A biocompatible cerasome based platform for direct electrochemistry of cholesterol oxidase and cholesterol sensing

Abstract

Cerasomes are a novel organic–inorganic hybrid material composed of spherical lipid bilayer vesicles with an internal aqueous compartment, which are similar to liposomes formed from phospholipids, with an inorganic silicate framework covering the vesicular surface. In this research, an anionic cerasome formed from N,N-dihexadecyl-N′-[(3-triethoxysilyl)propyl]urea was successfully prepared, and the cerasomes were characterized by scanning electron microscopy (SEM), behaving as a spherical-like structure. An electrochemical platform was constructed using a combination of the cerasomes and cholesterol oxidase (ChOx) on a glassy carbon electrode. Ultraviolet-visible (UV-vis) spectroscopy was used to monitor the assembly process and the electrochemical impedance spectroscopy (EIS) results demonstrated that ChOx had been successfully immobilized. The obtained enzyme-modified electrode exhibited both the effective direct electron transfer between the enzyme and electrode surface and the excellent electrochemical catalytic activity towards cholesterol with a wide linear range from 5.0 × 10⁻⁶ to 3.0 × 10⁻³ mol L⁻¹ and a low detection limit 1.7 × 10⁻⁶ mol L⁻¹ (S/N 3). The excellent catalytic performance of the modified electrode is attributed to the good biocompatibility of the cerasomes, which can provide a soft and morphologically stable interface for enzymatic immobilization, allowing the enzyme to retain its catalytic activity, along with their specific affinity (K_m 0.139 mmol L⁻¹) for water-insoluble cholesterol. The results indicate that cerasomes are useful as a platform for electrochemical sensing of cholesterol and have the potential to immobilize enzymes for bioelectrochemical applications.

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Introduction

In biological systems, cellular membranes play important roles in physiological processes. Liposomes are a kind of vesicular biomimetic cell membrane with good biological compatibility and bionic membrane structure that have been widely used in drug delivery and biosensing.^1–3 Liposomes are considered to be a promising biomimetic support matrix for active species. However, because of the fluidity of the lipid molecules in a membrane, the structure of liposomes tends to be unstable.⁴ Upon immobilization, liposomes tend to fracture and fuse with other vesicles to form a planar lipid bilayer membrane on a solid surface, which restricts their application in many fields.^5–10 For instance, for liposomes in electrochemical biosensors, the morphological change from a spherical structure to planar bilayer on the electrode surface results in a decrease of electric conductivity. Therefore, maintaining the vesicular structure of liposomal membranes to mimic the molecular interactions in cell membranes to express biological functions remains a challenge.¹¹

Kikuchi et al.^12,13 developed cerasomes, which are a novel kind of organic–inorganic nanohybrid material composed of a spherical lipid bilayer membrane with an internal aqueous phase similar to liposomes and then the surface of the lipid bilayer is covered with an ultrathin inorganic silicate framework. The inorganic framework increases the morphological stability of cerasomes compared with that of conventional liposomes.^14,15 Cerasomes have been widely applied in drug delivery,^16–18 gene transfection^19–21 and biomimetic functionalization for molecular information processing.²² Cerasomes maintain their vesicular forms not only in aqueous media but also on solid surfaces,^23,24 whereas conventional liposomes cannot retain their vesicular structures formed in aqueous media after transfer onto a solid surface. Like liposomes, cerasomes are expected to provide a biocompatible interface to immobilize biomolecules. By utilizing these advantages of cerasomes, we recently developed electrochemically active cerasomes on electrode surfaces.^25,26 That is, we were able to immobilize redox-active functional molecules such as a hydrophobic vitamin B₁₂ derivative and horse radish peroxidase on cerasomes through noncovalent interactions, and the resulting hybrid cerasomes exhibited good electrochemical performance on a glassy carbon electrode (GCE). Our results suggest that cerasomes provide a soft interface suitable to allow electrochemical sensing of various biologically important molecules.

