Preparation of Biocompatible Palladium-Fe3O4 Nanoparticles/Multiwalled Carbon Nanotubes Composite and its Electrocatalytic Activity towards Determination of Cholesterol on Screen Printed Electrode

Revanasiddappa Manjunatha; Gurukar S. Suresh; Jose S. Melo; Jakkid Sanetuntikul; Sangaraju Shanmugam

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Preparation of Biocompatible Palladium-Fe₃O₄ Nanoparticles/Multiwalled Carbon Nanotubes Composite and its Electrocatalytic Activity towards Determination of Cholesterol on Screen Printed Electrode

Revanasiddappa Manjunatha¹, Gurukar S. Suresh^{1, 2*}, Jose S. Melo^{3, 4*}, Jakkid Sanetuntikul⁵ and Sangaraju Shanmugam⁵

¹Chemistry Research Centre, S. S. M. R. V. Degree College, Jayanagar, Bangalore - 560041, India
²Department of Chemistry and Research Centre, N.M.K.R.V. College for Women, Jayangar, Bangalore -560011, India
³Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai - 400085, India
⁴Homi Bhabha National Institute, Anushakti Nagar, Mumbai- 400094, India
⁵Department of Energy Systems and Engineering, Daegu Gyeongbuk Institute of Science and Technology, Daegu 711-873, Republic of Korea

*Corresponding author: Jose Savio Melo, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay and HomiBhabha National Institute, Anushakti Nagar, Mumbai- 400 085, India, Telephone: 91-22-25592760; Fax no: 91-22-25505151.; E-mail:

Gurukar S. Suresh, Department of Chemistry and Research Centre, N.M.K.R.V. College for Women, Jayangar, Bangalore -560011, India, Telephone: 91 –80 – 26654920;Fax no: 91 – 80 – 22453665 ; E-mail:

Received: 03 October, 2016; Accepted: 04 January, 2017; Published: 10 February, 2017

Citation: Jose S. Melo (2017) Preparation of Biocompatible Palladium-Fe3O4 Nanoparticles/Multiwalled Carbon Nanotubes Composite and its Electrocatalytic Activity towards Determination of Cholesterol on Screen Printed Electrode. J of Biosens Biomark Diagn 2(1):1-10. DOI: 10.15226/2575-6303/2/1/00109

Abstract

A simple and facile microwave method was adopted to prepare Fe₃O₄ and Pd- Fe₃O₄ nanoparticles, which possess the mean particle diameter of 10 nm and 90 nm, respectively. Formation of Fe₃O₄ and Pd- Fe₃O₄ nanoparticles were confirmed from Powder X-ray diffraction, Transmission electron microscopy, Energy-dispersive X-ray spectroscopy, and FT-Infra red spectroscopy techniques. Negatively charged mutliwalled carbon nanotubes (COO--MWCNTs) were wrapped with positively charged poly (Diallyldimetheylammonium Chloride) (PDDA) followed by coating with Pd-Fe₃O₄ nanoparticle to get (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs) composite. This composite was used for the determination of cholesterol by using Cholesterol Oxidase (ChOx) enzyme on Screen Printed Electrode (SPE). (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs) composite provides biocompatible microenvironment for the ChOx to exhibit Direct Electron Transfer (DET) on electrode surface. A well defined redox peak at -0.365 and -0.443 V was observed, corresponding to the DET of the FAD/FADH2 of ChOx. Enzyme modified SPE was characterized by cyclic voltammetry and electrochemical impedance spectroscopy by means of Fe(CN)6 ^3-/4as an electrochemical probe. The linear range of the enzyme modified SPE was found to be 10-80 μm (R=0.9972) with detection limit of 1 μm of cholesterol. The sensitivity of the enzyme modified SPE was found to be 10.45 μA μM-1 cm^-2 for the determination of cholesterol, common interferents such as ascorbic acid, uric acid and glucose did not cause any interference because of low operating potential.

Keywords: Pd-Fe₃O₄; Nanoparticles; Screen Printed Electrode; Cholesterol Oxidase

Introduction

The magnetic nanoparticles in general, iron oxide (Fe₃O₄) have been attracted an increasing interest in the development of nanostructured materials and nanotechnology in biotechnology and medicine [1]. The main advantage of magnetic nanoparticles is that, they can easily and rapidly separate from their matrix by an external magnetic field. Some of the common features associated with the Fe₃O₄ nanoparticles are good biocompatibility, strong superparamagnetic property, high surface area, low toxicity and ease of preparation [2,3]. Thus, Fe₃O₄ nanoparticles have been used in a wide range of potential applications such as, electrochemical sensors/biosensors, catalysis, immunoassays, data storage [4-7]. Various methods have been used for the synthesis of magnetic nanoparticles which includes hydrothermal synthesis, co-precipitation, sol-gel method, microwave irradiation method [8-11]. The later method has significant advantage with respect to higher reaction rates and product yields in a shorter period of time.

