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TOPICS IN (NANO) BIOTECHNOLOGY Enzyme sensors 30th June PhD Course.

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Presentation on theme: "TOPICS IN (NANO) BIOTECHNOLOGY Enzyme sensors 30th June PhD Course."— Presentation transcript:

1 TOPICS IN (NANO) BIOTECHNOLOGY Enzyme sensors 30th June PhD Course

2 Communication between redoxenzyme and electrode



5 Electron transfer in biosensors _ First generation_ Second generation_ Third generation



8 Major groups of redox enzymes used in biosensor work



11 Electron transfer in biosensors _ First generation_ Second generation_ Third generation

12 _ First generation biosensors at conventional electrodes electrochemical oxidation of H 2 O 2 occurs at ≥ + 600 mV vs. Ag|AgCl › the system is open for interfering reactions › the response is unstable with time

13 Ways to reduce the potential for electrochemical conversion of H 2 O 2 i noble metal deposition on carbon electrodes i Prussian Blue deposition on conventional electrodes i peroxidase modified electrodes i other catalysts e.g. iron phthalocyanine

14 noble metal (Pt, Pd, Ru, Rh) deposition on carbon electrodes lack of selectivity - future???? A carbon electrode sputtered with palladium and gold for the amperometric detection of hydrogen peroxide. Gorton, L. Anal. Chim. Acta (1985), 178(2), 247-53 Catalytic Materials, Membranes, and Fabrication Technologies Suitable for the Construction of Amperometric Biosensors. Newman, J. D.; White, S. F.; Tothill, I. E.; Turner, A. P. F. Anal. Chem. (1995), 67(24), 4594-9. Remarkably selective metalized-carbon amperometric biosensors. Wang, J; Lu, F; Angnes, L; Liu, J; Sakslund, H; Chen, Q; Pedrero, M; Chen, L; Hammerich, O. Anal. Chim. Acta (1995), 305(1-3), 3-7 Electrochemical metalization of carbon electrodes. O'Connell, P. J.; O'Sullivan, C. K.; Guilbault, G. G. Anal. Chim. Acta (1998), 373(2-3), 261-270.

15 deposition of Prussian Blue and related catalysts on conventional electrodes + selective electroreduction of H 2 O 2 at around 0 mV vs. Ag|AgCl - lack of long term stability at pH > 7.5 Prussian Blue and its analogues: electrochemistry and analytical applications. Karyakin, A. A.. Electroanalysis (2001), 13(10), 813-819 Metal-hexacyanoferrate films: A tool in analytical chemistry. de Mattos, Ivanildo Luiz; Gorton, Lo. Quimica Nova (2001), 24(2), 200-205

16 peroxidase modified electrodes of great bioelectrochemical interest practical applications??? Peroxidase-modified electrodes: fundamentals and application. Ruzgas, T; Csöregi, E; Emnéus, J; Gorton, L; Marko-Varga, G. Anal. Chim. Acta (1996), 330(2-3)

17 Advantages with coimmobilising H 2 O 2 producing oxidases with peroxidases ¸ general approach for all H 2 O 2 producing oxidases ¸ allows the oxidase to use its natural reoxidising agent (electron-proton acceptor), molecular oxygen (O 2 ) › no competition between artificial mediator and O 2 ¸ some oxidases have no or very low reaction rates with artificial mediators ¸ allows the use of an applied potential within the "optimal potential range" (≈ -150 - +50 mV vs. SCE, pH 7) › less interfering reactions from complex matrices ¸ electron transfer between electrode and peroxidase can be either direct or mediated (control of response range and sensitivity)

18 Electron transfer in biosensors _ First generation_ Second generation_ Third generation

19 Mediators in bioelectrochemistry 1 e - acceptor/donors vs. 2 e - -H + acceptor/ donors

20 1 e - acceptor/donor 2 e - -H + acceptor/donor +E°’ does not vary with pH-E°’ varies with pH no H + participates1-2 H + participate + no radical intermediates-radical intermediates stable redox reactionunstable redox reaction -low reaction rates with NADH+ high reaction rates with NADH -moderate reaction rates with+ high reaction rates peroxidases with peroxidases


22 Marcus equation The rate of electron transfer between two redox species is expressed by: distance thermodynamic driving force reorganisation energy


24 Example of an Os 2+/3+ -based redox polymer, A. Heller, J. Phys. Chem., 96 (1992) 3579-3587

25 formal potential (E°’) of mediator?????? mediators are ”general” electrocatalysts new Os 2+/3+ -polymer, E°’ ≈ + 100 mV vs. Ag|AgCl can it be further improved (i.e., lowered)? for E°’-values below 0 mV: risk for electrocatalytic reduction of O 2

26 Which group(s) works best with mediators????

27 Dehydrogenases with bound cofactors are the ”best” to wire because: + bound cofactor ( c.f. NAD dehydrogenase ) + not oxygen dependent ( c.f. oxidase ) but - not so many (yet) - often not so stable ( c.f. GOx, HRP )

