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chitosan-g-polyaniline composite film electrodeposited on Pt electrode
Sensors and Actuators B 193 (2014) 608–615
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Construction and application of an amperometric uric acid biosensor based on covalent immobilization of uricase on iron oxide nanoparticles/chitosan-g-polyaniline composite film electrodeposited on Pt electrode Rooma Devi, C.S. Pundir ∗ Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, Haryana, India
a r t i c l e
i n f o
Article history: Received 30 August 2013 Received in revised form 2 December 2013 Accepted 3 December 2013 Available online 12 December 2013 Keywords: Uricase Uric acid biosensor Iron oxide nanoparticles Chitosan Polyaniline
a b s t r a c t Commercial uricase was immobilized covalently onto iron oxide nanoparticles/chitosangraft-polyaniline (Fe3 O4 -NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode. Transmission electron microscopy (TEM) was used for characterization of Fe3 O4 -NPs. A uric acid biosensor was fabricated using/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 1s at pH 7.5 and 30 ◦ C, when polarized at 0.4 V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on uric acid concentration ranging from 0.1 to 800 M. The sensitivity of the biosensor was 0.44 mA mM−1 cm−2 , with a detection limit of 0.1 M (S/N = 3). Apparent Michaelis–Menton (Km ) value for uric acid was 12.5 M, and Imax 0.008A. The biosensor showed only 10% loss in its initial response after 120 uses over 100 days, when stored at 4 ◦ C. The biosensor measured uric acid in the serum of apparently healthy persons, which correlated well with a standard enzymic colorimetric method (r = 0.98). © 2014 Elsevier B.V. All rights reserved.
Introduction Uric acid (2,6,8-trihydroxypurine) is the main final product of purine metabolism in humans. It is an important marker in clinical diagnosis, because the elevated uric acid concentration in blood (hyperuricemia) is associated with a number of diseases, such as gout, arthritis [1], cardiovascular diseases [2], neurological diseases [3], insulin resistance, hypertension, and renal insufficiency [4,5]. A healthy adult human excretes uric acid at a rate of 0.6 g/24 h; the excreted product arises in part from turnover of the purine nucleotide of nucleic acids. The normal level of uric acid in serum is between 0.13 and 0.46 mM (2.18–7.7 mg dl−1 ) [6,7]. Consequently, uric acid determination is of paramount importance in the diagnosis and medical management of diseases caused by disorder of purine metabolism. Various methods such as colorimetric [7], high performance liquid chromatography (HPLC) [8,9], chemiluminescence [10] and
∗ Corresponding author. Tel.: +91 1262 295480/+91 9416 492413; fax: +91 1262 295480. E-mail addresses: [email protected], [email protected] (C.S. Pundir). 0925-4005/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.12.010
fluorescence methods [11] have been reported for the measurement of uric acid. However, the colorimetric method is time consuming and sensitive to numerous metabolites and drugs found in biological samples, while the enzymatic colorimetric method is expensive and also prone to metabolites. The biosensing method has many advantages over these routine techniques in terms of simplicity, rapidity, sensitivity and cost of analysis. Various electrochemical methods have been proposed for routine uric acid analysis, including potentiometric biosensors [12–18]. However, these electrochemical biosensors had problems such as lack of stability, sensitivity and low reproducibility, which are still to be improved. To overcome these problems, the enzyme is required to be immobilized directly and covalently onto the film coated electrode. The emergence of nanotechnology offered great opportunities to improve the sensitivity, stability and anti-interference ability of biosensing systems. Applications of nanomaterials to biosensors have recently aroused much interest as these materials exhibit large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency and strong adsorption ability that are helpful for immobilization of biosensing molecules. Moreover, nanoparticles have a unique ability to promote fast electron transfer between the electrode and the active site of the enzyme [19]. In
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this context, different types of nanoparticles such as gold (AuNPs) [20], zinc-oxide (ZnO-NPs) [21], iron-oxide (Fe3 O4 -NPs) [22], have been suggested as promising matrices for enzyme immobilization to improve the stability and sensitivity of the biosensor. Among these metal oxide nanoparticles, Fe3 O4 -NPs have been considered as most interesting for the immobilization of desired biomolecules, due to their high biocompatibility and strong superparamagnetic behavior, which provide better contact and low toxicity[22]. Immobilization of bioactive molecules onto a surface charged with super para magnetic nanoparticles is of special interest, since the magnetic behavior of these bioconjugates may result in improved delivery and recovery of biomolecules for desired biosensing applications [23,24]. Another very important property of this type of nanoparticle for electrochemical biosensors is their ability to provide a favorable microenvironment for biomolecules to exchange electrons directly with an electrode, thus improving the sensitivity of electrochemical biosensors. Besides this, the existing problem of aggregation and rapid biodegradation of Fe3 O4 -NPs onto a given matrix containing biomolecules can perhaps be overcome by modifying these nanoparticles using chitosan (CHIT). CHIT is an abundant natural biopolymer with excellent film forming abilities, biocompatibility, nontoxicity, good water permeability, high mechanical strength [24] and susceptible to chemical modification, due to the presence of reactive hydroxyl and amino functional groups. CHIT can accumulate metal ions through various mechanisms, such as chelation, electrostatic attraction and ion exchange, depending on the nature of the metal ion and pH of the solution. CHIT along with polyaniline (PANI), a conducting polymer provides a suitable matrix for covalent immobilization and stabilization condition for the biomolecules [25]. PANI as a conducting polymer has become very attractive, because of its facile synthesis [25], stable chemical and environmental properties [26] and inherent reversible doping/dedoping states [27]. Therefore, it is a suitable matrix for achieving electromagnetic shielding or absorption materials by doping with magnetic Fe3 O4 -NPs. The present report describes herein the use of unique properties of Fe3 O4 -NPs, CHIT and PANI for fabrication of an improved uric acid biosensor.
Material and methods Uric acid, Sephadex G-100, glutaraldehyde (25%) and CHIT were from Sigma-Aldrich, USA. A uric acid kit for enzymatic colorimetric determination manufactured by Transasia Bio-medicals (Solan, India) was obtained from Scientific Emporium (Rohtak, India). Aniline (purified through vacuum distillation before use) and zinc nitrate were from SISCO Research Lab., Mumbai, India. All other chemicals were of analytical reagent (AR) grade. Double distilled water (DW) was used in all experiments.
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Preparation of uricase Uricase (UOx) was prepared by dissolving 3.0 mg of powder of enzymatic reagent 1 of the uric acid kit into 1 ml of 0.02 M sodium phosphate buffer (pH 7.0) and loading it on a Sephadex G-100 column (24 × 1 cm) preequilibrated with 0.02 M sodium phosphate buffer (pH 7.0). The column was run in the same buffer at a flow rate of 0.5 ml min−1 . The fractions (2 ml each) were collected after passing the void volume. The fractions were tested for protein by the Lowry method. Fractions showing the presence of protein were pooled and treated as purified enzyme and tested for its activity as given below. Assay of free uricase The assay of free uricase was carried out as described previously [28] with slight modification and based on a decrease in A293 due to uric acid. The reaction mixture contained 3.0 ml of 50 mM Tris–HCl buffer (pH 8.5), 0.1 ml of uric acid (10 mM) and 50 l of uricase (1.6 mg ml−1 ). The blank contained 3.05 ml of 50 mM Tris–HCl buffer (pH 8.5) and 0.1 ml of 10 mM uric acid. The control consisted of 3.0 ml of 50 mM Tris–HCl buffer (pH 8.5), 0.1 ml of 10 mM uric acid, and 50 l of heat-denatured uricase. The decrease in A293 due to uric acid was read in an ultraviolet (UV) spectrophotometer for 4 min at an interval of 1 min. The activity of enzyme was calculated as follows: Unit activity =
(A293 per min in test solution-blank) × test volume 12.6 × volume of enzyme taken
One enzyme unit (U) is defined as amount of enzyme required to oxidize 1 mol of uric acid into allantoin per minute per milliliter under standard assay conditions. The free uricase showed an activity of 0.2 U min−1 ml−1 . Preparation of iron oxide nanoparticles (Fe3 O4 -NPs) The Fe3 O4 -NPs were prepared by the co-precipitation of Fe(II) and Fe(III) under a alkaline condition as described by Kim et al. [29], with slight modification. Five milliliters of iron ion solution containing 0.25 M ferrous chloride and ferric chloride (1:1 ratio) was added drop wise into 50 mL NaOH (2 mol l−1 ) solution under vigorous mechanical stirring for 35 min at 80 ◦ C. The precipitates were washed firstly with DW and then ethanol 2–3 times and collected by applying an external magnetic field until the supernatant solution turned neutral and finally, these were dried in oven at 70 ◦ C. The morphology, particle size and structure of the Fe3 O4 -NPs were determined by a TEM and XRD.
