Iron oxide-chitosan nanobiocomposite for urea sensor

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Author's personal copy Sensors and Actuators B 138 (2009) 572–580

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Iron oxide-chitosan nanobiocomposite for urea sensor Ajeet Kaushik a,b , Pratima R. Solanki a , Anees A. Ansari a , G. Sumana a , Sharif Ahmad b , Bansi D. Malhotra a,∗ a b

Department of Science & Technology Centre on Biomolecular Electronics, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 110012, India Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, India

a r t i c l e

i n f o

Article history: Received 29 December 2008 Received in revised form 4 February 2009 Accepted 6 February 2009 Available online 20 February 2009 Keywords: Chitosan Fe3 O4 nanoparticles Nanobiocomposite Urea Biosensor

a b s t r a c t Urease (Ur) and glutamate dehydrogenase (GLDH) have been co-immobilized onto superparamegnatic iron oxide (Fe3 O4 ) nanoparticles-chitosan (CH) based nanobiocomposite film deposited onto indium-tinoxide (ITO) coated glass plate via physical adsorption for urea detection. The magnitude of magnetization (60.9 emu/g) of Fe3 O4 nanoparticles (∼22 nm) estimated using vibrating sample magnetometer (VSM) indicates superparamagnetic behaviour. It is shown that presence of Fe3 O4 nanoparticles results in increased active surface area of CH-Fe3 O4 nanobiocomposite for immobilization of enzymes (Ur and GLDH), enhanced electron transfer and increased shelf-life of nanobiocomposite electrode. Differential pulse voltammetry (DPV) studies show that Ur-GLDH/CH-Fe3 O4 /ITO bioelectrode is found to be sensitive in the 5–100 mg/dL urea concentration range and can detect as low as 0.5 mg/dL. A relatively low value of Michaelis–Menten constant (Km , 0.56 mM) indicates high affinity of enzymes (Ur and GLDH) for urea detection. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The increasing demand for clinical diagnostics relating to kidney and liver diseases has necessitated evolution of new methods for faster and accurate estimation of urea in desired samples including urine and blood samples. The increased urea concentration (normal level in serum is 8–20 mg/dL) causes renal failure (acute or chronic), urinary tract obstruction, dehydration, shock, burns and gastrointestinal bleeding. Moreover, decreased urea concentration causes hepatic failure, nephritic syndrome, cachexia (low-protein and high-carbohydrate diets) [1–3]. The conventional methods for urea detection including gas chromatography, calorimetry and fluorimetric analysis suffer from complicated sample pre-treatment and are unsuitable for on-line monitoring. Electrochemical biosensors have been considered to provide interesting alternatives due to their simplicity, low cost and high sensitivity [3,4]. Biosensors reported for urea detection are generally based on urease (Ur) that is often present in most biological systems [1–7]. Ur catalyzes decomposition of urea into hydrogen bicarbonate and ammonium ions (NH4 + ). NH4 + ions are known to be unstable and easily disperse in the environment. Keeping this in view, glutamate dehydrogenase (GLDH) along with Ur has been utilized for urea detection since GLDH immediately catalyzes the reaction

∗ Corresponding author. Tel.: +91 11 45609152; fax: +91 11 45609310. E-mail address: [email protected] (B.D. Malhotra). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.02.005

between NH4 + , ␣-ketoglutarate (␣-KG) and nicotinamide adenine di-nucleotide (NADH) to produce NAD+ and l-glutamate [5–7]. Immobilization of Ur onto a suitable matrix is crucial for the development of an electrochemical urea sensor [1–6]. In this context, organic–inorganic hybrid nanobiocomposites have attracted much interest as a new class of materials that utilize the synergy of organic and inorganic components to obtain improved biosensing characteristics. In hybrid nanobiocomposites, surface fuctionalization of nanoparticles allows their covalent attachment and self-assembly on surfaces that can be used for loading of desired biomolecules in a favourable microenvironment for development of a biosensor [8,9]. In this context, metal oxide nanoparticles-chitosan (CH) based hybrid composites have attracted much interest for the development of a desired biosensor [6–8]. Metal oxide nanoparticles such as iron oxide (Fe3 O4 ) [10–12], zinc oxide (ZnO) [13,14], cerium oxide (CeO2 ) [15,16] etc. have been suggested as promising matrices for the immobilization of desired biomolecules. These nanomaterials exhibit large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency and strong adsorption ability that can be helpful to obtain improved stability and sensitivity of a biosensor. Moreover, nanoparticles have a unique ability to promote fast electron transfer between electrode and the active site of an enzyme. Among various metal oxide nanoparticles, Fe3 O4 nanoparticles due to biocompatibility, strong superparamagnetic behaviour and low toxicity have been considered as interesting for immobilization of desired biomolecules [17–19]. Immobilization of bioactive molecules onto surface charged superparmagnetic nanoparticles (size ∼25 nm) is

