Electropolymerised o-phenylenediamine film as means of immobilising lactate oxidase for a L-lactate biosensor

The immobilisation of L-lactate oxidase at a platinum electrode was achieved by entrapping the enzyme within an o-phenylenediamine film at 0.65 V (vs. Ag/AgCl). Anodic detection of the product of the enzymatic reaction, i.e., hydrogen peroxide, at 0.75 V (vs. Ag/AgCl) was employed for the quantification of L-lactate using amperometric batch and flow injection methods. This technique allows the enzyme to be entrapped in a strongly adherent thin membrane. The sensor exhibits a very fast response time, an active enzyme loading of 5 mU/cm(2) electrode surface, and high sensitivity with a detection limit of 2.46 x 10(-7)M. A sample throughput of 180/hr, precision of 3.53% for 25 injections and linearity up to 1.5mM were obtained in flow injection analysis studies. The one-step procedure for sensor preparation requires 20 min, and the discriminative properties of the polymer film show great promise as a means of excluding interfering compounds commonly found in serum.     

纳米金-还原氧化石墨烯修饰葡萄糖氧化酶传感器的制备及其电流法检测饮料中的葡萄糖

利用纳米金(Au NPs)与还原氧化石墨烯(rGO)复合纳米材料制备了葡萄糖氧化酶生物传感器并用于饮料中葡萄糖含量的检测。将壳聚糖作为还原剂及稳定剂,通过一步法合成了Au NPs-rGO复合材料,并通过物理吸附固定葡萄糖氧化酶(GOx)来制作GOx生物传感器。该传感器在磷酸盐缓冲溶液(0.1 mol/L,p H6.0)中,-0.45 V(vs.Ag/Ag Cl)电位下电流法检测葡萄糖含量,线性检测范围为0.01~0.88 mmol/L,灵敏度为22.54μA·mmol-1·L·cm-2,检出限为1.01μmol/L,且表观米氏常数为0.497 mmol/L。该传感器用于多种饮料中葡萄糖含量的直接检测,结果满意。     

Sensing the Lactic Acid in Probiotic Yogurts Using an L-Lactate Biosensor Coupled with a Microdialysis Fiber Inserted in a Flow Analysis System

An amperometric biosensor for the determination of L-lactic acid in probiotic yogurts has been assembled using L-lactate dehydrogenase (EC 1.1.1.27, LDH) entrapped in 1% (v/v) neutralized Nafion® solution deposited on Variamine blue redox mediator modified screen-printed electrodes. The Variamine blue was previously covalently linked to oxidized single-walled carbon nanotubes and used for modifying screen-printed electrodes. The electrochemical cell, containing the L-lactate biosensor operating at an applied working potential of +200 mV vs. Ag|AgCl, was coupled with a microdialysis fiber and connected with a flow system, thus obtaining a microdialysis based sampling experimental set-up. Various analytical parameters, such as the cofactor concentration (2 mM, NAD⁺), the flow rate (10.5 μL/min), the applied working potential (+200 mV vs. Ag|AgCl), the working buffer (50 mM phosphate buffer +0.1 M KCl), and pH (7.5), were optimized in batch amperometric experiments. The dynamic linear working range was comprised between 2·10^?4 and 1·10^?3 M. The proposed biosensor was challenged with real samples of yogurt, properly diluted in working buffer, and the performances of the L-lactate biosensor were compared with a commercially available kit for the determination of L-lactic acid in foodstuffs from R-Biopharm GmbH, Germany, showing a good agreement.     

Glucose Biosensor Based on Multi‐Walled Carbon Nanotube Modified Glassy Carbon Electrode

An amperometric glucose biosensor based on multi‐walled carbon nanotube (MWCNT) modified glassy carbon electrode has been developed. MWCNT‐modified glassy carbon electrode was obtained by casting the electrode surface with multi‐walled carbon nanotube materials. Glucose oxidase was co‐immobilized on the MWCNT‐modified glassy carbon surface by electrochemical deposition of poly(o‐phenylenediamine) film. Enhanced catalytic electroreduction behavior of oxygen at MWCNT‐modified electrode surface was observed at a potential of ?0.40 V (vs. Ag|AgCl) in neutral medium. The steady‐state amperometric response to glucose was determined at a selected potential of ?0.30 V by means of the reduction of dissolved oxygen consumed by the enzymatic reaction. Common interferents such as ascorbic acid, 4‐acetamidophenol, and uric acid did not interfere in the glucose determination. The linear range for glucose determination extended to 2.0 mM and the detection limit was estimated to be about 0.03 mM.     

