Development of Impedimetric Biosensor for Total Cholesterol Estimation Based on Polypyrrole and Platinum Nanoparticle Multi Layer Nanocomposite

A novel impedimetric biosensor was fabricated for total cholesterol sensing based on platinum nanoparticle and polypyrrole multilayer nanocomposite electrode. The Pt nanoparticles (PtNP) electrochemically deposited between two polypyrrole layers on indium tin oxide (ITO) glass plates (PtNP/PPY/ITO) have offered high-electroactive surface area and favourable microenvironment for immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) resulting in enhanced electron transfer between the enzyme system and the electrode. Impedimetric response studies of the ChEt-ChOx/PtNP/ITO nanobioelectrode exhibit improved linearity (2.5 × 10 to 6.5 × 10 M/l), low detection limit (2.5 × 10 M/l), fast response time (25 s), high sensitivity (196 Ω/mM/cm) and a low value of the Michaelis-Menten constant (Km, 0.2 M/l) with a regression coefficient of 0.997.


Introduction
Innovations in the field of electrochemical biosensors' matrix are of much importance nowadays for the development of highly sensitive, selective, reliable and low cost biosensors for the clinical diagnosis [1].The metal nanoparticles have been widely exploited due to their ability as electrode modification materials to enhance the efficiency of electrochemical biosensor [2][3][4].This can be attributed to the ability to design novel sensing systems and enhance the performance of bioanalytical assays 1 .Pt is a well-known catalyst that has a high catalytic activity for hydrogen peroxide electro oxidation [5][6][7].Pt nanoparticles (PtNP) on the electronically conducting polypyrrole can enhance conduction and charge transport properties compared to the conventionally synthesized polypyrrole film [8][9][10].PtNP possess high surface area, nontoxicity, good biocompatibility and chemical stability, and also show fast electron communication features which provide a desirable microenvironment to enzyme for the direct electron transfer between the enzyme's active sites and the electrode.The PtNP not only retain the bioactivity of the immobilized enzyme but also enhance the sensing characteristics such as sensitivity, selectivity and low detection limit of the fabricated amperometric enzymatic biosensors [11].The PtNP have the advantages of easy electrochemical synthesis and reproducible preparation, long-term stability and ability to incorporate enzyme successfully via covalent bonding by using carbodiimide chemistry i.e.N-hydroxysuccinimide (NHS) and N-ethyl-N-(3-dimethylaminopropyl carbodiimide) (EDC).Chen et al. have developed a glucose biosensor based on conducting chitosan decorated with PtNP with a response time of 5 s [12].
Human serum contains a mixture of free and esterified cholesterol, both of which are measured to determine the total cholesterol level [13].The determination of cholesterol level is of importance in clinical diagnosis [14] because of diseases such as coronary heart disease, myocardial infarction and arteriosclerosis [15,16].It is important to note that 70% of the total blood cholesterol exists in ester form and 30% in free form.But the re-search work on total cholesterol estimation is limited.Most research work till date is focused on the estimation of free cholesterol [17][18][19][20].Singh et al. have utilized conducting polymers (CP) for the total cholesterol determination [21][22][23].However, these biosensors have been found to have low sensitivity (7.5 × 10 −4 nA/mg/dl) and a high response time, like 240 s.Li et al. have fabricated screen-printed MWCNT polycarbonate electrode to investigate total cholesterol and found that the CNT-modified biosensor offers a reliable calibration profile [24].Solanki et al. have immobilized cholesterol esterase (ChEt) and ChOx via glutaraldehyde as a cross-linker onto sol-gel-derived silica/chitosan/MWCNT-based nanobiocomposite electrode deposited onto ITO for the determination of esterified cholesterol [25].This bioelectrode shows a response time of 10 s, sensitivity of 3.8 μA/mM, and shelf life of about 10 weeks for the estimation of cholesterol oleate.The gold nanowire-modified microfluidic-based amperometric total cholesterol bioelectrode has linearity of 1 to 6 mM/l [26].Arya et al. have reported self-assembled monolayer-based cholesterol sensors and found linearity of 10 to 500 mg/dl [27,28].Basu et al. have fabricated a nanocomposite electrode comprising of polypyrrole (PPY) and carboxy functionalized multiwalled carbon nanotubes (MWCNT) to estimate total cholesterol in the range of 4 × 10 −4 to 6.5 × 10 −3 M/l and they have used to determine cholesterol in blood serum samples [29].Basu et al. have also developed a reusable total cholesterol electrode based on nano structured conducting polyaniline [30].
Electrochemical impedance spectroscopy (EIS) has recently been important as a nondestructive, sensitive and efficient means for characterization of electrical properties of materials in biological interfaces [31,32].In this study, an attempt has been made to develop PtNP and PPY based electrodeposited tri-layer nanocomposite film and the same has been evaluated as a transducer matrix for total cholesterol estimation using impedance spectroscopy.The change in impedance of nanobiocomposite electrodes with the change of different concentration of cholesterol oleate is used as a measure of biosensor performance.To the best of our knowledge, electrochemical impedance spectroscopy, as a measure of total cholesterol estimation, has not been exploited till today.The novelty of this study lies on the ease of fabrication of PtNP based PPY multilayer nanocomposite electrode which provides enzyme friendly environment, non-destructive measurement technique for total cholesterol, high sensitivity, reproducibility and shelf life of the biosensor for total cholesterol estimation.