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of special interest, since magnetic behaviour of these bioconjugates may result in improved delivery and recovery of biomolecules for desired biosensing applications [9–11]. Besides this, existing problem of aggregation and rapid biodegradation of Fe3 O4 nanoparticles onto a given matrix containing biomolecules can perhaps be overcome by modifying these nanoparticles using CH by preparing hybrid nanobiocomposite [9,20–26]. CH (N-deacetylated derivative of chitin) is a linear copolymer of glucosamine and N-acetylglucosamine units and has been found in the exoskeleton of crustaceans, in fungal cell walls and in other biological materials. It displays an excellent film-forming ability, good adhesion, biocompatibility, high mechanical strength and susceptibility to chemical modification due to the presence of reactive hydroxyl and amino functional groups. CH has also been exploited for application in contact lens, as a matrix for cell and enzyme immobilization, and as an artificial skin (e.g., CH-collagen composite) [27–30]. Efforts have recently been made to improve optical and electrical properties of CH for biosensor application by dispersing superparamagnetic Fe3 O4 nanoparticles [9,26]. Lin et al. (2005) have fabricated CH-Fe3 O4 nanocomposite based chemical sensor for cathodic estimation of hydrogen peroxide (H2 O2 ). Harbac et al. (2007) have reported carbon electrode modified by nanoscopic Fe3 O4 particles to assemble chemical sensor for the estimation of H2 O2 using amperometric technique. Zhang et al. (2007) have studied direct electrochemistry of hemoglobin immobilized on carbon coated Fe3 O4 nanoparticles for amperometric estimation of H2 O2 . Zhao et al. (2006) have reported multi-layer thin film of CH-Fe3 O4 nanocomposite that has been found to promote direct electron transfer of hemoglobin. Liang et al. (2007) have synthesized polysaccharide modified iron oxide nanoparticles as an effective magnetic adsorbent for bovine albumin serum. CHFe3 O4 nanocomposite has recently been utilized for the estimation of ferritin and glucose sensing [9,21]. In this manuscript, we report results of studies relating to the immobilization of Ur and GLDH onto CH-Fe3 O4 nanobiocomposite film for urea sensor. 2. Materials and method 2.1. Reagents and preparation of solutions Urease (Ur), glutamate dehydrogenase (GLDH), nicotinamide adenine dinucleotide (NADH), ␣-ketoglutarate (␣-KG) and chitosan have been procured from Sigma–Aldrich (USA). Ferrous chloride, ferric chloride and triethyl amine have been purchased from Sigma–Aldrich and have been used for preparation of Fe3 O4 nanoparticles. Indium-tin-oxide (ITO) coated glass plates have been obtained from Balzers, UK. All chemicals used are of molecular biology grade. The deionized water (Milli Q 10 TS) has been used for the preparation of reagents. All the solutions and glass wares are autoclaved prior to being used. 2.2. Preparation of CH-Fe3 O4 hybrid nanobiocomposite Fe3 O4 nanoparticles (∼22 nm) prepared using co-precipitation method [9] are dispersed into 10 mL of CH (0.5 mg/mL) solution in acetate buffer of 0.05 M at pH 4.2 under continuous stirring at room temperature after which it is sonicated for about 4 h. Finally, viscous solution of CH with uniformly dispersed Fe3 O4 nanoparticles is obtained. CH-Fe3 O4 hybrid nanobiocomposite films have been fabricated by uniformly dispersing 10 ␮L solution of CH-Fe3 O4 composite onto an ITO surface (surface area is 0.25 cm2 ) and allowing it to dry at room temperature for 12 h. These solution cast CHFe3 O4 hybrid nanobiocomposite films are washed repeatedly with deionized water to remove any unbound particles.