Use of Pyranose Oxidase Enzyme in Inhibitor Biosensing

Abstract

An inhibition based biosensing system was developed for the determination of glutathione (GSH) and ethanol (EtOH) as pyranose oxidase (PyOx) inhibitors. The PyOx was immobilized in carbon paste electrode and the amperometric detection of hydrogen peroxide through the enzymatic reaction was monitored at + 0.9 V versus Ag/AgCl. In addition to the optimization studies, analytical characteristics and the effect of various compounds on the biosensor response were researched. Finally, the proposed system was applied to analyze GSH and EtOH in real matrices.     

Design of an amperometric xanthine biosensor based on a graphite transducer patterned with noble metal microparticles

A mesoporous graphite material micro-structured with palladium-platinum deposits (mixed in the ratio of 70:30% Pd:Pt) has been used as a cost-effective electrode material for designing an amperometric biosensor for xanthine. The here reported biosensor shows significantly improved operational parameters as compared to previously published results. At a constant applied potential of −0.05 V (vs. Ag/AgCl) it is distinguished with enhanced selectivity of the determination: at the working potential the current from the electrochemical transformation of various electrochemically active substances usually attending biological fluids (incl. uric acid, L-ascorbic acid, glutathione and paracetamol) has been eliminated. The effect of both the temperature and buffer composition on the analytical performance of the sensor has been investigated. Under optimal operational conditions (25°C, −0.05 V vs. Ag/AgCl, phosphate buffer, pH 8.4), the following have been defined for the biosensor: sensitivity 0.39 μA μM⁻¹, strict linearity of the response up to xanthine concentration 70 μM, detection limit of 1.5 μM (S/N=3) and a response time of at most 60 s.     

Highly Sensitive Amperometric Glucose Biosensor Based on Glassy Carbon Electrode with Copper/Palladium Coating

A highly sensitive amperometric glucose biosensor based on immobilizing glucose oxidase in electropolymerized poly(o‐phenylenediamine) film on glassy carbon electrode coated sequentially with copper and palladium layers has been developed. The steady‐state amperometric response to glucose was determined by means of the oxidation of hydrogen peroxide generated by the enzymatic reaction at a potential of either +0.70 or +0.40 V (vs. Ag|AgCl reference). The deposited copper/palladium layer showed great enhancement in the performance of the enzyme electrode, possibly due to its better electrocatalytic activity for hydrogen peroxide oxidation and large surface area. Effects of the relative loading of palladium, enzyme and polymer on the electrode performance were examined in detail. Sensitivity and detection limit for glucose determinations at +0.70 V were about 7.3 μA/mM and 0.1 μM, respectively. A wide linear range up to 6.0 mM glucose could be achieved. Electrode performance was superior to similar works reported in the literature. The response time was less than 2 s and its lifetime was longer than three months. The permeable polyphenylenediamine film also offered good anti‐interference ability to ascorbic acid, uric acid and acetaminophen, especially when a detection potential of +0.40 V was employed.     

Electroanalytical Determination of Some Phenolic Acids by High‐Performance Liquid Chromatography at Gold Electrodes

The electrochemical and amperometric behavior of a gold electrode was investigated towards the oxidation of several common phenolic acids in neutral phosphate solutions. Au electrodes show an appreciable stability and reproducibility of the amperometric signals by using a constant applied potential of 1.0 V vs. Ag/AgCl. Separations of selected phenolic acids using a reverse phase C₁₈ analytical column with a mobile phase containing 10 mM NaH₂PO₄ plus 10 mM Na₂HPO₄ (pH 7) and methanol as organic modifier, are achieved isocratically in less than 30 min. The detection limits at the level of nmol/L and linear ranges of four‐five orders of magnitude are generally achieved. The proposed chromatographic strategy coupled with the electrochemical detection at the Au electrode was successful tested for the quantitative determination of phenolic acids in beer, red wine and brandy with good sensitivity and recovery.     