Instruments
Construction of iron oxide nanoparticles/chitosan-g-polyaniline/platinum electrode (Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode)
All electrochemical experiments and electrochemical impedance spectroscopic (EIS) measurements were performed at 25 ± 1 ◦ C using an potentiostat/galvanostat equipped with an Autolab PGSTAT-302N, GPES and FRA software (Eco-Chemie, Utrecht, The Netherlands) with a three electrode system consisting Uricase/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode used as working, Ag/AgCl as reference and Pt wire as auxillary electrode. Fourier transform infrared (FTIR) spectroscopy was recorded in FTIR spectrophotometer (Thermo Scientific, USA). Scanning electron microscopy (SEM) transmission electron microscopy (TEM) and X-ray diffraction (XRD) studies were carried out on commercial basis.
Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode was prepared by cyclic voltammetric method. CHIT solution was prepared by dissolving 2.0 g of CHIT flakes into 100 ml of 1.0% acetic acid and stirred for 3 h at room temperature until completely dissolved.The CHIT solution was stored in a refrigerator when not in use. Fe3 O4 -NPs(400 l) were dispersed into transparent CHIT solution and kept on magnetic stirring for about 30 min at room temperature followed by the sonication for 4 h. Finally a highly viscous solution of CHIT with uniformly dispersed Fe3 O4 -NPs was obtained. A solution for electrodeposition was prepared by adding aniline (180 l) + Fe3 O4 -NPs/CHIT (75 l) + 0.5 M HCl(10 ml) in a glass cell. A nanocomposite film of Fe3 O4 -NPs/CHIT-g-PANI
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was electrodeposited on Pt electrode by cyclic voltammetry using three electrode-systems. A Pt electrode (1.9 cm × 1 mm) (length × diameter) was ultrasonicated in 5.0 M HNO3 and acetone for 15 min, rinsed with DW, and then immersed into a electrodepositing solution, the potential scan was cycled for 30 times between −0.1 and 1.0 V vs Ag/AgCl at a scan rate of 50 mV s−1 and subsequently allowed to dry at room temperature. The resulting Fe3 O4 -NPs/CHITg- PANI/Pt electrode was washed with DW. Immobilization of uricase on Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode Uricase was immobilized onto the Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode surface through glutaraldehyde coupling. First 10 l of 2.5% glutaraldehyde solution in 0.05 M phosphate buffer (PB) pH 7.5 was spread over the Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode and kept for 5 h at room temperature, washed in 0.05 M PB, pH 7.5 and then 100 l uricase was mounted on surface of the electrode and dried. The resulting UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode was washed thoroughly with 0.05 M PB of pH 7.5 to rinse off any loosely bound enzyme from the electrode. The resulting enzyme electrode was dried at room temperature and then stored in a refrigerator at 4 ◦ C, when not in use. The fabricated electrode was characterized by cyclic voltammetric, SEM, FTIR and EIS studies. Cyclic voltammetric measurement and optimization of uric acid biosensor The sensitivity of uric acid biosensor was tested by measuring current using three electrode system. The working (UOx/Fe3 O4 NPs/CHIT-g-PANI/Pt) electrode along with Ag/AgCl as reference electrode and Pt wire as auxillary electrode were connected through potentiostat/galvanostat to construct uric acid biosensor. The electrode system was dipped into a reaction mixture containing 10 ml 0.05 M PB, pH 7.5 and 0.5 ml uric acid solution (50–250 M). The electrode current response was measured applying a potential range of −0.1 to 1.0 V vs Ag/AgCl. The steady state current response increased with increase in working potential and optimum current response was obtained at 0.4 V vs Ag/AgCl. Therefore, 0.4 V vs Ag/AgCl was selected as the working potential for amperometric detection of uric acid concentration. To optimize the working condition of electrode, the pH of reaction buffer was varied from 6.0 to 10.0 at an interval of pH 0.5 using 0.05 M PB in the pH range 6–8 and 0.05 M sodium carbonate/bicarbonate buffer in the pH range 8.5–10. The optimum temperature was studied by incubating the reaction mixture at different temperature (25–45 ◦ C at an interval of 5 ◦ C). Similarly the current response was measured at 1–10 s at an interval of 1s and uric acid concentration was varied from 0.1 to 1200 M in reaction buffer under optimal conditions. Amperometric determination of uric acid in serum The modified Pt electrode was employed for measuring uric acid in serum. Serum samples from apparently healthy persons of different age groups and sex were collected from hospital of PGIMS, Rohtak in tubes and stored at 4 ◦ C until use. The content of uric acid in sera was determined by the present biosensor as described for its testing under optimal working conditions except that uric acid solution was replaced by serum. The content of uric acid was determined from standard curve between uric acid conc. vs current (mA) prepared under optimal conditions. The following criteria were studied to evaluate the performance of this biosensor viz. linearity, analytical recovery, detection limit, sensitivity, precision and correlation with standard method.
Fig. 1. (A) Transmission electron microscopic (TEM) images of Fe3 O4 -NPs dispersed in CHIT. (B) X-ray diffraction (XRD) pattern of Fe3 O4 -NPs.
Results and discussion Characterization of Fe3 O4 -NPs A TEM image of synthesized Fe3 O4 -NPs dispersed in CHIT (Fig. 1A) indicates that Fe3 O4 -NPs were nanocrystalline and composed predominantly of a large number of well dispersed spherical nanoparticles with some hexagonal shaped nanoparticles. The average size of the spherical nanoparticles was ∼20 nm. Moreover, Fe3 O4 -NPs were agglomerates due to their high surface area and magnetic dipole–dipole interactions between the particles [30] which could be overcome after modifying these NPs with CHIT. The XRD pattern (Fig. 1B) of synthesized Fe3 O4 -NPs showed their polycrystalline nature. The peak positions were well in agreement with joint committee on powder diffraction standard (JCPDS) and indexed to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0), respectively. The results obtained agree with standard magnetite (Fe3 O4 ) XRD patterns and identified that the Fe3 O4 -NPs were in a cubic spinel structure [31]. Construction of UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode Fig. 2 displays the cyclic voltammograms of CHIT-g-PANI (curve a) and Fe3 O4 -NPs/CHIT-g-PANI (curve b) composite film. It can be seen that the surface charged Fe3 O4 -NPs interact with the cationic biopolymer matrix of CHIT via electrostatic interactions and hydrogen bonding with NH2 /OH groups to form a hybrid
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The following electrochemical reactions occur during response measurements of present biosensor: UOx
Uric acid + O2 + H2 O −→ Allantoin + H2 O2 + CO2 0.4
H2 O2 −→ 2H + + O2 + 2e− 2e−
Fe3 O4 −NPs/CHIT −g−PANI
−→
Pt electrode
The current generated at the electrode was measured by potentiostat, which is directly p proportional to the uric acid concentration. Surface characterization by SEM Fig. 2. Cyclic voltamogram of CHIT-g-PANI (curve a) and Fe3 O4 -NPs/CHIT-g-PANI composite (curve b) films at 10 M uric acid.
nanobiocomposite [32]. The Fe3 O4 -NPs/CHIT-g-PANI composite film exhibited higher currents than the CHIT-g-PANI composite film, which indicate that the Fe3 O4 -NPs/CHIT-g-PANI composite film, had a larger effective surface area than the CHIT-g-PANI composite film. The Fe3 O4 -NPs/CHIT-g-PANI composite film appeared to provide a conducting path through the composite matrix for faster kinetics. Hence, the Fe3 O4 -NPs acting as an electron transfer mediator may help in enhancing the sensor response of enzyme electrode and thus increase the sensitivity of the biosensor. These observations suggest the formation of Fe3 O4 -NPs/CHIT-g-PANI composite film which provides a large surface area for the immobilization of enzyme. A method is described for construction of an amperometric biosensor using UOx/Fe3 O4 -NPs/CHIT-g-PANI film electrodeposited onto Pt electrode. This enzyme electrode is summarized in Scheme 1. To achieve it, firstly a Fe3 O4 -NPs/CHIT-g-PANI composite film was electrodeposited onto Pt electrode using cyclic voltammetry. The electrodeposition method was selected to produce Fe3 O4 -NPs film onto electrode surfaces, due to its simplicity and to control the layer thickness. Secondly, commercially available UOx was immobilized covalently onto Fe3 O4 -NPs/CHIT-g-PANI composite film through glutaraldehyde coupling. One –CHO group of the glutaraldehyde was attached to the NH2 group of CHIT of Fe3 O4 -NPs/CHIT-g-PANI film. While other –CHO group of the glutaraldehyde was attached to enzyme via its free –NH2 on the surface of enzyme, which resulted in UOx/Fe3 O4 -NPs/CHIT-g-PANI electrode.
