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Biosensors A focus on peroxidase-modified electrodes and their practical applications by Ivo Frébort Biosensor - an analytical device that exploits a biocatalytic reaction Consists of: biocatalyst (enzyme, cells, tissue) transducer (converts the biological or biochemical signal into a quantifiable electrical or optical signal) First biosensor - Clark (1962): glucose sensor with glucose oxidase and oxygen electrode Glucose + O2 Gluconic acid + H2O2 Oxygen electrode (1956) working electrode: Pt cathode (-0.6 V) reference electrode: Ag/AgCl electrodes separated from measured solution with a gas permeable mebrane Leland C. Clark, Jr. with the first enzyme electrode Construction of the biosensors Sensing electrode: platinum, gold, various forms of carbon Immobilization techniques: general method doesn’t exist - enzyme physical entrapment - covalent crosslinking O CH (CH2)3 C H O Glutaraldehyde BSA E CH O + H2N electrode BSA o-ring E E BSA Schiff base E E E CH N BSA BSA Reduction with NaBH4 E E dialyzing E membrane C H2 NH Covalent attachment to a support membrane or the electrode R R B NH2 N O N + O COOH + C C C N O C NH B NH R NH R' C O R' N C N DCC Carbodiimide reaction NH R' CMC N C N CH2CH2 N O H3C tosyl- C H3C H2 N C N (CH2)3 N(C H3)2 EDC O CH (C H2)3 C H O B NH2 NH2 N CH (C H2 )3 CH O NaBH4 N CH (CH2)3 CH N B NH C H (CH2)3 CH NH B Glutaraldehyde reaction Adsorption - glass, carbon, Au, Pt - often activation needed Adsorption of thiols to a gold electrode Silanization of an oxidized metal electrode S (C H2)2 NH2 O S (C H2)2 NH2 R Si (OC2H5)3 O Si R + --OH S (C H2)2 NH2 S (CH2)2 NH2 Au Au O --OH O Si R O S X S S (C 2H5)3S i (C H2)3 NH2 APTES S X X S X S S X S (C H3)3Si (C H2)3 O C H2 CH C H2 S X S X S X S O CH3 GOPS Organized layer Dilution with an inert thiol C 2H5 O Si (CH2)3 NH2 CH3 APDMES Screen printing Mobile wiper Matrix carrier Paste Screen grid Screen print An enzyme electrode P1 P1 - S1 - Sample E Electrode S2 - P2 P2 - I P2* Protective Enzyme layer Permselective membrane membrane 1. Thin enzyme layer with high specific activity, 2. Good selection of membranes Response controlled by diffusion through the permselective membrane (not by enzyme kinetics) Enzyme activity low - thick membrane needed to achieve linear response, response slow Enzyme activity high - thin membrane OK, rapid response 3. Detection: Steady-state or flow injection analysis Biosensor parameters 1. Sensitivity dS/dt 2. Linear response Analyte Signal 3. Detection limit 4. Background noise DS 5. Baseline drift 6. Selectivity 7. Response Time Background noise 8. Operating stability 9. Shelf life Detection Analyte S limit N DS/Dc Linear response S=3N Assay of the detection limit c Type of measurement Analyte additions S Time Direct contact with the sample Solution placed in a chamber A Flow cell S Time B IN OUT First generation biosensors - response to the substrates in solution Glucose + O2 Gluconic acid + H2O2 1. Reduction of oxygen with a Clark type electrode at -0.6 V (vs SCE) 2. Oxidation of hydrogen peroxide at a Pt electrode at +0.7 V 3. Measuring of pH change Examples of hydrogen peroxide measurning biosensor Analyte Enzyme Reaction Alcohol Alcohol oxidase Ethanol + O2 Acetaldehyde + H2O2 D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2 Lactose Galactose oxidase Lactose + O2 Galactose dialdehyde der. + H2O2 L-Lactate L-Lactate oxidase L-Lactate + O2 Pyruvate + H2O2 Starch Amyloglucosidase Starch + H2O β-D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2 Sucrose Invertase Sucrose + H2O α-D-Glucose + β-D-Fructose Mutarotase α-D-Glucose β-D-Glucose Glucose oxidase β-D-Glucose + O2 Gluconic acid + H2O2 Types of transducers used in biosensors Type Detectable species Amperometric O2, H2O2, I2, NADH Ion-selective electrode H+, Na+, Cl- Field effect transistors H+, Na+, Cl- Gas sensing electrode CO2, NH3 Photomultiplier Light emission ATP/Luciferase/Luciferin, H2O2/Peroxidase/Luminol, etc. Thermistor Heat of reaction Second generation biosensors - mediated electron transfer between enzyme and