Optical determination of glucose utilizing boronic acid adducts

• US 20060083688A1
• Filed: 12/07/2005
• Published: 04/20/2006
• Est. Priority Date: 12/05/2000
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Singaram, Bakthan, Wessling, Ritchie A.
• Granted Patent: US 7,470,420 B2
• Time in Patent Office: Days
• US Class Current: 424/9.600
• CPC Class Codes: A61K 49/0019 characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07D 213/82 in position 3
• CPC Class Codes: C07F 5/025 Boronic and borinic acid compounds
• CPC Class Codes: C09K 2211/1007 Non-condensed systems
• CPC Class Codes: C09K 2211/1011 Condensed systems
• CPC Class Codes: C09K 2211/1029 containing one nitrogen atom as the heteroatom
• CPC Class Codes: C09K 9/02 Organic tenebrescent materials
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: Y10T 436/144444 Glucose

Abstract

The present invention concerns an improved optical method and optical sensing device for determining the levels of polyhydroxyl-substituted organic molecules in vitro and/or in vivo in aqueous media. The range of detection is between about 400 and 800 nm. In particular, a sensory devise is implemented in a mammal to determine sugar levels. Specifically, a dye is combined with a conjugated nitrogen-containing heterocyclic aromatic boronic acid-substituted bis-onium compound in the presence of a sugar, such as fructose or glucose. The viologens are preferred as the aromatic conjugated nitrogen-containing boronic acid substituted compounds. The method is useful to determine sugar levels in a human being.

Claims

1. A device for optically determining an analyte concentration, which device comprises:

an analyte permeable component;

a fluorophore associated with the analyte permeable component and configured to absorb light at a first wavelength and emit light at a second wavelength;

a quencher associated with the analyte permeable component and configured to modify the light emitted by the fluorophore by an amount related to the analyte concentration, wherein the quencher comprises a boronic acid substituted viologen;

a light source; and

a detector.

2. The device of claim 1, wherein the analyte permeable component comprises a polymer matrix which has long term compatability and stability in the presence of an in vivo physiological fluid.

3. The device of claim 2, wherein the polymer matrix is a hydrogel.

4. The device of claim 3, wherein the hydrogel is formed by polymerization of hydrophilic monomers or by cross-linking hydrophilic polymers.

5. The device of claim 4, wherein the hydrophilic monomers are selected from the group consisting of 2-hydroxyethyl-methacrylate, polyethylene glycol methacrylate, methacrylic acid, hydroxyethyl acrylate, N-vinyl pyrrolidone, acrylamide, N,N′

-dimethyl acrylamide, methacrylaminopropyl trimethylammonium chloride, diallyl dimethyl ammonium chloride, sodium sulfopropyl methacrylate, optionally having cross-linkers selected from ethylene dimethacrylate, PEGDMA, methylene-bis-acrylamide and trimethylolpropane triacrylate, and combinations thereof.

6. The device of claim 2, wherein the polymer matrix is insoluble in water.

7. The device of claim 6, wherein the water-insoluble polymer matrix is prepared from monomers selected from the group consisting of HPTS(Lys-MA)3, HPTS-MA, HPTS-CO₂-MA, APTS-BuMA, and APTS-DegMA.

8. The device of claim 7 wherein the water-insoluble polymer matrix comprises copolymers HEMA and polyethylene glycol dimethacrylate or N,N′

-dimethylacrylamide and methylene-bis-acrylamide.

9. The device of claim 1, wherein the analyte permeable component comprises a membrane, which confines the fluorophore and the quencher.

10. The device of claim 1, wherein the fluorophore comprises a substituted pyrene.

11. The device of claim 10, wherein the substituted pyrene comprises a pyranine sulfonate derivative.

12. The device of claim 11, wherein the pyrene is a pyrene sulfonate derivative is selected from the structure:

13. The device of claim 1, wherein the boronic acid substituted viologen further comprises an aromatic boronic acid moiety.