Carbon nanotubes are one of the new kinds of carbon material, discovered in the last decades of the 20th century [12]. Researchers have explored various potential characteristics of CNTs, which could be applicable to various fields [13]. It was found that CNTs have excellent electronic conductivity, mechanical strength, chemical stability and unique structural properties [14]. However, the main drawbacks exist in the processibility of CNTs in solution, in which they precipitate into ropes or bundles due to strong Van der Waals interactions [15]. To overcome this problem, surface modifications have been employed, such as chemical functionalization using strong acids, polymer wrapping of CNTs [16,17]. In later method i.e. chemical functionalization of CNTs using strong acid results in partial oxidation of the carbon atoms to produce oxygen containing groups such as carboxylic groups, especially in the open ends of CNTs [18]. These groups are negatively charged in the aqueous solution and can interact with positively charged poly electrolytes [19].

Cholesterol is one of the most important analyte in clinical analysis, because its assay is important for diagnosis and prevention of a numerous clinical disorders such as, hypertension, cerebral thrombosis, arteriosclerosis and coronary heart disease [20]. Recent studies explored that cholesterol plays a vital role in the brain synapses and also in the immune system including protection against cancer. In earlier days, cholesterol was determined by using non-enzymatic spectrophotometric techniques by using colored substances [21]. However, this technique suffers from low specificity, instability of reagents and high cost. These can be effectively addressed by using enzymatic cholesterol biosensor. Some of the advantages of enzymatic cholesterol biosensors are specificity, simplicity, rapidness and cost effectiveness [22]. The most commonly used enzyme for the construction of cholesterol biosensor is cholesterol oxidase. ChOx is a Flavin-Adenine-Dinucleotide (FAD) containing flavoenzyme. In the presence of oxygen, ChOx catalyzes two reactions; oxidation of cholesterol to cholest-5-en-3-one and subsequently the isomerization to cholest-4-en-3-one.

Direct electrochemistry of redox enzyme systems has gained increasing interest both for the study of the electron transport proteins as well as development of third generation reagent less electrochemical biosensors [23,24]. However, direct electron transfer between redox enzyme and electrode is generally difficult to observe due to several factors. Such as, enzyme active sites are deeply embedded in protein matrix, resulting in a long distance between active sites and underlying electrode. In addition, conformational changes or denaturation of redox enzyme often occur while immobilization of enzyme onto the electrode surface. Thus, to obtain DET, many techniques have been developed such as layer-by-layer technique, covalent binding using cross linkers, physical adsorption [25-27]. Physical adsorption method involves van der Waals forces, ionic binding or hydrophobic forces. The main advantage of this method is that it is simple and can be used under mild conditions. It requires only a minimum activation steps resulting in little or no conformational changes of the enzyme or destruction of its active centre [28].

In the present work, a simple and facile microwave method was adopted to prepare Fe₃O₄ and Pd- Fe₃O₄ nanoparticles by following the procedure given in the research article with slight modifications [29]. Formation of both Fe₃O₄ and Pd-Fe₃O₄ nanoparticles were confirmed by pXRD, TEM, EDX and FTIR analysis. Negatively charged Pd- Fe₃O₄ nanoparticles were mixed with positively charged PDDA wrapped MWCNTs to get negatively charged novel composite. This negatively charged novel composite was drop casted on the Screen Printed Electrode (SPE). Positively charged ChOx (pH 4. 0) was immobilized on the composite by physical adsorption method. Enzyme modified screen printed electrode showed electrocatalytic activity towards detection of cholesterol. Cyclic Voltammery (CV) and Electrochemical Impedance Spectroscopy (EIS) techniques were used to characterize the enzyme modified screen printed electrode. Above mentioned novel composite provides biocompatible microenvironment for the ChOx to exhibit DET on SPE. To the best of our knowledge, for the first time we have used this novel composite for detection of cholesterol.

Methods

Reagents

TritonX-100,Cholesterol, Pd(NH₃)4Cl₂•H₂O, FeSO₄•7H₂O, MWCNTs and PDDA (Mw: 200,000-350,000), were purchased from Sigma Aldrich. Cholesterol oxidase was procured from SRL, India. Screen printed electrodes of diameter 3 mm (0.071 cm2) were purchased from CH instruments (product no. TE 100). Phosphate Buffer Saline of pH 7. 0 (PBS) was prepared from stock solutions of 0.1 M KH₂PO₄, 0.1 M K2HPO4 and 0.1M KCl. All other chemicals used were of analytical reagent grade unless otherwise mentioned and used without further purification. All solutions were prepared with milli-Q water.