electrode - can be easily miniaturized blood glucose measuring system in situ Third generation biosensors - direct electron transfer between enzyme and electrode Cell-based based biosensors - cheaper than purified enzymes, Nocardia erythropolis cells immobilised in polyacrylamide or agar (cholesterol oxidase) Cholesterol + O2 Cholest-4-en-3-one + H2O2 Enzyme immunosensors - many types, based on ELISA techniques - often use chemiluminiscence or bioluminiscence human chorionic gonadotropin - pregnancy Examples of biosensors Analyte Biocatalyst Transducer Immobilization Shelf life Response Alcohol Alcohol oxidase O2 Glutaraldehyde 2 weeks 1-2 min Arginine Streptococcus faecium NH3 Entrapment 3 weeks 20 min Cholesterol Nocardia erythropolis O2 Entrapment 4 weeks 35-70 s D-Glucose Glucose oxidase O2 Covalent 3 weeks 1 min Glutamate Glutamate decarboxylase CO2 Glutaraldehyde 1 week 10 min NAD+ NADase + Escherichia coli NH3 Membrane 1 week 5-10 min Nitrate Azotobacter vinelandii NH3 Entrapment 2 weeks 7-8 min Penicillin Penicillinase H+ Polyacrylamide 2 weeks 15-30 s Urea Urease NH4+ Polyacrylamide 3 weeks 20-40 s Personal glucose meter for diabetics (Medisense Britain, Ltd.) Automated affinity systems Biacore 2000 (Biacore) KI 1 (BioTuL) www.bioacore.com www.biotul.com IAsys (Affinity Sensors) IBIS II (XanTec) www.affinity-sensors.com www.xantec.com Peroxidase-based electrodes PEROXIDASE (EC 22.214.171.124) Protein of 35-45 kDa, prosthetic group - heme, Mn2+ Convenient sources: horse radish root, soybean, tobacco leaves, various fungi Ruiz-Duenas, F. J., Martinez, M. J., Martinez, A. T.: Peroxidase from the ligninolytic fungus Pleurotus eryngii. Mol Microbiol 31 pp. 223 (1999) The catalytic cycle of peroxidase Native peroxidase + H2O2 Compound-I + H2O (Fe3+) (Fe4+=O, Por+) Compound-I + AH2 Compound-II + AH* (Fe4+=O, Por+) (Fe4+=O) Compound-II + AH2 Native peroxidase+ AH* + H2O (Fe4+=O) (Fe3+) Applications of peroxidase-based electrodes 1. Detection of hydrogen peroxide in aqueous solutions industry, environmental protection, clinical control photochemical smog, pathological processes in lungs, etc. 2. Detection of organic peroxides in water and organic solutions free radical injury, oxidative stress, autooxidation of unsaturated lipids 3. Detection of compounds based on peroxidase inhibition CN-, F-, hydroxylamine 4. Detection of aromatic amines and phenolic compounds environmental control: chlorophenols in water 5. Immunosensors based on peroxidase electrodes peroxidase conjugates with antibody, H2O2-producing enzyme conjugates 6. Detection of analytes based on peroxidase/oxidase-coupled reactions glucose, ethanol, lactate,choline, xanthine, cholesterol, bilirubin, glutamate Electrode designs A. Surface modified electrodes Electrode material: graphite, glassy carbon, gold, SnO2 Coupling: carbodiimide, glutaraldehyde, adsorption B. Polymer-based electrodes Crosslinking with Os(bpy)23+/2+ redox polymer, electropolymerized polypyrrole, o-phenylethylamine C. Bulk modified composite electrodes Graphite-silicone oil paste, paraffin oil paste, epoxy composite D. Tissue-modified carbon paste electrodes Asparagus tissue, tobacco callus tissue, horseradish root, kohlrabi skin Mechanism of direct biocatalytic reduction of hydrogen peroxide at peroxidase-modified electrodes Compound-I H2O (Fe 4+=O, Por+) e- Electrode Eappl< 0.6 V Compound-II (Fe 4+=O) e- vs SCE 2H+ H2O2 H2O Ferriperoxidase (Fe 3+) Mechanism of mediated biocatalytic reduction of hydrogen peroxide at peroxidase-modified electrodes Compound-I H2O (Fe 4+=O, P +) Mred Mox Compound-II Electrode (Fe 4+=O) Mred 2H + H2O2 H 2O Mox Ferriperoxidase (Fe 3+) Mediator: ferrocene, o-phenylenediamine, hydroquinone The mediators OH NH2 R + Fe NH2 OH Ferrocene o-Phenylenediamine Hydroquinone Detailed look at a practical example ... Copper amine oxidase-based electrodes for the assay of biogenic amines Monitoring the biomarkers of food freshness: histamine, putrescine, cadaverine Currently used methods: chromatographic techniques - they often require sample pre-treatment steps and skilled operators; the