Preparation of ChEt-ChOx/PtNP/PPY/ITO Nano-Bioelectrode
The PtNP/PPY/ITO nano composite electrode was fabricated in three steps.In the first step, pyrrole was electropolymerised onto ITO coated glass plates (PPY/ITO) in a three-electrode cell containing 0. The enzymes (ChOx and ChEt (1:1)) were covalently immobilized onto the PtNP/PPY/ITO nano composite electrode using EDC as the coupling agent and NHS as the activator.For enzyme immobilization, the optimal binding was achieved when 30 µl of solution of 5 µg of enzymes (1:1) mixed with 0.4 M EDC and 0.1 M NHS was dropped on 1 cm 2 of PtNP/PPY/ITO nano composite electrode and kept in a humid chamber for 3 h.Thus, fabricated ChEt-ChOx/PtNP/PPY/ITO (Scheme 1) was then washed with phosphate buffer saline (PBS) solution (50 mM, 0.9% NaCl, pH 7.0) to remove any unbound enzyme and was stored at 4˚C when not in use. the ITO electrode, so a thin inner layer of PPY is required.[33].The peaks at 1105 cm −1 , 1055 cm −1 and 1320 cm −1 are attributed to the =C-H in plane vibration [33].The broad band at 1178 cm -1 may be assigned to N-C stretching band (Nicho and Hu, 2000).All the characteristics peaks of PPY/ITO electrode are visible in the FTIR spectrum of PtNP/PPY/ITO (Figure 3(b)).The peak at 3416 cm -1 is attributed to N-H stretch vibration.The band at 1320 cm -1 corresponds to =C-H band in plane vibration.The FTIR spectrum of ChEt-ChOx/ PtNP/PPY/ITO nanobioelectrode (Figure 3(c)) also reflects the characteristic peaks of PPY/ITO electrode [34].The ChOx and ChEt binding is indicated by the appearance of additional absorption bands at 1646 and 1560 cm -1 assigned to the carbonyl stretch (amide I band) and -N-H bending (amide II band), respectively [35].Besides that, a broadband seen around 3457 cm -1 is attributed to amide bond present in nano bioelectrode.Figures 6A and B show the CV of PtNP/PPY/ITO electrode and ChOx-ChEt/PtNP/PPY/ITO bioelectrode as a function of scan rate (20 -160 mV/s).The relationships between the peak current and corresponding potential with scan rate are shown in Table 1.It can be seen that the magnitudes of cathodic peak and anodic peak current increase with increasing scan rate (Figures 6A(a)  and B(a)).This linear dependence can be expressed by the Equations ( 1) to (4) in Table 1.Both the anodic and c the scan rate, suggesting that electro-chemical reaction is a diffusion-controlled process for both nano composite and nano bioelectrode.The peak potential also increases linearly with log of scan rate (Figures 6A(b) and B(b)) indicating a diffusion controlled process.The values of slope obtained from the plots of the peak current against the scan rate for ChOx-ChEt/PtNP/PPY/ITO nanobioelectrode are higher than the slope of the PtNP/PPY/ITO electrode indicating a favorable microenvironment for enzymes with nanobioelectrode.Table 1 (Equations ( 1)-( 8)) shows linear equations, slope, Regression Coefficient, and Standard deviation for the electrochemistry of the electrodes.The characteristics features of the nanobioelectrode as shown in Table 1 reflect that good electron transport is retained in the ChOx-ChEt/PtNP/PPY/ ITO nano bioelectrode.

