Medisense, Inc.

Genetics International, Inc.

Other

National Research Development Corporation

Serono Diagnostics Ltd.

Hugh A Hill

Irving J Higgins

Graham Davis

Grenville Arthur Robinson

Gordon C Forrest

Simon J Rattle

James Michael McCann

Elliot V Plotkin

Monika J Green

David Whitford

Nicholas J Walton

David J Page

Susan I Libor

Frazer A Armstrong

Brian N Oliver

A sensor system that detects a current representative of a compound in a liquid mixture features a two-electrode strip adapted for releasable attachment to signal readout circuitry. The strip comprises an elongated support (preferably flat) adapted for releasable attachment to the readout circuitry; a first conductor and a second conductor each extend along the support and comprise means for connection to the circuitry. An active electrode, positioned to contact the liquid mixture and the first conductor, comprises a single layer deposit of an enzyme capable of catalyzing a reaction involving the compound, a conductive material, and an electron mediator, capable of transferring electrons between the enzyme-catalyzed reaction and the first conductor. A reference electrode is positioned to contact the mixture and the second conductor. The system includes circuitry adapted to provide an electrical signal representative of the current. The two-electrode strip is manufactured, e.g., by screen printing, an admixture of an enzyme a conductive material, and a mediator as a single layer onto the substrate.

Strip electrode with screen printing

A sensor system that detects a current representative of a compound in a liquid mixture features a two-electrode strip adapted for releasable attachment to signal readout circuitry. The strip comprises an elongated support (preferably flat) adapted for releasable attachment to the readout circuitry; a first conductor and a second conductor each extend along the support and comprise means for connection to the circuitry. An active electrode, positioned to contact the liquid mixture and the first conductor, comprises a deposit of an enzyme capable of catalyzing a reaction involving the compound and (preferably) an electron mediator, capable of transferring electrons between the enzyme-catalyzed reaction and the first conductor. A reference electrode is positioned to contact the mixture and the second conductor. The system includes circuitry adapted to provide an electrical signal representative of the current. The two-electrode strip is manufactured, e.g., by screen printing the enzyme (and preferably the mediator) onto the substrate.

A sensor system that detects a current representative of a compound in a liquid mixture features a two-electrode strip adapted for releasable attachment to signal readout circuitry. The strip comprises an elongated support (preferably flat) adapted for releasable attachment to the readout circuitry; a first conductor and a second conductor each extend along the support and comprise means for connection to the circuitry. An active electrode, positioned to contact the liquid mixture and the first conductor, comprises a deposit of an enzyme capable of catalyzing a reaction involving the compound and (preferably) an electron mediator, capable of transferring electrons between the enzyme-catalyzed reaction and the first conductor. A reference electrode is positioned to contact the mixture and the second conductor. The system includes circuitry adapted to provide an electrical signal representative of the current. The two-electrode strip is manufactured, e.g., by screen printing an admixture of enzyme, a conductive material, and a mediator as a single layer onto the substrate.

Strip electrode including screen printing of a single layer

An electrochemical specific binding assay of a ligand (e.g., antigen, hapten or antibody) wherein at least one of the components is enzyme-labelled, and which includes the step of determining the extent to which the transfer of electrons between the enzyme substrate and an electrode, associated with the substrate reaction, is perturbed by complex formation or by displacement of any ligand complex relative to unbound enzyme-labelled component. The electron transfer is aided by electron-transfer mediators which can accept electrons from the enzyme and donate them to the electrode or vice versa (e.g. ferrocene) or by electron-transfer promoters which retain the enzyme in close proximity with the electrode without themselves taking up a formal charge. The electrochemical apparatus will typically comprise two or three electrodes, including one working electrode onto which components may advantageously be immobilized. The use of direct electrochemical measurement of the enzyme label avoids the errors and inconvenience of the known indirect measurement techniques.

Methods of assay

In its broadest aspect, the present invention provides a method of effecting an electrochemical specific-binding assay of a ligand, either qualitatively or quantitatively, in an apparatus comprising at leasts one electrode, in which method a labelled component of the assay medium is, at least in part, magnetically held in the vicinity of the electrode. The electrochemical assay method may include a separation step, whereby bound label may be separated from free label in the assay medium. Preferably the electrochemical assay method will include the step of determining a perturbation in an electrochemical characteristic of components of the assay medium associated with a ligand complexing reaction. Such an assay method is applicable to, for example, antibodies and antigens and suitable labels include redox centers, enzyme labels in the presence of an electron-transfer mediator and electron-transfer mediator labels in the presence of an electron donor or acceptor.

Method of assay employing a magnetic electrode

A ligand in a sample can be assayed using electrochemical apparatus containing a working electrode and components comprising:

Electrochemical methods of assay

Electrochemical assay for nucleic acids and nucleic acid probes

An electrode has a graphite surface at least part of which is an edge-plane graphite surface to which Cr(III) complexes are attached by surface C--O groups. The graphite can be polished pyrolytic graphite. The complex coverage can be 5-20%. The complex may comprise proteins, enzyme substrates, specific binding agents, intermediate metabolites or the like, e.g. the protein plastocyanin, so that direct unmediated electrochemistry is possible at the electrode.

