Polyaniline–Carboxymethyl Cellulose Nanocomposite for Cholesterol Detection

Copyright © 2010 American Scientific Publishers All rights reserved Printed in the United States of America

Journal of Nanoscience and Nanotechnology Vol. 10, 1–10, 2010

Polyaniline–Carboxymethyl Cellulose Nanocomposite for Cholesterol Detection Abdul Barik1 , Pratima R. Solanki1 , Ajeet Kaushik1 , Azahar Ali1 , M. K. Pandey1 , C. G. Kim2 , and B. D. Malhotra1
2
∗ 1

Department of Science and Technology Centre on Bimolecular Electronics, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 110012, India, 2 Centre for Nanobioengineering and Spintronics, Chungnam National University, Daejeon, 305-764 Korea

Cholesterol oxidase (ChOx) has been covalently immobilized onto polyaniline–carboxymethyl cellulose (PANI–CMC) nanocomposite film deposited onto indium-tin-oxide (ITO) coated glass plate using glutaraldehyde as a cross-linker. Fourier transform infrared (FTIR) spectroscopic and electrochemical studies have been used to characterize the PANI–CMC/ITO nanocomposite electrode and ChOx/PANI–CMC/ITO bioelectrode. Scanning electron microscopy (SEM) studies reveal the formation of PANI–CMC nanocomposite fibers of size ∼150 nm in diameter. The ChOx/PANI–CMC/ITO bioelectrode exhibits linearity as 0.5–22 mM, detection limit as 1.31 mM, sensitivity as 0.14 mA/mM cm2 , response time as 10 s and shelf-life of about 10 weeks when bioelectrode is stored at 4  C. The low value of Michaelis-Menten constant (Km
obtained as 2.71 mM reveals high affinity of immobilized ChOx for PANI–CMC/ITO nanocomposite electrode.

Keywords: Polyaniline, Carboxymethyl Cellulose, Cholesterol, Cholesterol Oxidase, Biosensor, Electrochemical Polymerization.

Polyaniline (PANI) has recently emerged as an interesting conducting polymer due its optical, electrical and molecular properties1–3 and its application in actuators, artificial muscles, solar cells, electronic shielding, electrochemical displays,4 corrosion protection,5 rechargeable batteries6 and fabrication of bio/gas sensors. This has been ascribed to its excellent conductivity, signal amplification, high thermal and chemical stability as well as cost-effectiveness etc.7–9 Moreover, easy synthesis of PANI in desired shape and size at nanoscale has led to its application in biosensing.10 This is because nanostructured PANI (NanoPANI) exhibits large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency, and strong adsorption ability that are helpful for immobilization of desired biomolecules for biosensor development.11 However, PANI based conducting polymers have been found to have low physical and mechanical strength leading to limited commercial applications. Numerous methods have been developed to overcome these shortcomings. Many nanocomposite/hybrid materials involving PANI and ∗

Authors to whom correspondence should be addressed.

J. Nanosci. Nanotechnol. 2010, Vol. 10, No. xx

other biopolymer systems, pluronic polymers, polystyrene and natural rubber have been utilized for composite formation with improved mechanical and other properties. Recently, biological and electronic properties of PANI have been found to be improved by modifying it by different dopants. Such composites have found applications in magnetic materials, corrosion protection and as sensors. Besides this, these materials have been used for fabricating eco-friendly highly efficient electronic devices.12–13 The composite of PANI with biopolymers such as chitosan, carboxy methyl cellulose (CMC), acacia gum, etc.14–16 are a new class of advanced materials having unique physicochemical properties for development of a desired biosensor. Nanocomposite of PANI and CMC has recently been used to obtain improved properties17–18 such as electrical conductivity, chemical stability, biocompatibility of PANI resulting in enhanced biosensing performance. CMC exhibits excellent properties like film forming ability, resistance to oil grease and solvents, physiological inertness, anionic character, binding properties making it suitable for desired biosensor application. Moreover, CMC shows nontoxic and biocompatible properties that are desirable for enzyme immobilization. And PANI and CMC have been found to have excellent properties

