Microbial electrode sensor for vitamin B12

A microbial sensor consisting of immobilized Escherichia coli 215 and an oxygen electrode is described for the determination of vitamin B₁₂. When the sample solution is injected into the microbial electrode system, the increased consumption of oxygen by the micro-organisms causes a decrease in the dissolved oxygen around the porous membrane of the oxygen electrode and the current decreases gradually with time until a steady state is reached. The response time for a rate measurement is 2 h. When 0.5 mg of Escherichia coli 215 is immobilized, a linear relationship is obtained between the current decrease and the vitamin B₁₂ concentration between 5 × 10^?9 and 25 × 10^?9 g ml^?1.     

L-Malate-sensing electrode based on malate dehydrogenase and NADH oxidase

An L-malate-sensing electrode was constructed from an oxygen electrode and a layer containing immobilized malate dehydrogenase (MDH) and NADH oxidase. MDH catalyses the dehydrogenation of L-malate by NAD⁺ and NADH oxidase catalyses the regeneration of NAD⁺ with the use of oxygen. The regeneration enables the L-malate oxidation to proceed efficiently even in a medium of neutral pH. At pH 8.0, L-malate in the concentration range 5 μM–1.5 mM can be measured. The relative standard deviation for the measurement is 1.2% (L-malate concentration, 0.2 mM; n=10). The present L-malate-sensing electrode is stable for 8 weeks. A two-electrode sensor system consisting of the L-malate-sensing electrode and an L-lactate-sensing electrode based on lactate oxidase was prepared and applied to the simultaneous determination of the two components in wines.     

Trans-membrane electron transfer in red blood cells immobilized in a chitosan film on a glassy carbon electrode

We have studied the trans-membrane electron transfer in human red blood cells (RBCs) immobilized in a chitosan film on a glassy carbon electrode (GCE). Electron transfer results from the presence of hemoglobin (Hb) in the RBCs. The electron transfer rate (k _s) of Hb in RBCs is 0.42 s^?1, and <1.13 s^?1 for Hb directly immobilized in the chitosan film. Only Hb molecules in RBCs that are closest to the plasma membrane and the surface of the electrode can undergo electron transfer to the electrode. The immobilized RBCs displayed sensitive electrocatalytic response to oxygen and hydrogen peroxide. It is believed that this cellular biosensor is of potential significance in studies on the physiological status of RBCs based on observing their electron transfer on the modified electrode.

Amperometric Choline Sensor with Enzyme Immobilized by Gamma-Irradiation in a Biocompatible Membrane

Abstract

An amperometric choline biosensor was constructed using choline oxidase immobilized on poly(2-hydroxyethylmethacrylate) membranes obtained by gamma radiation-induced polymerization at low temperature. The measurements were carried out by Clark-type oxygen or hydrogen peroxide electrodes. Calibration curves were linear in the 10-200 umol · 1^?1 range for the oxygen probe and 5-250 umol · 1^?1 for the H₂O₂-based probe. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the sensor are summarized. The immobilized enzyme membranes stored in glycine buffer or in a dry state were very stable and no significant decrease in the electrode response was observed after three months. The biosensor was employed also to analyse a choline-containing pharmaceutical product and the results were compared to those obtained by enzymatic-spectrophotometric detection.     

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).

Novel CeO2–CuO-decorated enzymatic lactate biosensors operating in low oxygen environments

