Method and device for detecting biomolecules

• US 20070037153A1
• Filed: 03/15/2004
• Published: 02/15/2007
• Est. Priority Date: 03/14/2003
• Status: Active Grant

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Apibio SAS (Compagnie Merieux Alliance SAS)
• Inventors: Mandrand, Bernard, Dupont-Filliard, Agnes
• Granted Patent: US 7,879,594 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10S 977/70 Nanostructure
• CPC Class Codes: Y10S 977/702 having biological material component
• CPC Class Codes: Y10S 977/704 Nucleic acids, e.g. DNA or RNA
• CPC Class Codes: Y10S 977/705 Protein or peptide
• CPC Class Codes: Y10S 977/72 On an electrically conducting, semi-conducting, or semi-insulating substrate

Abstract

The present invention relates to a method for detecting a target biomolecule in a sample comprising a plurality of biomolecules, whereby the target biomolecule is provided with a tag, said tag comprising a catalytic active moiety which catalyses a reaction yielding an insoluble reaction product which precipitates on flexible electrically conductive nanoelectrodes. The precipitation onto said nanoelectrodes causes a change in their electroconductivity which is accessible to electroanalytical methods. The invention relates further to a biochip comprising a solid support with nanoelectrodes attached thereto and probe molecules bound to all or to a plurality of said nanoelectrodes which may be the same or different, a segment of said probe molecules being able to interact specifically with a segment of the target biomolecules.

Claims

1. Method for detecting a target biomolecule in a sample comprising a plurality of biomolecules, whereby the target biomolecule is provided directly or indirectly with a tag, said tag comprising a catalytic active moiety which catalyses the transformation of an additional soluble species to an insoluble precipitate, said method comprising the steps of:

a) providing a solid support with a plurality of spatially different locations arranged on its surface, whereby at least one location is electrically conducting and a plurality of individual flexible nanoelectrodes with probe biomolecules attached thereto being linked to said location,

b) bringing the sample in contact with the solid support comprising said probe biomolecules, thereby forming a recognition complex between a probe biomolecule and a target biomolecule,

c) adding an essentially soluble species,

d) transforming the soluble species into an insoluble precipitate forming a deposit essentially on or at the nanoelectrodes, thereby decreasing the flexibility of the nanoelectrodes

e) measuring the electrical conductivity of the plurality of the nanoelectrodes at at least one location.

2. Method according to claim 1, characterized in that the value for the electrical conductivity obtained in step e) is compared with a value obtained before step e).

3. Method according to claim 2, characterized in that the change in electrical-conductivity is measured by a method selected from the group consisting of amperometric measurements, differential pulse voltammetry, chronoamperometry, impedance measurements.

4. Method according to claim 1, characterized in that the catalytic active moiety is a biocatalyst.

5. Method according to claim 4, characterized in that the biocatalyst is an enzyme.

6. Method according to claim 5, characterized in that the enzyme is selected from the group consisting of oxidases, peroxidases, reductases and dehydrogenases, most preferred the enzyme is a peroxidase or phosphatase.

7. Method according to claim 1, characterized in that the tag comprises further a biotin/avidin or biotin/streptavidin complex.

8. Method according to claim 6, characterized in that one single probe biomolecule is attached to each nanoelectrode.

9. Method according to claim 8, characterized in that two or more probe biomolecules are attached to each nanoelectrode, whereby the probe biomolecules are the same or different.

10. Method according to claim 1, characterized in that the probe biomolecule is selected from the group consisting of oligonucleotides, polypeptides, proteins, antigenes, antibodies, enzymes, receptors and carbohydrates.

11. Method according to claim 1, characterized in that the probe biomolecule and/or all or a portion of the nanoelectrodes further comprise an electron-donor group.

12. Method according to claim 11, characterized in that the electron-donor group comprises a transition metal complex.

13. Method according to claim 1, characterized in that the solid support is electrically conductive or is an insulator deposited on a conductive layer.

14. Method according to claim 1, characterized in that the target biomolecule is an oligonucleotide comprising a segment which is partially complementary to the probe molecule.

15. Method according to claim 14, characterized in that the tag comprises further a first detection species.

16. Method according to claim 15, characterized in that the first detection species is an oligonucleotide.

17. Method according to claim 16, characterized in that the oligonucleotide comprises a segment which is partially complementary to the target biomolecule.

18. Method according to claim 1, characterized in that the essentially soluble species is selected from the group consisting of 5-Bromo-4chloro-3-phosphate-1H-indole-(BCIP), nitrobluetetrazoliumchloride (NBT), dimethylbenzidine, diaminobenzidine, 4-chloro-naphthole, 3-amino-3-ethylcarbazole, tetramethylbenzidime (TMB).

19. Biochip comprising a solid support with a plurality of flexible rod-shaped nanoelectrodes attached on specific essentially electrically conducting locations to said support, whereby the nanoelectrodes are the same of different for each location, and probe molecules which are the same or different, bound to all or to a plurality of said nanoelectrodes a segment of said probe molecules being able to interact specifically with a segment of target biomolecules

20. Biochip according to claim 19, wherein the probe molecules are selected from the group consisting of oligonucleotides, peptides, proteins, antigenes, antibodies, enzymes, receptors, carbohydrates, hormone receptors, lipids.

21. Biochip according to claim 20, wherein said probe molecules are oligonucleotides.

22. Biochip according to claim 21, wherein the target biomolecules are nucleic acid sequences of different pathogenic microorganisms.

23. Biochip according to claim 22, wherein said nucleic acid sequences are nucleic acid sequences related to different genetic diseases.

24. Biochip according to claim 23, wherein said nucleic acid sequences are nucleic acid sequences of different tissues.

25. Biochip according to claim 24, wherein said nucleic acid sequences are nucleic acid sequences related to different individuals.

26. Kit for the detection of target biomolecules in a sample comprising a plurality of biomolecules, further comprising:

a) a support partially functionalized with flexible rod-shaped nanoelectrodes and having attached probe biomolecules thereto,

b) an essentially soluble species transformable in an insoluble precipitate