Reagent for detecting an analyte

• US 7,892,748 B2
• Filed: 12/14/2004
• Issued: 02/22/2011
• Est. Priority Date: 12/16/2003
• Status: Active Grant

• Current Assignee: Medtronic Minimed Incorporated (Medtronic Plc)
• Original Assignee: Precisense A/S (Medtronic Plc)
• Inventors: Laursen, Bo Wegge, Norrild, Jens Christian
• Primary Examiner(s): Marx; Irene
• Assistant Examiner(s): HANLEY, SUSAN MARIE
• Application Number:
  US10/582,794
• Publication Number:
  US 20070148652A1
• Time in Patent Office: 2,261 Days
• Field of Search: 435/46, 435/7.1, 552/101
• US Class Current: 435/7.1
• CPC Class Codes: C07C 309/14 containing amino groups bound to the carbon skeleton
• CPC Class Codes: C07D 207/404 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C09B 11/10 Amino derivatives of triarylmethanes
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

A reagent for use in detecting an analyte comprises a fluorescent energy donor and an energy acceptor, wherein the energy acceptor is of the general formula:

and wherein the distance between the energy donor and the energy acceptor of the reagent is capable of modulation by a suitable analyte to be detected.

Claims

1. A reagent for use in detecting an analyte, comprising a fluorescent energy donor, an energy acceptor and optionally a counterion, the energy donor and the energy acceptor being such that when they are sufficiently close to one another energy is non-radiatively transferred from the energy donor following excitation thereof to the energy acceptor quenching fluorescence of the energy donor, wherein the energy acceptor is of the formula:

2. A reagent as claimed in claim 1, wherein the energy donor and energy acceptor are linked together by a covalent linkage.

3. A reagent as claimed in claim 2, wherein the covalent linkage between the energy donor and energy acceptor is cleavable to increase the distance between the energy donor and the energy acceptor of the reagent.

4. A reagent as claimed in claim 2, wherein the energy donor and energy acceptor are linked via a polynucleotide sequence or a polynucleotide analogue sequence or a polypeptide sequence, the sequence having a conformation which is capable of modulation by a suitable analyte to be detected so as to modulate the distance between the energy donor and the energy acceptor of the reagent.

5. A reagent as claimed in claim 1, wherein the energy donor and energy acceptor are linked together by non-covalent binding.

6. A reagent as claimed in claim 5 wherein the non-covalent binding exists between an analyte binding agent linked to one of the energy donor and the energy acceptor and an analyte analogue linked to the other of the energy donor and the energy acceptor, the non-covalent binding being disruptable by a suitable analyte so as to increase the distance between the energy donor and the energy acceptor of the reagent.

7. A reagent as claimed in claim 6, wherein the analyte binding agent is a lectin.

8. A reagent as claimed in claim 6, wherein the analyte analogue is a glucose analogue.

9. A reagent as claimed in claim 8, wherein the analyte analogue is dextran.

10. A reagent as claimed in claim 1, wherein a linker structure is attached to the energy acceptor at R³, or where a bridging group is present optionally the linker structure is attached to the energy acceptor at the bridging group.

11. A reagent as claimed in claim 1, wherein the electron donating substituents are selected from amino, primary amine, secondary amine, O-alkyl, alkyl, S-alkyl, amide, ester, OH and SH.

12. A reagent as claimed in claim 11, wherein one or more of R¹ to R³ is dimethylamino, diethylamino or methylethylamino, any of those groups being optionally substituted with one or more of SO₃⁻

, PO₃ ²⁻

, OH, O-alkyl, SH, S-alkyl, COOH, COO⁻

, ester, amide, halogen, SO-alkyl, SO₂-alkyl, SO₂NH₂, SO₂NH-alkyl, SO₂N-dialkyl, SO₃-alkyl, CN, secondary amine or tertiary amine.

13. A reagent as claimed in claim 1, wherein an electron withdrawing substituent is present, and the electron withdrawing substituent is selected from NO, NO₂, CN, COOH, ester, COO⁻

, amide, CHO, keto, SO-alkyl, SO₂-alkyl, SO₂NH₂, SO₂NH-alkyl, SO₂N-dialkyl, and SO₃-alkyl.

