A Cholesterol Biosensor Based on Entrapment of Cholesterol Oxidase in a Silicic Sol‐Gel Matrix at a Prussian Blue Modified Electrode

A cholesterol biosensors fabricated by immobilization of cholesterol oxidase (ChOx) in a layer of silicic sol‐gel matrix on the top of a Prussian Blue‐modified glassy carbon electrode was prepared. It is based on the detection of hydrogen peroxide produced by ChOx at ?0.05 V. The half‐lifetime of the biosensor is about 35 days. Cholesterol can be determined in the concentration range of 1×10^?6?8×10^?5 mol/L with a detection limit of 1.2×10^?7 mol/L. Normal interfering compounds, such as ascorbic acid and uric acid do not affect the determination. The high sensitivity and outstanding selectivity are attributed to the Prussian Blue film modified on the sensor.     

Silica‐Polyaniline Based Bienzyme Cholesterol Biosensor: Fabrication and Characterization

In the present investigation, silica‐polyaniline based bienzyme cholesterol biosensor is fabricated through a simple one‐step electrochemical method. The one‐step fabrication process involves electrochemical polymerization of N[3‐(trimethoxysilyl)propyl]aniline to result poly(N[3‐(trimethoxysilyl)propyl]aniline) (PTMSPA) and simultaneous immobilization of two enzymes, horseradish peroxidase (HRP) and cholesterol oxidase (ChOx) into PTMSPA matrix. The modified electrode is designated as PTMSPA‐HRP/ChOx‐ME. PTMSPA facilitates direct electron transfer between the electrode surface and the active redox centers of HRP. This enables the operation of a biosensor at a low working potential of about ?150 mV (vs. Ag/AgCl) for the detection of hydrogen peroxide. The PTMSPA‐HRP/ChOx‐ME demonstrates excellent analytical performance for the detection of cholesterol between 1 and 25 mM with high sensitivity and selectivity. PTMSPA possesses features suited for the fabrication of third‐generation biosensors.     

Design and application of a flow cell for carbon-film based electrochemical enzyme biosensors

A flow cell has been designed for use with an electrochemical enzyme biosensor, based on low-cost carbon-film electrodes. Three types of mediators were used: cobalt and copper hexacyanoferrates and poly(neutral red) (PNR), covered with glucose oxidase (GOx) immobilised by cross-linking with glutaraldehyde in the presence of bovine serum albumin or inside a oxysilane sol–gel network. Mixtures of sol–gel precursors were made from 3-aminopropyl-triethoxysilane (APTOS) together with methyltrimethoxysilane (MTMOS), methyltriethoxysilane (MTEOS), tetraethyloxysilane (TEOS) or 3-glycidoxypropyl-trimethoxysilane (GOPMOS), and the best chosen for encapsulation. Optimisation in batch mode, using amperometric detection at fixed potential, showed the PNR-GOx modified carbon-film electrodes to be best for flow analysis for both glutaraldehyde and sol–gel enzyme immobilisation. Both types of enzyme electrode were tested under flow conditions and the reproducibility and stability of the biosensors were evaluated. The biosensors were used for fermentation monitoring of glucose in grape must and interference studies were also performed.     

Electrochemical Cholesterol Sensor Based on Tin Oxide‐Chitosan Nanobiocomposite Film

A chitosan (CS)‐tin oxide (SnO₂) nanobiocomposite film has been deposited onto an indium‐tin‐oxide glass plate to immobilize cholesterol oxidase (ChOx) for cholesterol detection. The value of the Michaelis–Menten constant (K_m) obtained as 3.8 mM for ChOx/CS‐SnO₂/ITO is lower (8 mM) than that of a ChOx/CS/ITO bioelectrode revealing enhancement in affinity and/or activity of ChOx towards cholesterol and also revealing strong binding of ChOx onto CS‐SnO₂/ITO electrode. This ChOx/CS‐SnO₂/ITO cholesterol sensor retains 95% of enzyme activity after 4–6 weeks at 4 °C with response time of 5 s, sensitivity of 34.7 μA/mg dL^?1 cm² and detection limit of 5 mg/dL.     