1533-4880/2010/10/001/010

doi:10.1166/jnn.2010.2511

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1. INTRODUCTION

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Polyaniline–Carboxymethyl Cellulose Nanocomposite for Cholesterol Detection

as conducting and biocompatible material, respectively for biosensing application. However, limited conductivity of CMC and biocompatibility of PANI has led to intensive research for the past about ten years. There have been several attempts to prepare composite of PANI and CMC for obtaining desired properties.17 Lukasiewicz et al., have synthesized blends of PANI with CMC.18 PANI–CMC composite has been electropolymerized onto stainless steel (AISI-304) from aqueous phosphoric acid solution using cyclic voltammetry.19 PANI composite film formed by chemical oxidative polymerization of aniline with CMC film has been investigated and its percolation threshold has been calculated.20 Banerjee et al. have polymerized aniline and CMC using steric acid as a stabilizer yielding stable dispersion.21 PANIcellulose acetate blends have been synthesized and the effect of transport of the anionic sodium dodecyl sulfate has been investigated.22 Methyl cellulose has been used as a stabilizer during polymerization of aniline.23 Two types of nanocomposites have been synthesized using cellulose nanofibers and conducting polymers (polyaniline and p-phenylene ethylnylene) doped with camphorsulfonic acid.24 However, no attempt has as yet been to made to utilize composite of PANI and CMC for biosensor application. Among the various metabolites, cholesterol is known to be the main constituent of nerve cells, brain cells25 and is a precursor for other biological materials.26 And various clinical disorders such as heart diseases, coronary artery diseases, cerebral thrombosis etc. are caused by high cholesterol level in blood. It is thus important to develop a reliable and sensitive biosensor that can be used for rapid determination of cholesterol. Among the various matrices, it has been reported that nanostructured PANI(NanoPANI) has the unique ability to promote fast electron transfer between electrode and active sites of the desired enzyme [cholesterol oxidase (ChOx)] for analyte (cholesterol) detection.27–32 PANI has been utilized for development of cholesterol biosensor. Dhand et al. have electrophoretically fabricated nanocomposite film of PANI and multi-walled carbon nanotubes (MWCNTs) for covalent immobilization of ChOx. This bioelectrode exhibits linearity as 1.29 to 12.93 mM cholesterol with high sensitivity of 6800 nA mM−1
33 And a composite of biopolymer with a nanomaterial has been used for the immobilization of ChOx for cholesterol estimation. Nanocomposite film of ZnO nanoparticles and chitosan (CS) has been used to immobilize ChOx for cholesterol detection and the results obtained reveal linearity as 5 to 300 mgdl−1 and Michaelis–Menten constant (Km  value as 8.63 mgdl−1
34 A chitosan(CS)-tin oxide nanobiocomposite film has been deposited onto an ITO to immobilize ChOx for cholesterol detection. The value of the Km has been obtained as 3.8 mM with sensitivity as 34.7 mA/mg dL−1 cm2 and detection limit of 5 mg/dL.35 Sol–gel derived CSsilica/MWCNTs based nanocomposite has been developed 2

Barik et al.

to prepare cholesterol biosensor that reveals sensitivity and Km value as 1.55 A mM−1 and 0.41 mM, respectively.36 A platinum (Pt) decorated CNT–Pt electrode has been prepared by chemical reduction method and has been modified by ChOx to detect cholesterol. This biosensor shows the linearity from 4
0 × 10−6 mol/L to 1
0 × 10−4 mol/L with detection limit as 1
4 × 10−6 mol/L.37 There is thus a considerable scope to develop a composite of PANI with biopolymers for cholesterol detection. In this manuscript, we report results of studies relating to the immobilization of ChOx onto electrochemically prepared PANI–CMC nanocomposite onto ITO coated glass for cholesterol detection.

