Long wave fluorophore sensor compounds and other fluorescent sensor compounds in polymers

• US 6,766,183 B2
• Filed: 12/28/2001
• Issued: 07/20/2004
• Est. Priority Date: 11/22/1995
• Status: Expired due to Term

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America), Medtronic Minimed Incorporated (Medtronic Plc), Regents of the University of California (University of California)
• Original Assignee: Medtronic Minimed Incorporated (Medtronic Plc), Regents of the University of California (University of California)
• Inventors: Peyser, Thomas A., Walsh, Joseph C., Satcher, Joe H. Jr., Lane, Stephen M., Noronha, Glenn, Mastrototaro, John Joseph, Heiss, Aaron M., Vachon, David J., Van Antwerp, William Peter
• Primary Examiner(s): WINAKUR, ERIC FRANK
• Application Number:
  US10/033,240
• Publication Number:
  US 20020193672A1
• Time in Patent Office: 935 Days
• Field of Search: 600/310, 600/312, 600/317, 600/322, 600/341, 600/342, 436/94, 436/95, 436/172, 422/82.07, 568/1, 546/13
• US Class Current: 600/317
• CPC Class Codes: A61B 5/0071 by measuring fluorescence emission
• CPC Class Codes: A61B 5/14532 for measuring glucose, e.g. by tissue impedance measurement
• CPC Class Codes: A61B 5/1455 using optical sensors, e.g. spectral photometrical oximeters
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/144444 Glucose

Abstract

Fluorescent biosensor molecules, fluorescent biosensors and systems, as well as methods of making and using these biosensor molecules and systems are described. Embodiments of these biosensor molecules exhibit fluorescence emission at wavelengths greater than about 650 nm. Typical biosensor molecules include a fluorophore that includes an iminium ion, a linker moiety that includes a group that is an anilinic type of relationship to the fluorophore and a boronate substrate recognition/binding moiety, which binds glucose. The fluorescence molecules modulated by the presence or absence of polyhydroxylated analytes such as glucose. This property of these molecules of the invention, as well as their ability to emit fluorescent light at greater than about 650 nm, renders these biosensor molecules particularly well-suited for detecting and measuring in-vivo glucose concentrations.

Claims

1. A fluorescent compound comprising:

a fluorophore including a charged moiety;

an anilinic group in relation to the fluorophore;

a boronate moiety capable of binding glucose;

a linker moiety that links the fluorophore to the boronate binding moiety;

wherein the compound exhibits an alteration in fluorescent properties when the boronate moiety is bound to glucose.

2. The compound of claim 1, wherein the compound emits a fluorescent signal in the visible to near infrared region of the spectrum that can be correlated to the presence of glucose in a substantially aqueous medium.

3. The compound of claim 1, wherein the compound has at least one maximum wavelength in an emission spectrum of the fluorophore that is between about 600 nm and about 800 nm.

4. The compound of claim 1, wherein the excitation wavelength for the fluorophore is greater than about 600 nm.

5. The compound of claim 1, wherein the compound is attached to a polymer matrix.

6. The compound of claim 5, wherein the polymer matrix includes polyethylene glycol.

7. The compound of claim 1, wherein the compound further includes one or more functional groups selected from the group consisting of at least an aliphatic, an aromatic, a haloalkane, an alcohol, an ether, an amine, an imine, an aldehyde, a ketone, an ester, a carboxylic acid, a sulfonic acid a cyano group, a phosphoric acid, and salts thereof.

8. The compound of claim 1, wherein the charged moiety is an minimum ion.

9. The compound of claim 1, wherein the fluorophore is Nile Blue.

10. An implantable biosensor, the biosensor comprising the biosensor compound of claim 1 contained in a polymer matrix.

11. The implantable biosensor of claim 10, wherein the polymer matrix is biocompatible, water-soluble and permeable to the glucose.

