Voltammetric sequence-selective sensor for target polynucleotide sequences

• US 5,312,527 A
• Filed: 10/06/1992
• Issued: 05/17/1994
• Est. Priority Date: 10/06/1992
• Status: Expired due to Term

• Current Assignee: Concordia University
• Original Assignee: Concordia University
• Inventors: Mikkelsen, Susan R., Spurmanis, Aleksandrs J., Millan, Kelly M.
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Bell, Bruce F.
• Application Number:
  US07/957,602
• Time in Patent Office: 588 Days
• Field of Search: 204/403, 204/153.12, 204/412, 435/288, 435/291, 435/810, 435/817, 435/6, 435/91, 436/501, 436/94
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to a voltammetric sequence-selective sensor for target polynucleotide sequences which essentially comprises: an immobilized polynucleotide probe having one end covalently bound onto an amperometric electrode. The immobilized probe includes a target binding region capable of hybridizing to the target polynucleotide sequences forming an immobilized heteroduplex having at least a hybridized region. The sequence-selective sensor of the present invention also comprises means for voltammetrically detecting immobilized heteroduplexes. There is also provided a method for detecting the presence of a target polynucleotide analyte in a physiological sample, which comprises the steps of incubating the prepared physiological sample with the voltammetric sequence-selective sensor of the present invention; voltammetrically detecting immobilized heteroduplexes; and comparing the resulting voltammogram with a control voltammogram.

Claims

1. A voltammetric sequence-selective sensor for target polynucleotide sequences, comprising:

a) an amperometric electrode;

b) an immobilized polynucleotide probe having one end covalently bound onto said electrode and said immobilized probe having a target binding region is hybridized to said target polynucleotide sequences to form an immobilized heteroduplex having at least a hybridized region; and

c) means for voltammetrically detecting said immobilized heteroduplex.

2. The voltammetric sequence-selective sensor according to claim 1, wherein said means for voltammetrically detecting comprises at least one electroactive complex which reversibly binds with heteroduplexes.

3. The voltammetric sequence-selective sensor according to claim 2, wherein said electroactive complex is a cobalt polypyridine complex.

4. The voltammetric sequence-selective sensor according to claim 1, wherein said covalently bound end of said probe comprises at least an oligo-d-G(100) sequence.

5. The voltammetric sequence-selective sensor according to claim 1, wherein said electrode is a carbon electrode.

6. The voltammetric sequence-selective sensor according to claim 1, wherein said sensor is reusable by rinsing in hot water.

7. A method for detecting the presence of a target polynucleotide analyte in a physiological sample, which comprises the steps of:

a) incubating said physiological sample with the voltammetric sequence-selective sensor according to claim 1;

b) voltammetrically detecting said immobilized heteroduplex; and

c) comparing the resulting voltammogram with a control voltammogram.

9. Method of using a voltammetric sequence-selective sensor according to claim 1 for the detection of base-pair mutation-associated diseases.

10. Method for using a voltammetric sequence selective sensor according to claim 2, wherein said base-pair mutation-associated diseases are selected from the group consisting of sickle-cell anemia, muscular dystrophy, osteodystrophy, phenylketonuria, hemophilia, hypercholesterolemia, thalassemia and cystic fibrosis.

8. A kit for detecting the presence of a target polynucleotide analyte in a physiological sample, comprising:

a) an amperometric electrode;

b) an immobilized polynucleotide probe having one end covalently bound onto said electrode and said immobilized probe having a target binding region capable of hybridizing to said target polynucleotide sequences to form an immobilized heteroduplex having at least a hybridized region;

c) an electroactive complex which reversibly binds with heteroduplexes; and

d) control polynucleotide sequences.