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Original article
13 (
1
); 2001-2010
doi:
10.1016/j.arabjc.2018.02.018

Development of cholesterol biosensor using Au nanoparticles decorated f-MWCNT covered with polypyrrole network

, , ,
a Department of Electronics and Communication Engineering, PSG College of Technology, Coimbatore, Tamil Nadu 641 004, India
b Electrochemical Sensor and Energy Materials Laboratory & Department of Chemistry, PSG College of Technology, Coimbatore, Tamil Nadu 641 004, India
c Department of Biomedical Engineering, PSG College of Technology, Coimbatore, Tamil Nadu 641 004, India

⁎Corresponding authors. alagappanpsg@gmail.com (M. Alagappan), rss@psgias.ac.in (R. Sivasubramanian)

Received: , Accepted: ,
© The Author(s) 2018
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Cholesterol forms the basic structural components in the biological membrane and is present in several nerve tissues, brain and also serves as a source for the preparation of fatty acids. The determination of cholesterol level is significant in clinical diagnosis for heart problems, thrombosis etc. In this work, we report an electrochemical cholesterol biosensor based on cholesterol oxidase (ChOx) enzyme immobilized on gold nanoparticles – functionalized – multiwalled carbon nanotube (MWCNT) – polypyrrole (PPy) nanocomposite modified electrode. The sensor was fabricated by a two-step approach wherein the Au NPs-f-MWCNT was prepared by wet chemical method followed by electropolymerization of pyrrole. PPy act as a support matrix to hold ChOx and the presence of Au-f-MWCNT increases the electrical conductivity. Cyclic voltammetry (CV) exhibits a decrease in current with increasing cholesterol concentration which arises due to the competing action of Triton X100 surfactant employed for the preparation of cholesterol solution. The AuNPs-f-MWCNT-PPy-ChOx/GCE showed a linear response from 2 × 10−3 to 8 × 10−3 M in amperometry with a sensitivity and detection limit of 10.12 µA mM−1 cm−2 and 0.1 × 10−3 M respectively. Further, the detection of cholesterol using impedance spectroscopy was also demonstrated. The reproducibility, stability and the validity of the sensor in biological samples were also examined.

Keywords

Cholesterol sensor
Au nanoparticles
Polypyrrole network
Electrochemical sensor
Impedance spectroscopy
1

1 Introduction

Cholesterol is a steroid ubiquitously present as a constituent in plasma membrane and acts as a precursor for bile acids, vitamin D and glucocorticoids such as estrogen, progesterone, etc. The human blood contains 70% of cholesterol in ester form and 30% as free form which together gives the total cholesterol level (Schwartz et al., 2004). The normal cholesterol level in blood should be less than 5.2 × 10−3 M i.e. 200 mg/dL and level more than 6.2 × 10−3 M (240 mg/dL) is considered as hypercholesterolemia. Such high levels lead to several complications such as coronary heart disease, myocardial infarction and atherosclerosis hypertension. On the other hand low cholesterol level (hypocholesterolemia) results in anemia and hepatopathy (Bittman, 1997; Nauck et al., 2000). Hence, determination of cholesterol is vital to monitor the clinical disorders arising due to its abnormal levels. The determination of cholesterol was carried out using various techniques such as HPLC (Okazaki et al., 2008), colorimetry (Li et al., 2012), spectrophotometry (Sommers et al., 1975) and electrochemical methods (Wisitsoraat et al., 2009). Among them electrochemical method is highly preferred due to its rapid response time, small size and satisfactory reproducibility and stability.

Many electrochemical sensors were developed earlier by the researchers to detect cholesterol. Likely, enzymatic sensors were developed using the enzyme cholesterol oxidase (ChOx) (Alagappan et al., 2020) with selectivity and high sensitivity. The detection of cholesterol using enzyme is based on the reaction as follows (Yildirimoğlu et al., 2009)

(1)
Cholesterol + O 2 + ChOx → cholestene- 3 -one + H 2 O 2

The cholesterol generated from cholesterol ester is oxidized by oxygen in the presence of ChOx enzyme to cholestene-3-one and H2O2. The measurement of H2O2 by oxidation or reduction will be an indirect quantification of cholesterol levels. However such sensing methods will be carried out at high anodic potential and is vulnerable to the interference of other molecules such as ascorbic acid (AA) and uric acid (UA). The problem was offset by the use of redox mediator wherein the amperometric signal of redox probe was measured instead of H2O2 (Rahman and Asiri, 2015). The method was immensely useful and constitutes a second generation cholesterol biosensor.

