Development of non-enzymatic cholesterol electrochemical sensor based on CuO(NPs)-Polyaniline-Murexide composite

In this study, a novel non-enzymatic sensor based on copper oxide nanoparticles (CuO), polyaniline nanobers (PANI) and murexide (Mu) modied glassy carbon electrode was developed and used for the detection of cholesterol. Copper oxide nanoparticles were deposited on the glassy carbon electrode through electrodeposition and electrochemical oxidation followed by electrodeposition of PANI-Mu composite. The as prepared CuO-PANI-Mu sensor was characterized using electrochemical, optical and morphological methods such as cyclic voltammetry (CV), impedance spectroscopy (EIS), linear sweep voltammetry (LSV), UV-visible and scanning electron microscopy (SEM). The elaborated composite matrix was used for cholesterol detection employing the impedance spectroscopy method. As a result, good analytical performances were obtained for cholesterol quantication with good stability and high sensitivity (5575 Ω/M) with a wide linear range from 0.5nM to 50mM.


Introduction
Sensors are one of the most developed devices according to their potential applications in several elds like clinical diagnostics, environmental and bioprocess monitoring. Cholesterol is one of the main constituents of mammalian cell membranes as well as cholesterol is a precursor of other biological materials [1]. The detection of cholesterol has received a lot of attention because high cholesterol blood levels can increase risks of heart diseases such as atherosclerosis, high blood pressure and myocardial infraction [2]. As a result, most food producers are obliged to decrease the cholesterol level in food in order to control cardio-diseases. Therefore, there is an increasing demand for sensitive, selective, fast, inexpensive, and robust methods for cholesterol determination in food [3]. The progress in non-enzymatic electrochemical detection of cholesterol was highly accelerated according to the innovative and powerful new sensing material such as nanoparticles and conductive polymers [4][5][6]. Nanocomposites attracted a lot of attention due to their unique physical properties and wide range of applications. Polymer nanocomposites have captivated scientists considering their novel properties gathered from the successful combination of the characteristics of many constituents into a single material [7]. Conducting polymers such as polyaniline (PANI) are becoming increasingly important. PANI is a relatively air stable organic conducting polymer with high electrical conductivity, good environmental, chemical, electrical stability and easy synthesis [8,9]. The use of nanoparticles like Copper oxide (CuO), a versatile semiconductor material, has been attracting the attention due to the commercial demand for electronic devices 10 . Copper oxide has recently been used in several research due to its fascinating properties and wide range of applications such as heterogeneous catalysts, gas sensors, superconductors, lithium ion electrode materials, solar cells and sensors [10][11][12]. CuO nanostructures have been used as an active material for the elaboration of electrochemical sensors for the reason that they have excellent catalytic and electrochemical properties, low temperature and inexpensive [13,14]. The development of inorganic/organic hybrid materials on nano-meter scale have received signi cant attention thanks to their unique properties [15,16]. Overall, the synthesis of hybrid nanocomposites based on polymer and inorganic materials forms a new composite material having synergetic or complementary behaviours of the polymer and the inorganic material [17,18]. Murexide is the ammonium salt of purpuric acid, it has the appearance of a reddish purple powder and is used in analytical chemistry [19,20]. Murexide contributed to the sensor's biocompatibility, better dispersion ability, and hydrophilic characters by introducing more hydroxyl groups. The murexide's hydroxyl groups effectively compensated the positive charge on polymer chains and suppressed the deprotonation processes [21]. In this study, a modi ed glassy carbon electrode based on polyaniline nanostructures, CuO nanoparticles and Murexide molecules was developed for the detection of cholesterol.
Hydroquinone, sulfuric acid, Triton X-100 and Murexide were purchased from Merck (Country/Region: Tunisia). Chemicals were of analytical grade and were used without further puri cation. Aqueous solutions were prepared using distilled-deionised water.

