Immobilization Of Cholesterol Oxidase Research Articles

Ultrasensitive cholesterol biosensor based on enzymatic silver deposition on gold nanoparticles modified screen-printed carbon electrode

Cholesterol is one of the essential structural constituents of cell membranes. Determination of cholesterol is of great importance in clinical analysis because the level of cholesterol in serum is an indicator in the diagnosis and prevention of heart diseases. In this work, a simple and ultrasensitive cholesterol biosensor based on enzymatic silver deposition was designed by immobilizing cholesterol oxidase (CHOD) and cholesterol esterase (CHER) onto the surface of gold nanoparticles (Au NPs) modified screen-printed carbon electrode (SPE). By the catalytic action of CHER and CHOD, the cholesterol was hydrolyzed to generate hydrogen peroxide (H2O2) which can reduced the silver (Ag) ions in the solution for the deposition of metallic Ag on the surface of Au NPs modified SPE. The ultrasensitive detection of cholesterol was achieved by anodic stripping voltammetry (ASV) measurement of the enzymatically deposited Ag. The influence of relevant experimental variables was optimized. The anodic stripping peak current of Ag depended linearly on the concentration of cholesterol in the range of 5–5000μg/mL with the regression correlation coefficient of 0.9983. A detection limit of 3.0μg/mL was attained by 3 sigma-rule. In addition, the ultrasensitive cholesterol biosensor exhibited higher specificity, acceptable reproducibility and excellent recoveries for cholesterol detection.

Cu-Pt-Bi Nanocomposite Modified Glassy Carbon Electrode for Dual Mode H2O2 and Cholesterol Sensing

In this work, it has been demonstrated that the sequential inclusion of three catalytically active metals (Cu, Pt and Bi) in a glassy carbon (GC) matrix can ascribe a dual role to the trimetallic composite facilitating the oxidation and reduction of H2O2 under physiological conditions. The trimetal based nanosensing platform was constructed by sequential steps which included electrodeposition of Cu, galvanic replacement of Cu by Pt and irreversible adsorption of Bi over Pt. The electrocatalytic activity exhibited by the trimetallic composite was found to depend upon the composition and surface area. The Cu-Pt-Bi nanocomposite modified GC electrode (GC/Cu-Pt-Bi) effectively catalyzed the electrochemical oxidation and reduction of H2O2 at the potential of + 0.25 V and −0.01 V vs NCE (normal calomel electrode) respectively, with fast amperometric response, high sensitivity and low detection limit. Along with these attractive features, the modified electrode also displayed very high specificity to H2O2 with complete elimination of interference from uric acid (UA), ascorbic acid (AA), and glucose. These advantageous characteristics motivated us to build a dual mode cholesterol sensor based on H2O2 transduction by immobilizing cholesterol oxidase (ChOx) on GC/Cu-Pt-Bi. The enzyme modified nanostructured platform provided a highly sensitive and selective sensing of cholesterol coupled with a fast response time and low Km value.

Dependence of seed layer thickness on sensitivity of nano-ZnO cholesterol biosensor

The anemone-like ZnO nanostructures have been synthesized by hydrothermal method and were further adsorbed immobilized cholesterol oxidase (ChOx) as a nano-biosensor. In this study, the sensitivity of biosensor were improved by varying the thickness of the ZnO seed layer. The SEM analysis showed changes in thickness of seed layer will not affect the morphologies of anemone-like ZnO nanostructures. The X-ray Diffraction patterns showed that the (002) plane of anemone-like ZnO grown on various thickness of the seed layer was more prouded than other crystal plane. Abioelectrode (ChOx/ZnO/ITO/glass) grown on the 30nm of ZnO seed layer with high sensitivity of 57.533μAmM-1cm-2 (1.488 μA (mg/dl) -1cm-2), a wide sensitive range from 25 to 500 mg/dl. It is concluded that the thinner sputtered ZnO seed layer for growing anemone-like ZnO nanostructure can effectively improve the sensitivity of the ZnO biosensor.

Influence of immobilization strategies on biosensing response characteristics: A comparative study

The immobilization technique plays an important role in fabrication of a biosensor. NiO based cholesterol biosensor has been used to study the effect of various immobilization techniques on the biosensing response characteristics. The biosensors were fabricated by immobilizing cholesterol oxidase on NiO thin films by three different immobilization techniques viz. physisorption, cross-linking and covalent binding. The study reveals a strong dependence of biosensing response on corresponding immobilization technique. The biosensor based on immobilization by covalent bonding shows superior response characteristics as compared to others owing to its zero length. The results highlight the significance of immobilization technique for biosensor fabrication.

