Nafion (Sigma), α-Fe₂O₃ (Merck), Cholesterol (Sigma), Phosphate buffer solution (prepared using monosodium and disodium phosphate salts, in the lab), ferri and ferrocyanide (Sigma-Aldrich), Indium Tin Oxide (ITO) coated glass substrates, Cholesterol oxidase (Sigma), Alcohol, acetone and de-ionized water.

A composite film of Nafion/aα-Fe₂O₃ films deposited on ITO glass substrates were characterized by D8 X-ray diffractometer (with x-ray source of CuKα at 1.54 Å wavelength) from Bruker. The functional group study was carried out using Fourier transform infrared spectroscopy (FTIR) on Bruker model Tensor 37 FTIR spectrometer. The morphology of the composite films was observed using 25 keV electron beam in field emission scanning electron microscope (FESEM) from MIRA II LMH (TESCAN). The films were further characterized with Nanoscope IIIa Atomic Force Microscope (AFM) for the topological studies (Sulania et al., 2018). The contact angle measurements with water drop were performed using DSA3 contact angle measurement set-up from KRUSS. In bulk materials and polymers, the dynamics of the water droplet mostly hinge on the liquid-surface interaction. The curvature of the droplet varies when the water droplet falls on the surface of the thin film showing the interaction between them (Bico et al., 2002). A 3-electrode electro-chemical cell assembly was used for the Electrochemical studies with Pt wire as the counter electrode (CE), nafion-α-Fe₂O₃/ITO as the working electrode (WE) and Ag/AgCl as the reference electrode (RE) dipped into the solution of PBS (0.1M PBS, pH 7.4) comprising 5 mM [Fe(CN)₆]^3−/4− and the redox probe allied to CHI-600D electrochemical analyzer. Further, the Cyclic Voltammetry studies were performed to observe the redox behavior of the nafion-α-Fe₂O₃/ITO electrodes. The sensing measurements were performed on nafion-α-Fe₂O₃/ITO and nafion-α-Fe₂O₃-CHOx/ITO electrodes with change in concentration of the cholesterol using electrochemical differential pulse voltammetry method.

The surface morphology of α-Fe₂O₃-nafion nanocomposite films formed on ITO was observed using FESEM and AFM. SEM analysis confirms the rod type structure of the α-Fe₂O₃ nanoparticles and the images are shown in Figures 2a,b. A thin layer covering the rod type structures is observed at higher magnification image in Figure 2b showing nafion was surrounding the nanoparticles in the composite. The diameter of the rods is <50 nm including the nafion coating. The EDX analysis carried out on α-Fe₂O₃-nafion nanocomposite films during FESEM study showed the presence of the elements Fe, O, S, F, and C as shown in the Figure 2c, which is clearly indicating the formation of the nanocomposite.

Figure 3 shows the 2D, 3D AFM images of α-Fe₂O₃-nafion nanocomposite film and the sectional analysis of the single rod. These AFM images clearly indicate the rod like structures that were observed in SEM images also. The breadth of a rod has been calculated using section analysis. The horizontal diameter of the rods is found to be 33 nm which is in agreement with the diameter of the rods <50 nm that was observed in SEM images.

The composite films deposited on glass substrates were used for XRD measurements. XRD spectrum was carried out in the range of 2θ from 10° to 50° using Cu-Kα source with wavelength 1.54Å with scan rate 1° per sec and the spectrum was shown in Figure 4A. The peaks visible at 2θ ≈ 21.8°(111), 27.5°(012), 30.1°(220), 35°(104), 43°(113), confirms the formation of α-Fe₂O₃ phase in the composite film (Banerjee et al., 2011; Mallick and Dash, 2013).

From the Scherrer's formula the crystallite size can be estimated as:

where K = 0.98, D is the crystallite size, b is the FWHM of the highest intense peak and l is the incident x-ray wavelength. The crystallite size was obtained as 33.8 nm for the highest intense peak corresponding to (220).

