Method and apparatus for detecting the presence and/or concentration of biomolecules

• US 5,341,215 A
• Filed: 05/28/1992
• Issued: 08/23/1994
• Est. Priority Date: 06/08/1991
• Status: Expired due to Term

• Current Assignee: Koninklijke Philips N.V.
• Original Assignee: Hewlett-Packard Company (HP Inc.)
• Inventors: Seher, Jens-Peter
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US07/889,629
• Time in Patent Office: 817 Days
• Field of Search: 356/445, 422/82.05, 422/82.08, 422/82.09, 422/82.11, 422/4, 435/287, 435/291, 435/808, 435/321, 435/174, 435/3, 436/165, 436/172
• US Class Current: 356/445
• CPC Class Codes: G01N 21/553 and using surface plasmons fluorescence excitation G01N21/648; enhanced Raman G01N21/658
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus

Abstract

A method and apparatus for detecting the presence and/or concentration of biomolecules, in particular DNA, by providing a boundary surface between an optically denser medium and an optically rarer medium. Biomolecules adsorb to the boundary surface and alter the characteristics of impinging light. The method and apparatus use surface plasmon resonance (SPR) to excite the biomolecules. The angle of incidence of the impinging light is maintained at the angle at which SPR occurs, thereby controlling the energy transferred to the biomolecules. The energy transferred to the biomolecules causes the biomolecules to generate excitation light which is reflected and monitored at the boundary surface. The angle of incidence is controlled by rotating a desk such that the intensity of the reflected light is always maintained at a minimum which ensures that the angle of incidence is equal to the angle at which SPR occurs.

Claims

1. A method for detecting biomolecules, comprising the steps of:

(a) attaching a plurality of biomolecules to a boundary surface, said boundary surface being between an optically denser and an optically rarer medium and having a first side adjacent to said optically denser medium and a second side adjacent to said optically rarer medium, said plurality of biomolecules being attached by at least one of

(i) adsorbing to said boundary surface;

(ii) depositing on said boundary surface; and

(iii) physically or chemically bonding to said boundary surface, said boundary surface having a film, the film having a conductive characteristic being capable of conducting surface plasmon waves, comprising the steps of;

(b) irradiating p-polarized electromagnetic waves, using light through said optically denser medium onto said boundary surface, wherein the angle of incidence (θ

) of the p-polarized electromagnetic waves impinging onto said boundary surface is substantially equal to an angle (θ

_SPR) at which surface plasmon resonance occurs, surface plasmon waves capable of causing said plurality of biomolecules at said boundary surface to reflect or generate electromagnetic waves in response to said surface plasmon wave;

(c) monitoring at least one of the electromagnetic waves reflected and the electromagnetic waves generated by said plurality of biomolecules at said boundary surface and determining at least one of;

(i) the presence of said plurality of biomolecules; and

(ii) the concentration of said plurality of biomolecules by analysis of at least one of said reflected or generated electromagnetic waves, wherein the intensity of the electromagnetic waves reflected or generated by said plurality of biomolecules at said boundary surface is detected; and

(d) controlling the angle of incidence (θ

) of the impinging p-polarized electromagnetic waves depending on the intensity of reflected electromagnetic waves at said boundary surface such that said intensity is substantially maintained at a minimum corresponding to the occurrence of surface plasmon resonance.

2. The method according to claim 1, wherein said optically denser medium is a transparent prism.

3. The method according to claim 2, further comprising the step of:

actuating a rotatable support carrying said transparent prism depending on said intensity.

4. The method according to claim 3, wherein said rotatable support is rotated in a first direction and then in a second direction opposite to said first direction in response to an increase in said intensity, or in case a predetermined time period (T_max) has expired.

5. The method according to claim 1, wherein said electromagnetic waves reflected at said boundary surface are fed to an array of monitoring elements comprising at least a central monitoring element and at least a lateral element at either side of said central monitoring element and a control signal causing adjustment of said angle of incidence (θ

) is generated if the intensity detected by one of said lateral elements is lower than the intensity detected by said central element.

6. The method according to claim 1, further comprising at least one of:

(i) passing p-polarized electromagnetic waves through a dielectric layer before passing through said film, and

(ii) passing p-polarized electromagnetic waves through a dielectric layer after passing through said film.

7. The method according to claim 1, wherein said optically rarer medium is a solvent in which said plurality of biomolecules are dissolved such that at least some of said plurality of biomolecules adsorb to, deposit on, or are physically or chemically bound to said second side of said boundary surface.

8. The method according to claim 7, wherein at least one of said plurality of biomolecules and complementary biomolecules which are complementary to said plurality of biomolecules are labelled with a fluorescent, phosphorescent, chemiluminescent or electroluminescent substance.

