Amperometric determination of total cholesterol in serum with use of immobilized cholesterol esterase and cholesterol oxidase

Cholesterol esterase and cholesterol oxidase were immobilized on octyl-agarose gel, activated with cyanogen bromide and placed in a reactor. The sensor system for total cholesterol was assembled with the immobilized enzyme reactor, a hydrogen peroxide electrode and a peristaltic pump. Characteristics of the sensor system were investigated by using cholesterol palmitate as a standard substrate. A linear relationship was obtained between peak current and cholesterol palmitate concentration below 1000 mg dl^-1 (10.3 mM). A 10-μl sample could be assayed in 5 min. Total cholesterol in human serum was determined in the range 100–400 mg dl^-1. The standard deviation for the determination of 50 samples of 300 mg dl^-1 was 6 mg dl^-1 (2%). The system was used for 300 assays without loss of enzymatic activity. The correlation coefficient was 0.94 for 27 samples of human sera analyzed by the system proposed and by the conventional chemical method.     

A Fiber Optic Biosensor Based on Hydrogel-Immobilized Enzyme Complex for Continuous Determination of Cholesterol and Glucose

A multiparameter fiber optic biosensor for continuous determination of cholesterol and glucose was developed. This sensor was based on poly(N-isopropylacrylamide) (PNIPAAm)-immobilized glucose oxidase (GOx) complex (PIGC) and immobilized cholesterol oxidase (COD). The immobilized COD catalysis to the oxidation of cholesterol and PIGC catalysis to the oxidation of glucose could be performed at different temperatures. Therefore, the sensor could detect cholesterol and glucose continuously by changing temperature. The optimal detection conditions for glucose were achieved with pH 6.5, 30 °C, and 10 mg GOx (in 100-mg carrier), and those for cholesterol were achieved with pH 7.5, 33 °C, and 25 mg COD (in 250-mg carrier). The sensor has the cholesterol detection range of 20–250 mg/dL and the glucose detection range of 50–700 mg/dL. This biosensor has outstanding repeatability and selectivity, and the detection results of the practical samples are satisfactory.

     

Electrophoretically deposited polyaniline nanotubes based film for cholesterol detection

Polyaniline nanotube (PANI-NT) based films have been fabricated onto indium-tin-oxide (ITO) coated glass plates via electrophoretic technique. These PANI-NT/ITO electrodes have been utilized for covalent immobilization of cholesterol oxidase (ChOx) using glutaraldehyde (Glu) as cross-linker. Structural, morphological and electrochemical characterization of PANI-NT/ITO electrode and ChOx/Glu/PANI-NT/ITO bioelectrode have been done using FT-IR spectroscopy, SEM, electrochemical impedance spectroscopy and cyclic voltammetry techniques. Response studies of the ChOx/Glu/PANI-NT/ITO bioelectrode have been carried out using both linear sweep voltammetry and UV-Visible spectrophotometry. The results of the biosensing studies reveal that this bioelectrode can be used to detect cholesterol in wide detection range of 25-500 mg/dL with high sensitivity of 3.36 mA mg(-1) dL and fast response time of 30 s at pH 7.4. This bioelectrode exhibits very low value of Michaelis-Menten constant of 1.18 mM indicating enhanced interactions between cholesterol and ChOx immobilized onto this nanostructured PANI matrix.     

Dithiobissuccinimidyl propionate self assembled monolayer based cholesterol biosensor

A dithiobissuccinimidyl propionate (DTSP) self-assembled monolayer (SAM) prepared onto a gold (Au) surface has been utilized for covalent immobilization of cholesterol oxidase (ChOx) and cholesterol esterase (ChEt). These ChOx-ChEt/DTSP/Au bio-electrodes have been characterized using electrochemical impedance and cyclic voltammetric (CV) techniques, respectively. Differential pulse voltammetry (DPV) has been used for enzymatic assay of immobilized ChOx and ChEt onto the DTSP modified gold surface as a function of cholesterol oleate concentration. The response measurement conducted on ChOx-ChEt/DTSP/Au bio-electrode reveal the value of Michaelis-Menten constant (Km) as 0.95 mM suggesting enhanced affinity of enzymes (ChOx and ChEt). The ChOx-ChEt/DTSP/Au bio-electrodes show linearity in range of 50 to 400 mg dl(-1) of cholesterol oleate and the shelf-life of more than 50 days when stored at 4 degrees C. This biosensing electrode shows correlation coefficient of 0.9973 and standard deviation of regression as 0.859 microA.     