Figure 4B shows the FTIR spectra of nafion-α-Fe₂O₃/ITO (a) and nafion-α-Fe₂O₃/CHOx/ ITO (b) electrodes. The peaks at 440 and 520 cm⁻¹ were attributed to Fe-O bond vibrations. The peaks at 985, 1,065, 1,155, 1,227, 1,285 cm⁻¹ correspond to S-O, C-O-C, C-F, and SO₃H in sulfonic acids, C-F stretch bands respectively of nafion. The peak at 1,650 cm⁻¹ corresponding to C=O was observed in the enzyme electrode spectrum (b). The major changes observed in the peaks at 1,065 cm⁻¹ after the enzyme incorporation and appearance of peak at 1,650 cm⁻¹ confirming the successful immobilization (Chen et al., 2013; Singh et al., 2016). The functional groups on nafion helped in the immobilization of CHOx on the electrode.

It is well-known that nafion is a super hydrophobic polymer and therefore not useful for sensing. Whereas, when nafion is used in composite form, the properties such as hydrophobicity, photon conductivity, water swelling properties etc. of the composite films found much more different than their individual components (Paul and Karan, 2014; Paul et al., 2016; Kurihara et al., 2017; Singh et al., 2019). The composite films were formed on the ITO coated glass plates and CHOx enzyme was immobilized on one electrode. The contact angle of the composite films before (a) and after enzyme immobilization (b) of CHOx on the nanocomposite films are shown in Figure 5.

It was found that the contact angle was 98.64° ± 2.7° of the α-Fe₂O₃-nafion-nanocomposite films showing slightly hydrophobic nature due to the presence of nafion. Upon the immobilization of the enzyme CHOx, the contact angle was drastically reduced to 8.05° ± 1.46°. So, the films have become completely hydrophilic in nature, which is the foremost requirement for the biosensing applications. The contact angle changed due to the incorporation of nanoparticles that were present in the nanocomposite that modified the surface as of only nafion. Further, enzyme immobilization leaded to increase in water surface interaction which contributed to the decrease in CA (Singh et al., 2019).

The electrochemical properties of nafion-α-Fe₂O₃/ITO and nafion-α-Fe₂O₃/CHOx/ITO electrodes were studied by CV technique in a three-electrode cell of 7.4 pH PBS buffer solution comprising 5 mM [Fe(CN)₆]^3−/4− used as the redox species. The CV curves of nafion-α-Fe₂O₃/ITO and nafion-α-Fe₂O₃/CHOx/ITO obtained at various scan rates shown in Figures 6A,B, respectively.

Figure 6 shows the good oxidation and reduction peaks for both the nanocomposite films. The increase in current with the increase in the scan rate shows the favorable redox nature of the electrodes for electrochemical sensing applications. The anodic and cathodic peaks currents and the area of the CV curve were found to increase with scan rate. This increase implies the homogeneous electron transfer of the electrodes. The plot between scan rate^½ and oxidation and reduction peak currents and voltages were showed in the inset and the linearity was observed. The linearity of these curves indicates the facile charge transfer kinetics and the charge transfer process was diffusion controlled (Xiong et al., 2012) and these electrodes provide adequate conductivity for charge flow among electrode and electrolyte.

The different electrochemical parameters as Ea, Ia, Ec, Ic, and the diffusion co-efficient (D) of the Nafion-α-Fe₂O₃/ITO and Nafion-α-Fe₂O₃/CHOx/ITO electrodes found at the scan rate, 50 mV/s. The D value was assessed from the equation,

where C; the concentration of mediator buffer, A; the area of the working electrode, n; the number of electrons participated in redox process and v; the scan rate.

The surface concentration (γ^*) of the ionic species of the Nafion-α-Fe₂O₃/ITO and Nafion-α-Fe₂O₃/CHOx/ ITO electrodes were assessed by Brown-Anson model (Bard and Faulkner, 2001) using the equation given by

The values of the heterogeneous electron transfer rate constant (k_s) was found for bioelectrodes using Laviron's equation (Singh et al., 2013).

Where m is ΔE = E_pa–E_pc, n is the electrons participated in charge transfer, F; the Faraday constant, v; scan rate, R; the gas constant and T; room temperature.