The advent of nanotechnology has led to a rapid progress in the construction of electrochemical sensors. The nanomaterials incorporated sensors provide selective and sensitive detection towards the analyte (Luo et al., 2006). Several nanomaterials based on metals (Wu et al., 2016), metal oxides (Charan and Shahi, 2014), polymers (Dervisevic et al., 2016) and carbon nanostructures (Saha and Das, 2014; Tsai et al., 2008) have been reported for cholesterol detection. Among them multiwalled carbon nanotubes (MWCNTs) are highly exploited due to their remarkable electron conductivity that arises by the presence of defect sites on the walls and edge of the nanotubes. Some properties of CNTs are highly attractive; one such is that the adsorption of a small quantity of a chemical species can result in drastic change in the conductivity of the CNT. Hence CNTs can be used for low level detection of biomolecules (Espinosa et al., 2007; Golestanifar et al., 2015). Owing to their good conducting property, chemical stability and high surface area, they have been used to sense various other biomolecules such as dopamine, uric acid, ascorbic acid, lactate and nucleic acid (Karimi-Maleh et al., 2015; Wang, 2005). To take advantage of the outstanding properties of the CNTs, they need to be properly functionalized. Modification of CNT with metal nanoparticles like Au, Fe and Pt enhances the sensitivity of the sensor (Ertan et al., 2016; Yola et al, 2014). Au NPs in particular are used extensively due to their quantum confinements which thereby increase the electrical conductivity through improved electron conduction pathways between CNT and Au NPs. However, their electrocatalytic activity are size and shape dependent (Yola and Atar, 2014).

In order to impart specificity towards cholesterol detection, the fabrication of biosensors generally includes the immobilization of enzyme on to the electrode. Typically the immobilization of enzyme should be a simple step with retention of the biological activity. But they suffer from desorption of protein, pH changes, ionic strength and change in enzyme conformation during the process. Hence immobilization of enzyme on a suitable material (substrate) is required to increase its stability. Various supporting materials such as polymers (Tamer et al., 2011), ionic liquids (Gholivand and Khodadadian, 2014) have been explored earlier. For instance Gopalan et al. in 2009 reported a biosensor based on Au-MWCNT composite with chitosan-ionic liquid network for cholesterol detection wherein the ionic liquid acts as a stable matrix to hold the enzyme. The sensor showed a linear response from 0.5 × 10−3 M to 5 × 10−3 M with a sensitivity of 200 µA mM−1. Similarly, in Cai et al. (2013) constructed a poly (allylamine hydrochloride) wrapped layer-by-layer assembly of MWCNT-AuNPs nanocomposite to immobilize horseradish peroxidase (HRP) and ChOx. The bienzyme based biosensor showed a detection limit of 0.02 × 10−3 M and a linear range from 0.18 × 10−3 M to 11 × 10−3 M. In a significant improvement, Tan et al proposed a third generation biosensor based on direct electrochemistry between the enzyme and Au-MWCNT without employing support matrix or redox mediator (Zhu et al., 2013). The authors proved the redox process of ChOx (FAD) to ChOx (FADH2) through a direct electron transfer from the electrode to enzyme. Though the results seem to be promising the sensor showed poor reproducibility with only a satisfactory performance of the material prepared from a single batch. Recent reports with improved third generation cholesterol biosensor are found to be promising (Abdelwahab et al., 2010; Saxena et al., 2011; Saxena and Goswami, 2012; Das et al., 2016). In this paper we report a simple fabrication of Au NPs supported f-MWCNT nanocomposite for cholesterol detection with polypyrrole (PPy) as supporting matrix for enzyme immobilization. PPy has gained much attention due to its high conductivity, biocompatibility and mechanical stability. Hence the studies on the performance of PPy as supporting matrix with the fabricated nanocomposite as an excellent biosensor for cholesterol detection will attract wide attention. The nanocomposite was prepared by a simple wet chemical method. The functionalization of MWCNT induces hydroxyl/carboxyl functional groups which aid to anchor the Au NPs. A PPy film was coated over Au-MWCNT nanocomposite by electropolymerization of pyrrole monomers. This PPy film forms a mesh that aids in holding the ChOx enzyme and also acts as a conducting media for electron transfer. The sensor showed better performance in sensing cholesterol and the influence of surfactant during amperometric measurement was highlighted.

