Studies on clay-gelatin nanocomposite as urea sensor

Applied Clay Science 146 (2017) 297–305

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Applied Clay Science journal homepage: www.elsevier.com/locate/clay

Research paper

Studies on clay-gelatin nanocomposite as urea sensor a,b

Anshu Sharma a b

a

, Kamla Rawat , H.B. Bohidar

a,b

, Pratima R. Solanki

MARK a,⁎

Special Centre for Nanoscience, Jawaharlal Nehru University, New Delhi, India School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Lap Gelatin Clay organogel Urea sensor Cyclic voltammeter

Homogeneous gelatin organogel-based nanocomposite (GA-NC) was prepared by Lap as fillers. For customized thermal and viscoelastic properties, saturation binding of Lap to GA chain was probed in aqueous solution, and Lap = 0.03% (w/v), (glycerol) = 30% (v/v) and (GA) = 3% (w/v) was used for organogel nanocomposite. The Lap platelets were successfully exfoliated in the organogel matrix giving rise to a homogeneous phase. The interaction of GA-NC with urea was studied using electrochemical as well as optical technique. For electrochemical study, a thin film of this GA-NC was prepared onto indium tin oxide (ITO) coated glass plate via dropcasting. This GA-NC/ITO electrode was characterized by electrochemical, FTIR and SEM techniques. This GANC/ITO electrode exhibited electrochemical response specific for urea with sensitivity of 32.7 and 5.56 µA mM− 2 cm− 2 in the two concentration range of 0.1 to 2 and 2 to 20 mM, respectively. The electrochemical profile of this electrode was sensitive towards urea which opens up the possibility for development of strip-based enzyme-free sensors for field applications. This GA-NC material in solution phase also shows optical response for urea with binding constant of 63.93 M− 1.

1. Introduction Urea is a well-known bio-molecule that plays a variety of roles in the metabolic pathway of protein processing in the body, and it is also important as a fertilizer. The urea concentration in body fluid is inversely related to the rate of excretion of urea, depends on protein intake and nitrogen metabolism. An increase in urea level [normal range 8 to 20 mg dL− 1 (2.5–7.5 mM)] in body fluid cause kidney relating diseases, dehydration, shock, and can result in gastrointestinal bleeding and reduced urea level may also cause hepatic failure, nephritic syndrome, and cachexia (Singh et al., 2008). On the other hand, urea detection in the environment, drinking water, and food samples is also important because it is easily washed out into the water bodies from the herbicides applied on crops, but polluting the surface and groundwater. The techniques currently used for the estimation of urea in tissue, and body fluids include ion chromatography, fluorimetric analysis and electrochemical biosensing (Shukla et al., 2014). However, electrochemical analysis has the inherent advantage of simplicity, high sensitivity, relatively low cost, and rapid response as compared to other sophisticated methods (Song et al., 2014). The electrochemical nanostructured based urea biosensors utilized enzymes i.e. urease/glutamate dehydrogenase (GLDH) to detect urea levels (Luo and Do, 2004; Massafera and Torresi, 2009, Massafera and Torresi, 2011; Rajesh et al., 2005; Solanki et al., 2008) have reported. Among these enzyme based

⁎

Corresponding author. E-mail addresses: [email protected], [email protected] (P.R. Solanki).

http://dx.doi.org/10.1016/j.clay.2017.06.012 Received 25 February 2016; Received in revised form 12 June 2017; Accepted 13 June 2017 0169-1317/ © 2017 Elsevier B.V. All rights reserved.

urea sensors, some reports are available on non-enzyme based electrochemical urea sensor too (Dutta et al., 2014). Mondal and Sangaranarayanan (2013) have reported the potentiodynamic polymerized pyrrole film made on platinum (Pt) electrode, and utilized it for development of non-enzymatic urea sensor which, worked in mildly acidic conditions with sensitivity obtained as 1.11 μA μM− 1 cm− 1. Patzer et al. (1989) fabricated a Pt based urea sensor which works in the alkaline medium, and produce NH4+ ions with potentials ranging from 0.06 to 0.3 V. These Pt based electrode could affect real sample analysis, and are prone to toxicity upon continuous exposure to body fluids, are the major disadvantages of the previous reported urea sensor (Petrii and Vassina, 1993). Thus, there is urgent need to find a new material/ platform, which works at neutral pH, is biocompatible, inexpensive, and with fast detection potential accurate for determination of content of urea in real samples. Recently, organic and inorganic nanocomposite materials have attracted much attention because their synergetic combination offers enhanced properties at the nanoscale level (Herrera et al., 2005; Sigolaeva et al., 2014). Generally, nanocomposite (NC) materials comprise of three main constituents: the (bio)-polymer matrix, the nanomaterial (fillers), and the solvent (continuous phase). In most of the studies not much attention is paid to the role played by the interface. This interface region exchanges energy between the matrix and filler and is associated with properties different from that of the bulk