Graphite electrode with modified surface

The invention disclosed relates to an electrochemical assay which is particularly concerned with an assay for the presence of, or amount of, glycosylated haemoglobin in a blood sample, but which extends to a general assay for detecting the presence of, monitoring the level of or determining the concentration of compounds containing cis-diol groups, such as glycols, nucleic acid components, sugars, polyols, catechols and glycosylated proteins. When ferrocene boronic acid or a derivative thereof is present free in solution, it can act as a mediator for a wide range of oxido-reductases. However in the presence of, for example, sugars containing the cis-diol structure, the metallocene (such as ferrocene boronic acid) covalently binds to the sugar. When the ferrocene boronic acid is thus bound, it is essentially immobilized and its properties as a mediator and ability to diffuse will be markedly affected.

Electrochemical assay for cis-diols

This specification discloses methods of detection or measurement of an enzyme or of its specific substrate, and sensors used in such methods. The present invention is concerned with a multi-enzyme system, and the specification discloses, as one aspect of the invention a method of assay in which an electrode poised at a suitable potential is contacted with a system comprising a first enzyme, a cofactor linked with said enzyme and a mediator compound which transfers charge to the electrode from the first enzyme when its electrical state is changed by reaction of cofactor material. The cofactor may be NAD, NADP (both collectively referred to herein as NAD(P)), cAMP, ATP, GTP, TTP, or CTP. The specification particularly illustrates a method of assay in which an electrode (1) poised at a suitable potential is contacted with a system comprising a first enzyme E.sub.1 a nicotinamide adenine dinucleotide compound N linked with said enzyme E.sub.1 and a mediator compound F which transfers charge to the electrodes from the first enzyme when its electrical state is changed by a NAD(P) NAD(P)H reaction. The NAD compound may act as a 'bridge' between the said enzyme/mediator system and further NAD utilizing enzyme E2.

Assay systems using more than one enzyme

This invention relates to equipment and methods for detecting the presence of, measuring the amount of, and/or monitoring the level of, one or more selected components in a liquid mixture, employing an electrode sensing system. We have discovered that a class of mediating compounds has extremely useful properties for mediating the transfer of charge between enzyme-catalyzed reactions and electrode surfaces (15) in electrode sensing systems. Specifically, the specification discloses as electrode sensor mediators, organometallic compounds composed of at least two organic rings, each of which has at least two double bonds in a conjugated relationship; a metal atom is in electron-sharing contact with those rings. An enzyme capable of catalyzing a reaction at a rate representative of the selected compound concentration is in contact with an assay mixture, and the mediator compound transfers charge between the enzyme and the conductive surface of the electrode at a rate representative of the enzyme catalyzed reaction rate.

Sensor for components of a liquid mixture

In a bioelectrochemical process in which the electrons are transferred directly, without use of a mediator, such as a redox dye or cofactor, between an electrode and an electroactive biological material, such as an enzyme or a protein, in either direction, rapid electron transfer has previously been achieved between an electrode and the positively charged protein horse-heart cytochrome c by adding a surface-modifier such as 4,4'-bipyridyl or a derivative thereof. It has now been found possible to promote electron transfer to either a positively or a negatively charged protein using the same surface-modifier for either job, namely a compound of formula ##STR1## wherein: the pyridine ring shown is substituted in the 2-, 3- or 4-position by the (methylene)hydrazinecarbothioamide group shown; R.sup.1 represents hydrogen atom(s) or one or two methyl or ethyl groups in the 2-,3- or 4-position (when the said position is not substituted by the (methylene)hydrazinecarbothioamide group shown);

Surface-modified electrode and its use in a bioelectrochemical process

A sensor electrode to detect one or more components in a liquid mixture comprises an electrically conductive material having at least an an external surface, the combination of an enzyme catalytic for a reaction of the desired component, and a mediator compound which transfers electrons from the enzyme to the electrode when such catalytic activity takes place. It can be used as an in vivo glucose sensor either with a silver electrode coated with e.g. glucose oxidase and a polyviologen as the mediators, or with a particulate carbon electrode, glucose oxidase and chloranil or fluoranil as mediator. Another system is to use bacterial glucose dehydrogenase or glucose oxidase as the enzyme and/or ferrocene or a ferrocene derivative as the mediators compound to give electrodes with improved linearity, speed of response and insensitivity to oxygen.

There are provided processes for carrying out enzymatic reactions, especially processes for the oxidative or reductive transformation of organic compounds catalyzed by enzymes, in which the enzymes require a continuing supply of reducing equivalents to regenerate reduced enzyme species in enzymatically active form, and in which the reducing equivalents are derived electrochemically, including electrochemically from molecular hydrogen, the reducing equivalents preferably being passed directly to the enzyme from the cathode of an electrochemical system.

Growing community of inventors

Oxford, United Kingdom

Hugh A Hill