reactions with the advantages of entrapping biological material.
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Recently, nickel nanoparticles (NiNPs) have received special attention as a support for immobilization of biomolecules and offer potential applications in many fields such as electrochromics, electrocatalysis, supercapacitor, as NiNPs make electrode surface more porous thus, facilitating electron transfer and possess biocompatibility, irreversible magnetization and long stability [18–20]. We describe herein the construction of an amperometric biosensor for determination of total phenolic content by fabricating NiNPs/PANI/MWCNTs/Au electrode and then immobilizing covalently a laccase (purified from Ganoderma sps) onto this modified electrode. Such a nano-hybrid enzyme electrode is expected to provide a biosensor with highly improved electrocatalytic efficiency, sensitivity, storage stability and reusability. 2. Materials and methods 2.1. Reagents Sephadex G-100 (40–120 m bead size, 1 g swells to 15–20 mL gel) and DEAE-Sephacel (40–160 m (wet), exclusion limit ∼1,000,000 Da, capacity 100–140 eq/mL) from Sigma (Aldrich), St. Louis, USA. Aniline (purity 99%, irritant), nickel chloride (purity >97%, irritant and carcinogenic), sodium dodecyl sulfate (SDS) (purity 90%, irritant), hydrazine (purity >99.0%, irritant) and polyvinyl pyrrolidone (PVP) (purity 99.9%) from MERCK India Pvt. Ltd. Guaiacol (purity 98–99%, irritant), Potassium ferricyanide (purity 100%, irritant), potassium ferrocyanide (purity 100%, irritant) from Sisco Research Laboratory (SRL), Mumbai, India were used. Carboxylated multi-walled carbon nanotubes (c-MWCNT) (90%, 12 walls, 15–30 m length, nil metal content) (functionalized MWCNT) from Intelligent Materials Pvt. Ltd., Panchkula (Haryana), India were used. Gold wire (1.5 cm × 0.05 cm) (23 carat) was purchased from local market. All other chemicals used were of AR grade. Fruit juices of various commercial brands were purchased from local market. Double distilled water (DW) was used throughout the experiments. 2.2. Purification of laccase The cell free extract of Ganoderma sp. Rckk02 grown in malt extract broth (MEB) [21,22] was used as crude laccase. The crude enzyme was purified by 0–80% ammonium sulfate precipitation, gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-Sephacel using linear gradient of KCl (0.1–0.6 M) in succession at 4–10 ◦ C. 2.3. Assay of laccase Assay of laccase was based on the oxidative polymerization of guaiacol [21]. The reaction mixture contained 0.2 mol of guaiacol, 230 mol acetate buffer (pH 5.0) and enzyme in a total volume of 2.5 mL. Change in absorbance of 0.01 min−1 mL−1 at 470 nm was studied. One enzyme unit is defined as unit activity=
A470 /min × total volume of reaction mixture extinction coefficient of guaiacol×volume of enzyme
Protein content in various enzyme preparations was determined by Lowry method using bovine serum albumin as standard protein. 2.4. Preparation of nickel nanoparticles (NiNPs) NiNPs were prepared as described in ref. [23]. Fifty mL of SDS solution (20 g/L) and 50 mL of PVP solution (20 g/L) were added to 100 mL of 10% NiCl2 solution in a glass beaker kept at 40 ◦ C. The pH of the nickel solution was then increased to 10.2 by adding saturated Na2 CO3 solution and heated to 60 ◦ C. Hydrazine (150 mL) was
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added to this heated solution slowly, under constant stirring. The appearance of gray/black precipitates in the beaker indicated the formation of NiNPs. This reaction was not instantaneous and took about 4–5 h for the nickel reduction. 2.5. Preparation of NiNPs/cMWCNT/PANI/Au electrode Prior to electrodeposition, the gold electrode was polished with alumina slurry. One mg of c-MWCNT was suspended in 1 mL mixture of concentrated H2 SO4 and HNO3 in 3:1 ratio and ultrasonicated for 2 h to get a finely dispersed black colored solution of c-MWCNTs. Aniline (50 L) was added to 10.0 mL of 1 M HCl and mixed with 1.0 mL finely dispersed solution of c-MWCNT in a glass cell. c-MWCNT and aniline were electrodeposited, through cyclic voltammetry (CV), onto Au electrode by immersing it in 25 mL of 1 M KCl solution by applying 30 polymerization cycles at 0.0–1.5 V [24]. The resulting cMWCNT/PANI/Au modified electrode was washed thoroughly with distilled water to remove unbound matter and kept in dry petri plate at 4 ◦ C. The electrodeposition of NiNPs onto the cMWCNT/PANI modified Au electrode was carried out by immersing the modified electrode into a mixture of 23 mL of 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) and 2 mL of NiNPs solution and then applying potential between 0–1.5 V (vs. Ag/AgCl) for 25 cycles at a scan rate of 0.02 V s−1 . After rinsing with DW, the NiNPs/cMWCNTs/PANI/Au electrode was dried in air. K3 Fe(CN)6 /K4 Fe(CN)6 was used as an electrolyte, as it acts as a very good redox probe, which is known to exhibit nearly a reversible electrode reaction without any complications of post chemical reactions. 2.6. Preparation of working electrode (Lac/NiNPs/cMWCNT/PANI/Au) The purified laccase was immobilized onto the surface of NiNPs/cMWCNTs/PANI/Au electrode through covalent coupling by layering 10 L of enzyme solution (protein 40 mg mL−1 ) in 0.1 M acetate buffer (pH 5.0) and keeping it undisturbed for about 12 h at 4 ◦ C. The electrode was finally washed with distilled water to remove unbound enzyme. The protein concentration in wash out solution was determined. The principle of working of this biosensor was as follow: guaiacol (standard phenolic substrate) was oxidized to its corresponding o-quinone by NiNPs/cMWCNT/PANI/Au electrode bound laccase and then regenerated through electrochemical reduction of oquinone, thus forming a bioelectrocatalytic amplification cycle and thereby generating electrons, which were passed to working electrode from solution and relayed to potentiometer, in which it was read as current in A (Ampere). The electrons then reached Ag/AgCl electrode, where Ag+ ions were reduced to Ag metal. The current was measured, which was directly proportional to guaiacol concentration. The resulting enzyme electrode was stored in refrigerator at 4 ◦ C, when not in use. 2.7. Characterization of enzyme electrode The modified Au electrode, at different stages of its construction was characterized by studying its scanning electron microscopic (SEM) images in scanning electron microscope (make: Joel Japan, model: JSM-6510, at Advanced Instrumentation Research Facility, JNU, New Delhi). Fourier transform infrared (FTIR) spectra in FTIR spectometer (make: Thermoelectron, USA, model: iS10) and cyclic voltammetric and electrochemical impedance spectroscopic (EIS) studies on potenitostat/galvanostat (make: Autolab, Eco Chemie BV, Netherlands, model: AUT83785 with the GPES 4.9 software) with three electrode system, Lac/NiNPs/cMWCNT/PANI/Au
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electrode as the working electrode, a Pt wire as the auxiliary electrode and an Ag/AgCl (saturated 3 M KCl) electrode as the reference electrode. The EIS measurements were carried out in 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] in the 0.1 M PB pH 7.0 at ambient temperature, at the polarization potential of 0.2 V (vs Ag/AgCl) in the frequency range 0.1–104 Hz and with the amplitude of 10 mV. The resulting enzyme electrode (Lac/NiNPs/cMWCNT/PANI/Au electrode) was stored in refrigerator at 4 ◦ C, when not in use. To record FTIR spectra of hybrid material deposited onto Au electrode, it was scrapped off the Au electrode, mixed with dried KBr and its pellet was formed by hydraulic pellet press. Then this pellet was kept into the socket of the FTIR spectrometer and its spectrum was recorded.