12. The implantable biosensor of claim 11, wherein the polymer matrix is selected from at least polyethylene glycol (amino-terminated), polyethylene glycol (hydroxy terminated), Jeffamine polymers (2-propyl amino terminated block-polypropylene oxide block-polyethylene oxide block-polypropylene oxide), poly(vinyl alcohol), poly(acrylic acid), and mixtures of these polymers.

13. The implantable biosensor of claim 10, wherein the biosensor is contained in a biocompatible, water-insoluble material that is permeable to glucose.

14. The biosensor of claim 13, wherein the water-insoluble material is selected from at least polyesters of carbonic acid, poly(vinylchloride), polyamides, polychloroethers, poly(urethanes), poly(imides) and mixtures of these materials.

15. The biosensor of claim 10, wherein the polymer matrix is a multiple attachment-point polymer.

16. The biosensor of claim 15, wherein the multiple attachment-point polymer is selected from at least cross-linked poly(vinyl alcohol), cross-linked poly(acrylic acid), star dendrimers and mixtures of these polymers.

17. The biosensor of claim 10, wherein the polymer matrix is polycarboxystyrene.

18. The biosensor of claim 16, wherein the polymer matrix includes a blend of polymers selected to affect the pH and/or solubility of the environmental milieu surrounding the biosensor molecule.

19. The biosensor of claim 16, wherein the polymer matrix includes grafted polymers.

20. A ditopic biosensor molecule, the biosensor molecule comprising:

a first biosensor molecule of claim 1;

a second biosensor molecule of claim 1; and

a bridging linker element, wherein the bridging linker element forms a bridge between the first and the second biosensor molecules and wherein the first and second biosensor molecules are the same or different.

21. The ditopic biosensor molecule of claim 20, wherein the ditopic biosensor binds glucose below about 100 mg/dL of analyte and emits a detectable emission signal that correlates to the glucose binding below about 100 mg/dL.

22. The ditopic biosensor molecule of claim 21, wherein the ditopic sensor also binds glucose up to about 1000 mg/dL of analyte and emits a detectable emission signal that correlates with glucose binding up to about 1000 mg/dL of analyte.

23. The ditopic biosensor molecule of claim 20, wherein the ditopic biosensor emits light at about 600 nm or greater.

24. The ditopic biosensor molecule of claim 20, further comprising a functional group capable of forming a covalent linkage to a polymer matrix.

25. The ditopic biosensor molecule of claim 24, wherein the ditopic biosensor molecule is attached to a polymer matrix.

26. The ditopic biosensor molecule of claim 20, wherein one of the biosensor molecules includes a Nile Blue fluorophore.

27. The ditopic biosensor molecule of claim 20, wherein the ditopic molecule emits a fluorescent signal at about 600 nm or greater.

28. The ditopic biosensor molecule of claim 20, wherein the bridging linker element includes 2-10 carbons.

29. A compound having a formula comprising:

wherein;

F is a fluorophore including an iminium ion;

B is a boron atom;

R¹ is selected from the group consisting of hydrogen, aliphatic and aromatic functional groups, wherein the functional group (R¹O)₂B is capable of binding glucose;

R², R³ and R⁴ are optional and independent functional groups selected from the groups consisting of hydrogen, aliphatic and aromatic functional groups, further functionalized aliphatic and aromatic groups, functional groups that alter the electronic properties of the group to which they are attached and functional groups that are capable of forming covalent linkages to a polymer matrix;

L¹ is an optional linking group having from zero to four atoms selected from the group consisting of nitrogen, carbon, oxygen, sulfur and phosphorous; and

Z is a heteroatom selected from the group consisting of nitrogen, phosphorous, sulfur, and oxygen;

wherein the heteroatom is in an anilinic-type relationship to the fluorophore;

wherein the fluorescence of the compound increases in the presence of bound glucose.

30. The compound of claim 29, wherein Z is nitrogen.

31. The biosensor molecule of claim 29, wherein R³ is a hydroxyl carbonyl, a carboxylic acid, carboxylate or an amine functional group.

32. The compound of claim 31, wherein the hydroxyl, carbonyl, a carboxylic acid, carboxylate or amine functional group includes an aliphatic linker with 2 or more carbon atoms.

