Electrochemical detection of cholesterol based on competitive host–guest recognition using a β-cyclodextrin/poly(N-acetylaniline)/graphene-modified electrode

Abstract

A sensitive and selective electrochemical approach for cholesterol sensing based on a competitive host–guest recognition between β-cyclodextrin (β-CD) and a signal probe (methylene blue)/target molecule (cholesterol) using a β-CD/poly(N-acetylaniline)/graphene (β-CD/PNAANI/Gra)-modified electrode was developed. Due to the host–guest interaction, MB molecules can enter into the hydrophobic inner cavity of β-CD, and the β-CD/PNAANI/Gra modified glassy carbon electrode displays a remarkable anodic peak. In the presence of cholesterol, a competitive interaction to β-CD occurs and the MB molecules are displaced by cholesterol. This results in a decreased oxidation peak current of MB as MB is a well known redox probe and hence can be easily detected using the differential pulse voltammetery technique. A linear response range of 1.00 to 50.00 μM for cholesterol with a low detection limit of 0.50 μM (S/N = 3) was obtained by using the indirect method. The proposed method could be successfully utilized to detect cholesterol in serum samples, and may be expanded to analysis of other non-electroactive species. Besides, the host–guest interaction between cholesterol and β-CD was studied by molecular modeling calculations, which revealed that the complexation could reduce the energy of the system and the complex of 2 : 1 host–guest stoichiometry had the lowest ΔE value of −10.45 kcal mol⁻¹. The molecular docking studies suggested that hydrogen bonding, electrostatic interactions, and hydrophobic interactions should be the major driving forces for the formation of the inclusion complex.

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1. Introduction

Cholesterol is a vital component in cells and tissues of humans, playing a functional role in construction of cell membranes or serving as a biosynthetic precursor of bile acids, vitamin D, steroid hormones etc.¹ The normal level of total cholesterol in healthy human serum is ∼200 mg dL⁻¹.² Excess cholesterol in blood serum forms plaques in the arteries of blood vessels which prevent blood circulation and cause cardiovascular diseases.³ Thus the levels of total cholesterol in serum and food are major parameters for diagnostic treatment. Over various analytical methodologies, the electrochemical approach of different kinds of cholesterol biosensing platforms has received great attention in the past few decades as reviewed by Arya et al.⁴ Most of the cholesterol biosensors reported till date are based on the detection of electrooxidation of hydrogen peroxide produced during the catalysis of cholesterol by cholesterol oxidase.⁵ This requires a high anodic potential that can induce simultaneous oxidation of other electrochemically active species present in samples leading to false positive signals. Besides, detection selectivity in these methods relies on the use of cholesterol selective enzymes which are expensive, unstable, and prone to denaturation.³ Therefore, an alternative, simple, and cost effective method for the sensitive and selective detection of cholesterol is highly desirable.

The concept of indicator displacement assay (IDA) has received considerable interest with the development of supramolecular chemistry, which exploit the potential of artificial receptors, particularly macrocyclic hosts, for its promising applications in molecular recognition and sensing.^6,7 The sensing principle of IDA relies on the competition between a test substance and an indicator for the same binding site on the host.⁸ When an analyte is added to a solution containing host–indicator complex, the analyte displaces the indicator from the binding site. Upon displacement of the indicator, a change in signal is observed. Cyclodextrins (CDs) as the typical macrocyclic molecules have toroidal shapes with hydrophobic inner cavities and hydrophilic exteriors.⁹ The interesting characteristics can enable them to bind a wide variety of hydrophobic guest molecules to form stable host–guest complex in their hydrophobic cavity.¹⁰ The host–guest interaction between β-CD and cholesterol has been used for cholesterol extraction from bioenvironment such as food, cell membranes, blood serum and cultured cells.³ This suggests that β-CD can be an alternative to cholesterol enzyme for selective recognition of cholesterol. However, this interaction does not produce any signal change. In addition, cholesterol is one of the non-electroactive species, resulting its direct electrochemical determination is very difficult. Alternatively, designing a competitive host–guest recognition system by selecting an electroactive probe as signal indicator might have promising applications for the analysis of non-electroactive species. Mondal and Jana³ have recently carried out fluorescent detection of cholesterol using graphene-β-CD (Gra-β-CD) hybrid system, where the optical detection of cholesterol was carried out using rhodamine 6G (R6G) dye as a fluorophore. Although the competitive host–guest interaction system has been widely applied in the fluorescent sensing filed,^3,11–15 it is rarely investigated for electrochemical sensing applications except few researchers contributed to this area.^16,17