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2.3. Immobilization of Ur and GLDH onto CH-Fe3 O4 hybrid nanobiocomposite film 10 ␮L of bienzyme solution containing Ur (10 mg/ml) and GLDH (1 mg/ml) in 1:1 ratio [prepared in Tris buffer (5 mM)] is immobilized onto CH-Fe3 O4 nanobiocomposite/ITO electrode. The Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrodes are kept undisturbed for about 12 h at 4 ◦ C. Finally, the dry bioelectrode is immersed in 50 mM PBS (pH 7.0) in order to wash out any unbound enzymes from the electrode surface. 2.4. Characterization The structure and particle size of Fe3 O4 nanoparticles have been investigated using X-ray diffraction (XRD) studies. The superparamagnetism of the Fe3 O4 nanoparticles has been measured using vibrating sample magnetometer (VSM, Lakeshore-7304). FTIR (PerkinElmer, Spectrum BX II) spectrophotometer has been used to characterize CH-Fe3 O4 hybrid nanobiocomposite and its interaction with Ur. The surface morphological studies have been investigated using scanning electron microscopy (LEO-440). The cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) measurements have been recorded on an Autolab Potentiostat/Galvanostat (Eco Chemie, Netherlands). The electrochemical measurements have been conducted on a three-electrode cell in phosphate buffer (50 mM, pH 7.0, 0.9% NaCl containing 50 mM [Fe(CN)6 ] 3−/4− as the electrolyte. 3. Results and discussion 3.1. Characterization of Fe3 O4 nanoparticles X-ray diffraction (XRD, Fig. 1A) pattern of synthesized Fe3 O4 nanoparticles reveals reflection planes that are consistent with Fe3 O4 . However, the broad reflection planes are perhaps due to the nano-size of Fe3 O4 particles. The average particle size of Fe3 O4 nanoparticles estimated using Scherrer formula has been estimated as 22 nm. However, broadening of the reflection peak indicates formation of fine nanocrystalline particles [9,31–32]. The superparamagnetism of Fe3 O4 nanoparticles and CH-Fe3 O4 has been estimated using Langevin equation as M = Ms [(Coth(mH/kb T ) − kb T/mH],

(1)

where Ms is the saturated magnetization of the nanoparticles, m is the average magnetic moment of an individual particle in the sample, H is the applied magnetic field, T is absolute temperature and kb is the Boltzmann constant [31]. The VSM plot between magnetizatioin M and applied magnetic field H (Fig. 1B) reveals superparamagnetic hysteresis of Fe3 O4 nanoparticles (curve a). It can be seen that the coercive force (Hc) is small (65.84 Oe and Hc is 500–800 Oe for bulk Fe3 O4 ), and this value does not decrease to zero due to small particle size indicating super paramagnetic behaviour. Interestingly, anisotropy energy is less than the heat disturbance energy of ions indicating that magnetized direction is no longer fixed and the movement of ions is random [31,32]. It may be noted that the value of Ms is considerably lower (60.91 emu/g) than that of bulk magnetite particle (65–84 emu/g) and low retentivity of 4.88 emu/g. However, the value of Hc, Ms and retentivity are found to decrease to 60.93, 48.67 and 3.98 emu/g, respectively for CH-Fe3 O4 nanobiocomposite (curve b). These results reveal that Fe3 O4 nanoparticles embedded in CH matrix in which each Fe3 O4 nanoparticle acts as single magnetic domain with least aggregates with a little change in magnetization and significant decrease in the retentivity.

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3.3. Characterization of CH-Fe3 O4 nanobiocomposite and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrodes

Fig. 1. (A) X-ray diffraction pattern of Fe3 O4 nanoparticles. (B) Magnetization curve of Fe3 O4 nanoparticles and CH-Fe3 O4 nanobiocomposite.

3.2. Mechanism of immobilization of Ur-GLGH onto CH-Fe3 O4 nanobiocomposite film The proposed mechanism relating to the preparation of CHFe3 O4 nanobiocomposite film and immobilization of Ur-GLDH onto CH-Fe3 O4 nanobiocomposite film is shown in Fig. 2. It can be seen that surface charged Fe3 O4 nanoparticles [33] interact with cationic biopolymer matrix of CH via electrostatic interactions and hydrogen bonding with –NH2 /OH group to form hybrid nanobiocomposite [22]. These electrostatic interactions between Fe3 O4 nanoparticles and CH have been confirmed by FTIR spectroscopic studies [9]. Ur is a multi-active sites enzyme composed of 3:3 (␣:␤) stoichiometry with a 2-fold symmetric structure and GLDH is a branch-point enzyme composed of 18 ␣ helices and ␤ sheet that are both parallel and anti-parallel. The Ur-GLDH molecules exist in anionic form at pH 7 because pH of solution is above isoelectric point (5.5) of Ur-GLDH molecules that facilitate [34] interactions with positively charged CH of nanobiocomposite via electrostatic interactions (Fig. 2). In the nanobiocomposite, presence of Fe3 O4 nanoparticles results in increased electroactive surface area of CH for loading of the enzymes due to affinity of the Fe3 O4 nanoparticles towards oxygen atoms of enzymes. This suggests that Ur-GLDH molecules easily bind with charged CH-Fe3 O4 hybrid nanobiocomposite matrix via electrostatic interactions and this is further confirmed by optical and electrochemical studies.