The detection of the lactate level in blood plays a key role in diagnosis of some pathological conditions including cardiogenic or endotoxic shocks, respiratory failure, liver disease, systemic disorders, renal failure, and tissue hypoxia. Here, we described for the first time the use of a novel mixed metal oxide solution system to address the oxygen dependence challenge of first generation amperometric lactate biosensors. The biosensors were constructed using ceria-copper oxide (CeO₂–CuO) mixed metal oxide nanoparticles for lactate oxidase immobilization and as electrode material. The oxygen storage capacity (OSC, 492 μmol-O₂/g) of these metal oxides has the potential to reduce the oxygen dependency, and thus eliminate false results originated from the fluctuations in the oxygen concentration. In an effort to compare the performance of our novel sensor design, ceria nanoparticle decorated lactate sensors were also constructed. The enzymatic activity of the sensors were tested in oxygen-rich and oxygen-lean solutions. Our results showed that the OSC of the electrode material has a big influence on the activity of the biosensors in oxygen-lean environments. While the CeO₂ containing biosensor showed an almost 21% decrease in the sensitivity in a O₂-depleted solution, the CeO₂–CuO containing electrode, with a higher OSC value, experienced no drop in sensitivity when moving from oxygen-rich to oxygen-lean conditions. The CeO₂–CuO decorated sensor showed a high sensitivity (89.3 ± 4 μA mM⁻¹ cm⁻²), a wide linear range up to 0.6 mM, and a low limit of detection of 3.3 μM. The analytical response of the CeO₂–CuO decorated sensors was studied by detecting lactate in human serum with good selectivity and reliability. The results revealed that CeO₂–CuO containing sensors are promising candidates for continuous lactate detection.     

Studies on Microbial Biosensor for DL-Phenylalanine and Its Dynamic Response Process

Abstract

A biosensor for the determination of DL-phenylalanine, based on immobilized bacteria P. Vulgaris in calcium alginate gel coupled with an amperometric oxygen electrode, was designed and constructed. The effect of the culture medium, the response character of the electrode and its kinetic mechanism were studied, and the activity of phenylalanine deaminase was tested by biochemical method. The biosensor exhibited longer lifetime, higher sensitivity and faster response to the Phe in the linear range of 2.5×10^?5 to 2.5×10^?3 M for the optimum analytical performance. The relationship between the response velocity and the substrate concentration was also discussed. The results indicate that the dynamic response process of the reaction catalyzed by bacteria is similar to that by isolated enzymes. The apparent Michaelis constant. K_m ^app the apparent activation energy. E_a and the temperature coefficient, Q₁₀ for deaminase in the immobilized bacterial membrane were calculated to be 2.0×10^?3 M, 49.5kJ/mol and 1.86 respectively.     

A Biosensor for ADP with Internal Substrate Amplification

Abstract

An enzyme electrode was constructed from an oxygen electrode and a layer incorporating four enzyme systems for the sensitive determination of ADP and ATP. The cofactor is cycled between pyruvate kinase and hexokinase under formation of pyruvate which is detected by the coimmobilized, sequentially acting enzymes lactate dehydrogenase and lactate monooxygenase. The enzymatic recycling results in a 220-fold increased sensitivity to ADP compared to the unamplified reaction.     

Immobilized enzyme electrode for the determination of salicylate in blood serum

This determination of salicylate in blood serum is based on application of an immobilized enzyme electrode. Salicylate hydroxylase (E.C.1.14.13.1) is chemically immobilized onto a pig intestine mounted on an oxygen electrode. The signals are monitored amperometrically and the resulting output voltage is read using a simple adapter. The experimental parameters and possible interferences are discussed. Samples containing 1.0 × 10^?5?1.87 × 10^?3 M (1.6–300 μg ml^?1) salicylate were assayed with relative standard deviations between 1.3% and 6% and recoveries between 98.7 and 103%. Results obtained by the proposed method and by the established clinical method for randomly spiked pooled serum samples correlated well (r = 0.99).     

Immobilization of horseradish peroxidase to a nano-Au monolayer modified chitosan-entrapped carbon paste electrode for the detection of hydrogen peroxide

A procedure for fabricating an enzyme electrode has been described based on the effective immobilization of horseradish peroxidase (HRP) to a nano-scaled particulate gold (nano-Au) monolayer modified chitosan-entrapped carbon paste electrode (CCPE). The high affinity of chitosan entrapped in CCPE for nano-Au associated with its amino groups has been utilized to realize the use of nano-Au as an intermediator to retain high bioactivity of the enzyme. Hydrogen peroxide (H₂O₂) was determined in the presence of hydroquinone as a mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano-Au displayed excellent electrocatalytical activity to the reduction of H₂O₂. The effects of experimental variables such as the operating potential of the working electrode, mediator concentration and pH of measuring solution were investigated for optimum analytical performance by using an amperometric method. The enzyme electrode provided a linear response to hydrogen peroxide over a concentration range of 1.22×10⁻⁵-2.43×10⁻³ mol l⁻¹ with a sensitivity of 0.013 A l mol⁻¹ cm⁻² and a detection limit of 6.3 μmol l⁻¹ based on signal per noise =3. The apparent Michaelis-Menten constant (K_m^app) for the sensor was found to be 0.36 mmol l⁻¹. The lifetime, fabrication reproducibility and measurement repeatability were evaluated with satisfactory results. The analysis results of real sample by this sensor were in satisfactory agreement with those of the potassium permanganate titration method.     