2.8. Response measurement of Lac/NiNPs/cMWCNT/PANI/Au All electrochemical characterizations and measurements were carried out using a conventional three-electrode system with the Lac/NiNPs/cMWCNT/PANI/Au electrode as the working electrode, a Pt wire as the auxiliary electrode and an Ag/AgCl (saturated 3 M KCl) electrode as the reference electrode. Cyclic voltammetric measurements were carried out in a three-electrode cell containing 20 mL electrolyte [2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1)], 5 mL acetate buffer (0.1 M, pH 5.0) and 0.1 mL of guaiacol (10 M). Current measurements were performed applying CV in the potential range, 0 and +1.5 V (vs Ag/AgCl). To record cyclic voltammograms, the following instrumental parameters were used: step potential +6 mV and scan rate 20 mV s−1 . All electroanalytical measurements were carried out at room temperature.
2.9. Optimization of Lac/NiNPs/cMWCNT/PANI/Au electrode To determine the optimum working conditions of the enzyme electrode, the pH of reaction buffer (0.1 M) was varied from pH 3.0 to 7.0 at an interval of pH 0.5 using acetate buffer in acidic pH range (pH 3.0–5.5) and phosphate buffer in pH range 6.0–7.0. Similarly, to determine optimum temperature, the reaction mixture was incubated at 15–50 ◦ C at an interval of 5 ◦ C. To determine the optimum response time, the current response was measured from 2 s to 12 s at an interval of 2 s. To study the effect of substrate concentration, different guaiacol concentration, ranging from 0.1 to 500 M were used.
Fig. 1. The calibration curve for phenolic biosensor Inset: Calibration curve at a concentration below 10 M guaiacol. Error bars represent the standard deviation for three independent measurements.
2.11. Amperometric measurement of total phenolic content in fruit juices The biosensor was employed for determination of phenolic content in five commercial brands of fruit juices. The fruit juice samples were diluted 10 times with acetate buffer (pH 5.5). Total phenolic content in fruit juice samples was determined by the biosensor in the same manner as described above for its response measurement under its optimal working conditions except that guaiacol was replaced by these sample. The current (A) was recorded and the amount of total phenolic content was interpolated from standard curve between guaiacol concentrations and current (A) prepared under optimal working conditions (Fig. 1).
3. Results and discussion 3.1. Characterization of NiNPs The morphological characterization of nickel nanoparticles was carried out by Transmission electron microscope (TEM) at SAIF, Punjab University, Chandigarh. TEM image of prepared NiNPs showed their spherical shape with different diameter in the range, 10–100 nm revealing their different size but not shape (Fig. 2).
2.10. Evaluation The following criteria was studied to evaluate the analytic performance of the biosensor, e.g. linearity, analytical recovery, detection limit, precision and correlation. Analytical recovery was studied using red wines with two different standard concentrations (0.1 mM) of guaiacol. To test the reproducibility and reliability of the present biosensor, phenolic content in six fruit juice samples was measured five times on single day (within batch) and five times again after their storage at −20 ◦ C for one week (between batch). To study the correlation the phenolic content was measured in 10 brands of fruit juice samples by the standard spectrophotometric method and the present method and their correlation was studied using regression equation. To examine the long-term storage stability, the activity of enzyme electrode was tested upto three months at an interval of one week, when stored at 4 ◦ C. To reuse the enzyme electrode, it was washed 3–4 times with reaction buffer before its use in next assay.
Fig. 2. Transmission electron microscopic (TEM) image of nickel nanoparticles.
S. Chawla et al. / Biochemical Engineering Journal 68 (2012) 76–84
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Fig. 3. Scanning electron micrographs of (a) bare gold electrode (b) cMWCNT/PANI/Au electrode (c) Lac/NiNPs/cMWCNT/PANI/Au electrode.
3.2. Electrode surface characterization by SEM and FTIR Fig. 3 shows morphology of bare Au electrode, cMWCNTs/PANI/Au electrode and Lac/NiNPs/cMWCNT/PANI/Au electrode as characterized by scanning electron micrographs (SEM) studies. The SEM of the bare Au electrode (Fig. 3a) showed a homogeneous surface. The cMWCNTs/PANI composite film showed net like structure (Fig. 3b). Film was more uniform and porous and hence effective surface area was larger. The SEM images for Lac/NiNPs/cMWCNT/PANI/Au showed a regular globular structural morphology (Fig. 3c), indicating the successful immobilization of laccase to the surface of cMWCNT/PANI layer. Fig. 4 displays the FTIR spectra for cMWCNT/PANI/Au (curve a) and Lac/NiNPs/cMWCNT/PANI Au (curve b). The FTIR spectrum of electrodeposited cMWCNT/PANI composite (curve a) showed benzenoid and quinoid ring stretching bands (C C) present at 1447 and 1595 cm−1 . The presence of peaks at 1129 and 3402 cm−1 could be attributed to B N+ Q stretching and N H stretching vibrations of PANI in the composite. The peak at 1710 cm−1 is attributed to C O stretching vibrations indicating the presence
of carboxyl group ( COOH). The presence of these peaks reveals the existence of both PANI and cMWCNT on the Au electrode. The Lac/NiNPs/cMWCNT/PANI/Au electrode (curve b) showed characteristic peaks at 1550 and 1600 cm−1 which could be attributed to the primary and secondary amide linkages between NH2 groups on the surface group on the surface of enzyme and free COOH groups of MWCNT. Also the peaks for free COOH groups were not found in curve b, which confirms that the free COOH groups have been occupied by NH2 group of enzyme, thus conforming the formation of a strong covalent bond. method is described for construction of a A Lac/NiNPs/cMWCNT/PANI/Au electrode. Scheme 1 summarizes the different chemical reactions involved in the fabrication of lac electrode based on covalent immobilization of lac onto NiNPs/cMWCNT/PANI/Au electrode through amide bond formation between the free COOH groups of carboxylated cMWCNT of NiNPs/cMWCNT/PANI/Au electrode and free NH2 groups on surface of enzyme. Our results showed that NiNPs and cMWCNTs provided a remarkable synergistic effect toward the oxidation of guaiacol.
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Fig. 4. The infrared spectra of (a) c-MWCNT/PANI/Au electrode and (b) Lac/NiNPs/cMWCNT/PANI/Au electrode.
3.3. Cyclic voltammetry study To evaluate the charge-transfer properties on the surface of the modified electrodes, cyclic voltammetry technique was employed, using potassium ferricyanide–potassium ferrocyanide mixture as electrolyte. Cyclic voltammograms were recorded in 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] and 0.1 M acetate buffers (pH 5.5). CV of cMWCNT/PANI/Au revealed two redox peaks for
oxidation of aniline to radical cation and then to radical dication [25]. The electrodeposition of NiNPs onto cMWCNT/PANI modified Au electrode surface leads to increase in current intensity, as a result of increase in electroactive area. The preservation of a quasi reversible electrode process and the large increase in peak currents for the nanocomposite film modified electrode proved that NiNPs exerted an obvious improvement effect on cMWCNT/PANI/Au electrode.
Scheme 1. Scheme of chemical sequence of electropolymerization of NiNPs/cMWCNT/PANI onto Au electrode and chemical reaction of immobilization of laccase on modified electrode.
S. Chawla et al. / Biochemical Engineering Journal 68 (2012) 76–84
525
(A)
(c)
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i / μA
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i / μA
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Fig. 6. The Nyquist plots of EIS of comparison of bare Au electrode (curve a) c-MWCNT/PANI/Au electrode (curve b) NiNPs/cMWCNT/PANI/Au electrode (curve c) and Lac/NiNPs/cMWCNT/PANI/Au electrode (curve d) in 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 .
50 25 0
(b)
-25 -50
0
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Fig. 5. (A) Comparative cyclic voltammogram of (a) c-MWCNT/PANI/Au electrode (b) NiNPs/cMWCNT/PANI/Au electrode (c) Lac/NiNPs/cMWCNT/PANI/Au electrode. (B) Cyclic voltammetric (CV) curves of Lac/NiNPs/cMWCNT/PANI/Au electrode in 0.1 M acetate buffer (pH 6.0) (a) with and (b) without 10 M guaiacol solution. Supporting electrolyte: 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)]; scan rate: 20 mV s−1 .
