Elektrochemické biosenzory s prostorově oddělenou enzymatickou a detekční části pro selektivní analýzu v průtokovém uspořádání

This dissertation thesis presents the newly developed four highly reusable, stable as well as simple, and cost-effective electrochemical (bi)enzymatic biosensors for the selective and reliable determination of choline, acetylcholine, uric acid, and L-lactic acid in flow injection analysis. All biosensors are based on the concept of the spatial separation of the biorecognition part from detection one and amperometric monitoring of the enzymatically consumed oxygen via its four-electron reduction at the highly negative detection potential. In this way, the design of the biosensors includes an easily replaceable enzymatic mini-reactor(s) connected upstream to the flow cell that contains the appropriate silver amalgam-based transducer. The enzymatic mini-reactor based on choline oxidase, uricase, or lactate oxidase was used for choline, uric acid, or L-lactic acid biosensors, respectively. The acetylcholine bienzymatic biosensor includes the consequently connected choline oxidase- and acetylcholinesterase-based mini-reactors. The first part of this thesis focuses on the construction of two different silver amalgam-based electrodes. Specifically, this section discusses the fabrication of a silver solid amalgam electrode covered by mercury film operating in a wall-jet cell and also highlights the...Předpokládaná disertační práce pojednává o čtyřech nově vyvinutých, opakovaně použitelných, stabilních a zároveň jednoduchých a cenově výhodných elektrochemických (bi)enzymatických biosenzorech pro selektivní a spolehlivé stanovení cholinu, acetylcholinu, močové kyseliny a L-mléčné kyseliny. Všechny biosenzory jsou založeny na koncepci prostorového oddělení biorozpoznávací části od detekční a amperometrického monitorování enzymaticky spotřebovaného kyslíku prostřednictvím jeho čtyřelektronové redukce při vysoce záporném detekčním potenciálu. Konstrukce biosenzorů zahrnuje snadno vyměnitelný enzymatický/é mini-reaktor(y) připojený/é před průtokovou celou, která obsahuje příslušný převodník na bázi pevného stříbrného amalgámu. Enzymatické mini-reaktory založené na cholin oxidase, urikase nebo laktát oxidase byly použity pro biosenzory na cholin, močovou kyselinu nebo L-mléčnou kyselinu. Bienzymatický biosenzor na acetylcholin zahrnuje sériově propojené mini-reaktory na bázi acetylcholinesterasy a cholin oxidasy. První část této práce se zaměřuje na konstrukci dvou různých elektrod na bázi stříbrného amalgámu. Konkrétně tato část pojednává o výrobě stříbrné pevné amalgámové elektrody pokryté rtuťovým filmem pro wall-jet průtokovou celu a navíc, zdůrazňuje rozdíly mezi jejím využitím jako...Department of Analytical ChemistryKatedra analytické chemiePřírodovědecká fakultaFaculty of Scienc

A comparative study of covalent glucose oxidase and laccase immobilization techniques at powdered supports for biosensors fabrication

In order to develop the optimal strategy and to deepen the knowledge in the field of enzyme immobilization, three different techniques of covalent binding for two enzymes (glucose oxidase and laccase) at powdered surfaces were compared. Immobilization protocol was optimized by changing supports (two mesoporous silica powders (SBA−15, MCM−41) and a cellulose powder), the functionalized\ngroups introduced at support surfaces (−NH and −COOH), and the methods of activation (glutaraldehyde and carbodiimide). Amino and carboxyl functionalized mesoporous silica and cellulose powders\nwere prepared by silanization using (3-aminopropyl)triethoxysilane and carboxyethylsilanetriol, respectively. It was found that coupling of both enzymes by their –NH groups through glutaraldehyde to -NH functionalized supports, in particular SBA15−NH and cellulose−NH for glucose oxidase, MCM41−NH for laccase, showed the highest activity and the best stability

Comparison of the Covalent Laccase Immobilization at Amino- and Carboxylfunctionalized Mesoporous Silica, Glassy Carbon, and Graphite Powders using Different Coupling Agents for Optimal Biosensor Preparation

In order to find the most suitable immobilization protocol, a comparison of three strategies\nbased on the application of –NH2 and –COOH functionalized supports with the different\nactivation agents (glutaraldehyde and carbodiimide) have been conducted for the covalent\nenzyme (laccase) attachment. Two kinds of the supports, namely mesoporous silica (SBA−15,\nMCM−41) and carbonaceous (glassy carbon, graphite) powders, have been used. It was found\nthat a biosensor consisted of tubular detector of silver solid amalgam as a working electrode\nand the enzymatic mini-reactor with laccase covalently attached to glutaraldehyde activated\n–NH2 functionalized MCM−41 shows the best results regarding sensitivity and stability for\ndopamine detection

An Enzymatic Biosensor with Amperometric Detection in a Flow Injection Analysis for the Determination of L-lactic Acid: Development and Application

An amperometric biosensor consisting of an enzymatic mini-reactor (lactate oxidase covalently\nattached to −NH2 functionalized mesoporous silica powder SBA−15 using glutaraldehyde) and\na silver amalgam-based screen-printed electrode acting as a transducer was developed for the\ndetermination of L-lactic acid (LA) in FIA. The detection potential of −0.9 V vs. Ag pseudoreference\nelectrode was applied for cathodic detection of enzymatically consumed oxygen.\nUnder the optimized conditions, the constructed biosensor enabled selective determination of\nLA with a micromolar limit of detection. Importantly, the proposed biosensor represented\nexcellent operational stability after ≥350 measurements. Finally, it was successfully applied to\nreal sample analysis

Construction and Application of Flow Amperometric Biosensor Based on Enzymatic Reactor for Determination of Choline

In this contribution, a novel fast, simple and stable biosensor with the enzymatic reactor based on choline oxidase (ChOx) was developed and applied for the determination of choline using flow injection analysis with amperometric detection. The preparation of the reactor is based on the covalent immobilization of ChOx with glutaraldehyde to mesoporous silica powder (SBA-15) previously covered by NH2-groups. The experimental parameters affecting the sensitivity and stability of the biosensor were optimized. The proposed biosensor with the newly developed ChOx-based reactor possesses good repeatability, reproducibility, long-term stability, and reusability. It was successfully applied for the determination of choline in commercial pharmaceuticals

Construction and application of the amperometric uric acid biosensors based on the covalent immobilization of uricase by different strategies

In this work, a promising combination of a biosensor based on the\nenzymatic mini-reactor with the detection principle of four-electron\nreduction of the consumed oxygen at highly negative potential has\nbeen developed for uric acid determination using flow injection\nanalysis. The construction of the biosensor provides a spatial\nsegregation of the biorecognition (uricase-based mini-reactor) and\ndetection (tubular detector of silver solid amalgam (TD-p-AgSA))\nparts. To find out the most appropriate enzyme immobilization\nprotocol, three different strategies of the covalent attachment for\nuricase from Bacillus fastidiosus have been compared. It was found\nthat the biosensor with the mini-reactor based on the covalent\nattachment of uricase via glutaraldehyde to -NH2 functionalized\nmesoporous silica powder MCM-41 showed extremely high stability\n(>1 year) and reusability (at least 600 measurements) The biosensor's\npractical applicability was confirmed by successful determination\nof uric acid in human urine