The surface concentration of the redox species of electrodes was calculated using Brown–Anson model (Bard and Faulkner, 2001).

Where the slope of the scan rate vs. I_pc is I_p/V and the surface concentration of the electrode is I^*. All the electrochemical parameters are tabulated in Table 1.

For cholesterol response measurements, nafion-α-Fe₂O₃/ITO and nafion-α-Fe₂O₃/CHOx/ ITO electrodes were positioned in the buffer and various concentrations of cholesterol solutions from 4 to 770 mg dl⁻¹ were added and DPV for each concentration was recorded. There is an increase in the peak current of Nafion-Fe₂O₃/ITO electrodes with increased concentrations was observed as shown in Figure 7A. The linear calibration plot of peak current with concentration was plotted and shown in inset of the Figure 7A.

The electrodes are suitable for sensing of cholesterol without any enzyme immobilization with sensitivity of 0.012 μA (mg/dl)⁻¹ cm⁻². But, the crucial limitation of the enzyme less biosensor is the selectivity or specificity. The electrode was verified for selectivity with common interfering agents such as Ascorbic Acid (AA), Urea (U), 4-Acetymanophenol (4-AP), Citric Acid (CA), and glucose (Glu) at 4 mM concentrations and the results are shown in Figure 8A. Unfortunately, the Naf-Fe₂O₃/ITO electrode did not show much change in the peak currents as compared to cholesterol at 4 mM concentration implies the enzyme less sensing is not possible with real samples. Therefore, the enzyme cholesterol oxidase was immobilized on the electrode's surface by casting method through adsorption, dried, washed and dried again. The enzyme was immobilized without any cross-linking agent. The DPV response of different cholesterol concentrations on enzyme electrodes was tested. The DPV curves with different concentrations from a very low concentration from about 4–770 mg dl⁻¹ was carried out as given in Figure 7B. The peak currents vs. the concentration was plotted against and the obtained linearity plot was shown in the inset. The electrode sensitivity of was estimated as 64.93 × 10⁻² μA (mg/dl)⁻¹ cm⁻². The enzyme-substrate interaction (Michaelis–Menten) constant K_m, was obtained to be 19 mg/dl. The lower detection limit (LOD) is obtained as 2 mg dl⁻¹. The specificity or the selectivity was verified with the general interfering agents such as U, CA, 4-AP, AA, and glu at 4 mM concentrations, and the DPV peak currents of each of these analytes were less than the peak current of 4 mM cholesterol (Figure 8B) which confirmed the specificity of the developed electrode.

The synthesized nanocomposite was found suitable for the cholesterol sensing without the help of enzyme but without selectivity. Therefore, the enzymatic electrode was fabricated. The current values were found to increase after the enzyme immobilization with cholesterol concentration by showing the increase in the DPV peak current. The immobilization of CHOx on magnetic nanoparticles showed the improved biochemical activity and the immobilized CHOx proved to be the effective biocatalyst to catabolize cholesterol (Ghosh et al., 2018). The increased stability and activity of CHOx leading to the suitable application in biosensors (Ghosh et al., 2018). The enzyme mediated electrochemical reaction took place at the electrode surface (Nakaminami et al., 1997) causes the change in the current with the addition of cholesterol. The catabolic reaction of CHOx combined with the electrochemical oxidation of mediator at the surface of the electrode produces reduced CHOx [CHOx (red)] and cholestenon when cholesterol was added in the first step. When CHOx (red) was combined with the mediator (M) (ferri/ferro) at oxidation potential it produces CHOx (ox) and M_red in the second step. In the third step, M_red becomes again M_ox by releasing electrons. The first and second steps utilize O₂/H₂O₂ which is available in the buffer solution and forms an intermediate in the reaction. The H₂O₂ produced during second step gets electrochemically oxidize by giving the anodic current, which is the final measure of the cholesterol concentration. The schematic representation of the developed biosensing platform is given in Figure 9. The enzyme electrodes were found stable for almost 8 weeks when stored in the refrigerator, and after that the peak current started decreasing as shown in Figure 10.