Use of a copper electrode in alkaline medium as an amperometric sensor for sulphite in a flow-through configuration

A flow injection analysis (FIA) method has been developed for the determination of sulphite in beverages. The method is based on the amperometric detection (0.60 V vs Ag/AgCl (sat. NaCl)) of the analyte at a copper surface in an alkaline medium (1 M NaOH solution) with a manifold that incorporates flow extraction of sulphite as SO2 through a PTFE membrane. Under optimal experimental conditions the peak current response increases linearly with sulphite concentration over the range from 1.0 to 5.0 mM. The repeatability of the electrode response in the FIA configuration was evaluated as 4% ( n =20), the limit of detection of the method was 0.04 mM (S/N =3) and the analytical frequency was 50 h(-1). Since ethanol is also electroactive and permeates through the PTFE membrane, a strategy involving in a first step measurements of only ethanol by manipulating the pH of the donor stream was employed for wine samples. Then, both ethanol and sulphite were measured at the copper electrode at 0.40 V vs Ag/AgCl (sat. NaCl) and the sulphite concentration was determined by difference. Results for 3 different beverage samples (alcoholic and non-alcoholic) showed excellent agreement with the ones obtained by using a recommended procedure for sulphite analysis.     

Chronoamperometric Biosensor for Protease Activity Assay and Inhibitor Screening

Herein, we report an electrochemical biosensor for the measurement of proteins and protease activity using carbon nanopowder paste electrode modified with tyrosinase. The measurement of proteins is based on the amperometric measurement of the enzymatic product, tyrosine, at low applied potential (i.e +0.05 V vs. Ag/AgCl), in a linear range from 1 μg/mL to 10 μg/mL for casein and albumin bovine serum. This biosensor is also used to assess the enzymatic activity of the enzymes namely trypsin and proteinase K, observing a detection limit of 0.02 mU/mL (S/N=3). The ability of this assay to monitor the trypsin activity is exploited to investigate its inhibition by leupeptin. The trypsin kinetic interactions reveal uncompetitive binding of leupeptin with 50 % inhibition equal to 3.75 μM, a dynamic range of 0.25–10 μM, and a detection limit of 0.25 μM. The assay developed can be considered as general activity assay of any protease with the advantages of good storage stability (few weeks), low detection limit and ability to analyse turbid and colored samples.     

Determination of breath alcohol using a differential-type amperometric biosensor based on alcohol dehydrogenase

A differential-type amperometric biosensor based on conventional thick-film technology has been developed for breath alcohol measurement. The amperometric breath alcohol biosensor utilizes the alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) cofactor which produce reduced NADH as a product of the oxidation of alcohol. The biosensor was designed in a differential format consisting of a common Ag/AgCl reference electrode, an active working electrode containing the ADH, and the inactive working electrode containing only bovine serum albumin instead of the ADH. The differential signal between the active working electrode and the inactive working electrode minimized the interference from a large number of oxidizable species present in a person's breath. Prior to the amperometric measurement the biosensor was hydrated simply by dipping it into a phosphate buffer solution at pH 7.4. The NADH produced from the enzymatic reaction was oxidized at the working electrode biased at a potential of 470 mV vs. an on-board Ag/AgCl reference electrode. The biosensor can measure a person's breath alcohol over the concentration range 20–800 ppm routinely required in a test of drunken driving.     