Electrochemical
ChOx-ChEt/PtNP/PPY/ITO ing the last decade, Electrochemcial Impedan troscopy (EIS) has been widely used various types of biomolecular interactions based immunosensors, DNA hybridization, rapid biomolecular screening, cell culture monitoring and relevant literature has been comprehensively reviewed [31,[36][37][38].EIS is a measure of the response of an electrochemical cell subjected to a small amplitude sinusoidal voltage signal as a function of frequency [39,40].The resulting sinusoidal current expressed with respect to the perturbing (voltage) wave, and the ratio ( ) ( ) V t I t is defined as the impedance (Z).Impedance is expressed as a complex number, where the real com easures the ohmic resistances and the imaginary one is the capacitive reactance.f Nyquist format, the imaginary impedance component (Zʹ out-of-phase) is plotted against the real impedance component (Zʹʹ in-phase) at each excitation frequency, whereas in the latter format, both the logarithm of the absolute impedance, |Z| and the phase shift, are plotted against the logarithm of the excitation frequency [40] 2 .
The complex impedance can be presented as the real (Zʹ) and imaginary (Zʹʹ) components that mainly originate from the resistance and capacitance of the cell and can be calculated using Equation (9) (for parallel circuit).

(
) ( ) where, R s = Ohmic resistance of the electrolyte solution; C dl calculated by following Equation (10).
and R CT , depend on the dielectric and te interface.The semircle diameter of EIS spectra gives value of R CT that reveals charge-transfer resistance of redox probe at the electrode interface.However, R s represents bulk properties of the electrolyte solution and diffusion of applied redox probe are not affected by biochemical reaction occurring at the electrode interface 3 .The Equations ( 9) and (10) represent relationship between the total impedance and other electrochemical parameters such as R CT and C dl .The interfacial properties of the electrode in the electrolyte depend on charge transfer resistance (R CT ) and capacitance (C dl ).
The Similarly, the value of charge transfer resistance (586.8Ω, R CT ) of PtNP/PPY/ITO electrode is lower (Figure 7, curve c) than PPY/ITO electrode, revealing that PtNP increases the electroactive surface area of electrode resulting in easier electron transfer from the medium to electrode due to increased diffusion of redox species [Fe(III)/Fe(IV)] onto surface charged nanocomposite PtNP/PPY/ITO film.Interestingly, R CT value further decreases to 353.92 Ω (Figure 7, curve d) after the immobilization of ChOx-ChEt onto PtNP/PPY/ITO revealing that PtNP provides a desirable microenvironment for the immobilization of ChOx-ChEt [41].It can be assumed that ChOx-ChEt rearrange their structure and presents better conformation onto PtNP/PPY/ITO electrode.Thus ChOx-ChEt/PtNP/PPY/ITO nano bioelectrode provides easier electron transfer due to increased active sites for electrical contact between electrode and the redox label in solution.In practice, an ideal semicircle characteristics is generally not observed in impedance spectra.It is normally an inclined semicircle with its centre depressed below the real axis by a finite angle referred to as the depression angle.The depression angle (θ) by which such a semicircular is displaced below the real axes, is related to the width of the relaxation time distribution and is an important parameter.It can be seen that arc of the semicircle with least q value, that is less distorted from centre of the real axis can be calculated using Equation (11).

Depression angle
( ) where, n ponent and c the standard commercial software available with the in-hEt/PtNP/PPY/ IT is the fractional ex alculated form strument.From the above discussion, it is clear that it is necessary to find out the optimum condition at which the minimum depression angle is obtained for both type of electrodes and the cholesterol nano biosensor should be evaluated at that optimum condition.The EIS spectra of PtNP/PPY/ITO electrode and ChEt-ChOx/PtNP/PPY/ ITO bioelectrodes are studied as a function of potential (0 -0.20 V) to locate an ideal condition.
The Figures 8A and B, shows the EIS spectra of PtNP/PPY/ITO electrode and ChOx-C O bioelectrode, respectively as a function of potential (0.0 to 0.2 V).The R CT values both for the PtNP/PPY/ ITO electrode and ChOx-ChEt/PtNP/PPY/ITO nano bio- electrode have been found to decrease linearly with inc ey follow Equations ( 12) and ( 13).