Enzyme solution preparation

100 U/ml ChOx solution was prepared in 0.1 M acetate buffer solution of pH 4. A stock solution of 10 mM cholesterol was prepared by dissolving 0.0967 g of cholesterol in a mixture of 1 mL Triton X-100 and 0.5 mL isopropanol at 65°C and diluting the resulting solution to 25 mL in a standard flask using hot PBS of pH 7.0. The solution was stored at 4°C in the dark and was stable for two weeks (until a slight turbidity was observed).

Synthesis of Fe₃O₄ and Pd- Fe₃O₄ nanoparticles

As stated earlier, Fe₃O₄ and Pd- Fe₃O₄ nanoparticles were prepared according to the procedure described elsewhere [29]. In brief, 2.0 mm FeSO₄.7H₂O was dissolved in 100 ml distilled water with continuous stirring. The pH of the solution was adjusted to 11 by conc. ammonia solution resulting in the formation of black precipitation. This suspension was transferred into microwave oven, in which microwave radiation of high energy was applied for one minute. The product Fe₃O₄ nanoparticles were separated by centrifugation method. Finally Fe₃O₄ nanoparticles washed thoroughly with water followed by ethanol and dried at 50°C in vacuum oven.

Similarly, Pd- Fe₃O₄ nanoparticles were prepared by coprecipitation method. 0.5 mm Pd (NH₃)4Cl₂•H₂O, and 2.0 mmol FeSO₄•7H₂O were dissolved in 100 ml distilled water with constant stirring. The pH of the solution was adjusted to about 11 by conc. ammonia solution. Then the reaction mixer was taken into microwave oven for microwave irradiation. The product was isolated by using above descried procedure.

Electrochemical measurements

Cyclic voltammetry, electrochemical impedance spectroscopy, differential pulse voltammetry experiments were carried out with Versa stat 3 (Princeton Applied Research, USA). The microwave oven used in the present study was a domestic microwave oven (LG, intellowave, MS-2342 AE). Powder X-Ray Diffraction (pXRD) patterns of the samples were recorded using a Philips X’pert Pro diffractometer with CuKα (λ = 1.5418 Ǻ). FT-IR experiments were carried out with Bruker Alpha-T FTIR spectrometer (ATR mode, diamond crystal, resolution 4 cm-1, 400-4000 cm-1). The morphology of the samples were analyzed by the Field emission scanning electron microscopy (Hitachi, S4800 FE-SEM) and the Field emission transmission electron microscopy (Hitachi, HF 3600 FE-TEM). TEM experiments were performed at an acceleration voltage of 300 kV. For the elemental mapping study, an Energy Dispersive X-Ray Spectroscopy (EDXS) connected to a TEM was used in scanning mode. TEM samples were prepared by dropping ultrasonically dispersed isopropyl alcohol solution of nanoparticles on a copper grid coated with amorphous carbon film. All experiments were done in an electrochemical cell consisting of SPE with an unmodified or modified carbon working electrode, a carbon counter electrode and Ag/AgCl reference electrode.

Preparation of enzyme modified screen printed electrode

As we already discussed in the introduction, CNTs are insoluble in most of the solvents because they precipitate into ropes are bundles due to strong Vander Waals interactions. To overcome this difficulty, we introduced carboxylic groups on MWCNTs surface by refluxing with conc. nitric acid for 5 h, followed by filtration and washed with pure water until the filtrate become neutral. Finally the product was dried in vacuum at 50°C [30]. PDDA is a water soluble, quaternary ammonium cationic polyelectrolyte. It is positively charged colloid when dissolved in aqueous solutions [31,32]. The positively charged PDDA polymer can be easily wrapped/coated on negatively charged MWCNTs [33]. 1 mg/ ml carboxylated MWCNTs dispersed in 0.2% PDDA solution, ultrasonicalted for 20 min. followed by stirred at 50°C for 12 h. To this composite 0.5 mg/ml Pd- Fe₃O₄ nanoparticles were added and stirred for 12 h at room temperature. In this stage negatively charged Pd- Fe₃O₄ nanoparticles coated on positively charged MWCNTs wrapped with PDDA as shown in figure 1.

Figure 1:Schematic representation for the fabrication of enzyme electrode based on the (PdFe₃O₄/PDDA/COO^- _-MWCNTs) composite.

2.5 μl of Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs composite was drop casted on screen printed electrode, dried at ambient temperature. 5 μl of positively charged ChOx (pH 4.0) drop casted on composite, dried at 4°C. Hereafter, the enzyme modified electrode was denoted as ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE.

