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

Communication between redoxenzyme and electrode

Electron transfer in biosensors First generation Second generation Third generation An enzyme can communicate with the electrode in different ways In the 1st generation of biosensors oxidases were trapped at the electrode surface and the enz reaction was monitored by measuring the consumption of oxygen or production of hydrogenperoxide by direct measurements thus at a low or high potential respectively in 2nd generation biosensors an artificial mediator molecule shuttles the electrons between the enz and the electrode In the third generation are based on enzymes that can exchange electrons directly with the electrode

Major groups of redox enzymes used in biosensor work

Electron transfer in biosensors First generation Second generation Third generation

First generation biosensors at conventional electrodes electrochemical oxidation of H2O2 occurs at ≥ + 600 mV vs. Ag|AgCl › the system is open for interfering reactions › the response is unstable with time

Ways to reduce the potential for electrochemical conversion of H2O2 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

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.

deposition of Prussian Blue and related catalysts on conventional electrodes + selective electroreduction of H2O2 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

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)

Advantages with coimmobilising H2O2 producing oxidases with peroxidases ¸ general approach for all H2O2 producing oxidases ¸ allows the oxidase to use its natural reoxidising agent (electron-proton acceptor), molecular oxygen (O2) › no competition between artificial mediator and O2 ¸ 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)

Electron transfer in biosensors First generation Second generation Third generation

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

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

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

Example of an Os2+/3+-based redox polymer, A. Heller, J. Phys. Chem

formal potential (E°’) of mediator formal potential (E°’) of mediator?????? mediators are ”general” electrocatalysts new Os2+/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 O2

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

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)

NAD-dependent dehydrogenase

Electrocatalytic oxidation of NAD(P)H on mediator- modified electrodes 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)

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

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

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.

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

Electron transfer in biosensors First generation Second generation Third generation

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

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

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

Self-assembled monolayers as an orientation tool - Reconstitution + apo-HRP + hemin (and EDC) + diaminoalkane Formation of a mixed monolayer with suitable dilution of activated headgroups addition of diaminoalkanes coupling of hemin groups to the amino group by carbodiimide addition of apoHRP and reconstitution mixed SAM e.g., GOx, GDH-PQQ H. Zimmermann, A. Lindgren, W. Schuhmann, L. Gorton, Chem. Eur. J. 6 (2000) 592-599

Peroxidase Peroxidases are found in Cofactor  heme Plants Bacteria Structure of horseradish peroxidase (HRP) C Peroxidases are found in Plants Bacteria Fungi Animal tissues Cofactor  heme The first is peroxidase which is a common enzyme in biotechnology, it is often used as an label in immunoassays and is also commonly used in biosensors. POD is found in ….. most common POD is HRP (this short will be used from now on), which structure is shown with glukans M. Gajhede, et.al., Nature Structural Biology, 4 (1997) 1032.

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

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

ket 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%) CHisrec HRP/gold ≈ 35 s-1 (75%) NHisrec HRP/gold ≈ 30 s-1 (65%)

Direct electron transfer In the presence of enzyme substrate In the absence of enzyme substrate I want to specify that I will be distinguish between two ways of observing direct ET In the presence of substrate- electrocatalytic current In the absence of substrate observed as an redox-wave seen for fewer enzymes

Direct electron transfer of CDH Cyclic voltammetry of CDH Electrocatalytic current Current/µA + 3.8 mM cellobiose pH 4.4, scanrate 50 mV/s Current/µA E°’=-41±3 mV This can be exemplified with CDH show a very nice redox wave of the enzyme in the absence of substrate and clear catalytic current by addition of substrate (starting at the potential of the redox wave) CDH trapped under a membrane at a gold electrode (modified with cystamine) in 50 mM Ac-buffer, pH 5.1. A. Lindgren, T. Larsson, T. Ruzgas, L. Gorton, J. Electroanal. Chem., 494 (2000) 105-113

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 pH 3.6 pH 4.4 Current/µA pH 5.1 pH 6.0 Current/µA With 3.8 mM cellobiose, without cellobiose 50 mM Ac-buffer, scan rate 50 mV s-1.