A Fiber Optic Lactate Biosensor with an Oxygen Optrode as the Transducer

Abstract

A biosensor for continuous determination of lactate is presented. Lactate monooxygenase was immobilized covalently on nylon membranes, and the consumption of oxygen was measured by following, via a fiber optic bundle, the changes in the fluorescence of an oxygen-sensitive dye dissolved in 10- and 25-um silicone membranes placed beneath the enzyme layer. Oxygen is consumed as a result of the oxidation of lactate by the enzyme, and the decrease in its partial pressure is indicated by the fluorescent dye. For two types of sensors (with different nylon membranes and different thicknesses of the indicator layer) the analytical ranges were 2–50 mM and 0.3–6.0 mM, with response times (t₉₀) of 2.3–3.0 and 4.0–6.0 min, respectively.     

Amino oxidase amperometric biosensor for polyamines

An improved amino oxidase enzyme electrode has been constructed and applied to the determination of the amount of polyamines present in real samples. The electrode is based on the amperometric detection of H₂O₂ produced in the enzymatic oxidation of polyamines by amino oxidase. Amino oxidase from soybean seedlings, characterized by an extremely high activity for cadaverine and putrescine, was used. The enzyme was immobilized in an agarose matrix in the presence of glutaraldehyde and bovine serum albumin on the surface of a Pt electrode. Cadaverine, in concentrations between 0.5 and 500 μM, can be quantitatively determined by use of the amino oxidase electrode, the linear calibration range being 0.5–10 μM. The lower detection limit was 0.2 μM and the response time was 15 to 60 s. Putrescine showed similar behaviour. The maximum current response for cadaverine was 5.1 μA/cm², with an apparent Michaelis-Menten constant (K_m′) of 0.175 mM. The sensor response was stable for more than 32 hours of continuous operation at room temperature and, in the presence of fish or meat homogenates, no change in the signal-to-noise ratio was observed. The long-term stability, pH and temperature response of the biosensor has also been studied.     

A Novel Amperometric Immunosensor for Phytohormone Abscisic Acid Based on In Situ Chemical Reductive Growth of Gold Nanoparticles on Glassy Carbon Electrode

Abstract

An amperometric immunosensor for phytohormone abscisic acid was developed based on in situ chemical reductive growth of gold nanoparticles on glassy carbon electrode. First, an approximate 10 nm gold layer was sputtered uniformly onto the electrode surface, and then gold nanoparticles were grown directly on the gold layer for antibody adsorption by immersing the electrode into the H₂AuCl₄ solution. Determination was based on an enzyme-linked competitive immunoreaction between free and enzyme-labeled abscisic acid to bind on immobilized antibody on electrode. The linear response was from 10 ng/ml to 10 µg/ml with a detection limit of 5 ng/ml.     

Lactate biosensor based on a bionanocomposite composed of titanium oxide nanoparticles, photocatalytically reduced graphene, and lactate oxidase

We have developed a lactate biosensor based on a bionanocomposite (BNC) composed of titanium dioxide nanoparticles (TiO₂-NPs), photocatalytically reduced graphene, and lactate oxidase. Graphene oxide was photochemically reduced (without using any chemical reagents) in the presence of TiO₂-NPs to give graphene nanosheets that were characterized by atomic force microscopy, Raman and X-ray photoelectron spectroscopy. The results show the nanosheets to possess few oxygen functionalities only and to be decorated with TiO₂-NPs. These nanosheets typically are at least 1 μm long and have a thickness of 4.2 nm. A BNC was obtained by mixing lactate oxidase with the nanosheets and immobilized on the surface of a glassy carbon electrode. The resulting biosensor was applied to the determination of lactate. Compared to a sensor without TiO₂-NPs, the sensor exhibits higher sensitivity (6.0 μA mM^?1), a better detection limit (0.6 μM), a wider linear response (2.0 μM to 0.40 mM), and better reproducibility (3.2 %).