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because of its active coupling to the filler surface (Sudha et al., 2011). In most of the cases, the secondary forces between fillers, and the matrices, enhance interfacial adhesion, and improve their physical and electrochemical properties of NC materials. The exfoliated systems, being more homogeneous, produce enhanced physical, mechanical properties and stability which are most suitable for sensor application. The Lap, mono-dispersed layers consisting of numerous negative charge on the surface and residual positive charge at the edges (Fatnassi and Es-Souni, 2015). The Lap is easily modified with polymer to improve their properties, such as hydrophobicity and affinity for adsorbents, because the intercalated molecules can expand the layered structure in such a way that the exfoliation occurs (separation of the individual layers), and subsequently yielding functionalized leaflets with high surface area (Wagner and Vaia, 2004). However, Lap contains a relatively low amount of hydroxyls located at the edge of the individual particles, which can bind only to a small proportion of organics (Rausell-Colom and Serratosa, 1987). To overcome the low reactivity of Lap surfaces towards organic molecules, several authors have used different strategies to enhance the reactivity. Herrera et al. (2005) reported Lap particles grafted with monofunctional c-methacryloxypropyl dimethyl methoxysilane (c-MPTDES), and trifunctional c-methacryloxy propyl trimethoxysilane (c-MPTMS) coupling agents. Few reports have reported using Lap in the form of NC coupled with different polymers/ bio-polymers tailored for biomedical applications. A nanocomposite of Lap/chitosan was used to immobilize polyphenol oxidase on the surface of a glassy carbon electrode for phenol determination (Fan et al., 2007). The non-enzymatic electrode sensor comprising of Lap-ionic liquid was fabricated for oxalic acid detection using electrochemical techniques (Joshi et al., 2015). Recently, the change in photo-physical and biocidal properties of cationic p-phenyleneethynylenes oligomers (OPEs) with Lap was studied (Hill et al., 2015). Thus, there is a wide scope to explore the Lap based biocompatible NC for non-enzymatic electrochemical sensor development. In the present manuscript, gelatin (GA) obtained by partial degradation of collagen was used as matrix because the material has gained attention as edible films for its abundance, hydrophilicity, biodegradability, relatively low cost, good affinity, compatibility and excellent functional properties (Rivero et al., 2009). The physical properties of GA organogel have been exhaustively studied by Sanwlani et al. (2011). This type of NCs is useful due to their unexpected marketable potential for exploitation in electrochemical sensors, batteries, enzyme immunoassay etc. (Shukla et al., 2014). Further, it provides thermal stability, chemical inertia, well-defined layered structure, ionexchange properties and low cost. Moreover, the permeability of this material provides electrostatic interactions due to the charge, and cationic-exchange properties of the clay minerals, which greatly affects the biosensor performance (Hu et al., 2002). Keeping these points in view, a thin film of gelatin-organogel with Lap filler onto indium tin oxide (ITO) substrate by drop casting method was fabricated. Glycerol was added as a plasticizer to enhance the functional properties of films, and improved film flexibility. The results compared based on GA-NC are compared to different enzymatic and non-enzymatic urea biosensors reported in the literature.