Herein, a non-enzymatic electrochemical approach for cholesterol sensing based on a competitive host–guest recognition between β-CD and signal probe/target molecules using β-CD/poly(N-acetylaniline)/Gra (β-CD/PNAANI/Gra)-modified electrode was proposed. Methylene blue (MB) and cholesterol were selected as the probe and target molecules, respectively. Gra was chosen here considering that it can enhance the electrode conductivity and facilitate the electron transfer. The introduction of PNAANI film is used to steadily immobilize β-CD and to avoid the non-specific adsorption of MB on the Gra film by a π–π stacking interaction. Due to the host–guest interaction, MB molecules can enter into the hydrophobic inner cavity of β-CD, and the β-CD/PNAANI/Gra modified glassy carbon electrode displays a remarkable anodic peak. In the presence of cholesterol, competitive interaction to β-CD occurs and the MB molecules are displaced by cholesterol. This results in a decreased oxidation peak current of MB. As MB is a well known redox probe and hence can be easily detected using differential pulse voltammetery (DPV) technique. Agnihotri et al.¹⁷ reported an electrochemical detection of cholesterol using Gra-β-CD hybrid as the sensing matrix, while the host–guest interaction in their work did not occur on the electrode surface (in an electrochemical cell). Compared with this previous report, the host–guest interaction in the present work occurred completely on the β-CD/PNAANI/Gra-modified electrode surface, which is less expensive as an electrochemical cell needs 3 mL materials. And the non-specific adsorption of MB on the Gra film was avoided by the PNAANI film. The proposed method could be successfully utilized to detect cholesterol in serum samples, and exhibited a promising application in practice. In addition, the host–guest interaction between cholesterol and β-CD was investigated by molecular modeling calculations.

2. Materials and methods

2.1. Chemicals

Graphite oxide was purchased from Nanjing XFNANO Materials Tech Co., Ltd (Nanjing, China). Cholesterol, MB, β-CD, and N-acetylaniline (NAANI) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade. Phosphate buffer (PBS, 0.1 M pH 7.0) was used as working solution. All aqueous solutions were prepared with deionized water (DW, 18 MΩ cm).

2.2. Apparatus

Electrochemical impedance spectroscopy (EIS) and DPV experiments were performed with a CHI 660E Electrochemical Workstation from Shanghai Chenhua Instrument (Shanghai, China) and conducted using a three-electrode system, with the modified GCE as working electrode, a platinum wire as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode. The morphologies of the prepared samples were characterized by a QUNT200 scanning electron microscopy (SEM, USA). UV-visible spectra were analyzed in a U-2001 Hitachi (Tokyo, Japan) UV spectrophotometer. Fourier transform infrared (FTIR) study was performed over the wavenumber, range of 4000–400 cm⁻¹ by a Thermo Fisher SCIENTIFIC Nicolet IS10 (Thermo Fisher, Massachusetts, USA) FTIR impact 410 spectrophotometer using KBr pellets. Raman spectra were obtained on a 400F PERKINELMER Raman spectrometer (USA) with 514.5 nm wavelength incident laser light.

2.3. Molecular docking

The crystal structures of β-CD (ID: IHEGEI) and cholesterol (ID: CHOEST21) were obtained from Cambridge Crystallographic Data Centre (CCDC) and optimized using molecular dynamics simulation with the Gaussian 03 program. Both the optimized structures were used as a starting structure in the docking study. AutoDock4.2 with Lamarckian Genetic Algorithm (LGA) was used for docking study. An initial population of 150 individuals with a maximum number of energy evaluations of 25 000 000 and a maximal number of generations of 27 000 were used as an end criterion. An elitism value of one was used and a probability of mutation and crossing-over of 0.02 and 0.8 was used, respectively. We have defined the conformational search space implementing an 60 × 60 × 60 grid and 0.375 Å spacing between each point in such a way that it covered both the external surface and the internal cavity of the β-CD. A total of 50 docking runs were carried out. At the end of each run, the solutions were separated into clusters according to their lowest RMSD and the best energy score value based on an empirical free energy function. Clustering was performed on the docked complexes with a cut-off of 2 Å. From the docking calculations, the lowest energy conformation was selected as the cholesterol/β-CD binding mode, and the binding free energy of the cholesterol/β-CD complex was calculated by using the semi-empirical method PM3.