2. METHODS AND APPROACHES 2.1. Reagents Cholesterol oxidase (ChOx), cholesterol, Na2 HPO4 and NaH2 PO4 has been procured from Sigma Aldrich (USA). Cholesterol and cholesterol oxidase (3 U/ml) have been purchased from Sigma–Aldrich (Germany). Aniline and sodium carboxymethylcellulose (CMC) is of analytical grade. Aniline is distilled prior to electropolymerization. Other chemicals are of analytical grade and are used without further purification. ITO coated glass substrates have been obtained from Balzers, UK. The deionized water obtained from Millipore water purification system (Milli Q 10 TS) is used for the preparation of solutions and buffers. For the preparation of cholesterol stock solution (25 mM), Triton X-100 is used as surfactant and is kept at 4  C. This stock solution is further diluted to make different concentration of cholesterol. 2.2. Polymerization of Polyaniline–Carboxymethylcellulose 180 l of aniline (1 M) monomer and 5 l of CMC (1% in deionized water) are dissolved in 10 ml of 1 M hydrochloric acid and sonicated for about 10 minutes. Prior to polymerization of aniline and CMC film, indium-tin-oxide (ITO) coated glass plate is cleaned with a solution comprising of water, hydrogen peroxide and ammonium hydroxide (5:2:2) and is then rinsed thoroughly with deionized water. Electrochemically polymerization of aniline and CMC onto ITO (0.25 cm2 and resistance as 35 /cm2  electrode has been carried out at 150 A (chronopotentiometric) for about 10 min at a scan rate of 50 mV/s using a three-electrode (ITO as working, platinum foil as counter and Ag/AgCl as a reference electrode) cell. These PANI–CMC/ITO nanocomposite films are rinsed with distilled water to remove any oligomers and dried at room temperature for 24 hours. During electrochemical synthesis of PANI, a large number of positive charges are generated on nitrogen atoms in J. Nanosci. Nanotechnol. 10, 1–10, 2010

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Polyaniline–Carboxymethyl Cellulose Nanocomposite for Cholesterol Detection

3. RESULTS AND DISCUSSION

the polymeric chains. The negative charges of CMC moieties interact with positively charge PANI backbone via electrostatic interactions (Fig. 1).

3.1. Optical Characterization

2.3. Immobilization of ChOx on PANI–CMC/ITO Electrodes The PANI–CMC/ITO nanocomposite electrode is modified with glutaraldehyde by spreading 0.1% of glutaraldehyde on PANI–CMC/ITO electrode for about 2 h, after which it is washed with deionized water a number of times. 10 L solution of ChOx (1.0 mg/mL, in PB, 50 mM, pH 7.0) is immobilized onto modified PANI–CMC/ITO electrode. Prior to being used, ChOx/PANI–CMC/ITO bioelectrode is allowed to dry overnight under desiccated conditions and is then washed with phosphate buffer saline (PBS, 50 mM, pH 7.0, 0.9% NaCl) to remove any unbound ChOx and is stored in a refrigerator at 25  C when not in use.

2.4. Characterization Scanning electron microscopy (SEM, Leo 440) has been used for surface morphology of PANI, PANI– CMC/ITO nanocomposite, ChOx/PANI–CMC/ITO bioelectrode, respectively. This electrode is characterized by Fourier transform infrared (FTIR) spectrophotometer (Perkin-Elmer, Model 2000) in 400–4000 cm−1 wavelength range. PANI, PANI–CMC/ITO nanocomposite, ChOx/PANI–CMC/ITO bioelectrode have been characterized with a UV–vis spectrophotometer (Shimadzu 160 A). Electrochemical studies have been carried out on an Autolab Potentiostat/Galvanostat (Eco Chemie, Netherlands) in PBS solution.

H

H

N

N y

O O–

O H HO H

H OH

O

H

N+ H 1–y

Polyaniline (emaraldine salt) OH

H

H

O

Carboxylic methylic cellulose

OH OH H

N+ H

H

n

H O

O– O

N+ H 1–y Fig. 1.

N+

N H

Polyaniline (emaraldine salt)

N y

H

Proposed mechanism of PANI–CMC naocomposite during electrochemical polymerization.