Semiconducting tin oxide electrodes in molten sodium chloroaluminate at 175°C

Electrode behavior of Sb-doped poly-crystalline tin oxide electrodes has been investigated by means of current and differential capacity measurements in molten chloroaluminate melts (AlCl₃+NaCl) with different pCl values. The SnO₂ is stable in the melts consisting of near equimolar composition, being used as an indicator electrode possessing a polarizable potential region between chlorine evolution and its cathodic decomposition. The differential capacity is assigned to the space charge layer capacity of the electrode side and its potential dependence is explained by using the Mott-Schottky equation. It is found that the flat band potential does depend on pCl (=?log a_Cl^?) at a rate of 2(2.3kT/e) per pCl unit. This anomaly is attributed to the specific adsorption of Cl^? ions on the oxide electrode.     

Enzyme Sensor for the Detection of Herbicides Inhibiting Acetolactate Synthase

Abstract

An enzyme sensor for the detection of sulfonylurea herbicides inhibiting acetolactate synthase II (ALS) was developed using an oxygen electrode. ALS, which has an oxygen consumption side reaction, was entrapped in the matrix of PVA-SbQ polymer, and the enzyme membrane was attached to the electrode. The inhibition of side oxygen reaction of ALS II is measured as decreased consumption of O₂ monitored by an oxygen electrode. Preliminary results show that 10^?6 M herbicide concentration can be determined by this method.     

An improved electrode for the assay of urea in blood

An improved urea enzyme electrode is applied for the determination of urea in blood samples. The electrode is based on the enzymatic hydrolysis of urea, and potentiometric detection of the ammonium ion produced. A silicone rubber-based nonactin ammonium ion-selective electrode serves as the sensor. The selectivity coefficients of this electrode were 6.5 for NH₄⁺/K⁺; 750 for NH₄⁺/Na⁺, and much higher for other cations. The reaction layer of the electrode was made of urease enzyme chemically immobilized on polyacrylic gel. The prepared gel was stable at 4° for over four months. The electrodes retained their activity for over one month. A three-electrode system, which allowed dilution to a constant interference level, was applied to avoid interfering effects in blood samples. Analyses of blood sera showed good agreement with a standard spectrophotometric method. Routine clinical assays of blood urea are feasible.     

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H₂O₂ product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit was 0.02 ng mL⁻¹. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.     

Layer by layer assembly of catalase and amine-terminated ionic liquid onto titanium nitride nanoparticles modified glassy carbon electrode: Study of direct voltammetry and bioelectrocatalytic activity

A novel, simple and facile layer by layer (LBL) approach is used for modification of glassy carbon (GC) electrode with multilayer of catalase and nanocomposite containing 1-(3-Aminopropyl)-3-methylimidazolium bromide (amine terminated ionic liquid (NH₂-IL)) and titanium nitride nanoparticles (TiNnp). First a thin layer of NH₂-IL is covalently attached to GC/TiNnp electrode using electro-oxidation method. Then, with alternative self assemble positively charged NH₂-IL and negatively charged catalase a sensitive H₂O₂ biosensor is constructed, whose response is directly correlated to the number of bilayers. The surface coverage of active catalase per bilayer, heterogeneous electron transfer rate constant (k_s) and Michaelis–Menten constant (K_M) of immobilized catalase were 3.32 × 10⁻¹² mol cm⁻², 5.28 s⁻¹ and 1.1 mM, respectively. The biosensor shows good stability, high reproducibility, long life-time, and fast amperometric response with the high sensitivity of 380 μA mM⁻¹ cm⁻² and low detection limit of 100 nM at concentration range up to 2.1 mM.     

Amperometric determination of cholinesterase with use of an immobilized enzyme electrode

A choline-sensitive electrode consisting of an immobilized choline oxidase layer and an oxygen electrode is described. Cholinesterase (0.5–60 I.U. l^-1) is measured by addition of acetylcholine, and detection of the choline produced. The precision is 3%, and the electrode is stable for more than 2 weeks (140 assays).