2.4. Preparation of the Gra

The graphite oxide was exfoliated into graphene oxide (GO) sheets by ultrasonication at room temperature for one hour. The as-obtained yellow-brown aqueous suspension of GO was stored at room temperature and used for further experiment. Compared with the traditional procedure using highly toxic hydrazine as reductant, ascorbic acid (AA) was used as reducing agent to prepare Gra in DW at room temperature. In a typical experiment, 50 mg of AA was added into 10.0 mL of 0.5 mg mL⁻¹ GO aqueous suspension and stirred for 48 h at room temperature. After centrifuging and washing with DW for three times, the resulting Gra material was obtained by freeze-drying.

2.5. Preparation of the modified electrodes

Glassy carbon electrode (GCE, 3 mm in diameter) was polished with 0.3 and 0.05 μm Al₂O₃ powder respectively and subsequently sonicated in ethanol and DW to remove the adsorbed substance and dried in air. The Gra was dissolved in DW at a concentration of 0.5 mg mL⁻¹ with the aid of ultrasonic agitation for 20 min, resulting in a homogeneous suspension. To prepare the Gra modified electrode, 5 μL of the Gra suspension was dropped onto the electrode surface and dried at room temperature. The obtained electrode was noted as Gra/GCE. In order to prepare the PNAANI/Gra/GCE, the Gra/GCE was held at a constant voltage of 1.0 V for 1 min in a mixture solution containing 0.1 M NAANI and 1.0 M HClO₄ aqueous solution, and then swept from −0.2 to 1.0 V for 25 cycles at a scan rate of 100 mV s⁻¹. Finally, by electro-oxidation of the PNAANI/Gra/GCE at a constant potential of 1.2 V for 12 min by amperometric i–t curve technique in a mixture solution containing 0.05 M β-CD and 0.1 M LiClO₄ in DMSO, the β-CD/PNAANI/Gra/GCE was obtained successfully. For comparison, a similar procedure was used to prepare PNAANI/Gra/GCE and β-CD/PNAANI/GCE.

2.6. Electrochemical measurements

DPV was applied in 0.1 M pH 7.0 PBS from −0.7 to 0.7 V with a pulse amplitude of 0.05 V and a pulse width of 0.05 s. EIS was recorded in the frequency range from 10⁻¹ to 10⁵ Hz with an amplitude of 5 mV using 2.0 mM [Fe(CN)₆]^3−/4− redox couple (1 : 1) with 0.1 M KCl as supporting electrolyte. All the measurements were carried out at room temperature. As cholesterol has very low solubility in water, 1.0 mM stock solution of cholesterol was prepared in ethanol and diluted to different concentrations by 0.1 M pH 7.0 PBS for further use. Before electrochemical measurements, the β-CD/PNAANI/Gra/GCE was incubated with 1.0 mM MB solution (in 0.1 M pH 7.0 PBS) for a definite time, and rinsed gently with ultrapure water. Then, the electrode was further incubated with different concentrations of cholesterol solution. After that, the electrode was rinsed gently with ultrapure water and the current response of the MB-bound β-CD/PNAANI/Gra/GCE were investigated by DPV in 0.1 M pH 7.0 PBS.

3. Results and discussion

3.1. Design strategy of the electrochemical sensor

Scheme 1 illustrates the assay protocol of the proposed electrochemical sensor based on the competitive host–guest interaction between β-CD and MB (signal probe)/cholesterol (target). MB molecules can enter into the inner cavity of β-CD due to the host–guest interaction, and the MB-bound β-CD/PNAANI/Gra/GCE displays a remarkable oxidation peak due to MB. However, in the presence of cholesterol, competitive association to the β-CD occurs and the MB molecules are displaced by cholesterol. This results in a decrease of the oxidation peak current of the MB probe.