purchased from Sisco Research Laboratory Pvt. Ltd. India. 2.2. Sample preparation To prepare solutions, GA powder (3% w/v) was dissolved in the required glycerol solutions (30% v/v) and mixed under continuous stirring for 1 h at 50 °C to homogenize. The Lap powder was dried at 120 °C for 4 h to remove the moisture (Pujala et al., 2011). The Lap concentration 0.03% (w/v) with GA powder (3% w/v) in glycerol solutions (30% v/v) was used for electrode fabrication and found film stable. Different concentrations of urea (Ur) were freshly prepared in deionized water from stock solution (20 mM), and stored in refrigerator (4 °C). 2.3. Preparation of GA/ITO and GA-NC/ITO electrodes The dispersion of GA prepared with glycerol solution [30% (v/v)] and Lap [0.03% (w/v)] was used for GA-NC/ITO electrode fabrication. GA-NC/ITO electrode was prepared by drop-casting of 10 μL of the above dispersion onto an ITO electrode surface (0.25 cm2), then dried at room temperature (20 °C) for about 24 h. Similarly, a thin film of GA in glycerol [30% (v/v)] solution without Lap, GA/ITO was also made. To remove any unbound particles from the GA/ITO and GA-NC/ITO electrodes, the electrodes were washed with deionized water and stored in refrigerator (4 °C) when not in use. Scheme 1 shows the different steps of GA-NC/ITO electrode fabrication. 2.4. Optical studies The photometric response of GA-NC was monitored as a function of substrate concentration using UV–Vis spectrophotometer Sigma protocol with slight modification to ascertain the selectivity and sensitivity of the matrix (GA-NC) with Ur in the solution phase. UV–Vis titration measurements were conducted with the addition of different concentrations of Ur from 0 to 20 mM. 2.5. Characterization GA, Lap and GA-NC were characterized using X-ray diffraction (XRD, PANalyticalX'pert PRO 2200 diffractometer with CuKα radiation at λ = 1.5406 Å) with 2θ range from 3 to 33°, step size of 0.22 and scan rate of 1°/min with an accelerating voltage of 40 KV. The availability of functional group on GA, Lap, GA-NC and changes in GA-NC after interaction with Ur were recorded by Fourier transforms infrared spectroscopic (FTIR, 600 UMA Varian spectrometer) with in the range of 400–4000 cm− 1 and number of scan 65. All the samples individually were prepared by drop-casting of 10 μL of each samples (GA, Lap, GANC) onto an ITO electrode surface (0.25 cm2), then dried at room temperature (25 °C) for about 24 h. The surface morphology of GA-NC/ ITO electrode before and after interaction with Ur was recorded using Scanning electron microscopy (SEM, Lvo 40 Zeiss instrument). During the SEM measurement, the GA-NC/ITO electrode was attached on double stick carbon tape and fixed on aluminum stubs. The photometric response studies of GA-NC nanocomposite as a function of Ur concentration were investigated by UV/Vis Spectrophotometer (PG Instruments T90 + Double Beam Scanning) at spectrum range of 200–800 nm. The electrochemical studies [cyclic voltammetry (CV) in the potential range of −0.1 to 0.4 V and impedance spectroscopy (FRA) in the frequency range 0.01–105 Hz] were conducted on Autolab Potentiostat/Galvanostat Model PGSTAT302N (Eco Chemie, Netherlands) using three electrode cell system; where GA-NC/ITO was used as working electrode, platinum (Pt) wire was the auxiliary electrode and Ag/AgCl as reference electrode in phosphate buffer saline (PBS, pH 7.0, 0.9% KCl) containing 3.3 mM [Fe(CN)6]3 −/4 −. All the experiments were repeated three times under the same experimental conditions to check the reliability and reproducibility of the results.

2. Materials and methods 2.1. Materials Gelatin-A (GA) (Extracted from Porcine skin and bloom number 300), having nominal molecular weight 50 KDa bought from Sigma–Aldrich, USA, was used for sample preparation. The Lap was purchased from Southern Clay Products, USA. For solution preparation de-ionized water was used, purchased from Organo Biotech Laboratories, India. Indium tin oxide (ITO) coated glass plates having a resistance of 25 Ω sq− 1, the transmittance of 90% and thickness of 1.1 mm was procured from Blazers, UK. Urea and glycerol were 298

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Scheme 1. shows the possible interactions between the gelatin and Lap and electrochemical interaction of GA-NC/ITO electrode with Ur.

3. Results and discussion 3.1. Gelatin lap interaction

Intensity /a.u.

The GA is a polyampholyte biopolymer having positive and negative charge with some hydrophobic amino acid groups present in an approximate ratio of 1:1:1, which makes this polypeptide extraordinary (Veis, 1964). It can easily interact via electrostatic interaction with the Lap layer containing negative charge on the face and positive charge at the edge. However, glycerol is a nonsolvent, so it is selectively excluded from the nonpolar domains of the GA surface, and binds preferentially to the polar regions in direct proportion to their concentration (Sanwlani et al., 2011). GA molecules tend to fold in glycerol solutions because glycerol raises the chemical potential of the GA protein and makes the situation thermodynamically unfavorable. The shrinking of hydrodynamic radius/size in glycerol solution as the concentration of glycerol increases was reported by Sanwlani et al. (2011). Scheme 1 shows the possible interactions between the GA and Lap.

(b)

(a) (c)

5

10

15

20

25

30

2 θ (Degree)

3.2. X-ray diffraction

Fig. 1. XRD spectra of (a) GA and (b) Lap and (c) GA-NC electrode.

The diffraction pattern obtained from thin films of aqueous dispersion of GA (a), Lap (b) and GA-NC (c) are shown in Fig. 1. The XRD of GA (a) shows reflection at 2θ = 7.5° indicate partially crystalline in nature (Asma et al., 2014). The Lap XRD show reflection at 2θ = 4.8°, which corresponds to (001) (Fatnassi and Es-Souni, 2015). However, GA-NC (c) does not show any reflection of Lap at 2θ = 4.8°, due to low concentration of Lap in GA-NC composite.

(a)

(b)

3.3. Scanning electron microscopy SEM images of GA-NC/ITO electrode revealed morphological changes occurring on the surface morphology after reacting with Ur [Fig. 2(a–b)]. Image (a) shows a rough and uneven surface morphology of GA-NC. This rough surface of GA-NC facilitates the interaction of Ur onto electrodes surfaces [image (b)], resulting in a change in surface

Fig. 2. SEM images of (a) GA-NC/ITO and (b) Ur/GA-NC/ITO electrodes.