3.3.1. Optical studies The FTIR spectra (Fig. 3A) of pure CH (curve a,) display bands at 3200–3400 cm−1 due to the stretching vibration mode of OH and NH2 groups. The peaks seen at 2950–3000 and 2870 cm−1 are due to sp3 and sp2 hybridization, respectively (corresponding to C–H group). The peak due to C–O stretching along with N–H deformation mode has been observed at 1615 cm−1 . The bands at 1550, 1430 and 1340 cm−1 are attributed to N–H deformation, symmetrical deformation of CH3 and CH2 group, COO− group in carboxylic acid salt. The IR peaks seen at 1150 and 925 cm−1 are assigned to ␤ (1–4) glucosidic band in the polysaccharide unit, 1080 cm−1 is attributed to stretching vibration mode of the hydroxyl group and 1020 cm−1 stretching vibration is due to C–O–C in the glucose unit of CH [9,35]. The IR bands corresponding to –NH/OH stretching modes are found to be shifted in the IR spectra of CH-Fe3 O4 hybrid nanobiocomposite film (curve b) as compared to that of pure CH. This indicates that amine group of CH binds with Fe3 O4 nanoparticles via electrostatic interactions. The presence of IR band at 580 cm−1 (curve b) pertaining to the stretching vibration mode and the torsional vibration mode of Fe–O bonds in the tetrahedral sites and octahedral site reveal the formation of complex between surface charged Fe3 O4 nanoparticles and cationic CH matrix, indicating the formation of CH-Fe3 O4 hybrid nanobiocomposite [9]. However, shape of absorption peak in the CH-Fe3 O4 hybrid nanobiocomposite becomes broader due to overlapping of the functional groups of Ur-GLDH and CH-Fe3 O4 nanobiocomposite film (curve c) indicating immobilization of Ur-GLDH onto hybrid nanobiocomposite matrix. The surface morphologies of CH-Fe3 O4 nanobiocomposite/ITO electrode and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode have been investigated using scanning electron microscopy (SEM, Fig. 3B). Globular porous morphology of CH-Fe3 O4 nanobiocomposite reveals incorporation of the Fe3 O4 nanoparticles in CH, indicating the formation of CH-Fe3 O4 hybrid nanobiocomposite. This may be attributed to electrostatic interactions between cationic CH and the surface charged Fe3 O4 nanoparticles. However, after the immobilization of Ur-GLDH onto CH-Fe3 O4 nanobiocomposite/ITO (image d) electrode, the globular morphology changes to regular form. This suggests that Fe3 O4 nanoparticles provide a favourable environment for high loading of Ur-GLDH moieties. These results are further supported by electrochemical and FTIR studies. 3.3.2. Electrochemical studies Fig. 4A shows cyclic voltammograms of CH/ITO electrode, CHFe3 O4 nanobiocomposite/ITO electrode and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode recorded in phosphate buffer saline (PBS, 50 mM, pH, 7.0, 0.9% NaCl) containing 5 mM [Fe (CN)6 ]3−/4− and in the potential range, −0.3 to 0.6 V at 20 mV/s rate. CH/ITO shows a well-defined redox behaviour (curve a) at 0.257 V (Epc) and the anodic peak potential (Epa) at 0.052 V. This may be due to cationic CH that accepts electrons from ferricyanide species (that are negatively charged in PBS) resulting in enhanced redox current. The magnitude of current response for CH-Fe3 O4 nanobiocomposite/ITO electrode (curve b) increases in comparison to that of CH/ITO electrode. This may be due to the induced magnetization of magnetic domains (Fe3 O4 nanoaparticles) on application of electrical field. It appears that the electric field induces alignment of magnetic nanoparticles in a particular direction and facilitates electron flow resulting in increased value of current. These results suggest that the presence of Fe3 O4 superparamagnetic nanoparticles results in increased electroactive surface area of CH and enhanced electron transfer. In the CH-Fe3 O4 nanobiocomposite, the electrocatalytic activity of Fe3 O4 superparamagnetic

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Fig. 2. Proposed mechanism for preparation of CH-Fe3 O4 nanobiocomposite and immobilization of Ur-GLDH onto CH-Fe3 O4 nanobiocomposite film.