Fig. 5A reveals the comparative cyclic voltammograms of the c-MWCNT/PANI modified Au electrode (curve a) and NiNPs/cMWCNT/PANI (curve b) in a solution of 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] and acetate buffer (0.1 M, pH 5.0) at 20 mV s−1 . The rise in current clearly indicated the role of NiNPs in increase of conductivity of modified electrode. 3.4. Response toward guaiacol at the Lac/NiNPs/cMWCNT/PANI/Au Electrode To evaluate the catalytic activity of laccase immobilized onto NiNPs/cMWCNT/PANI/Au electrode, the modified electrode was characterized by a cyclic voltammogram in the presence of guaiacol in the potential range from 0 to +1.5 V (vs Ag/AgCl). Fig. 5B shows cyclic voltammograms of the NiNPs/cMWCNT/PANI/Au electrode in an unstirred 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] solution and 0.1 M acetate buffer (pH 5.5) with (curve a) and without guaiacol solution (curve b) at scan rate 20 mV s−1 . It was observed that with the addition of 2 M guaiacol, both the reduction current and oxidation current increased, which showed the improved catalytic properties of modified electrode to the reduction of guaiacol. 3.5. Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) provides useful information on the impedance changes of the electrode surface during the fabrication process. The semicircular portion at higher frequencies corresponds to the electron-transfer-limited process and its diameter is equal to the electron transfer resistance, which controls the electron transfer kinetics at the electrode interface. Meanwhile, the linear part at lower frequencies corresponds to the diffusion process [26]. Fig. 6 displays the Nyquist plots of the EIS of (a) bare Au electrode (b) cMWCNT/PANI/Au electrode (c) NiNPs/cMWCNT/PANI/Au electrode and (d) Lac/NiNPs/cMWCNT/PANI/Au electrode in 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] in solution of 0.1 M PB pH 7.0, at the
polarization potential of 0.2 V in the frequency range 0.1–104 Hz. RCT value ∼50 correspond to the bare Au (curve a). Nyquist diameter cMWCNT/PANI/Au (curve (b), RCT 400 ), was much larger than NiNPs/cMWCNT/PANI/Au electrode (curve (c), RCT 192.85 ) which suggests that cMWCNT/PANI modified electrode increased the resistance of the electrodes [27] whereas NiNPs act as a nanoscale electrode, promoting electron transfer between the redox probe and the electrode. However, after immobilization of laccase onto NiNPs/cMWCNT/PANI/Au electrode, RCT was found to increase to 570.50 (curve d). This suggests that immobilized molecules strongly bind with hybrid nanobiocomposite and block charge carriers in the nanobiocomposite matrix. Also this increase could be attributed to the fact that most biological molecule, including enzyme, are poor electrical conductors at low frequency and cause hindrance to electron transfer. These results confirm the success of the assembly on the modified electrode. The change in the RCT value reflecting the increase or decrease in the diameter of the semicircle at high frequencies in the impedance spectra is associated with the blocking behaviour of the electrode surface for the charge transfer to the redox probe. 3.6. Optimization of experimental conditions of biosensor To optimize the biosensor (NiNPs/cMWCNT/PANI/Au electrode) the effect of pH, incubation temperature and scan rate were studied. The CV at different pH (Fig. 7a) showed that the current response resulting from the NiNPs/cMWCNT/PANI/Au bound enzyme-catalyzed reaction achieved a maximum value at pH 5.5 (Fig. 7b), which is higher than that immobilized onto different supports, e.g. Lentinula edodes laccase immobilized onto chitosan (pH 4.0) [28], Pycnoporus sanguineus laccase immobilized onto magnetic chitosan microspheres (pH 3.0) [29] but lower than that of Trametes versicolor laccase immobilized onto platinum nanoparticles (pH 6.5) [30]. Therefore, a pH of 5.5 was used in further experiments. The CV at different temperature (Fig. 8a) exhibited the optimum temperature at 35 ◦ C (Fig. 8b) which is lower than that immobilized on magnetic chitosan microspheres (55 ◦ C) [29] but higher than that carbon paste modified graphite electrode [31] and nanoparticles (25 ◦ C) [32]. Polyaniline being a good thermal insulator for enzymes might have provided the microenvironment to protect the enzyme from environmental and thermal variations, which favours the repeated use of NiNPs/cMWCNT/PANI electrode [15]. The biosensor showed maximum response at 8 s (Fig. 9). The effect of the scan rate from 10 to 100 mV s−1 on the biosensor response using 2 M guaiacol in 0.1 M acetate buffer solution (pH 5.5) was also investigated. A potential scan rate of 20 mV s−1 was
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Table 1 A comparison of various analytical and kinetic properties of polyphenol biosensors based on laccase immobilized on different supports. Property
Jiang et al., 2005 [29]
Liu et al., 2006 [9]
Vianello et al., 2006 [10]
Kochana et al., 2008 [8] Corman et al., 2010 [32]
Chawla et al., 2011 [36] Present
– Silane-modified platinum surface Adsorption
– Carbon nanotubes chitosan composite Cross-linking
– Hydrophilic matrix
– Titania gel matrix
Trametes versicolor Nanoparticles
Ganoderma lucidium NC membrane
Ganoderma lucidium NiNPs/cMWCNT/Au
Covalent coupling
Adsorption
Adsorption
5.5 NR
Pycnoporus sanguineus Magnetic chitosan microspheres Glutaraldehyde cross-linking 3.0 55
6.0 NR
5.0 22
6.0 25
6.0 25
Covalent coupling and adsorption 6.0 35
Covalent coupling and adsorption 5.5 35
Current
Current
Current
Current
Current
Current
Current
Current
60
150
150
100
40
125
120
120
NC: Nitrocellulose; NiNPs: Nickel nanoparticles; cMWCNT: carboxylated multiwalled carbon nanotubes
(a)
(b)
Ι / μA
i / μA
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8
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Fig. 7. CV curves (a) and graphical representation (b) for effect of pH on the current response of Lac/NiNPs/cMWCNT/PANI/Au electrode. Error bars represent the standard deviation for three independent measurements.
20 25 30 35 40 45 50
0.250
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selected, since with these experimental conditions, the highest analytical signal and very good cyclic voltammogram profiles were obtained.
0
30
Temperature / ºC
3.7. Evaluation of biosensor
275
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0
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3.7.1. Linearity There was a linear relationship between current (A) and guaiacol concentration ranging from 0.1–10 M (lower
i / μA
(a) 300
(b) 350
300
250
200
150
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0 10
Fig. 8. CV curves (a) and graphical representation (b) for effect of temperature on the current response of Lac/NiNPs/cMWCNT/PANI/Au electrode. Error bars represent the standard deviation for three independent measurements.
I/μA
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Source of laccase Support for immobilization Method of immobilization Optimum pH Optimum temperature (◦ C) Mode of measurement Storage life at 4 ◦ C (days)
Quan et al., 2004 [7]
S. Chawla et al. / Biochemical Engineering Journal 68 (2012) 76–84
500
Table 2 Comparison of total phenolic content in different brands of fruit juices as measured by NiNPs/cMWCNT/PANI/Au electrode and standard spectrophotometric method.
450
Current (μA)
400 350 300 200 150 100 50 0
2.5
5
7.5
10
12.5
Fig. 9. Time course study of Lac/NiNPs/cMWCNT/PANI/Au electrode. Error bars represent the standard deviation for three independent measurements. *
Phenolic content (μmoles) as measured by present method
Samples Fruit juices
Total phenolic content by present method Mean ± SD* (n = 4) (mol guaiacol/g)
Brand a Brand b Brand c Brand d Brand e Brand f Brand g Brand h Brand i Brand j
0.498 0.520 0.772 0.642 0.494 0.616 0.580 0.672 0.657 0.798
250
0
0.9 0.8
83
± ± ± ± ± ± ± ± ± ±
0.008 0.012 0.013 0.014 0.013 0.008 0.012 0.013 0.014 0.013
Total phenolic content by standard spectrophotometric method Mean ± SD* (n = 4) (mol guaiacol/g) 0.490 0.510 0.765 0.636 0.490 0.606 0.568 0.660 0.650 0.790
± ± ± ± ± ± ± ± ± ±
0.012 0.020 0.022 0.031 0.019 0.022 0.027 0.020 0.019 0.026
p > 0.05.
method. Within and between coefficients of variation were <2.03% and <3.82% respectively. The high precision indicated the good reproducibility and consistency of the present method.
y = 0.9991x - 0.0078 R² = 0.9994
3.7.3. Correlation A Comparison of phenolic content in 10 fruit juice samples as measured by the present method (y) with those obtained by standard spectrophotometric method (x) showed a good correlation with r = 0.99, regression equation being, y = 0.994x + 9.6785 (Fig. 10). These results indicate the high accuracy of the method.