Immobilization of cholesterol oxidase on magnetic fluorescent core-shell-structured nanoparticles.

The magnetic fluorescent core-shell structured nanoparticles, Fe3O4@SiO2(F)@meso-SiO2 nanoparticles, were prepared. Cholesterol oxidase (COD) was immobilized on their surface to form Fe3O4@SiO2(F)@meso-SiO2@COD nanoparticles. Optimal immobilization was achieved with 2.5% (v/v) APTES, 2.0% (v/v) GA, 10mg COD (in 15 mg carrier) and solution pH of 7.0. Fe3O4@SiO2(F)@meso-SiO2@COD nanoparticles showed maximal catalytic activity at pH7.0 and 50°C. The thermal, storage and operational stabilities of COD were improved greatly after its immobilization. After the incubation at 50°C for 5h, the nanoparticles and free COD retained 80% and 46% of its initial activity, respectively. After kept at 4°C for 30 days, the nanoparticles and free COD maintained 86% and 65% of initial activity, respectively. The nanoparticles retained 71% of its initial activity after 7 consecutive operations. Since Fe3O4@SiO2(F)@meso-SiO2@COD nanoparticles contained tris(2,2-bipyridyl)dichloro-ruthenium(II) hexahydrate (Ru(bpy)3Cl2) and were optical sensitive to oxygen in solution, it might be used as the sensing material and has the application potential in multi parameter fiber optic biosensor based on enzyme catalysis and oxygen consumption.

Electrochemical and optical properties of a conducting polymer and its use in a novel biosensor for the detection of cholesterol

A simple and robust cholesterol biosensor was designed by immobilizing cholesterol oxidase (ChOx) onto a conducting polymer modified graphite electrode. For this purpose, monomer, (Z)-4-(4-(9H-carbazol-9-yl) benzylidene)-2-(4-nitrophenyl) oxazol-5(4H)-one (CBNP), was synthesized and electrochemically polymerized on an electrode to achieve an effective immobilization platform for enzyme immobilization. After electropolymerization of the monomer (CBNP), electrochemical and spectroelectrochemical properties were investigated. Through the presence of nitro group on the polymer backbone hydrogen-bonding between enzyme molecules and polymer was achieved. Moreover, strong π–π stacking between aromatic moities in the polymer and aromatic residues of the enzyme enables a sensitive and reliable biosensor by conserving the crucial structure of biological molecules during the enzymatic reaction. The efficient interaction of the enzyme with the polymer coated surface brings easy and long-life detection of the substrate, cholesterol. After successful immobilization of ChOx with the help of glutaraldehyde as the crosslinking agent, amperometric biosensor responses were recorded at −0.7V vs Ag wire in phosphate buffer (pH 7.0). KMapp (37.3μM), Imax (3.92μA), LOD (0.4063μM) and sensitivity (1.49μAμM−1cm−2) values were determined. Finally, the prepared biosensor was successfully applied for determination of cholesterol content in real blood samples.

Direct electrochemistry of cholesterol oxidase immobilized on chitosan–graphene and cholesterol sensing

A novel cholesterol biosensor (ChOx/CS–GR/GCE) with enhanced sensitivity and low detection limit was investigated. The biosensor based on direct electrochemistry of cholesterol oxidase with an apparent rate constant (ks) of 2.69s−1 was fabricated by immobilizing cholesterol oxidase (ChOx) on Glassy carbon electrode (GCE) functionalized by chitosan–graphene (CS–GR) nanocomposites. Graphene oxide (GO) was synthesized by a novel microwave method and CS–GR was prepared by simple situ reduction of chitosan–graphene oxide (CS–GO). The results of transmission electron microscopy (TEM) and FT-IR spectroscopy showed that the graphene oxide (GO) was successfully prepared and deoxygenized. The presence of the CS–GR nanocomposites not only accelerated direct electron transfer from the enzyme to the electrode surface, but also enhanced the immobilized amount and stability of cholesterol oxidase (ChOx). The fabricated electrode exhibited a linear response to cholesterol in the range of 0.005–1.0mM with a detection limit of 0.715μM (S/N=3). The Michaelis–Menten constant (kmapp) was found as 17.39μM. In addition, the biosensor also exhibited excellent reproducibility, stability and very high specificity to cholesterol with complete elimination of interference from UA, AA, DA and glucose.