Synthetic nanocrystalline diamond as a third-generation biosensor support

Horseradish peroxidase (HRP) has been immobilized on the surface of functionalized nanocrystalline diamond (NCD) thin films. The structure of the modified NCD surface as well as the electrochemical behavior of the whole system was characterized by impedance spectroscopy and cyclic voltammetry. The proximity of HRP heme groups to the NCD surface allowed direct electron transfer between them, resulting in two separated one-electron-transfer peaks at 0.05 V and 0.29 V vs Ag/AgCl, corresponding to the cathodic and anodic process, respectively. The heterogeneous electron-transfer constant for both processes was calculated to be 0.066 s(-1), the charge-transfer coefficient alpha = 0.49, and the immobilized enzymatic layer about 2.10(-10) mol/cm2. The modified NCD electrode was used as a third-generation biosensor for hydrogen peroxide determination showing a linear response in the 0.1-45 mM H2O2 range, at +0.05 V vs Ag/AgCl.     

Amperometric Biosensor for Adenosine-5′-Triphosphate Based on a Platinum-Dispersed Carbon Paste Enzyme Electrode

Abstract

A new amperometric biosensor for adenosine-5′-triphosphate (ATP) was designed using a platinum-dispersed carbon paste into which glycerol kinase and glycerol-3-phosphate oxidase were incorporated. The biosensor is based on the detection of hydrogen peroxide produced by the enzymatic reaction of ATP with glycerol and the subsequent oxidation of glycerol-3-phosphate. The use of the platinum-dispersed carbon paste electrode lowered the oxidation potential for hydrogen peroxide, permitting the sensitive detection of ATP at 0.4 V vs. Ag/AgCl. A linear response to ATP was observed in the concentration range of 1 x 10^?5 to 2.5 x 10^?3 M.

     

Sub-millimolar determination of formalin by pulsed amperometric detection

Formalin, formaldehyde in the presence of methanol, was determined by pulsed amperometric detection (PAD). A triple waveform using E_det=−0.3 V (t_det=30 ms), E_oxd=+0.8 V (t_oxd=200 ms), and E_red=−0.8 V (t_red=350 ms) versus Ag/AgCl was applied at a Au electrode for detection in a flow injection (FI) system. The approach was rapid and yielded a sub-millimolar detection limit (0.0129 mM) with a dynamic range up to 100 mM. A precision of 8.8% R.S.D. at 1.0 mM for two hundred repetitive injections by the FI-PAD was obtained, whereas holding at a constant potential (−0.3 V versus Ag/AgCl) for anodic oxidation of formaldehyde caused the response to decrease dramatically after a few measurements. The method developed was used to analyze the formalin contents of water from rinsed samples of vegetables and fruit and ice-melt from seafood, and the method showed good agreement with the liquid chromatography (LC) method.     

Amperometric Glucose Sensor Fabricated by Combining Glucose Oxidase Micelle Membrane and Aminated Glassy Carbon Electrode

Abstract

An amperometric glucose biosensor based on the detection of the reduction of oxygen has been developed by combining an aminated glassy carbon electrode with a polystyrene (PS) membrane containing glucose oxidase (GOD) micelles. The structure of GOD micelles contained in PS membrane was observed by scanning electron microscope. The micelle has a roughly spherical shape, and the enzyme colony is contained inside the micelle. This glucose sensor exhibited good sensitivity with short response time (within 2 min). A good linear relationship was observed in the concentration range of 0.2 mM to 2.6 mM when the applied potential was ? 0.45 V vs. Ag/AgCl.     

Direct Electrochemistry of Glucose Oxidase at Carbon Nanotube‐gold Colloid Modified Electrode with Poly(diallyldimethylammonium chloride) Coating

Glucose oxidase showed direct electrochemical transfer at glassy carbon electrodes immobilized with carbon nanotube‐gold colloid (CNT‐Au) composites with poly(diallydimethylammonium chloride) (PDDA) coatings. The modified electrode (GC/CNT/Au/PDDA‐GOD) was employed for the amperometric determination of glucose. Under optimal conditions, the biosensor displayed linear response to glucose from 0.5 to 5 mM with a sensitivity of 2.50 mA M^?1 at an applied potential of ?0.3 V (vs. Ag|AgCl reference).     