2

2 Experimental

2.1

2.1 Materials

All the chemicals such as HAuCL4, trisodium citrate (Merck > 99%), functionalized MWCNT, Pyrrole, KH2PO4, K2HPO4, K4[Fe(CN)6],Cholesterol and ChOx (17 unit/mg) were purchased from Sigma Aldrich and employed as received. Millipore water was used for the preparation of all the solutions.

2.2

2.2 Preparation of Au NPs – f-MWCNT

To a 5 mL of 1 mM HAuCl4, 38 mM of trisodium citrate was added in drops under constant stirring. The solution was then heated to 90 °C for 15 min and the colour change from pale yellow to dark blue was noticed. For the preparation of Au-f-MWCNTNPs, 2.5 mg of commercially purchased f-MWCNT was added to a 5 mL of 1 mM HAuCl4 and the procedure was repeated as above. Then the solution was subjected to centrifugation at 5000 rpm for 15 min. The concentrated solution was washed and dispersed in 2 mL of distilled water.

2.3

2.3 Material characterization

The morphologies of the nanocomposite were characterized using transmission electron microscope (TEM) from JEOL JEM 2100, Japan. The X-ray diffraction (XRD) pattern was recorded from Bruker DS Advance with Cu Kα as radiation source and the structural analysis was performed using Fourier Transform Infrared Spectroscopy (FTIR) from Schimadzu, IR Affinity 1s where the sensor was fabricated on an ITO electrode and the spectrum was recorded using ATR mode fitted with ZnSe crystal. The resolution of the FTIR spectrometer being 0.5 cm−1. For TEM, the sample was dispersed in ethanol and drop casted on carbon coated copper grid.

2.4

2.4 Electrochemical measurements

All the electrochemical studies such as CV, CA and EIS were carried out from a three electrode cell configuration using CH instruments USA (CHI 600 E). The glassy carbon electrode (GCE) with a geometric surface area of 0.07 cm2, Pt wire and Ag/AgCl (3 M KCl) were used as working, counter and reference electrodes respectively. The prepared nanomaterial was coated on the GCE electrode for further studies. Prior to modification, the GCE was cleaned using various grades of alumina (0.5 µm and 1 µm) followed by ultra-sonication for 15 min. For modification, 7 µL of well dispersed f-MWCNT-Au solution was drawn and drop-casted on the surface of GCE, followed by drying at ambient temperature.

2.5

2.5 Electropolymerization of pyrrole and enzyme immobilization on Au-f-MWCNT/GCE

The electropolymerization of Pyrrole was performed on Au-f-MWCNT/GCE electrode in a solution of 0.1 M SDS and 10 µL of pyrrole. The polymerization was carried out using CV within the potential range of 0.4 V to −0.8 V (scan rate - 50 mV s−1) for 20 cycles (Fig. S1 of Supporting Information). The electropolymerized film was washed with water and the modification was further continued using immobilization of ChOx enzyme. Typically 3 µL of the enzyme was drop-casted on Au-f-MWCNT-PPy/GCE and dried at room temperature. Thus the Au-f-MWCNT-PPy-ChOx/GCE constitutes the modified electrode for the detection of cholesterol.

2.6

2.6 Preparation of cholesterol solution

To a 5 mM cholesterol in PBS solution, 3 mL of triton-X100 and 1 mL of ethanol was added, heated to 60 °C for 1 h and cooled till the solution becomes clear without any turbidity. The as prepared cholesterol solution was employed for sensor studies.

2.7

2.7 Electrochemical studies

The electrocatalytic cholesterol oxidation was investigated using CV on Au-f-MWCNT-PPy-ChOx/GCE in a mixture of 0.1 PBS, 5 mM K4 [Fe(CN)6] and 1 mM cholesterol in the potential range of −0.4 V to 0.4 V at a scan rate of 50 mV s−1. The detection of cholesterol was carried out using CA at a constant potential of 0.3 V from 0.1 M PBS and 5 mM K4 [Fe(CN)6]. The CA curves were recorded upto 50 s for successive addition of cholesterol. The detection of cholesterol using EIS was performed from a solution of 0.1 M PBS and 5 mM K4[Fe(CN)6] between the frequency range of 104 Hz to 1 Hz at their open circuit potential. The charge transfer resistance (Rct) values were determined for successive addition of cholesterol.