28 NAD-dependent dehydrogenase

29 Electrocatalytic oxidation of NAD(P)H on mediator- modified electrodes. obstacles to solve to make electrochemical sensors based on these enzymes: 1. both NAD(P) + and NAD(P)H suffer from severe electrochemical irreversibility 2. enzyme depends on a soluble cofactor 3. the equilibrium of the reaction for most substrates favours the substrate NOT the product side NAD + has a LOW oxidising power (E°' pH 7 = -560 mV vs. SCE)

30 Dehydrogenase with bound cofactor, e.g., glucose PQQ-dehydrogenase

31 Engineered new enzymes tailormade for biosensor applications i GDH-PQQ membrane bound enzyme i PQQ loosely bound to the enzyme i Different GDH-PQQ have different selectivities i Different GDH-PQQ have different pH optima => through genetic engineering combine the ”best” properties of each of several GDH-PQQs and produce a new ”optimal” glucose oxidising enzyme

32 Bioengineered (new) enzymes Construction of multi-chimeric pyrroloquinoline quinone glucose dehydrogenase with improved enzymatic properties and application in glucose monitoring. Yoshida, H; Iguchi, T; Sode, K. Biotechnology Letters (2000), 22(18), 1505-1510. Secretion of water soluble pyrroloquinoline quinone glucose dehydrogenase by recombinant Pichia pastoris. Yoshida, H; Araki, N; Tomisaka, A; Sode, K. Enzyme Microb. Technol. (2002), 30(3), 312-318.

33 New electrode materials Walcarius, Alain. Electrochemical Applications of Silica-Based Organic-Inorganic Hybrid Materials. Chemistry of Materials (2001), 13(10), 3351-3372 Walcarius, Alain. Electroanalysis with pure, chemically modified, and sol-gel-derived silica-based materials. Electroanalysis (2001), 13(8-9), 701-718 Walcarius, Alain. Zeolite-modified electrodes in electroanalytical chemistry. Anal. Chim. Acta (1999), 384(1), 1-16. Walcarius, Alain. Analytical applications of silica-modified electrodes. A comprehensive review. Electroanalysis (1998),10(18), 1217-1235

34 Electron transfer in biosensors _ First generation_ Second generation_ Third generation



37 L.-H. Guo and H. A. O. Hill, Adv. Inorg. Chem., 36 (1991) 341-373


39 Random adsorption/orientation on carbon < 100% of enzyme molecules in direct ET contact with the electrode

40 ordered orientation on thiol modified gold high % (≈ 100%) of enzyme molecules in DET contact with the electrode

41 Self-assembled monolayers as an orientation tool - Reconstitution mixed SAM + diaminoalkane + hemin (and EDC) + apo-HRP e.g., GOx, GDH-PQQ H. Zimmermann, A. Lindgren, W. Schuhmann, L. Gorton, Chem. Eur. J. 6 (2000) 592-599

42 Peroxidases are found in –Plants –Bacteria –Fungi –Animal tissues Cofactor  heme Structure of horseradish peroxidase (HRP) C M. Gajhede,, Nature Structural Biology, 4 (1997) 1032. Peroxidase

43 Structural Models of Recombinant (left) and Native Glycosylated (right) Horseradish Peroxidase C Hydrophobic residues are coloured in red and hydrophilic in blue

44 Structural Model of Recombinant Horseradish Peroxidase C with a His-tag located at either the C- or the N-terminus

45 k et and % in DET between HRP and electrode native HRP/graphite ≈ 2 s -1 (50% DET) rec HRP/graphite ≈ 8 s -1 (65%) rec HRP/gold ≈ 18 s -1 (60%) C His rec HRP/gold ≈ 35 s -1 (75%) N His rec HRP/gold ≈ 30 s -1 (65%)

46 Direct electron transfer In the presence of enzyme substrate In the absence of enzyme substrate

47 Direct electron transfer of CDH Electrocatalytic current Cyclic voltammetry of CDH CDH trapped under a membrane at a gold electrode (modified with cystamine) in 50 mM Ac-buffer, pH 5.1. Current/µA E°’=-41±3 mV Current/µA + 3.8 mM cellobiose pH 4.4, scanrate 50 mV/s A. Lindgren, T. Larsson, T. Ruzgas, L. Gorton, J. Electroanal. Chem., 494 (2000) 105-113

48 Electrocatalysis at the CDH electrode Electrocatalytic current was observed in the presence of the enzyme substrate, cellobiose. At high pH the internal ET is decreased Low pH High pH With 3.8 mM cellobiose, without cellobiose 50 mM Ac-buffer, scan rate 50 mV s -1. pH 3.6 pH 4.4 pH 5.1pH 6.0 Current/µA

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