As well known, cholesterol is an essential material in animal tissue, and is widely found in the kidneys, spleen, liver and bile, which have important physiological functions.^27,28 When there is excess cholesterol in the body, hypercholesterolemia occurs, which has adverse effects and can result in diseases such as atherosclerosis, venous thrombosis, myocardial infarction and gallstones.²⁹ Therefore, the development of novel methods for cholesterol determination has attracted wide attention.^30–32 In particular, technologies that allow simple, rapid and highly sensitive detection of cholesterol need to be developed. Electrochemical biosensors based on cholesterol oxidase (ChOx) that measure cholesterol concentration in solution exhibit high selectivity and convenience, making them a promising method to detect cholesterol.^28,33 In electrochemical biosensing based on enzymes, the support matrix use to immobilize the enzyme on the electrode surface is very important to obtain good detection performance. Many endeavors have been made to develop conformable support matrices for enzymatic immobilization to maintain biological activity and augment the performance of biosensors.^34–37 Singh et al.³⁸ synthesized an ultrafine monodispersed cuprous oxide (Ufm-Cu₂O) nanoparticles by a facile wet chemical method using poly-N-vinylpyrrolidone (PVP) as a capping agent. This colloidal solution of Ufm-Cu₂O and chitosan (CS) were electrophoretically deposited (EPD) onto an indium tin-oxide (ITO) glass substrate. This novel biomedical nanocomposite platform has been explored to fabricate a cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto the Ufm-Cu₂O–CS/ITO electrode surface. The proposed biocompatible ChEt–ChOx/Ufm-Cu₂O–CS/ITO bioelectrode shows fast response time (<5 s), good reproducibility, and long-term stability. Kumar et al.³⁹ developed an electrochemical cholesterol biosensor, which was fabricated via covalent immobilization of ChOx and ChEt onto a biocompatible self assembled monolayer (SAM) of (3-glycidoxypropyl)trimethoxysilane (GPTMS) fabricated onto an indium tin oxide (ITO) electrode. Thus the biosensing electrode can be used to estimate the total cholesterol from 1.5 to 6.1 mmol L⁻¹, with a sensitivity of 0.351 mA (mg dL⁻¹)⁻¹, and can be used more than 10 times with a shelf life of up to 10 weeks; Singh et al.⁴⁰ developed a new hybrid nanocomposite based on hydrothermally synthesized nanostructured NiFe₂O₄ (n-NiFe₂O₄) and chitosan (CH), which has been explored for bienzyme (ChEt and ChOx) immobilization for application as total cholesterol biosensor.

Herein, negatively charged cerasomes composed of N,N-dihexadecyl-N′-[(3-triethoxysilyl)propyl]urea (1) are prepared in aqueous media, and ChOx is immobilized on a GCE modified with the cerasomes through hydrophobic interactions (Fig. 1). The electrochemical catalytic activity of the obtained enzyme-modified electrode towards cholesterol is assessed by comparison with the performance of the corresponding enzyme-modified electrodes in which the cerasomes are replaced by liposomes formed from phospholipids and silica (SiO₂) nanoparticles. Cerasomes acted as ChOx immobilized matrix with the good structural stability and biocompatibility, were modified on the surface of electrode to fabricate the biocompatible interface. The nature structure and electrochemical catalytic activity of immobilized ChOx were primely maintained owing to the good biocompatibility. In addition, the liposomal bilayer membrane structure of cerasomes probably has specific affinity for water-insolube substrate cholesterol which was advantageous to the enzyme catalytic reaction. The direct electron transfer was well realized between ChOx and electrode surface. Hence, a novel way to broaden the application of cerasomes and a new platform to sensitive determination of cholesterol were developed.

Fig. 1 Schematic illustration of the preparation of a glassy carbon electrode modified with cerasomes and cholesterol oxidase.

Experimental

Materials

ChOx (EC 1.1.3.6, 500U), cholesterol esterase (CHE, EC 3.1.1.13, 500U), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DMPG) were purchased from Sigma. Cholesterol, 1-hexadecylamine, 1-bromohexadecane, (3-triethoxysilyl)propyl isocyanate, Triton X-100, isopropanol, ascorbic acid (AA), glucose (Glc), uric acid (UA) and lactic acid (LA) were obtained from Alfa Aesar and used without further purification. Phosphate buffer solution (PBS; 0.1 mol L⁻¹) was prepared from stock solutions of 0.2 mol L⁻¹ NaH₂PO₄ and 0.2 mol L⁻¹ Na₂HPO₄. Human serum samples were obtained from normal healthy volunteers at the Affiliated Hospital of China Medical University, from whom informed consent was obtained respectively, and all the experiments were performed in accordance with approval from the Ethics Committee of China Medical University. All aqueous solutions were prepared with ultrapure water. A stock solution of 0.1 mmol L⁻¹ cholesterol was prepared in PBS at pH 7.0 containing 0.5% (v/v) isopropanol and 0.2% (v/v) Triton X-100 by heating in a water bath at 60 °C. The cholesterol solution was stored at 4 °C until use.