33. The compound of claim 29, wherein the excitation wavelength for the fluorophore is greater than about 600 nm and wherein the compound is able to emit a fluorescent signal having a wavelength of greater than about 600 nm.

34. A fluorescent biosensor system for measuring levels of glucose, the biosensor system comprising:

the fluorescent compound of claim 29 attached to, or contained within, a polymer matrix to form the biosensor of the biosensor system;

an optical light source; and

a detector which detects a fluorescent signal, wherein the signal can be correlated to in-vivo levels of glucose.

35. The biosensor system of claim 34, wherein the biosensor can be subcutaneously implanted in a person'"'"'s body.

36. The biosensor system of claim 35, wherein an implanted biosensor can be transdermally excited by an excitation signal from the light source and in response to the excitation signal can emit a fluorescent signal that is detectable by the detector.

37. The biosensor system of claim 34, wherein the biosensor can be injected subcutaneously beneath a person'"'"'s skin.

38. The biosensor system of claim 34, further comprising an optical fiber for percutaneous implantation.

39. The biosensor system of claim 34, further comprising at least one biocompatible coating on the surface of the biosensor.

40. The biosensor of claim 39, further comprising an at least an adhesion coating, an angiogenic coating and mixtures thereof.

41. The biosensor system of claim 34, wherein the fluorescent compound includes a Nile Blue moiety.

42. A fluorescent compound of the general formula:

wherein;

F is a Nile Blue moiety;

B is a boron atom;

R¹ is selected from the group consisting of hydrogen, aliphatic and aromatic functional groups, wherein the functional group (R¹O)₂B is capable of binding glucose;

R², R³ and R⁴ are optional and independent functional groups selected from the groups consisting of hydrogen, aliphatic and aromatic functional groups, further functionalized aliphatic and aromatic groups, functional groups that alter the electronic properties of the group to which they are attached and functional groups that are capable of forming covalent linkages to a polymer matrix;

L¹ and L² are optional linking groups having from zero to four atoms selected from the group consisting of nitrogen, carbon, oxygen, sulfur and phosphorous; and

Z is a heteroatom selected from the group consisting of nitrogen, phosphorous, sulfur, and oxygen wherein the heteroatom is in an anilinic-type relationship to the Nile Blue moiety.

43. A method of quantifying the amount of glucose, the method comprising:

interrogating a subcutaneously implantable biosensor which includes biosensor molecules contained in or attached to a polymer matrix, with a light source to produce an excited state of the biosensor molecules that yield an emission signal that can be correlated to the amount of glucose in body fluids surrounding an implanted biosensor, wherein the biosensor molecules include an anilinic group and a fluorophore that includes an iminium ion;

detecting the emission signal; and

quantifying the amount of glucose from the emission signal.

44. The method of claim 43, wherein the biosensor molecules have an emission signal at about 650 nm or greater.

45. The method of claim 43, wherein the biosensor molecules comprise a molecule of the formula:

wherein;

F is the fluorophore including the iminium ion;

B is a boron atom;

R¹ is selected from the group consisting of hydrogen, aliphatic and aromatic functional groups, wherein the functional group (R¹O)₂B is capable of binding polyhydroxylate analyte;

R², R³ and R⁴ are optional and independent functional groups selected from the groups consisting of hydrogen, aliphatic and aromatic functional groups, further functionalized aliphatic and aromatic groups, functional groups that alter the electronic properties of the group to which they are attached and functional groups that are capable of forming covalent linkages to a polymer matrix;

L¹ is an optional linking group having from zero to four atoms selected from the group consisting of nitrogen, carbon, oxygen, sulfur and phosphorous; and

Z is a heteroatom selected from the group consisting of nitrogen, phosphorous, sulfur, and oxygen, wherein the heteroatom is in an anilinic-type relationship to the fluorophore and wherein the fluorescence of the biosensor molecule increases in the presence of bound glucose.

46. The method of claim 43, wherein quantifying the amount of glucose involves lifetime measurements of the biosensor molecules.