Development of an Amperometric Biosensor Platform for the Combined Determination of <Emphasis Type="SmallCaps">l</Emphasis>-Malic,Fumaric, and <Emphasis Type="SmallCaps">l</Emphasis>-Aspartic Acid

Three amperometric biosensors have been developed for the detection of l-malic acid, fumaric acid, and l -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD⁺) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for l-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM^?1 (l-malate biosensor) and 0.4 μA mM^?1 (fumarate biosensor). The l-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM^?1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.     

Flow injection analysis for glucose by the combined use of an immobilized glucose oxidase reactor and a peroxidase electrode

The amperometric peroxidase electrode measures hexacyanoferrate(III), produced by hydrogen peroxide, which is generated by injecting a 2μl sample into a reactor of immobilized glucose oxidase covalently bound to silica gel. The peak current is linearly related to the glucose concentration in the range 0.05–10 g l^?1; sample throughput is about 100 h^?1. Ascorbic acid (? 0.5 mM) does not interfere.     

Amperometric Biosensor for Lactate Based on Meldola's Blue Adsorbed on Silica Gel Modified with Niobium Oxide

A reagentless amperometric biosensor sensitive to lactate was developed. This sensor comprises a carbon paste electrode modified with lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD⁺) cofactor and Meldola's blue (MB) adsorbed on silica gel coated with niobium oxide. The amperometric response was based on the electrocatalytic properties of MB to oxidize NADH, which was generated in the enzymatic reaction of lactate with NAD⁺ under catalysis of LDH. The dependence on the biosensor response was investigated in terms of pH, supporting electrolyte, ionic strength, LDH and NAD⁺ amounts and applied potential. The biosensor showed an excellent operational stability (95% of the activity was maintained after 250 determinations) and storage stability (allowing measurements for over than 2.5 months, when stored in a refrigerator). The proposed biosensor also presented good sensitivity allowing lactate quantification at levels down to 6.5×10^?6 mol L^?1. Moreover, the biosensor showed a wide linear response range (from 0.1 to 14 mmol L^?1 for lactate). These favorable characteristics allowed its application for direct measurements of lactate in biological samples such as blood. The precision of the data obtained by the proposed biosensor show reliable results for real complex matrices.     

Determination of Glycerol in Biodiesel Using a Nickel(II) Oxyhydroxide Chemically Modified Electrode by Cyclic Voltammetry

A glassy carbon electrode chemically modified with nickel oxyhydroxide from a nickel hexacyanoferrate (NiHCF) film was used to determine glycerol in biodiesel by cyclic voltammetry. The modified electrode exhibited a linear response to glycerol concentration in the range from 0.05 to 0.35 mmol L^?1, and a detection limit of 0.030 mmol L^?1. The glycerol concentration found in the biodiesel sample was 0.156 mmol L^?1. The method developed in this study showed a recovery of (100.3±5.0)%.     

Development of a Carbon Paste Electrode for Lactate Detection Based on Meldola’s Blue Adsorbed on Silica Gel Modified with Niobium Oxide and Lactate Oxidase

A reagentless amperometric biosensor sensitive to lactate was developed. The sensor employs a carbon paste electrode modified with lactate oxidase (LOx) and Meldola’s Blue (MB) adsorbed on silica gel coated with niobium oxide. The dependence on the biosensor response was investigated in terms of pH, supporting electrolyte, ionic strength, lactate oxidase (LOx) amounts and applied potential. The biosensor showed an excellent operational stability (96 % of the activity was maintained after 150 determinations) and storage stability (allowing measurements for more than 1.5 months, when stored in a refrigerator). The proposed biosensor also presented good sensitivity allowing lactate quantification at levels down to 6.5×10^?7 mol L^?1. Moreover, the biosensor showed a good linear response range (from 0.1 to 5.0 mmol L^?1 for lactate). Lactate analysis in biological samples such as blood was also performed. The precision of the data obtained by the proposed biosensor showed reliable results for real complex matrices.     