The surface morphologies of Bare Pt electrode, Fe3 O4 NPs/CHITg-PANI/Pt and UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt were investigated by SEM (Fig. 3). Globular porous morphology of Fe3 O4 -NPs/CHIT-g-PANI (Fig. 3B) biocomposite film reveals incorporation on Pt electrode (Fig. 3A), indicating the formation of Fe3 O4 -NPs/CHIT-g-PANI biocomposite film. However, after the immobilization of UOx onto Fe3 O4 -NPs/CHIT-g-PANI composite film, the globular morphology changed to regular form (Fig. 3C). This suggests that Fe3 O4 NPs provided a favorable environment for high loading of UOx. FTIR spectra Fig. 4A showed the FTIR spectra obtained for CHIT-g-PANI, Fe3 O4 NPs/CHIT-g-PANI and UOx/Fe3 O4 NPs/CHIT-g-PANI composites. The FTIR spectrum of the CHIT-g-PANI composite (curve i) illustrated the characteristic peaks of PANI, as well as CHIT [33]. The stretching bands of benzenoid and quinoid ring were observed at 1528 and 1604 cm−1 . The absorption band of the N Q N bending vibration of protonated PANI was observed at 1194 cm−1 in the CHIT-g-PANI copolymer. The FTIR spectrum of Fe3 O4 NPs/CHIT-gPANI composite (curve ii) exhibits characteristic IR bands of the functional group corresponding to CHIT-g-PANI and the Fe3 O4 NPs. The absorption band of Fe3 O4 NPs appears at 581 cm−1 belonging to the stretching vibration mode and the torsional vibration mode of Fe–O bonds in the tetrahedral sites and in the octahedral sites. The FTIR spectrum of the UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode (curve iii) showed peaks broadening at 3098–3315 cm−1 (addition of N H stretching vibration) due to the attachment of enzymes with the Fe3 O4 NPs/CHIT-g-PANI composite. Hence, FTIR
Scheme 1. Chemical reactions involved in immobilization of uricase on Fe3 O4 -NPs/CHITg-PANI modified Pt electrode.
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Fig. 3. (A) SEM image of bare Pt electrode. (B) SEM image of Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode. (C) SEM image of UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode.
spectra confirmed the covalent immobilization of UOx onto the Fe3 O4 NPs/CHIT-g-PANI/Pt electrode. Electrochemical impedance spectroscopy (EIS) studies EIS is an effective method for probing the features of surface modified electrodes. The Nyquist plot of impedance spectra includes a semicircle portion and a linear portion, with the former at higher frequencies corresponding to the electron transfer limited process and the latter at lower frequencies corresponding to the
diffusion process. The electron transfer resistance (RCT ) at electrode surface is equal to the semicircle diameter, which can be used to describe the interface properties of the electrode. Fig. 5 presents the Nyquist plot of the impedance spectroscopy of CHITg-PANI/Pt, Fe3 O4 NPs/CHIT-g-PANI/Pt and UOx/Fe3 O4 NPs/CHIT-gPANI/Pt electrode in 5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.05 M PB, pH 7.5. The diameter of semicircle for Fe3 O4 NPs/CHITg-PANI composite film (curve b) was smaller than that of CHIT-g-PANI composite film (curve a) which suggested that electron transfer in Fe3 O4 NPs/CHIT-g-PANI composite film was
Fig. 4. (A) FTIR spectra obtained for CHIT-g-PANI (curve i), Fe3 O4 -NPs/CHIT-g-PANI (curve ii), UOx/Fe3 O4 -NPs/CHIT-g-PANI (curve iii).
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613
250
(c)
Z" (ohms)
200 150 100
b
50
(a)
0 0
100
200
300
400
500
600
Z' (ohms) Fig. 5. The Nyquist plot of the EIS of (a) CHIT-g-PANI/Pt, (b)Fe3 O4 NPs/CHIT-g-PANI/Pt and (c) UOx/Fe3 O4 NPs/CHIT-g-PANI/Pt electrode in 5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.05 M PB, pH 7.5.
easier between the solution and electrode i.e. Fe3 O4 NPs which not only provided the hydrophilic surface but also acted as a nanoscale electrode and promoted electron transfer due to permeable structure of CHIT-g-PANI/Pt. However the diameter of the semicircle for UOx/Fe3 O4 NPs/CHIT-g-PANI/Pt electrode (curve c) was further increased. This increase in diameter can be attributed to the fact that most biological molecules, including enzymes, are poor electrical conductor at low frequencies and cause hindrance to electron transfer. These results also indicate binding of enzyme onto Fe3 O4 NPs/CHIT-g-PANI composite film.
of 0.025–0.1 mM [30], 0.05–0.5 mM [31] and 0.0–0.30 mM [32]. The calibration curve was linear up to 0.1 M with a sensitivity of 0.44 mA mM−1 cm−2 , which is higher than that reported previously (0.024 mA mM−1 cm−2 ).
Optimization of the biosensor (UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode) The experimental conditions affecting the biosensor response were studied in terms of the effects of pH, incubation temperature, time and substrate (uric acid) concentration. The optimal current was obtained within 1s at pH 7.5, which is higher than that of free enzyme (pH 7.0) [27]. The change in optimal pH of enzyme after immobilization might be due to an improved stability at that pH, the prevention of dissociation, an internal pH difference from the external pH, and/or even a chemical modification of the enzyme. The immobilized enzyme had an optimal temperature of 35 ◦ C, which is slightly higher than that of free enzyme (37 ◦ C) [27] UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt modified electrode achieved 95% of steady state currents within 1s, which is lower than 8s [18] 60s [16] and 70s [17] reported previously. There was a linear relationship between biosensor response and uric acid concentration up to a final concentration of 0.1 M (Fig. 6A–C). The Lineweaver–Burk plot between 1/I (mA) and 1/[uric acid] yielded an apparent Km of 0.17 mM, and Imax 0.008A, which is lower than that for free enzyme (0.90 mM) [34], previous enzyme electrodes 0.34 mM [35] and polyaniline polypyrrole modified electrode (1.57 mM) [17], indicating increased affinity of enzyme toward substrate (uric acid) after immobilization, which might be due to enhanced diffusion of uric acid through Fe3 O4 -NPs/CHIT-gPANI composite. Evaluation of uricase biosensor Linearity and sensitivity There was a linear relationship between biosensor response (i.e. current) and uric acid concentration ranging from 0.1 to 800 M in Tris–HCl buffer (pH 7.5), which is better than previous reports
Fig. 6. Effect of substrate (uric acid) concentration on response of the uric acid biosensor (A) and the standard curve of uric acid at lower and higher concentration range showing the linear relationship by the uric acid biosensor based on UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode (B and C).
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Fig. 7. Correlation (0.98) between serum samples (mg l−1 ) determinations by standard enzymic colorimetric method (x) and present amperometric method (y).
Detection limit The detection limit of the biosensor was 0.1 M at a signal-to noise ratio of 3, which is lower than that of the previously reported uric acid biosensor of 0.05 mM [19]. Recovery and precision The analytical recoveries of added uric acid in serum samples at 1 mg dl−1 and 2 mg dl−1 concentrations were 94.0% to 98.9%, respectively, demonstrating the good reliability of the present method (Table 1). Within and between coefficients of variation were 1.58% and 2.07%, respectively. The high precision indicated the good reproducibility and consistency of the present method (Table 2). Correlation A Comparison of uric acid values in 15 serum samples, as measured by the present method (y) with those obtained by standard enzymic colorimetric method (x) showed a good correlation with r = 0.98, regression equation being, y = 0.9598x + 0.7474. These results indicate the high accuracy of the method (Fig. 7).
Table 3 Effect of potential interferents on UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode based uric acid biosensor response. Compound added
Concentration (physiological conc. in serum)
% Relative response
None Ascorbic acid Urea Cholesterol EDTA Lactic acid Glucose Pyruvate
– 3.40 mM 0.10 g l−1 2.00 g l−1 2.00 mM 0.50 mM 0.90 g l−1 0.01 g l−1
100.00 ± 0.01 92.40 ± 0.64 95.10 ± 0.51 91.70 ± 0.35 94.34 ± 0.57 96.60 ± 0.72 97.89 ± 0.36 98.90 ± 0.30
Storage stability and reusability of biosensor To examine the long-term storage stabilities, the activity of UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode was tested with respect to storage time. After each experiment, the sensor was washed with working buffer solution and stored at 4 ◦ C. Studies revealed that the electrode lost only 10% loss in its initial response after 120 uses over 100 days. Conclusion
Interference study The effect of various interferents found in serum such as ascorbic acid, urea, cholesterol, EDTA, Lactic acid, glucose and pyruvate were studied on the response of UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt at their physiological concentrations. All these metabolites had practically no effect (Table 3). Table 1 Analytical recovery of added uric acid in serum sample, by uric acid biosensor based on UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode.