299

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% Transmittance

A

Gelatin A

6

B (i) Laponite

C-H

(b)

2656

4

2

0

4000

3000 2000 Wavenumber (cm-1)

Wavenumber (cm-1)

1000

(ii)

(iii) (b)

C C

(a)

amide I, C=O str

2124

Wavenumber (cm-1) Fig. 3. FTIR spectra of (A) commercially available Lap and GA and (B; i–iii) GA-NC/ITO electrode and after its interaction with Ur.

2003). The bands near the region of 3300 cm− 1 assigned to NH2 antisymmetric stretch of primary amide of GA and also corresponding to OeH stretching due to adsorbed water. In the case of GA-NC/ITO electrode [curve a; Fig. 3B (i-iii)] IR band at 2964 cm− 1 refers to the CeH stretching vibrations, at 1700–1600 cm− 1 assign to amide-I band originated from C]O stretching (hydrogen bonding in combination with COO) (Kong and Yu, 2007; Li et al., 2015). The IR bands observed at around 1620–1460 cm− 1 for amide II results from NeH bending and CeN stretching vibrations (Liu et al., 2004). The signature bands appeared at 1004 cm− 1 correspond to the SieO bonds and at 660 cm− 1 assigned to the MgeO bond of clay (Joshi et al., 2015) which confirm the presence of Lap in GA-NC. However, after the interaction of the GANC with Ur, the IR bands intensity [curve b; Fig. 3B (i-iii)] increased and become sharp, indicating interaction among them.

morphology. 3.4. Fourier transforms infrared spectroscopic The FTIR spectra of commercial Lap, commercial GA and GA-NC/ ITO electrode (before and after interaction with Ur) are shown in Fig. 3. FTIR spectrum of commercial Lap (Fig. 3A) exhibits a broad range of bands at 3700–3400 cm− 1 which assigned to the stretching of hydroxyl groups and adsorbed and interlayer water. The strong bands observed at 1004 and 460 cm− 1 were assigned to bending vibrations of SieO bond. The bands observed at 650 cm− 1 indicate MgeO bonding. Another band at 1638 cm− 1 was also ascribed to OH-bending vibration of Lap (Joshi et al., 2015; Paul et al., 2013). FTIR spectra of commercial GA (Fig. 3A) shows the characteristics bands of amide I, amide II and amide III at 1670, 1578 and 1265 cm− 1, respectively; which were assigned due to C]O stretching, NeH in-plane bending, CeH stretching and CeN and NeH in-plane stretching band, respectively (Rawat et al., 2014, Gates et al., 2017). The bands at 1028 cm− 1 were assigned due to the vibrational modes of eCH2OH groups and CeO stretching vibration. The bands at 785 and 510 cm− 1 frequency ranges were assigned due to CeH rocking of CH2 and CeNeC of amines of GA. The bands at 2950 and 3081 cm− 1 assigned due to eCH anti-symmetric and symmetric stretching of eCH3, CH2 and CeH, respectively (Cheng et al.,

3.5. Photometric response study The photometric response of GA-NC was monitored as a function of substrate concentration using UV–Vis spectrophotometer Sigma protocol with slight modification. To ascertain the selectivity and sensitivity of the matrix (GA-NC) with Ur in the solution phase, UV–Vis titration measurements were conducted with the addition of different concentrations of Ur from 0 to 20 mM. The characteristic absorption 300

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A. Sharma et al.

A

(a)

(c)

0.25

(a)

200 0.01 mM

Current (μ A)

(b)

Abs

0.20

0.15 20 mM

100 0

-100

0.10

-200 0.05 260

280

300

0.0

320

0.2

λ (nm)

B

0.220

(b)

4

0.215

(c)

(b)

0.210

3

Z"(kΩ)

Absorbance (a.u.)

0.4

Potential (V)

0.205 0.200

(a)

2

0.195 0.190

1

0.185 0

5

10

15

20

0 0.0

Urea (mM)

1.5

3.0

4.5

6.0

7.5

9.0

Z'(kΩ )

C

(c)

200

ia

100

-0.6

Current (μ A)

100

-0.8

-1.0

0 R2=0.99

-100

ic

-200 3

0

4

5 6 7 8 Scan rate (mV/s)1/2

-6.2

-6.0

-5.8 -5.6 log[Q]

-5.4

-5.2

10

10 mV/s Ea Ec

0.25

-100 -6.4

9

Potential (V)

log[(A0-A)/A0]

R2= 0.97

100 mV/s 200 Current (μ A)

-0.4

0.20 0.15

R2=0.98

0.10 0.05

-5.0

3

-200 -0.1

Fig. 4. (a) The absorbance spectra, (b) maxima of absorbance and (c) plot between log [(A0-A)/A0] and log (Q) obtained for GA-NC matrix as a function of urea concentration (0 to 20 mM).