An amperometric uric acid biosensor based on chitosan-carbon nanotubes electrospun nanofiber on silver nanoparticles

A novel amperometric uric acid biosensor was fabricated by immobilizing uricase on an electrospun nanocomposite of chitosan-carbon nanotubes nanofiber (Chi–CNTsNF) covering an electrodeposited layer of silver nanoparticles (AgNPs) on a gold electrode (uricase/Chi–CNTsNF/AgNPs/Au). The uric acid response was determined at an optimum applied potential of ?0.35 V vs Ag/AgCl in a flow-injection system based on the change of the reduction current for dissolved oxygen during oxidation of uric acid by the immobilized uricase. The response was directly proportional to the uric acid concentration. Under the optimum conditions, the fabricated uric acid biosensor had a very wide linear range, 1.0–400 μmol L^?1, with a very low limit of detection of 1.0 μmol L^?1 (s/n?=?3). The operational stability of the uricase/Chi–CNTsNF/AgNPs/Au biosensor (up to 205 injections) was excellent and the storage life was more than six weeks. A low Michaelis–Menten constant of 0.21 mmol L^?1 indicated that the immobilized uricase had high affinity for uric acid. The presence of potential common interfering substances, for example ascorbic acid, glucose, and lactic acid, had negligible effects on the performance of the biosensor. When used for analysis of uric acid in serum samples, the results agreed well with those obtained by use of the standard enzymatic colorimetric method (P?>?0.05).

Nickel Hexacyanoferrate‐Based Sensor Electrode for the Detection of Nitric Oxide at Low Potentials

Recently transition metal hexacyanoferrates, analogues of Prussian Blue, have found application in electroanalysis for the detection of biologically relevant species. Our study describes the development of a novel electrode based on nickel hexacyanoferrate (NiHCF) for the sensorial NO determination. A NiHCF layer was deposited on platinum by cyclic voltammetry in a solution of nickel (II) chloride and potassium hexacyanoferrate (III). The electrode was found to be active for NO reduction. The interaction with the radical was studied voltammetrically within the range from 0 V up to +0.4 V vs. Ag/AgCl/1 M KCl. The most appropriate potential for an amperometric detection was determined to be +0.25 V due to the advantageous signal/noise ratio. The sensitivity of the electrodes was found to be 2.0–2.3 A M^?1 cm^?2. The sensor response of the most important interferents for NO analysis, hydrogen peroxide, ascorbic acid and nitrite, was measured and determined to be sufficiently low.     

Immobilization of lactate as an electroactive indicator on pencil graphite electrode for the development of a new electrochemical biosensor for the detection of lactate dehydrogenase

A biosensor was developed for the detection of lactate dehydrogenase (LDH) enzyme using a lactate modified pencil graphite electrode (PGE). The sensor relies on the immobilization of the lactate on PGE, and LDH detection is based on the decrease of lactate peak current following oxidation to pyruvate in the presence of LDH. Square wave voltammetric technique was used for the assay of signals in the range of ?0.6 to 0.8 V and a frequency of 25 Hz for the determination of LDH. The dependence of the response was investigated in terms of reaction time, washing time and LDH and NAD⁺ amounts. Also, the electrochemical behavior of LDH treatment on the lactate modified PGE was studied. The electrode showed good selectivity, repeatability and an operational stability of about 90% of its original response for two weeks. Moreover, the sensor displayed a linear response range from 0.36?C2.13 U ??l^?1 for LDH with a detection limit of 0.16 U ??l^?1. The response time of the LDH-treated lactate modified PGE was found to be 2 s. The relative standard deviation (RSD) obtained was 3.5% (for LDH 0.71 U ??l^?1 and n = 3).     

Simultaneous Determination of Ascorbic Acid and Uric Acid by Using a PVC/TTF‐TCNQ Composite Electrode as Detector in a FIA System

A PVC/TTF‐TCNQ composite electrode has been employed as detector in a flow injection system. The proposed method allows the simultaneous detection of ascorbic acid (AA) and uric acid (UA) in mixtures by using a FIA system in a simple manner, without pre‐treatment or modified electrode. This method is based on the amperometric determination of (a) ascorbic acid at 0.15 V and (b) both analytes at 0.35 V, being the response linear in the range 1×10^?2–4×10^?4 M for both analytes with detection limits (S/N=3) of 1.2×10^?4 M and 8.1×10^?5 M for AA and UA, respectively.     

Immobilization of uricase in layer-by-layer films used in amperometric biosensors for uric acid

The layer-by-layer technique was exploited to immobilize the enzyme uricase onto indium tin oxide substrates coated with a layer of Prussian Blue. Uricase layers were alternated with either poly(ethylene imine) or poly(diallyldimethylammoniumchloride), and the resulting films were used as amperometric biosensors for uric acid. Biosensors with optimum performance had a limit of detection of 0.15 μA μmol l⁻¹ cm⁻² with a linear response between 0.1 and 0.6 μM of uric acid, which is sufficient for use in clinical tests. Bioactivity was preserved for weeks, and there was negligible influence from interferents, as detection was carried out at 0.0 V vs saturated calomel electrode. This paper is dedicated to the memory of Francisco C. Nart.     