0.7 0.6 0.5 0.4
Fig. 10. Correlation between total phenolic content values determined by standard spectrophotometric method (x-axis) and present biosensor employing MnNPs/cMWCNT/PANI/Au electrode method (y-axis). Error bars represent the standard deviation for three independent measurements.
concentration range) with linear equation being y = 0.923x + 49.96 and 10–500 M (higher concentration range) with linear equation being y = 5.826x + 1.192 (Fig. 1), which is better than earlier biosensor based on carbon paste modified graphite electrode (1.97 × 10−4 to 3.24 × 10−3 M)] [31], polyether sulfone membrane [1 × 10−5 to 8 × 10−5 M] [33], based on chitosan membrane modified with tripolyphosphate [5.99 × 10−7 to 3.92 × 10−6 M] [34] and carbon nanotubes–chitosan composite [3 × 10−7 to 1.2 × 10−6 M] [35]. The detection limit of the biosensor was 0.05 (S/N = 3). 3.7.2. Recovery and precision The analytical recovery of added guaiacol in fruit juice (0.1 mM) was 95.61%, demonstrating the satisfactory accuracy of the present
3.7.4. Interference study The addition of the following interferrants such as ascorbic acid (4.8 M), cysteine (0.24 M), fructose (60 M), glucose (22.2 M), citric acid (100 M) and ethanol (70 M) evoked a negligible interference (a decrease of only 9.76% for glucose, 10.63% for citrate, 5.99% for fructose, 9.13% for cysteine and 8.66% for ascorbic acid on the biosensor response). 3.7.5. Storage stability and reusability of biosensor The electrode lost 15% of its initial activity after its 200 uses during the span of four months when stored at 4 ◦ C. The properties of present biosensor have been compared with previous biosensors (Table 1). 3.7.6. Determination of total phenolic content in fruit juice samples Total phenolic content as measured from CV curves (Fig. 11) by the present sensor ranged from 0.494 to 0.798 mol guaiacol/g in ten different fruit samples of different brands (Table 2). 4. Conclusion
300 a b c d e f g
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i / μA
250 225 200
h i j
0 -200 -225
0
0.250
0.500
0.750
1.000
E/V
1.25
1.500
1.750
Fig. 11. Cyclic voltammetric (CV) curves for determination of total phenolic content in different fruit juice samples.
We constructed a novel composite of NiNPs and c-MWCNT/PANI at Au electrode for electrocatalysis, which is stable for a long time at pH 5.5 in 0.1 M acetate buffer solution. Furthermore the composite provides covalent immobilization of laccase onto modified electrode as confirmed by FTIR. This Lac/NiNPs/cMWCNT/PANI modified Au electrode exhibited improved performance of biosensor in terms of wide linear range viz. 0.1–10 M (lower concentration range) and 10–500 M (higher concentration range), response time (8 s), detection limit (0.05 mM), sensitivity (0.694 A M−1 cm−2 ), reusability (more than 200 times) and stability (four months). Thus this work illustrates a simple and novel approach for the development of an improved amperometric enzyme sensor for determination of phenolic compounds.
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[20] N. Zhang, C. Xu, Q. Wei, B. Du, R. Li, T. Zhang, D. Wu, Y. Dai, Studies on the interaction of DNA with vitamin B12 based on the immobilization of dsDNA on nano-scale hydroxyapatite coating, Adv. Mater. Lett 1 (2010) 34–39. [21] K. Vasdev, R.C. Kuhad, Induction of laccase production in C. bulleri under shaking and static culture conditions, Folia Microbiol. 39 (1994) 326–330. [22] S. Dhawan, R. Lal, R.C. Kuhad, Ethidium bromide stimulated hyper laccase production from birds nest fungus Cyathus bulleri, Lett. Appl. Microbiol. 36 (2003) 64–67. [23] M.S. Hussain, K.M.A. Haque, Synthesis of nano-sized nickel particles by a bottom-up approach in the presence of an anionic surfactant and a nonionic polymer, J. Sci. Res. 2 (2010) 313–321. [24] S. Chawla, R. Rawal, C.S. Pundir, Fabrication of polyphenol biosensor based on laccase immobilized on copper nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode, J. Biotechnol. 156 (2011) 39–45. [25] D.L. Feldheim, C.A. Foss Jr., in: D.L. Feldheim, C.A. Foss Jr. (Eds.), Metal Nanoparticles: Synthesis, Characterization and Applications, Dekker, New York, 2002, pp. 1–14. [26] J.J. Feng, G. Zhao, J.J. Xu, H.Y. Chen, Direct electrochemistry and electrocatalysis of heme proteins immobilized on gold nanoparticles stabilized by chitosan, Anal. Biochem. 342 (2005) 280–286. [27] Y.Q. Miao, S.N. Tan, Amperometric hydrogen peroxide biosensor based on immobilization of peroxidase in chitosan matrix crosslinked with glutaraldehyde, Analyst 125 (2000) 1591–1594. [28] A. D’Annibale., S.R. Stazi, V. Vinciguerra, E.D. Mattia, G.G. Sermann, Characterization of immobilized laccase from Lentinula edodes and its use in olive-mill wastewater treatment, Process Biochem. 34 (1999) 697–706. [29] D.S. Jiang, S.Y. Long, J. Huang, H.Y. Xiao, J.Y. Zhou, Immobilization of Pycnoporus sangunieus laccase on magnetic chitosan microspheres, Biochem. Eng. J. 25 (2005) 15–23. [30] D. Brondani, C.W. Scheeren, J. Dupont, I.C. Vieira, Biosensor based on platinum nanoparticles dispersed in ionic liquid and laccase for determination of adrenaline, Sens. Actuat. B 140 (2009) 252–259. [31] M. Santhiago, I.C. Vieira, l-cysteine determination in pharmaceutical formulation using a biosensor based on laccase from Aspergillus oryzae, Sens. Actuat. B 128 (2007) 279–285. [32] M.E. Corman, N. Öztürk, N. Bereli, S. Akgölc, A. Denizli, Preparation of nanoparticles which contains histidine for immobilization of Trametes versicolor laccase, J. Mol. Catal. B: Enzym. 63 (2010) 102–107. [33] S.A.S.S. Gommes, M.J.F. Rebelo, A new laccase biosensor for polyphenols determination, Sensors 3 (2003) 166–175. [34] S.C. Fernandes, I.R.W.Z de Oliveira, O.A. Fatibello-Filho Spinelli, I. Cruz Vieira, Biosensor based on laccase immobilized on microspheres of chitosan crosslinked with tripolyphosphate, Sens. Actuat. B: Enzym. 133 (2008) 202–207. [35] G. Singh, A. Bhalla, N. Capalash, P. Sharma, Characterization of immobilized laccase from ␥-proteobacterium JB: approach towards the development of biosensor for the detection of phenolic compounds, Ind. J. Sci. Technol. 3 (2010) 48–52. [36] C.S. Pundir, R. Rawal, S. Chawla, Renuka, Development of an amperometric polyphenol biosensor based on fungal laccase immobilized on nitrocellulose membrane, Artif. Cells Blood Substit. Immobil. Biotechnol. 40 (2012) 163–170.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
An amperometric biosensor based on laccase immobilized onto nickel nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode for determination of phenolic content in fruit juices Sheetal Chawla, Rachna Rawal, Swati Sharma, Chandra Shekhar Pundir ∗ Department of Biochemistry, M.D. University, Rohtak 124001, Haryana, India
a r t i c l e
i n f o
Article history: Received 16 January 2012 Received in revised form 23 May 2012 Accepted 11 July 2012 Available online 20 July 2012 Keywords: Biosensors Enzymes Immobilisation Optimisation NiNPs cMWCNT