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FTIR spectra (Fig. 2(A)) of PANI/ITO electrode (Curve (a)), CMC/ITO (Curve (b)), PANI–CMC/ITO nanocomposite electrode (Curve (c)) and ChOx/PANI– CMC/ITO bioelectrode (Curve (d)) are shown in Figure 2(B). Curve (a) for PANI exhibits IR bands in the region from 3200–3800 cm−1 assigned to N–H stretching vibration mode of the NH2 group. The infrared band markers seen at 2923 and 2853 cm−1 can be assigned to aliphatic CH2 or CH3 stretching vibration mode, originating due to the presence of dopants in polyaniline. The peak seen at 1540 cm−1 is assigned to C C stretching mode of quinoid ring in the emeraldine salt and absorption band at 1480 cm−1 is assigned to C C stretching vibration mode in the benzenoid ring. The C–N stretching vibration mode in secondary aromatic amine nitrogen (quinoid system) in doped polyaniline found at 1310 cm−1 corresponds to the oxidation or protonation state. The in-plane vibration of C–H bending mode in NQN, Q–N + H–B or B–N + H–B (where Q = quinoid and B = benzenoid) is observed at 1175 cm−1 in the FTIR spectra. The band at 1133 cm−2 is assigned to aromatic C–H in-plane bending. The absorption band seen at 802 cm−1 (Curve (a)) is attributed to aromatic ring and out-of-plane C–H deformation vibrations for 1,4-disubstituted aromatic ring system. The IR band at 670 cm−1 is assigned C–N stretching arising due to in plane ring deformation vibration characteristics of para substituted benzene. FTIR spectra of CMC (Curve (b)) exhibit all the bands corresponding to the functional groups available on CMC. The peak 3400 cm−1 due to O–H stretching vibration mode of the –OH group and a band at 2925 cm−1 is attributed to C–H stretching vibration. The IR band at 1600 cm−1

Polyaniline–Carboxymethyl Cellulose Nanocomposite for Cholesterol Detection

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(A) 92

(d)

90

(c) 88

(a)

T (%)

86

(b) 84

82

80 a = PANI/ITO electrode b = CMC/ITO electrode

78

c = PANI-CMC/ITO electrode d = ChOx/PANI-CMC/ITO bioelectrode

76 4000

3500

3000

2500

2000

Wavenumber

1500

1000

500

(cm–1)

(B)

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(a)

(b)

(c)

Fig. 2. (A) FTIR spectra of (a) NanoPANI/ITO electrode, (b)CMC/ITO electrode, (c) PANI–CMC/ITO nanocomposite electrode and (c) ChOx/NanoPANI–CMC/ITO nanocomposites; (B) Scanning electron micrographs of (a) NanoPANI/ITO (b) PANI–CMC/ITO naocomposite electrode (c) ChOx/PANI–CMC/ITO bioelectrodes.

is assigned to COOH group due to ring stretching of glucose. The bands seen around 1420 and 1315 cm−1 are assigned to C–H scissoring and –OH bending vibration modes in CH2 and COH groups, respectively. The band at 1035 cm−1 is assigned to C–O stretching vibration modes due to primary alcoholic –CH2 OH stretching mode. The weak band at around 700 cm−1 is due to ring stretching and ring deformation of -D-(1–4) and -D-(1–6) linkages.38–39 It has been found that the band found at 3400 cm−1 , 1600 and 1420 cm−1 in FTIR spectra of CMC (curve (b)) and band at 3410 cm−1 in PANI (curve (a)) disappears in the IR spectra of PANI–CMC nanocomposite (curve (c)) revealing that NH2 group of PANI interacts with OH/COO− group of CMC via electrostatic interactions (Fig. 1). Moreover, the band seen at 1480, 1310, 1133 and 802 cm−1 shifts to higher frequency region in the IR spectra of PANI–CMC nanocomposite due to interaction between PANI and CMC revealing the 4

formation nanocomposite. However, in the FTIR spectra of PANI–CMC nanocomposite the peak intensity of 670 cm−1 band at becomes weaker than that of the PANI spectra and presence of a sharp band at 830 cm−1 indicates that the amount of linear chain structure corresponding to the 1,4-para-disubstitution mode in PANI–CMC is dominant. It can be seen that ChOx/PANI/CMC/ITO bioelectrode (Curve (c)) exhibits all bands of PANI–CMC nanocomposite including two new bands at 3250 cm−1 assigned to the NH2 group in protein and at 1580 cm−1 are attributed to amide I bond in enzyme revealing immobilization of ChOx onto the PANI–CMC/ITO nanocomposite electrode. The surface morphologies of PANI/ITO electrode (image (a)), PANI–CMC/ITO nanocomposite electrode (image (b)), and ChOx/PANI–CMC/ITO bioelectrode (image (c)) have been investigated using scanning electron microscopy (SEM, Fig. 2(B)). Morphology of PANI appears as a three-dimensional, inter-connected J. Nanosci. Nanotechnol. 10, 1–10, 2010