A Multicommuted Flow‐based System for Hydrogen Peroxide Determination by Chemiluminescence Detection Using a Photodiode

Abstract

A simple, rapid, and automated assay for hydrogen peroxide in pharmaceutical samples was developed by combining the multicommutation system with a chemiluminescence (CL) detector. The detection was performed using a spiral flow‐cell reactor made from polyethylene tubing that was positioned in front of a photodiode. It allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light. The chemiluminescence was based on the reaction of luminol with hydrogen peroxide catalyzed by hexacyanoferrate(III).

The feasibility of the flow system was ascertained by analyzing a set of pharmaceutical samples. A linear response within the range of 2.2–210 µmol l^?1 H₂O₂ with a LD of 1.8 µmol l^?1 H₂O₂ and coefficient of variations smaller than 0.8% for 1.0×10^?5 mol l^?1 and 6.8×10^?5 mol l^?1 hydrogen peroxide solutions (n=10) were obtained. Reagents consumption of 90 µg of luminol and 0.7 mg of hexacyanoferrate(III) per determination and sampling rate of 200 samples per hour were also achieved.     

Determination of sulphur dioxide by flow injection analysis with amperometric detection

Sulphur dioxide can be determined at a sampling rate of 120 h^?1, with amperometric detection after separation in a diffusion cell with a teflon membrane. At 25°C, the calibration graph shows two linear ranges, between 0.06 and 6 mg l^?1 and 12 and 110 ml l^? sulphur dioxide, with a detection limit of 0.03 mg l^?1. At 50°C, the liner range is 04–5 mg l^?1, with a detection limit of mg of l^?1. The procedure has been applied to the determination of sulphur dioxide in wines.     

Amperometric determination of formaldehyde via the hexacyanoferrate(III) -coupled dehydrogenase reaction

An enzymatic method with amperometric detection was developed for the determination of formaldehyde. Formaldehyde is first oxidized by reaction with NAD⁺ in the presence of formaldehyde dehydrogenase. The resulting NADH is then oxidized by hexacyanoferrate(III) in the presence of diaphorase to produce hexacyanoferrate(II). The anodic current generated by oxidation of the hexacyanoferrate(II) at the surface of a glassy carbon working electrode, held at a potential of 0.40 V vs. an Ag/AgCl reference electrode, is measured. The effects of solution conditions are examined and a linear relationship between rate of current change and formaldehyde concentration is obtained from 0.01 to 0.5 μg ml^?1 with a correlation coefficient of 0.9998. The relative standard deviation for the proposed method is 6.4% at 0.01 μg ml^?1 formaldehyde and 0.88% at 0.5 μg ml^?1.     

Amperometric Sensor for Hydroxylamine Based on Hybrid Nickel‐Cobalt Hexacyanoferrate Modified Electrode

Hybrid nickel‐cobalt hexacyanoferrate (NiCoHCF) particles were immobilized onto a glassy carbon electrode by cyclic voltammetry. Characterization of these particles by Fourier transform infrared spectroscopy, X‐ray photoelectron spectroscopy, X‐ray diffraction and electrochemistry revealed that NiCoHCF was a substitution‐type hybrid hexacyanoferrate rather than a simple mixture system. As an important reducing agent, hydroxylamine could be electrocatalytically oxidized at the NiCoHCF modified electrode. The effects of the solution pH and the applied potential on the amperometric response of hydroxylamine were examined. Under optimum conditions, the catalytic peak current was proportional to the concentration of hydroxylamine in the range 2.0×10^?5–1.0×10^?2 mol/L with a detection limit of 2.3×10^?7 mol/L. Furthermore, detection results obtained with this sensor showed high sensitivity, fast response time, good stability and anti‐interference ability.     

Flow-through sensors for enhancing sensitivity and selectivity of FTIR spectroscopy in aqueous media