Results and discussion

Characterization of Fe₃O₄ and Pd- Fe₃O₄ nano particles using pXRD, TEM, EDX and FT-IR techniques

Figure 2a shows XRD patterns of the synthesized Fe₃O₄ nanoparticles. Diffraction peaks of Fe₃O₄ nanoparticles were obtained at 30.18°, 35.54°, 43.29°, 53.69°, 57.29° and 62.96° corresponding to the index planes (220), (311), (400), (422), (511) and (440) respectively. This is quite identical to pure Fe₃O₄ nanoparticles and well matched with that of JCPDS no. 82-1533. This revealed that the Fe₃O₄ nanoparticles have a cubic spinel structure [34,35]. Also, no characteristic peaks of impurities were observed. From the XRD data, the mean particle diameter of Fe₃O₄ nanoparticles was calculated from index planes (220), (311) and (400) by using Debye-Schererrer’s equation, which is given below.)

D = 0.94 λ / β c o s θ (1)

Figure 2:A. XRD patterns of Fe₃O₄ (a) and Pd- Fe₃O₄ (b) nanoparticles. B. FT-IR spectrum of Fe₃O₄ nanoparticles.

Where, λ- wavelength of X-ray, β- full width at half maximum,θ- Bragg’s diffraction angle. The mean particle diameter of Fe₃O₄ nanoparticles was found to be 10 nm. These results depicts that the Fe₃O₄ nanoparticles can be rapidly synthesized with 5-10 minutes. Usually most of the Fe₃O₄ nanoparticles synthetic methods need more than an hour [36].

Formation of Fe₃O₄ nanoparticles by microwave irradiation can be explained as follows

{Fe}^{2 +} + {2NH}_{3} + H_{2} O \to Fe {(OH)}_{2 +} {2NH}_{4}^{+} (2)

When ammonia is added to the FeSO₄ solution Fe(OH)2 is formed according to the equation 2, which is oxidized to Fe₃O₄ nanoparticles und the influence of microwave radiation as follows

3Fe {(OH)}_{2 +} 1 / {2O}_{2} \to_{Radiations}^{Microwave} {Fe}_{3} O_{4} + {3H}_{2} O (3)

Similarly figure 2b shows XRD patterns of the Fe₃O₄ nanoparticles decorated on Pd. The addition diffraction peaks at 40.07°, 46.54° and 68.09° corresponding to the (111), (2000) and (220) lattice planes were attributed to formation of Pd nanoparticles [37,38]. The mean particle diameter of Pd-Fe₃O₄ nanoparticles was found to 90 nm according to equation 1. The possible mechanism for the formation of Pd-Fe₃O₄ shown as below,

{2 Fe}^{2 +} + {[Pd {({NH}_{3})}_{4}]}^{+} + {2OH}^{-} + {4H}_{2} O \to 2 Fe {(OH)}_{3} + Pd +_{} {4NH}_{4}^{+} (4)

{Fe}^{2 +} + {2NH}_{3} + H_{2} O \to_{Radiations}^{Microwave} Fe {(OH)}_{2 +} {2NH}_{4}^{+} (5)

2 Fe {(OH)}_{3} + Fe {(OH)}_{2} + Pd \to Pd - {Fe}_{3} O_{4} + {4H}_{2} O (6)

FT-IR data of Fe₃O₄ nanoparticles is shown in figure 2B. It is noteworthy that in figure 2B, peak at 545 cm-1 is attributed to the Fe-O bond vibration of Fe₃O₄ [39]. The broad peak at 3346 cm-1 is due to stretching vibrations of –OH bond, which is absorbed by Fe₃O₄ nano particles. Also, the peak at ∼1610 cm-1 may be assigned to the deformation vibrations of water molecules trapped onto the magnetic nanoparticles [40]. These results confirm the formation of Fe₃O₄ nanoparticles. There was no major change in the FT-IR spectrum of Pd-Fe₃O₄ (results not shown).

Figure 3:(a) TEM (b) HRTEM images of Fe₃O₄ nanoparticles. B. (a) TEM (b) HRTEM Images of Pd-Fe₃O₄ nanoparticles. C. (a) Bright field TEM image Pd-Fe₃O₄ nanoparticles, the corresponding EDX maps depict the distribution of constituting individual elements within the structure as shown in (b-d). The images correspond to the (b) Fe, (c) Pd, and (d) O.