Apparatus and measurements

Ultraviolet-visible (UV-vis) absorption spectra were measured by a spectrophotometer (Lambda 25, Perkin Elmer, USA) in the range from 250 to 600 nm. Fourier transform infrared (FT-IR) spectra were recorded using a FT-IR spectrophotometer (Thermo Nicolet 5700, Nicolet, USA) with an average of 100 scans and resolution of 4 cm⁻¹ in KBr pellets. Morphological studies were performed on a scanning electron microscope (SEM) (SU8010, Hitachi, Japan) and an atomic force microscope (AFM; 5100, Agilent, USA). Dynamic light scattering (DLS) and zeta-potential measurements were conducted using a particle analyzer (Zetasizer Nano-ZS, Malvern, England). The specific surface area was measured using Tristar 3020 volumetric adsorption analyzers manufactured by Micromeritics (Norcross, GA).

Cyclic voltammetry was performed on an electrochemical workstation (BAS100B, Bioanalytical Systems Inc., USA). All electrochemical experiments used a conventional three-electrode system consisting of a modified GCE as the working electrode, a platinum wire as the counter electrode and an Ag/AgCl electrode as the reference electrode. All electrochemical experiments were carried out in PBS at pH 7.0 deaerated by highly purified N₂ gas for at least 30 min prior to measurement. Impedance measurements were carried out on an electrochemical workstation (CHI 660E, Shanghai Chenhua Instruments Co., China) in the frequency range of 0.1 Hz to 100 kHz using an ac voltage of 5 mV in 5.0 mmol L⁻¹ K₃[Fe(CN)₆]/K₄[Fe(CN)₆] solution containing 0.1 mol L⁻¹ KCl.

Preparation of cerasomes, liposomes and silica nanoparticles

1 was prepared from (3-triethoxysilyl)propyl isocyanate via one-step condensation with dihexadecylamine.¹⁴ Cerasomes were prepared from lipid 1 by the ethanol sol injection method according to previous reports.^14,41 Lipid 1 was hydrolyzed with a mixture of EtOH, H₂O and HCl using a vortex agitator at room temperature. The molar ratio of 1/EtOH/H₂O/HCl was 1 : 200 : 19 : 0.03. The hydrolyzed lipid solution (30 μL) was injected into ultrapure water (5 mL) to give a lipid concentration of 0.5 mmol L⁻¹. The solution was incubated for 24 h to form the polysiloxane surface network structure. The SiO₂ nanoparticles and liposomes formed from DMPC/DMPG with a molar ratio of 7 : 3 were prepared according to reported methods.^42,43

Fabrication of modified electrodes

A GCE modified with ChOx and cerasomes (denoted ChOx/cerasome/GCE) was fabricated as shown in Fig. 1. First, a bare GCE was successively polished with 1.0, 0.3 and 0.05 μm alumina slurry. The GCE was thoroughly rinsed with ultrapure water to remove the alumina residue, followed by sonication in acetone, ethanol and ultrapure water sequentially. To prepare ChOx/cerasome/GCE, cerasome aqueous dispersion (7 μL, 0.5 mmol L⁻¹) was added onto the surface of the pretreated GCE with a microsyringe. The water in the dispersion was evaporated slowly in air by covering the electrode with a beaker. The cerasome-modified electrode was immersed in PBS containing 0.5 mg mL⁻¹ ChOx at pH 4 overnight to obtain ChOx/cerasome/GCE. The isoelectric point value of the enzyme is 4.4–5.1.²⁸ Therefore, ChOx solution was positive charged in the pH 4.0 PBS. The driving force of adsorption between ChOx and cerasome/GCE is mainly the electrostatic interaction. For comparison, SiO₂/GCE, liposome/GCE, ChOx/SiO₂/GCE, ChOx/liposome/GCE were prepared by similar procedures to that described above.

Results and discussion

Morphological characteristics of cerasomes

The morphology of the cerasomes formed with lipid 1 in aqueous media and on a solid surface was characterized by DLS and SEM, respectively, and compared with those of SiO₂ nanoparticles and liposomes formed from DMPC/DMPG (Fig. 2). The hydrodynamic diameters of the cerasomes, SiO₂ nanoparticles and liposomes in aqueous media evaluated by DLS were 290, 270 and 220 nm, respectively, so these particles are comparable in size. The zeta potentials of the aqueous dispersions of cerasomes, SiO₂ nanoparticles and liposomes at pH 7.0 were −40.7, −34.6 and −41.7 mV, respectively, indicating that the surfaces of these particles are negatively charged to a similar degree.

Fig. 2 Particle size distribution evaluated by (a–c) DLS and (d–f) zeta potentials of cerasomes formed from lipid 1, SiO₂ nanoparticles and liposomes formed from DMPC/DMPG (7 : 3 mol mol⁻¹) in aqueous dispersion states at pH 7.0, respectively, and (g–i) SEM images after casting aqueous dispersions of the materials on a silicon chip and subsequent drying. Concentrations: 1 = 0.5 mmol L⁻¹, SiO₂ = 0.03 mg mL⁻¹ and phospholipid = 0.5 mmol L⁻¹.