a b s t r a c t A method is described for construction of an enzyme electrode for detection of phenolic compounds based on covalent immobilization of laccase onto nickel nanoparticles (NiNPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto gold (Au) electrode. The modified electrode was characterized at different stages of its construction by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammograms and electrochemical impedance spectroscopy (EIS). An amperometric biosensor for phenolic compounds was fabricated by connecting enzyme electrode (Lac/NiNPs/cMWCNTs/PANI/AuE) as working electrode, with Ag/AgCl as reference electrode and Pt wire as auxiliary electrode through potentiostat. The biosensor showed optimum response at pH 5.5 (0.1 M acetate buffer) and 35 ◦ C, when operated at a scan rate of 20 mV s−1 . Linear range, response time, detection limit and sensitivity of biosensor were 0.1–10 M (lower concentration range) and 10–500 M (higher concentration range), 8 s, 0.05 M and 0.694 A M−1 cm−2 respectively. The biosensor measured total phenolic content in fruit juices. The enzyme electrode was used 200 times over a period of four months, when stored at 4 ◦ C. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Polyphenols are the most abundant antioxidants in our diet. Diets containing an abundance of antioxidants are protective against a variety of diseases, particularly cardiovascular diseases and cancer. A number of analytical techniques are available for determination of phenolic content such as spectrophotometry, gas chromatography, liquid chromatography and capillary electrophoresis [1]. However, some of these techniques (e.g. spectrophotometry) are not sensitive and specific, while others (e.g. gas chromatography, liquid chromatography and capillary electrophoresis) are cumbersome and require time consuming sample pre-treatment, expensive equipments and skilled persons to operate. Therefore, there is an interest in developing a simple, sensitive, rapid, cost-effective and portable system such as biosensor for determination of phenolic compounds in important areas such as clinical, biomedical, environmental contamination, industrial and pharmaceutical analysis [2–4]. A number of biosensors for detection of phenolic compounds have been reported based on immobilization of laccase onto various supports such as graphite electrode [5,6], silane-modified
∗ Corresponding author. Tel.: +91 9416492413. E-mail address: [email protected] (C.S. Pundir). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.07.008
platinum surface [7], titania gel matrix [8] through adsorption; carbon nanotubes–chitosan composite [9] by crosslinking; hydrophilic matrix [10] by covalent coupling. All these biosensors suffer from leakage of enzyme except that based on covalent coupling and had a drawback of limited electron flow and storage stability. In recent years, the use of nanomaterials has led to improvement of the analytic performance of enzyme electrodes. Carbon nanotubes (CNTs) are known for their good electrocatalytic properties, unique structure, high surface-to-volume ratio, metallic, semiconducting and superconducting electron transport and high capacity for storing guest molecules [11–14], which make them extremely attractive nanomaterial for electrochemical biosensors. Composite materials based on integration of CNT with some conducting polymer have been exploited further for improvement in the analytic performance of electrochemical biosensors. Polyaniline (PANI) is unique among conducting polymers, primarily due to its high conductivity and chemical durability, good environmental stability and reversible control of conductivity both by protonation and by charge-transfer doping [15,16]. Thus, PANI/CNT composite film can be a useful platform for immobilizing biomolecules to improve analytic performance of biosensor [17]. Such a composite-modified electrode combines the ability of CNTs and conductive polymer to promote electron transfer reactions with the advantages of entrapping biological material.
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Recently, nickel nanoparticles (NiNPs) have received special attention as a support for immobilization of biomolecules and offer potential applications in many fields such as electrochromics, electrocatalysis, supercapacitor, as NiNPs make electrode surface more porous thus, facilitating electron transfer and possess biocompatibility, irreversible magnetization and long stability [18–20]. We describe herein the construction of an amperometric biosensor for determination of total phenolic content by fabricating NiNPs/PANI/MWCNTs/Au electrode and then immobilizing covalently a laccase (purified from Ganoderma sps) onto this modified electrode. Such a nano-hybrid enzyme electrode is expected to provide a biosensor with highly improved electrocatalytic efficiency, sensitivity, storage stability and reusability. 2. Materials and methods 2.1. Reagents Sephadex G-100 (40–120 m bead size, 1 g swells to 15–20 mL gel) and DEAE-Sephacel (40–160 m (wet), exclusion limit ∼1,000,000 Da, capacity 100–140 eq/mL) from Sigma (Aldrich), St. Louis, USA. Aniline (purity 99%, irritant), nickel chloride (purity >97%, irritant and carcinogenic), sodium dodecyl sulfate (SDS) (purity 90%, irritant), hydrazine (purity >99.0%, irritant) and polyvinyl pyrrolidone (PVP) (purity 99.9%) from MERCK India Pvt. Ltd. Guaiacol (purity 98–99%, irritant), Potassium ferricyanide (purity 100%, irritant), potassium ferrocyanide (purity 100%, irritant) from Sisco Research Laboratory (SRL), Mumbai, India were used. Carboxylated multi-walled carbon nanotubes (c-MWCNT) (90%, 12 walls, 15–30 m length, nil metal content) (functionalized MWCNT) from Intelligent Materials Pvt. Ltd., Panchkula (Haryana), India were used. Gold wire (1.5 cm × 0.05 cm) (23 carat) was purchased from local market. All other chemicals used were of AR grade. Fruit juices of various commercial brands were purchased from local market. Double distilled water (DW) was used throughout the experiments. 2.2. Purification of laccase The cell free extract of Ganoderma sp. Rckk02 grown in malt extract broth (MEB) [21,22] was used as crude laccase. The crude enzyme was purified by 0–80% ammonium sulfate precipitation, gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-Sephacel using linear gradient of KCl (0.1–0.6 M) in succession at 4–10 ◦ C. 2.3. Assay of laccase Assay of laccase was based on the oxidative polymerization of guaiacol [21]. The reaction mixture contained 0.2 mol of guaiacol, 230 mol acetate buffer (pH 5.0) and enzyme in a total volume of 2.5 mL. Change in absorbance of 0.01 min−1 mL−1 at 470 nm was studied. One enzyme unit is defined as unit activity=
A470 /min × total volume of reaction mixture extinction coefficient of guaiacol×volume of enzyme
Protein content in various enzyme preparations was determined by Lowry method using bovine serum albumin as standard protein. 2.4. Preparation of nickel nanoparticles (NiNPs) NiNPs were prepared as described in ref. [23]. Fifty mL of SDS solution (20 g/L) and 50 mL of PVP solution (20 g/L) were added to 100 mL of 10% NiCl2 solution in a glass beaker kept at 40 ◦ C. The pH of the nickel solution was then increased to 10.2 by adding saturated Na2 CO3 solution and heated to 60 ◦ C. Hydrazine (150 mL) was