Scheme 1 Illustration of the strategy of the proposed electrochemical sensor based on the competitive host–guest interaction between β-CD and MB (signal probe)/cholesterol (target).

3.2. Molecular docking

In recent years, widespread use has been made of computer aided molecular modeling to rapidly and simply obtain a three dimensional image of the most likely structure of the inclusion complex. Typically, the more negative the binding energy is, the stronger interaction is between the host and guest molecules. As listed in Table 1, the lowest binding free energy (ΔE) was −6.68 kcal mol⁻¹ for the host–guest complex of cholesterol and β-CD with 1 : 1 stoichiometry calculated by PM3 method. The lowest energy docked conformation for 1 : 1 complex of cholesterol and β-CD, shown in Fig. 1A, reveals that the B-, C-, and D-rings of cholesterol molecule inserted into β-CD cavity due to hydrophobic interactions, however, the cyclohexanol part (A-ring) and the alkyl chain of the cholesterol molecule located just outside the β-CD host. The A-, B-, C-, and D-rings of cholesterol were indicated in Scheme 2. Among all the docking conformations of cholesterol and β-CD, two other conformations capture our attention. One is the cyclohexanol part (A-ring) that inserted into the cavity of β-CD, the other is the alkyl chain of the cholesterol that inserted into β-CD cavity. Thus, we speculated that one cholesterol molecule may bind with two β-CD molecules to form a 1 : 2 inclusion complex. So we have further studied the interaction between one cholesterol and two β-CD molecules. The crystal structure of two adjacent β-CDs (ID: WISRIZ) was obtained from Cambridge Crystallographic Data Centre (CCDC) and also optimized using molecular dynamics simulation with the Gaussian 03 program. The docked conformation was displayed in Fig. 1B, indicating that the cyclohexanol (A-ring) and the alkyl chain of cholesterol molecule inserted into the cavities of two β-CD molecules, respectively. The ΔE calculated by PM3 method was −10.45 kcal mol⁻¹, which is much lower than that that of a 1 : 1 inclusion complex, indicating that a 1 : 2 guest–host binding mode is more stable. Analysis of host–guest interaction as obtained from the docking studies reveals that hydrogen bonding, electrostatic interactions, and hydrophobic interactions are the predominant driving forces of the 1 : 2 guest–host complex. Firstly, the hydroxyl on A-ring of the cholesterol molecule formed hydrogen bonding with the hydroxyl of β-CD and the bond length is 2.0 Å. Secondly, as shown in Fig. 1C, the surface of cholesterol molecule is mainly negatively charged, while the internal cavities of the two adjacent β-CDs are mainly positively charged. Thus, strong electrostatic interactions formed between cholesterol molecule and the two β-CDs. Thirdly, as revealed in Fig. 1D, strong hydrophobic interactions also formed between cholesterol molecule and the two β-CDs. The cyclohexanol and the alkyl chain groups of the cholesterol molecule inserted into the hydrophobic cavities of two β-CD molecules, respectively. Recently, Cheng et al.¹⁸ proposed a similar 1 : 2 cholesterol/β-CD complex structure based on the 2D ¹H ROESY results. This result is in accordance with that of our obtained from molecular docking.

Table 1 The interaction energy between cholesterol and β-CD for 1 : 1 stoichiometry calculated by PM3 method

System	Cluster rank	Number in cluster	ΔE (kcal mol^−1)
Cholesterol/β-CD	1	23	−6.68
	2	7	−6.45
	3	8	−6.45
	4	4	−6.39
	5	2	−6.32
	6	1	−6.32
	7	2	−6.25
	8	2	−6.24
	9	1	−5.71

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Fig. 1 Lowest energy cholesterol/β-CD docked complex for 1 : 1 (A) and 2 : 1 (B) host–guest stoichiometry (left is the side view, right is the top view). The electrostatic forces (C, left is the bottom view, right is the top view; red represents the strongest positively charged, blue represents the strongest negatively charged) and hydrophobic forces (D, left is the side view, right is the bottom view; brown represents the strongest hydrophobic, blue represents the strongest hydrophilic) of cholesterol/β-CD docked complex for 2 : 1 host–guest stoichiometry.

Scheme 2 The chemical structure of cholesterol.