Figure 3A, 3B shows TEM images of Fe₃O₄ and Pd- Fe₃O₄ nanoparticles respectively. Both Fe₃O₄ and Pd- Fe₃O₄ nanoparticles appeared to be almost spherical in shape. TEM image of figure 3B (a) clearly depicts the presence of slightly bigger Pd nanoparticles (dark contrast) surrounded by Fe₃O₄ nanoparticles, which is evidenced in the difference in contrast. The average diameter of Fe₃O₄ nanoparticles was found to be 12.8 nm, whereas, the average particle size of Pd- Fe₃O₄ nanoparticles was found to be 92.4 nm. These results are in well agreement with XRD results shown in figure 3A. The high resolution TEM (HRTEM) images of Fe₃O₄ and Pd-Fe₃O₄ nanoparticles are shown in figure 3A (b) & 3B (b), respectively. The distance between two lattice planes of the Fe₃O₄ crystallite was 0.252 nm, which corresponds to the (311) plane of spinal Fe₃O₄. In the same way, HRTEM image of Pd- Fe₃O₄ nanoparticle showed well resolved lattice fringes with a distance of 0.225 nm, corresponding to the (111) plane of cubic Pd. Furthermore, the presence of Pd nanoparticles in Fe₃O₄ was also confirmed using scanning transmission electron microscope (STEM) coupled with elemental mapping analysis (STEM-EDS). Figure 3C (a) shows a bright field TEM image of Pd-Fe₃O₄ nanoparticles. The corresponding elemental maps are given in figure 3C (b-d). The EDS mapping results suggest that metallic Pd core is surrounded by several Fe₃O₄ nanoparticles.

Figure 4:SEM images of unmodified SPE (a), (PDDA/COO^- _-MWCNTs)/ SPE (b), (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE(c)andChOx-(Pd-Fe₃O₄/ PDDA/COO^- _-MWCNTs)/SPE (d).

Figure 4 displays the SEM images of unmodified SPE (a), (PDDA/COO^- _-MWCNTs)/SPE (b), (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE (c) and ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/ SPE (d). The morphology of the unmodified SPE exhibits rough surface having different size grains of several microns. PDDA coated MWCNTs composite is uniformly deposited on SPE, which can be seen in image (b). Nano sized Pd-Fe₃O₄ particles appear as small white grains which are incorporated into (PDDA/COO^- _-MWCNTs) composite clearly visible in image (c). Image (d) shows uniform immobilization of ChOx enzyme on (Pd-Fe₃O₄/ PDDA/COO^- _-MWCNTs) composite. Inset shows the different size granules of ChOx enzyme.

Characterization of ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE using CV and EIS

Fe(CN)6 ^3-/4- redox couple is widely used as an electrochemical probe to characterize the property of unmodified/modified electrodes. Figure 5

Figure 5:Cyclic voltammograms of unmodified SPE (a), Pd- Fe₃O₄/ PDDA/COO^- _-MWCNTs)/SPE (b) and ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/ SPE (c) in 0.1 M PBS containing 5 mM Fe(CN)64−/3− (pH 7.0); scan rate: 50 mVs-1.

Illustrates, the cyclic volt ammogramms of SPE, (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE and ChOx-(Pd-Fe₃O₄/PDDA/ COO- -MWCNTs)/SPE in 5 mM Fe(CN)6^3-/4- containing PBS (pH 7.0) at scan rate of 50 mVs-1. Irreversible voltammogram was observed at SPE (curve a, dotted line) with minimal cathodic peak current (Ipc) and anodic peak current (Ipa). The cathodic peak potential (Epc) and anodic peak potential (Epa) were found at 521 mV and -256 mV respectively with peak to peak separation (ΔEp) 777 mV. However, SPE modified with (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs) composite, a well redox peak of Fe(CN)6^3-/4- with the ΔEp 48 mV (curve b) was obtained. These results depict that the over potential decreased by 729 mV and around 2 fold of increase in current was observed for 5 mM Fe(CN)6^3-/4- at (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE than that of SPE. This shows that Electrocatalytic activity of (Pd- Fe₃O₄/ PDDA/COO^- _-MWCNTs) composite. After immobilization of ChOx enzyme on (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE, decreased in peak current was observed (curve c). This could be attributed to macromolecular non conducting enzymatic structure, impede electrochemical redox reaction of Fe(CN)6^3-/4 -at electrode surface. This demonstrates that ChOx enzyme successfully immobilized on (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs) composite by means of electrostatic attraction.

Elctrochemical Impedance Spectroscopy (EIS) is a powerful and sensitive characterization tool for studying the charge transfer process at electrode/electrolyte interface [41]. Hence, Characterization of SPE, (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE and ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE was further investigated using EIS. EIS was carried out in the presence of 5 mM Fe (CN)64−/3−as a electrochemical redox probe, in the frequency range of 100 kHz to 0.1 Hz with amplitude of 5 mV as shown in figure 6A The equivalent circuit shown in the inset of figure 6B was used to fit experimental data. The simulated curve of experimental data and best fitting equivalent circuit are shown in the figure 6C. The obtained impedance data are shown in Table 1.