0.0

0.1 0.2 Potential (V)

4 5 6 7 1/2 8 Scan rate (mV/s)

0.3

9

10

0.4

Fig. 5. (A). Cyclic voltammograms of (a) bare ITO; (b) GA/ITO and (c) GA-NC/ITO electrodes at scan rate of 50 mV/s in phosphate buffer saline (PBS, pH 7.0, 0.9% KCl) containing [Fe(CN)6]3 −/4 − solution. 5(B). Nyquist plot of (a) bare ITO electrode; (b) GA/ ITO and (c) GA-NC/ITO electrode. Inset shows the equivalent circuit diagram. (C). Cyclic voltammograms of GA-NC/ITO electrode using an increasing scan rate from 10 to 100 mV/s. Upper and lower inset shows magnitude of the current and potential vs square root of scan rate (10–100 mV/s), respectively.

band of GA-NC at 265 nm gradually decreased without producing any other observable changes i.e. there was no shift in wavelength. The matrix GA-NC interacts with Ur without forming any complexes [Fig. 4(a)]. The maxima of absorbance Amax at 265 nm as a function of the Ur concentration are shown in Fig. 4(b). The Amax exhibited a linear behavior (linear regression coefficient 0.97) with the concentration of Ur with a slope of 0.0014 mM− 1. A plot between log [(A0 − A) / A0] 301

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down the electron transfer from electrolyte medium to electrode surface. These electrodes were characterized by analyzing the key parameters such as the time constant (τ). The value of RCT is dependent on the electrochemical reaction time constant (τ) = [1/2 π fmax = RCT·Cp, where fmax is the frequency (Hz) at which maximum Z″ obtained, RCT (Ω) is the charge transfer resistance and Cp (F) is the double layer capacitance]. The ionic conductivity of ITO and GA-NC/ITO electrodes were l calculated from the Rs values by employing the formula σ = R A , where S 2 l is the thickness (cm) of electrode and A (cm ) is the contact area between the electrode and electrolyte. A high ionic conduction value of 7 × 10− 5 S/cm at 25 °C for GA-NC/ITO electrode was obtained. In curve b, the value of RCT for GA/ITO (8.65 kΩ) is greater than that for hydrolyzed ITO (curve a, 7.33 kΩ). The higher value of RCT was observed due to the hindrance in electron transfer, caused by the macromolecular structure of GA. The value of RCT in curve c (6.65 kΩ) is less than that of the hydrolyzed ITO (curve a, 7.33 kΩ), indicating the film formation and it can be attributed to the effect of ionic species present in the solution. It is more conducting (higher anodic peak current) due to negatively charged Lap this was also confirmed from CV graph. The higher electric double layer formed between the conductive ITO electrode and an adjacent electrolyte as compared to GA/ITO electrode was confirmed from the low value of CP for GA-NC/ITO electrode. The heterogeneous charge transfer constant (k0) in the presence of [Fe(CN)6]− 3/− 4 mediator was determined for various fabricated electrodes as shown in Table 1. The value of k0 can be estimated using following Eq. (2).

and log (Q), used to obtain binding constant K, from the intercept (Hu et al., 2005) is shown in Fig. 4(c). Here Q is the concentration of Ur, A is absorbance measured with analyte (Ur), and A0 is the absorbance prior to complex formation. The binding constant of GA-NC for Ur was found to be 63.93 M− 1. 3.6. Electrochemical studies The CV studies (Fig. 5A) on bare ITO [curve (a)], GA/ITO electrode [curve (b)] and GA-NC/ITO electrode [curve (c)] were conducted to monitor the electrochemical behavior in PBS having [Fe(CN)6]3 −/4 − in the potential range of −0.05 to +0.40 V. Well-defined oxidation and reduction peaks were found in all samples. The CV of bare ITO (curve a) shows well-defined electrochemical characteristics with a couple of redox peaks of [Fe(CN)6]3 −/4 −, and shows an oxidation peak current (Ipa) of 241.4 μA. The magnitude of current of GA/ITO electrode [curve (b)] was lower as compared to bare ITO electrode that confirmed the presence of the film on ITO surface. However, after the incorporation of Lap in GA-glycerol solution the magnitude of anodic peak current increased, and peak potential shifted towards higher potential (Epa ~ 0.25 V) as compared to that of GA/ITO electrode. The potential separation (ΔE) of the two peaks for GA/ITO and GA-NC/ITO are 140 and 184 mV respectively. The GA/ITO [curve (b)] and GA-NC/ITO [curve (c)] shows shift in anodic peak potential by 2.0 and 2.3 mV respectively, with respect to bare ITO. The GA-NC/ITO has a higher electrocatalytic activity than GA/ITO electrode. The increase of oxidation peak current and shift in peak potential of GA-NC/ITO electrode could be due to faster electron transfer and electrostatic attraction occurring between positive charge of Lap and negative charges of redox species in electrolyte. The Lap particles must play a significant role in facilitating electron exchange and provided sufficient accessibility to electrons between the electrolyte and the electrode (Joshi et al., 2015). The diffusion co-efficient (D) of electrolyte ionic species of GA/ITO and GA-NC/ITO electrodes were calculated using the Randles–Sevcik equation (Bard and Faulkner, 1980).

