EP0149339A2
Expired / Lapsed
EP
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Assay for nucleic acids
Assignee: Medisense Inc
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Filing: 1984-12-14
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Granted: —
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Expires: 2004-12-14
Abstract
Abstract The present invention discloses an assay for nucleic acid which comprises the steps of; (a) providing a probe material comprising; (i) a sequence of nucleic acids complementary to a given target sequence, and, (ii) a first ligand chemically linked thereto and capable of a specific binding reaction with an antiligand; (b) contacting the said probe material with an assay system comprising; (i) a suitable mediator, enzyme, substrate system capable of transferring charge to an electrode surface when the enzyme is catalyticaly active, and; (ii) a second ligand chemically linked to one of said mediator, enzyme or substrate, wherein the second ligand is capable of a competitive binding reaction with the antiligand, and; (iii) the said antiligand, Whereby the said first ligand competes with the said second ligand in a specific binding reaction with the antiligand, and; (c) contacting the above system with a solution suspected of containing the said target sequence whereby the binding of any of the said target sequence present to the probe affects the availability of the first ligand and therefore alters the rate of charge transfer to the electrode.
Inventors
- 👤 Hugh Allen Oliver Hill
- 👤 Susan Liona Libor
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 18:30
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; G01N33/581; C; C12; C12Q; C12Q1/00; C12Q1/001; C12Q1/004; C12Q1/54; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6825; G01N33/53; G01N33/543; G01N33/54366; G01N33/54373; G01N33/5438; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/817; Y10S436/00; Y10S436/806; Y10S436/904
Claims Analysis
Total Claims: 53
Claim 1
Independent Claim
179 words
1) An assay for nucleic acid which comprises the steps of; (a) providing a probe material comprising; (i) a sequence of nucleic acids complementary to a given target sequence and, (ii) a first ligand chemically linked thereto and capable of a specific binding reaction with an antiligand; (b) contacting the said probe material with an assay system comprising; (i) a suitable mediator, enzyme, substrate system capable of transferring charge to an electrode surface when the enzyme is catalyticaly active, and; (ii)a second ligand chemically linked to one of said mediator, enzyme or substrate, wherein the second ligand is capable of a competitive binding reaction with the antiligand, and; (iii) the said antiligand, whereby the said first ligand competes with the said second ligand in a specific binding reaction with the antiligand, and; (c) contacting the above system with a solution suspected of containing the said target sequence whereby the binding of any of the said target sequence present to the probe affects the availability of the first ligand and therefore alters the rate of charge transfer to the electrode.
Claim 2
Dependent Claim
179 words
1) An assay for nucleic acid which comprises the steps of; (a) providing a probe material comprising; (i) a sequence of nucleic acids complementary to a given target sequence and, (ii) a first ligand chemically linked thereto and capable of a specific binding reaction with an antiligand; (b) contacting the said probe material with an assay system comprising; (i) a suitable mediator, enzyme, substrate system capable of transferring charge to an electrode surface when the enzyme is catalyticaly active, and; (ii)a second ligand chemically linked to one of said mediator, enzyme or substrate, wherein the second ligand is capable of a competitive binding reaction with the antiligand, and; (iii) the said antiligand, whereby the said first ligand competes with the said second ligand in a specific binding reaction with the antiligand, and; (c) contacting the above system with a solution suspected of containing the said target sequence whereby the binding of any of the said target sequence present to the probe affects the availability of the first ligand and therefore alters the rate of charge transfer to the electrode.
Claim 3
Dependent Claim
13 words
2) The assay of claim 1, wherein the nucleic acid is deoxyribonucleic acid.
Claim 4
Dependent Claim
13 words
3) The assay of claim 1, wherein the nucleic acid is ribonucleic acid.
Claim 5
Dependent Claim
65 words
4) The assay of claim 1, wherein the specific binding reaction between either the first or the second ligand and the antiligand is characteristic of the specific binging reaction between a pair of reagents or derivatives thereof selected from the group of pairs comprising; biotin and avidin, antigen and antibody, hormone and receptor, lectin and carbohydrate, cofactor and enzyme, nucleic acid and complementary nucleic acid.
Claim 6
Dependent Claim
29 words
5) The assay of claim 4, wherein both the first ligand and the second ligand are biotin or derivatives thereof and the antiligand is avidin or a derivative thereof.
Claim 7
Dependent Claim
13 words
6) The assay of claim 1, wherein the mediator is an organometallic compound.
Claim 8
Dependent Claim
39 words
7) The assay of claim 6, wherein the mediator comprises at least one organic ring, which is characterised by at least two double bonds that are conjugated and a metal atom in electron-sharing contact with each of the rings.