New Amperometric Cholesterol Biosensors Using Poly(ethyleneoxide) Conducting Polymers

Accumulation of cholesterol in human blood can cause several health problems such as heart disease, coronary artery disease, arteriosclerosis, hypertension, cerebral thrombosis, etc. Therefore, simple and fast cholesterol determination in blood is clinically important. In this study, two types of amperometric cholesterol biosensors were designed by physically entrapping cholesterol oxidase in conducting polymers; thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemically and cholesterol oxidase was immobilized by entrapment during electropolymerization. The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H₂O₂ at +0.7 V in the absence of a mediator. Kinetic parameters, such as Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. Km values were found as 1.47 and 5.16 mM for PEO-co-PPy and CP-co-PPy enzyme electrodes, respectively. By using these Km values, it can be observed that ChOx immobilized in PEO-co-PPy shows higher affinity towards the substrate.     

Direct Electrochemistry of Cholesterol Oxidase Immobilized on a Conducting Polymer: Application for a Cholesterol Biosensor

Direct electrochemistry of cholesterol oxidase (ChOx) immobilized on the conductive poly‐3′,4′‐diamine‐2,2′,5′,2″‐terthiophene (PDATT) was achieved and used to create a cholesterol biosensor. A well‐defined redox peak was observed, corresponding to the direct electron transfer of the FAD/FADH₂ of ChOx, and the rate constant (k_s) was determined to be 0.75 s^?1. Glutathione (GSH) covalently bonded with PDATT was used as a matrix for conjugating AuNPs, ChOx, and MP, simultaneously. MP co‐immobilized with ChOx on the AuNPs‐GSH/PDATT exhibited an excellent amperometric response to cholesterol. The dynamic range was from 10 to 130 μM with a detection limit of 0.3±0.04 μM.     

Hemin Functionalized Multiwalled Carbon Nanotubes as a Matrix for Sensitive Electrogenerated Chemiluminescence Cholesterol Biosensor

Based on hemin‐MWCNTs nanocomposite and hemin‐catalyzed luminol‐H₂O₂ reaction, a sensitive electrogenerated chemiluminescence (ECL) cholesterol biosensor was proposed in this paper. Firstly, hemin‐MWCNTs was prepared via π–π stacking and modified on the surface of GCE. Subsequently, cholesterol oxidase (ChOx) was adsorbed on the modified electrode to achieve a cholesterol biosensor. Hemin‐MWCNTs nanocomposite provided the electrode with a large surface area to load ChOx, and endowed the nanostructured interface on the electrode surface to enhance the performance of biosensor. The biosensor responded to cholesterol in the linear range from 0.3 µM to 1.2 mM with a detection limit of 0.1 µM (S/N=3).     

Characterisation of poly(neutral red) modified carbon film electrodes; application as a redox mediator for biosensors

The polymer redox mediator, poly(neutral red) (PNR), has been synthesised and characterised electrochemically to investigate the best electropolymerisation and mediation conditions for application in enzyme biosensors and to clarify the mechanism of action. Neutral red was electropolymerised by potential cycling on carbon film electrode substrates by allowing the monomer to be oxidised during the full 20 cycles of polymerisation or reducing the positive limit of the potential window after the first 2 cycles to impede monomer oxidation with a view to obtaining longer polymer chains and a lesser degree of branching. Comparison was made with glassy carbon substrates. The PNR films on carbon film electrodes were characterised using cyclic voltammetry and electrochemical impedance spectroscopy, as well as in glucose biosensors prepared with PNR. Glucose oxidase enzyme was immobilised by encapsulation in silica sol-gel and compared with that obtained by cross-linking with glutaraldehyde. The biosensors were evaluated by chronoamperometry in 0.1 M phosphate buffer saline solution, pH 7.0, and showed evidence of electron transfer between the enzyme cofactor flavin adenine dinucleotide and PNR dissolved in the enzyme layer competing with PNR-mediated electrochemical degradation of H₂O₂ formed during the enzymatic process. This paper is dedicated to Professor Dr. Algirdas Vaskelis on the occasion of his 70th birthday.     