The Study of a Disposable Reagentless Biosensor for Fast Test of Aspartate Aminotransferase

A reagentless biosensor for amperometric measurement of aspartate aminotransferase (AST) has been developed. The three‐electrode biosensor was modified with two enzyme membrane with an integrated planar Ag|AgCl reference electrode. With a cellulose membrane containing catalytic substance coated, the reagentless biosensor was used for measurement of serum AST. The optimal sensitive membrane composition and operation conditions were studied. With low polarizing potential (0 V vs. integrated Ag|AgCl) and appropriate substrate concentration (20 mM α‐ketoglutarate acid), the response of the biosensor to AST activity was linear over the range of 25–1000 U/L with good relativity (R=0.9904 and 0.9823 in standard AST solutions and AST serum respectively) and fast response time (120 s). The biosensor also indicated good stability of more than 90% of its original activity for 60 days stored in 4 °C, and 90% of the activity was retained after a storage period of 15 days at room temperature (25 °C). The biosensor is expected to be used for the diagnosis of heart and liver disease in the future.     

Poly (4-methoxyphenol) film as a galactose-sensing material

Poly (4-methoxyphenol)-galactose oxidase biosensor for galactose is reported. This amperometric biosensor was prepared in a one-step procedure by electrochemical polymerization of the relevant monomer in the presence of galactose oxidase on Pt electrode surface in a KCl solution at potential of 0.6 V vs. Ag/AgCl. From the steady-state amperometric responses to galactose of the resulting polymeric biosensor, its sensor characteristics such as feasibility of preparation, linear range, response time, selectivity and stability were evaluated.     

Electrocatalytic Reduction of H2O2 and Oxygen on the Surface of Thionin Incorporated onto MWCNTs Modified Glassy Carbon Electrode: Application to Glucose Detection

A new H₂O₂ enzymeless sensor has been fabricated by incorporation of thionin onto multiwall carbon nanotubes (MWCNTs) modified glassy carbon electrode. First 50 μL of acetone solution containing dispersed MWCNTs was pipetted onto the surface of GC electrode, then, after solvent evaporations, the MWCNTs modified GC electrode was immersed into an aqueous solution of thionin (electroless deposition) for a short period of time <5–50 s. The adsorbed thin film of thionin was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase enzyme. Also the modified electrode shows excellent catalytic activity for oxygen reduction at reduced overpotential. The rotating modified electrode shows excellent analytical performance for amperometric determination of hydrogen peroxide, at reduced overpotentials. Typical calibration at ?0.3 V vs. reference electrode, Ag/AgCl/3 M KCl, shows a detection limit of 0.38 μM, a sensitivity of 11.5 nA/μM and a liner range from 20 μM to 3.0 mM of hydrogen peroxide. The glucose biosensor was fabricated by covering a thin film of sol–gel composite containing glucose oxides on the surface of thionin/MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 1 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. In addition biosensor can reach 90% of steady currents in about 3.0 s and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) is eliminated. The usefulness of biosensor for direct glucose quantification in human blood serum matrix is also discussed. This sensor can be used as an amperometric detector for monitoring oxidase based biosensors.     

Amperometric lactate biosensor for flow injection analysis based on a screen-printed carbon electrode containing Meldola's Blue-Reinecke salt, coated with lactate dehydrogenase and NAD

A biosensor for the measurement of lactate in serum has been developed, which is based on a screen-printed carbon electrode, modified with Meldola's Blue-Reinecke Salt (MBRS-SPCE), coated with the enzyme lactate dehydrogenase NAD⁺ dependent (from Porcine heart), and NAD⁺. A cellulose acetate layer was deposited on the top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V vs. Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer pH 10 containing 0.1 M potassium chloride solution; a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range 0.55-10 mM lactate; the former represents the detection limit. The precision of the system was determined by carrying out 10 repeat injections of 10 mM l(+)lactic acid standard; the calculated coefficient of variation was 4.28%. It was demonstrated that this biosensor system could be applied to the direct measurement of lactate in serum without pre-treatment; therefore, this would allow high throughput-analysis, at low cost, for this clinically important analyte.