14. A reagent as claimed in claim 1, wherein one or more pairs of groups R⁴ and R¹⁰ and/or R⁵ and R¹¹ and/or R⁶ and R¹² and/or R⁷ and R¹³ and/or R⁸ and R¹⁴ and/or R⁹ and R¹⁵ is a bridging group consisting of alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO₃⁻

, PO₃²⁻

, OH, O-alkyl, SH, S-alkyl, COOH, COO⁻

, ester, amide, halogen, SO-alkyl, SO₂-alkyl, SO₂NH₂, SO₂NH-alkyl, SO₂N-dialkyl, SO₃-alkyl, CN, secondary amine or tertiary amine.

15. A reagent as claimed in claim 1, wherein R¹⁰ to R¹⁵ are each O-methyl or O-ethyl.

16. A reagent as claimed in claim 1, further comprising one or more counterions selected from halide, BF₄⁻

, PF₆⁻

, NO₃⁻

, carboxylate, ClO₄⁻

, Li⁺, Na⁺, K⁺, Mg²⁺ and Zn²⁺.

17. A reagent as claimed in claim 1, wherein a linker structure is present, and is formed by reaction of a linker element selected from an active ester, an isothiocyanate, an acid chloride, an aldehyde, an azide, an a-halogenated ketone and an amine with a reaction partner.

18. A reagent as claimed in claim 17, wherein the reaction partner is selected from a polysaccharide, a polynucleotide and a protein.

19. A reagent as claimed in claim 17, wherein the linker element is an active ester, and is selected from succinimidyl and pentafluorophenyl active esters.

20. A reagent as claimed in claim 1, wherein the energy donor is a dye that absorbs at 594 nm and fluoresces at 620 nm.

21. A reagent as claimed in claim 1, in which:

R¹ and R² are both NMe₂ or both MeNCH₂CH₂SO₃⁻

R³ is a linker element which is a succinimidyl ester

R^4-9 are all H

R^10-15 are all OMe;

the energy donor and energy acceptor are linked together by non-covalent binding which exists between an analyte binding agent linked to one of the energy donor and the energy acceptor and an analyte analogue linked to the other of the energy donor and the energy acceptor, the non-covalent binding being disruptable by the analyte so as to increase the distance between the energy donor and the energy acceptor of the reagent.

22. A reagent as claimed in claim 21, wherein the analyte is glucose.

23. A reagent as claimed in claim 22, wherein the analyte analogue is dextran.

24. A reagent as claimed in claim 22, wherein the analyte binding agent is a lectin.

25. A reagent as claimed in claim 21, wherein the energy donor is a dye that absorbs at 594 nm and fluoresces at 620 nm.

26. A method of detecting or measuring an analyte with a reagent as claimed in claim 1, comprising the steps of:

contacting the reagent with a sample;

illuminating the reagent and sample with light of wavelength within the absorption spectrum of the fluorescent energy donor;

detecting non-radiative energy transfer between the energy donor and energy acceptor by measuring the fluorescence of the energy donor; and

correlating the fluorescence measurements with the presence or concentration of the analyte.

27. A method as claimed in claim 26, wherein the fluorescence of the energy donor is measured by making intensity based or time resolved fluorescence measurements.

28. A method as claimed in claim 26, wherein the analyte is measured by comparing sample fluorescence measurements with fluorescence measurements made using known concentrations of analyte.

29. A dye compound having the formula:

30. A dye compound as claimed in claim 29, wherein at least one of R¹⁰, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ is alkyl.

31. A dye compound as claimed in claim 30, wherein one or more pairs of groups R⁴ and R¹⁰ and/or R⁵ and R¹¹ and/or R⁶ and R¹² and/or R⁷ and R¹³ and/or R⁸ and R¹⁴ and/or R⁹ and R¹⁵ is a bridging group consisting of alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO₃, PO₃²⁻

, OH, O-alkyl, SH, S-alkyl, COOH, COO³¹ , ester, amide, halogen, SO-alkyl, SO₂NH₂, SO₂NH-alkyl, SO₂N-dialkyl, SO₃-alkyl, CN, secondary amine or tertiary amine.

32. A dye compound as claimed in claim 29, wherein R¹⁰ to R¹⁵ are each O-methyl or O-ethyl.

33. A dye compound as claimed in claim 29, further comprising one or more counterions selected from halide, BF₄⁻

, PF₆⁻

, NO₃⁻

, carboxylate, ClO₄⁻

, Li⁺, Na⁺, K⁺, Mg²⁺ and Zn²⁺.