14. The device of claim 13, wherein the quencher is prepared from a precursor selected from the group consisting of:

15. The device of claim 1, wherein the viologen is prepared from a precursor selected from the group consisting of:

16. The device of claim 1, wherein the quencher is configured to bind an amount of the analyte.

17. The device of claim 15, wherein the quencher is configured to reduce the light emitted by the fluorophore.

18. The device of claim 17, wherein the quencher is further configured to reduce the light emitted by the fluorophore by an amount inversely related to the amount of bound analyte.

19. The device of claim 1, wherein the analyte comprises a polyhydroxyl-substituted organic molecule.

20. The device of claim 19, wherein the analyte is glucose.

21. The device of claim 1, wherein the light source is a blue light emitting diode (LED).

50. The device for optically determining an analyte concentration of claim 49 which is further defined by claims 2, 3, 5, 6, 7, and 8 to 20.

53. The device of claim 1 wherein the analyte permeable component includes quantum dot moieties.

54. The device of claim 1 wherein the analyte permeable component is a polymer independently selected from the group consisting of HEMA, PEGMA, methacrylic acid, hydroxyethyl acrylate, N-vinyl pyrrolidone, acrylamide, N,N′

-dimethyl acrylamide, methacryloylaminopropyl trimethylammonium chloride, diallyl dimethyl ammonium. chloride, vinyl benzyl trimethyl ammonium chloride, sodium sulfopropyl methacrylate with crosslinkers including ethylene dimethacrylate, PEGDMA, methylene bis acrylamide, trimethylolpropane triacrylate, and combinations thereof.

56. The device of claim 1 wherein the fluorophore is present having at least one negative charge.

22. A device for optically determining an analyte concentration in a physiological fluid, comprising:

an analyte permeable component;

a fluorophore associated with the analyte permeable component and configured to absorb light at a first wavelength and emit light at a second wavelength, wherein the fluorophore comprises a substituted pyrene,

a quencher associated with the analyte permeable component and configured to modify the light emitted by the fluorophore by an amount related to the analyte concentration;

a light source; and

a detector.

23. An analyte sensor, comprising:

a fluorophore configured to absorb light at a first wavelength and emit light at a second wavelength; and

a quencher configured to modify the light emitted by the fluorophore by an amount related to the analyte concentration, wherein the quencher comprises a boronic acid substituted viologen.

24. The analyte sensor of claim 23, wherein the fluorophore comprises a substituted pyrene.

25. The analyte sensor of claim 24, wherein the fluorophore is a pyrene sulfonate derivative is selected from the structures:

26. An analyte sensor, comprising:

a fluorophore dye comprising a pyrene derivative configured to absorb light at a first excitation wavelength and emit light at a second emission wavelength; and

a quencher configured to bind an analyte, wherein the quencher is operably coupled to the fluorophore dye, and wherein the quencher is configured to modulate the light emitted by the fluorophore dye in relation to the binding of the analyte.

27. A method for optically determining the concentration of an analyte in a sample, which method comprises:

contacting the analyte sensor of claim 26 with the sample;

applying light to the sensor;

detecting emitted light; and

determining the concentration of the analyte.

28. The method of claim 27, wherein the concentration of analyte is determined continuously over a period of time.

29. The method of claim 28, wherein the sample is a fluid.

30. The method of claim 29, wherein the sample is a physiological fluid.

31. The method of claim 30, wherein the sample is a physiological fluid in a living mammal.

32. The method of claim 27, wherein the light is applied at a first excitation wavelength.

33. The method of claim 32, wherein the light is applied by a light emitting diode (LED).

34. The method of claim 27, wherein the light is detected at a second emission wavelength.

35. The method of claim 27, wherein the light is applied substantially continuously.

36. The method of claim 27, wherein the light is applied periodically.

37. The method of claim 27, wherein the analyte comprises a polyhydroxyl-substituted organic molecule.

38. The method of claim 37, wherein the polyhydroxyl-substituted organic molecule is glucose.

39. The method of claim 27, wherein the contacting step further comprises implanting the analyte sensor subcutaneously.

40. The method of claim 27, wherein the contacting step further comprises implanting the analyte sensor within a blood vessel.

