Flow-injection determination of l-tyrosine in serum with an immobilized tyrosinase reactor and fluorescence detection

Tyrosinase is immobilized on controlled-pore glass beads and packed into a stainless-steel column (5 cm × 4 mm i.d.). Serum is deproteinized with tungstate and sulphuric acid. The carrier stream is 0.3 M phosphate buffer (pH 7.2), and is mixed with 5 M potassium hydroxide after the enzyme reactor. The fluorescent dihydroxyindole formed is detected at 490 nm (excitation at 375 nm). The calibration graph is linear for 1 × 10^?7 ?1 × 10^?4 M tyrosine; the detection limit is 2 × 10^?8 M.     

Determination of pesticides in vegetable samples using an acetylcholinesterase biosensor based on nanoparticles ZrO2/chitosan composite film

An amperometric pesticides inhibition biosensor has been developed and used for determination of pesticides in vegetable samples. To eliminate the interference of ascorbic acid, multilayer films of polyelectrolyte (chitosan/polystyrensulfonate) were coated on the glass carbon electrode. Then, acetylcholinesterase was immobilized on the electrode based on surface-treated nanoporous ZrO₂/chitosan composite film as immobilization matrix. As a modified substrate, acetylthiocholine was hydrolysed by acetylcholinesterase and produced thiocholine which can be oxidized at +700?mV vs. SCE. Pesticides inhibit the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to acetylthiocholine within 9.90?×?10^?6 to 2.03?×?10^?3?M. The electrode provided a linear response over a concentration range of 6.6?×?10^?6 to 4.4?×?10^?4?M for phoxim with a detection limit of 1.3?×?10^?6?M, over a range of 1.0?×?10^?8 to 5.9?×?10^?7?M for malathion, and over a range of 8.6?×?10^?6 to 5.2?×?10^?4?M for dimethoate. This biosensor has been used to determine pesticides in a real vegetable sample.     

Flow-injection amperometric determination of oxalate with immobilized oxalate oxidase

Oxalate is immobilized on controlled-pore glass and is used on-line in a glass minicolumn (2.5×25 mm). The hydrogen peroxide formed is detected amperometrically. Oxalate (6×10^?6?9×10^?4 M) is determined in a flowing stream of pH 3.5 citrate (or succinate) buffer. As little as 20 ng (in 40 μl; 5.7×10^?6 M) of oxalate can be detected. Copper inhibition can be removed either by adding EDTA to the carrier stream or incorporating a chelating-resin minicolumn into the flow system prior to the enzyme column.     

An amperometric monoamine oxidase biosensor for determining some antidepressants

An amperometric biosensor based on a platinum screen-printed electrode and immobilized monoamine oxidase is developed to determine antidepressants of different classes. Petylyl, pyrazidol, and flu-oxetine can be determined with determination limits of 8 × 10^?9, 8 × 10^?7, and 8 × 10^?10 M, respectively. A procedure is proposed for determining fluoxetine in tablets. It is shown that petylyl can be selectively determined by an immunochemical technique using the developed biosensor and immobilized antibodies in the concentration range from 1 × 10^?4 to 1 × 10^?8 M.     

Flow injection analysis for traces of zinc with immobilized carbonic anhydrase

A small column packed with immobilized bovine carbonic anhydrase is used for determination of traces of zinc in aqueous solution, based on the measurement of recovered esterase activity of the metal-free apoenzyme after taking up zinc from the sample solution. Conditions for the removal of zinc from the immobilized enzyme and for activity measurement are established. A linear calibration graph is obtained between 1 × 10^?8 and 4 × 10^?7 g of zinc. Then the method is successfully applied to the determination of zinc in tea, sediment and spring water.     

Determination of acetaldehyde by flow injection analysis with soluble or immobilized aldehyde dehydrogenase

Acetaldehyde (0.18–7.7 × 10^?4 M) in water is determined by using a double injection technique with the soluble enzyme or with a mini-column of aldehyde dehydrogenase immobilized on cyanogen-activated Sepharose 4B. The NADH produced is monitored spectrophotometrically. The sample throughput is ca. 40 h^?1, and the immobilized enzyme is stable for at least a month. Ethanol up to 5% (v/v) does not interfere.     

