Immunoassays involving surface enhanced Raman scattering

• US 6,750,065 B1
• Filed: 11/20/2000
• Issued: 06/15/2004
• Est. Priority Date: 02/26/1998
• Status: Expired due to Term

• Current Assignee: Renishaw Diagnostics Limited (Renishaw PLC)
• Original Assignee: University of Strathclyde
• Inventors: White, Peter Cyril, Sadler, Daran Antony, Athmani, Salah, Smith, William Ewen
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): CHEU, CHANGHWA J
• Application Number:
  US09/623,033
• Time in Patent Office: 1,303 Days
• Field of Search: 435/7.95, 435/7.98, 435/6, 436/164, 436/173, 436/518, 436/525, 436/801, 436/805, 436/536, 436/541
• US Class Current: 436/518
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/587 Nanoparticles
• CPC Class Codes: G01N 33/946 CNS-stimulants, e.g. cocaine, amphetamines
• CPC Class Codes: Y10S 436/801 Electron dense compounds, e.g. ferritin
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Immunoassay displacement methods for detecting (preferably identifying) the presence or amount of a target analyte (e.g. drug) in a sample comprising the steps of: (a) exposing the sample to a complex comprising an immobilized antibody capable of specifically binding the analyte, said antibody being specifically bound to a displacement agent, wherein said agent comprises: (I) an analyte analog capable of specifically binding the antibody, (ii) a surface enhanced resonance raman scattering (SERRS) or a surface enhanced raman scattering (SERS) active label, (iii) a SERRS or SERS surface-seeking group (e.g. benztriazole), whereby any analyte present in the sample causes the displacement of agent from the antibody; (b) exposing any displaced agent to a SERRS or SERS surface; and, (c) detecting any displaced agent associated with said surface using SERRS or SERS. In preferred forms of the invention several target analytes are detected from a single source or sample simultaneously. Also provided are agents per se e.g. for use in drug or explosive detection. These may include chromophores and linking groups.

Claims

1. A method for detecting the presence or amount of a target analyte in a sample comprising the steps of:

(a) exposing the sample to a complex comprising an immobilized antibody capable of specifically binding the analyte, said antibody being specifically bound to a displacement agent, wherein said agent comprises;

(i) an analyte analog portion, capable of specifically binding the antibody,

(ii) a label, which has surface enhanced Raman scattering (SERS) activity, or surface enhanced resonance Raman scattering (SERRS) activity,

(iii) a SERRS or SERS surface-seeking group,

whereby the analyte if present in the sample causes the displacement of the agent from the antibody;

(b) exposing the agent displaced from the antibody by the analyte to a SERRS or SERS surface such that said displaced agent becomes adsorbed on said surface; and

(c) detecting displaced agent associated with said surface using SERRS or SERS.

2. A method as claimed in claim 1 wherein the method is used to identify the analyte in the sample.

3. A method as claimed in claim 1 wherein more than one target analyte is detected from the sample simultaneously.

4. A method as claimed in claim 3 wherein multiple antibodies are used, each of said multiple antibodies is specific for a target analyte or class of analytes, and wherein each of said multiple antibodies is respectively bound to a corresponding agent having a distinctive SERRS or SERS spectrum .

5. A method as claimed in claim 4 wherein the SERRS or SERS spectrum of the agent is dependent on inherent properties of the analyte analog in said agent.

6. A method as claimed in claim 5 wherein the SERRS or SERS surface is a metal surface and the analyte analog portion of the agent is brought into close proximity to the metal SERRS or SERS surface during detection in step (c).

7. A method as claimed in claim 4 wherein the multiple antibodies are cross-specific for the multiple target analytes, and each agent comprises the same analyte analog and wherein the distinctive SERRS or SERS spectrum of each agent is dependent on a distinctive label present in said agent.

8. A method as claimed in claim 1 wherein a full SERRS or SERS spectrum across a range of wavelengths is obtained in step (c).

9. A method as claimed in claim 1 wherein the SERRS or SERS surface is an aggregation of metal colloid particles, wherein the metal is selected from silver, gold or copper.

10. A method as claimed in claim 9 wherein the metal comprises an organic coating selected from the group consisting of citrate;

a polyamine and polyphenol.

11. A method as claimed in claim 1 wherein the antibody is immobilised in a glass or plastic capillary tube.

12. A method as claimed in claim 11 wherein multiple antibodies are present in two or more capillary tubes linked together in series.

13. A method as claimed in claim 1 wherein the antibody is immobilised on a bead or microtitre plate.

14. An agent for use in a method as claimed in claim 1, the agent being capable of specifically binding an antibody raised to an analyte and being SERRS or SERS active, wherein said agent comprises an analyte-analog portion and a SERRS or SERS surface-seeking group.

15. An agent as claimed in claim 14 wherein the analyte analog portion is selected from the group consisting of a drug or pharmaceutical or metabolite thereof;

an explosive;

a pesticide; and

an environmental pollutant.

16. An agent as claimed in claim 15 wherein the drug is selected from the group consisting of amphetamines, opiates, benzodiazepines, barbiturates, cannabinoids, cocaine, lysergic acid diethylamide and their metabolites.

17. An agent as claimed in claim 14 wherein the SERRS or SERS spectrum of the agent is dependent on inherent properties of the analyte analog in the agent.

18. An agent as claimed in claim 17 wherein the analyte-analog portion of the agent is derivitised to the surface seeking group either directly or via an azo group.

19. An agent as claimed in claim 18 wherein the agent contains at least one of a chromophore and a fluorophore to enhance the SERRS or SERS activity of the agent.

20. An agent as claimed in claim 19 comprising an azo group.

21. An agent as claimed in claim 14 wherein the agent contains a linking group.

22. An agent as claimed in claim 21 wherein the linking group alters the resonance frequency of the agent.

23. An agent as claimed in claim 22 wherein the linking group is dimethoxyaniline.

24. An agent as claimed in claim 14 wherein the SERRS or SERS surface-seeking group is capable of immobilizing the agent to the SERRS or SERS surface.

25. An agent as claimed in claim 14 wherein the SERRS or SERS surface-seeking group comprises a heterocyclic compound which contains at least two heteroatoms which may be the same or different selected from:

nitrogen, sulphur or phosphorous.

26. An agent as claimed in claim 25 wherein the SERRS or SERS surface-seeking group is the benzotriazole group or a derivative thereof.

27. An agent as claimed in claim 14 wherein the SERRS or SERS surface-seeking group comprises a group selected from any one of formulae A1 to A25 as shown in FIG. 6A.

28. An agent as claimed in claim 27 wherein the SERRS or SERS surface-seeking group comprises a group selected from either formula A5 or formula A6 as shown in FIG. 6A.

29. An agent as claimed in claim 14 bound to an antibody.

30. An agent as claimed in claim 14 bound to a SERRS or SERS surface via a SERRS or SERS surface-seeking group.

31. A method as claimed in claim 4, wherein each antibody is immobilised in a glass or plastic capillary tube.

32. A method as claimed in claim 4, wherein each antibody is immobilised on a bead or microtitre plate.