While the size and surface charge state of the present cerasomes, SiO₂ nanoparticles and liposomes were analogous in aqueous media, marked differences were observed when they were transferred onto solid surfaces. A SEM image of the cerasomes obtained after casting the aqueous dispersion on a silicon chip and subsequent drying revealed that the cerasomes maintained their spherical shape in a dry state on the solid surface. The SiO₂ nanoparticles also maintained their spherical shape when dried on a solid surface. On the contrary, the vesicular structure of the liposomes in aqueous media ruptured to form a planar lipid bilayer membrane on the solid surface, which is typical for conventional liposomal membranes.⁴⁴ The specific surface area and the total pore volume of cerasomes were 0.05 m² g⁻¹ and 0.02 cm³ g⁻¹ respectively, according to Brunauer–Emmett–Teller (BET) method. The pores came from the stack of the cerasomes. These results indicate that the cerasomes possess much higher morphological stability than the liposomes, allowing them to keep their vesicular structure even on a solid surface. In FT-IR spectra of the cerasomes in the dry state, the stretching bands originating from Si–OH and Si–O–Si bonds were observed at 950 and 1100 cm⁻¹, respectively, indicating that the cerasomes possess siloxane frameworks like those of SiO₂ nanoparticles on their vesicular surface.¹⁵

For electrochemical biosensing, a supporting matrix with good conductivity and biocompatibility is needed to modify the electrode surface to allow attachment of redox-active biomolecules. In general, liposomes have good compatibility with biomolecules, but the planar lipid bilayer structure formed from liposomes on the electrode surface acts as an insulating layer that hinders electron transfer between the redox-active biomolecules and electrode. SiO₂ nanoparticles have been also used as a support material for redox-active biomolecules, but their biocompatibility is poor because they cause denaturation of biomolecules. It is hoped that the cerasomes can combine the biocompatibility of liposomes and conductivity of SiO₂ to achieve high-performance electrochemical biosensing.

Fabrication of ChOx/cerasome/GCE

ChOx is a bifunctional enzyme containing flavin adenine dinucleotide (FAD) as a cofactor that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one and reduces oxygen (O₂) to hydrogen peroxide (H₂O₂) as a by-product. ChOx binds to lipid bilayers mainly through hydrophobic interactions.⁴⁵ Because cerasomes provide a biocompatible interface to bind enzymes such as nicotinamide adenine dinucleotide-dependent L-lactate dehydrogenase and horse radish peroxidase,^22,26 ChOx should also be immobilized on the cerasomes formed using lipid 1, while keeping its catalytic activity.

UV-vis absorption spectra of the cerasomes, ChOx and ChOx immobilized on the cerasomes (ChOx/cerasome), whose aqueous solutions were cast on a quartz slide and then dried, are shown in Fig. 3A. The cerasomes has no obvious absorption peak in the wavelength region over 250 nm, but weak turbidity arising from the Tyndall effect. Meanwhile, ChOx exhibited a strong absorption from the protein and cofactor at 275 nm and weak absorptions from the cofactor around 350 and 450 nm. The absorption spectra of the ChOx/cerasome composite contained absorptions similar to those of ChOx alone, indicating that ChOx was immobilized on the cerasomes without denaturation.

Fig. 3 (A) UV-vis absorption spectra of cerasomes formed with lipid 1 (dotted line), cholesterol oxidase (ChOx) (dashed line) and ChOx/cerasome (solid line) on a quartz slide. (B) Electrochemical impedance spectra of (a) bare glassy carbon electrode (GCE), (b) cerasome/GCE and (c) ChOx/cerasome/GCE in 5.0 mmol L⁻¹ K₃[Fe(CN)₆]/K₄[Fe(CN)₆] solution containing 0.1 mol L⁻¹ KCl.

The morphology of prepared ChOx/cerasome/GCE was characterized by SEM and AFM in Fig. S1.† As can be seen, after the immobilization of ChOx onto the cerasome/GCE, the spherical morphology of cerasome changed into homogeneous aligned structures which may be formed by the assembling of globular ChOx molecules on the cerasomes.

Electrochemical impedance spectroscopy (EIS) is an effective method to investigate the electron transfer properties of modified electrodes.⁴⁶ Fig. 3B shows the Nyquist plots of a bare GCE, cerasome/GCE and ChOx/cerasome/GCE recorded in 0.1 mol L⁻¹ aqueous KCl solution containing 5 mmol L⁻¹ [Fe(CN)₆]^3−/4− as a redox couple probe. In the Nyquist plots, Z_re and Z_im are the real and imaginary components of the complex impedance, and the electron transfer resistance (R_et) can be estimated from the semicircle diameter. We found that R_et of cerasome/GCE was much higher than that of the bare GCE, indicating that the electrode was successfully modified with the cerasomes. Furthermore, R_et of ChOx/cerasome/GCE was further increased compared with that of cerasome/GCE. This reveals that the integration of the protein on the surface of cerasome/GCE inhibited the electron transfer of the Fe(CN)₆^3−/4− redox couple from the solution to the electrode. The results indicate that ChOx was successfully self-assembled on cerasome/GCE.