Flow injection potentiometry for enzymatic assay of cholesterol with a tungsten electrode sensor

Flow injection potentiometry (FIP) for the enzymatic determination of cholesterol is reported. The assay utilises a combination of three enzymes: cholesterol esterase (CE), cholesterol oxidase (COD) and peroxidase (POD). The method is developed by the use of a tungsten wire electrode as a sensor vs. Ag/AgCl in conjunction with a redox mediator ferrocyanide. CE converts esterified cholesterol to free cholesterol, which is then oxidised by COD with hydrogen peroxide as product. Ferrocyanide is converted to ferricyanide by hydrogen peroxide, catalysed by POD, and the tungsten electrode responds to the ratio of ferricyanide to ferrocyanide. Flow injection potentiometry gave well-defined peaks for cholesterol samples with a fast response (30 s). Linear calibration was obtained from 0.05 to 3.0 mM cholesterol, with a slope of 60.2 mV/decade change in cholesterol concentration, and detection limit 0.01 mM cholesterol (S/N=3). Repeatability was 3% (CV). Interferences from commonly found species were shown to be negligible. The sensor cell is simple to construct, and it was free from surface contamination problems over long periods of use. The application of the sensor for the determination of serum cholesterol was demonstrated.     

A biocompatible needle-type glucose sensor based on platinum-electroplated carbon electrode

A biocompatible needle-type glucose sensor with a 3-electrode configuration was constructed. A platinum-electroplated carbon stick was used as the working electrode, Ag/AgCl as the reference electrode, and a disposable hypodermic needle made of stainless steel as the counter electrode. A Nafion membrane, an immobilized glucose oxidase (GOD) membrane, and a biocompatible membrane with diffusion-limiting effect were coated successively onto the working electrode. The sensor showed a rapid response (< 120 s in batch operation), good reproducibility (RE < 3%), good stability (over 36 h in control serum), a wide dynamic range (5-600 mg/dL glucose), and superior biocompatibility. It was used to determine glucose in serum. The data obtained from the sensor showed good agreement with that from a clinical autoanalyzer (R > 0.95).     

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.     

壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮-多壁碳纳米管复合物固定漆酶基化学传感器的性能

通过壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮(CTS-g-N-CSIDZ)非共价功能化多壁碳纳米管(MWCNTs)的方式制备固定漆酶载体,该复合物载体主要通过物理吸附和漆酶活性中心与载体上配体之间的配位作用来固定漆酶,较大程度地保持了游离漆酶活性位原始构象.将固定了漆酶的复合物附着在裸玻碳电极上便构筑了复合物固定漆酶修饰玻碳电极.在以分光光度法测定了这种复合物载体对漆酶的担载量、固定漆酶比活力、稳定性、重复使用性及其催化2,6-二甲氧基苯酚(DMP)氧化动力学参数的基础上,还对基于此种复合物固定漆酶修饰玻碳电极作为化学传感器(以DMP作为底物)的性能进行了研究.结果表明,该复合物具有较高的固酶担载量(81.7 mg/g)和固定漆酶比活力(1.33 U/mg);而作为电化学传感器的复合物固定漆酶修饰玻碳电极对底物DMP具有较高的亲和力(对DMP的米氏常数KM是0.0918 mmol/L),较高的灵敏度( 3680 mA· L/mol),较低的检测限(3.3×10-4 mmol/L),较高的响应选择性,良好的重现性、重复使用性和长期稳定性.这种漆酶基电极有望用作电流型特定结构的酚类传感器.     

A novel electrochemical biosensor for detection of cholesterol

We report on a highly sensitive electrochemical biosensor for determination of cholesterol. The biosensor was fabricated by co-immobilizing bi-enzymes, cholesterol oxidase (ChOx), and horseradish peroxidase (HRP). Voltammetric technique such as cyclic voltammetry and impedance experiment were used to study the characterization of modified electrode step by step. The developed sensor is cheap, disposable, portable and exhibits higher sensitivity. The biosensor expressed a wide linear range up to 300 mg dL^–1 in a physiological condition (pH 7.0), with a correlation coefficient of 0.9969. A sensitivity of 13.28 μA mg^–1 dL cm^?2 which makes it very promising for the clinical determination of cholesterol.     