Claim 9
Dependent Claim
13 words
8) The assay of claim 7, wherein the metal is a transition metal.
Claim 10
Dependent Claim
19 words
9) The assay of claim 8, wherein the metal is selected from the group comprising iron, chromium and ruthenium.
Claim 11
Dependent Claim
12 words
10) The assay of claim 9, wherein the mediator is a ferrocene.
Claim 12
Dependent Claim
26 words
11) The assay of claim 1, wherein the mediator is selected from the group comprising viologens, poly-viologens, phenazines, ferricyanide and derivatives therof, metalloporphyrins and one-dimensional conductors.
Claim 13
Dependent Claim
12 words
12) The assay of claim 1, wherein the mediator is a carboborane.
Claim 14
Dependent Claim
13 words
13) The assay of claim 1, wherein the mediator is a ruthenium compound.
Claim 15
Dependent Claim
39 words
14) The assay of claim 10, wherein the mediator is selected from the group consisting of ferrocene; chloroferrocene; methyl-trimethylaminoferrocene ; 1,1-dimethylferrocene; 1,1 -dicarboxyferrocene; carboxyferrocene; uinylferrocene; trimethylaminoferrocene; 1,1 dimethylferrocene; polyvinylferrocene; ferrocene monocarboxylic acid; hydroxyethylferrocene; acetoferrocene; and 1, 1 bishydroxymethyl ferrocene.
Claim 16
Dependent Claim
12 words
15) The assay of claim 11, wherein the mediator is a cytochrome.
Claim 17
Dependent Claim
11 words
16) The assay of claim 15, wherein the mediator is cytochrome-C.
Claim 18
Dependent Claim
11 words
17) The assay of claim 11, wherein the mediator is TCNQ.
Claim 19
Dependent Claim
19 words
18) The assay of any one of claims 1-17, wherein the mediator is confined to an electrically conductive surface.
Claim 20
Dependent Claim
17 words
19) The assay of any one of claims 1-17, wherein the enzyme is a non-oxygen specific flavo-protein.
Claim 21
Dependent Claim
55 words
20) The assay of claim 19, wherein said flauo-protein is selected from the group consisting of methanol oxidase, pyruvate oxidase (EC 1.2.3.3.), xanthine oxidase (EC 1.2.3.2.), sarcosine oxidase (EC 1.5.3.1), lipoamide dehydrogenase (EC 1.6.3.4.), glutathione reductase (EC 1.6.4.2.), carbonmonoxide oxido-reductase, glucose oxidase, glycollate oxidase (EC 1.1.3.1.) L-amino acid oxidase (EC 1.4.3.2.) and lactose oxidase .
Claim 22
Dependent Claim
15 words
21) The assay of any one of claims 1-17, wherein the enzyme is a quino-protein.
Claim 23
Dependent Claim
23 words
22) The assay of claim 21, wherein said quino-protein is selected from the group consisting of glucose dehydrogenase alcohol dehydrogenase and methanol dehydrogenase.
Claim 24
Dependent Claim
19 words
23) The assay of any one of claims 1-17, wherein the enzyme is confined to an electrically conductive surface.
Claim 25
Dependent Claim
16 words
24) The assay of any one of claims 1-17, wherein the enzyme is a haem-containing enzyme.
Claim 26
Dependent Claim
25 words
25) The assay of claim 24, wherein the haem-containing enzyme is chosen from the group consisting of lactate dehydrogenase yeast cytochrome-C peroxidase and horseradish peroxidase.
Claim 27
Dependent Claim
15 words
26) The assay of any one of claims 1-17, wherein the enzyme is a cuproprotein.
Claim 28
Dependent Claim
12 words
27) The assay of claim 26, wherein the cuproprotein is galactose oxidase.
Claim 29
Dependent Claim
31 words
28) The assay of any one of claims 1-17, wherein the electrode is made of a material chosen from the group comprising; gold, platinum, silver, carbon or a - one-dimensional conductor.
Claim 30
Dependent Claim
73 words
29) A nucleic acid sequence probe for establishing the presence and/or copy number of a given sequence in a single-stranded nucleic acid molecule, wherein a probe nucleic acid sequence is linked to; a)either a mediator compound or an enzyme electrochemically coupled by the said mediator compound, and, b)biotin, wherein binding of the probe nucleic acid sequence to the target nucleic acid sequence modifies the electrochemical coupling between the mediator compound and the enzyme.