Fig. 3. (A) FTIR spectra of CH/ITO electrode (a), CH-Fe3 O4 nanobiocomposite/ITO electrode (b) and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode. (B) Scanning electron microscopy images of CH-Fe3 O4 nanobiocomposite/ITO electrode (a) Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO electrode.

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Fig. 4. (A) Cyclic voltammograms of CH/ITO electrode (a), CH-Fe3 O4 nanobiocomposite/ITO electrode (b) and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode (c). (B) Cyclic voltammograms of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode at different scan rate. (C) Electrochemical impedance spectroscopic studies of CH/ITO electrode (a), CH-Fe3 O4 nanobiocomposite/ITO electrode (b) and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode (c). (D) Differential pulse voltammograms of CH/ITO electrode (a), CH-Fe3 O4 nanobiocomposite/ITO electrode (b) and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode (c).

nanoparticles and the accumulation ability of CH due to amino groups of the negatively charged ferricyanide ions have been found to be responsible for enhanced electrochemical properties due to increased surface concentration. The surface concentrations of redox species onto CH/ITO, CH-Fe3 O4 nanobiocomposite/ITO electrodes have been estimated using Eq. (2) [36]. ip = 0.227 nFA C0∗ k0 exp

 −˛n F a RT



(Ep − E0 )

(2)

where, ip is the anodic peak current, n is the number of electrons transferred (1), F is the Faraday constant (96,485.34 C mol−1 ), A is surface area (0.25 cm2 ), R is the gas constant (8.314 J mol−1 K−1 ), C0∗ is surface concentration of the ionic species of film surface (mol cm−2 ), Ep is the peak potential and E0 is the formal potential. –␣na F/RT and k0 (rate constant) corresponding to the slope and intercept of ln (ip ) verses Ep − E0 curve at different scan rates. It may be noted that surface concentration of redox species onto CHFe3 O4 nanobiocomposite/ITO bioelectrode (2.6 × 10−6 mol cm−2 ) is higher than that of CH/ITO (1.25 × 10−6 mol cm−2 ) revealing the interaction between CH and Fe3 O4 nanoparticles that increase the electroactive surface area of CH-Fe3 O4 nanocomposite. The increased surface concentration of redox species onto CH-Fe3 O4 electrode reveals that larger number of redox moieties are available for oxidation leading to higher faradic current [37] wherein, the presence of Fe3 O4 nanoparticles results in increased electron transport between redox species and electrode. However, after the

immobilization of Ur-GLDH onto CH-Fe3 O4 nanobiocomposite/ITO electrode, the magnitude of the response current decreases (curve c), indicating strong binding of Ur-GLDH with CH-Fe3 O4 nanobiocomposite/ITO electrode that blocks transport of charge carriers. This may be attributed to the insulating characteristics of enzymes (Ur-GLDH) that may perturb the transfer of electrons between medium and electrode. Fig. 4B shows cyclic voltammograms of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode in PBS (50 mM, pH 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6 ]3−/4− as a function of scan rate from 10 to 100 mV s−1 . It can be seen that both cathodic (Ip ) and anodic (Ic ) peak currents of the electrode increase linearly and are proportional to the scan rate (inset, Fig. 4B) according to Eqs. (3) and (4). Ip (A) = 0.77 ␮A(s/mV) × scan rate [mV/s] − 0.11 ␮(A) with value of regression coefficient as 0.999

(3)

Ic (A) = 0.6 ␮A(s/mV) × scan rate (mV/s) − 0.514 ␮(A) with value of regression coefficient as 0.996

(4)

It has been shown that Ur-GLDH adsorbed onto CH-Fe3 O4 nanobiocomposite/ITO electrode undergoes reversible electron transfer with nanobiocomposite film. It is found that both catiodic (Ep ) and anodic (Ec ) peak potentials increase linearly and are proportional

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to the logarithm of scan rates and obey Eqs. (5) and (6): Ep (V) = 0.489 (s) × scan rate (mV/s) + 0.518 (V) with value of regression coefficient as 0.996

(5)

Ec (V) = −0.043 (s) × scan rate (mV/s) + 0.143 (V) with value of regression coefficient as 0.993

(6)