The use of Fe3 O4 -NPs/CHIT-g-PANI composite film has resulted into an improved analytical performance of the uric acid biosensor in terms of low response time (1s), lower/better detection limit, 0.1 M (S/N = 3), higher storage stability (100 days), linear range (0.1–800 M) and no interference by various metabolites. Thus this work illustrates a simple and novel approach for the development of an amperometric biosensor for determination of serum uric acid concentration in gout patients, employing UOx/Fe3 O4 -NPs/CHIT-gPANI/Pt nanocomposite.
Uric acid added (mg dl−1 )
Uric acid found (mg dl−1 ) mean (n = 6)
% Recovery mean ± SD
Acknowledgment
– 1.0 2.0
1.04 1.98 3.00
– 94.00 ± 0.67 98.90 ± 1.1
Rooma Devi is thankful to the Indian Council of Medical Research for the award of Senior Research Fellowship (SRF) and Research Associateship (RA).
Table 2 Within batch and between batch coefficients of variation for determination of uric acid, by uric acid biosensor based on UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode. (n)
Uric acid (mg dl−1 ) mean ± SE
Coefficient of variation (%)
Within assay (5) Between assay (5)*
2.31 ± 0.04 2.45 ± 0.02
1.58 2.07
*
After storage at -20 degree for one week.
References [1] H.A. Harper (Ed.), Review of Physiological Chemistry, 16th ed., Lange Medical Publications, San Francisco, CA, 1977. [2] R.H. Heptinstall (Ed.), Gout in Pathology of the Kidney, second ed., Little Brown, Boston, MA, 1966. [3] F. Arellano, J.A. Sacristan, Allopurinol hypersensitivity syndrome: a review, Ann. Pharmacother. 27 (1993) 337–343. [4] Y.S. Zhao, F.H. Liao, Serum uric acid and hyperuricemia associated disease, Int. J. Intern. Med. 34 (2007) 41–47.
R. Devi, C.S. Pundir / Sensors and Actuators B 193 (2014) 608–615 [5] M.A. Becker, M. Jolly, Hyperuricemia and associated diseases, Rheum. Dis. Clin. North Am. 32 (2006) 275–293. [6] C.R. Raj, T. Ohsaka, Voltammetric detection of uric acid in the presence of ascorbic acid at a gold electrode modified with a self-assembled monolayer of heteroaromatic thiol, J. Electroanal. Chem. 540 (2003) 69–77. [7] I.C. Isdale, M.J. Buchanan, B.S. Rose, Serum uric acid estimation: a modified chemical method, Ann. Rheum. Dis. 25 (1966) 184–185. [8] K. Lorentz, W. Berndt, Enzymic determination of uric acid by a colorimetric method, Anal. Biochem. 18 (1967) 58–63. [9] D. Yao, A.G. Vlessidis, N.P. Evmriridis, Monitoring reactive oxygen species in vivo using microdialysis sampling and chemiluminescence detection as an alternative global method for determination of total antioxidant capacity, Anal. Chim. Acta 467 (2002) 133–144. [10] X. Dai, X. Fang, C. Zhang, R. Xu, B. Xu, Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID–MS) as a candidate reference method, J. Chromatogr. B 1 (2007) 287–295. [11] J. Galban, Y. Andreu, M.J. Almenara, S. de Marcos, J.R. Castillo, Direct determination of uric acid in serum by a fluorometric–enzymatic method based on uricase, Talanta 54 (2001) 847–854. [12] J. Kan, X. Pan, C. Chen, Polyaniline–uricase biosensor prepared with template process, Biosens. Bioelectron. 19 (2004) 1635–1640. [13] Y.C. Luo, J.S. Do, C. Liu, An amperometric uric acid biosensor based on modified Ir–C electrode, Biosens. Bioelectron. 22 (2006) 482–488. [14] S. Cete, A. Yasar, F. Arslan, An amperometric biosensor or uric acid determination prepared from uricase immobilised in polypyrrole film, Artif. Cells, Blood Substitues, Immobilization Biotechnol. 34 (2006) 367–380. [15] F. Zhang, X. Wang, A.I. Shiyun, Z. Sun, Q. Wan, Z. Zlu, Y. Xian, L. Jin, Immobilization of uricase on ZnO nanorods for a reagentless uric acid biosensor, Anal. Chim. Acta 519 (2004) 155–160. [16] K. Arora, G. Sumana, V. Saxena, R.K. Gupta, S.K. Gupta, J.V. Yakhmi, M.K. Pandey, S. Chand, B.D. Malhotra, Improved performance of polyaniline–uricase biosensor, Anal. Chim. Acta 594 (2007) 17–23. [17] F. Arslan, An amperometric biosensor for uric acid determination prepared from uricase immobilized in polyaniline–polypyrrole film, Sensors 8 (2008) 5492–5500. [18] M. Bhambi, G. Sumana, B.D. Malhotra, C.S. Pundir, An amperometric uric acid biosensor based on immobilization of uricase onto polyaniline–multiwalled carbon nanotube composite film, Artif. Cells, Blood Substitues, Immobilization Biotechnol. 38 (2010) 178–185. [19] P. Pandey, M. Datta, B.D. Malhotra, Prospects of nanomaterials in biosensors, Anal. Lett. 41 (2008) 159–209. [20] P. Pandey, S.P. Singh, S.K. Arya, V. Gupta, M. Datta, S. Singh, Application of thiolated gold nanoparticles for the enhancement of glucose oxidase activity, Langmuir 23 (2007) 3333–3337. [21] A. Wei, X.W. Sun, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Enzymatic glucose biosensor based on ZnO nanorod array grown by hydrothermal decomposition, Appl. Phys. Lett. 89 (2006) 123902.
615
[22] J.X. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Zinc oxide nanocomb biosensor for glucose detection, Appl. Phys. Lett. 88 (2006) 233106. [23] L.M. Rossi, A.D. Quach, Z. Rosenzweig, Glucose oxidase–magnetite nanoparticle bioconjugate for glucose sensing, Anal. Bioanal. Chem. 380 (2004) 606–613. [24] B.W. Lu, W.C. Chen, A disposable glucose biosensor based on drop-coating of screen-printed carbon electrodes with magnetic nanoparticles, J. Magn. Mater. 304 (2006) 400–402. [25] A.K. Gupta, M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications, Biomaterials 26 (2005) 3995–4021. [26] A. Tiwari, S.K. Shukla, Synthesis and characterization of electrical conducting chitosan-graft-polyaniline, Polym. Lett. 3 (2009) 553–559. [27] J. Anand, S. Palaniappan, D.N. Sathyanarayan, Conducting polyaniline blends and composites, Prog. Polym. Sci. 23 (1998) 993–1018. [28] K. Yasuji, T. Ichikawa, E. Nakano, Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase), J. Biochem. 120 (1996) 969–973. [29] D.K. Kim, M. Mikhaylova, Y. Zhang, M. Muhammed, Protective coating of superparamagnetic iron oxide nanoparticle, Chem. Mater. 15 (2003) 1617–1627. [30] K.R. Reddy, K.P. Lee, A.G. Lyengar, Synthesis and characterization of novel conducting composites of Fe3 O4 nanoparticles and sulfonated polyanilines, J. Appl. Polym. Sci. 104 (2007) 4127–4134. [31] F.Y. Cheng, C.H. Su, Y.S. Yang, C.S. Yeh, C.Y. Tsai, C.L. Wu, Characterization of aqueous dispersions of Fe3 O4 nanoparticles and their biomedical applications, Biomaterials 26 (2005) 729–738. [32] Y.Y. Liang, L.M. Zhang, W. Li, R.F. Chen, Polysaccharide-modified iron oxide nanoparticles as an effective magnetic affinity adsorbent for bovine serum albumin, Colloid Polym. Sci. 285 (2007) 1193–1199. [33] A. Tiwari, V. Singh, Synthesis and characterization of electrical conducting chitosan-graft-polyaniline, Polymer. Lett. 1 (2007) 308–317. [34] I.S. Kuwabata, T.S. Nakminami, H. Yoneyama, Preparation and properties of amperometric uric acid sensors, Sens. Actuators, B 52 (1998) 72–77. [35] Y. Jiang, A. Wang, J. Kan, Selective uricase biosensor based on polyaniline synthesized in ionic liquid, Sens. Actuators, B 124 (2007) 529–534.
