Design of a Multilayer Cholesterol Amperometric Biosensorfor Preparation and Use in Flow Systems (original) (raw)
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Analytica Chimica Acta, 2001
A rapid, two-step method for constructing cholesterol biosensors by entrapment of cholesterol oxidase (ChOx) within a composite poly(2-hydroxyethyl methacrylate) (p(HEMA))/polypyrrole (p(pyrrole)) membrane has been developed. Platinum electrode-supported polymer films were prepared by UV polymerization of the hydrogel component containing dissolved enzyme followed immediately by electrochemical polymerization of entrapped pyrrole monomer (Py) within the performed hydrogel network. The optimized cholesterol biosensor exhibited a linear response range from 5 × 10 −4 to 1.5 × 10 −2 M and detection limit of 120 M toward cholesterol. The response time of the biosensor was 30 s. The analytical recovery of cholesterol in serum samples ranged from 97 to 103% with mean coefficients of variation of 3% (within-day analyses) and 3.9% (day-to-day analyses). Up to 60 samples/h can be manually analyzed. The cholesterol biosensor retained 80% of initial activity after 12 months when stored desiccated in the absence of buffer. When applied to the analysis of cholesterol in serum samples of clinical patients, >0.998 correlation was obtained between the biosensor results and those obtained by a standard hospital method using an Abbott V.P. Chemistry Analyzer.
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 hydrogenbonding between enzyme molecules and polymer was achieved. Moreover, strongstacking 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 −0.7 V vs Ag wire in phosphate buffer (pH 7.0). K app M (37.3 M), I max (3.92 A), LOD (0.4063 M) and sensitivity (1.49 A M −1 cm −2 ) values were determined. Finally, the prepared biosensor was successfully applied for determination of cholesterol content in real blood samples.
Electroanalysis, 2004
Different cholesterol amperometric biosensors were developed based on entrapment of cholesterol esterase and/or cholesterol oxidase in polymer films of diaminonaphthalene isomers, electrochemically synthesised from aqueous solutions of the monomers and enzymes in phosphate buffer at neutral pH. These conditions permit the growth of films with extraordinary selective properties which allow the preparation of interference-free biosensors for application in biological media without the response being affected by the presence of either endogenous species (ascorbic and uric acid) or exogenous species like 4-acetamidophenol. These selective properties were evaluated for the different monolayer and bilayer configurations proposed in function of the film permeation factor. All the steps involved in the preparation of the biosensors and determination of free or total cholesterol were carried out in a flow system. A comparative study was made of the analytical properties of each of the configurations developed and their application to the flow-injection determination of cholesterol in a synthetic serum.
Cholesterol biosensors prepared by layer-by-layer technique
Biosensors and Bioelectronics, 2001
The analysis of formation, deposition and characterization of cholesterol oxidase (COX) layer-by-layer films were performed. Initially, a layer of polyanion, poly(styrene sulfonate) (PSS) was adsorbed followed by a layer of polycation, poly(ethylene imine) (PEI) on each solid substrate from aqueous solutions. The alternating layers were formed by consecutive adsorption of polycations (PEI) and negatively charged proteins (COX) and cholesterol esterase (CE). A strong interaction between protein and polyelectrolyte improves the stability of the alternating multilayer; however, it can change a native protein conformation and impair the protein activity. The PSS/PEI/COX, PSS/PEI/COX/PEI/CE, PSS/PEI/COX-CE/PEI etc. layered structures were prepared on the surface of a platinum electrode, ITO coated glass plate, quartz crystal microbalance, quartz plates, mica and silicon substrates. Optical and gravimetric measurements based on an ultraviolet-visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of molecular layered of the proteins. The surface morphology of such bilayer films was investigated by using atomic force microscopy. The electrochemical redox processes of the enzyme-layered films deposited either on platinum or ITO coated glass plate were investigated. The response current of cholesterol oxidase electrode with concentration of cholesterol was investigated at length.
Analytical Sciences, 2002
The preparation of a cholesterol amperometric biosensor using a platinized Pt electrode as a support for the electropolymerization of a polypyrrole film, in which cholesterol oxidase and ferrocene monocarboxylic acid (electrontransfer mediator) were co-entrapped, is described. All the biosensor preparation steps (platinization and electropolymerization) and the cholesterol determination take place in the same flow system. The presence of the mediator enhances the sensitivity and selectivity of the platinized biosensor without modifying the dynamic parameters of the response, and the platinized layer improves the operational lifetime of the mediated sensor. The sensitivity obtained was 88.51 nA mM-1 and the limit of detection was 12.4 µM of cholesterol. The analytical properties of the biosensor for the flow-injection determination of cholesterol were studied and compared with those of other more simple amperometric biosensor configurations.
A new amperometric cholesterol biosensor based on poly(3,4-ethylenedioxypyrrole)
Sensors and Actuators B: Chemical, 2009
Cholesterol oxidase (ChOx) was physically entrapped in poly(3,4-ethylenedioxypyrrole) (PEDOP) to construct an amperometric cholesterol biosensor. The responses of the enzyme electrodes were measured via monitoring oxidation current of H 2 O 2 at +0.7 V in the absence of a mediator. Kinetic parameters, operational and storage stabilities, pH and temperature dependencies were determined. K m , I max and sensitivity (I max /K m ) were calculated as 3.4 mM, 34 A cm −2 and 10 A mM −1 cm −2 , respectively. The minimum detectable substrate concentration was 0.4 mM and for a period of 20 days the biosensor showed the maximum relative activity.