The values of heterogeneous electron transfer rate constant (ks ) of Ur-GLDH immobilized CH-Fe3 O4 nanobiocomposite/ITO electrode have been calculated using the Laviron model [38]. ks =

mnF RT

(7)

where m is peak-to-peak separation, F is Faraday constant,  is scan rate (mV/s), n is the number of transferred electrons and R is gas constant. The value of ks obtained as 4.2 s−1 (T = 298 K, n = 1, m = 0.103 V and  = 100 mV) is higher than that of other nanoparticles based bioelectrodes [39–42] indicating fast electron transfer between immobilized Ur-GLDH and electrode due to the presence of Fe3 O4 nanoparticles in the CH-Fe3 O4 nanobiocomposite. Electrochemical impedance spectroscopy (EIS) technique measures impedance of the electrode surface as a function of frequency due to variation in interfacial properties of the interface of the electrode and solution (Rs ), including electrode impedance (W), capacity of the electric double layer (Cdl ), and surface electron transfer resistance (RCT ). The modification of electrode surface results in change in the value of RCT . In the EIS, semicircle part corresponds to electron-transfer limited process as its diameter is equal to the electron transfer resistance (RCT ) that controls electron transfer kinetics of the redox probe at the electrode interface [15,26]. Fig. 4C shows the Nyquist diagrams of CH/ITO electrode, CH-Fe3 O4 nanobiocomposite/ITO electrode and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode in PBS solution [50 mM, pH, 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6 ]3−/4− in the frequency range from 0.01 to 105 Hz. It can be seen (Fig. 4C) that RCT value (1.31 K, curve a) obtained from the semicircle of CH/ITO electrode, characteristic of a diffusion limiting step of the electrochemical process, decreases for the CH-Fe3 O4 nanobiocomposite/ITO electrode (RCT = 1.11 K curve c). This result suggests that electron transfer in the CH-Fe3 O4 nanobiocomposite film is easier between solution and the electrode i.e. Fe3 O4 nanoparticles not only provide the hydrophilic surface, but also act as a nanoscale electrode and promote electron transfer due to permeable structure of CH/ITO. However, after the immobilization of Ur-GLDH onto and CH-Fe3 O4 nanobiocomposite/ITO, RCT is found to increase to 1.47 . This suggests that immobilized Ur-GLDH molecules strongly bind with hybrid nanobiocomposite and block charge carriers in the nanobiocomposite matrix. These results clearly indicate the immobilization of Ur-GLDH onto CHFe3 O4 nanobiocomposite/ITO electrode. Fig. 4D shows differential pulse voltammograms (DPV) of CH/ITO electrode (curve a), CH-Fe3 O4 nanobiocomposite/ITO electrode (curve b) and Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode (curve c) in PBS solution {50 mM PBS (pH 7, 0.9% NaCl) containing 5 mM [Fe(CN)6 ]3−/4− }. CH/ITO electrode (curve a) shows well-defined DPV characteristics suggesting that CH promotes electron transfer due to cationic characteristics and accepts electrons from the medium and transfer these to the electrode. Moreover, magnitude of the current peak further increases for the CH-Fe3 O4 nanobiocomposite/ITO electrode (curve b) suggesting that Fe3 O4 nanoparticles promote electron transfer due to uniform dispersion throughout the CH network on the electrode [26]. Moreover, Fe3 O4 nanoparticles provide favourable environment for the immobilization of Ur-GLDH onto electrode resulting in enhanced electron