Electrochemical redox behavior of enzyme-modified electrodes

The electrochemical behavior of cerasome/GCE, SiO₂/GCE and liposome/GCE modified with ChOx was investigated by cyclic voltammetry. First, cyclic voltammograms of these electrodes without ChOx were recorded in O₂-free PBS at pH 7.0 (Fig. 4A). No clear redox peak was observed at a scan rate of 200 mV s⁻¹, which indicated that the cerasomes, liposomes and SiO₂ nanoparticles were not electroactive in the potential window between −0.8 and 0.2 V.

Fig. 4 Cyclic voltammograms of cerasome/glassy carbon electrode (GCE) (solid line), SiO₂/GCE (dotted line) and liposome/GCE (dashed line) (A) without and (B) with cholesterol oxidase in 0.1 mol L⁻¹ PBS at pH 7.0. Scan rate, 200 mV s⁻¹.

Although none of the modified electrodes without a redox enzyme gave a response, markedly different electrochemical behavior was observed for the individual electrodes after modification with ChOx. As shown in Fig. 4B, a pair of well-defined and nearly symmetrical redox peaks was observed for ChOx/cerasome/GCE, indicating that the enzyme assembled on the surface of cerasome/GCE retained its catalytic activity. Thus, direct electron transfer (DET) took place between the cofactor FAD in the active site of ChOx and the electrode surface without the assistance of an electron mediator. In contrast, no obvious redox peak was observed for ChOx/SiO₂/GCE or ChOx/liposome/GCE, demonstrating that the cerasomes aided DET more than the SiO₂ nanoparticles and liposomes alone. The increased DET in ChOx/cerasome/GCE compared with that in ChOx/SiO₂/GCE and ChOx/liposome/GCE presumably originates from the good biocompatibility of the vesicular cerasomes along with their moderate conductivity. In contrast, the SiO₂ nanoparticles with their bioincompatible rigid surface and liposomes that formed a ruptured, non-conductive planar lipid bilayer surface are unfavorable interfaces for the integration of ChOx while keeping its enzymatic activity.

The cathodic peak potential (E_pc) and anodic peak potential (E_pa) of ChOx/cerasome/GCE were −0.373 and −0.305 V, respectively, with a peak potential separation of 68 mV. These values suggest that the immobilized enzyme underwent a reversible electrochemical reaction. The formal potential (E_p), the average of E_pc and E_pa, was −0.339 V. Thus, the cerasomes provided an appropriate hydrophobic microenvironment for the immobilized ChOx to realize DET.

Effect of scan rate on the electrochemical behavior of ChOx/cerasome/GCE

The influence of scan rate on the voltammetric response of ChOx/cerasome/GCE was also investigated. Fig. 5A depicts cyclic voltammograms of ChOx/cerasome/GCE at different scan rates from 100 to 800 mV s⁻¹. The cathodic (I_c) and anodic (I_a) peak currents rose in proportion to the increase of scan rate (Fig. 5B). The linear regression equations were I_c = 0.008 − 0.979v (R = 0.999) and I_a = 0.038 + 1.162v (R = 0.998). These results indicate that the redox reaction of ChOx on the cerasome-modified electrode was a surface-controlled electrochemical process, exhibiting that the rate of the electrode reaction was slower than the diffusion velocity of the substance to the surface of electrode and the cerasomes did not hinder the diffusion process. Furthermore, the surface concentration (Γ*) of electroactive enzyme on ChOx/cerasome/GCE was calculated according to the charge integration of the cathodic peak.⁴⁷ According to Faraday's law, Q = nFAΓ*, in which Q is calculated by integrating the cathodic peak of ChOx; n is the number of electrons transferred; F is the Faraday constant; and A represents the area of the electrode surface. The calculated Γ* for ChOx/cerasome/GCE was 1.72 × 10⁻¹¹ mol cm⁻². The peak-to-peak separation (ΔE_p) of ChOx/cerasome/GCE was 68 mV at a scan rate of 200 mV s⁻¹. The electron transfer rate constant (k_s) of ChOx calculated by measuring the variation of peak potential with scan rate was 4.67 s⁻¹, which is larger than that reported for ChOx immobilized on gold nanoparticles (0.35 s⁻¹).⁴⁸

Fig. 5 (A) Cyclic voltammograms of cholesterol oxidase (ChOx)/cerasome/glassy carbon electrode (GCE) at scan rates of (a) 100, (b) 200, (c) 300, (d) 400, (e) 500, (f) 600, (g) 700 and (h) 800 mV s⁻¹ in 0.1 mol L⁻¹ PBS at pH 7.0. (B) Plots of oxidation peak current I_a (filled squares) and reduction peak current I_c (filled circles) versus scan rate for ChOx/cerasome/GCE.