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Polyaniline–Carboxymethyl Cellulose Nanocomposite for Cholesterol Detection

network of smooth nanofiber with average diameter of 120–160 nm and length of several microns. However, morphology of PANI changes into nanofibers (average diameter of 150–200 nm) with globular appearance on addition of CMC in PANI backbone.s This suggests that CMC binds on the nanofibers of PANI via electrostatic interactions. It can be seen that rough nanofibrous morphology of PANI–CMC nanocomposite further changes into planner morphology after the immobilization of ChOx revealing immobilization of ChOx onto PANI– CMC nanocomposite. Moreover, globular nanofibrous

morphology of PANI–CMC provides added advantage for adsorption of ChOx. 3.2. Electrochemical Studies The concentration of CMC of PANI–CMC nanocomposite has been optimized using cyclic voltametric (CV) technique in phosphate buffer (50 mM, pH 7.0, 0.9% NaCl) at scan rate 30 mv/s. Figure 3(A) shows CV of PANI with different concentration of CMC varying from 0.5–11.25 M revealing that magnitude of

(A) 2.5 × 10–4 2.0 × 10

–4

1.5 × 10–4

Current (A)

2.5×10 2.0×10 1.5×10 1.0×10 5.0×10

1.0 × 10–4

(c)

–4

(b)

–4 –4 –4

(a)

–5

(d)

0.0 0.000 0.002 0.004 0.006 0.008 0.010 0.012

(e)

Current (A)

Cellulose concentration (mM)

5.0 × 10

(f)

–5

0.0 –5.0 × 10–5 –1.0 × 10–4

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–1.5 × 10–4 –2.0 × 10–4 –2.5 × 10–4 –0.8

–0.6

–0.4

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0.0

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Potential (V) 16.0k

(B)

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(a)

(C)

(c)

14.0k –4

3.0 × 10 12.0k

(b) 2.0 × 10–4

Current (A)

–Z′′/ohm

10.0k 8.0k 6.0k 4.0k

(b)

(a)

1.0 × 10–4 0.0 –1.0 × 10–4

2.0k

(c)

0.0 0.0

–2.0 × 10–4

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Z′/ohm

–0.8 –0.6 –0.4 –0.2

0.0

0.2

0.4

0.6

0.8

Potential (V)

Fig. 3. (A) Cyclic voltammograms of PANI–CMC naocomposite electrode at different concentration of cellulose (a) 0.5, (b) 1.25, (c) 2.5, (d) 5, (e) 7.5, (f) 11.25 M in phosphate buffer (pH 7.0, 50 mM, 0.9% NaCl) at scan rate 30 mV/s (inset: plot of current vs. cellulose concentration); (B) Impedance spectra of (a) NanoPANI/ITO, (b) PANI–CMC/ITO naocomposite electrode and (c) PANI–CMC-ChOx/ITO bioelectrodes in phosphate buffer (50 mM, pH 7.0, 0.9% NaCl); (C) Cyclic voltammograms of (a) NanoPANI/ITO electrode, (b) PANI–CMC/ITO naocomposite electrode and (c) ChOx/PANI–CMC/ITO bioelectrodes at scan rate 30 mV/sec in phosphate buffer [PH 7.0, 50 mM, 0.9% NaCl].

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4.0 × 10–4 3.5×10

–1

(c) 3.0 × 10–4

2.0 × 10–4

Current (mA)

3.0×10

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–1

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(b)

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(d)

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–0.6

–0.4

–0.2

0.0

0.2

0.4

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0.8

Potentail (V)

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Fig. 4. Cyclic voltammograms of the ChOx/PANI–CMC/ITO bioelectrode at different pH as (a) pH 6.0, (b) pH 6.5, (c) pH 7.0 and (d) pH 8.0 at scan rate 30mV/sec in phosphate buffer (50 mM) (inset: variation of obtained current vs. different pH).