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added to this heated solution slowly, under constant stirring. The appearance of gray/black precipitates in the beaker indicated the formation of NiNPs. This reaction was not instantaneous and took about 4–5 h for the nickel reduction. 2.5. Preparation of NiNPs/cMWCNT/PANI/Au electrode Prior to electrodeposition, the gold electrode was polished with alumina slurry. One mg of c-MWCNT was suspended in 1 mL mixture of concentrated H2 SO4 and HNO3 in 3:1 ratio and ultrasonicated for 2 h to get a finely dispersed black colored solution of c-MWCNTs. Aniline (50 L) was added to 10.0 mL of 1 M HCl and mixed with 1.0 mL finely dispersed solution of c-MWCNT in a glass cell. c-MWCNT and aniline were electrodeposited, through cyclic voltammetry (CV), onto Au electrode by immersing it in 25 mL of 1 M KCl solution by applying 30 polymerization cycles at 0.0–1.5 V [24]. The resulting cMWCNT/PANI/Au modified electrode was washed thoroughly with distilled water to remove unbound matter and kept in dry petri plate at 4 ◦ C. The electrodeposition of NiNPs onto the cMWCNT/PANI modified Au electrode was carried out by immersing the modified electrode into a mixture of 23 mL of 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) and 2 mL of NiNPs solution and then applying potential between 0–1.5 V (vs. Ag/AgCl) for 25 cycles at a scan rate of 0.02 V s−1 . After rinsing with DW, the NiNPs/cMWCNTs/PANI/Au electrode was dried in air. K3 Fe(CN)6 /K4 Fe(CN)6 was used as an electrolyte, as it acts as a very good redox probe, which is known to exhibit nearly a reversible electrode reaction without any complications of post chemical reactions. 2.6. Preparation of working electrode (Lac/NiNPs/cMWCNT/PANI/Au) The purified laccase was immobilized onto the surface of NiNPs/cMWCNTs/PANI/Au electrode through covalent coupling by layering 10 L of enzyme solution (protein 40 mg mL−1 ) in 0.1 M acetate buffer (pH 5.0) and keeping it undisturbed for about 12 h at 4 ◦ C. The electrode was finally washed with distilled water to remove unbound enzyme. The protein concentration in wash out solution was determined. The principle of working of this biosensor was as follow: guaiacol (standard phenolic substrate) was oxidized to its corresponding o-quinone by NiNPs/cMWCNT/PANI/Au electrode bound laccase and then regenerated through electrochemical reduction of oquinone, thus forming a bioelectrocatalytic amplification cycle and thereby generating electrons, which were passed to working electrode from solution and relayed to potentiometer, in which it was read as current in A (Ampere). The electrons then reached Ag/AgCl electrode, where Ag+ ions were reduced to Ag metal. The current was measured, which was directly proportional to guaiacol concentration. The resulting enzyme electrode was stored in refrigerator at 4 ◦ C, when not in use. 2.7. Characterization of enzyme electrode The modified Au electrode, at different stages of its construction was characterized by studying its scanning electron microscopic (SEM) images in scanning electron microscope (make: Joel Japan, model: JSM-6510, at Advanced Instrumentation Research Facility, JNU, New Delhi). Fourier transform infrared (FTIR) spectra in FTIR spectometer (make: Thermoelectron, USA, model: iS10) and cyclic voltammetric and electrochemical impedance spectroscopic (EIS) studies on potenitostat/galvanostat (make: Autolab, Eco Chemie BV, Netherlands, model: AUT83785 with the GPES 4.9 software) with three electrode system, Lac/NiNPs/cMWCNT/PANI/Au
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electrode as the working electrode, a Pt wire as the auxiliary electrode and an Ag/AgCl (saturated 3 M KCl) electrode as the reference electrode. The EIS measurements were carried out in 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] in the 0.1 M PB pH 7.0 at ambient temperature, at the polarization potential of 0.2 V (vs Ag/AgCl) in the frequency range 0.1–104 Hz and with the amplitude of 10 mV. The resulting enzyme electrode (Lac/NiNPs/cMWCNT/PANI/Au electrode) was stored in refrigerator at 4 ◦ C, when not in use. To record FTIR spectra of hybrid material deposited onto Au electrode, it was scrapped off the Au electrode, mixed with dried KBr and its pellet was formed by hydraulic pellet press. Then this pellet was kept into the socket of the FTIR spectrometer and its spectrum was recorded.
2.8. Response measurement of Lac/NiNPs/cMWCNT/PANI/Au All electrochemical characterizations and measurements were carried out using a conventional three-electrode system with the Lac/NiNPs/cMWCNT/PANI/Au electrode as the working electrode, a Pt wire as the auxiliary electrode and an Ag/AgCl (saturated 3 M KCl) electrode as the reference electrode. Cyclic voltammetric measurements were carried out in a three-electrode cell containing 20 mL electrolyte [2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1)], 5 mL acetate buffer (0.1 M, pH 5.0) and 0.1 mL of guaiacol (10 M). Current measurements were performed applying CV in the potential range, 0 and +1.5 V (vs Ag/AgCl). To record cyclic voltammograms, the following instrumental parameters were used: step potential +6 mV and scan rate 20 mV s−1 . All electroanalytical measurements were carried out at room temperature.
2.9. Optimization of Lac/NiNPs/cMWCNT/PANI/Au electrode To determine the optimum working conditions of the enzyme electrode, the pH of reaction buffer (0.1 M) was varied from pH 3.0 to 7.0 at an interval of pH 0.5 using acetate buffer in acidic pH range (pH 3.0–5.5) and phosphate buffer in pH range 6.0–7.0. Similarly, to determine optimum temperature, the reaction mixture was incubated at 15–50 ◦ C at an interval of 5 ◦ C. To determine the optimum response time, the current response was measured from 2 s to 12 s at an interval of 2 s. To study the effect of substrate concentration, different guaiacol concentration, ranging from 0.1 to 500 M were used.
Fig. 1. The calibration curve for phenolic biosensor Inset: Calibration curve at a concentration below 10 M guaiacol. Error bars represent the standard deviation for three independent measurements.
2.11. Amperometric measurement of total phenolic content in fruit juices The biosensor was employed for determination of phenolic content in five commercial brands of fruit juices. The fruit juice samples were diluted 10 times with acetate buffer (pH 5.5). Total phenolic content in fruit juice samples was determined by the biosensor in the same manner as described above for its response measurement under its optimal working conditions except that guaiacol was replaced by these sample. The current (A) was recorded and the amount of total phenolic content was interpolated from standard curve between guaiacol concentrations and current (A) prepared under optimal working conditions (Fig. 1).
3. Results and discussion 3.1. Characterization of NiNPs The morphological characterization of nickel nanoparticles was carried out by Transmission electron microscope (TEM) at SAIF, Punjab University, Chandigarh. TEM image of prepared NiNPs showed their spherical shape with different diameter in the range, 10–100 nm revealing their different size but not shape (Fig. 2).
2.10. Evaluation The following criteria was studied to evaluate the analytic performance of the biosensor, e.g. linearity, analytical recovery, detection limit, precision and correlation. Analytical recovery was studied using red wines with two different standard concentrations (0.1 mM) of guaiacol. To test the reproducibility and reliability of the present biosensor, phenolic content in six fruit juice samples was measured five times on single day (within batch) and five times again after their storage at −20 ◦ C for one week (between batch). To study the correlation the phenolic content was measured in 10 brands of fruit juice samples by the standard spectrophotometric method and the present method and their correlation was studied using regression equation. To examine the long-term storage stability, the activity of enzyme electrode was tested upto three months at an interval of one week, when stored at 4 ◦ C. To reuse the enzyme electrode, it was washed 3–4 times with reaction buffer before its use in next assay.
Fig. 2. Transmission electron microscopic (TEM) image of nickel nanoparticles.
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Fig. 3. Scanning electron micrographs of (a) bare gold electrode (b) cMWCNT/PANI/Au electrode (c) Lac/NiNPs/cMWCNT/PANI/Au electrode.