Apparatus
Electrochemical data were obtained in a standard three-electrode cell using a potentiostat (DY200 POT Eco-chimie/Maldova) and an Autolab PGSTAT 320N potentiostat (Metrohm/Switzerland). A glassy carbon electrode (GCE, 3 mm diameter) was used as a working electrode, platinum (Pt) wire was used as counter electrode and an Ag/AgCl(KCl) electrode as a reference. Permeability measurements are performed on a rotating working electrode. The UV-Visible measurements were carried out in a spectrophotometers models 6705 UV/Vis (JENWAY/UK). The morphological characterization of the modi ed electrodes was carried out by a scanning electron microscope JSM 5100 from JEOL/USA using carbon screen printed electrode. Cholesterol solutions were freshly prepared using deionized water and 10% triton-X-100. Cholesterol detection was performed employing EIS measurement in phosphate buffer pH=7.4.

Electrode modi cation
Before measurements, the working electrode was polished with 0.3 μm alumina powders, sonicated in deionized water, rinsed with water and then electrochemically activated in a 0.5 mol.L −1 NaOH solution at a scan rate of 50 mV.s −1 using repetitive cyclic potential sweeps in the range of 0.6 to 1.2 V. Figure 1 shows the different steps adopted to elaborate the modi ed electrode. In brief, the electrode was dried before use. The Cu nanoparticles were electrodeposited on the surface of the electrode at a constant potential of -0.6 V. Following this step, in order to obtain copper oxide nanoparticles (Cu x O) ( Figure 2A and 2B), cyclic voltammetery was performed in NaOH solution (0.1M) at the applied potential range from -0.5 to 0.3 V with a scan rate of 50 mV.s-1 for 40 cycles [22]. An electrolyte containing H 2 SO 4 and aniline was used for the electrodeposition of PANI nano bers on the electrode [23]. PANI-Mu composite was prepared by chronoamperometry at U = 0.85 V versus SCE for 1500 s in 1.00 M H 2 SO 4 solution containing 0.025M aniline and 0.00125M murexide.

Electrode characterization
The characterization of bare and modi ed GC electrodes was investigated by cyclic voltammetry (CV), impedance spectroscopy (EIS), linear sweep voltammetry (LSV) measurements and scanning electron microscopy (SEM). In order to characterize the formation of the composite layer on the GC electrode surface, cyclic voltammograms were applied in the presence of hydroquinone 10 -3 M in phosphate buffer solution (Pbs) at pH=7,4. Figure 3 shows the cyclic voltammograms recorded for the modi ed and the bare electrodes. The electron transfer is more e cient at modi ed electrodes if compared with the bare GC electrode. An increase in anodic and cathodic current response is observed after electrode modi cation indicating that the modi ed electrode surface was electrochemically active. The EIS was employed in order to investigate the modi cation effect of GC electrodes surfaces by the composite lm. Figure 4A represents typical Nyquist plots for bare GC, GC/Cu(NPs), GC/CuO(NPs), GC/CuO(NPs)/PANI and GC/CuO (NPs)/PANI/Mu electrodes. The Nyquist plots of the bare GC represents at low frequency a straight line with a very small semi-circle at high frequency region. On the other hand, the Nyquist plots of the modi ed electrodes represents a semi-circle at high frequency region, which is related to higher charge transfer resistance (R Ct ) than it was estimated for the bare GCE. This change indicates the passivation of the GC electrode. To simulate the Nyquist plots a standard randles equivalent model circuit was used to estimate the analytical parameters as presented in Figure 4B. The proposed equivalent circuit includes the solution resistance (R s ), the charge transfer resistance (R Ct ), the Warburg resistance Where R 0ct is the charge transfer resistance of the bare GC electrode and R Ct is the charge transfer resistance of the modi ed electrode. The obtained results from Eq.(1) are gathered in Table 1. It was demonstrated that EIS signal is equivalent to 86 % coverage of the GC electrode surface. In fact, when the electrode modi cation was performed, a dense and better-packed layer of CuO(NPs)/PANI/Mu was established. Therefore, it could be concluded that nanoparticules were intercalated in the conducting polymer lm. The permeability P m of the elaborated lms under optimal conditions was investigated using a rotating disk electrode (RDE) as shown in Figures 5A. The rotating disk voltammograms were recorded for the modi ed electrodes at different rotation rates in an aqueous solution of hydroquinone as electroactive permeant and compared to the results obtained from a bare glassy carbon electrode. The calculation was done according to the equation introduced by Gough and Leypoldt (Gough and Leypoldt, 1979;Gough, 1980). The equation relates the variation of limiting current i lim with the mass transport for a functionalized rotating disk electrode [26]. P m was calculated using this equation: Where, n is the number of electrons transferred, F is the Faraday constant, A is the electrode radius, D is the diffusion coe cients for the substrate in the bulk solution, C is the hydroquinone concentration, ν is the kinematic viscosity of the solution and ω is the rotation rate of the RDE. The plot of 1/i lim versus ω1/2 presents a linear behavior with a positive intercept that depends on the permeability P m of the lm ( Figure 5B). Permeability values of 0.0039 and 0.0027 cm.s -1 were calculated for the GC/CuO(NPs)/PANI and the GC/CuO(NPs)/PANI/Mu electrodes respectively. UV visible spectroscopy was also done in order to evaluate the optical activity of the developed matrix. Figure 6 shows the spectra of the PANI layer containing two distinctive peaks at 326 nm and 644 nm which are attributed to π-π* and polaron-π* transitions respectively [27]. The UV-visible spectra of the composite lm presents three distinctive peaks localized at 259nm, 292nnm and 452nm. From literature [15] and  Figure 7. As it can be seen from Figure 7, either bare or modi ed GC electrode surfaces are different from each other. Figure 7 (b) shows the formation of Cuo nanoparticules on the electrode surface. In previous work, the same procedure of deposition was investigated to elaborate CuO nanoparticules and con rm the formation of nano-copper [28]. The SEM image in Figure 7(c and d) shows that polymeric layer is uniformly distributed on the GC electrode surface. The layer is composed of PANI nano bers as it can be seen in Figure 7 and as discussed by Haibin Zhang and all [29]. They estimate that the applied potential resulted in different PANI morphologies. This phenomenon can be attributed to the decomposition of PANI controlled by potentials. As we know, the decomposition of PANI occurred simultaneously with the polymerization and there is a competition between the polymerization and the decomposition. When the applied potentials reached 1.00 V versus SCE, the decomposition of PANI was not negligible. However, in present experiments, the applied potentials did not exceed 0.85 V versus SCE, and the decomposition of PANI can be neglected. Therefore, the PANI nano bers can be obtained easily [29]. From the SEM image, we can observe better bers using murexide molecules.