[ ] [ ]
where, R = Gas constant, T = Absolute temperatur F = Faraday constant, A = Electrode area (cm 2 ), S = Bulk Pt e (K), concentration of redox probe (mol•cm 3 ) and n = Number of transferred electrons per molecule of the redox probe.
Figure 9 shows the variation of depression angle (θ) obtained for PtNP/PPY/ITO electrode and ChOx-ChEt/ NP/PPY/ITO bioelectrode as a function of applied potential.It is observed that values of depression angle (θ) for PtNP/PPY/ITO electrode and ChOx-ChEt/PtNP/PPY/ ITO bioelectrode decrease on increasing the potential from 0 to 0.2 V.However, 0.06 V is the minimum potential at which the values of (θ) obtained for PtNP/PPY/ ITO electrode (0.4˚) and ChOx-ChEt/PtNP/PPY/ITO bioelectrode (0.3˚) are close to the values of depression angle (θ) at higher potential (0.2 V).This suggests that 0.06 V can be chosen as the working potential for electro-chemical sensing wherein the semicircle of the experimental data is near the centre of real axes with com-  It is necessary to mention here that impedance behavior of an ele order to find out the best fitted circuit, impedance spectra of PtNP/PPY/ITO has been compared with computer simulated spectra using six different electronic circuits (EC) based on Randles and Ershler theoretical model.Figure 10 shows that the Faradic impedance spectra of PtNP/PPY/ITO electrode, fitted with computer simulated spectra using six different electronic circuits (EC).The best fit between the simulated electronic circuit and experimental spectra is obtained for electronic circuit EC 4 i.e., EC 4 . And the minimum error has been observed in EC4 as compared to other circuits.
Figure 11 shows EIS spectra simulated by EC 4 for PtNP/PPY/ITO electrode and ChOx-ChEt/PtNP/PPY/ITO nano bioelectrode at poten tial 0.06 V.It can be seen that R CT value of for ChOx-ChEt/PtNP/PPY/ITO bioelectrode (264.98Ω) is lower than that of the PtNP/PPY/ITO electrode (475.04Ω) revealing that PtNP provides a desired micro-environment for immobilization of ChOx-ChEt where in the immobilized enzyme has better conformation and it retains its natural activity.But the values of double layer capacitance, C dl are almost identical for the electrodes (Table 2).
Figure 12 shows the enzyme activities of the ChOx-ChEt/PtNP/PPY/ITO nano bioelectrodes, evaluated as a function of pH varying from 6.0 to 7.8 at room temperature (25˚C) with 100 mg/dl cholesterol oleate solution.The least value of R CT obtained at pH 7.0 (Figure 12    [ ] with a correlation coefficient of 0.99.The results of experiments carried out in triplicate sets reveal reproducibility of the system within 5%.It has been shown that the ChOx-ChEt/PtNP/PPY/ITO bi as low detection oelectrodes represents characteristics such limit (0.25 mM), fast response time (20 s), high sensitivity (196 Ω/mM/cm 2 ).The detection limit is determined by the minimum concentration of cholesterol oleate at which decrease in R CT value as compared to bioelectrode is observed.As impedance measurement is carried out at 0.06 V, the developed nano bioelectrode shows excellent reusability (data not shown).The shelf-life of the ChOx-ChEt/PtNP/PPY/ITO nanobioelectrode measured after an interval of 1 week has been estimated as 7 weeks.The decrease in the value of R CT has been found to be about 10% up to about 7 weeks after which the current decreases sharply resulting in about 70% loss in about 10 weeks (data not shown).The value of the Michaelis-Menten constant (Km) obtained as 0.2 mM for ChOx-ChEt/PtNP/PPY/ITO using Lineweaver-Burke plot reveals that PtNP/PPY/ITO matrix facilitates enzymatic reaction and helps the immobilized enzyme to achieve better conformation for faster enzymatic reaction resulting in enhanced enzymatic activity.The conformational changes are known to affect a biochemical reaction at bioelectrode that in turn may be influenced by the surface morphology and the nature of immobilization matrix.The double layer capacitance (C dl ) generally increases with the increase of charge transfer resistance (R CT ).The same trend is observed with the C dl value of the nanobioelectrode with the concentration of cholesterol oleate (Figure 13A(a) and (b)).
Figure 14 shows the selectivity of ChOx-ChEt/PtNP/ PPY/ITO nanobioelectrode, estimated by comparing magnitude of R CT by adding normal concentration of interferents such as glucose (5 mM), ascorbic acid (0.05 mM), uric acid (0.1 mM), urea (5 mM), sodium pyruvate acid (0.5 mM) and sodium ascorbate (0.05 mM).The role of interferents has been examined by mixing equal volume of desired interferent with cholesterol.In Figure 14, the first bar (Cho-oleate) shows the value of charge transfer resistance (R CT ) obtained with 3.6 mM cholesterol oleate.The remaining bars show the variation of the R CT corresponding to the mixture of cholesterol oleate and interferents in a 1:1 ratio.The straight line parallel to the X-axis shows the observed R CT in the presence of desired interferents, revealing a maximum of 7.4% interference.The percentage interference (% inter) has been  Et/PtNP/PPY/ITO nano bioelectrode is not significantly affected by the presence of interferents The biosensing characteristics hEt-ChOx/PtNP/PPY/ITO bioelectrode has been compared with other total cholesterol biosenosrs, reported in literature have been summarized in Table 3.It can be noted that the Km value obtained by the impedance measurement reflects actual interaction between enzyme and the substrate as the impedance spectra depends on interfacial property.