A New Enzyme Immobilization Technique Based on Thionine‐Bovine Serum Albumin Conjugate and Gold Colloidal Nanoparticles for Reagentless Amperometric Biosensor Applications

A novel enzyme immobilization technique based on thionine‐bovine serum albumin conjugate (Th‐BSA) and gold colloidal nanoparticles (nano‐Au) was developed. Thionine was covalently bound onto the BSA film with glutaraldehyde(GA) as cross‐linker to achieve Th‐BSA conjugate. The free amino groups of thionine were then used to attach nano‐Au for the immobilization of horseradish peroxidase (HRP). Such nano‐Au/Th‐BSA matrix shows a favorable microenvironment for retaining the native activity of the immobilized HRP and thionine immobilized in this way can effectively shuttle electrons between the electrode and the enzyme. The proposed biosensor displays excellent catalytic activity and rapid response for H₂O₂. The linear range for the determination of H₂O₂ is from 4.9×10^?7 to 1.6×10^?3 M with a detection limit of 2.1×10^?7 M at 3σ and a Michaelies‐Menten constant K value of 0.023 mM.     

Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon paste electrode

An amperometric enzyme electrode for the determination of hypoxanthine in fish meat is described. The hypoxanthine sensor was prepared from xanthine oxidase immobilized by covalent binding to cellulose triacetate and a carbon paste electrode containing hydroxymethylferrocene. The xanthine oxidase membrane was retained behind a dialysis membrane at a carbon paste electrode. The sensor showed a current response to hypoxanthine due to the bioelectrocatalytic oxidation of hypoxanthine, in which hydroxymethyiferrocene served as an electron-transfer mediator. The limit of detection is 6 × 10^?7 M, the relative standard deviation is 2.8% (n=28) and the response is linear up to 7 × 10^?4 M. The sensor responded rapidly to a low hypoxanthine concentration (7 × 10^?4 M), the steady-state current response being achieved in less than 1 min, and was stable for more than 30 days at 5 ° C. Results for tuna samples showed good agreement with the value determined by the conventional method.     

Flow-injection determination of malate with immobilized malate dehydrogenase

Malate dehydrogenase (MDH) is immobilized chemically on controlled-pore glass and used on-line in a glass minicolumn (25×2.5 mm i.d.). Malate solution passes through the minicolumn of immobilized MDH and the NADH formed is monitored spectrophotometrically from 9 × 10-^?4 down to 7 × 10^?6 M (36 ng in 40 μl) at 50 samples h^?1.     

Flow-injection determination of d-mannitol with immobilized mannitol dehydrogenase

A flow-injection system is described for the determination of d-mannitol. Mannitol dehydrogenase is immobilized on poly(vinyl alcohol) beads and packed in a column (5 cm × 4 mm i.d.). The NADH formed is detected fluorimetrically. The response is linear between 5 × 10^?7 and 1 × 10^?4 M mannitol and the detection limit is 1 × 10^?7 M. The throughput is 30 samples per hour. The reactor is stable for at least 8 weeks.     

Direct Electrochemistry of Glucose Oxidase Immobilized on Titanium Carbide‐Au Nanoparticles‐Fullerene C60 Composite Film and Its Biosensing for Glucose