Electrochemical Study of Chitosan‐SiO2‐MWNT Composite Electrodes for the Fabrication of Cholesterol Biosensors

We report a novel composite electrode made of chitosan‐SiO₂‐multiwall carbon nanotube (CHIT‐SiO₂‐MWNT) composite coated on the indium‐tin oxide (ITO) glass substrate. Cholesterol oxidase (ChOx) was covalently immobilized on the CHIT‐SiO₂‐MWNT/ITO electrode that resulted in a ChOx/CHIT‐SiO₂‐MWNT/ITO cholesterolactive bioelectrode. The CHIT‐SiO₂‐MWNT/ITO and ChOx/CHIT‐SiO₂‐MWNT/ITO electrodes were characterized with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The influence of various parameters was investigated, including the applied potential, pH of the medium, and the concentration of the enzyme on the performance of the biosensor. The cholesterol bioelectrode exhibited a sensitivity of 3.4 nA/ mgdL^?1 with a response time of five seconds. The biosensor using ChOx/CHIT‐SiO₂‐MWNT/ITO as the working electrode retained its original response after being stored for six months. The biosensor using ChOx/CHIT‐SiO₂‐MWNT/ITO as the working electrode showed a linear current response to the cholesterol concentration in the range of 50–650 mg/dL.     

An Electrochemical Biosensor with Cholesterol Oxidase/ Sol‐Gel Film on a Nanoplatinum/Carbon Nanotube Electrode

The carbon nanotubes decorated nanoplatinum (CNT‐Pt) were prepared using a chemical reduction method and a novel base electrode was constructed by intercalating CNT‐Pt on the surface of a waxed graphite electrode. The results showed that the nano‐particles of platinum at a waxed graphite electrode exhibits high catalytic activity for the reduction of hydrogen peroxide. The cholesterol oxidase (ChOx), chosen as a model enzyme, was immobilized with sol‐gel on the CNT‐Pt base electrode to construct a biosensor. The current response of the biosensor for cholesterol was very rapid (<20 s). The linear range for cholesterol measurement was 4.0×10^?6 mol/L ?1.0×10^?4 mol/L with a detection limit of 1.4×10^?6 mol/L. The experiments also showed that the ChOx/sol‐gel/CNT‐Pt biosensor was sensitive and stable in detecting cholesterol in serum samples.     

Polyaniline／Prussian Blue Composite Film Electrochemical Biosensors for Cholesterol Detection

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Fabrication of a Sensitive Cholesterol Biosensor Based on Cobalt‐oxide Nanostructures Electrodeposited onto Glassy Carbon Electrode

Electrodeposited cobalt oxide (CoOx) nanomaterials are not only used for immobilization of cholesterol oxidase (ChOx) but also as electron transfer mediator for oxidation of H₂O₂ generated in the enzymatic reaction. Voltammetry and flow injection analysis (FIA) were used for determination of cholesterol. FIA determination of cholesterol with biosensors yielded a calibration curve with the following characteristics: linear range up to 50 μM, sensitivity of 43.5 nA μM^?1 cm^?2 and detection limit of 4.2 μM. The apparent Michaelis‐Menten constant and the response time of the biosensor are 0.49 mM and 15 s, respectively. This biosensor also exhibits good stability, reproducibility and long life time.     

Bioanalytical device based on cholesterol oxidase-bonded SAM-modified electrodes

A rapid, simple and reproducible two-step method for constructing cholesterol biosensors by covalently bonding cholesterol oxidase (ChOx) to a 3,3′-dithiodipropionic acid di(N-succinimidyl ester) (DTSP)-modified gold electrode is described. Exhaustive characterizations of both the immobilization process and the morphological properties of the resulting ChOx monolayer were performed via a quartz crystal microbalance (QCM) and atomic force microscopy (AFM) operated under liquid conditions, respectively. In addition, scanning electrochemical microscopy (SECM) measurements were performed in order to check that the immobilized enzyme retains its catalytic activity. The replacement of the natural electron acceptor (O₂) in the enzymatic reaction with an artificial mediator, hydroxymethylferrocene (HMF), was also studied. Finally, cholesterol was amperometrically determined by measuring the hydrogen peroxide produced during the enzymatic reaction at +0.5 V. The optimized cholesterol biosensor exhibited a sensitivity of 54 nA mM⁻¹ and a detection limit of 22 μM.     