Conclusion
The matrices of Pt-nanoparticle (PtNP) and PPY multilayer electrodes have been fabricated for the development of total cholesterol biosensor.The nov Open Access IJOC emonst friendly microenvironment to retain the bioactivity of the enzyme system.The above nano bioelectrodes ChOx-ChEt/PtNP/PPY/ITO offer an excellent performance in terms of linearity, sensitivity, detection limit, response time and shelf life.This is attributed to the presence of PtNP along with PPY to enhance the overall biochemical reaction.The unique features of the ChOx-ChEt/PtNP/ PPY/ITO nanobioelectrode lie on the novelty of fabrication process, measurement technique, reusability, minimum interference and very low K m value.The limitation of the impedance measurement is the semi-logarithm relationship of the calibration curve and selection of the representative circuit from the various Nyquist plots.
PtNP/PPY/ITO and ChOx-ChEt/PtNP/PPY/ITO Electrode The morphology of PPY/ITO, PtNP/PPY/ITO and ChOx-ChEt/PtNP/PPY/ITO was studied by scanning electron Open Access IJOC microscope (SEM).The SEM images are shown in Figures 2A-C.The Figure 2A shows that the bulk polymer tends to aggregate in large particles in the globular morphology which becomes more evident with spherical ball shapes.The PtNP, grown in between the PPY thin layers structure during electrodeposition, has been observed in the SEM image of PtNP/PPY/ITO (Figure 2B).The SEM micrograph shows that PtNP grown on PPY/ITO electrode (Figure 2B) exhibits a granular form like a ball structure along with few cylindrical rod-like structure arrayed in a disordered way.The size of the spherical nanoparticles varies from ca.50 to 80 nm with average particle size of ca.70 nm.Moreover, PtNP are seen to agglomerate due to high surface energy and strong inter atomic interactions.However, after the immobilization of ChEt and ChOx, the surface morphology of PtNP/PPY/ ITO film changes into well-arranged regular morphology and surface roughness decreases revealing that the enzymes are adsorbed onto PtNP/PPY/ITO (Figure 2C) via covalent bonding and electrostatic interactions.The SEM images of ChEt-ChOx/PtNP/PPY/ITO nanobio-electrode exhibit uniform distribution of the enzymes indicating that PtNP/PPY/ITO film provides a desired microenvironment for strong adsorption of enzymes.

Figure 6 .
Figure 6. A. Cyclic voltammograms of (a) PtNP/PPY/ITO nano electrode as a function of scan rate (20 to 200 mV/s).Inset: (a) R p = Polarization resistance [R p at 0 potential describes as (10) insulating features at the electrode/electroly ci electron charge transfer resistance (R CT )], w = Radial frequency and C dl = Double layer capacitance.

Figure 7
shows the impedance spectra of (a) bare ITO (b) PPY/ITO (c) PtNP/PPY/ITO and (d) ChOx-ChEt/PtNP/PPY/ITO.It has been observed that charge transfer resistance (R CT ) value for PPY/ITO electrode (821.3Ω, Figure 7, curve b) at 0 V bias potential is smaller than that of the bare ITO electrode (1044.8Ω).

Scheme 1. The
deposition potential and concentration of hexa chloroplatonic acid solution, and deposition time of each layer were varied to achieve the optimum electrochemical properties of PtNP/PPY/ITO nano composite electrode.The PtNP/PPY/ITO nano composite electrode was cleaned with deionized water.