Optimization of experimental conditions

The influence of pH on the voltammetric response of the modified electrode was determined to investigate the electrochemical mechanism of ChOx on the modified electrode. Cyclic voltammograms of ChOx/cerasome/GCE in PBS at different pH are presented in Fig. 6A. The cathodic and anodic peak potentials shifted to more negative potential as pH was increased from 4.0 to 8.0, implying that proton interchange occurred in the electrode reaction of ChOx. There was a linear relationship between the formal potentials and pH with a slope of −59.8 mV/pH (Fig. 6B), which is close to the theoretical value of −58.6 mV/pH.^32,49 This indicates that a reversible two-proton, two-electron transfer process between FAD and its reduced form (FADH₂) occurs in the enzymatic electrode reaction as follows:^32,50

ChOx(FAD) + 2H⁺ + 2e⁻ ⇌ ChOx(FADH₂) (1)

Fig. 6 (A) Cyclic voltammograms of cholesterol oxidase (ChOx)/cerasome/glassy carbon electrode (GCE) in N₂-saturated 0.1 mol L⁻¹ PBS at pH (a) 4.0, (b) 5.5, (c) 5.9, (d) 7.0 and (e) 8.0. Scan rate, 200 mV s⁻¹. (B) Relationships of the formal potential E_p (filled circles) and the cathodic peak current I_c (open circles) with pH. (C) Plots of the cathodic peak current (I_c) vs. the concentration of ChOx. The cerasome/GCE immersed in the different concentration of ChOx: 0.1, 0.3, 0.5, 0.8, 1.0 and 1.5 mg mL⁻¹.

In addition, the maximum response for the cathodic peak current was observed at pH 7.0 (Fig. 6B), and was comparable to those reported for immobilized ChOx entrapped in a variety of matrices.^34,51 Therefore, the following catalytic behavior was investigated at pH 7.0 to achieve optimum enzymatic activity.

Moreover, the effect of ChOx concentration applied for the construction of the electrode on the current response has been studied. As can be seen in Fig. 6C, the cathodic peak current of the ChOx/cerasome/GCE increased with the augmenter concentration of ChOx and then reached a maximum value at the concentration of 0.5 mg mL⁻¹. When the ChOx surpassed the concentration, the cathodic peak current started to slightly decrease, illustrating that the adsorption of enzyme was attained saturation near the concentration. Therefore, the concentration of ChOx was selected at 0.5 mg mL⁻¹ as the optimal condition for the preparation of ChOx/cerasome/GCE in all electrochemical experiments.

Electrochemical catalysis of cholesterol by ChOx/cerasome/GCE

Because O₂ participates in the ChOx-catalyzed reaction,⁵⁰ cyclic voltammetric measurements of ChOx/cerasome/GCE were carried out under N₂- and O₂-saturated conditions to evaluate the catalytic activity of immobilized ChOx toward cholesterol as a substrate. As illustrated in Fig. 7, a pair of well-defined redox peaks appeared in N₂-saturated PBS in the absence of substrate, which was ascribed to the DET of ChOx as mentioned above. In O₂-saturated PBS, the enzymatic reaction contends with the electrochemical oxidation of ChOx(FADH₂) and results in the decrease of oxidation peak current of ChOx(FADH₂). Afterwards, the ChOx(FAD) that are generated by the reaction with O₂ can be reduced on the surface of the modified GCE. So, the reduction peak current increased substantially, accompanied with the disappearance of the oxidation peak. These results indicate that the ChOx immobilized on the cerasomes remains high catalytic activity, as expressed by reaction (1) above and the following reaction (2).^32,50

ChOx(FADH₂) + O₂ → ChOx(FAD) + H₂O₂ (2)

Fig. 7 Cyclic voltammograms of cholesterol oxidase (ChOx)/cerasome/glassy carbon electrode (GCE) in (a) N₂-saturated and (b) O₂-saturated 0.1 mol L⁻¹ PBS at pH 7.0 without cholesterol, and (c) in O₂-saturated 0.1 mol L⁻¹ PBS at pH 7.0 with 1.625 mmol L⁻¹ cholesterol. Scan rate, 200 mV s⁻¹.