The direct electrochemistry of glucose oxidase (GOD) immobilized on the designed titanium carbide‐Au nanoparticles‐fullerene C₆₀ composite film modified glassy carbon electrode (TiC‐AuNPs‐C₆₀/GCE) and its biosensing for glucose were investigated. UV‐visible and Fourier‐transform infrared spectra of the resulting GOD/TiC‐AuNPs‐C₆₀ composite film suggested that the immobilized GOD retained its original structure. The direct electron transfer behaviors of immobilized GOD at the GOD/TiC‐AuNPs‐C₆₀/GCE were investigated by cyclic voltammetry in which a pair of well‐defined, quasi‐reversible redox peaks with the formal potential (E^0′) of ‐0.484 V (vs. SCE) in phosphate buffer solution (0.05 M, pH 7.0) at the scan rate of 100 mV·s^?1 were obtained. The proposed GOD modified electrode exhibited an excellent electrocatalytic activity to the reduction of glucose, and the currents of glucose reduction peak were linearly related to glucose concentration in a wider linearity range from 5.0 × 10^?6 to 1.6 × 10^?4 M with a correlation coefficient of 0.9965 and a detection limit of 2.0 × 10^?6 M (S/N = 3). The sensitivity and the apparent Michaelis‐Menten constant (K_M^app) were determined to be 149.3 μA·mM^?1·cm^?2 and 6.2 × 10^?5 M, respectively. Thus, the protocol will have potential application in studying the electron transfer of enzyme and the design of novel electrochemical biosensors.     

The Influence of Malathion and Its Decomposition Products on Free and Immobilized Acetylcholinesterase1

The influence of malathion and its four main degradation products found in irradiated solutions (malaoxon, isomalathion, diethyl maleate and O,O-dimethyl phosphate) on acetylcholinesterase (AChE) of free and immobilized bovine erythrocytes was investigated. The concentration-dependent responses to malathion and related organophosphates, malaoxon and isomalathion, of both AChE bioassays used were obtained. The IC ₅₀ values for free and immobilized AChE (3.7 ± 0.2) × 10⁻⁴ M/(1.6 ± 0.1) × 10⁻⁴, (2.4 ± 0.3) × 10⁻⁶/(3.4 ± 0.1) × 10⁻⁶ M, and (3.2 ± 0.3) × 10⁻⁶ M/(2.7 ± 0.2) × 10⁻⁶ M were obtained in the presence of malathion, malaoxon and isomalathion, respectively. However, diethyl maleate inhibited AChE activity at concentrations ≥ 10 mM, while O,O-dimethyl phosphate did not noticeably affect enzyme activity at all investigated concentrations. The relation between the structure of the compounds and their ability to inhibit enzyme activity was discussed. The article is published in the original.     

An optical-fibre sensor for fluoride

The optical sensor is based on the formation of a fluoride ternary complex from an immobilized fluorescent binary zirconium-calcein blue chelate at pH 2.2. The enhanced fluorescence enables fluoride to be determined in the range 2.63 × 10^?5 ?4.21 × 10^?4 M (0.5–8.00 mg l^?1) with a limit of detection of 2.63 × 10^?5 M. The response of the sensor to fluoride is affected by ionic strength and temperature. The response time depends on the particle size and the packing density of the polymer (XAD-4) substrate used.     

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.     

Sol‐Gel‐Derived Biosensor Based on Plant Tissue: The Inhibitory Effect of Atrazine on Polyphenol Oxidase Activity for Determination of Atrazine

This work presents a sol‐gel based biosensor for atrazine determination which has been obtained by introducing the enzyme polyphenol oxidase from apple tissue in a sol‐gel matrix. Apple tissue acts as a molecular recognition element. Atrazine is an inactive compound electrochemically; redox coupling of dopamine was used for studying atrazine behavior. Atrazine was determined by monitoring the inhibition power of polyphenol oxidase activity. The measurements were performed in 0.1 M KH₂PO₄‐NaOH buffer (pH 7.5). The effect of various experimental parameters such as pH, concentration of buffer, concentration of dopamine, incubation time and matrix composition has been investigated for optimum analytical performance. The biosensor consisted of 10.3% (w/w) of apple tissue. The bioelectrode exhibits a linear response for dopamine and atrazine concentrations in the range of 5.66 × 10^?6?2.27 × 10^?3M and 1 × 10^?5 ?1 × 10^?4 M with a detection limit of 4.2 × 10^?6 and 5.5 × 10^?6 M, respectively. A correlation coefficient of 0.9945 and a relative standard deviation (R.S.D.) of 3.29% for dopamine, 0.9944 and 3.69% for a trazine were achieved.     