3.3. Characterization of the β-CD/PNAANI/Gra/GCE film

The reduction of the GO was characterized by UV-visible spectroscopy, FTIR spectroscopy, and Raman spectroscopy, respectively. Initially, UV-visible spectroscopy was used to study the reduction of GO. As shown in Fig. 2A, the GO shows a strong absorption at 230 nm and a shoulder at 300 nm, which correspond to the π–π* transition of the aromatic C=C bond and the n–π* transition of the C=O bond, respectively. After reduction, the peak at 230 nm shifts to 260 nm indicating the restoration of the π-conjugation network of the Gra. The disappearance of the peak at 300 nm reflects the effect of deoxygenation. To further illustrate the formation of Gra, FTIR spectra were employed to investigate the reduction of GO. Fig. 2B shows the FTIR spectra of the GO and Gra. As observed, the GO displays several characteristic bands at approximately 3425, 1724, 1617, 1213, and 1052 cm⁻¹, which are caused by the stretching vibrations of –OH, C=O, C=C, C–O–C, and C–O, respectively. After reduction, the band at 1724 cm⁻¹ corresponding to the C=O stretch vanishes, and the bands at 3425 and 1213 cm⁻¹ decreases dramatically, confirming that the GO was reduced to Gra by AA. Raman spectroscopy is one of the most widely used techniques to characterize the structural and electronic properties of Gra including disordered and defective structures, defect density, and doping levels. Fig. 2C shows the typical Raman spectra of GO and Gra. As expected, GO displays two prominent peaks at 1360 and 1602 cm⁻¹ corresponding to the D and G bands, respectively. The Gra shows two prominent peaks at 1355 and 1589 cm⁻¹, corresponding to the breathing mode of k-point phonons of A_1g symmetry (D band) and the E_2g phonons of C sp² atoms (G band) of Gra, respectively. The intensity ratio of the D band to the G band (I_D/I_G) is clearly higher when compared with that of GO (0.97 vs. 0.78), suggesting a decrease in sp² domains and a partially ordered crystal structure of Gra induced by AA reduction.

Fig. 2 UV-vis absorption spectra (A), FTIR spectra (B), and Raman spectra (C) of GO and Gra.

Because the PNAANI and β-CD were immobilized on the Gra/GCE by electro-polymerization electro-oxidation, respectively. It is difficult to characterize by FTIR. Thus, the Gra/GCE, PNAANI/Gra/GCE, and β-CD/PNAANI/Gra/GCE films were investigated using SEM. As shown in Fig. 3A, the microstructure image reveals that after reduction the Gra material consists of randomly aggregated thin, wrinkled sheets closely associated with each other. It is noted that after polymerization of NAANI on the surface of Gra/GCE, a layer of PNAANI was densely covered on the surface of Gra/GCE (Fig. 3B). Besides, it can be seen from Fig. 3C that a layer of β-CD thin films was uniformly covered on the surface of PNAANI/Gra/GCE, indicating that the β-CD/PNAANI/Gra/GCE film was successfully obtained.

Fig. 3 SEM images of Gra/GCE (A), PNAANI/Gra/GCE (B), and β-CD/PNAANI/Gra/GCE (C).

3.4. Electrochemical characterization of the modified electrodes

EIS was performed at the potential of 0.1 V and the frequency ranges was from 10¹ to 10⁵ Hz, using 2.0 mM [Fe(CN)₆]^3−/4− redox couple (1 : 1) with 0.1 M KCl as supporting electrolyte. The value of the electron-transfer resistance (R_ct) of the modified electrode was estimated by the semicircle diameter. Fig. 4A illustrates the EIS of the bare GCE, Gra/GCE, β-CD/PNAANI/GCE, and β-CD/PNAANI/Gra/GCE. Obviously, the bare GCE exhibited a semicircle portion and the value of R_ct was estimated to be 750 Ω. While the R_ct decreased remarkably at Gra/GCE, indicating that Gra had good conductivity and improved obviously the diffusion of ferricyanide toward the electrode interface. In the case of β-CD/PNAANI/GCE, its R_ct further increased to 1500 Ω due to the poor conductivity of β-CD, suggesting that large amount of β-CD molecules were successfully immobilized on the surface of PNAANI/GCE. Furthermore, the R_ct of the β-CD/PNAANI/GCE is much larger than that of the β-CD/PNAANI/Gra/GCE, although the introduction of β-CD results in the increase of the semicircle diameter. These results indicate that the Gra film can significantly accelerate the interfacial charge transfer between the electrochemical probe [Fe(CN)₆]^3−/4− and the modified electrode, which will be beneficial to the good analytical performance of the electrode.