Ip = (2.69 × 105) n3 2 A D1 2Cv1

2

k 0 = RT n2F 2AR CTC

(2)

where, n is the number of electron (1) involved in the redox process, F (96,485.34 mol− 1) is Faraday constant, A is the area of the electrode (0.25 cm− 2), R is the gas constant, T is the temperature (298 K), RCT (Ω) is the charge transfer resistance and C (Mol cm− 3) is the concentration of the ionic species (Ali et al., 2015). The electrochemical reaction time constant (τ) is higher for GA/ITO electrode due to smallest value of k0. The higher ionic conductivity of GA-NC/ITO electrodes was due to removable of protons associated with amino and hydroxyl groups from small chain fragments of GA, which migrate through electrode under the influence of electric field (Bard and Faulkner, 1980). The electrochemical reaction time constant, τ is higher for GA/ITO electrode due to smallest value of k0. The highest value of k0 and a high ionic conduction value of 7 × 10− 5 S/cm at 25 °C was obtained for GA-NC/ITO electrode, which indicated fast electron transfer occur (in short time period) between the electrolyte and electrode for GA-NC/ITO electrode as compared to GA/ITO. The similar results were also found from CV graph. This GA-NC/ITO electrode shows higher values of these electrochemical parameters such as: anodic peak current, diffusion coefficient, heterogeneous charge transfer constant and ionic conductivity and lower value of charge transfer resistance and time constant. Thus, GA-NC/ITO electrode shows highest electrochemical activity and faster electron transfer as compared to GA/ITO. The equivalent circuit elements of bare ITO and GA-NC/ITO electrodes are shown in Table 1. The effect of scan rate was monitored onto GA-NC/ITO electrode, by

(1)

where Ip is the peak current of the electrode (Ipa anodic and Ipc cathodic), n is the number of electrons [Fe(III)/Fe(II)] involved (1), A is the surface area of electrode (0.25 cm2), D is the diffusion co-efficient, C is the concentration of ionic species [Fe(CN)6]− 3/− 4 and v is scan rate (50 mV/s). The D value was obtained for GA/ITO and GA-NC/ITO electrode found as 1.15 × 10− 11 and 1.29 × 10− 11 cm2 s− 1, respectively. Higher value of D for GA-NC/ITO electrode is due to high aspect ratio of Lap that provides an electron conduction path for ion-transportation from bulk solution towards the electrode. The Faradic impedance spectra, called Nyquist plots obtained from real (Z′) and imaginary (Z″) impedance in the frequency range 0.01–105 Hz are shown in Fig. 5(B). The shape of the Nyquist, composed of three regions, at low frequency capacitive behavior is characterized by a nearly vertical line, a high to medium frequency a sloping (at 45°) linear region is related to diffusion resistance, and at high frequency a semicircle part represents the pseudo charge-transfer resistance (Choudhury et al., 2008). The decreased radius of the semicircle at high frequency means that the pseudo charge-transfer resistance is markedly decreased, consistent with the CV curves (Fig. 5A). The electron transfer resistance (RCT), measured from diameter of the semicircle part of the Nyquist plot, is related to the electron-transfer limited process and controls electron transfer kinetics of the ionic species at the electrode interface. The semicircle of ITO (curve a) exhibits charge transfer phenomena between electrode and electrolyte. Compared to bare ITO (curve a), RCT value found for GA-NC/ITO electrode (curve b) decreased resulting in conductive pathway or enhanced electron transfer towards electrode. However, RCT value obtained for GA/ITO electrode (curve c) increases resulting in slowing

Table 1 Equivalent circuit elements of GA-NC/ITO electrodes.

302

EIS values

Bare ITO

GA/ITO electrode

GA-NC/ITO electrode

RCT (kΩ) Rs (Ω) Cp (μF) n τ (ms) σ (S/cm) k0 (cm/s)

7.33 298.20 2.64 0.99 19.40 6 × 10− 5 4.40 × 10− 4

8.65 016.42 3.93 0.99 33.97 5 × 10− 5 3.73 × 10− 4

6.65 110.80 3.78 0.99 25.00 7 × 10− 5 4.85 × 10− 4

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using CV technique and scan rate was varied from 10 to 100 mV/s. The magnitude of both peak currents that is, anodic (Ipa) and cathodic (Ipc) increased linearly with square root of the scan rate (v1/2), indicates that electrochemical activity was a diffusion-controlled mass transfer reactions [Fig. 5(C)]. The anodic (Epa) and cathodic (Epc) peak potentials and potential peak shifts (ΔEp = Epa – Epc) also exhibited a linear relationship (linear regression coefficient 0.99) with the square root of scan rate and shifts towards higher and lower value of potential respectively, indicating facile charge transfer kinetics in the scan rate range from 10 to 100 mV/s [Fig. 5(C)]. The GA-NC/ITO electrode provided sufficient accessibility to electrons between the electrolyte and the electrode. The surface concentration of ionic species of GA/ITO and GA-NC/ITO electrode was estimated using Brown–Anson model (Eq. (3)) (Ali et al., 2015; Bard and Faulkner, 1980; Singh et al., 2013).