Amperometric Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized on Fe3O4/Chitosan Modified Glassy Carbon Electrode

A new type of amperometric hydrogen peroxide biosensor was constructed based on horseradish peroxidase (HRP) immobilized on Fe₃O₄/chitosan modified glassy carbon electrode. The effects of some experimental variables such as the concentration of supporting electrolyte, pH, enzyme loading, the concentration of the mediator of methylene blue (MB) and the applied potential were investigated. The linear range of the calibration curve for H₂O₂ was 2.0×10^?4–1.2×10^?2 M with a detection limit of 1.0×10^?4 M (S/N=3). The response time was less than 12 s. The apparent Michaelis‐Menten constant K_m was 21.4 mM and it illustrated the excellent biological activity of the fixed enzyme. In addition, the biosensor had long‐time stability and good reproducibility. And this method has been used to determine H₂O₂ concentration in the real sample.     

Xanthine Sensors Based on Anodic and Cathodic Detection of Enzymatically Generated Hydrogen Peroxide

A xanthine biosensor was fabricated by the covalent immobilization of xanthine oxidase (XO) onto a functionalized conducting polymer (Poly‐5, 2′: 5′, 2″‐terthiophine‐3‐carboxylic acid), poly‐TTCA through the formation of amide bond between carboxylic acid groups of poly‐TTCA and amine groups of enzyme. The immobilization of XO onto the conducting polymer (XO/poly‐TTCA) was characterized using cyclic voltammetry, quartz crystal microbalance (QCM), and X‐ray photoelectron spectroscopy (XPS) techniques. The direct electron transfer of the immobilized XO at poly‐TTCA was found to be quasireversible and the electron transfer rate constant was determined to be 0.73 s^?1. The biosensor efficiently detected xanthine through oxidation at +0.35 V and reduction at ?0.25 V (versus Ag/AgCl) of enzymatically generated hydrogen peroxide. Various experimental parameters, such as pH, temperature, and applied potential were optimized. The linear dynamic ranges of anodic and cathodic detections of xanthine were between 5.0×10^?6?1.0×10^?4 M and 5.0×10^?7 to 1.0×10^?4 M, respectively. The detection limits were determined to be of 1.0×10^?6 M and 9.0×10^?8 M with anodic and cathodic processes, respectively. The applicability of the biosensor was tested by detecting xanthine in blood serum and urine real samples.     

Flow-injection analysis for l-glutamate using immobilized l-glutamate oxidase: comparison of an enzyme reactor and enzyme electrode

An enzyme electrode and enzyme based on immobilized l-glutamate oxidase are used for the determination of l-glutamate in a flow-injection system. The hydrogen peroxide produced is monitored amperometrically. The enzyme reactor system surpasses the enzyme electrode system with regard to sensitivity and analytical speed. For both systems, the peak current is linearly related to the l-glutamate concentration in the range 5 × 10^?6-1 × 10^?3 M. l-Glutamate in seasoning can be determined very selectively with < 0.7% r.s.d.     

Self-assembly of ultra-small gold nanoparticles on an indium tin oxide electrode for the enzyme-free detection of hydrogen peroxide

A novel enzyme-free electrochemical sensor for H₂O₂ was fabricated by modifying an indium tin oxide (ITO) support with (3-aminopropyl) trimethoxysilane to yield an interface for the assembly of colloidal gold. Gold nanoparticles (AuNPs) were then immobilized on the substrate via self-assembly. Atomic force microscopy showed the presence of a monolayer of well-dispersed AuNPs with an average size of ~4 nm. The electrochemical behavior of the resultant AuNP/ITO-modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. This non-enzymatic and mediator-free electrode exhibits a linear response in the range from 3.0?×?10^?5 M to 1.0?×?10^?3 M (M?=?mol?·?L^?1) with a correlation coefficient of 0.999. The limit of detection is as low as 10 nM (for S/N?=?3). The sensor is stable, gives well reproducible results, and is deemed to represent a promising tool for electrochemical sensing.

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).