When 1.625 mmol L⁻¹ cholesterol was added to the O₂-saturated PBS, the reduction peak current obviously decreased, demonstrating that the immobilized ChOx on the cerasomes had good catalytic performance toward cholesterol.

The catalytic behavior of ChOx/cerasome/GCE toward different concentrations of cholesterol was studied in detail by cyclic voltammetry. Due to cholesterol can be primarily oxidized by ChOx(FAD) (reaction (3)), which would contend with the electrochemical reduction of ChOx(FAD). Therefore, the reduction peak current decreased with increasing cholesterol concentration in O₂-saturated PBS (Fig. 8A), indicating that the immobilized ChOx exhibited good catalytic activity toward the reduction of cholesterol. In contrast, no obvious catalytic reduction peak was observed for ChOx/cerasome/GCE in N₂-saturated PBS in the presence of cholesterol under similar experimental conditions. Thus, the catalytic process of the immobilized ChOx in the presence of cholesterol is described as follows:^32,50

Cholesterol + ChOx(FAD) → cholest-4-en-3-one + ChOx(FADH₂) (3)

Fig. 8 (A) Cyclic voltammograms of cholesterol oxidase (ChOx)/cerasome/glassy carbon electrode (GCE) in O₂-saturated 0.1 mol L⁻¹ PBS at pH 7.0 containing (a) 0, (b) 0.005, (c) 0.015, (d) 0.055, (e) 0.275 and (f) 2.825 mmol L⁻¹ cholesterol. Scan rate, 200 mV s⁻¹. (B) Dependence of the electrocatalytic current change ΔI before and after addition of cholesterol on cholesterol concentration for ChOx/cerasome/GCE. Inset: relationship between the cholesterol concentration/ΔI and cholesterol concentration. Scan rate, 200 mV s⁻¹.

The relationship between the electrocatalytic current change ΔI, which is the difference between the current without cholesterol and the current with cholesterol, at −0.38 V and the concentration of cholesterol (C_cholesterol) is displayed in Fig. 8B. The ΔI value increased linearly at lower cholesterol concentration and became saturated at higher cholesterol concentration. This result demonstrates that the present electrocatalytic behavior can be analyzed by Michaelis–Menten kinetics,⁵² namely, 1/ΔI = 1/ΔI_max + K_m/(ΔI_maxC_cholesterol), where ΔI_max and K_m are the maximum ΔI value when the concentration of cholesterol is saturated and the apparent Michaelis constant, respectively.

A linear relationship between C_cholesterol/ΔI and C_cholesterol was derived from the Michaelis–Menten equation; the data are plotted in the inset of Fig. 8B. Good linearity was obtained over the concentration range of cholesterol from 5.0 × 10⁻⁶ to 3.0 × 10⁻³ mol L⁻¹, indicating that ChOx/cerasome/GCE effectively catalyzed the enzymatic oxidation of cholesterol. The linear regression equation was C_cholesterol/ΔI = 0.004 + 0.218C_cholesterol with a low detection limit of 1.7 × 10⁻⁶ mol L⁻¹ (S/N 3) and high correlation coefficient of 0.999 (n = 15). The linear range observed for ChOx/cerasome/GCE is much wider than those of other cholesterol biosensors.^31,53,54 The value of K_m depends on various factors such as the properties of matrix and immobilized method of enzymes, etc. The K_m of immobilized ChOx was calculated to be 0.139 mmol L⁻¹ by Michaelis–Menten kinetics, which was much higher for the immobilized enzyme compared to the value for the soluble one, owing to a higher concentration of substrate was required to overcome the effects of the nanostructured film.⁵⁵ The small K_m value, compared with those reported in the literature (0.29 mmol L⁻¹, 0.64 mmol L⁻¹),^50,56 indicates the high affinity of the immobilized enzymes of ChOx/cerasome/GCE, which is attributed to the favorable orientation of ChOx. Appropriate orientation of the enzyme is important for improving electronic communication between the electrode surface and the immobilized enzyme, and improper orientation may lead to decreased active enzyme concentration on the electrode surface. The present biosensor research is concentrated upon developing favorable matrix using novel biocompatible materials that can fabricate a stable biosensor and improving the electron transfer between enzyme and the electrode.⁵⁷ The experimental results showed that the orientation of ChOx on the cerasomes is beneficial. The low K_m suggests that the cerasomes provide a biocompatible soft interface that allows ChOx to maintain its catalytic activity and an effective hydrophobic binding site for cholesterol to increase the local substrate concentration.