Activity of cholesterol oxidase immobilized on Layered Double Hydroxide nanomaterials for biosensor application: Acacia salicina scavenging power of hypercholesterolemia therapy

In the current study, we aimed to immobilize cholesterol oxidase (ChOx) on Layered Double Hydroxide (LDH) for cholesterol sensing. Obtained results have shown that immobilized ChOx keeps its activity even after several uses. However, some free radicals, such as hydrogen peroxide (H2O2), were generated through the enzymatic activity. In this work, we investigated the effects of hexane extract from Acacia salicina toward free radical scavenging activity. Hexane extract was an effective inhibitor of H2O2 generated by the enzymatic cholesterol/cholesterol oxidase catabolism (inhibition percentage ≈ 40%). This activity could be attributed to the presence of phytosterols in this extract. The present study demonstrates that extracts of A.salicina leaves are a potential source of antioxidant agents (most likely sterols compounds), their antioxidant activity might be used in chemoprevention trials.

Electroactive Prussian Blue Encapsulated Iron Oxide Nanostructures for Mediator‐Free Cholesterol Estimation

AbstractIron oxide nanoparticles of size ∼10 nm have been encapsulated into four nanometer thick shells of Prussian blue and were then electrophoretically deposited onto an indium tin oxide substrate. The immobilization of cholesterol oxidase has been done onto the nanostructured film to investigate the kinetic parameters and biosensing characteristics. The fabricated bioelectrode exhibits an electron transfer coefficient and a charge transfer rate constant of 0.45 and 45.15 s−1, respectively. Direct electron transfer properties of the nanostructured film result in 3rd generation cholesterol biosensor. The bioelectrode exhibits high sensitivity (2.15 mAM−1 cm−2), a low Kmapp value (0.07 mM), good stability and high selectivity towards cholesterol.

A cholesterol biosensor based on a bi-enzyme immobilized on conducting poly(thionine) film

A simple and cheap cholesterol biosensor was designed by immobilizing cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) onto a poly(thionine)-modified glassy carbon electrode (GCE/PTH). Being mediated by hydroquinone (HQ), the immobilized HRP exhibited excellent electrocatalytic activity in reducing H2O2, which was produced from cholesterol by the enzymatic reaction of ChOx. The linear detection range for cholesterol was 25–125μM, with a detection limit (S/N=3) and a sensitivity of 6.3μM and 0.18μA/cm2/μM, respectively, under optimal conditions. The highly reproducible and sensitive GCE/PTH/ChOx/HRP sensor exhibited an interference-free signal for cholesterol detection with excellent recoveries for real sample analysis.

Bi-pseudoenzyme synergetic catalysis to generate a coreactant of peroxydisulfate for an ultrasensitive electrochemiluminescence-based cholesterol biosensor

A novel electrochemiluminescence (ECL) enzyme biosensor for the ultrasensitive detection of cholesterol was designed based on a bi-pseudoenzymatic reaction to generate a coreactant of peroxydisulfate for signal amplification. In this work, hemin-functionalized graphene (hemin-GR) was synthesized and used to immobilize cholesterol oxidase (COx) to construct an ECL biosensor for cholesterol. When cholesterol was added to the detection solution, COx catalyzed the oxidation of cholesterol to generate H2O2, which could be further catalyzed by hemin to produce O2 as the coreactant in the peroxydisulfate system for signal amplification. The linear range for cholesterol detection was 3.3–1,500nM, with a lower detection limit of 1.0nM (signal to noise ratio=3). Therefore, the detection limit and sensitivity of the biosensor were improved. This novel strategy offers advantages of simplicity, improved sensitivity, good selectivity, and repeatability, and therefore, holds promise for use in sensitive bioassays for clinical determination of cholesterol levels.

High performance cholesterol sensor based on ZnO nanotubes grown on Si/Ag electrodes

Zinc oxide nanotube (ZNT) arrays were grown on Si/Ag substrate by one-step chemical process in an aqueous solution and further used as a working electrode to fabricate an enzyme-based cholesterol biosensor through immobilization of cholesterol oxidase (ChOx). The fabricated biosensors exhibit high and reproducible sensitivity of 79.40μA/mM/cm2, wide linear range from 1.0μM to 13.0mM, fast response time of ~2s and ultra-low detection limit of 0.5nM (S/N=3) for cholesterol sensing. The anti-interference ability and long-term stability of the biosensor were also assessed. Finally, the biosensor was applied to analyze cholesterol concentration in human serum samples.