2.8

2.8 Analysis of real sample

Serum was separated using ultracentrifugation method from 5 mL of blood sample collected from healthy volunteers of age group between 25 and 30. The amount of cholesterol in the serum was determined using the Au-f-MWCNT-PPy-ChOx/GCE electrode from a solution of 0.1 M PBS and K4 [Fe(CN)6] containing 500 µL of the serum using CA at a constant potential of 0.3 V up to 50 s. The CA curves were also recorded for the successive addition of cholesterol solution to the electrolyte containing serum.

3

3 Results and discussion

3.1

3.1 Construction of the biosensor

The Au-f-MWCNT-PPy-ChOx/GCE based biosensor for cholesterol detection was constructed based on the electrostatic interaction between the moieties. The functionalization of MWCNT imparts hydroxyl or carboxyl functional group on the CNT network which increases the hydrophilicity of the substrate. The Au NPs were prepared by the in-situ reduction of HAuCl4 by trisodium citrate in the presence of f-MWCNT at elevated temperature. The NPs were anchored on the f-MWCNT matrix through electrostatic interaction between the functional groups of f-MWCNT and Au NPs. Further the Au NPs-f-MWCNT was coated with polypyrrole prepared via electropolymerization of pyrrole. The polymerization was carried out over the Au-f-MWCNT layer which can act as a sheet like structure. The addition of polymer will contribute to the increase in the conductivity and will also act as a support matrix for the deposition of ChOx enzyme. The presence of amine and carboxyl functional groups in the enzyme aid in the electrostatic attraction with the polypyrrole matrix and thereby helps in the immobilization of the enzyme without any change in the structural conformation of the protein. Such reports where polymer act as support matrix were also reported earlier (Dhand et al., 2007). Scheme 1 shows the schematic depiction of the construction of Au-f-MWCNT-PPy-ChOx/GCE biosensor for cholesterol detection.

Schematic illustration of the fabrication of the Au-f-MWCNT-PPy-ChOx/GCE biosensor.
Scheme 1 Schematic illustration of the fabrication of the Au-f-MWCNT-PPy-ChOx/GCE biosensor.

3.2

3.2 Morphological characterization

Fig. 1 shows the TEM images of f-MWCNT, Au NPs, Au-f-MWCNT and Au-f-MWCNT-PPy composite respectively. The TEM image of f-MWCNT in Fig. 1a shows interlocked bundles of nanotubes with an average diameter of ∼73.71 nm. The HR-TEM image shown in the inset of Fig. 1a gives a lattice fringe distance of 0.38 nm which corresponds to (0 0 2) plane of f-MWCNT. The average size of Au-NPs synthesized by a citrate reduction method was found to be ∼12 nm in diameter (Fig. 1b) and the particles were found to be decorated on f-MWCNT and were rarely observed in the background of Fig. 1c, which indicates the anchoring of Au NPs on f-MWCNT matrix. The presence of Au and carbon was also confirmed through EDX analysis (Fig. S2 of Supporting Information). From Fig. 1d it is inferred that the sheet like morphology of PPy covered the Au-f-MWCNT. The composite structure could provide good electron conductivity and formation of more active sites which can play a crucial role in the electrocatalytic studies. Also the polymer matrix facilitates the immobilization of ChOx enzyme.

TEM images of (a) f-MWCNT, (b) Au Nps, (c), f-MWCNT-Au and (d) f-MWCNT-Au-PPy nanocomposite.
Fig. 1 TEM images of (a) f-MWCNT, (b) Au Nps, (c), f-MWCNT-Au and (d) f-MWCNT-Au-PPy nanocomposite.

Fig. 2(a and b) depicts the XRD pattern and the FTIR spectrum of Au-f-MWCNT-PPy composite. The XRD pattern in Fig. 2a shows a broad peak at 25.2° which indicates the amorphous nature of PPy and the sharp peak at 43.5° corresponds to the (2 0 0) plane of Au NPs. The FTIR spectrum in Fig. 2b depicts the characteristic bands at 3426 cm−1, 1632 cm−1 and 1220 cm−1. The band at 3426 cm−1 and 1632 cm−1 belongs to the stretching vibration of —OH group. The band at 1220 cm−1 is related to C⚌C stretching vibration. Moreover the small peaks around 3000 cm−1 belong to C—N vibration of the PPy.