current response is maximum for 2.5 M PANI–CMC nanocomposite (Curve (c)). It is observed that PANI– CMC nanocomposite with 1.25 M CMC (curve (b)) exhibits oxidation and reduction peaks at 0.25 V and −0.24 V, respectively indicating a reversible system. Thus PANI–CMC nanocomposite consisting of 1.25 M CMC concentration has been selected for fabrication of biosensor electrode. It appears that as amount of CMC increases from 0.5 to 2.5 M, the emeraldine form of PANI increases resulting in increased conductivity. However, when amount of CMC further increases above 2.5 M, the emeraldine form of PANI is oxidized to pernigraniline form resulting in decrease electrical conductivity. Electrochemical impedance spectroscopy (EIS) technique has been used to measure resistance of the modified electrode as a function of frequency due to variation in the interfacial properties. Figure 3(B) shows Faradic impedance spectra in the frequency range 0.01–105 Hz, represented by the Nyquist plots of the PANI/ITO electrode, PANI–CMC/ITO nanocomposite electrode and ChOx/PANI–CMC/ITO bioelectrode (Curve (a), (b) and (c) respectively) obtained in phosphate buffer [50 mM, pH 7.0, 0.9% NaCl]. The semicircle diameter of EIS spectra gives value of charge transfer resistance (RCT  revealing electron-transfer kinetics at the electrode interface. It is observed that RCT value of PANI–CMC/ITO nanocomposite electrode (10.3 k is lower than that of the PANI/ITO 6

(41.5 k indicating that CMC increases the electroactive surface area of PANI and facilitates electron transport between the medium and electrode. Interestingly, after the immobilization of ChOx onto PANI–CMC/ITO nanocomposite electrode, RCT value decreases to 6.5 k. These results suggest that electron transfer in the ChOx/PANI– CMC/ITO electrode is easier between the solution and electrode leading to improved diffusion of molecules towards the electrode surface. Cyclic voltammetric (CV) studies of PANI/ITO electrode, PANI–CMC/ITO nanocomposite electrode and ChOx/PANI–CMC/ITO bioelectrode (Curves (a), (b) and (c) in Fig. 3(C)) have been conducted in phosphate buffer (50 mM, pH 7.0, 0.9% NaCl) at scan rate of 30 mV/s. It is observed that PANI/ITO electrode (Curve (a)) shows well-defined oxidation peak at 0.28 V and reduction peak at −0.24 V, indicating a reversible system. The magnitude of current response for PANI– CMC/ITO nanocomposite electrode (Curve (b), 0.22 mA) is higher than that of PANI/ITO electrode (curve (a), 0.34 mA). This suggests that negatively charged CMC molecules diffuse into PANI matrix resulting in change in the oxidation state (increased emeraldine salt form) that may result in enhanced electrical conductivity of PANI. It appears that presence of CMC results in increased electroactivity of the PANI resulting in enhanced electron transport between medium and the electrode. Further, magnitude of the response current increases (0.86 mA) after J. Nanosci. Nanotechnol. 10, 1–10, 2010

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Polyaniline–Carboxymethyl Cellulose Nanocomposite for Cholesterol Detection (A) 4.5 × 10–4 4.5×10–4

3.5 × 10–4 3.0 × 10–4 2.5 × 10–4

g

4.0×10–4 Change in current (A)

4.0 × 10

–4

3.5×10–4 3.0×10–4

2.0×10–4 1.5×10–4

a

Current (A)

1.0×10–4

2.0 × 10

–4

a = 0.55 mM b = 5.55 mM c = 11.1 mM d = 13.86 mM e = 16.65 mM f = 19.45 mM g = 22.25 mM

2.5×10–4

5.0×10

–5

0

1.5 × 10–4

5 10 15 20 Cholesterol concentartion (mM)

25

1.0 × 10–4 5.0 × 10–4 0.0 –5.0 × 10–5 –1.0 × 10–4 –1.2

–0.9

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Potential (V)

(B) 2.5 × 10–4

2.4×10–4

a b

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1.8×10 –4 1.6×10

a = CH b = CH+AA

c

c = CH+Glu

d

Current (A)

2.0 × 10

–4

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2.2×10–4

1.4×10–4

d = CH+U

1.2×10–4

e

e = CH+UA

1.0×10–4

1.5 × 10–4

8.0×10–5 6.0×10–5

Current (A)