The circuit includes the solution resistance (Rs), charge transfer resistance (Rct), double layer capacitance (Qdl), Warburg impedance (Zw), Faradaic resistance, (Rf) and Faradaic capacitance (Cf). At SPE, big semicircle having a Rct of 5714 Ω was observed for Fe(CN)64−/3− (curve a), which suggests that unmodified SPE exhibits sluggish and unfavorable Fe(CN)64−/3− electron transfer. However, SPE modified with (Pd- Fe₃O₄/

Table 1: EIS data of unmodified SPE, (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE and ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE in 5 mM Fe(CN)6 ^3-/4

Electrode	Rs /Ω	n	Q/µF	Rct /Ω	W /Ω
SPE	189	0.95	0. 49	5714	0.0009
Pd- Fe₃O₄/PDDA/ COO--MWCNTs)/SPE	198	0.97	0. 23	0.19	0.006
ChOx-(Pd- Fe₃O₄/PDDA/ COO--MWCNTs)/SPE	196	0.78	14.5	557	0.0016

Figure 6:Nyquist impedance plots of unmodified SPE (a), Pd- Fe₃O₄/ PDDA/COO^- _-MWCNTs)/SPE (b) and ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/ SPE (c) in 0.1 MPBS containing 5 mM Fe(CN)64−/3− (pH 7.0). The frequency range is from 100 kHz to 0.1 Hz and amplitude 5 mV. B. The equivalent circuit used to fit experimental data. (C) Nyquist impedance plots of experimental data of ChOx-(Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/ SPE and best fitting by using equivalent.

PDDA/COO- -MWC) composite, big semicircle was replaced with straight line, which have a Rct of 0.19 Ω as shown in figure 5A inset (curve b). This shows (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs) composite facilitates fast and favorable electron transfer towards electrode surface. Small semicircle was observed (curve c) with a Rct of 557 Ω, when ChOx enzyme immobilized on (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE, which illustrates that non conducting macromolecular ChOx was successfully immobilized modified SPE and it oppose the electron transfer towards electrode surface.

Direct electrochemistry of ChOx on (Pd- Fe₃O₄/PDDA/ COO- -MWCNTs)/SPE

Figure 7 shows, cyclic voltammogram of ChOx on unmodified SPE (curve a) and (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/ SPE (curse b). When ChOx immobilized on unmodified SPE, which does not facilitate DET. This illustrates that unmodified SPE does not provide micro environment for the immobilization of ChOx. However, ChOx immobilized on (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE exhibits a pair of well defined redox peaks at -0.365 and -0.443, these peaks are assigned for FAD/FADH2, which could be ascribed to electron transfer between ChOx and under laying electrode [19,22,27]. The potential difference between the two peaks ΔEp was 78

Figure 7: Cyclic voltammograms of ChOx-unmodified SPE (a) and ChOx- (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE in 0.1M PBS (pH 7.0) scan rate: 50mVs-1.

mV at a scan rate of 50 mVs-1, which suggests that ChOx has undergone a quasi-reversible redox reaction (Pd-Fe₃O₄/PDDA/ COO- -MWCNTs)/SPE. The surface area (τ) of ChOx-(Pd-Fe₃O₄/ PDDA/COO^- _-MWCNTs)/SPE was calculated using the following equation.

τ = \frac{Q}{n F A}

Where, Q is the charge, n is the number of electrons transferred, F is the Faraday constant. Therefore (τ) was found to be 5.38 × 10-9 mol cm^-2.

Effect of scan rate and pH at ChOx-(Pd-Fe₃O₄/PDDA/ COO- -MWCNTs)/SPE

To determine the kinetics of electrode reactions, the effect of scan rate on the voltammetric response of ChOx-(Pd-Fe₃O₄/ PDDA/COO^- _-MWCNTs)/SPE in a 0.1 M PBS of pH 7 was studied in the range of 5-75 mVs-1 as shown in figure 8A. The linear regression equations are as given below

Ipa = -6.6628E-7 + 5.4642E-7 ν (Vs-1); R = 0.9981 (8)

Ipc = -1.4345E-5 - 6.3799E- 7 ν (Vs-1); R = 0.9932 (9)

The redox peak current of ChOx increased linearly with increasing scan rate (Figure 8B) and the peak to peak separation also increased, indicating that surface controlled quasi-reversible process is involved.