(a) XRD and (b) FTIR spectrum of Au-f-MWCNT-PPy nanocomposite.
Fig. 2 (a) XRD and (b) FTIR spectrum of Au-f-MWCNT-PPy nanocomposite.

The EIS was utilized to study the interfacial properties of bare GCE, Au-f-MWCNT-PPy/GCE and Au-f-MWCNT-PPy-ChOx/GCE. Fig. 3 shows the Nyquist plot recorded from 0.1 M PBS and 5 mM K4[Fe(CN)6] in the frequency range 103 Hz to 1 Hz. The Nyquist plot is characterized by the semicircle at high frequency end and vertical line at low frequency end. The diameter of the semicircle indicates the charge transfer resistance (Rct) and the vertical spike denotes the Warburg impedance due to diffusion. The Nyquist plot obtained for different electrodes was fitted using equivalent circuits as shown in the inset of Fig. 3 and Table S1 of the Supporting Information. The values obtained from the fitting shows that the Rct for bare GCE, PPy/GCE, Au-f-MWCNT-PPy/GCE and Au-f-MWCNT-PPy/ChOx/GCE were found to be ∼1272.0 Ω, 3393.0 Ω, 496.2 Ω and 609.1 Ω respectively as shown in Fig. 3. The Rct value is less for Au-f-MWCNT-PPy/GCE compared to bare GCE which indicates improved conductivity of the fabricated sensor. However upon enzyme immobilization the Rct increases which is attributed to the poor electronic conductivity of the coated enzyme and its repulsion towards [Fe(CN)6]2+ ions.

Nyquist plot of the modified electrodes obtained from a solution of 0.1 M PBS and 5 mM of K4[Fe(CN)6] in the frequency range of 103 Hz to 1 Hz. The inset depicts the corresponding equivalent circuit obtained for the Nyquist plot.
Fig. 3 Nyquist plot of the modified electrodes obtained from a solution of 0.1 M PBS and 5 mM of K4[Fe(CN)6] in the frequency range of 103 Hz to 1 Hz. The inset depicts the corresponding equivalent circuit obtained for the Nyquist plot.

3.3

3.3 Performance of Au-f-MWCNT-PPy-ChOx/GCE as a cholesterol biosensor

Fig. 4a shows the CV curves recorded for Au-f-MWCNT-ChOx/GCE and Au-f-MWCNT-PPy-ChOx/GCE in 0.1 PBS of pH 7.0 at a cholesterol concentration of 1 mM in the presence of redox mediator K4[Fe(CN)6]. It is inferred that the anodic and cathodic peak current decreases with increasing cholesterol concentration. However without the polymer film the sensor performance degrades with time. Hence, the experiment was continued with the polypyrrole coated biosensor. Further, the CV was recorded for the addition of different concentration of cholesterol, Fig. 4b. It was noticed that the current sequentially decreased with increasing concentration of cholesterol. This behavior was in contrast to those reported earlier where there is an increase in current for each addition of cholesterol concentration (Gopalan et al., 2009). As reported by Vidal et al (1999), there is a trade off in current values which depends on the surfactant and cholesterol addition. The current increases with cholesterol addition but decreases with increase in triton-X 100 concentration. As a result of both the process, an average value of current was observed in successive addition of cholesterol. The concentration of triton–X 100 is crucial which dictates the response of the biosensor (Vidal et al., 1999; Gopalan et al., 2009).

(a) CV curves recorded for ( ) f-MWCNT-Au-PPy/GCE and (-) f-MWCNT-Au-PPy-ChOx/GCE from a solution of 0.1 M PBS, 5 mM of K4[Fe(CN)6] and 1 mM of cholesterol and (b) CV curves for varying concentration of cholesterol from 1 mM to 10 mM on f-MWCNT-Au-PPy-ChOx/GCE. Scan rate = 100 mV s−1.
Fig. 4 (a) CV curves recorded for ( ) f-MWCNT-Au-PPy/GCE and (-) f-MWCNT-Au-PPy-ChOx/GCE from a solution of 0.1 M PBS, 5 mM of K4[Fe(CN)6] and 1 mM of cholesterol and (b) CV curves for varying concentration of cholesterol from 1 mM to 10 mM on f-MWCNT-Au-PPy-ChOx/GCE. Scan rate = 100 mV s−1.