4.0×10–5

1.0 × 10–4

2.0×10–5 0.0 a

b

c d Interferents (mM)

e

5.0 × 10–5

0.0

CH = cholesterol; U = Urea; UA = Uric Acid –5.0 × 10–5

Glu = Glucose; AA = Ascorbic Acid

–1.0 × 10–4 –1.0

–0.8

–0.6

–0.4

–0.2

0.0

0.2

0.4

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0.8

1.0

Potential (A) Fig. 5. (A) Cyclic voltammograms of the ChOx/PANI–CMC/ITO bioelectrode as a function of cholesterol concentration (a) 0.55, (b)5.55, (c) 11.1, (d) 13.86, (e) 16.65, (f) 19.45, (g) 22.25 mM at scan rate 30mV/sec in phosphate buffer [pH 7, 50 mM, 0.9% NaCl] (inset: Change in current obtained as a function of cholesterol concentration); (B) Interferent studies of the ChOx/PANI–CMC/ITO bioelectrode.

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Table I. Characteristics of ChOx/PANI–CMC/ITO bioelectrode along with those reported for various nanocomposites utilized for cholesterol estimation in literature.

Electrode

Linearity

Detection limit

Km

Sensitivity

NanoPANI-CNT 1.29–12.93 mM — — 6.8 A/mM ChOx/NanoZnO-CHIT 5–300 mg/dl 8.63 mgdl−1 5 mgdl−1 1
41 × 10−4 Abs mgdl−1 ChOx/Chitosan-tin oxide 0.26–10.4 mM 3.8 mM 5 mg/dL. 34.7 mA/mg dL−1 cm2 0.24 mM ChOx/CS/MWCNT 4
0 × 10−6 –7
0 × 10−4 mM — — — ChOx/NanoPt/CNT 4.0×10−6 –1.0×10−4 mol l−1 ChOx/Polyaniline-Cellulose 0.5–22 mM 2.7 mM 1.31 mM 0.140 mA/mM

the immobilization of ChOx onto PANI–CMC/ITO electrode (Curve (c)) revealing that PANI–CMC/ITO electrode plays an important role in accelerated electron transfer between ChOx and electrode. Moreover, PANI–CMC/ITO nanocomposite electrode provides a three-dimensional structure and some of the restricted orientation that favors direct and faster electron communication between enzyme and the electrode surface. 3The surface concentrations of redox species onto PANI/ITO electrode, PANI–CMC/ITO nanocomposite electrode and ChOx/PANI–CMC/ITO bioelectrode have been calculated using Brown-Anson model.40

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Ip = N 2 F 2 CAV /4RT

(1)

where N is the number of electrons transferred (2), F is Faraday constant (96485.3 C/mol), C is the surface concentration of the electrode (mol/cm−2 , A is the surface area of the electrode (0.25 cm−2 , V is the scan rate (30 mV/s), R is the gas constant (8.314 J/mol K), T is the absolute temperature (293 K) and Ip is the peak anodic current of the electrode. The values of surface concentration C of anions in PANI/ITO electrode, PANI–CMC/ITO nanocomposite electrode and ChOx/PANI–CMC/ITO bioelectrodes have been obtained as 41
5 × 10−6 mmol/cm−2 , 3
8 × 10−6 mmol/cm−2 and 5
8 × 10−6 mmol/cm−2 , respectively. It has been found that the surface concentration of the PANI–CMC/ITO nanocomposite is higher than that of PANI/ITO electrode suggesting that CMC loading increases electroactive surface area of the PANI– CMC nanocomposite. The higher surface concentration of ChOx/PANI–CMC/ITO bioelectrode implies high loading of ChOx on the PANI–CMC nanocomposite matrix. 3.3. Activity of ChOx/PANI–CMC/ITO Nanocomposite Bioelectrode Figure 4 shows effect of pH (6 to 8) on the electrochemical behavior of ChOx/PANI–CMC/ITO bioelectrode using CV technique. The magnitude of current response is maximum at pH 7. This value of pH is optimum for catalytic activity of ChOx for the hydrolysis of cholesterol. This suggests that ChOx/PANI–CMC/ITO bioelectrode shows maximum activity at pH 7 at which ChOx retains its natural structure and is responsible for low detection limit and high sensitivity for cholesterol detection. 8

Response Shelf time(s) life (Weeks) — 15 s 5 s,