Amperometric Biosensors for Glucose Based on Layer‐by‐Layer Assembled Functionalized Carbon Nanotube and Poly (Neutral Red) Multilayer Film

Abstract

Multiwalled carbon nanotubes (MWNTs) were treated with a mixture of concentrated sulfuric and nitric acid to introduce carboxylic acid groups to the nanotubes. Conducting polymer film was prepared by electrochemical polymerization of neutral red (NR). By using a layer‐by‐layer method, homogeneous and stable MWNTs and poly (neutral red) (PNR) multilayer films were alternately assembled on glassy carbon (GC) electrodes. With the introduction of PNR, the MWNTs/PNR multilayer film system showed synergy between the MWNTs and PNR, with a significant improvement of redox activity due to the excellent electron‐transfer ability of carbon nanotubes (CNTs) and PNR. The electropolymerization is advantageous, providing both prolonged long‐term stability and improved catalytic activity of the resulting modified electrodes. The MWNTs/PNR multilayer film modified glassy carbon electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. As compared to MWNTs and PNR‐modified GC electrodes, the magnitude of the amperometric response of the MWNTs/PNR composite‐modified GC electrode is more than three‐fold greater than that of the MWNTs modified GC electrode, and nine‐fold greater than that of the PNR‐modified GC electrode. With the immobilization of glucose oxidase onto the electrode surface using glutaric dialdehyde, a biosensor that responds sensitively to glucose has been constructed. In pH 6.98 phosphate buffer, nearly interference‐free determination of glucose has been realized at ?0.2 V vs. SCE with a linear range from 50 µM to 10 mM and response time <10s. The detection limit was 10 µM glucose (S/N=3).     

Carbon Nanotubes‐Based Amperometric Cholesterol Biosensor Fabricated Through Layer‐by‐Layer Technique

A carbon nanotubes‐based amperometric cholesterol biosensor has been fabricated through layer‐by‐layer (LBL) deposition of a cationic polyelectrolyte (PDDA, poly(diallyldimethylammonium chloride)) and cholesterol oxidase (ChOx) on multi‐walled carbon nanotubes (MWNTs)‐modified gold electrode, followed by electrochemical generation of a nonconducting poly(o‐phenylenediamine) (PPD) film as the protective coating. Electrochemical impedance measurements have shown that PDDA/ChOx multilayer film could be formed uniformly on MWNTs‐modified gold electrode. Due to the strong electrocatalytic properties of MWNTs toward H₂O₂ and the low permeability of PPD film for electroacitve species, such as ascorbic acid, uric acid and acetaminophen, the biosensor has shown high sensitivity and good anti‐interferent ability in the detection of cholesterol. The effect of the pH value of the detection solution on the response of the biosensor was also investigated. A linear range up to 6.0 mM has been observed for the biosensor with a detection limit of 0.2 mM. The apparent Michaelis‐Menten constant and the maximum response current density were calculated to be 7.17 mM and 7.32 μA cm^?2, respectively.     

Mediator free cholesterol biosensor based on self-assembled monolayer platform

Self-assembled monolayer (SAM) of 4-aminothiophenol (4-ATP) has been investigated for immobilization of bi-enzymes (ChOx and ChEt) towards development of enzyme biosensors for detection of free and total cholesterol. This enzyme immobilized SAM surface has been characterized by scanning electron microscopy and electrochemical measurements. The results of electrochemical response studies reveal fast enzymatic reaction in phosphate buffer saline solution without using any artificial mediator. This may be attributed to the molecular wire type behavior of short 4-ATP molecule that promotes electron transfer between enzyme and the electrode surface due to its conjugated structure. Interference free estimation of free and total cholesterol has been realized at low operating potential of 0.33 V with range of detection as 25 to 400 mg dl(-1), sensitivity of 542.3 nA mM(-1) (for ChOx/4-ATP/Au) and 886.6 nA mM(-1) (for ChEt-ChOx/4-ATP/Au) with a response time of 20 s at pH 7.4.     

Development of electrochemical biosensors based on sol-gel enzyme encapsulation and protective polymer membranes

Protective polymer coatings have been used to enhance the retention of enzymes in sol-gel films as immobilisation phases in electrochemical biosensors. Carbon film electrodes were electrochemically modified with poly(neutral red) (PNR). These electrodes were coated with oxysilane sol-gels incorporating glucose oxidase and an outer coating of carboxylated PVC (CPVC) or polyurethane (PU), with and without Aliquat-336 or isopropyl myristate (IPM) plasticizer, was applied. The biosensors were characterised electrochemically using cyclic voltammetry and amperometry, electrochemical impedance spectroscopy and scanning electron microscopy. Impedance spectra showed that the electrode surface is most active when the sol-gel–GOx layer is not covered with a membrane. However, membranes without plasticizer extend the lifetime of the biosensor to more than 2 months when PU is used as an outer membrane. The linear range of the biosensors was found to be 0.05–0.50 mM of glucose and the biosensor with PU outer membrane exhibited higher sensitivity (ca.117 nA mM⁻¹) in the region of linear response than that with CPVC. The biosensors were applied to glucose measurement in natural samples of commercial orange juice.     