In order to understand the mechanism of cholesterol oxidation, the CV experiment was carried out for cholesterol oxidation on the Au-f-MWCNT-PPy-ChOx/GCE in the absence of the redox mediator. Fig. S3 of the supporting information shows the CV response for cholesterol in the absence of redox mediator. It is seen that in the absence of redox mediator the response for the modified electrode is very poor which indicates that the oxidation response of the cholesterol is almost negligible. On the other hand the same experiment was repeated in the absence of ChOx (Fig. S4 of the Supporting Information). The CV curves showed the response of the redox mediator and as the concentration of cholesterol is increased there is not much change in the peak current. This suggests that the oxidation of cholesterol in the absence of enzyme is negligible. From these studies it is found that the enzyme imparts specificity for cholesterol oxidation and the addition of redox mediator helps in the electron transport (electron hopping) from the analyte to the enzyme modified electrode. Hence the redox mediator plays a major role in the oxidation of cholesterol and the fast reversible electron transfer increases the kinetics of the electrochemical reaction.

In order to study the kinetics of cholesterol oxidation, the CV curves with varying scan rate was recorded. The plot between peak current vs scan rate showed linear relation which indicates that the reaction is an adsorption controlled process (Fig. S5 of Supporting Information). The number of electrons involved was calculated using Laviron’s equation

(2)
I p = nFQv / 4 RT and estimated to 1.86 which is close to the theoretical value 2. The surface coverage was calculated using the formula
(3)
τ = Q / nFA where Q is the charge (C), n denotes the number of electrons, F is the faraday constant (96,500 C mol−1), A is the area of the electrode (cm2), ν is the scan rate (V s−1), R is the gas constant (J K−1 mol−1) and T is the temperature (°C), which was found to be 2.28 × 10−9 mol cm−2 which implies that the enzyme immobilization was near to the monolayer level. The charge transfer coefficient α was calculated using the slope obtained from the plot of Ep Vs ln ν (Fig. S5 of Supporting Information) and was found to be 0.56. The electron transfer rate constant was deduced as 1.51 s−1 using the following equation
(4)
E pa = E o ′ + RT ( 1 - α ) nF ln ( 1 - α ) nF RTk s + RT ( 1 - α ) nF ln ν
(5)
E pc = E o ′ - RT α nF ln α nF RTk s - RT α nF ln ν
(6)
Δ E p = RT ( 1 - α ) α nF α ln ( 1 - α ) + ( 1 - α ) ln α - RT nF - ln k s + RT ( 1 - α ) α nF ln ν where E o ′ is the formal reduction potential (V), α denotes the transfer coefficient, n denotes the number of electrons and ks is the standard heterogeneous rate constant (s−1) respectively.

3.4

3.4 Cholesterol detection using amperometry and EIS

3.4.1

3.4.1 Amperometry

Fig. 5 shows the amperometric response of Au-f-MWCNT-PPy-ChOx/GCE from a solution of 0.1 M PBS for various cholesterol concentrations from 1 × 10−3 M to 10 × 10−3 M. In order to determine the optimum potential to be applied for cholesterol detection, the amperometric current response was recorded at various applied potentials in presence of 1 mM cholesterol (Fig. S6 of Supporting Information). The potential 0.3 V showed maximum current and hence chosen as the optimum potential for cholesterol detection. Prior to measurements, the initial blank spectrum was run so as to obtain a uniform steady current. The current response was recorded for 50 s for each cholesterol addition. It is seen that the current decreases steadily for successive addition of cholesterol from 1 × 10−3 M to 10 × 10−3 M. The calibration curve was plotted by using the current value obtained at the 50th second with respect to the concentration of cholesterol. The linear relationship was obtained from 2 × 10−3 to 8 × 10−3 M with a correlation coefficient of 0.9897 and a sensitivity of 10.12 µA mM−1 cm−2. The limit of detection was determined using the formula LOD = 3 S/m where S is the standard deviation of blank without the addition of cholesterol and m is the slope of the calibration curve, which is 0.1 × 10−3 M. The performance of the proposed sensor was compared with other Au and MWCNT based cholesterol biosensor as shown in Table 1. The present work is placed suitably among the biosensor reported with low detection limit and long linear range.

