Method and device for detecting biomolecules

• US 7,879,594 B2
• Filed: 03/15/2004
• Issued: 02/01/2011
• Est. Priority Date: 03/14/2003
• Status: Expired due to Fees

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Mandrand, Bernard, Dupont-Filliard, Agnes
• Primary Examiner(s): Yu; Melanie
• Application Number:
  US10/548,869
• Publication Number:
  US 20070037153A1
• Time in Patent Office: 2,514 Days
• Field of Search: 435/6, 435/7.1, 435/7.5, 435/7.9, 435/7.92, 435/283.1, 435/287.2, 436/518, 436/524, 436/525, 422/50, 422/68.1, 422/78, 422/82.01, 977/700, 977/702, 977/704, 977/705, 977/720, 977/904, 977/920
• US Class Current: 435/287.2
• 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. A 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, which is linked to a signal generating agent 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 biomolecule attached thereto being linked to said location,

b) bringing the sample in contact with the solid support comprising said probe biomolecules, thereby the probe biomolecule and the target biomolecule interacting together,

c) adding the soluble species,

d) transforming the soluble species into the 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 least one location, wherein a decrease in the flexibility of the electrode causes a decrease in the electrical conductivity to indicate the presence of the target biomolecule.

2. The method according to claim 1, wherein the value for the electrical conductivity obtained in step e) is compared with a value obtained before step e).

3. The method according to claim 2, wherein a change in electrical-conductivity is measured by amperometric measurements, differential pulse voltammetry, chronoamperometry, or impedance measurements.

4. The method according to claim 1, wherein the catalytic active moiety is a biocatalyst.

5. The method according to claim 4, wherein the biocatalyst is an enzyme.

6. The method according to claim 5, wherein the enzyme is oxidases, peroxidases, reductases or dehydrogenases.

7. The method according to claim 1, wherein the tag comprises further a biotin/avidin or biotin/streptavidin complex.

8. The method according to claim 1, wherein the probe biomolecule is oligonucleotides, polypeptides, proteins, antigenes, antibodies, enzymes, receptors or carbohydrates.

9. The method according to claim 1, wherein the probe biomolecule and/or all or a portion of the nanoelectrodes further comprise an electron-donor group.

10. The method according to claim 9, wherein the electron-donor group comprises a transition metal complex.

11. The method according to claim 1, wherein the solid support is electrically conductive or is an insulator deposited on a conductive layer.

12. The method according to claim 1, wherein the target biomolecule is an oligonucleotide comprising a segment which is partially complementary to the probe molecule.

13. The method according to claim 12, wherein the tag comprises is linked to a first detection species.

14. The method according to claim 13, wherein the first detection species is an oligonucleotide.

15. The method according to claim 14, wherein the oligonucleotide comprises a segment which is partially complementary to the target biomolecule.

16. The method according to claim 1, wherein the soluble species is 5-Bromo-4chloro-3-phosphate-1H-indole-(BCIP), nitrobluetetrazoliumchloride (NBT), dimethylbenzidine, diaminobenzidine, 4-chloro-naphthole, 3-amino-3-ethylcarbazole, or tetramethylbenzidime (TMB).

17. A 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, which is linked to a signal generating agent 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 electropolymerized nanoelectrodes with probe biomolecule attached thereto being linked to said location,

b) bringing the sample in contact with the solid support comprising said probe biomolecules, thereby the probe biomolecule and the target biomolecule interacting together,

c) adding the soluble species,

d) transforming the soluble species into the 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 least one location, wherein a decrease in the flexibility of the electrode causes a decrease in the electrical conductivity to indicate the presence of the target biomolecule.

18. A 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, which is linked to a signal generating agent 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, which nanoelectrodes are optionally substituted polypyrroles or copolymers thereof, with probe biomolecule attached thereto being linked to said location,

b) bringing the sample in contact with the solid support comprising said probe biomolecules, thereby the probe biomolecule and the target biomolecule interacting together,

c) adding the soluble species,

d) transforming the soluble species into the 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 least one location, wherein a decrease in the flexibility of the electrode causes a decrease in the electrical conductivity to indicate the presence of the target biomolecule.

19. The method according to claim 18, wherein the nanoelectrodes are electropolymerized.