Polyaniline Langmuir-Blodgett film based cholesterol biosensor

Cholesterol oxidase (ChOx) has been covalently linked to Langmuir-Blodgett (LB) monolayers of polyaniline (PANI)-stearic acid (SA) prepared onto indium-tin-oxide (ITO) coated glass plates via glutaraldehyde (Glu) chemistry. These ChOx/Glu/PANI-SA LB film/ITO electrodes have been characterized by FT-IR, cyclic voltammetry, and scanning electron microscopy, respectively. The results of response measurements carried out on these bioelectrodes using linear sweep voltammetry (LSV) reveal linearity from 25 to 400 mg/dL of cholesterol concentration with sensitivity of 88.9 nA mg(-1) dL. The linear regression analysis of bioelectrode reveals standard deviation and correlation coefficient of 0.737 microA and 0.9988, respectively. The low value of the Michaelis-Menten constant of these bioelectrodes obtained as 1.21 mM for the immobilized enzyme indicates increased interaction between ChOx and cholesterol in the PANI-SA LB film.     

Amperometric biosensor for ethanol based on immobilization of alcohol dehydrogenase on a nickel hexacyanoferrate modified microband gold electrode

Alcohol dehydrogenase (ADH) has been immobilized on a nickel hexacyanoferrate modified microband gold electrode surface by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new amperometric sensor for the assay of ethanol. The resulting enzyme electrode exhibits excellent electrocatalysis for the oxidation of reduced nicotinamide-adenine dinucleotide (NADH). The amperometric determination is based on the electrochemical detection of NADH which is generated in the enzymatic reaction of ethanol with NAD(+) under catalysis of ADH. The influence of various experimental conditions was examined for the determination of the optimum analytical performance. The sensor responds rapidly to ethanol with a detection limit of (5.0 +/- 0.3) x 10(-7) mol 1(-1). The response current increases linearly with ethanol concentration up to 5 mmol 1(-1). The sensor remains relatively stable for about 1 week.     

钴原卟啉附修饰玻碳电极为基底的胆固醇酶电极

本文利用钴原卟啉对H₂O₂氧化有很高催化活性的特点,以钴原卟啉修饰玻碳电极为基底,戊二醛交联法固定胆固醇氧化酶,以电聚合邻苯二胺膜抗干扰、抗毒化.得到了线性范围为0.1～2mmol/L,响应时间在30s以内,可连续使用150次的胆固醇酶电极.     

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.     

Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis-Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.     

A fast and highly sensitive detection of cholesterol using polymer microfluidic devices and amperometric system

In this work, the rapid detection of cholesterol using poly(dimethylsiloxane) microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, was developed. Direct amperometric detection for poly(dimethylsiloxane) (PDMS) microchip capillary electrophoresis was successfully applied to quantify cholesterol levels. Factors influencing the performance of the method (such as the concentration and pH value of buffer electrolyte, concentration of cholesterol oxidase enzyme (ChOx), effect of solvent on the cholesterol solubility, and interferences) were carefully investigated and optimized. The migration time of hydrogen peroxide, product of the reaction, was less than 100 s when using 40 mM phosphate buffer at pH 7.0 as the running buffer, a concentration of 0.68 U/mL of the ChOx, a separation voltage of +1.6 kV, an injection time of 20 s, and a detection potential of +0.5 V. PDMS microchip capillary electrophoresis showed linearity between 38.7 μg/dL (1 μM) and 270.6 mg/dL (7 mM) for the cholesterol standard; the detection limit was determined as 38.7 ng/dL (1 nM). To demonstrate the potential of this assay, the proposed method was applied to quantify cholesterol in bovine serum. The percentages of recoveries were assessed over the range of 98.9-101.8%. The sample throughput was found to be 60 samples per hour. Therefore, PDMS microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, is very rapid, accurate and sensitive method for the determination of cholesterol levels.     