In addition, the anodic peak potential shifted to a more potential value with increasing scan rate, where as the cathodic peak potential shifted in a negative direction. The pH of the electrolyte solution has a significant influence on the redox reaction of FAD/FADH2 of ChOx with respect to peak current and peak potential. Figure 9A Shows cyclic voltammograms of effect of pH of electrolyte in the range 4 to 8 studied on the response of ChOx-(Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE. The electrochemical response of enzyme immobilized on the electrode surface is due to redox reaction of its active site, i.e. FAD/FADH2. Where, FAD is known to undergo redox reaction involving two electrons with two protons to form FADH2.The redox peak current of ChOx increased linearly with increasing scan rate (Figure 8B) and the peak to peak separation also increased, indicating that surface controlled quasi-reversible process is involved.

Figure 8: Cyclic voltammograms of ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/ SPE in PBS (pH 7.0) at different scan rates: (5-75 mV-1). B. The plot of peak current vs. scan rate.

Figure 9: Cyclic voltammograms of ChOx-(Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs)/ SPE at various pHs of the solution (pH 4–8) scan rate: 50mVs-1. (B) Plots of potential vs. E1/2 at pH (4–8).

Due to protons involve in the reaction, the acidity of the solution has a significant effect on the redox potential of ChOx. Thus, the anodic and cathodic peak potentials of ChOx immobilized on the (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE should be pH dependent. It was observed that, redox peak potential of the enzyme shifted towards negative with increase in pH as shown in figure 8B indicating that protons are involved in the redox reaction. A good linear relationship was obtained between half wave potential (E1/2) and the solution pH. The corresponding linear regression equation is given as

E1/2 = -0.025 - 0.058 pH; R = 0.9908 (10)

From the above equation, slope of E1/2 is 58 mV, which is close to the theoretical value (59 mV pH-1) for a classical Nernstian two electrons and protons process. Hence, ChOx redox system is a two proton participated two electron redox processes.

Determination of cholesterol based on the direct electrochemistry of ChOx on the (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE

In the this protocol, the direct electrochemistry of ChOx is based on the redox reaction of its active center, i.e. FAD, in the absence of oxygen, direct electron transfer of immobilized ChOx can be expressed as follows

ChOx - FAD + {2H}^{+} + {2e}^{-} \leftrightarrow ChOx - {FADH}_{2} (11)

In the presence of oxygen, the reduced enzyme is oxidized very quickly at the electrode surface. Electron transfer turnover rate of the molecular oxygen is about 700 s-1 to accept electrons [42]. This is much faster than that of ChOx on the (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE. As a result, obvious electrostatic process towards the reduction of dissolved oxygen, which is given below

ChOx - {FADH}_{2} + O_{2} \to ChOx - FAD + H_{2} O_{2} (12)

The catalytic regeneration of the enzyme in its oxidized form causes the loss of reversibility as a result increase in the size of the reduction peak as shown in figure 10 (curve b) [24]. By the addition of cholesterol, a competitive reaction take place at the vicinity of the enzyme modified electrode surface. Thus, leading to the decrease of reduction peak current (curve c), as a result the sensitive determination of cholesterol. In other words, in the presence of oxygen, ChOx on modified electrode will catalyze the oxidation of cholesterol according to the following enzymatic reaction.

ChOx - FAD + Cholesterol \to ChOx - {FADH}_{2} + Choles - 4 - en - 3 - one (13)

Figure 10:Cyclic voltammograms obtained at ChOx-(Pd- Fe₃O₄/PDDA/ COO—MWCNTs)/SPE in nitrogen saturated and oxygen saturated PBS (a and b), after addition of 50 μM cholesterol to oxygen saturated PBS (c). Scan rate: 50mVs-1.

By the addition of cholesterol, a competitive reaction take place at the vicinity of the enzyme modified electrode surface. Thus, leading to the decrease of reduction peak current (curve c), as a result the sensitive determination of cholesterol. In other words, in the presence of oxygen, ChOx on modified electrode will catalyze the oxidation of cholesterol according to the following enzymatic reaction.

The reduction peak of the ChOx-(Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE in oxygen saturated PBS (pH 7. 0), decreased with addition of cholesterol, which suggested that the immobilized ChOx still retained its enzymatic activity. This could be due to the biocompatible, microenvironment provided by (Pd-Fe₃O₄/PDDA/ COO- -MWCNTs) composite. Thus, the addition of cholesterol restrains Electrocatalytic reaction between the oxidized form of ChOx i.e. ChOx-FAD and cholesterol, which attenuates the concentration of the ChOx-FAD. This causes decrease in the reduction peak current of the enzyme [27]. Also, the dissolved oxygen mediates the enzymatic oxidation of cholesterol by ChOx. Therefore, the depletion of the oxygen proximal to the electrode surface makes the reduction of the oxidized form of ChOx less favorable, leading to the decrease of the reduction peak current of the enzyme [43].

Eventually, cholesterol is determined by measuring the decreased reduction peak current, by the addition of cholesterol in oxygen saturated PBS (pH 7.0).