3.2. Electrode surface characterization by SEM and FTIR Fig. 3 shows morphology of bare Au electrode, cMWCNTs/PANI/Au electrode and Lac/NiNPs/cMWCNT/PANI/Au electrode as characterized by scanning electron micrographs (SEM) studies. The SEM of the bare Au electrode (Fig. 3a) showed a homogeneous surface. The cMWCNTs/PANI composite film showed net like structure (Fig. 3b). Film was more uniform and porous and hence effective surface area was larger. The SEM images for Lac/NiNPs/cMWCNT/PANI/Au showed a regular globular structural morphology (Fig. 3c), indicating the successful immobilization of laccase to the surface of cMWCNT/PANI layer. Fig. 4 displays the FTIR spectra for cMWCNT/PANI/Au (curve a) and Lac/NiNPs/cMWCNT/PANI Au (curve b). The FTIR spectrum of electrodeposited cMWCNT/PANI composite (curve a) showed benzenoid and quinoid ring stretching bands (C C) present at 1447 and 1595 cm−1 . The presence of peaks at 1129 and 3402 cm−1 could be attributed to B N+ Q stretching and N H stretching vibrations of PANI in the composite. The peak at 1710 cm−1 is attributed to C O stretching vibrations indicating the presence
of carboxyl group ( COOH). The presence of these peaks reveals the existence of both PANI and cMWCNT on the Au electrode. The Lac/NiNPs/cMWCNT/PANI/Au electrode (curve b) showed characteristic peaks at 1550 and 1600 cm−1 which could be attributed to the primary and secondary amide linkages between NH2 groups on the surface group on the surface of enzyme and free COOH groups of MWCNT. Also the peaks for free COOH groups were not found in curve b, which confirms that the free COOH groups have been occupied by NH2 group of enzyme, thus conforming the formation of a strong covalent bond. method is described for construction of a A Lac/NiNPs/cMWCNT/PANI/Au electrode. Scheme 1 summarizes the different chemical reactions involved in the fabrication of lac electrode based on covalent immobilization of lac onto NiNPs/cMWCNT/PANI/Au electrode through amide bond formation between the free COOH groups of carboxylated cMWCNT of NiNPs/cMWCNT/PANI/Au electrode and free NH2 groups on surface of enzyme. Our results showed that NiNPs and cMWCNTs provided a remarkable synergistic effect toward the oxidation of guaiacol.
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Fig. 4. The infrared spectra of (a) c-MWCNT/PANI/Au electrode and (b) Lac/NiNPs/cMWCNT/PANI/Au electrode.
3.3. Cyclic voltammetry study To evaluate the charge-transfer properties on the surface of the modified electrodes, cyclic voltammetry technique was employed, using potassium ferricyanide–potassium ferrocyanide mixture as electrolyte. Cyclic voltammograms were recorded in 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] and 0.1 M acetate buffers (pH 5.5). CV of cMWCNT/PANI/Au revealed two redox peaks for
oxidation of aniline to radical cation and then to radical dication [25]. The electrodeposition of NiNPs onto cMWCNT/PANI modified Au electrode surface leads to increase in current intensity, as a result of increase in electroactive area. The preservation of a quasi reversible electrode process and the large increase in peak currents for the nanocomposite film modified electrode proved that NiNPs exerted an obvious improvement effect on cMWCNT/PANI/Au electrode.
Scheme 1. Scheme of chemical sequence of electropolymerization of NiNPs/cMWCNT/PANI onto Au electrode and chemical reaction of immobilization of laccase on modified electrode.
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525
(A)
(c)
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(b)
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i / μA
81
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25 -75
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E/V
1.500
1.750
125 100
(a)
i / μA
75
Fig. 6. The Nyquist plots of EIS of comparison of bare Au electrode (curve a) c-MWCNT/PANI/Au electrode (curve b) NiNPs/cMWCNT/PANI/Au electrode (curve c) and Lac/NiNPs/cMWCNT/PANI/Au electrode (curve d) in 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 .
50 25 0
(b)
-25 -50
0
0.250
0.500
0.750
1.000
E/V
1.250
1.500
1.750
Fig. 5. (A) Comparative cyclic voltammogram of (a) c-MWCNT/PANI/Au electrode (b) NiNPs/cMWCNT/PANI/Au electrode (c) Lac/NiNPs/cMWCNT/PANI/Au electrode. (B) Cyclic voltammetric (CV) curves of Lac/NiNPs/cMWCNT/PANI/Au electrode in 0.1 M acetate buffer (pH 6.0) (a) with and (b) without 10 M guaiacol solution. Supporting electrolyte: 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)]; scan rate: 20 mV s−1 .
Fig. 5A reveals the comparative cyclic voltammograms of the c-MWCNT/PANI modified Au electrode (curve a) and NiNPs/cMWCNT/PANI (curve b) in a solution of 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] and acetate buffer (0.1 M, pH 5.0) at 20 mV s−1 . The rise in current clearly indicated the role of NiNPs in increase of conductivity of modified electrode. 3.4. Response toward guaiacol at the Lac/NiNPs/cMWCNT/PANI/Au Electrode To evaluate the catalytic activity of laccase immobilized onto NiNPs/cMWCNT/PANI/Au electrode, the modified electrode was characterized by a cyclic voltammogram in the presence of guaiacol in the potential range from 0 to +1.5 V (vs Ag/AgCl). Fig. 5B shows cyclic voltammograms of the NiNPs/cMWCNT/PANI/Au electrode in an unstirred 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] solution and 0.1 M acetate buffer (pH 5.5) with (curve a) and without guaiacol solution (curve b) at scan rate 20 mV s−1 . It was observed that with the addition of 2 M guaiacol, both the reduction current and oxidation current increased, which showed the improved catalytic properties of modified electrode to the reduction of guaiacol. 3.5. Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) provides useful information on the impedance changes of the electrode surface during the fabrication process. The semicircular portion at higher frequencies corresponds to the electron-transfer-limited process and its diameter is equal to the electron transfer resistance, which controls the electron transfer kinetics at the electrode interface. Meanwhile, the linear part at lower frequencies corresponds to the diffusion process [26]. Fig. 6 displays the Nyquist plots of the EIS of (a) bare Au electrode (b) cMWCNT/PANI/Au electrode (c) NiNPs/cMWCNT/PANI/Au electrode and (d) Lac/NiNPs/cMWCNT/PANI/Au electrode in 2.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 [(1:1)] in solution of 0.1 M PB pH 7.0, at the
polarization potential of 0.2 V in the frequency range 0.1–104 Hz. RCT value ∼50 correspond to the bare Au (curve a). Nyquist diameter cMWCNT/PANI/Au (curve (b), RCT 400 ), was much larger than NiNPs/cMWCNT/PANI/Au electrode (curve (c), RCT 192.85 ) which suggests that cMWCNT/PANI modified electrode increased the resistance of the electrodes [27] whereas NiNPs act as a nanoscale electrode, promoting electron transfer between the redox probe and the electrode. However, after immobilization of laccase onto NiNPs/cMWCNT/PANI/Au electrode, RCT was found to increase to 570.50 (curve d). This suggests that immobilized molecules strongly bind with hybrid nanobiocomposite and block charge carriers in the nanobiocomposite matrix. Also this increase could be attributed to the fact that most biological molecule, including enzyme, are poor electrical conductors at low frequency and cause hindrance to electron transfer. These results confirm the success of the assembly on the modified electrode. The change in the RCT value reflecting the increase or decrease in the diameter of the semicircle at high frequencies in the impedance spectra is associated with the blocking behaviour of the electrode surface for the charge transfer to the redox probe. 3.6. Optimization of experimental conditions of biosensor To optimize the biosensor (NiNPs/cMWCNT/PANI/Au electrode) the effect of pH, incubation temperature and scan rate were studied. The CV at different pH (Fig. 7a) showed that the current response resulting from the NiNPs/cMWCNT/PANI/Au bound enzyme-catalyzed reaction achieved a maximum value at pH 5.5 (Fig. 7b), which is higher than that immobilized onto different supports, e.g. Lentinula edodes laccase immobilized onto chitosan (pH 4.0) [28], Pycnoporus sanguineus laccase immobilized onto magnetic chitosan microspheres (pH 3.0) [29] but lower than that of Trametes versicolor laccase immobilized onto platinum nanoparticles (pH 6.5) [30]. Therefore, a pH of 5.5 was used in further experiments. The CV at different temperature (Fig. 8a) exhibited the optimum temperature at 35 ◦ C (Fig. 8b) which is lower than that immobilized on magnetic chitosan microspheres (55 ◦ C) [29] but higher than that carbon paste modified graphite electrode [31] and nanoparticles (25 ◦ C) [32]. Polyaniline being a good thermal insulator for enzymes might have provided the microenvironment to protect the enzyme from environmental and thermal variations, which favours the repeated use of NiNPs/cMWCNT/PANI electrode [15]. The biosensor showed maximum response at 8 s (Fig. 9). The effect of the scan rate from 10 to 100 mV s−1 on the biosensor response using 2 M guaiacol in 0.1 M acetate buffer solution (pH 5.5) was also investigated. A potential scan rate of 20 mV s−1 was
82
Table 1 A comparison of various analytical and kinetic properties of polyphenol biosensors based on laccase immobilized on different supports. Property
Jiang et al., 2005 [29]
Liu et al., 2006 [9]
Vianello et al., 2006 [10]
Kochana et al., 2008 [8] Corman et al., 2010 [32]
Chawla et al., 2011 [36] Present
– Silane-modified platinum surface Adsorption
– Carbon nanotubes chitosan composite Cross-linking
– Hydrophilic matrix
– Titania gel matrix
Trametes versicolor Nanoparticles
Ganoderma lucidium NC membrane
Ganoderma lucidium NiNPs/cMWCNT/Au
Covalent coupling
Adsorption
Adsorption
5.5 NR
Pycnoporus sanguineus Magnetic chitosan microspheres Glutaraldehyde cross-linking 3.0 55
6.0 NR
5.0 22
6.0 25
6.0 25
Covalent coupling and adsorption 6.0 35
Covalent coupling and adsorption 5.5 35
Current
Current
Current
Current
Current
Current
Current
Current
60
150
150
100
40
125
120
120
NC: Nitrocellulose; NiNPs: Nickel nanoparticles; cMWCNT: carboxylated multiwalled carbon nanotubes
(a)
(b)
Ι / μA
i / μA
300
275
250
225
0
200
-200
-225
350
300
250
200
150
100
50
0
0
4
4.5 5.0 5.5 6.0 6.5 7.0 7.5
0.250
5 0.500
0.500
7
1.000
E/V
0.750
6
pH
1.000
E/V
0.750
1.250
1.250
50
1.500
8
1.500
60
1.750
1.750
Fig. 7. CV curves (a) and graphical representation (b) for effect of pH on the current response of Lac/NiNPs/cMWCNT/PANI/Au electrode. Error bars represent the standard deviation for three independent measurements.