Flow injection analysis of cholesterol using FFT admittance voltammetric biosensor based on MWCNT–ZnO nanoparticles

A new electrochemical method was designed for determination of cholesterol by combination of continuous admittance square wave voltammetry method and a highly sensitive biosensor. The new biosensor was constructed using immobilization of cholesterol oxidase (ChOx) on multiwalled carbon nanotubes (MWCNT) and ZnO nanoparticles (ZnO). The biosensor was used in a flow injection analysis system. The special square wave voltammetric method was established based on application of fast Fourier transform (FFT) and continuously calculating the biosensor admittance in a time window. The response of the injected cholesterol samples was calculated by background subtraction and integration of the biosensor signal in the recorded voltammograms in form of coulomb. The method is very sensitive and the response time is less than 8s. The technique was applied as an electrochemical detector in a flow injection analysis system. Detection limit of the cholesterol was 0.05nM in a linear range from 0.2 to 60.0nM (R2=0.994).

Chemochromic properties of neutral polyaniline throughout cholesterol exposure

This paper presents a novel biosensing platform for cholesterol accomplished by the spin-coating deposition of neutral polyaniline on poly(methyl methacrylate) support and the subsequent covalent immobilization of cholesterol oxidase onto polyaniline surface. The polyaniline film entraps the enzyme and it also acts as an active layer for optical sensing of free cholesterol. The effects of immobilization process of the enzyme over the oxidation state of polyaniline backbone and the optimum pH and temperature conditions for immobilized cholesterol oxidase were studied. The sensibility of the system to cholesterol in concentrations equivalent to those present in human serum evidences its potentiality in an optical method simply based on chemochromic properties of polyaniline.

Potentiometric Cholesterol Biosensor Based on ZnO Nanowalls and Stabilized Polymerized Lipid Film

AbstractA novel potentiometric cholesterol biosensor was fabricated by immobilization of cholesterol oxidase into stabilized lipid films using zinc oxide (ZnO) nanowalls as measuring electrode. Cholesterol oxidase was incorporated into the lipid film prior polymerization on the surface of ZnO nanowalls resulting in a sensitive, selective, stable and reproducible cholesterol biosensor. The potentiometric response was 57 mV/ decade concentration. The sensor response had no interferences by normal concentrations of ascorbic acid, glucose, and urea, proteins and lipids. The present biosensor could be implanted in the human body because of the biocompatibility of the lipid film.

A novel ternary NiFe2O4/CuO/FeO-chitosan nanocomposite as a cholesterol biosensor

We report the results of studies relating to the in situ synthesis of a novel ternary NiFe2O4/CuO/FeO-chitosan nanocomposite, which could be utilized as a cholesterol biosensor. The phase identification, morphology and particle size of the NiFe2O4/CuO/FeO nanocomposite have been investigated via X-ray diffraction pattern (XRD), scanning electron microscopy (SEM), high resolution transmission electron microscope (HR-TEM) and Fourier transform infrared (FTIR) spectroscopy. The quantification of cholesterol was accomplished by immobilizing cholesterol oxidase (ChOx) onto a chitosan-NiFe2O4/CuO/FeO nanocomposite (NiFe2O4/CuO/FeO-CH NC) deposited onto an indium-tin-oxide (ITO) glass substrate via the sol–gel technique. The electrochemical study results of the biocompatible ChOx/NiFe2O4/CuO/FeO-CH/ITO electrode reveal good linearity (50–5000mg/L), a low detection limit (313mg/L), high sensitivity (0.043μA/(mg/Lcm−2)), a fast response time (10s) and a shelf-life of 3 months. The low Michaelis–Menten constant (Km) of 80mg/L (0.21mM) indicates the high affinity of ChOx for the analytes. Further, this bioelectrode has been used in clinical applications to estimate cholesterol levels with negligible interference (2%) from analytes present in human serum samples.

A nanocomposite based biosensor for cholesterol determination

An electrochemical biosensor was prepared by immobilizing cholesterol oxidase (ChOx) on a nanocomposite consisting of amine functionalized multi-walled carbon nanotubes and a room temperature ionic-liquid (1-butyl-3-methylimidazolium tetrafluoroborate). The biosensor was examined for cholesterol quantification in clinical serum samples. ChOx on the modified electrode exhibited a couple of quasi-reversible redox peaks corresponding to the direct electron transfer of FAD/FADH2 buried in the enzyme structure. A formal potential of −400 mV was obtained for the immobilized enzyme in phosphate buffer solution (0.1 M, pH 7.4). Under the optimized experimental conditions, the biosensor exhibited a linear response towards cholesterol concentration from 26 nM to 3.4 μM, with a sensitivity of 540 μA mM−1and a detection limit of 12 nM.