Biographies C.S. Pundir Biochemistry and Biotechnology educator, Head, Dept. of Biochemistry and Former Dean, Faculty of Life Sciences, M.D. University, Rohtak India. B.Sc. Hons. in Chemistry, M.S. & Ph.D. in Biochemistry, G.B. Pant U. Agr. and Tech., Pantnagar, India. Rooma Devi received Ph.D. degree in Jan. 2013 from Department of Biochemistry, M.D. University, Rohtak 124001, India, under the supervision of Prof. C.S. Pundir. Her interested fields include electrochemistry, chemically modified electrode, enzymebased biosensor and immobilization of enzymes.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Construction and application of an amperometric uric acid biosensor based on covalent immobilization of uricase on iron oxide nanoparticles/chitosan-g-polyaniline composite film electrodeposited on Pt electrode Rooma Devi, C.S. Pundir ∗ Department of Biochemistry, Maharshi Dayanand University, Rohtak 124001, Haryana, India
a r t i c l e
i n f o
Article history: Received 30 August 2013 Received in revised form 2 December 2013 Accepted 3 December 2013 Available online 12 December 2013 Keywords: Uricase Uric acid biosensor Iron oxide nanoparticles Chitosan Polyaniline
a b s t r a c t Commercial uricase was immobilized covalently onto iron oxide nanoparticles/chitosangraft-polyaniline (Fe3 O4 -NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode. Transmission electron microscopy (TEM) was used for characterization of Fe3 O4 -NPs. A uric acid biosensor was fabricated using/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 1s at pH 7.5 and 30 ◦ C, when polarized at 0.4 V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on uric acid concentration ranging from 0.1 to 800 M. The sensitivity of the biosensor was 0.44 mA mM−1 cm−2 , with a detection limit of 0.1 M (S/N = 3). Apparent Michaelis–Menton (Km ) value for uric acid was 12.5 M, and Imax 0.008A. The biosensor showed only 10% loss in its initial response after 120 uses over 100 days, when stored at 4 ◦ C. The biosensor measured uric acid in the serum of apparently healthy persons, which correlated well with a standard enzymic colorimetric method (r = 0.98). © 2014 Elsevier B.V. All rights reserved.
Introduction Uric acid (2,6,8-trihydroxypurine) is the main final product of purine metabolism in humans. It is an important marker in clinical diagnosis, because the elevated uric acid concentration in blood (hyperuricemia) is associated with a number of diseases, such as gout, arthritis [1], cardiovascular diseases [2], neurological diseases [3], insulin resistance, hypertension, and renal insufficiency [4,5]. A healthy adult human excretes uric acid at a rate of 0.6 g/24 h; the excreted product arises in part from turnover of the purine nucleotide of nucleic acids. The normal level of uric acid in serum is between 0.13 and 0.46 mM (2.18–7.7 mg dl−1 ) [6,7]. Consequently, uric acid determination is of paramount importance in the diagnosis and medical management of diseases caused by disorder of purine metabolism. Various methods such as colorimetric [7], high performance liquid chromatography (HPLC) [8,9], chemiluminescence [10] and
∗ Corresponding author. Tel.: +91 1262 295480/+91 9416 492413; fax: +91 1262 295480. E-mail addresses: [email protected], [email protected] (C.S. Pundir). 0925-4005/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.12.010
fluorescence methods [11] have been reported for the measurement of uric acid. However, the colorimetric method is time consuming and sensitive to numerous metabolites and drugs found in biological samples, while the enzymatic colorimetric method is expensive and also prone to metabolites. The biosensing method has many advantages over these routine techniques in terms of simplicity, rapidity, sensitivity and cost of analysis. Various electrochemical methods have been proposed for routine uric acid analysis, including potentiometric biosensors [12–18]. However, these electrochemical biosensors had problems such as lack of stability, sensitivity and low reproducibility, which are still to be improved. To overcome these problems, the enzyme is required to be immobilized directly and covalently onto the film coated electrode. The emergence of nanotechnology offered great opportunities to improve the sensitivity, stability and anti-interference ability of biosensing systems. Applications of nanomaterials to biosensors have recently aroused much interest as these materials exhibit large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency and strong adsorption ability that are helpful for immobilization of biosensing molecules. Moreover, nanoparticles have a unique ability to promote fast electron transfer between the electrode and the active site of the enzyme [19]. In
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this context, different types of nanoparticles such as gold (AuNPs) [20], zinc-oxide (ZnO-NPs) [21], iron-oxide (Fe3 O4 -NPs) [22], have been suggested as promising matrices for enzyme immobilization to improve the stability and sensitivity of the biosensor. Among these metal oxide nanoparticles, Fe3 O4 -NPs have been considered as most interesting for the immobilization of desired biomolecules, due to their high biocompatibility and strong superparamagnetic behavior, which provide better contact and low toxicity[22]. Immobilization of bioactive molecules onto a surface charged with super para magnetic nanoparticles is of special interest, since the magnetic behavior of these bioconjugates may result in improved delivery and recovery of biomolecules for desired biosensing applications [23,24]. Another very important property of this type of nanoparticle for electrochemical biosensors is their ability to provide a favorable microenvironment for biomolecules to exchange electrons directly with an electrode, thus improving the sensitivity of electrochemical biosensors. Besides this, the existing problem of aggregation and rapid biodegradation of Fe3 O4 -NPs onto a given matrix containing biomolecules can perhaps be overcome by modifying these nanoparticles using chitosan (CHIT). CHIT is an abundant natural biopolymer with excellent film forming abilities, biocompatibility, nontoxicity, good water permeability, high mechanical strength [24] and susceptible to chemical modification, due to the presence of reactive hydroxyl and amino functional groups. CHIT can accumulate metal ions through various mechanisms, such as chelation, electrostatic attraction and ion exchange, depending on the nature of the metal ion and pH of the solution. CHIT along with polyaniline (PANI), a conducting polymer provides a suitable matrix for covalent immobilization and stabilization condition for the biomolecules [25]. PANI as a conducting polymer has become very attractive, because of its facile synthesis [25], stable chemical and environmental properties [26] and inherent reversible doping/dedoping states [27]. Therefore, it is a suitable matrix for achieving electromagnetic shielding or absorption materials by doping with magnetic Fe3 O4 -NPs. The present report describes herein the use of unique properties of Fe3 O4 -NPs, CHIT and PANI for fabrication of an improved uric acid biosensor.
Material and methods Uric acid, Sephadex G-100, glutaraldehyde (25%) and CHIT were from Sigma-Aldrich, USA. A uric acid kit for enzymatic colorimetric determination manufactured by Transasia Bio-medicals (Solan, India) was obtained from Scientific Emporium (Rohtak, India). Aniline (purified through vacuum distillation before use) and zinc nitrate were from SISCO Research Lab., Mumbai, India. All other chemicals were of analytical reagent (AR) grade. Double distilled water (DW) was used in all experiments.
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Preparation of uricase Uricase (UOx) was prepared by dissolving 3.0 mg of powder of enzymatic reagent 1 of the uric acid kit into 1 ml of 0.02 M sodium phosphate buffer (pH 7.0) and loading it on a Sephadex G-100 column (24 × 1 cm) preequilibrated with 0.02 M sodium phosphate buffer (pH 7.0). The column was run in the same buffer at a flow rate of 0.5 ml min−1 . The fractions (2 ml each) were collected after passing the void volume. The fractions were tested for protein by the Lowry method. Fractions showing the presence of protein were pooled and treated as purified enzyme and tested for its activity as given below. Assay of free uricase The assay of free uricase was carried out as described previously [28] with slight modification and based on a decrease in A293 due to uric acid. The reaction mixture contained 3.0 ml of 50 mM Tris–HCl buffer (pH 8.5), 0.1 ml of uric acid (10 mM) and 50 l of uricase (1.6 mg ml−1 ). The blank contained 3.05 ml of 50 mM Tris–HCl buffer (pH 8.5) and 0.1 ml of 10 mM uric acid. The control consisted of 3.0 ml of 50 mM Tris–HCl buffer (pH 8.5), 0.1 ml of 10 mM uric acid, and 50 l of heat-denatured uricase. The decrease in A293 due to uric acid was read in an ultraviolet (UV) spectrophotometer for 4 min at an interval of 1 min. The activity of enzyme was calculated as follows: Unit activity =
(A293 per min in test solution-blank) × test volume 12.6 × volume of enzyme taken
One enzyme unit (U) is defined as amount of enzyme required to oxidize 1 mol of uric acid into allantoin per minute per milliliter under standard assay conditions. The free uricase showed an activity of 0.2 U min−1 ml−1 . Preparation of iron oxide nanoparticles (Fe3 O4 -NPs) The Fe3 O4 -NPs were prepared by the co-precipitation of Fe(II) and Fe(III) under a alkaline condition as described by Kim et al. [29], with slight modification. Five milliliters of iron ion solution containing 0.25 M ferrous chloride and ferric chloride (1:1 ratio) was added drop wise into 50 mL NaOH (2 mol l−1 ) solution under vigorous mechanical stirring for 35 min at 80 ◦ C. The precipitates were washed firstly with DW and then ethanol 2–3 times and collected by applying an external magnetic field until the supernatant solution turned neutral and finally, these were dried in oven at 70 ◦ C. The morphology, particle size and structure of the Fe3 O4 -NPs were determined by a TEM and XRD.