The principle of novel flow-through sensor systems with FTIR spectroscopic detection is presented on the example of the determination of organic acids in aqueous solution. The constructed flow-through sensor system is based on trapping of derivatized porous polymer beads in a conventional IR transmission cell and integration of the flow cell into a sequential injection (SI) manifold. By the SI-manifold sample pre-conditioning, sample-sensor interaction and sensor regeneration were performed in an automated and highly reproducible way. The polymer beads used in this study contained anion exchanger groups so that negatively charged molecules such as organic acids present in the anionic form could selectively interact with the polymer beads. Upon pumping a sample through the sensor cell organic acids were retained on the polymer beads whereas non-ionic matrix molecules passed hence allowing to separate the target analytes form the matrix. Apart from that the organic acids were also concentrated onto the polymer beads so that absolute analyte amounts in the low μg range could easily be detected. Linear calibration curves from 0 to 1 mmol l⁻¹ were recorded for acetic and malic acid using a sample volume of 500 μl (s_x0: 0.032 mmol l⁻¹ acetic acid and 0.031 mmol l⁻¹ malic acid). Mixtures of both acids were analyzed as well and it could be shown that by application of multivariate data evaluation procedures (PLS) simultaneous quantification of both acids could be performed successfully using the developed flow-through sensor system.     

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.     

Pure copper vs. mixed copper and palladium hexacyanoferrates for glucose biosensing applications

A glucose amperometric biosensor was developed. Glucose oxidase enzyme was immobilized by means of a Nafion membrane on glassy carbon modified with an electrochemically deposited mixed Cu and Pd hexacyanoferrate (CuPdHCF). According to the data provided by X-ray atomic spectroscopy measurements, this Cu- and Pd-based hexacyanoferrate is likely to be a mixture of single CuHCF and PdHCF pure phases. The biosensor performances were evaluated by recording the steady-state currents due to submillimolar additions of glucose to a potassium buffer solution (pH 5.5) and exploiting the electrocatalytic reduction of the enzymatically produced hydrogen peroxide. The CuPdHCF-based biosensor exhibited a sensitivity of 8.1?±?0.6 A M^?1 m^?2, a limit of detection of 1.4?×?10^?5 M, and a linear response range extending between 5?×?10^?5 and 4?×?10^?4 M, with a dynamic response range up to 4?×?10^?3 M glucose. Electrode sensitivity and signal stability resulted more satisfactory as compared to those of a CuHCF-based biosensor fabricated according to the same procedure. The selectivity was investigated through an interference study. The response to easily oxidizable species was found to be low enough to allow glucose determination in biological samples.     

Direct electrochemical non-enzymatic assay of glucose using functionalized graphene

A non-enzymatic amperometric sensor is developed based on the graphite electrode modified with functionalized graphene for the determination of β, d (+)-glucose. Cyclic voltammetry and electrochemical impedance spectroscopy techniques are used to study the behavior. Atomic force microscopy was used to study the surface topography of the working electrode before and after its modification. The sensor enabled the direct electrochemical oxidation of β, d (+)-glucose in alkaline medium and responded linearly to the analyte over the range from 0.5?×?10^?3 to 7.5?×?10^?3?M with a limit of detection of 10?μM. The sensor is found to exhibit a better sensitivity of 28.4?μA?mM^?1?cm^?2, good stability, and shelf life. The sensitivity of the sensor to β, d (+)-glucose was not affected by the commonly co-existing interfering substances such as l-ascorbic acid, dopamine, uric acid, and acetaminophen.     

Determination of selenium in human body fluids by hydride-generation atomic absorption spectrometry : Optimization of sample decomposition

The accuracy of the determination of selenium in human body fluids by hydride-generation a.a.s. depends critically upon the sample decomposition-method used. Digestion with HNO₃ alone gave low selenium recoveries, but with nitric, sulfuric and perchloric acids at a final temperature of 310°C gave results that agreed with those obtained by other techniques. The recovery of selenomethionine added to whole blood and of trimethylselenonium iodide added to urine was 97–104%. The average selenium values found for 6 healthy individuals were 88 μg l^?1 in whole blood, 75 μg l^?1 in blood plasma and 307 μg (kg Hb)^?1 in erythrocytes. A detection limit of 5 μg l^?1 Se in body fluids was found under routine conditions.     

Immobilization of Horseradish Peroxidase on a Biocompatible Titania Layer–Modified Gold Electrode for the Detection of Hydrogen Peroxide

Abstract

A procedure for fabricating an enzyme electrode has been described based on the effective immobilization of horseradish peroxidase to an ultrathin titania layer–modified self‐assembled gold electrode. The resulting electrode exhibits excellent electrocatalytical activity to hydrogen peroxide in the presence of hydroquinone as a mediator. The analytical conditions were studied in detail by using an amperometric method. Under the optimized conditions, a detection limit of 7.1×10^?7 mol l^?1 and a linear response to hydrogen peroxide that ranged from 1×10^?6 mol l^?1 to 7.6×10^?4 mol l^?1 were obtained. The reproducibility and stability were examined with satisfactory results.