20 25 30 35 40 45 50
0.250
40
selected, since with these experimental conditions, the highest analytical signal and very good cyclic voltammogram profiles were obtained.
0
30
Temperature / ºC
3.7. Evaluation of biosensor
275
250
225
0
200
-200
-225
20
3.7.1. Linearity There was a linear relationship between current (A) and guaiacol concentration ranging from 0.1–10 M (lower
i / μA
(a) 300
(b) 350
300
250
200
150
100
50
0 10
Fig. 8. CV curves (a) and graphical representation (b) for effect of temperature on the current response of Lac/NiNPs/cMWCNT/PANI/Au electrode. Error bars represent the standard deviation for three independent measurements.
I/μA
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Source of laccase Support for immobilization Method of immobilization Optimum pH Optimum temperature (◦ C) Mode of measurement Storage life at 4 ◦ C (days)
Quan et al., 2004 [7]
S. Chawla et al. / Biochemical Engineering Journal 68 (2012) 76–84
500
Table 2 Comparison of total phenolic content in different brands of fruit juices as measured by NiNPs/cMWCNT/PANI/Au electrode and standard spectrophotometric method.
450
Current (μA)
400 350 300 200 150 100 50 0
2.5
5
7.5
10
12.5
Fig. 9. Time course study of Lac/NiNPs/cMWCNT/PANI/Au electrode. Error bars represent the standard deviation for three independent measurements. *
Phenolic content (μmoles) as measured by present method
Samples Fruit juices
Total phenolic content by present method Mean ± SD* (n = 4) (mol guaiacol/g)
Brand a Brand b Brand c Brand d Brand e Brand f Brand g Brand h Brand i Brand j
0.498 0.520 0.772 0.642 0.494 0.616 0.580 0.672 0.657 0.798
250
0
0.9 0.8
83
± ± ± ± ± ± ± ± ± ±
0.008 0.012 0.013 0.014 0.013 0.008 0.012 0.013 0.014 0.013
Total phenolic content by standard spectrophotometric method Mean ± SD* (n = 4) (mol guaiacol/g) 0.490 0.510 0.765 0.636 0.490 0.606 0.568 0.660 0.650 0.790
± ± ± ± ± ± ± ± ± ±
0.012 0.020 0.022 0.031 0.019 0.022 0.027 0.020 0.019 0.026
p > 0.05.
method. Within and between coefficients of variation were <2.03% and <3.82% respectively. The high precision indicated the good reproducibility and consistency of the present method.
y = 0.9991x - 0.0078 R² = 0.9994
3.7.3. Correlation A Comparison of phenolic content in 10 fruit juice samples as measured by the present method (y) with those obtained by standard spectrophotometric method (x) showed a good correlation with r = 0.99, regression equation being, y = 0.994x + 9.6785 (Fig. 10). These results indicate the high accuracy of the method.
0.7 0.6 0.5 0.4
Fig. 10. Correlation between total phenolic content values determined by standard spectrophotometric method (x-axis) and present biosensor employing MnNPs/cMWCNT/PANI/Au electrode method (y-axis). Error bars represent the standard deviation for three independent measurements.
concentration range) with linear equation being y = 0.923x + 49.96 and 10–500 M (higher concentration range) with linear equation being y = 5.826x + 1.192 (Fig. 1), which is better than earlier biosensor based on carbon paste modified graphite electrode (1.97 × 10−4 to 3.24 × 10−3 M)] [31], polyether sulfone membrane [1 × 10−5 to 8 × 10−5 M] [33], based on chitosan membrane modified with tripolyphosphate [5.99 × 10−7 to 3.92 × 10−6 M] [34] and carbon nanotubes–chitosan composite [3 × 10−7 to 1.2 × 10−6 M] [35]. The detection limit of the biosensor was 0.05 (S/N = 3). 3.7.2. Recovery and precision The analytical recovery of added guaiacol in fruit juice (0.1 mM) was 95.61%, demonstrating the satisfactory accuracy of the present
3.7.4. Interference study The addition of the following interferrants such as ascorbic acid (4.8 M), cysteine (0.24 M), fructose (60 M), glucose (22.2 M), citric acid (100 M) and ethanol (70 M) evoked a negligible interference (a decrease of only 9.76% for glucose, 10.63% for citrate, 5.99% for fructose, 9.13% for cysteine and 8.66% for ascorbic acid on the biosensor response). 3.7.5. Storage stability and reusability of biosensor The electrode lost 15% of its initial activity after its 200 uses during the span of four months when stored at 4 ◦ C. The properties of present biosensor have been compared with previous biosensors (Table 1). 3.7.6. Determination of total phenolic content in fruit juice samples Total phenolic content as measured from CV curves (Fig. 11) by the present sensor ranged from 0.494 to 0.798 mol guaiacol/g in ten different fruit samples of different brands (Table 2). 4. Conclusion
300 a b c d e f g
275
i / μA
250 225 200
h i j
0 -200 -225
0
0.250
0.500
0.750
1.000
E/V
1.25
1.500
1.750
Fig. 11. Cyclic voltammetric (CV) curves for determination of total phenolic content in different fruit juice samples.
We constructed a novel composite of NiNPs and c-MWCNT/PANI at Au electrode for electrocatalysis, which is stable for a long time at pH 5.5 in 0.1 M acetate buffer solution. Furthermore the composite provides covalent immobilization of laccase onto modified electrode as confirmed by FTIR. This Lac/NiNPs/cMWCNT/PANI modified Au electrode exhibited improved performance of biosensor in terms of wide linear range viz. 0.1–10 M (lower concentration range) and 10–500 M (higher concentration range), response time (8 s), detection limit (0.05 mM), sensitivity (0.694 A M−1 cm−2 ), reusability (more than 200 times) and stability (four months). Thus this work illustrates a simple and novel approach for the development of an improved amperometric enzyme sensor for determination of phenolic compounds.
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