Instruments
Construction of iron oxide nanoparticles/chitosan-g-polyaniline/platinum electrode (Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode)
All electrochemical experiments and electrochemical impedance spectroscopic (EIS) measurements were performed at 25 ± 1 ◦ C using an potentiostat/galvanostat equipped with an Autolab PGSTAT-302N, GPES and FRA software (Eco-Chemie, Utrecht, The Netherlands) with a three electrode system consisting Uricase/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode used as working, Ag/AgCl as reference and Pt wire as auxillary electrode. Fourier transform infrared (FTIR) spectroscopy was recorded in FTIR spectrophotometer (Thermo Scientific, USA). Scanning electron microscopy (SEM) transmission electron microscopy (TEM) and X-ray diffraction (XRD) studies were carried out on commercial basis.
Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode was prepared by cyclic voltammetric method. CHIT solution was prepared by dissolving 2.0 g of CHIT flakes into 100 ml of 1.0% acetic acid and stirred for 3 h at room temperature until completely dissolved.The CHIT solution was stored in a refrigerator when not in use. Fe3 O4 -NPs(400 l) were dispersed into transparent CHIT solution and kept on magnetic stirring for about 30 min at room temperature followed by the sonication for 4 h. Finally a highly viscous solution of CHIT with uniformly dispersed Fe3 O4 -NPs was obtained. A solution for electrodeposition was prepared by adding aniline (180 l) + Fe3 O4 -NPs/CHIT (75 l) + 0.5 M HCl(10 ml) in a glass cell. A nanocomposite film of Fe3 O4 -NPs/CHIT-g-PANI
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was electrodeposited on Pt electrode by cyclic voltammetry using three electrode-systems. A Pt electrode (1.9 cm × 1 mm) (length × diameter) was ultrasonicated in 5.0 M HNO3 and acetone for 15 min, rinsed with DW, and then immersed into a electrodepositing solution, the potential scan was cycled for 30 times between −0.1 and 1.0 V vs Ag/AgCl at a scan rate of 50 mV s−1 and subsequently allowed to dry at room temperature. The resulting Fe3 O4 -NPs/CHITg- PANI/Pt electrode was washed with DW. Immobilization of uricase on Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode Uricase was immobilized onto the Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode surface through glutaraldehyde coupling. First 10 l of 2.5% glutaraldehyde solution in 0.05 M phosphate buffer (PB) pH 7.5 was spread over the Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode and kept for 5 h at room temperature, washed in 0.05 M PB, pH 7.5 and then 100 l uricase was mounted on surface of the electrode and dried. The resulting UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode was washed thoroughly with 0.05 M PB of pH 7.5 to rinse off any loosely bound enzyme from the electrode. The resulting enzyme electrode was dried at room temperature and then stored in a refrigerator at 4 ◦ C, when not in use. The fabricated electrode was characterized by cyclic voltammetric, SEM, FTIR and EIS studies. Cyclic voltammetric measurement and optimization of uric acid biosensor The sensitivity of uric acid biosensor was tested by measuring current using three electrode system. The working (UOx/Fe3 O4 NPs/CHIT-g-PANI/Pt) electrode along with Ag/AgCl as reference electrode and Pt wire as auxillary electrode were connected through potentiostat/galvanostat to construct uric acid biosensor. The electrode system was dipped into a reaction mixture containing 10 ml 0.05 M PB, pH 7.5 and 0.5 ml uric acid solution (50–250 M). The electrode current response was measured applying a potential range of −0.1 to 1.0 V vs Ag/AgCl. The steady state current response increased with increase in working potential and optimum current response was obtained at 0.4 V vs Ag/AgCl. Therefore, 0.4 V vs Ag/AgCl was selected as the working potential for amperometric detection of uric acid concentration. To optimize the working condition of electrode, the pH of reaction buffer was varied from 6.0 to 10.0 at an interval of pH 0.5 using 0.05 M PB in the pH range 6–8 and 0.05 M sodium carbonate/bicarbonate buffer in the pH range 8.5–10. The optimum temperature was studied by incubating the reaction mixture at different temperature (25–45 ◦ C at an interval of 5 ◦ C). Similarly the current response was measured at 1–10 s at an interval of 1s and uric acid concentration was varied from 0.1 to 1200 M in reaction buffer under optimal conditions. Amperometric determination of uric acid in serum The modified Pt electrode was employed for measuring uric acid in serum. Serum samples from apparently healthy persons of different age groups and sex were collected from hospital of PGIMS, Rohtak in tubes and stored at 4 ◦ C until use. The content of uric acid in sera was determined by the present biosensor as described for its testing under optimal working conditions except that uric acid solution was replaced by serum. The content of uric acid was determined from standard curve between uric acid conc. vs current (mA) prepared under optimal conditions. The following criteria were studied to evaluate the performance of this biosensor viz. linearity, analytical recovery, detection limit, sensitivity, precision and correlation with standard method.
Fig. 1. (A) Transmission electron microscopic (TEM) images of Fe3 O4 -NPs dispersed in CHIT. (B) X-ray diffraction (XRD) pattern of Fe3 O4 -NPs.
Results and discussion Characterization of Fe3 O4 -NPs A TEM image of synthesized Fe3 O4 -NPs dispersed in CHIT (Fig. 1A) indicates that Fe3 O4 -NPs were nanocrystalline and composed predominantly of a large number of well dispersed spherical nanoparticles with some hexagonal shaped nanoparticles. The average size of the spherical nanoparticles was ∼20 nm. Moreover, Fe3 O4 -NPs were agglomerates due to their high surface area and magnetic dipole–dipole interactions between the particles [30] which could be overcome after modifying these NPs with CHIT. The XRD pattern (Fig. 1B) of synthesized Fe3 O4 -NPs showed their polycrystalline nature. The peak positions were well in agreement with joint committee on powder diffraction standard (JCPDS) and indexed to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0), respectively. The results obtained agree with standard magnetite (Fe3 O4 ) XRD patterns and identified that the Fe3 O4 -NPs were in a cubic spinel structure [31]. Construction of UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode Fig. 2 displays the cyclic voltammograms of CHIT-g-PANI (curve a) and Fe3 O4 -NPs/CHIT-g-PANI (curve b) composite film. It can be seen that the surface charged Fe3 O4 -NPs interact with the cationic biopolymer matrix of CHIT via electrostatic interactions and hydrogen bonding with NH2 /OH groups to form a hybrid
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The following electrochemical reactions occur during response measurements of present biosensor: UOx
Uric acid + O2 + H2 O −→ Allantoin + H2 O2 + CO2 0.4
H2 O2 −→ 2H + + O2 + 2e− 2e−
Fe3 O4 −NPs/CHIT −g−PANI
−→
Pt electrode
The current generated at the electrode was measured by potentiostat, which is directly p proportional to the uric acid concentration. Surface characterization by SEM Fig. 2. Cyclic voltamogram of CHIT-g-PANI (curve a) and Fe3 O4 -NPs/CHIT-g-PANI composite (curve b) films at 10 M uric acid.
nanobiocomposite [32]. The Fe3 O4 -NPs/CHIT-g-PANI composite film exhibited higher currents than the CHIT-g-PANI composite film, which indicate that the Fe3 O4 -NPs/CHIT-g-PANI composite film, had a larger effective surface area than the CHIT-g-PANI composite film. The Fe3 O4 -NPs/CHIT-g-PANI composite film appeared to provide a conducting path through the composite matrix for faster kinetics. Hence, the Fe3 O4 -NPs acting as an electron transfer mediator may help in enhancing the sensor response of enzyme electrode and thus increase the sensitivity of the biosensor. These observations suggest the formation of Fe3 O4 -NPs/CHIT-g-PANI composite film which provides a large surface area for the immobilization of enzyme. A method is described for construction of an amperometric biosensor using UOx/Fe3 O4 -NPs/CHIT-g-PANI film electrodeposited onto Pt electrode. This enzyme electrode is summarized in Scheme 1. To achieve it, firstly a Fe3 O4 -NPs/CHIT-g-PANI composite film was electrodeposited onto Pt electrode using cyclic voltammetry. The electrodeposition method was selected to produce Fe3 O4 -NPs film onto electrode surfaces, due to its simplicity and to control the layer thickness. Secondly, commercially available UOx was immobilized covalently onto Fe3 O4 -NPs/CHIT-g-PANI composite film through glutaraldehyde coupling. One –CHO group of the glutaraldehyde was attached to the NH2 group of CHIT of Fe3 O4 -NPs/CHIT-g-PANI film. While other –CHO group of the glutaraldehyde was attached to enzyme via its free –NH2 on the surface of enzyme, which resulted in UOx/Fe3 O4 -NPs/CHIT-g-PANI electrode.