Figure 11A shows the differential pulse voltammograms of various cholesterol concentrations at ChOx-(Pd-Fe₃O₄/ PDDA/COO^- _-MWCNTs)/SPE in oxygen saturated PBS (pH 7. 0). The reduction current decreased gradually upon increasing cholesterol concentration.

Figure 11: Differential pulse voltammetric measurements at ChOx- (Pd- Fe₃O₄/PDDA/COO-MWCNTs)/SPE at oxygen saturated PBS(pH 7.0), without cholesterol (a) and (b–j) with cholesterol of 10, 20, 30, 40, 50, 60, 70, 80 and 90 μM. DPV parameters; scan rate: 20 mV s−1, pulse height: 200 mV, pulse width: 0.05 s, step height: 10 mV and step width:0.5 s. B. shows relationship between Ipc and concentrations of cholesterol.

Figure 11B shows the calibration current corresponding to decrease of reduction current and concentration of cholesterol. The linear regression equation is given by

Ipc (Cholesterol) (μA) = -4.9712E-4 + 7.4202E-7 C (Cholesterol) (μA); R = -0.9972 ..................... (14)

Using slope of the above equation, the sensitivity of the enzyme modified SPE was calculated to be 0.742 μA μM-1 or 10.45 μA μM-1 cm^-2 (area of electrode surface is 0.071 cm2). Comparison of the enzyme modified SPE with other cholesterol determination based on SPEs are given in Table 2 [44-46].

Results shown in table 2, depicts that the sensitivity of the ChOx-(Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE much better when compare to other determinationofcholesterol based on SPEs. Furthermore, applied potential and detection limit of the ChOx-(Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE is quiet comparable with respect to other determination of cholesterol based on SPEs.

Table 2: Comparison of the ChOx enzyme SPE with other ChOx basedSPEs

Cholesterol Biosensor	Sensitivity μA μMˉ1	Potential applied (mV)	Linear range (µM)	Detection limit (µM)	Reference
GNS-nPt/SPE	-	400	0-35	0.2	[45]
SP-rhodium-graphite- -Au-P450scc	0.13	-400	10-70	-	[46]
SP-RP450scc	0.0138	-600	50-300	-	[47]
ChOx-(Pd-Fe₃O₄/PDDA /COO--MWCNTs)/SPE	0.742	-380	10-80	1	Present work

Stability and reproducibility of the ChOx-(Pd-Fe₃O₄/ PDDA/COO^- _-MWCNTs)/SPE

Direct electron transfer of ChOx on (Pd-Fe₃O₄/PDDA/ COO- -MWCNTs)/SPE is very stable. When twenty consecutive CV curves obtained at ChOx-(Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE in 0.1 M PBS pH 7.0 at scan rate of 50mVs-1, there was no change in the peak to peak separation. However, peak current gradually decreased. The electrode retained 86.6% of its initial response after twenty consecutive cycles. These results shows that ChOx binds strongly on (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs) composite. To ascertain fabrication reproducibility, five sets of ChOx on (Pd-Fe₃O₄/PDDA/COO^- _-MWCNTs)/SPE were fabricated for the determination of cholesterol. The results show that the enzyme modified SPE had satisfying reproducibility with the Relative Standard Deviation (RSD) of 9.5%.

Conclusion

Fe₃O₄ and Pd-Fe₃O₄nanoparticles were synthesized by simple and facile microwave method. Formation of Fe₃O₄and Pd- Fe₃O₄ nanoparticles were confirmed from powder X-ray diffraction and FT-IR techniques. Pd- Fe₃O₄ nanoparticles used for the preparation of biocompatible composite which consists of negatively charged mutliwalled carbon nanotubes (COO- -MWCNTs) wrapped with positively charged poly diallyldimethyl ammonium chloride. This composite was successfully used for the determination of cholesterol by using cholesterol oxidase enzyme on screen printed electrode. DET of ChOx was observed on (Pd- Fe₃O₄/PDDA/COO^- _-MWCNTs) composite which shows that the composite provides biocompatiable microenvironment for the ChOx. The linear range of the enzyme modified SPE was found to be 10-80 μM (R=9972) with detection limit of 1 μM. Common interferents such as ascorbic acid, uric acid and glucose did not cause any interference because of low operating potential.

Acknowledgements

The authors gratefully acknowledge the financial support from Vision Group on Science and Technology, Government of Karnataka. R. Manjunatha thanks Council of Scientific and Industrial Research, New Delhi for the award of Senior Research Fellowship. We thank Sri. A. V. S. Murthy, honorary secretary, Rashtreeya Sikshana Samiti Trust, Bangalore for his continuous support and encouragement.

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