Reagentless oxidoreductase sensors

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Biosemors & Bioelectronics

Vol. 11, No. l/2, pp. 127-135, 1996

@) 19% Elsevier Science Limited Printed in Great Britain. All rights reserved 09565663/96/$15.00

ELSEVIER ADVANCED TECHNOLOGY

Reagentless Hanns-Ludwig Lehrstuhl

oxidoreductase Schmidt*

& Wolfgang

Schuhmann

fur Allgemeine Chemie und Biochemie, Technische Universitat Vottingerstr. 40, D-85354 Freising-Weihenstephan, Germany. Tel: [49] (0) 8161-713253 Fax: [49] (0) 8161-713583. (Received 30 November

sensors

Miinchen.

1994; accepted 4 April 1995)

Abstract:

The function of oxidoreductase biosensors is in genera1 dependent on charge transport between the enzyme and an electrode surface by means of coenzymes or redox mediators. Reagentless biosensors should integrate these components within the very electrode area simultaneously preventing their leaking into the analyte medium and conserving their shuttle function. Concepts and strategies for the realization of this demand are outlined based on the structure of the active sites of the enzyme types in question and the reaction mechanism to be catalyzed. Experiences with different combinations of enzyme- and electrode-integrated mediators are reported. Keywords: biosensors, reagentless biosensors, transfer mechanism, oxidoreductases

INTRODUCTION Oxidoreductases in amperometric

serve

as recognition

elements

biosensors by catalyzing the oxidation or dehydrogenation of their specific substrate and transferring redox equivalents to suitable electron acceptors such as 02, an oxidized coenzyme or an artificial redox mediator. The reoxidation of these auxiliary compounds at an appropriate electrode surface then leads to a substrate-proportional current through the electrochemical cell. Only in a few exceptional cases can a direct or protein-mediated communication between enzyme and electrode be attained (Cass et al., 1985). A ‘reagentless biosensor’ as used throughout this communication is defined as an amperometric enzyme electrode, which provides a substrateproportional current signal without being depen-

* To whom correspondence

should be addressed.

enzyme

electrodes,

electron-

dent on a mediator or coenzyme This does not exclude the existence of such compounds fixed in proximity to the electrode or within a limited electrode chamber. Our definition is therefore a challenge and a task from where the following questions arise: How can we immobilize redox mediators or coenzymes close to the electrode surface without preventing their shuttle function for the transfer of redox equivalents between the active site of the oxidoreductase and the electrode surface? How can we preserve high electrontransfer rates using immobilized redox relays? A practically useful solution meeting these demands are carbon-paste electrodes, integrating the enzyme and suitable redox mediators in a semi-solid single phase (Kulys et al., 1992). The main advantage of these sensors is their easy preparation by simply mixing the enzyme, the redox mediator, graphite powder and paraffin oil to a homogeneous paste. Although it has been shown that a wide variety of different biosensors can be advantageously developed using 127

Biosensors & Bioelectronics

H.-L. Schmidt & W. Schuhmann

this concept, their electron-transfer principle is not yet fully elucidated. Most probably, the surface-adsorbed enzyme transfers electrons via the redox mediator which leaks continuously from the electrode forming a small and constant mediator concentration near the electrode surface (Schuhmann et al., 1993a; Wittstock et al., 1994). Thus, carbon-paste electrodes are partially ‘reagentless’ in the sense of our definition, however, they do not meet the complete demand because they contaminate the analyte solution by leakage of low-molecular weight sensor components. A practical possibility proposed for the integration of all sensor components in an electrode chamber and for preventing the exchange of mediator or coenzyme molecules with the electrolyte is their binding to high-molecular weight carriers, which can be physically entrapped behind a semipermeable membrane with an appropriate molecular-weight cut-off. We have investigated the applicability of polyethylene glycol-bound NAD+ (Lammert et al., 1989) and of polyethylene glycol-bound ferrocene derivatives (Schuhmann, 1993). We have found that, due to the slow diffusion of these mediators within the electrode chamber, corresponding electrodes in general show very long response times. However, the primary aim of the present communication is not the development of new and practically applicable reagentless biosensors, but rather a fundamental study of possible ways for the construction of defined reagentless biosensors using individual types of oxidoreductases. Based on the one hand on Marcus’ electrontransfer theory (Marcus & Sutin, 1985; Marcus, 1993) and on the other hand on the partially available X-ray structures of the enzymes (Brookhaven), giving details on the conformation of the active site and the nature of the redox center, possibilities for the realization of such reagentless amperometric enzyme electrodes are discussed and tested. The study deals with flavoproteins, representing a group of enzymes with a tightly bound coenzyme, often buried in a deep cleft, with NAD+-dependent dehydrogenases, which bind the coenzyme only transiently, with pyrroloquinoline quinone (PQQ) enzymes, which may release their cofactor, often bound near the protein surface, and with heme-proteins, which use their tightly bound cofactor only for electron transport. To these enzymes the most suitable principles of integration of corresponding 128

sensor components on the biosensor surface listed below are applied (Fig. l), namely: (a) Covalent binding of redox mediators at the electrode surface and, concomitantly, adsorption of the enzyme. (b) Modification of the enzyme itself, either with redox mediators or the coenzyme, and covalent binding of the modified enzyme at electrode surfaces. (c) Entrapment of a modified enzyme within conducting polymer films. (d) Entrapment of a (modified) enzyme within redox polymer films. (e) Covalent binding of the coenzyme at the electrode surface and reconstitution of the holoenzyme with the apoenzyme. (f) Orientated binding of an enzyme to modified electrode surface to allow direct electrical communication.