The surface morphologies of Bare Pt electrode, Fe3 O4 NPs/CHITg-PANI/Pt and UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt were investigated by SEM (Fig. 3). Globular porous morphology of Fe3 O4 -NPs/CHIT-g-PANI (Fig. 3B) biocomposite film reveals incorporation on Pt electrode (Fig. 3A), indicating the formation of Fe3 O4 -NPs/CHIT-g-PANI biocomposite film. However, after the immobilization of UOx onto Fe3 O4 -NPs/CHIT-g-PANI composite film, the globular morphology changed to regular form (Fig. 3C). This suggests that Fe3 O4 NPs provided a favorable environment for high loading of UOx. FTIR spectra Fig. 4A showed the FTIR spectra obtained for CHIT-g-PANI, Fe3 O4 NPs/CHIT-g-PANI and UOx/Fe3 O4 NPs/CHIT-g-PANI composites. The FTIR spectrum of the CHIT-g-PANI composite (curve i) illustrated the characteristic peaks of PANI, as well as CHIT [33]. The stretching bands of benzenoid and quinoid ring were observed at 1528 and 1604 cm−1 . The absorption band of the N Q N bending vibration of protonated PANI was observed at 1194 cm−1 in the CHIT-g-PANI copolymer. The FTIR spectrum of Fe3 O4 NPs/CHIT-gPANI composite (curve ii) exhibits characteristic IR bands of the functional group corresponding to CHIT-g-PANI and the Fe3 O4 NPs. The absorption band of Fe3 O4 NPs appears at 581 cm−1 belonging to the stretching vibration mode and the torsional vibration mode of Fe–O bonds in the tetrahedral sites and in the octahedral sites. The FTIR spectrum of the UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode (curve iii) showed peaks broadening at 3098–3315 cm−1 (addition of N H stretching vibration) due to the attachment of enzymes with the Fe3 O4 NPs/CHIT-g-PANI composite. Hence, FTIR
Scheme 1. Chemical reactions involved in immobilization of uricase on Fe3 O4 -NPs/CHITg-PANI modified Pt electrode.
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Fig. 3. (A) SEM image of bare Pt electrode. (B) SEM image of Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode. (C) SEM image of UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode.
spectra confirmed the covalent immobilization of UOx onto the Fe3 O4 NPs/CHIT-g-PANI/Pt electrode. Electrochemical impedance spectroscopy (EIS) studies EIS is an effective method for probing the features of surface modified electrodes. The Nyquist plot of impedance spectra includes a semicircle portion and a linear portion, with the former at higher frequencies corresponding to the electron transfer limited process and the latter at lower frequencies corresponding to the
diffusion process. The electron transfer resistance (RCT ) at electrode surface is equal to the semicircle diameter, which can be used to describe the interface properties of the electrode. Fig. 5 presents the Nyquist plot of the impedance spectroscopy of CHITg-PANI/Pt, Fe3 O4 NPs/CHIT-g-PANI/Pt and UOx/Fe3 O4 NPs/CHIT-gPANI/Pt electrode in 5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.05 M PB, pH 7.5. The diameter of semicircle for Fe3 O4 NPs/CHITg-PANI composite film (curve b) was smaller than that of CHIT-g-PANI composite film (curve a) which suggested that electron transfer in Fe3 O4 NPs/CHIT-g-PANI composite film was
Fig. 4. (A) FTIR spectra obtained for CHIT-g-PANI (curve i), Fe3 O4 -NPs/CHIT-g-PANI (curve ii), UOx/Fe3 O4 -NPs/CHIT-g-PANI (curve iii).
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250
(c)
Z" (ohms)
200 150 100
b
50
(a)
0 0
100
200
300
400
500
600
Z' (ohms) Fig. 5. The Nyquist plot of the EIS of (a) CHIT-g-PANI/Pt, (b)Fe3 O4 NPs/CHIT-g-PANI/Pt and (c) UOx/Fe3 O4 NPs/CHIT-g-PANI/Pt electrode in 5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.05 M PB, pH 7.5.
easier between the solution and electrode i.e. Fe3 O4 NPs which not only provided the hydrophilic surface but also acted as a nanoscale electrode and promoted electron transfer due to permeable structure of CHIT-g-PANI/Pt. However the diameter of the semicircle for UOx/Fe3 O4 NPs/CHIT-g-PANI/Pt electrode (curve c) was further increased. This increase in diameter can be attributed to the fact that most biological molecules, including enzymes, are poor electrical conductor at low frequencies and cause hindrance to electron transfer. These results also indicate binding of enzyme onto Fe3 O4 NPs/CHIT-g-PANI composite film.
of 0.025–0.1 mM [30], 0.05–0.5 mM [31] and 0.0–0.30 mM [32]. The calibration curve was linear up to 0.1 M with a sensitivity of 0.44 mA mM−1 cm−2 , which is higher than that reported previously (0.024 mA mM−1 cm−2 ).
Optimization of the biosensor (UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode) The experimental conditions affecting the biosensor response were studied in terms of the effects of pH, incubation temperature, time and substrate (uric acid) concentration. The optimal current was obtained within 1s at pH 7.5, which is higher than that of free enzyme (pH 7.0) [27]. The change in optimal pH of enzyme after immobilization might be due to an improved stability at that pH, the prevention of dissociation, an internal pH difference from the external pH, and/or even a chemical modification of the enzyme. The immobilized enzyme had an optimal temperature of 35 ◦ C, which is slightly higher than that of free enzyme (37 ◦ C) [27] UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt modified electrode achieved 95% of steady state currents within 1s, which is lower than 8s [18] 60s [16] and 70s [17] reported previously. There was a linear relationship between biosensor response and uric acid concentration up to a final concentration of 0.1 M (Fig. 6A–C). The Lineweaver–Burk plot between 1/I (mA) and 1/[uric acid] yielded an apparent Km of 0.17 mM, and Imax 0.008A, which is lower than that for free enzyme (0.90 mM) [34], previous enzyme electrodes 0.34 mM [35] and polyaniline polypyrrole modified electrode (1.57 mM) [17], indicating increased affinity of enzyme toward substrate (uric acid) after immobilization, which might be due to enhanced diffusion of uric acid through Fe3 O4 -NPs/CHIT-gPANI composite. Evaluation of uricase biosensor Linearity and sensitivity There was a linear relationship between biosensor response (i.e. current) and uric acid concentration ranging from 0.1 to 800 M in Tris–HCl buffer (pH 7.5), which is better than previous reports
Fig. 6. Effect of substrate (uric acid) concentration on response of the uric acid biosensor (A) and the standard curve of uric acid at lower and higher concentration range showing the linear relationship by the uric acid biosensor based on UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode (B and C).
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Fig. 7. Correlation (0.98) between serum samples (mg l−1 ) determinations by standard enzymic colorimetric method (x) and present amperometric method (y).
Detection limit The detection limit of the biosensor was 0.1 M at a signal-to noise ratio of 3, which is lower than that of the previously reported uric acid biosensor of 0.05 mM [19]. Recovery and precision The analytical recoveries of added uric acid in serum samples at 1 mg dl−1 and 2 mg dl−1 concentrations were 94.0% to 98.9%, respectively, demonstrating the good reliability of the present method (Table 1). Within and between coefficients of variation were 1.58% and 2.07%, respectively. The high precision indicated the good reproducibility and consistency of the present method (Table 2). Correlation A Comparison of uric acid values in 15 serum samples, as measured by the present method (y) with those obtained by standard enzymic colorimetric method (x) showed a good correlation with r = 0.98, regression equation being, y = 0.9598x + 0.7474. These results indicate the high accuracy of the method (Fig. 7).
Table 3 Effect of potential interferents on UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode based uric acid biosensor response. Compound added
Concentration (physiological conc. in serum)
% Relative response
None Ascorbic acid Urea Cholesterol EDTA Lactic acid Glucose Pyruvate
– 3.40 mM 0.10 g l−1 2.00 g l−1 2.00 mM 0.50 mM 0.90 g l−1 0.01 g l−1
100.00 ± 0.01 92.40 ± 0.64 95.10 ± 0.51 91.70 ± 0.35 94.34 ± 0.57 96.60 ± 0.72 97.89 ± 0.36 98.90 ± 0.30
Storage stability and reusability of biosensor To examine the long-term storage stabilities, the activity of UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode was tested with respect to storage time. After each experiment, the sensor was washed with working buffer solution and stored at 4 ◦ C. Studies revealed that the electrode lost only 10% loss in its initial response after 120 uses over 100 days. Conclusion
Interference study The effect of various interferents found in serum such as ascorbic acid, urea, cholesterol, EDTA, Lactic acid, glucose and pyruvate were studied on the response of UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt at their physiological concentrations. All these metabolites had practically no effect (Table 3). Table 1 Analytical recovery of added uric acid in serum sample, by uric acid biosensor based on UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode.