relatively long analysis time and high costs make these methods not suitable for routine use Aim of the work: design and construction of the amperometric biosensors for monitoring of biomarkers Two biosensor designs: monoenzymatic and bienzymatic, using both the direct and mediated electron transfer pathways Biological recognition element: copper amine oxidase (EC 126.96.36.199) Mediator: poly(1-vinylimidazole) complexed with [Os(4,4'- dimethylbipyridine)2Cl]+/2+ (PVI13-dmeOs) Assay system: The biosensors were used in a flow-injection analysis (FIA) line The biogenic amines: histamine, putrescine and cadaverine NH2 N H2N-(CH2)n-NH2 N n=4: Putrescine; n=5: Cadaverine H Histamine Formed by the decarboxylation Formed by the biodegradation of histidine biocatalysed by of the aminoacids ornithine and various microorganisms lysine by the action of putrefactive bacteria Stimulates smooth muscle contraction and relaxation, Oversaturate the histamine- including heart motions detoxifying enzymes, enhancing the toxicity of histamine Stimulates sensory and motory neurons Controls acid gastric secretion Copper amine oxidase (AO) Redox active polymer (PVI13-dmeOs) Biological sources: bacteria, fungi, plants, animals a b Biological functions: involved in cell N N growth, proliferation and differentiation Cofactors: N N O H N N N Os2+/ 3+ & Cu(II) HO O N Cl N O Topa quinone (TPQ) Copper Catalyzed reaction: R-CH2-NH2 + H2O + O2 R-CHO + NH3 + H2O2 Flow-injection system used Working mechanism for monoenzymatic electrodes H N AOox Electrode N H2 C C H2 NH2 NH3 2e- Eappl.= +200 mV Histamine vs. Ag/AgCl H2O H N AOred N C CHO H2 Imidazoleacetaldehyde Working mechanism for bienzymatic electrodes H Electrode N AOox H2O2 HRPred 2 Os2+ 2e- 2e- Eappl.= -50 mV H2 N (TOPA -native) (Fe3+- native) vs. Ag/AgCl C C H2 H2O 2 Os3+ H2N H2O HRPox Histamine (Fe4+ = O, P+ inactive) NH3 H N AOred O2 N (TOPA -inactive) OHC C H2 Imidazoleacetaldehyde Electrode type C No Os2+/3+ Electrode type D Biosensors characteristics ELECTRODE ANALYTE Kmapp Imax S DL DR TYPE (µM) (µA) (mA/Mcm2) (µM) (µM) TYPE A HISTAMINE 375±34 0.164±0.06 5.99±0.09 2.7 10-100 TYPE B HISTAMINE 730±33 0.360±0.08 6.76±0.05 2.2 10-200 TYPE C HISTAMINE 332±17 1.34±0.02 55.29±0.73 0.16 1-100 PUTRESCINE 227±16 3.01±0.07 181.64±1.01 0.06 1-100 H2O2 112±8 2.70±0.06 330.14±1.02 1-100 TYPE D HISTAMINE 901±85 4.85±0.41 73.74±1.73 0.33 1-150 PUTRESCINE 512±40 7.26±0.53 194.11±1.37 0.17 1-400 H2O2 977±92 22.8±1.68 319.59±1.63 1-250 Native enzyme Km - putrescine 0.2 mM, histamine 0.35 mM Monitoring real samples - turbot fish 30 fish kept at -20°C fish kept at 25°C 25 g histamine/kg fish 20 15 10 5 0 0 2 4 6 8 10 12 Days •Amine content from fish kept in different conditions was extracted with 0.1M potassium phosphate buffer, pH 7.2, and analyzed by direct injection in the FIA system using the bienzymatic mediated electrode Comparison of selectivity of the developed systems 350 AO biosensor 300 AO-HRP biosensor Relative response (%) 250 200 150 100 50 0 Hsm TDAB CDAB Csm Trm EDA Agm Spd Put Cad Amine substrate Further oxidation of the histamine reaction product H N H2O + O2 AOred N H2 C C H2 NH2 Histamine H N NH3 + H2O2 AOox Electrode N C CHO H2 2e- Eappl.= +200 mV vs. Ag/AgCl Imidazoleacetaldehyde H N N C COOH H2 Imidazoleacetic acid + NH3 AO O + O2 + H2O + H2O2 + NH4+ NH2 NH2 Putrescine - H2O Putrescine and cadaverine form cyclic products - cannot be N further oxidized !!! 1-Pyrroline + NH3 AO O + O2 + H2O + H2O2 + NH4+ NH2 NH2 Cadaverine - H2O N 1-Piperideine Conclusions 1. Combination of the monoenyzmatic (AO) and bienzymatic (AO- HRP) electrode can be used for selective detection of histamine and diamines (putrescine and cadaverine). 2. The biosensors were tested for detection of fish product poisoning by putrefactive amines. 3. The monoenzymatic electrode (AO) is the first example of DET with copper amine oxidase, which can proceed anaerobically. 4. With histamine as an analyte, both DET and further oxidation of the product aldehyde contribute to the biosensor response.
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