Interference study

Typical interferents ordinarily considered to accompany cholesterol are LA, UA, Glc and AA. Fig. 9 shows the time course of the reduction current at −0.38 V for ChOx/cerasome/GCE when 0.3 mmol L⁻¹ cholesterol, 0.2 mmol L⁻¹ LA, 0.2 mmol L⁻¹ UA, 2.0 mmol L⁻¹ Glc and 2.0 mmol L⁻¹ AA were sequentially added to PBS at pH 7.0. A fast, obvious response signal was obtained when cholesterol was added to the solution. In contrast, the current was not strongly influenced by the addition of any of these interferents. These results indicate that the ChOx/cerasome/GCE biosensor has high selectivity for cholesterol.

Fig. 9 Time course of amperometric current at −0.38 V for cholesterol oxidase (ChOx)/cerasome/glassy carbon electrode (GCE) in O₂-saturated 0.1 mol L⁻¹ PBS at pH 7.0 upon addition of 0.3 mmol L⁻¹ cholesterol, 0.2 mmol L⁻¹ lactic acid, 0.2 mmol L⁻¹ uric acid, 2 mmol L⁻¹ glucose and 2 mmol L⁻¹ ascorbic acid.

Detection of cholesterol in serum

To demonstrate its practical application, the present biosensor was used to detect the total cholesterol in human serum. Cholesterol mainly exists as free cholesterol and cholesterol ester in serum.⁵⁸ The total cholesterol content in serum is the sum of free and esterified cholesterol. Accordingly, before the detection, 0.5 mg mL⁻¹ CHE was added to a serum sample at 37 °C and incubated for 30 min to hydrolyze the esterified cholesterol. Then, 0.1 mL of the sample was added to PBS (10 mL) at pH 7.0 to ensure the cholesterol concentration was in the linear range of ChOx/cerasome/GCE. As shown in Table 1, the determined cholesterol concentrations of the serum samples were consistent with the values evaluated by utilizing automatic biochemical analyzer. The control experiment was carried on by adding various concentrations of cholesterol into the real serum samples. The recoveries were obtained in the range of 96–106%, indicated that the biosensor was satisfactory for the analysis of total cholesterol in the serum samples. Furthermore, the experimental results exhibited the substances that exist in the plasma did not significantly affect the detection process. Thus, ChOx/cerasome/GCE acts as a useful electrochemical sensor for biological samples.

Table 1 Application of the as-prepared biosensor in human serum

Sample	Added cholesterol (mmol L^−1)	Found cholesterol (mmol L^−1)	Recovery (%)	Hospital data (mmol L^−1)
1	—	5.77 ± 0.25	—	6.02
	0.2	5.96 ± 0.12	96.0
	0.5	6.29 ± 0.08	103.0
	0.8	6.62 ± 0.05	106.0
2	—	3.52 ± 0.09	—	3.61
	0.2	3.73 ± 0.14	105.0
	0.5	4.04 ± 0.09	104.0
	0.8	4.29 ± 0.07	96.3

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Repeatability and stability of ChOx/cerasome/GCE

The repeatability of the present enzyme modified electrode was evaluated by determining the responses to 0.5 mmol L⁻¹ cholesterol of six different modified electrodes under identical conditions. The relative standard deviation (RSD) for the reduction peak current was found to be 4.2%. The RSD of five successive determinations was 3.8%. Thus, ChOx/cerasome/GCE exhibited good reproducibility.

The stability of the enzyme-modified electrode was also investigated. The redox peak current of the enzyme in the absence of substrate showed no obvious change during successive sweeps in PBS at pH 7.0 for one week. The long-term stability of ChOx/cerasome/GCE was also evaluated by measuring its response to cholesterol after storage. The modified electrodes were stored in PBS at pH 7.0 and 4 °C when not in use. As shown in Fig. S2,† the response of ChOx/cerasome/GCE to 0.5 mmol L⁻¹ cholesterol remained at nearly 97% and 94% of the initial response after 15 days and 1 month, respectively. Accordingly, ChOx/cerasome/GCE has satisfactory repeatability and stability for biosensing of cholesterol.

Conclusions

In summary, we applied the cerasomes to electrochemical biosensing of cholesterol for the first time. The results indicated that the cerasomes provided a favorable soft interface for ChOx to construct an enzyme-modified electrode that showed effective DET between the enzyme and electrode. The enzyme-modified electrode acted as a biosensor, exhibiting catalytic activity toward cholesterol with a wide linear range and low detection limit, as well as good selectivity. Overall, these findings demonstrate that cerasomes are a promising biomimetic material for construction of biosensors and biomolecular electronic devices.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (51273087) (XS), the Natural Science Foundation of Shenyang (F16-103-4-00), the Scientific Research Fund of Liaoning Provincial Education Department (LT2015012, LJQ2014003) (QZ) and a Grant-in-Aid for Scientific Research B (25288077) from the Japan Society for the Promotion of Science (JSPS) (JK).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra06368c

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