Covalent immobilization of cholesterol esterase and cholesterol oxidase on polyaniline films for application to cholesterol biosensor

Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) have been covalently immobilized on electrochemically prepared polyaniline (PANI) films. These PANI/ChEt/ChOx enzyme films have been characterized using UV-visible, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). Electrochemical behavior of these films has been studied using cyclic voltammetry (CV) and amperometric techniques, respectively. The PANI/ChEt/ChOx enzyme films show broad oxidation peak from 0.2 to 0.5 V. These PANI/ChEt/ChOx biosensing electrodes have a response time of about 40s, linearity from 50 to 500 mg/dl of cholesterol oleate concentration. These PANI/ChEt/ChOx films are thermally stable up to 46 degrees C. This polyaniline based cholesterol biosensor has optimum pH in the range of 6.5-7.5, sensitivity as 7.5x10(-4) nA/mg dl and a lifetime of about 6 weeks.     

Nanostructured Iron Oxide Platform for Impedimetric Cholesterol Detection

A nanostructured iron oxide (NanoFe₃O₄, particle size ca. 25 nm and roughness ca. 21 nm) film deposited onto a hydrolyzed indium‐tin‐oxide (ITO) coated glass plate has been used to immobilize cholesterol oxidase (ChOx) to fabricate an impedimetric cholesterol sensor. Electrochemical studies reveal that surface charged Fe₃O₄ nanoparticles provide better conformation for ChOx loading resulting in enhanced electron transfer between ChOx and the electrode. Impedimetric response studies of the ChOx/NanoFe₃O₄/ITO bioelectrode exhibit improved linearity (2.5–400 mg/dL), low detection limit (0.25 mg/dL), fast response time (25 s), high sensitivity (86 Ω/mg dL^?1/cm^?2) and a low value of the Michaelis‐Menten constant (K_m, 0.8 mg/dL) with a regression coefficient of 0.997.     

Amperometric hydrogen peroxide and cholesterol biosensors designed by using hierarchical curtailed silver flowers functionalized graphene and enzymes deposits

Novel flower-like silver particles with triangular plates as building block along with functionalized graphene (straggled sheets) and enzymes horseradish peroxidase (HRP) or cholesterol oxidase (ChOx), were obtained on graphite electrode by galvanostatic electrodeposition method. The morphology of the electrodeposits has been characterized using scanning electron microscopy and energy-dispersive analysis of X-ray. The resulting biosensors named Nf/(HRP-f-graphene-Ag)/Gr and Nf/(ChOx-f-graphene-Ag)/Gr were evaluated for electrochemical activity using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperometry. Optimization of the interdependent experimental parameters such as pH and temperature were achieved and maintained constant throughout the experiments. An activation energy of 2.5 kJ mol^?1 was obtained for Nf/(HRP-f-graphene-Ag)/Gr electrode while Nf/(ChOx-f-graphene-Ag)/Gr showed an activation energy of 2.06 and 3.12 kJ mol^?1. Furthermore, the former electrode demonstrated a good linear range of 25 μM to 19.35 mM with rapid response time of 3 s and detection limit of 5 μM for hydrogen peroxide. Similarly, the Nf/(ChOx-f-graphene-Ag)/Gr electrode revealed a linear range of 0.1–4.5 mM with rapid response time of 3 s and an excellent detection limit of 0.514 mM for cholesterol. Besides this, the Nf/(HRP-f-graphene-Ag)/Gr and Nf/(ChOx-f-graphene-Ag)/Gr electrodes displayed a Michaelis–Menten constant of 0.26 and 0.57 mM, respectively, suggesting high affinity and enzymatic activity. The enhanced performance of biosensors towards detection of substrate and rejection of interferents, provided an evidence for its high anti-interference ability. Additionally the biosensors exhibit long term storage stability and reproducibility with antifouling properties.