Multienzymatic-rotating biosensor for total cholesterol determination in a FIA system

Fabrication of an amperometric-rotating biosensor for the enzymatic determination of cholesterol is reported. The assay utilizes a combination of three enzymes: cholesterol esterase (ChE), cholesterol oxidase (ChOx) and peroxidase (HRP); which were co-immobilizing on a rotatory disk. The method is developed by the use of a glassy carbon electrode as detector versus Ag/AgCl/3 M NaCl in conjunction with a soluble-redox mediator 4-tert-butylcatechol (TBC). ChE converts esterified cholesterol to free cholesterol, which is then oxidized by ChOx with hydrogen peroxide as product. TBC is converted to 4-tert-butylbenzoquinone (TBB) by hydrogen peroxide, catalyzed by HRP, and the glassy carbon electrode responds to the TBB concentration. The system has integrated a micro packed-column with immobilized ascorbate oxidase (AAOx) that works as prereactor to eliminate l-ascorbic acid (AA) interference. This method could be used to determine total cholesterol concentration in the range 1.2 μM-1 mM (r = 0.999). A fast response time of 2 min has been observed with this amperometric-rotating biosensor. Lifetime is up to 25 days of use. The calculated detection limits was 11.9 nM. Reproducibility assays were made using repetitive standards solutions (n = 5) and the percentage standard error was less than 4%.     

Wireless biosensor system for real-time cholesterol monitoring in fish “Nile tilapia”

The rapidly increasing demand for cultured fish as a food resource requires simple, effective methods for controlling fish health in culture conditions. Plasma total cholesterol levels are significantly related to fish mortality following bacterial challenge, and are thus a good indicator of the general health of fish. We developed a wireless biosensor system to continuously monitor the total cholesterol concentration in fish (Nile tilapia, Oreochromis niloticus). The biosensor was constructed with Pt-Ir wire (φ0.178 mm) as the working electrode and Ag/AgCl paste as the reference electrode. Cholesterol oxidase and cholesterol esterase were immobilized on the working electrode using glutaraldehyde. The sensor output was linear and strongly correlated with the cholesterol level (R = 0.9970) in the range of 2.65-403 mg dl⁻¹. This range covers the range of total cholesterol levels in fish. To avoid blood coagulation and proteins coalescing on the sensor, we implanted the sensor in the fluid under the scleral surface of the eyeball (EISF). The EISF is presumed to reflect the levels of most blood components and does not include the substances contained in blood that inhibit sensor measurement. Total cholesterol concentrations in blood and EISF were strongly correlated (R = 0.8818, n = 72) in the blood total cholesterol range of 74-480 mg dl⁻¹. Therefore, we used EISF as an alternative to blood and performed continuous in vivo-monitoring of the total cholesterol concentration in fish. We also investigated the application of the calibration method and wireless monitoring system. These applications enabled us to securely monitor total cholesterol levels in free-swimming fish in an aquarium for over 40 h. Thus, our newly developed sensor provided a rapid and convenient method for real-time monitoring of total cholesterol concentrations in free-swimming fish.     

Growth of non-enzymatic cholesterol biosensor using TiO2 decorated graphene oxide with bare GCE and PPy-GCE

In this work, we report an electrochemical cholesterol biosensor based on cholesterol oxidase(ChOx) enzyme immobilized on TiO₂- nanoparticles – reduced graphene oxide(rGO) – polypyrrole (PPy) nanocomposite modified electrode.The electrochemical properties of GCE modified PPy (PPy-GCE) were studied using CV (Cyclic Voltammetry) and DPV (Differential Pulse Voltammetry). The developed sensor exhibited piecewise linearity from 0.1 μM to 1 μM and from 1 μM to 600 μM with the sensitivity of 61.665 and 0.1466 (2 mA mM × cm) respectively. The limit of detection of the sensor was found to be 32 nm.     

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.     

A Selective Cholesterol Biosensor Based on Composite Film Modified Electrode for Amperometric Detection

An amperometric cholesterol biosensor based on immobilization of cholesterol oxidase in a Prussian blue (PB)/polypyrrole (PPy) composite film on the surface of a glassy carbon electrode was fabricated. Hydrogen peroxide produced by the enzymatic reaction was catalytically reduced on the PB film electrode at 0 V with a sensitivity of 39 μA (mol/L)^?1. Cholesterol in the concentration range of 10^?5 ? 10^?4 mol/L was determined with a detection limit of 6 × 10^?7 mol/L by amperometric method. Normal coexisting compounds in the bio‐samples such as ascorbic acid and uric acid do not interfere with the determination. The excellent properties of the sensor in sensitivity and selectivity are attributed to the PB/PPy layer modified on the sensor.