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transfer phenomena. The magnitude of current response is found to decrease for Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode (curve c) due to insulating characteristics of Ur-GLDH that hinders transfer of electrons at bioelectrode. The effect of pH on Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode has been carried out using DPV. It is observed that magnitude of the current is maximum at pH 7 (inset Fig. 4D). This value of pH is optimum for catalytic activity of Ur for the decomposition of urea [35]. This suggests that Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode shows maximum activity at pH 7 at which Ur retains its natural structure and is responsible for low detection limit and high sensitivity for urea detection. The results of electrochemical experiments repeated at least 20 times have been found to be reproducible. 3.4. Electrochemical response studies of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode Electrochemical response studies of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode have been carried out as a function of urea concentration in the presence of 30 ␮L of nicotinamide adenine dinucleotide (NADH, 3.7 mg/dL) and 70 ␮L of ␣-Keto glutamate (␣-KG, 47.5 mg/dL) using DPV in PBS solution {50 mM PBS (pH 7, 0.9% NaCl) containing 5 mM [Fe(CN)6 ]3−/4− }. It is observed that magnitude of current obtained for the Ur-GLDH/CHFe3 O4 nanobiocomposite/ITO bioelectrode increases on addition of urea (Fig. 5A). The response time of the Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode found to be about 10 s is attributed to faster electron communication feature of CH-Fe2 O3 nanobiocomposite. It is revealed that Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode (inset Fig. 5A) can be used to estimate urea from 5 to 100 mg/dL. The sensitivity of the Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode calculated from the slope of curve has been found to be 12.5 ␮A/(mM cm−2 ). It has been shown that good linearity is found in the low concentration range (5–40 mg dL−1 ) and the current varies as I (A) = 186 ␮A +293 ␮A (mg/dL) × urea concentration (mg/dL) with the value of correlation coefficient as 0.996 (inset b, Fig. 5A) and standard deviation of 0.46 ␮A/mg/dL. When urea concentration is larger than 40 mg/dL, the response current starts to level off. The observed sluggish increase in the current for solutions at higher concentration is likely to be due to restriction of the enzymatic reaction. At higher urea concentration, the original firstorder enzymatic reaction appears to have changed to 0th order reaction at which the reaction rate becomes independent of substrate concentration [5]. Interestingly, in the concentration range (60–100 mg dL−1 ), the variation of current follows I (A) = 2.82 ␮A +0.9 ␮A (dL/mg) × urea concentration (mg/dL) with correlation coefficient of 0.999 and standard deviation of 0.46 ␮A/mg/dL. The detection limit of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode is estimated to be about 5 mg/dL. The reproducibility of response of the bioelectrode has been investigated at 10 mg/dL urea concentration. No significant decrease in current is observed after using at least 6 times (data not shown). This bioelectrode achieves 95% of steady state current in less than 10 s indicating fast electron exchange between Urs-GLDH and CH-Fe3 O4 nanobiocomposite/ITO electrode. The proposed biochemical reaction during the urea detection is shown in Fig. 5B. Ur catalyzes hydrolysis of urea to carbamine acid that gets hydrolyzed to ammonia (NH3 ) and carbon dioxide (CO2 ). GLDH catalyzes the reversible reaction between ␣-KG and NH3 to NAD+ and linked oxidative deamination of l-glutamate in two steps. The first step involves a Schiff base intermediate being formed between NH3 and ␣-KG. The second step involves the Schiff base intermediate being protonated due to the transfer of the hydride ion from NADH resulting in l-glutamate. NAD+ is utilized in the forward

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Fig. 5. (A) Electrochemical response of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode as a function of urea concentration (5–100 mg/dL). (B) Biochemical reaction during electrctrochemical detection of urea using Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode. (C) The effect of interferents on electrctrochemical response of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode. (D) Shelf-life curve for Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode as a function of time.

reaction of ␣-KG and free NH3 that are converted to l-glutamate via hydride transfer from NADH to glutamate. NAD+ is utilized in the reverse reaction, involving l-glutamate being converted to ␣KG and free (NH3 ) via oxidative deamination reaction. The electrons generated from the biochemical reactions are transferred to the CHFe3 O4 /ITO electrode through the Fe(III)/Fe(IV) couples that help in amplifying the electrochemical signal resulting in increased sensitivity of the sensor. The value of the apparent Michaelis–Menten constant (Km ) has been calculated to show suitability of the enzyme in the hybrid nanobiocomposite matrix to urea. Using Lineweaver–Burke plot (1/I versus 1/[C]), Km value has been found to be 0.56 mM for the immobilized Ur-GLDH indicating maximal catalytic activity of the enzyme at low substrate concentration. The selectivity of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode has been determined by measuring its electrochemical response by adding normal concentration of different interferents (Fig. 5C) such as cholesterol (5 mM), ascorbic acid (0.05 mM), uric acid (0.1 mM) and glucose (100 mg/dL) along with urea (1 mM) in phosphate buffer (50 mM, pH 7, 0.9% NaCl). It can be seen that value of the electrochemical response current remains nearly same except for lactic acid wherein there is a decrease of about 6%. The shelf-life of Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO bioelectrode has been monitored by measuring electrochemical current response with respect to time, with regular interval of 1

