Method and system for the simultaneous and multiple detection and quantification of the hybridization of molecular compounds such as nucleic acids, dna rna, pna, and proteins

• US 20030143575A1
• Filed: 10/11/2002
• Published: 07/31/2003
• Est. Priority Date: 03/07/2000
• Status: Abandoned Application

• Current Assignee: Mario Raimondo Caria
• Original Assignee: Mario Raimondo Caria
• Inventors: Caria, Mario Raimondo
• Application Number:
  US10/220,958
• Publication Number:
  US 20030143575A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00

Abstract

The method is used for detecting a position of several hybridization sites on a support (2001) containing probes (3002) having hybridized targets (3003) remaining attached thereto after a washing step. It comprises the steps of: emitting a radiation (3004) from a source (3001) towards the support (2001); receiving the radiation coming from the support on a microelectrode detector (1002) sensitive to the radiation; and quantifying the targets in different sites of the support at the same time.

Claims

1. A method for detecting a position of several hybridization sites on a support (2001) containing probes (3002;

4002) possibly having hybridized targets (3003;

4003) remaining attached thereto after a washing step, characterized in that it comprises the steps of;

emitting a radiation (3004;

4004) from a source (3001;

4001) towards the support (2001);

receiving a radiation coming from the support on a microelectrode detector (1002) sensitive to the radiation; and

quantifying different sites of the support at the same time concerning possible hybridized targets.

2. The method of claim 1, characterized in that it comprises the step of receiving a radiation from a site of the support at different moments, and preferably quantifying the evolution of hybridization at this site along time.

3. The method of claim 1 or 2, characterized in that the reception step comprises the step of receiving the radiation after it passed through the support.

4. The method of any of claims 1 to 3, characterized in that the quantification step comprises the step of determining the amount of hybridized targets at some sites.

5. The method of any of claims 1 to 4, characterized in that the quantification step comprises the step of counting a number of photons arriving to the detector from a site.

6. The method of any of claims 1 to 5, characterized in that the radiation is emitted directly onto the support.

7. The method of any of claims 1 to 6, characterized in that the targets and/or probes are taken from the group consisting in:

DNA fragments, RNA fragments hybrid systems such as PNA (Peptide Nucleic Acid) protein fragments, synthetic oligonucleotides, and synthetic oligopeptides.

8. The method of any of claims 1 to 7, characterized in that it comprises the steps of amplifying and transforming a signal emitted by the detector and digitally reading thereof.

9. The method of any of claims 1 to 8, characterized in that the radiation is electromagnetic and lays in the energy interval going from 1 eV to 6 eV.

10. The method of any of claims.1 to 9, characterized in that the radiation is a laser beam.

11. The method of any of claims 1 to 10, characterized in that the radiation comes from a radioactive source.

12. The method of any of claims 1 to 11, characterized in that the targets contain substances, such as fluorescent or radio-excitable substances, arranged to react to the radiation.

13. The method of any of claims 1 to 12, characterized in that the targets do not contain a substance arranged to react to the radiation.

14. The method of any of claims 1 to 13, characterized in that the radiation comprises an ultraviolet radiation.

15. The method of any of claims 1 to 14, characterized in that the radiation has wavelengths situated between 190 nm and 700 nm.

16. The method of any of claims 1 to 15, characterized in that the radiation is received by different sites of the support under the form of a common beam.

17. The method of any of claims 1 to 16, characterized in that the targets are purified molecules.

18. The method of any of claims 1 to 17, characterized in that the targets are PCR(“

Polymerase Chain Reaction”

)-amplified molecules.

19. The method of any of claims 1 to 18, characterized in that the targets are untreated molecules such as simple cellular lysate extract.

20. A device for detecting a position of several hybridization sites on a support (2001) containing deposited probes (3002;

4002) possibly having hybridized targets (3003;

4003) remaining attached thereto after a washing step, characterized in that it comprises;

a support (2001) for the probes;

a source (3001;

4001) for emitting a radiation (3004;

4004) towards the support;

a microelectrode detector (1002) arranged to receive a radiation coming from the support and sensitive thereto; and

means (3006, 3007;

4006, 4007) for quantifying different sites of the support at the same time concerning possible hybridized targets.

21. The device of claim 20, characterized in that the detector is arranged to receive the radiation after it passed through the support.

22. The device of any of claims 20 to 21, characterized in that the means for quantifying are arranged for determining the amount of hybridized targets at some sites.

23. The device of any of claims 20 to 22, characterized in that the means for quantifying are arranged to count a number of photons arriving to the detector from a site.

24. The device of any of claims 20 to 23, characterized in that the source is arranged to emit a radiation on a site of the support at different moments, and the quantifying means are arranged to quantify the evolution of hybridization at this site along time.

25. The device of any of claims 20 to 24, characterized in that the source is a gas discharge lamp.

26. The device of any of claims 20 to 25, characterized in that the source is a laser source, preferably a semiconductor one or a gas one.

27. The device of any of claims 20 to 26, characterized in that the source is arranged to emit an ultraviolet radiation.

28. The device of any of claims 20 to 27, characterized in that the source is arranged to emit a radiation having a plurality of wavelengths situated between 190 nm and 700 nm.

29. The device of any of claims 20 to 28, characterized in that it is arranged so that the radiation is received by the different sites of the support under the form of a common beam.

30. The device of any of claims 20 to 29, characterized in that it comprises a lens or a system of lenses arranged in the path of the radiation, before or after the support.

31. The device of any of claims 20 to 30, characterized in that it comprises a micro-lenses system to allow the passage of the maximum intensity of the incident radiation, arranged in the path of the radiation before or after the support.

32. The device of any of claims 20 to 31, characterized in that it comprises a monochromator or filter system for the selection of the passing energy of the incident radiation before or after the support.

33. The device of any of claims 20 to 32, characterized in that the means for quantifying comprises an electronic reading circuit connected to the detector, welded or glued directly to the detector.

34. The device of any of claims 20 to 33, characterized in that the means for quantifying comprises an electronic reading circuit connected to the detector and grown with the detector.

35. The device of claims 33 or 34, characterized in that the electronic reading circuit is of the VLSI (“

Very Large Scale Integrated”

) design type.

36. The device of any of claims 20 to 35, characterized in that the microelectrode detector is formed by junctions on a semiconductor material.

37. The device of any of claims 20 to 36, characterized in that the semiconductor material is chosen from the group consisting of:

high resistivity Silicon, synthetic Diamond, a Gallium-based compound, or a compound containing Gallium and Aluminum.

38. The device of any of claims 20 to 37, characterized in that the semiconductor has contacts implanted to form junctions in diode type configurations.

39. The device of any of claims 20 to 38, characterized in that the distance between the microelectrodes is substantially the same, center to center, as the distance between the hybridization sites.

40. The device of any of claims 20 to 39, characterized in that the distance between the sites and/or between the microelectrodes ranges from 1 micrometer to 1 centimeter.

41. The device of any of claims 20 to 40, characterized in that the means for quantifying is arranged to transform the charge into electric current.

42. The device of any of claims 20 to 41, characterized in that means for quantifying comprises an amplifying system.

43. The device of any of claims 20 to 42, characterized in that the support for the probes is made of glass with thin films of another material.

44. The device of any of claims 20 to 43, characterized in that the support for the probes is made of a plastic polymer.