RESULTS AND DISCUSSION In the following sections, some of these principles are applied to or tested with enzymes which appear to be the most promising and suitable. Flavoproteins The tertiary structures of many flavoproteins are known (Muller, 1992; Hech et al., 1993). Their active sites often integrate-in addition to often two flavin molecules---other redox centers such as sulfhydryl groups or metal cations. Substrate and electron acceptor enter the active site sequentially in a so-called ping-pong mechanism. Based on preliminary results with glucose oxidase and high molecular-weight ferrocene derivatives mentioned above (Schuhmann, 1993), we concentrated on new possibilities to retain the redox mediator within the electrode compartment, simultaneously optimizing the probability for electron-transfer reactions between the active site of the enzyme and the mediator molecules. In cooperation with Adam Heller and his group in Austin we succeeded in covalent binding of spacer-linked ferrocene derivatives to the outer surface of glucose oxidase (Schuhmann et al., 1991). It could be demonstrated, that this ferrocene-functionalised enzyme used-in the absence of oxygen-its own bound redox mediators for electrical communication with a glassy carbon electrode, and provided a glucose-concentration proportional current. The observed current response was dependent on the length of the spacer between the redox mediator and the

Biosensors & Bioelectronics

Reagentless oxidoreductase sensors

Fig. 1. Schematic representation of possibilities for the development of reagentless amperometric explanation of (a) to (f) see text.

enzyme surface. Intermolecular charge transport was exclusively observed with short spacer chains, while with long and flexible spacer chains an intramolecular charge transport was found. The latter electron-transfer mechanism has been called ‘whip-mechanism’. The logical subsequent step was the immobilization of these mediator-modified enzymes onto the electrode surface in such a way as to establish a high probability for electron transfer according to the whip-mechanism. In order to entrap the modified glucose oxidase within a conducting polymer film, pyrrole was polymerized in the presence of the ferrocene-modified enzyme, and, as expected, with the electrode obtained, a glucose-proportional current could be observed in the absence of oxygen (Schuhmann, 1994). The electron-transfer mechanism from the polymerentrapped modified glucose oxidase to the electrode surface was dependent on the morphology of the polypyrrole network. While within a polymer film with large pores the enzyme-bound

biosensors. For

ferrocene derivatives seemed to move freely, giving rise to charge transfer according to the whip-mechanism, in dense and rigid polymer networks the electron transfer seemed to occur via the conducting polymer film itself. Presuming that the movement of a mediator bound on top of a flexible spacer chain within a given radius is a general prerequisite for the enhancement of electron-transfer reactions, it should also be possible to use suitable redox mediators or mediator-modified enzymes bound via long and flexible spacer:chains directly to the electrode surface. This has successfully been accomplished by covalent binding of ferrocenemodified glucose oxidase to thiol-monolayer modified gold electrodes (Lotzbeyer et al., to be published). Again, a glucose-proportional current could be observed in the absence of oxygen. Quinoproteins

Quinoproteins have in common quinone cofactors in their active site, among which topaquinone 129

H.-L.

Schmidt & W. Schuhmann

Biosensors & Bioelectronics

(TQQ) and tryptophane-tryptophane quinone (TTQ) are covalently bound, while pyrroloquinoline quinone (PQQ) is (in many cases) reversibly exchangeable (Duine, 1991; Davidson, 1993). As the enzymes are often part of bacterial electrontransport chains, many of them use proteins such as cytochromes or copper proteins as electron acceptors. However, they are also capable of reacting with a number of artificial acceptors such as quinones, dichlorophenol-indophenol and phenazine methosulfate. It must also be pointed out that the catalytic activity of some of them is dependent on Ca2+ ions, which are probably involved in the binding of the cofactor. So far, the tertiary structure of only one of these enzymes (methylamine dehydrogenase) has been elucidated (Matthews and Hol, 1993). From its structure and the knowledge about the size and conformation of the natural electron acceptors, it can be derived that the cofactor must be situated close to the protein surface. Thus, this class of redox enzymes should be predestined for the construction of reagentless biosensors, because from the short distance between cofactor and electrode surface, fast electron-transfer processes can be expected. In order to evaluate the possibilities of achieving reagentless quinoprotein electrodes, the electrochemical behaviour of the free cofactor at modified electrode surfaces was studied. For this investigation, gold electrodes were modified with chemisorbed monolayers of thiol-derivatives, which suggested the possibility of producing different charges towards the electrolyte solution (Katz ef al., 1994a) (Scheme 1).

Au

I-

-S-(CH,),-C

z”_‘

repulsion,

PQQ+

0

Au1 - -S-(CH,),-OH

=f=

PQ&

completely

AU - -S-(CH,),I

CH,

-H-l-

PQQ8

blocked

A+

-S-(CH&-

A+

-S-(CH,),-E;

NH,

PaW ; blockd

PQQ

3-

pH>9

pH
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