Fig. 4 (A) EIS characterization of GCE, Gra/GCE, β-CD/PNAANI/GCE, and β-CD/PNAANI/Gra/GCE using 2.0 mM [Fe(CN)₆]^3−/4− redox couple (1 : 1) with 0.1 M KCl as supporting electrolyte. (B) DPV responses of the Gra/GCE (a), PNAANI/Gra/GCE (b), β-CD/PNAANI/GCE (c), and β-CD/PNAANI/Gra/GCE (d) in 0.1 M pH 7.0 PBS after incubation with 1.0 mM MB. (C) DPV response of the β-CD/PNAANI/Gra/GCE in 0.1 M pH 7.0 PBS (a), incubated in 1.0 mM MB solution for 25 min (b), and further incubated in 10 μM cholesterol solution for 30 min then tested in 0.1 M pH 7.0 PBS (c).

Fig. 4B shows the DPV responses of different electrodes in 0.1 M pH 7.0 PBS after incubation with 1.0 mM MB. On the Gra/GCE (curve a), an oxidation peak was observed due to the non-specific adsorption of MB on Gra via π–π stacking. However, there is no oxidation peak of MB on the PNAANI/Gra/GCE (curve b), indicating that the adsorption of MB was restrained by PNAANI film. Furthermore, on the β-CD/PNAANI/Gra/GCE (curve d), the oxidation peak current of MB increases obviously due to the good molecular recognition property and high enrichment capability of β-CD compared to all the other electrodes. As a comparison, the DPV response of MB on the β-CD/PNAANI/GCE was also investigated (curve c). It is noted that the peak current is smaller than that on the β-CD/PNAANI/Gra/GCE. These imply that the large specific surface area and good electron transfer property of Gra are important to improve the electrochemical performance of the modified electrode. In general, it can be concluded that the introduction of Gra film is beneficial to enhancing the electron transfer of the electrochemical sensor, and the PNAANI film can avoid successfully the non-specific adsorption of MB on Gra film via π–π stacking interaction.

3.5. Feasibility of the electrochemical sensor

To demonstrate the assay feasibility of the proposed sensor, DPV response of the β-CD/PNAANI/Gra/GCE was investigated in 0.1 M pH 7.0 PBS. As can be seen from Fig. 4C, no detectable signal (curve a) is observed for the β-CD/PNAANI/Gra/GCE in 0.1 M pH 7.0 PBS due to the absence of the redox mediator MB. After incubated in 1.0 mM MB solution for 25 min, the MB-bound β-CD/PNAANI/Gra/GCE was then tested in 0.1 M pH 7.0 PBS and an obvious oxidation peak of MB (curve b) can be observed at about −0.1 V. When the β-CD/PNAANI/Gra/GCE was first incubated in 1.0 mM MB solution for 25 min and further incubated in 10 μM cholesterol solution for 30 min, then tested in 0.1 M pH 7.0 PBS, a decreased oxidation peak (curve c) was obtained due to competitive association of cholesterol/MB to the β-CD occurs. This is because cholesterol has higher binding affinity to β-CD cavity due to its hydrophobic nature. This suggests that the MB molecules present inside the β-CD/PNAANI/Gra/GCE host can be replaced by cholesterol and the MB-bound β-CD/PNAANI/Gra/GCE can be used to sensitively detect cholesterol by the competitive electrochemical sensing strategy.