Electrochemical performances of the sensing matrix for cholesterol quanti cation
The deposited CuO(NPs)/PANI/Mu composite layer was used for cholesterol detection using electrochemical impedance spectroscopy measurements (Figure 8). EIS parameters was calculated using NOVA software by tting the obtained diagrams (Table 2). An increase in charge transfer resistance (R Ct ) is observed with the increase of cholesterol concentration on the electrode surfaces. We can also observe a decrease in Warburg values with increasing cholesterol concertation. These changes in impedance parameters indicate the electrode passivation. A signi cant increase in the charge transfer resistance as a function of cholesterol concentration is observed. The calculation of the surface coverage of the electrode after each addition shows an increase in the covered electrode surface from 9% to 83%. This increase proves that cholesterol molecules adheres to the surface of the electrode and increases its concentration in the solid-liquid interface. The cholesterol adhesion on the electrode surface is probably due to its hydrophobic property [30]. Figure 9 shows  Table 3. Selectivity is an important parameter for non-enzymatic cholesterol sensor. It is well known that ascorbic acid (AA), uric acid (UA) and glucose interfere with the detection of cholesterol [31]. Therefore, in this study, concentrations of these molecules are 10 times higher than

Conclusion
A CuO(NPs)/polyaniline/murexide nanocomposite deposited onto a glassy carbon electrode was used to investigate the non-enzymatic detection of cholesterol. The SEM analysis showed that CuO nanoparticles were incorporated onto the polyaniline-murexide composite. The electrochemical response for cholesterol quanti cation was evaluated by Impedance spectroscopy method. The non-enzymatic sensor demonstrates a high sensitivity and good stability in a wide range of concentrations from 1nM to 1mM. The developed bioanalytical system was successful applied for cholesterol determination in milk with high recovery and selectivity.

Con icts of interest
There are no con icts of interest to declare Tables