week. It is observed that this bioelectrode retains about 85% of enzyme (Ur-GLDH) activity even after about 8 weeks when stored in refrigerated conditions (4 ◦ C) after which the current response decreases to 75% in about 10 weeks (Fig. 5D). 4. Conclusions Nanobiocomposite of CH and superparamagnetic Fe3 O4 , (magnetisation: 60.8 emu/g) nanoparticles have been prepared and used for urea detection via immobilization of Urs and GLDH. Ur-GLDH/CH-Fe3 O4 nanobiocomposite/ITO based urea biosensor shows linearity of 5–100 mg/dL, lower detection limit of 2 mg/dL, response time of 10 s and sensitivity of 12.5 ␮A/mM cm−2 . A relatively low value of the Michaelis–Menten constant obtained as 0.56 mM indicates enhanced enzyme affinity of Ur to urea. The wide range of detection and high sensitivity may be assigned to amplification of the magnitude of current due to the alignment of Fe3 O4 nanoparticles in CH-Fe3 O4 nanobiocomposite matrix. This biosensor shows negligible influence of interferents and can be useful to detect urea in samples containing other analytes. This interesting CH-superparamagnetic Fe3 O4 nanobiocomposite platform should be utilized for estimation of urea in serum samples and to develop other biosensors. Besides this, efforts should be made to utilize these materials for in vivo sensing.

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Acknowledgements We thank Dr. Vikram Kumar, Director, National Physical Laboratory, New Delhi, India for providing facilities. We are thankful to Dr. R. K. Kotnala for VSM measurement. PRS and A K are thankful to Council of Scientific Industrial Research (CSIR), India for the award of Senior Research Associate ship and Senior Research Fellowship. Financial support received under the Department of Science and Technology(DST) projects [DST/TSG/ME/2008/18 and GAP- 070932], in-house project (OLP-070632D) and the DBT project (GAP-070832) is gratefully acknowledged. References [1] G. Dhawan, G. Sumana, B.D. Malhotra, Recent development in urea biosensor, Biochem. Engin. J. 44 (2009) 42–52. [2] R. Singhal, A. Gambhir, M.K. Pandey, S. Annapoorni, B.D. Malhotra, Immobilization of urease on poly (N-vinyl carbazole)/stearic acid Langmuir-Blodgett films for application to urea biosensor, Biosens. Bioelectron. 17 (2002) 697–703. [3] Rajesh, V. Bisht, W. Takashima, K. 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Biographies Mr. Ajeet Kaushik received his M.Sc. degree in chemistry (Organic) from the Meerut University, Utter Pradesh 2002. He is working as a Senior Research Fellow in the Bimolecular Electronics and Conducting Polymer Research Group at the National Physical Laboratory, New Delhi, India in the area of development of biosensors based on nanomaterials, bionanocomposites and conducting polymers for healthcare. Dr. Pratima R. Solanki obtained her M.Sc. and Ph.D degrees from Maharishi Dayanand University, Rohtak (Haryana) during 1995 and 2000 in Bioscience. She is working as a Senior Research Associate (CSIR) with the Biomolecular Electronics and Conducting Polymer Research Group at the National Physical Laboratory, New Delhi, India. She is actively engaged in the technical development of biosensors for healthcare. Dr. Anees A. Ahmed received his M.Sc. in 1998 from Rohel Khand University, Breily and Ph.D degree from the Jamia Milia University, Delhi (2004) in chemistry (inorganic). He is working as Senior Research Associate (CSIR) in the Bimolecular Electronics and Conducting Polymer Research Group at the National Physical Laboratory, New Delhi, India in the area of development of biosensors based on metal oxide nanoparticles as well as nanocomposites for healthcare. Dr. G. Sumana received her Ph.D. (1998) from Jiwji University in chemistry. She is currently working as scientist in biomolecular electronics and conducting polymer research group, National Physical Laboratory, New Delhi. She has a research experience of 10 years in controlled drug delivery, liquid crystal polymers, polymer dispersed liquid crystals and biosensors. Prof. Sharif Ahmad received his M.Sc. degree in chemistry (Organic) from the Aligarh Muslim, Utter Pradesh 1981. He is working as a Senior Professor in the Department of Chemistry at Jamia Millia Islamia, New Delhi, India. He has research experience of about 30 years in the area of corrosion materials based on nanomaterials, nanocomposites and conducting polymers has guided 15 Ph.D. students.

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Dr. B. D. Malhotra received his Ph.D degree in physics from University of Delhi, Delhi, India in 1980. He has published 159 papers, has filed 9 patents, has edited/co-edited books on biosensors and polymer electronics, and is currently the Scientist G and Head of the Biomolecular Electronics & Conducting Polymer Research Group at the

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National Physical Laboratory, India. He has research experience of about 25 years in the field of biomolecular electronics and has guided 14 Ph.D students till date. His current activities including biosensors, conducting polymers, Langmuir–Blodgett films, self-assembled monolayers and nanomaterials, etc.