Ip =

n2F 2I∗AV 4RT

(NH2 )2 CO + 3H2 O + H+ →

ITO

(3)

= 17.27 μA + 2.35 (μA mV 1

s ) [scan rate (mV s)] 2 with R2 = 0.99

Ipc (μA)GA

ITO

(4)

= −15.28 μA − 16.87(μA mV s) [scan rate (mV s)]1

2

with R2 = 0.99 (5)

Ipa (μA)GA − NC

ITO

= 20.5 μA + 31.39 (μA mV 1

s ) [scan rate(mV s)] 2 with R2 = 0.99 Ipc (μA)GA − NC

ITO

(8)

During the electrochemical measurement, the magnitude of anodic current increased, and the potential shifted towards higher potential as the urea concentration was raised. The electrochemical mechanism of GA-NC/ITO electrode with Ur is shown in Scheme 1. The results indicate that interaction occurred between Ur and GA-NC/ITO electrode. This compound decomposes and release NH3 species and carbamic acid, followed by spontaneous decomposition of carbamic acid into ammonia (NH3) and carbon dioxide. The generated NH3, immediately converted to the NH4+ via proton generated amino, and hydroxyl group of GA, and from glycerol. It has been reported that glycerol oxidation to tartronate is an intermediate complex which further oxidizes to mesoxalate at low potentials (Srivastava et al., 2013; Zhang et al., 2012). This mechanism is similar to hydrolysis reaction of urease based sensor. The possible interaction between the urea and GA-NC/ITO electrode were shown in Scheme 1. The detection range of Ur was observed as 0.01 to 20 mM with standard deviation and correlation coefficient of 0.2 μA, and 0.97. The sensitivity of the GA-NC/ITO electrode was determined from the slope of linear calibration curve, and was found as 32.7 and 5.56 μA/ mM cm− 2 in two concentration ranges from 0.1 to 2 and 2 to 20 mM, respectively for Ur. A single GA-NC/ITO electrode was used for electrochemical response studies as a function of variable concentration of urea various from 0.1 to 20 mM. The lower detection limit obtained as 0.69 and 0.34 mM for Ur in concentration range of 0.1 to 2 and 2 to 20 mM, respectively, was calculated by using the 3 σb/m equations, where m is the slope of the calibration curve, and σb is the standard deviation of the signal without analyte. The sensing parameters of the GA-NC/ITO electrode are summarized in Table 3 along with polymerbiopolymer NCs based urea sensors reported in the literature. The sensitivity of GA-NC/ITO electrode was higher than all enzymatic as well as non-enzymatic urea sensors (Luo and Do, 2004; Massafera and Torresi, 2009, 2011; Mondal and Sangaranarayanan, 2013; Rajesh et al., 2005; Solanki et al., 2008).

where n is the number of electrons transferred which is 1 in this case, F is Faraday constant (96485.34 C mol− 1), A is surface area of the electrode (0.25 cm2), R is gas constant, I* is surface concentration of ionic species (mol/cm2), T is 298 K, and Ip/V is the slope of calibration plot (scan rate value). The surface concentration of ionic species of GA/ITO and GA-NC/ITO electrode was estimated from plot of Ip versus scan rate (v1/2) and found to be 1.79 × 10− 7 and 1.52 × 10− 8 mol/cm2, respectively. On basis of scan rate dependent data the values of the slope, intercept and correlation coefficient of GA/ITO and GA-NC/ITO electrode is given in following equations from (4) to (7).

Ipa (μA) GA

H CO3− + 2NH+4

(6)

3.8. Interference study

= −14.9 μA

The selectivity of GA-NC/ITO electrode was monitored by potential interferents at their normal physiological concentrations present in real samples (blood/serum). These were studied by taking a solution containing a 1:1 ratio of urea (4 mM) and the normal physiological concentration (4 mM) of interferents ascorbic acid (AA), citric acid (CA), oxalic acid (OA), cholesterol (ChO) and glucose (Glu) by using CV measurements at a scan rate of 50 mV/s [Fig. 6(B)]. The present electrode was specific to urea, and did not show significant response to the presence of other analytes which indicated that GA-NC/ITO electrode was highly selective towards Ur detection. The reproducibility of the GA-NC/ITO electrode was investigated under similar conditions. The changes in magnitude of anodic peak current for different electrodes (n = 5) was negligible, as indicated by the relative standard deviation (RSD) data which was below 5%. It is single time use sensor. The shelf-life of the GA-NC/ITO electrode was observed by measuring electrochemical current response at the regular intervals of one week for about 6 weeks. It was observed that GA-NC/ ITO electrode retained about 90% of its activity even after 6 weeks of preparation when stored under refrigerated conditions (4 °C).