Claim 31
Dependent Claim
73 words
29) A nucleic acid sequence probe for establishing the presence and/or copy number of a given sequence in a single-stranded nucleic acid molecule, wherein a probe nucleic acid sequence is linked to; a)either a mediator compound or an enzyme electrochemically coupled by the said mediator compound, and, b)biotin, wherein binding of the probe nucleic acid sequence to the target nucleic acid sequence modifies the electrochemical coupling between the mediator compound and the enzyme.
Claim 32
Dependent Claim
107 words
30) A method of assay for nucleic acids wherein an electrode is placed in contact with an enzyme and a substrate of the enzyme, and a mediator compound is associated with the enzyme to transfer charge from the enzyme to the electrode as the conversion of substrate into product proceeds, and, a probe nucleic acid sequence is linked to either the enzyme or the mediator compound, whereby the activity the enzyme and the mediator compound and hence the quantity of charge being transferred to the electrode is modified by the presence or absence of a complementary target nucleic acid sequence bound to the probe nucleic acid sequence.
Claim 33
Dependent Claim
107 words
30) A method of assay for nucleic acids wherein an electrode is placed in contact with an enzyme and a substrate of the enzyme, and a mediator compound is associated with the enzyme to transfer charge from the enzyme to the electrode as the conversion of substrate into product proceeds, and, a probe nucleic acid sequence is linked to either the enzyme or the mediator compound, whereby the activity the enzyme and the mediator compound and hence the quantity of charge being transferred to the electrode is modified by the presence or absence of a complementary target nucleic acid sequence bound to the probe nucleic acid sequence.
Claim 34
Dependent Claim
28 words
31) The assay of claim 29 or 30, wherein the mediator compound, is selected from the group comprising; transition metal compounds, organic conductors, phenazines, carbon-boron compounds and viologens.
Claim 35
Dependent Claim
13 words
32) The assay of claim 31, wherein the mediator compound is biotin-labled cytochrome-C.
Claim 36
Dependent Claim
33 words
33) The assay of claim 31 or 32, wherein either avidin or streptavidin is added to the reaction mixture and binds to the mediator, thereby reducing the transfer of charge to the electrode.
Claim 37
Dependent Claim
24 words
34) The assay of claim 31 or 32, wherein the electrode detects and measures, in the presence of a mediator, an enzyme-complex catalysed reaction.
Claim 38
Dependent Claim
16 words
35) The assay of claim 32 and 33, wherein the enzyme complex comprises a biotin-peroxidase-avidin complex.
Claim 39
Dependent Claim
21 words
36) The assay of claim 35, wherein the enzyme complex is capable of binding further biotin or biotin labled nucleic acid.
Claim 40
Dependent Claim
14 words
37) The assay of claim 29, wherein the mediator is immobillized on a surface.
Claim 41
Dependent Claim
11 words
38) The assay of claim 37, wherein the surface comprises nitrocellulose.
Claim 42
Dependent Claim
15 words
39) A nucleic acid probe comprising; a) nucleic acid, b) a mediator chemically linked thereto.
Claim 43
Dependent Claim
15 words
39) A nucleic acid probe comprising; a) nucleic acid, b) a mediator chemically linked thereto.
Claim 44
Dependent Claim
26 words
40) The probe of claim 39, wherein the mediator compound, is selected from the group comprising; transition metal compounds, organic conductors, phenazines, carbon-boron compounds and viologens.
Claim 45
Dependent Claim
11 words
41) The probe of claim 40, wherein the mediator is cytochrome-C.
Claim 46
Dependent Claim
11 words
42) The probe of claim 40, wherein the mediator is ferrocene.
Claim 47
Dependent Claim
13 words
43) The probe of claim 40, wherein the mediator is a ruthenium compound.
Claim 48
Dependent Claim
17 words
44) The probe of claim 43, wherein the mediator comprises [Ru(NH 3 ) 5 py] 2+ .
Claim 49
Dependent Claim
20 words
45) An assay for nucleic acids substantially as hereinbefore described with reference to and as illustrated in the accompanying figures.
Claim 50
Dependent Claim
20 words
45) An assay for nucleic acids substantially as hereinbefore described with reference to and as illustrated in the accompanying figures.
Claim 51
Dependent Claim
25 words
46) A probe for use in an assay for nucleic acids substantially as hereinbefore described with reference to and as illustrated in the accompanying figures.
Claim 52
Dependent Claim
25 words
46) A probe for use in an assay for nucleic acids substantially as hereinbefore described with reference to and as illustrated in the accompanying figures.
Claim 53
Dependent Claim
20 words
47) A kit of reagents and/or apparatus for carrying out a method of assay as claimed in any previous claim.
References Cited (Backward Citations)
No citation records indexed.
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