Preparation of Biocompatible Palladium-Fe 3 O 4 Nanoparticles / Multiwalled Carbon Nanotubes Composite and its Electrocatalytic Activity towards Determination of Cholesterol on Screen Printed Electrode

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.


Introduction Preparation of Biocompatible Palladium-Fe 3 O 4 Nanoparticles/Multiwalled Carbon Nanotubes Composite and its Electrocatalytic Activity towards Determination of Cholesterol on Screen Printed Electrode
The magnetic nanoparticles in general, iron oxide (Fe 3 O 4 ) 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 Fe 3 O 4 nanoparticles are good biocompatibility, strong superparamagnetic property, high surface area, low toxicity and ease of preparation [2,3].Thus, Fe 3 O 4 nanoparticles have been used in a wide range of potential applications such as, electrochemical sensors/biosensors, catalysis, immunoassays, data storage [4][5][6][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][9][10][11].The later method has significant advantage with respect to higher reaction rates and product yields in a shorter period of time.

Synthesis of Fe 3 O 4 and Pd-Fe 3 O 4 nanoparticles
As stated earlier, Fe 3 O 4 and Pd-Fe 3 O 4 nanoparticles were prepared according to the procedure described elsewhere [29].In brief, 2.0 mm FeSO 4 .7H 2 O 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 Fe 3 O 4 nanoparticles were separated by centrifugation method.Finally Fe 3 O 4 nanoparticles washed thoroughly with water followed by ethanol and dried at 50°C in vacuum oven.
Similarly, Pd-Fe 3 O 4 nanoparticles were prepared by coprecipitation method.0.5 mm Pd (NH 3 )4Cl 2 •H 2 O, and 2.0 mmol FeSO 4 •7H 2 O 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 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][26][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 Fe 3 O 4 and Pd-Fe 3 O 4 nanoparticles by following the procedure given in the research article with slight modifications [29]

Aplastic anemia in systemic lupus erythematosus: A better prognosis acquired aplastic anemia
Copyright: © 2016 Pannu and Varma 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 = 1.5418Ǻ).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-Fe 3 O 4 nanoparticles were added and stirred for 12 h at room temperature.In this stage negatively charged Pd-Fe 3 O 4 nanoparticles coated on positively charged MWCNTs wrapped with PDDA as shown in figure 1.

Characterization of
Where, λ-wavelength of X-ray, β-full width at half maximum,θ-Bragg's diffraction angle.The mean particle diameter of Fe 3 O 4 nanoparticles was found to be 10 nm.These results depicts that the Fe 3 O 4 nanoparticles can be rapidly synthesized with 5-10 minutes.Usually most of the Fe 3 O 4 nanoparticles synthetic methods need more than an hour [36].It is noteworthy that in figure 2B, peak at 545 cm -1 is attributed to the Fe-O bond vibration of Fe 3 O 4 [39].The broad peak at 3346 cm -1 is due to stretching vibrations of -OH bond, which is absorbed by Fe 3 O 4 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 Fe 3 O 4 nanoparticles.There was no major change in the FT-IR spectrum of Pd-Fe 3 O 4 (results not shown).  3/4-redox couple is widely used as an electrochemical probe to characterize the property of unmodified/modified electrodes.( ) ( )

Aplastic anemia in systemic lupus erythematosus: A better prognosis acquired aplastic anemia
Copyright 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-Fe 3 O 4 /PDDA/COO --MWCNTs)/SPE and ChOx-(Pd-Fe 3 O 4 /PDDA/COO --MWCNTs)/SPE was further investigated using EIS.EIS was carried out in the presence of 5 mM Fe (CN)6 4−/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.
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
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/FADH 2 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-Fe 3 O 4 /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/FADH 2 .Where, FAD is known to undergo redox reaction involving two electrons with two protons to form FADH 2.
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.
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-Fe 3 O 4 /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 From the above equation, slope of E 1/2 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-Fe
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.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

Aplastic anemia in systemic lupus erythematosus: A better prognosis acquired aplastic anemia
Copyright: © 2016 Pannu and Varma accept electrons [42].This is much faster than that of ChOx on 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.The reduction peak of the ChOx-(Pd-Fe 3 O 4 /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-Fe 3 O 4 /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).
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 cm 2 ).Comparison of the enzyme modified SPE with other cholesterol determination based on SPEs are given in Table 2 [44][45][46].
Results shown in table 2, depicts that the sensitivity of the ChOx-(Pd-Fe 3 O 4 /PDDA/COO --MWCNTs)/SPE much better when compare to other determinationofcholesterol based on SPEs.Furthermore, applied potential and detection limit of the ChOx-(Pd-Fe 3 O 4 /PDDA/COO --MWCNTs)/SPE is quiet comparable with respect to other determination of cholesterol based on SPEs.

Conclusion
Fe3O4 and Pd-Fe 3 O 4 nanoparticles were synthesized by simple and facile microwave method.Formation of Fe 3 O 4 and Pd-Fe 3 O 4 nanoparticles were confirmed from powder X-ray diffraction and FT-IR techniques.Pd-Fe 3 O 4 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-Fe 3 O 4 /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.
and Varma Similarly figure 2b shows XRD patterns of the Fe 3 O 4 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-Fe 3 O 4 nanoparticles was found to 90 nm according to equation 1.The possible mechanism for the formation of Pd-Fe 3 O 4 shown as below, FT-IR data of Fe 3 O 4 nanoparticles is shown in figure 2B.

Figure
Figure 3A, 3B shows TEM images of Fe 3 O 4 and Pd-Fe 3 O 4 nanoparticles respectively.Both Fe 3 O 4 and Pd-Fe 3 O 4 nanoparticles appeared to be almost spherical in shape.TEM image of figure 3B (a) clearly depicts the presence of slightly

Figure 3 :
Figure 3: (a) TEM (b) HRTEM images of Fe 3 O 4 nanoparticles.B. (a) TEM (b) HRTEM Images of Pd-Fe 3 O 4 nanoparticles.C. (a) Bright field TEM image Pd-Fe 3 O 4 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.
-Fe 3 O 4 /PDDA/COO --MWCNTs)/SPE.As a result, obvious electrostatic process towards the reduction of dissolved oxygen, which is given below