Phospholipid–Sepiolite Biomimetic Interfaces for the Immobilization of Enzymes

Biomimetic interfaces based on phosphatidylcholine (PC) assembled to the natural silicate sepiolite were prepared for the stable immobilization of the urease and cholesterol oxidase enzymes. This is an important issue in practical advanced applications such as biocatalysis or biosensing. The supported lipid bilayer (BL-PC), prepared from PC adsorption, was used for immobilization of enzymes and the resulting biomimetic systems were compared to several other supported layers including a lipid monolayer (ML-PC), a mixed phosphatidylcholine/octyl-galactoside layer (PC-OGal), a cetyltrimethylammonium monolayer (CTA), and also to the bare sepiolite surface. Interfacial characteristics of these layers were investigated with a focus on layer packing density, hydrophilicity/hydrophobicity, and surface charge, which are being considered as key points for enzyme immobilization and stabilization of their biological activity. Cytoplasmic urease and membrane-bound cholesterol oxidase, which served as model enzymes, were immobilized on the different PC-based hybrid materials to probe their biomimetic character. Enzymatic activity was assessed by cyclic voltammetry and UV-vis spectrophotometry. The resulting enzyme/bio-organoclay hybrids were applied as active phase of a voltammetric urea biosensor and cholesterol bioreactor, respectively. Urease supported on sepiolite/BL-PC proved to maintain its enzymatic activity over several months while immobilized cholesterol oxidase demonstrated high reusability as biocatalyst. The results emphasize the good preservation of bioactivity due to the accommodation of the enzymatic system within the biomimetic lipid interface on sepiolite.

Potassium-doped carbon nanotubes toward the direct electrochemistry of cholesterol oxidase and its application in highly sensitive cholesterol biosensor

We demonstrate herein a newly developed serum total cholesterol biosensor by using the direct electron transfer of cholesterol oxidase (ChOx), which is based on the immobilization of cholesterol oxidase and cholesterol esterase (ChEt) on potassium-doped multi-walled carbon nanotubes (KMWNTs) modified electrodes. The KMWNTs accelerate the electron transfer from electrode surface to the immobilized ChOx, achieving the direct electrochemistry of ChOx and maintaining its bioactivity. As a new platform in cholesterol analysis, the resulting electrode (ChOx/KMWNTs/GCE) exhibits a sensitive response to free cholesterol, with a linear range of 0.050–16.0 μmol L −1 and a detection limit of 5.0 nmol L −1 (S/N = 3). Coimmobilization of ChEt and ChOx (ChEt/ChOx/KMWNTs/GCE) allows the determination of both free cholesterol and esterified cholesterol. The resulting biosensor shows the same linear range of 0.050–16.0 μmol L −1 for free cholesterol and cholesteryl oleate, with the detection limit of 10.0 and 12.0 nmol L −1 (S/N = 3), respectively. The concentrations of total (free and esterified) cholesterol in human serum samples, determined by using the techniques developed in the present study, are in good agreement with those determined by the well-established techniques using the spectrophotometry.

Development of an Amperometric Cholesterol Biosensor Based on Graphene−Pt Nanoparticle Hybrid Material

We describe the development of a highly sensitive amperometric biosensor based on the hybrid material derived from nanoscale Pt particles (nPt) and graphene for the sensing of H2O2 and cholesterol. The biosensing platform was developed using the hybrid material and enzymes cholesterol oxidase and cholesterol esterase. Chemically synthesized graphene has been decorated with nanosized Pt particles. The electron microscopic measurements show that the Pt nanoparticles on graphene have an average size of 12 nm and are randomly distributed throughout the surface. The Pt nanoparticle based hybrid material modified electrode efficiently catalyzes the electrochemical oxidation of H2O2 at the potential of 0.4 V, which is >100 mV less positive with respect to the bulk Pt electrode. The sensing platform is highly sensitive and shows linear response toward H2O2 up to 12 mM with a detection limit of 0.5 nM [S/N (signal-to-noise ratio) = 3] in the absence of any redox mediator or enzyme. The combination of electronically highly conductive graphene and catalytically active Pt nanoparticle favors the facilitated electron transfer for the oxidation of H2O2. The cholesterol biosensor was developed by immobilizing cholesterol oxidase and cholesterol esterase on the surface of graphene−nanoparticle hybrid material. The bienzyme integrated nanostructured platform is very sensitive, selective toward cholesterol, and it has a fast response time. The sensitivity and limit of detection of the electrode toward cholesterol ester are 2.07 ± 0.1 μA/μM/cm2 and 0.2 μM, respectively. The apparent Michaelis−Menten constant (Kmapp) was calculated to be 5 mM. The sensor does not suffer from the interference due to other common electroactive species and is highly stable. The analytical performance of the hybrid material was further evaluated using screen-printed electrodes with 50 μL of electrolyte.