(a) Amperometric response for varying concentration of cholesterol on f-MWCNT-Au-PPy-ChOx/GCE from a solution of 0.1 M PBS and 5 mM of K4[Fe(CN)6] and (b) their corresponding calibration plot.
Fig. 5 (a) Amperometric response for varying concentration of cholesterol on f-MWCNT-Au-PPy-ChOx/GCE from a solution of 0.1 M PBS and 5 mM of K4[Fe(CN)6] and (b) their corresponding calibration plot.
Table 1 Performance comparison various enzymatic cholesterol biosensors.
Sensing electrode Type of detection Detection limit (M) Linear range (×10−3 M) Ref.
MWCNT–Au/chitosan/IL Amperometry 0.35 × 10−3 0.5–5 Gopalan et al. (2009)
MWCNT-PANI Amperometric – 1.29–12.93 Dhand et al. (2008)
MWCNTs-MnO2 Cyclic Voltammetry 0.3 × 10−9 1 – 100 Norouzi et al. (2010)
ALP-MWCNT Amperometric – 0.2–20 Zhang et al. (2012)
MWCNT-PPy Amperometry – 0.4–6.5 Singh et al. (2012)
NiO/MWCNT Amperometry 0.03 × 10−3 2.59–5.18 Ali et al. (2013)
Au/Propolis/MWCNT Amperometry 49 × 10−6 0.15–0.55 Molaei et al. (2014)
Graphene/PVP/PANI Amperometry 1 × 10−6 0.05–10 Ruecha et al. (2014)
MWCNT/Au Amperometry 0.01 × 10−3 0.01–5.83 Lata et al. (2016)
Au-f-MWCNT-PPy Amperometry 0.1 × 10−3 2–8 This work

MWCNT – Multiwalled carbon nanotube, IL – Ionic liquid, PANI – polyaniline, PPy – polypyrrole, PVP – polyvinylpyrolidone.

The enzyme substrate kinetics of the fabricated biosensor was studied using the apparent Michaelis-Menton equation constant (Kmapp) from Lineweaver-Burk equation 1/i = Kapp/imax(1/C) + (1/imax) where I is the current (A), I max is the maximum current (A) and C is the concentration of cholesterol (mM). The plot between 1/i and 1/C provides a slope from which Kapp can be deduced. Fig. S7 of Supporting Information shows the L-B plot between 1/i and 1/C and the slope was chosen from the steady state values of the cholesterol concentration. The Kmapp was determined to be 1.66 mM which indicates a greater enzyme-substrate interaction. Lower the Kmapp higher is the affinity of cholesterol towards cholesterol oxidase enzyme. The Kmapp value for the f-MWCNT-PPy-ChOx/GCE was smaller than other previously reported cholesterol biosensors (Singh et al., 2004; Umar et al., 2009).

3.4.2

3.4.2 Impedimetric analysis

It is well-known that EIS is a non-destructive technique and is being employed to study the electrical changes in the biological interface. Hence, impedimetric analysis has been used for the detection of cholesterol which was carried out at 0.3 V in the frequency range of 105 Hz to 1 Hz. Fig. 6a shows the variation of charge transfer resistance with the concentration of cholesterol and Fig. 6b shows the corresponding calibration plot. The charge transfer resistance and concentration are related as (Shervedani et al., 2006)

