OPTICAL BIOSENSORS

• US 20110159519A1
• Filed: 01/24/2008
• Published: 06/30/2011
• Est. Priority Date: 01/24/2007
• Status: Active Grant

• Current Assignee: Carnegie Mellon University
• Original Assignee: Carnegie Mellon University
• Inventors: Bruchez, Marcel P., Jarvik, Johathan W., Schmidt, Brigitte F., Szent-Gyorgyi, Christopher S., Waggoner, Alan S., Berget, Peter B.
• Granted Patent: US 8,664,364 B2
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0023 Di-or triarylmethane dye xanthene dyes A61K49/0041
• CPC Class Codes: A61K 49/0032 Methine dyes, e.g. cyanine dyes
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: A61K 49/0058 Antibodies
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Provided are biosensors, compositions comprising biosensors, and methods of using biosensors in living cells and organisms. The biosensors are able to be selectively targeted to certain regions or structures within a cell. The biosensors may provide a signal when the biosensor is targeted and/or in response to a property of the cell or organism such as membrane potential, ion concentration or enzyme activity.

Claims

1. A ligand-dye complex, comprising a cognate ligand of a dye, wherein a ten fold increase in a detectable fluorescence signal occurs upon non covalent binding of the dye to the cognate ligand.

2. The ligand-dye complex of claim 1, wherein the cognate ligand is a protein.

3. The ligand-dye complex of claim 2, wherein the cognate ligand is an antibody.

4. The ligand-dye complex of claim 3, wherein the antibody is an scFv antibody.

5. The ligand-dye complex of claim 1, wherein the dye is stimulated to fluoresce upon binding the ligand, said complex having an extinction coefficient of greater than 30,000 and a maximum absorption at a wave length of greater than 350 nm.

6. The ligand-dye complex of claim 1, wherein the dye emits photons and the polarization of the emitted photons changes upon binding the ligand.

7. The ligand-dye complex of claim 6 wherein the polarization change is greater than or equal to 20%.

8. The ligand-dye complex of claim 1, wherein the emission wave length of the dye increases by at least 10 nm upon binding the ligand.

9. The ligand-dye complex of claim 1, wherein the dye is protonated or deprotonated upon binding the ligand to produce the increase in fluorescence signal.

10. The ligand-dye complex of claim 1, wherein the dye fluorescence increases in response to association with the hydrophobic microenvironment of the cognate ligand upon binding to the cognate ligand.

11. The ligand-dye complex of claim 1, wherein the dye is a single methine dye.

12. The ligand-dye complex of claim 11 wherein the dye is selected from the group consisting of a mono-methin cyanine dye, a malachite green dye, a thiazole orange dye, a merocyanine dye, a styryl dye and a mono-methin oxynol dye or analogs thereof.

13. The ligand-dye complex of claim 1 wherein the dye has the general structure

A-B=A′

wherein A is selected from

14. The ligand-dye complex of claim 1 wherein the dye is selected from the group consisting of

15. The ligand-dye complex of claim 13 further comprising at least one of a linker, a photoreactive group, and a reactive group.

16. The ligand-dye complex of claim 15 wherein said reactive group contains isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, phosphoramidite, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, axidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal, haloacetamido, or aldehyde.

17. The ligand-dye complex of claim 1, wherein the dye is a multi-methine dye.

18. The ligand-dye complex of claim 1, wherein the ligand reversibly binds a monomethin cyanine dye.

19. The ligand-dye complex of claim 1, wherein the ligand reversibly binds Malachite Green or an analog thereof.

20. The ligand-dye complex of claim 1, wherein the ligand reversibly binds thiazole orange or an analog thereof.

21. The ligand-dye complex of claim 1, wherein the ligand is fixed to a substrate.

22. The ligand-dye complex of claim 21, wherein the substrate comprises molecules of formula X(a)-R(b)-Y(c), wherein R is a spacer, X is a functional group that binds R to a surface, Y is a functional group for binding to the ligand, (a) is an integer from 0 to about 4, (b) is an integer from 0 or 1, and (c) is an integer not equal to 0.

23. The ligand-dye complex of claim 21, wherein the substrate comprises a tiled array.

24. An antibody capable of binding a dye as recited in claim 13.

25. The antibody of claim 24, wherein the antibody comprises a polypeptide sequence having at least 85% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

7, SEQ ID NO;

9, SEQ. ID NO. 11, SEQ. ID NO. 13, SEQ. ID NO. 15, SEQ. ID NO. 17, SEQ. ID NO. 19, and SEQ. ID NO. 21.

25-1. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

35. A method of analyzing a region of a cell of interest with a fluorescent dye, comprising:

(a) expressing a ligand in the region of the cell of interest; and

(b) contacting the cell of interest with a dye comprising a moiety that is specific for the ligand;

(c) incubating the cell of interest after said contacting under conditions sufficient to allow the dye to interact with the ligand, wherein interaction of the dye with the ligand produces a wherein a ten fold increase in a detectable fluorescence signal of the dye; and

(d) detecting the signal of the dye.

36. (canceled)