3.6. Optimization of experimental conditions

Several control experiments were carried out to determine the optimum reaction conditions. Fig. 5A shows the effect of the potential cycle number of the electrodeposition of PNAANI film on the DPV peak current of the PNAANI/Gra/GCE in 0.1 M pH 7.0 PBS after incubation in 1.0 mM MB solution. It is noted that the oxidation peak current of MB decreases with an increase of the cycle number and reaches about 0 at 25 cycles, indicating that the surface of Gra/GCE is covered completely by PNAANI film and that the PNAANI/Gra/GCE can successfully avoid the non-specific adsorption of MB on the surface of Gra by π–π stacking interactions. Therefore, a cycle number of 25 was selected for further study. Moreover, the effect of the electro-oxidation time of β-CD on the DPV peak current of the β-CD/PNAANI/Gra/GCE was also investigated (Fig. 5B). Due to the host–guest interaction between MB and β-CD, it was found that the peak current of MB increases with the increase of the deposition time and a maximum is observed at 12 min. This suggests that the immobilized amount of β-CD is at a maximum at 12 min. Thus, this time was selected as the optimum electro-oxidation time for β-CD on the PNAANI/Gra/GCE. In addition, the incubation time of the modified electrode in MB solution is one of the key parameters that will affect the response performance of the electrode. Fig. 5C shows that the oxidation peak current of the electrode increases with the increase of the incubation time and reaches a maximum at 25 min for MB incubation. Therefore, 25 min is selected as the optimum incubation time for the as-prepared electrode in MB solution. Similarly, the effect of the incubation time of the modified electrode in cholesterol solution was also studied. Fig. 5D shows that the oxidation peak current of the electrode decreases with the increase of the incubation time and reaches about 0 at 30 min for cholesterol incubation. Therefore, 30 min is selected as the optimum incubation time for the as-prepared electrode in cholesterol solution.

Fig. 5 Effects of the cycle number of the CV deposition of PNAANI (A) and the electro-oxidation time of β-CD (B) on the DPV peak currents of the PNAANI/Gra/GCE in 0.1 M pH 7.0 PBS after incubation in 1.0 mM MB solution. Effects of incubation time on the DPV peak currents of the β-CD/PNAANI/Gra/GCE in 0.1 M pH 7.0 PBS after incubation in 1.0 mM MB solution (C) and further incubated in 50 μM cholesterol solution (D).

3.7. Quantitative analysis of the electrochemical sensor toward cholesterol

Under optimal conditions, DPV was used to determine the concentrations of cholesterol because it is a highly sensitive and low-detection limit electrochemical method. Fig. 6A shows the DPV curves of electrochemical signal on the MB-bound β-CD/PNAANI/Gra/GCE under different concentrations of cholesterol solution. The oxidation peak currents of MB decreased with increased cholesterol concentrations. Fig. 6B shows the corresponding calibration curve for cholesterol quantification. The peak currents were proportional to the cholesterol concentrations between 1.00 and 50.00 μM with a detection limit of 0.50 μM (S/N = 3). The corresponding regression equation was calculated as ΔI (μA) = 0.154C (μM) + 0.588 with correlation coefficients of 0.998. Detection limit was less than 1.0 μM which was quite low and satisfactory with respect to other recently reported articles. Table 2 illustrates few of the recent literatures on cholesterol sensing platforms, through both enzymatic and non-enzymatic sensing routes. The detection limit and sensitivity of the present sensing strategy is comparatively better than the reported ones.

Fig. 6 (A) DPV curves of the proposed sensing platform under different concentrations of cholesterol. (B) Calibration curves for the determination of cholesterol using the proposed sensor. The error bars represent the standard deviations of three parallel tests. (C) Interference studies using different species in the developed cholesterol detection method, using DPV and keeping all the parameters constant. The cholesterol concentration is 30 μM against the concentration of all other substances, which is kept at 2.0 mM.

Table 2 Comparison of the present work with other recent literatures, using various electrode or matrix for cholesterol sensing

Electrode or matrix	Method	Liner range (μM)	LOD (μM)	Ref.
Nafion/ChOx/GNPs-MWCNTs/GCE	DPV	10.0–5000.0	4.3	19
Chit–Hb/Chit–ChOx	Amperometry	10.0–600.0	9.5	20
ChEt–ChOx/ZnO–CuO/ITO/glass	CV	500.0–12 000.0	500.0	1
ChOx/PANI/PVP/graphene	Amperometry	50.0–10 000.0	1.0	21
ChOx/nano-ZnO/ITO	CV	130.0–10 360.0	13.0	22
ChOx/ZnO(T)/CT/GCE	CV	400.0–4000.0	200.0	23
Nafion/ChOx/Fe2O3	CV	100.0–8000.0	18.0	24
AuE/dithiol/AuNPs/MUA/ChOx	CV	40.0–220.0	34.6	5
Grp/β-CD/methylene blue	DPV	1.0–100.0	1.0	17
Grp/β-CD/rhodamine 6G	Fluorescence	5.0–30.0	5.0	3
β-CD/PNAANI/Gra/GCE	DPV	1.0–50.0	0.50	This work

