A Biosilification Fusion Protein for a ‘Self‐immobilising’ Sarcosine Oxidase Amperometric Enzyme Biosensor

Monomeric sarcosine oxidase (mSOx) fusion with the silaffin peptide, R5, designed previously for easy protein production in low resource areas, was used in a biosilification process to form an enzyme layer electrode biosensor. mSOx is a low activity enzyme (10–20 U/mg) requiring high amounts of enzyme to obtain an amperometric biosensor signal, in the clinically useful range <1 mM sarcosine, especially since the K_m is >10 mM. An amperometric biosensor model was fitted to experimental data to investigate dynamic range. mSOx constructs were designed with 6H (6×histidine) and R5 (silaffin) peptide tags and compared with native mSOx. Glutaraldehyde (GA) cross‐linked proteins retained ～5 % activity for mSOx and mSOx‐6H and only 0.5 % for mSOx‐R5. In contrast R5 catalysed biosilification on (3‐mercaptopropyl) trimethoxysilane (MPTMS) and tetramethyl orthosilicate (TMOS) particles created a ‘self‐immobilisation’ matrix retaining 40 % and 76 % activity respectively. The TMOS matrix produced a thick layer (>500 μm) on a glassy carbon electrode with a mediated current due to sarcosine in the clinical range for sarcosinemia (0–1 mM). The mSOx‐R5 fusion protein was also used to catalyse biosilification in the presence of creatinase and creatininase, entrapping all three enzymes. A mediated GC enzyme linked current was obtained with dynamic range available for creatinine determination of 0.1–2 mM for an enzyme layer ～800 nm.     

Activation of peroxyl and molecular oxygen using bis -benzimidazole diamide copper (II) compounds

New tetradentatebis-benzimidazole ligands have been synthesized and utilized to prepare copper (II) complexes. Some of these copper (II) complexes have been characterized structurally. The copper (II) in these complexes is found to possess varying geometries. A distorted octahedral geometry is found with a highly unsymmetrical bidentate nitrate group. An unusual polymeric one-dimensional structure is observed where copper (II) is in a distorted square pyramidal geometry with a monodentate nitrate ion, having long Cu-O bond, while a distorted triagonal bipyramidal geometry is found with two carbonyl O atoms and a Cl atom in the equatorial plane, and two benzimidazole imine N atoms occupy the axial position. These compounds are found to activate the cumylperoxyl group, and this has been utilized in the facile oxidation of aromatic alcohols to aldehydes, where they act as catalysts with large turnovers. The yields of the respective products vary from 32 to 65%. The role of molecular oxygen has been studied and an attempt has been made to identify the “active copper species”. Activation of molecular oxygen has also been observed and has been used for oxidative dealkylation of a hindered phenol, producing di-butyl quinones with yields of 20–25% and 10–12 fold catalytic turnover. Dihydroxybenzenes and substituted catechols are also readily oxidized to the corresponding quinones, in oxygen-saturated solvents. Yields of 84% have been observed with 34-fold catalyst turnover, with di-t-butylcatechol. The activity of these copper (II) —bis-benzmidazolediamide compounds is reminiscent of the functioning of copper centres in galactose oxidase, tyrosinase and catechol oxidase.     

Evaluation of Polycation-Stabilized Lactate Oxidase in a Silica Sol–Gel as a Biosensor Platform

 Whereas glucose oxidase and related proteins are encapsulated readily in silica sol–gels, α-hydroxy enzymes such as lactate oxidase (LOx), are reported to be damaged by electrostatic interaction with these matrices. Based on a previous report, poly(ethyleneimine), PEI, was evaluated as a protecting compound under conditions suited to analytical measurements. With LOx and PEI co-encapsulated in a silica sol–gel, the enzyme retained 62% of its initial activity after 20 days. In the absence of PEI, activity was lost during the processing. Batch analytical measurements with enzyme-doped sol–gel yielded a linear response over the range 0.5–2.0 mM lactate and a detection limit of 0.03 mM lactate. Both simple incorporation of LOx in a silica sol–gel and an alternative protection method, blocking the ion-exchange sites on silica with La(III), failed. These negative results supported the hypothesis that the efficacy of PEI was related to its formation of a protective sheath around the enzyme. Author for correspondence. E-mail: coxja@muohio.edu Received July 29, 2002; accepted December 15, 2002 Published online May 19, 2003     

Direct Electron Communication Between Glucose Oxidase and Carbon Electrode Covered with Polypyrrole

IntroductionDirectelectrochemistryofenzymeshasarousedincreasinginterestofmanyresearchersasitisapreferablewayforproducingarealreagent1essbiosensor.Glucoseoxidase(GOD),beingaflavoprotein,iswell-knownduetoitswidespreaduseinthebiosensors'Recently,agreatnumberofpeoplehaveat-temptedtoachievedirectelectrontransferbetweenGODandvariouselec-trodes[l-19].Szucselal.determinedtheshapeandsizeoftheGODmoleculead-sorbedonagoldelectrodebyel1ipsometry['jandexamineddirectelectrontrans-ferbetweentheadsorbedenz…     

Development and application of an integrated system for monitoring ethanol content of fuels

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-aminophenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05–1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91-mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05–1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.     

L—氨基酸氧化酶电极的制作及电极特性的研究

报道了一种新的Ｌ－氨基酸氧化酶电极，这种酶电极系由氨气敏电极和以氨基化玻璃布为载体的酶膜所组成；研究了固定化条件对酶膜活性的影响以及底物浓度、温度和ｐＨ对电极响应特性的影响。该电极在６．０×１０＾－５～４．０×１０＾－３ｍｏｌ／Ｌ的底物浓度范围内呈良好的线性关系，检测下限为５．０×１０＾－５ｍｏｌ／Ｌ。在最宜条件下，酶电极具有良好的稳定性。     

植物生物分子的电分析化学研究——Ⅸ.多酚氧化酶活性的脉冲极谱法测定

本文用微分脉冲极谱法研究了多酚氧化酶的活性测定和酶的反应动力学。应用于马铃薯等10余种植物样品的酶活力测定,结果良好。方法简便快速,重现性好。     

Immobilization of Glucose Oxidase on Cellulose／Cellulose Acetate Membrane and its Detection by Scanning Electrochemical Microscope （SECM）

Cellulose/cellulose acetate membranes were prepared and functionalized by introducingamino group on it, and then immobilized the glucose oxidase (Gox) on the functionalizd membrane.SECM was applied for the detection of enzyme activity immobilized on the membrane.Immobilized biomolecules on such membranes was combined with analysis apparatus and can beused in bioassays.     

Application of amperometric sol-gel biosensor to flow injection determination of glucose

A glucose biosensor with enzyme immobilised by sol–gel technology was constructed and evaluated. The glucose biosensor reported is based on encapsulated GOX within a sol–gel glass, prepared with 3-aminopropyltriethoxy silane, 2-(3,4-epoxycyclohexyl)-ethyltrimetoxy silane and HCl. A flow system incorporating the amperometric biosensor constructed was developed for the determination of glucose in the 1×10⁻⁴–5×10⁻³ mol l⁻¹ range with a precision of 1.5%. The results obtained for the analysis of electrolytic solution for iv administration and human serum samples showed good agreement between the proposed method and the reference procedure, with relative error <5%.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.