9. The method according to claim 8, wherein said second side of said boundary surface is coated with capture molecules complementary to said plurality of biomolecules, said solvent comprising said plurality of biomolecules and labelled biomolecules complementary to said biomolecules.

10. The method according to claim 9, wherein said second side of said boundary surface is coated with capture molecules complementary to said plurality of biomolecules, said solvent comprising said plurality of biomolecules and labelled biomolecules complementary to said biomolecules.

11. The method according to claim 1, wherein at least one of said plurality of biomolecules and complementary biomolecules which are complementary to said plurality of biomolecules are labelled with a fluorescent, phosphorescent, chemiluminescent or electroluminescent substance.

12. The method according to claim 1, wherein said optically rarer medium is a solvent in which said plurality of biomolecules are dissolved such that at least some of said plurality of biomolecules adsorb to, deposit on, or are physically or chemically bound to said second side of said boundary surface.

13. The method according to claim 12, wherein at least one of said plurality of biomolecules and complementary biomolecules which are complementary to said plurality of biomolecules are labelled with a fluorescent, phosphorescent, chemiluminescent or electroluminescent substance.

14. An apparatus for detecting biomolecules at a boundary surface, said boundary surface being between an optically denser medium and an optically rarer medium, said biomolecules being attached on or to said boundary surface, and said boundary surface having a film with a conductive characteristic coupled thereto, said film being capable of conducting surface plasmon waves, the apparatus comprising:

a source of electromagnetic waves;

polarizing means for p-polarizing the electromagnetic waves emitted by said source of electromagnetic waves;

means for directing said p-polarized electromagnetic waves through an optically denser medium onto said boundary surface, said boundary surface being between said optically denser medium and an optically rarer medium;

a first monitoring means for monitoring radiation reflected or generated by said biomolecules at said boundary surface;

a second monitoring means for monitoring electromagnetic waves reflected at said boundary surface;

an intensity detection means coupled to said first and second monitoring means for detecting an intensity of said electromagnetic waves reflected at said boundary surface; and

a control means coupled to said intensity detection means and coupled to said means for directing said p-polarized electromagnetic waves, said control means controlling the angle of incidence of the impinging p-polarized electromagnetic waves depending on the intensity of the electromagnetic waves reflected at said boundary surface such that said intensity is substantially maintained at a minimum corresponding to an occurrence of surface plasmon resonance.

15. The apparatus according to claim 14, wherein said second monitoring means comprises a detector, the detector being responsive only in the spectrum of said p-polarized electromagnetic waves.

16. The apparatus according to claim 14, wherein said first monitoring means together with said second monitoring means comprise a detector selectively responsive to said p-polarized electromagnetic waves and said electromagnetic waves reflected at said boundary surface.

17. The apparatus according to claim 14, wherein said second monitoring means comprises an array of monitoring elements, with at least a central monitoring element and at least a lateral element at either side of said central monitoring element, a control signal generating means are providing a control signal if the intensity detected by one of said lateral elements is lower than the intensity detected by said central monitoring element, said control signal being fed to said control means.

18. The apparatus according to claim 14, wherein said means for directing said p-polarized electromagnetic waves onto the boundary surface comprises a transparent prism.

19. The apparatus according to claim 18, wherein said transparent prism is mounted on a support rotatable by operating means, controlled by said control means, wherein said transparent prism is rotated an angle (Δ

θ

).

20. The apparatus according to claim 19, further comprising:

transmission means for rotating said support around first angle (θ

) and for rotating at least one of said first and second monitoring means twice said first angle (2Δ

θ

).

21. The apparatus according to claim 14, wherein said source of electromagnetic waves is adjustable.

22. A method for detecting biomolecules, comprising the steps of:

(a) attaching a plurality of biomolecules to a boundary surface, said boundary surface being between an optically denser and an optically rarer medium and having a first side adjacent to said optically denser medium and a second side adjacent to said optically rarer medium, said plurality of biomolecules being attached by at least one of

(i) adsorbing to said boundary surface;

(ii) depositing on said boundary surface; and

(iii) physically or chemically bonding to said boundary surface, said boundary surface having a film, the film having a conductive characteristic being capable of conducting surface plasmon waves;

(b) irradiating said plurality of biomolecules so attached to said boundary surface with impinging electromagnetic waves so that said impinging electromagnetic waves pass through said optically denser medium onto said boundary surface, wherein the angle of incidence (θ