1

− 50(μA mV s)[scan rate(mV s)] 2 with R2 = 0.99 (7) The electrochemical parameters for respective electrodes are shown in Table 2. 3.7. Electrochemical response studies The electrochemical response studies of GA-NC/ITO bioelectrode was monitored through CV at scan rate of 50 mV/s in phosphate buffer saline of pH 7.0, containing [Fe(CN)6]3 −/4 − with 0.9% KCl as a supporting electrolyte solution, as a function of Ur concentration range varying from 0.01 to 20 mM. It was found that the anodic peak current increased and gradually shifts in potential towards higher side as increased concentration of Ur from 0.01 to 20 mM [Fig. 6(A)]. Generally, the determination of urea is based on hydrolysis process in presence of urease in aqueous solutions and successive measurement of ions consumed or produced in catalytic reaction as mentioned in Eq. (8). Table 2 Shows the electrochemical parameters of respective electrodes. Electrodes

Ipa (μA)

Ipc (μA)

Ks (s− 1)

D (cm2 s− 1)

A (mm2)

γ (mol cm− 2)

GA/ITO GA-NC/ITO

168.5 180.0

− 153.00 − 158.0

0.292 0.36

1.15 × 10− 11 1.29 × 10− 11

0.71 0.58

1.79 × 10− 7 1.52 × 10− 8

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(A) (a)

150

R2=0.92

(b) 170

20 mM

50

Current (μ A)

Current(μA)

100

160

0.01 mM

0

150

-50

R2=0.81

140 -100

130

-150

-0.1

0.1 0.2 Potential (V)

0.3

0

0.4

0.240

6

8

10 12 14 16 18 20

150

Ea

0.245

4

(B) 200

2.72x10-4

(c)

2

[Urea]/ (mM)

Current (μ A)

Potential (V)

0.250

0.0

3.8x10-3

100

50

0.235 0

5

10 [Urea]/ (mM)

15

20

0 Ur

Ur+AA

Ur+OA

Ur+CA

Ur+glu

Ur+ChO

Fig. 6. (A). (a) The electrochemical response studies of GA-NC/ITO electrode as function of Ur concentration range from 0.01 to 20 mM. (b) Shows the plot between current and Ur concentration and (c) shows plot between potential shifts with concentration of Ur. (B). Effect of interference of GA-NC/ITO electrode in presence of other analytes.

4. Conclusions

concentration from 0.1 to 2 and 2 to 20 mM, respectively, and GA-NC shows binding constant of 63.93 M− 1 for urea. This electrode is highly selective for urea detection. This opens up the possibility for generating new customized soft materials that may find applications in pharmaceutics, personal care and packing products. The most important conclusion of this work was the clear demonstration of possibility of development of enzyme-free sensors in future starting from easily available, and cheap raw material. This GA-NC/ITO and other clays

The present study is centered on the formation of NC gels where colloidal platelets of Lap were present in the organogel matrix. A thin film of this NC was deposited uniformly onto indium tin oxide (ITO) coated glass plate could acts as an excellent urea sensor. This GA-NC/ ITO electrode exhibited electrochemical response more specific in terms of higher sensitivity as 32.7 and 5.56 µA mM− 1 cm− 2 in two range of

Table 3 Shows the sensing properties of GA-NC/ITO electrode along with nanocomposite of polymer-biopolymers based enzymatic and non-enzymatic based urea sensor those reported in the literature. S. no

Electrode composition

Linear range

Sensitivity

Detection limit

stability

Ref.

0.8–16.6 mM 0.16–5.02 mM 3–30 mg/dL 1.53–6 mmol− 1 L

0.13 μA/mM cm− 2 0.47 mA mM− 1 cm− 2 5.27 μA (mg dl− 1)− 1 cm− 2 0.35 μA cm− 2 m mol− 1 L

3 mg/dL 0.02 mM 0.3 mg/dL –

3 months 2 months – –

Solanki et al. (2008) Rajesh et al. (2005) Luo and Do (2004) Massafera and Torresi (2011)

0.58 × 10− 3–0.04 mol L− 1

3.44 μA cm− 2 mmol− 1 L mg− 1

–

2 weeks

Massafera and Torresi (2009)

Non-enzymatic urea sensor 6 Pyrrole/Pt

80–1440 μM

1.11 μA− 1 μM− 1 cm− 2

40 μM

2 weeks

7

0.1–20 mM

7.7 µA mM− 1 cm− 2

0.3 mM

6 weeks

Mondal and Sangaranarayanan (2013) Present work

Enzymatic urea sensor 1 Urs-GLDH/ZnO-Ch/ITO 2 Urs/PAPCP/ITO 3 Nafion(urease)/PANi-Nafion 4 Urs-poly(5-amino-1-naphtol)/bulk PPy 5 poly(5-amine-1-naphtol)

GA-NC/ITO

Abbreviation: Chitosan (CH); zinc oxide (ZnO); PAPCP (poly (N-3-aminopropyl pyrrole-co-pyrrole); polyaniline (PANi).

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