Journal of the Brazilian Chemical Society, 2012
Um eletrodo enzimático amperométrico baseado em polímero foi preparado para detecção amperométrica de colesterol livre. Inicialmente, o filme polimérico poli(o-fenilenodiamina) (PoPD) foi eletropolimerizado no meio acetonitrila-água contendo o monômero o-fenilenodiamina (oPD) e ácido canforsulfônico em eletrodo de Pt através da técnica de voltametria cíclica. A enzima oxidase colesterol foi imobilizada sobre a superfície do eletrodo Pt/PoPD. A determinação de colesterol foi realizada por maio do monitoramento da corrente de oxidação de H 2 O 2 produzido enzimaticamente a +0,7 V vs. Ag/AgCl. A concentração da solução tampão, o valor de pH e a temperatura de trabalho otimizados foram 0,05 mol L −1 , 7,5 and 40 °C, respectivamente. O intervalo de trabalho do eletrodo de enzima para colesterol foi 9,8 × 10 −3-11 µmol L −1 e tempo de resposta 150 s. Os efeitos de possíveis interferências presentes em amostras de soro em resposta ao eletrodo de enzima foram examinados. A determinação do colesterol total em amostras de soro foi realizada usando o eletrodo de enzima Pt/PoPD/ChOx proposto e os resultados foram em bom acordo com os obtidos pelo método espectrofotométrico. A polymer-based amperometric enzyme electrode (Pt/PoPD/ChOx) was prepared for the amperometric detection of free cholesterol. Firstly, poly(o-phenylenediamine) (PoPD) polymer film was prepared in acetonitrile-water medium containing o-phenylenediamine (oPD) monomer and (±)-10-camphorsulfonic acid (HCSA) on Pt electrode by the use of cyclic voltammetry technique. Cholesterol oxidase (ChOx) enzyme was immobilized onto Pt/PoPD electrode surface. The determination of cholesterol was performed via monitoring of the oxidation current of enzymatically produced H 2 O 2 at +0.7 V vs. Ag/AgCl. Optimum buffer concentration, pH and working temperature were found as 0.05 mol L −1 , 7.5 and 40 °C, respectively. The working range of enzyme electrode to cholesterol was 9.8 × 10 −3-11 µmol L −1 and response time 150 s. The effects of possible interferences present in serum samples on response of enzyme electrode were examined. The determination of total cholesterol in serum samples was performed by using proposed Pt/PoPD/ChOx enzyme electrode and results were in good agreement with those obtained by spectrophotometric method.
2013
In the present work the development of an amperometric transducer in order to build a free cholesterol biosensor in planar configuration is reported. The one single use disposable biosensors were constructed by screen printing process which is compatible with automated methodologies of production. Based in the incorporation of tetracyanoquinodimethane (TCNQ), Prussian blue (PB) or ferrite (Fe 3 O 4 ) as mediator or electrocatalytic agent, three types of electrochemical transducers were evaluated. The cholesterol biosensors require a sample volume of 7.2 µL, exhibits good reproducibility and selectivity and cover a lineal answer of 2-16 mM with detection limits from 0.3 to 1.6 mM. The characteristics of biosensors are satisfactory for the decentralized analysis of the lipid in blood since enclose the range of clinical interest (3.5-6.5 mM).
New redox and conducting polymer modified electrodes for cholesterol biosensing
Analytical Methods, 2013
New enzyme biosensors for cholesterol detection based on a recently developed transduction platform constituted of a GCE modified with polymer redox mediator poly(methylene blue) (PMB) and conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) were for the first time prepared and evaluated. The enzyme cholesterol oxidase (ChOx) was immobilized by simple adsorption, the ChOx/ PEDOT/PMB/GCE biosensor being applied for amperometric determination of cholesterol by monitoring the peroxide reduction produced by the enzymatic reaction at À0.4 V vs. SCE. Amperometric studies at fixed potential reveal that almost interference-free cholesterol determination can be achieved at the newly developed biosensor in a range between 10 and 220 mM with a sensitivity of 79.0 mA cm À2 mM À1 and a detection limit of 1.6 mM. The recovery and storage stability of the biosensor were evaluated and the biosensor was applied to cholesterol detection in whole cow milk and chicken egg yolk.
Analytica Chimica Acta, 2004
Fabrication of an amperometric cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto conducting polypyrrole (PPY) films using electrochemical entrapment technique is described. Electrochemical polymerization was carried out using a two-electrode cell configuration at 0.8 V. Characterization of resulting amperometric biosensor for the estimation of cholesterol has been experimentally determined in terms of linear response range, optimum pH, applied potential, temperature, and shelf-life. These PPY/ChEt/ChOx electrodes can be used for cholesterol ester estimation from 1 to 8 mM and have shelf-life of about 4 weeks at 4 °C during which about 15 estimations of cholesterol ester could be made. The sensitivity of PPY/ChEt/ChOx electrode has been found to be 0.15 μA/mM and the apparent Km value for this electrode is 9.8 mM. Conductivity of the polymer films found to be about 3×10−3 S/cm.