(7)
R ct = RT ( n 2 f 2 AK et [ C ] ) - 1 where Ket is the electron transfer rate constant (s−1) and [C] is the concentration of cholesterol (mM), n is the number of electron transfer, f is the frequency (Hz), A is the area of the electrode (cm2), T is the temperature (K) and R is the gas constant (8.314 J K−1 mol−1). From Eq. (7), it is seen that the concentration takes an inverse trend with respect to Rct i.e the Rct should decrease with increase in concentration. However in the present case the Rct increases with concentration of cholesterol which may arise due to the effect of surfactant employed in cholesterol preparation as mentioned earlier. The impedance plot was fitted with the suitable equivalent circuit and the parameters are listed in Table S2 of the supporting information. The values of Rct for constructing the calibration plot were obtained from the fitting parameters. The linear relationship between 1/Rct and concentration was obtained in the range of 2 × 10−3 M and 9 × 10−3 M. Similar studies on cholesterol detection has also been made by Malhotra et al using nanostructured Fe3O4 modified electrode (Kaushik et al., 2010).
Nyquist plot recorded for varying concentration of cholesterol on f-MWCNT-Au-PPy-ChOx/GCE from a solution of 0.1 M PBS and 5 mM of K4[Fe(CN)6] and (b) the calibration plot of 1/Rctvs concentration. The inset in (a) depicts the corresponding equivalent circuit obtained for the Nyquist plot.
Fig. 6 Nyquist plot recorded for varying concentration of cholesterol on f-MWCNT-Au-PPy-ChOx/GCE from a solution of 0.1 M PBS and 5 mM of K4[Fe(CN)6] and (b) the calibration plot of 1/Rctvs concentration. The inset in (a) depicts the corresponding equivalent circuit obtained for the Nyquist plot.

3.5

3.5 Reproducibility and stability

The reproducibility of the biosensor was tested by recording the amperometric current for 3 × 10−3 M cholesterol on the same modified electrode. The current response was found to be satisfactory for the same batch of fabricated sensor. The stability test was conducted by storing the sensor for a longer period in PBS buffer and recording the current response for every 2 days. The sensor retained 76% of the initial current after 7 days with slight decrease in catalytic activity. The above results suggest an excellent stability and reproducibility of the fabricated cholesterol biosensor.

3.6

3.6 Interference studies

The effect of interfering agents such as AA, UA, KCl, NaCl, lactate and glucose in the detection of cholesterol was studied using the fabricated biosensor as shown in Fig. 7. It was found that the amperometric response for cholesterol had a negligible effect upon addition of AA, UA, KCl and NaCl. But there is a significant reduction in current when lactate and glucose were added. As the detection was performed at more positive potentials there is a strong influence of interfering agents such as glucose and lactate.

Bar plot depicting the interference of various species in the detection of cholesterol.
Fig. 7 Bar plot depicting the interference of various species in the detection of cholesterol.

3.7

3.7 Analysis of cholesterol in serum samples

The Au-f-MWCNT-PPy-ChOx/GCE biosensor was employed to determine free cholesterol level in serum sample. The serum sample was diluted in 0.1 M PBS containing triton-X 100 and ethanol. The analysis was done using standard addition method and the results are compiled in Table 2. Initially the cholesterol level in serum was determined to be 0.5 × 10−3 M and cross checked with spectroscopic methods (procedure detailed in Supporting Information) which gave a total cholesterol level to be 1 × 10−3 M. This was followed by the addition of cholesterol at various concentration. The values were predicted with good accuracy recovery values. Hence the sensor can be employed to determine cholesterol in biological samples

Table 2 Estimation of cholesterol from serum samples.
S. No Amount of Cholesterol added (mM) Amount of Cholesterol predicted (mM) % Recovery
1 0 0.5 –
2 2 3.19 127.6
3 3 3.82 109.1
4 4 4.21 93.5

4

4 Conclusion

In summary, Au-f-MWCNT-PPy/ChOx based biosensor was constructed for electrochemical sensing of cholesterol. The sensor was fabricated by a two step approach, by combining chemical and electrochemical method and was characterized using TEM, XRD and FTIR spectroscopy. The sensor showed a good selectivity, sensitivity and reproducibility. The increased electrical conductivity due to Au-f-MWCNT and the robust PPy matrix to hold the enzyme led to excellent performance of the sensor. The work provides a good platform for development of other metal–carbon-polymer nanocomposite framework for cholesterol biosensor.

Acknowledgements

This work was financially supported by the University Grants Commission (UGC) New Delhi, Grant No. 42-907/2013 (SR) and the Department of Science and Technology-SERB (Grant No. ECR/2015/000099), Government of India. The authors wish to acknowledge the facilities and support provided by the Management of PSG & Sons’ Charities, Coimbatore, India.

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Appendix A

Supplementary material

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.arabjc.2018.02.018.

Appendix A

Supplementary material

Supplementary Figs. S1–S9 and Tables S1 and S2

Supplementary Figs. S1–S9 and Tables S1 and S2


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