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3.8. Selectivity, reproducibility, and stability

As we know, human blood serum contains many more biocomponents like salts, amino acids, carbohydrates, lipids etc., those can interfere with cholesterol detection and hamper the selectivity of the electrochemical sensor. Therefore, we have tested interference from common molecules present in human blood serum and found very negligible interference. As shown in Fig. 6C, some salts, carbohydrates, protein, anionic surfactant, etc. including glucose, AA, bovine serum albumin (BSA), sodium dodecyl sulphate (SDS), NaCl, KCl, and, MgCl₂ showed negligible interference even at the concentration of 2.0 mM, compared to cholesterol detected for only 30 μM concentration. Six equal MB-bound β-CD/PNAANI/Gra/GCEs were used to evaluate the fabrication reproducibility of the present method for cholesterol detection. The six modified electrodes exhibited similar signals with a relative standard deviation of 3.9%, indicating satisfactory reproducibility. Additionally, a long-term stability experiment was performed intermittently (every 5 days) and used to examine the stability of the MB-bound β-CD/PNAANI/Gra/GCE. The constructed sensor was stored in a refrigerator at 4 °C when not in use. Initial responses of over 94.3% and 85.6% remained after storage for 15 and 30 days, respectively, indicating an acceptable stability of the MB-bound β-CD/PNAANI/Gra/GCE.

3.9. Real sample analysis

The proposed method was used to detect cholesterol in serum samples using standard addition methods to evaluate the feasibility of the MB-bound β-CD/PNAANI/Gra/GCE for real sample analysis. The serum sample was diluted fifty times with 0.1 M pH 7.0 PBS. Results showed recoveries ranging from 98.4% to 105.2% and RSDs ranging from 2.6% to 4.5% (Table 3). The results demonstrated that this method can be extended for cholesterol detection in blood.

Table 3 Determination of cholesterol in human serum samples (n = 3)

Sample	Added (μM)	Founded (μM)	RSD (%)	Recovery (%)
1	5.0	4.92 ± 0.22	4.47	98.4
2	10.0	10.15 ± 0.35	3.45	101.5
3	20.0	21.03 ± 0.54	2.59	105.2

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The proposed sensing platform may also be expanded to wide and potential applications in biological and environmental samples. It is worthy note that, as an oligosaccharide, β-CD is more stable than cholesterol selective enzymes (mostly oxidase) under complex conditions. Thus the present sensing platform seems to be more suitable for analysis of practical cholesterol samples than traditional enzyme-based biosensor.

4. Conclusions

In conclusion, based on a competitive host–guest interaction between β-CD and signal probe/target molecules, a new electrochemical approach for cholesterol sensing using β-CD/PNAANI/Gra-modified electrode was developed. Due to the good electron transfer property of the Gra, the excellent inhibiting ability of PNAANI film for the non-specific adsorption of MB, and the excellent host–guest recognition of β-CD, the developed β-CD/PNAANI/Gra/GCE displays excellent analytical performance for the electrochemical detection of cholesterol: the linear response range is 1.00–50.00 μM and the LOD is 0.50 μM (S/N = 3). In addition, the developed electrochemical sensing platform is important as it does not use any enzyme or antibody for detection of cholesterol efficiently with outstanding selectivity over the common interfering species. Besides, the host–guest interaction between cholesterol and β-CD was studied by molecular modeling calculations, which revealed that the complexation could reduce the energy of the system and the complex of 2 : 1 host–guest stoichiometry had the lowest ΔE value of −10.45 kcal mol⁻¹. The molecular docking studies suggested that hydrogen bonding, electrostatic interactions, and hydrophobic interactions should be the predominant driving forces for the formation of the inclusion complex.

Acknowledgements

This work was supported by the Natural Science Foundation of China (31160334) and the Natural Science Foundation of Yunnan Province (2012FB112, 2014RA022), People's Republic of China.

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Footnote

† These authors contributed equally to this work.

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