) of the impinging electromagnetic waves is substantially equal to an angle (θ

_SPR) at which surface plasmon resonance occurs, surface plasmon waves capable of causing said plurality of biomolecules at said boundary surface to at least reflect or generate electromagnetic waves in response to said surface plasmon wave;

(c) detecting the intensity of at least one of the electromagnetic waves reflected and the electromagnetic waves generated by said plurality of biomolecules at said boundary surface;

(d) determining based upon said intensity so detected at least one of;

(i) the presence of said plurality of biomolecules; and

(ii) the concentration of said plurality of biomolecules; and

(e) monitoring shifts in the angle θ

_SPR by detecting the intensity of the reflected electromagnetic waves;

(f) controlling the angle of incidence (θ

) of the impinging p-polarized electromagnetic waves depending on the intensity of reflected electromagnetic waves so detected to substantially optimize the energy of the impinging p-polarized electromagnetic waves coupled into said plurality of biomolecules for making said determination of step (d).

23. The method according to claim 22, wherein said optically denser medium is a transparent prism.

24. The method according to claim 22, further comprising the step of:

actuating a rotatable support carrying said transparent prism depending on said intensity.

25. The method according to claim 24, wherein said rotatable support is rotated in a first direction and then in a second direction opposite to said first direction in response to an increase in said intensity, or in case a predetermined time period (T_max) has expired.

26. The method according to claim 22, wherein said electromagnetic waves reflected at said boundary surface are fed to an array of monitoring elements comprising at least a central monitoring element and at least a lateral element at either side of said central monitoring element and a control signal causing adjustment of said angle of incidence (θ

) is generated if the intensity detected by one of said lateral elements is lower than the intensity detected by said central element.

27. The method according to claim 22, further comprising at least one of:

(i) passing said impinging electromagnetic waves through a dielectric layer before passing through said film, and

(ii) passing impinging electromagnetic waves through a dielectric layer after passing through said film.

28. The method according to claim 22, wherein at least one of said plurality of biomolecules and complementary biomolecules which are complementary to said plurality of biomolecules are labelled with a fluorescent, phosphorescent, chemiluminescent or electroluminescent substance.

29. An apparatus for detecting biomolecules at a boundary surface, said boundary surface being between an optically denser medium and an optically rarer medium, said biomolecules being attached on or to said boundary surface, and said boundary surface having a film with a conductive characteristic coupled thereto, said film being capable of conducting surface plasmon waves, the apparatus comprising:

a source of electromagnetic waves;

polarizing means for p-polarizing the electromagnetic waves emitted by said source of electromagnetic waves;

means for directing said p-polarized electromagnetic waves through an optically denser medium onto said boundary surface to effect surface plasmon resonance whereby energy is coupled into said biomolecules, said boundary surface being between said optically denser medium and an optically rarer medium and having a first side adjacent to said optically denser medium and a second side adjacent to said optically rarer medium;

a first monitoring means for monitoring radiation reflected or generated by said biomolecules at said boundary surface to determine at least one of a presence and a concentration of said biomolecules;

an intensity detection means coupled to said first monitoring means for detecting an intensity of said electromagnetic waves reflected at said boundary surface;

a second monitoring means coupled to said intensity detection means for monitoring, based on said intensity, a shift in an angle of incidence at which surface plasmon resonance occurs (θ

_SPR); and

a control means coupled to said second monitoring means and coupled to said means for directing said p-polarized electromagnetic waves, said control means controlling the angle of incidence (θ

) of the impinging p-polarized electromagnetic waves depending on the intensity of the electromagnetic waves reflected at said boundary surface to substantially optimize the energy of the impinging p-polarized electromagnetic waves coupled into said biomolecules for said determination of said presence or concentration of said plurality of biomolecules.

30. The apparatus according to claim 29, wherein said second monitoring means comprises a detector, the detector being responsive only in the spectrum of said p-polarized electromagnetic waves.

31. The apparatus according to claim 29, wherein said intensity detection means comprises an array of detection elements, with at least a central detection element and at least a lateral detection element at either side of said central detection element, said second monitoring means monitoring said intensity detected at said detection elements and providing a control signal if the intensity detected by one of said lateral elements is lower than the intensity detected by said central monitoring element, said control signal being fed to said control means.

32. The apparatus according to claim 29, wherein said means for directing said impinging electromagnetic waves onto said boundary surface comprises a transparent prism.

33. The apparatus according to claim 32, wherein said transparent prism is mounted on a support rotatable by operating means, controlled by said control means, wherein said transparent prism is rotated an angle (Δ

θ

).

34. The apparatus according to claim 33, further comprising:

transmission means for rotating said support around angle (Δ

θ

) and for rotating at least one of said first and second monitoring means twice said angle (2Δ

θ

).