The use of Fe3 O4 -NPs/CHIT-g-PANI composite film has resulted into an improved analytical performance of the uric acid biosensor in terms of low response time (1s), lower/better detection limit, 0.1 M (S/N = 3), higher storage stability (100 days), linear range (0.1–800 M) and no interference by various metabolites. Thus this work illustrates a simple and novel approach for the development of an amperometric biosensor for determination of serum uric acid concentration in gout patients, employing UOx/Fe3 O4 -NPs/CHIT-gPANI/Pt nanocomposite.
Uric acid added (mg dl−1 )
Uric acid found (mg dl−1 ) mean (n = 6)
% Recovery mean ± SD
Acknowledgment
– 1.0 2.0
1.04 1.98 3.00
– 94.00 ± 0.67 98.90 ± 1.1
Rooma Devi is thankful to the Indian Council of Medical Research for the award of Senior Research Fellowship (SRF) and Research Associateship (RA).
Table 2 Within batch and between batch coefficients of variation for determination of uric acid, by uric acid biosensor based on UOx/Fe3 O4 -NPs/CHIT-g-PANI/Pt electrode. (n)
Uric acid (mg dl−1 ) mean ± SE
Coefficient of variation (%)
Within assay (5) Between assay (5)*
2.31 ± 0.04 2.45 ± 0.02
1.58 2.07
*
After storage at -20 degree for one week.
References [1] H.A. Harper (Ed.), Review of Physiological Chemistry, 16th ed., Lange Medical Publications, San Francisco, CA, 1977. [2] R.H. Heptinstall (Ed.), Gout in Pathology of the Kidney, second ed., Little Brown, Boston, MA, 1966. [3] F. Arellano, J.A. Sacristan, Allopurinol hypersensitivity syndrome: a review, Ann. Pharmacother. 27 (1993) 337–343. [4] Y.S. Zhao, F.H. Liao, Serum uric acid and hyperuricemia associated disease, Int. J. Intern. Med. 34 (2007) 41–47.
R. Devi, C.S. Pundir / Sensors and Actuators B 193 (2014) 608–615 [5] M.A. Becker, M. Jolly, Hyperuricemia and associated diseases, Rheum. Dis. Clin. North Am. 32 (2006) 275–293. [6] C.R. Raj, T. Ohsaka, Voltammetric detection of uric acid in the presence of ascorbic acid at a gold electrode modified with a self-assembled monolayer of heteroaromatic thiol, J. Electroanal. Chem. 540 (2003) 69–77. [7] I.C. Isdale, M.J. Buchanan, B.S. Rose, Serum uric acid estimation: a modified chemical method, Ann. Rheum. Dis. 25 (1966) 184–185. [8] K. Lorentz, W. Berndt, Enzymic determination of uric acid by a colorimetric method, Anal. Biochem. 18 (1967) 58–63. [9] D. Yao, A.G. Vlessidis, N.P. Evmriridis, Monitoring reactive oxygen species in vivo using microdialysis sampling and chemiluminescence detection as an alternative global method for determination of total antioxidant capacity, Anal. Chim. Acta 467 (2002) 133–144. [10] X. Dai, X. Fang, C. Zhang, R. Xu, B. Xu, Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID–MS) as a candidate reference method, J. Chromatogr. B 1 (2007) 287–295. [11] J. Galban, Y. Andreu, M.J. Almenara, S. de Marcos, J.R. Castillo, Direct determination of uric acid in serum by a fluorometric–enzymatic method based on uricase, Talanta 54 (2001) 847–854. [12] J. Kan, X. Pan, C. Chen, Polyaniline–uricase biosensor prepared with template process, Biosens. Bioelectron. 19 (2004) 1635–1640. [13] Y.C. Luo, J.S. Do, C. Liu, An amperometric uric acid biosensor based on modified Ir–C electrode, Biosens. Bioelectron. 22 (2006) 482–488. [14] S. Cete, A. Yasar, F. Arslan, An amperometric biosensor or uric acid determination prepared from uricase immobilised in polypyrrole film, Artif. Cells, Blood Substitues, Immobilization Biotechnol. 34 (2006) 367–380. [15] F. Zhang, X. Wang, A.I. Shiyun, Z. Sun, Q. Wan, Z. Zlu, Y. Xian, L. Jin, Immobilization of uricase on ZnO nanorods for a reagentless uric acid biosensor, Anal. Chim. Acta 519 (2004) 155–160. [16] K. Arora, G. Sumana, V. Saxena, R.K. Gupta, S.K. Gupta, J.V. Yakhmi, M.K. Pandey, S. Chand, B.D. Malhotra, Improved performance of polyaniline–uricase biosensor, Anal. Chim. Acta 594 (2007) 17–23. [17] F. Arslan, An amperometric biosensor for uric acid determination prepared from uricase immobilized in polyaniline–polypyrrole film, Sensors 8 (2008) 5492–5500. [18] M. Bhambi, G. Sumana, B.D. Malhotra, C.S. Pundir, An amperometric uric acid biosensor based on immobilization of uricase onto polyaniline–multiwalled carbon nanotube composite film, Artif. Cells, Blood Substitues, Immobilization Biotechnol. 38 (2010) 178–185. [19] P. Pandey, M. Datta, B.D. Malhotra, Prospects of nanomaterials in biosensors, Anal. Lett. 41 (2008) 159–209. [20] P. Pandey, S.P. Singh, S.K. Arya, V. Gupta, M. Datta, S. Singh, Application of thiolated gold nanoparticles for the enhancement of glucose oxidase activity, Langmuir 23 (2007) 3333–3337. [21] A. Wei, X.W. Sun, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Enzymatic glucose biosensor based on ZnO nanorod array grown by hydrothermal decomposition, Appl. Phys. Lett. 89 (2006) 123902.
615
[22] J.X. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Zinc oxide nanocomb biosensor for glucose detection, Appl. Phys. Lett. 88 (2006) 233106. [23] L.M. Rossi, A.D. Quach, Z. Rosenzweig, Glucose oxidase–magnetite nanoparticle bioconjugate for glucose sensing, Anal. Bioanal. Chem. 380 (2004) 606–613. [24] B.W. Lu, W.C. Chen, A disposable glucose biosensor based on drop-coating of screen-printed carbon electrodes with magnetic nanoparticles, J. Magn. Mater. 304 (2006) 400–402. [25] A.K. Gupta, M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications, Biomaterials 26 (2005) 3995–4021. [26] A. Tiwari, S.K. Shukla, Synthesis and characterization of electrical conducting chitosan-graft-polyaniline, Polym. Lett. 3 (2009) 553–559. [27] J. Anand, S. Palaniappan, D.N. Sathyanarayan, Conducting polyaniline blends and composites, Prog. Polym. Sci. 23 (1998) 993–1018. [28] K. Yasuji, T. Ichikawa, E. Nakano, Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase), J. Biochem. 120 (1996) 969–973. [29] D.K. Kim, M. Mikhaylova, Y. Zhang, M. Muhammed, Protective coating of superparamagnetic iron oxide nanoparticle, Chem. Mater. 15 (2003) 1617–1627. [30] K.R. Reddy, K.P. Lee, A.G. Lyengar, Synthesis and characterization of novel conducting composites of Fe3 O4 nanoparticles and sulfonated polyanilines, J. Appl. Polym. Sci. 104 (2007) 4127–4134. [31] F.Y. Cheng, C.H. Su, Y.S. Yang, C.S. Yeh, C.Y. Tsai, C.L. Wu, Characterization of aqueous dispersions of Fe3 O4 nanoparticles and their biomedical applications, Biomaterials 26 (2005) 729–738. [32] Y.Y. Liang, L.M. Zhang, W. Li, R.F. Chen, Polysaccharide-modified iron oxide nanoparticles as an effective magnetic affinity adsorbent for bovine serum albumin, Colloid Polym. Sci. 285 (2007) 1193–1199. [33] A. Tiwari, V. Singh, Synthesis and characterization of electrical conducting chitosan-graft-polyaniline, Polymer. Lett. 1 (2007) 308–317. [34] I.S. Kuwabata, T.S. Nakminami, H. Yoneyama, Preparation and properties of amperometric uric acid sensors, Sens. Actuators, B 52 (1998) 72–77. [35] Y. Jiang, A. Wang, J. Kan, Selective uricase biosensor based on polyaniline synthesized in ionic liquid, Sens. Actuators, B 124 (2007) 529–534.
Biographies C.S. Pundir Biochemistry and Biotechnology educator, Head, Dept. of Biochemistry and Former Dean, Faculty of Life Sciences, M.D. University, Rohtak India. B.Sc. Hons. in Chemistry, M.S. & Ph.D. in Biochemistry, G.B. Pant U. Agr. and Tech., Pantnagar, India. Rooma Devi received Ph.D. degree in Jan. 2013 from Department of Biochemistry, M.D. University, Rohtak 124001, India, under the supervision of Prof. C.S. Pundir. Her interested fields include electrochemistry, chemically modified electrode, enzymebased biosensor and immobilization of enzymes.