41. The method of claim 40, wherein the blood vessel is an artery.

42. The method of claim 40, wherein the blood vessel is a vein.

43. The method of any of claims 39 to 42, wherein the analyte sensor is implanted in a human.

44. The method of claim 27, wherein the analyte sensor further comprises a biocompatible coating.

48. The method of claim 47 where it is further defined by any of claims 37, 38, 40, 43 and 44.

55. The method of claim 27 wherein the viologen comprises two or more boronic acid moieties.

45. A method of making an analyte sensor, comprising:

reacting a dipyridyl with an alkylating agent comprising an arylboronic acid to produce a N,N′

-bis-benzylboronic acid viologen; and

operably coupling the N,N′

-bis-benzylboronic acid viologen to a fluorophore capable of being quenched by a viologen.

46. The method of claim 45, wherein said alkylating agent is a halomethylphenylboronic acid wherein halo is chloro or bromo.

47. An optical method for the in vivo detection of polyhydroxyl-substituted organic molecules as the analyte between about 400 and 800 nm detection, which method comprises:

A. Obtaining a fluorophore dye D, which is compatible with the analyte solution, wherein D is selected from;

(a) D¹ which is a fluorophore dye having the properties of

i. A fluorophore,

ii. An excitation in the range greater than 400 nm and less than 800 nm,

iii. Resistant to photobleaching under the conditions of analysis,

iv. A Stokes shift of about or greater than 30 nm,

v. Compatibility with said analyte solution, and wherein said

vi. Dye D¹ is quenched by methyl viologen to produce an experimentally determined apparent Stern-Volmer quenching constant (Ksv) greater than or equal to 50,

wherein the fluorophore dye D¹ which is neutral or negatively charged is;

(i) a discrete compound having a molecular weight of 1,000 daltons or greater, with the proviso that if the dye is substituted with negatively charged groups the molecular weight is 500 daltons or greater;

(ii) a pendant group or chain unit in a water-soluble or dispersible polymer having a molecular weight greater than about 10,000 daltons, and

optionally said polymer is non-covalently associated with a water-insoluble polymer matrix M¹ and is physically immobilized within said polymer matrix M¹

wherein

said polymer matrix M¹ is permeable to or in contact with said analyte solution; and

optionally where dye D¹ is negatively charged and the polymer is immobilized as a complex with a cationic water-soluble polymer, said complex formed is permeable to or

in contact with said analyte solution;

(b) D² is a fluorophore dye having the properties of

i. A fluorophore,

ii. An excitation in the range greater than 400 nm and less than 800,

iii. A Stokes shift of about or greater than 30 nm,

iv. Resistant to photobleaching under the conditions of analyses,

v. Compatibility in the analyte solution, and wherein

vi. Said Dye D² is quenched by methyl viologen to produce an apparent Stern-Volmer quenching constant (Ksv) greater than or equal to 50, wherein D² is covalently bonded to an insoluble polymer matrix M¹ wherein said polymer matrix M¹ is permeable to or in contact with said analyte;

wherein said fluorophore dye D² is a part of the structure;

M¹-L¹-D² with the proviso that D² which is polyfunctional is bonded to matrix M¹ at one, two or three sites;

L¹ is a hydrolytically stable covalent linking group selected from the group consisting of a direct bond, lower alkylene having 1 to 8 carbon atoms optionally terminated with or including one or more divalent connecting groups selected from sulfonamide, amide, ester, ether, sulfide, sulfone, phenylene, urethane, urea, and amine, and

B. Combining with a boronic acid-containing quencher moiety Q, wherein Q is comprised of a conjugated nitrogen-containing heterocyclic, aromatic bis-onium salt having the properties of compatibility in said analyte solution and produces a detectable change in the emission of the fluorophore dye (D¹ or D²) in the presence of said analyte, selected from;

(i) quencher Q¹ which is a discrete compound having a molecular weight of about 400 daltons or greater or is a pendant group or a chain unit in a water-soluble or water-dispersible polymer having a molecular weight greater than 10,000 daltons and said polymer optionally is non-covalently associated with the optional polymer matrix M¹ when present, and is physically immobilized in said polymer matrix, or optionally said polymer is immobilized as a complex with a negatively charged water-soluble polymer, or

(ii) quencher Q² which is covalently bonded by linking group L² to M¹ or to a second water insoluble polymer matrix M² producing M²-L²-Q² wherein L² is selected from the group consisting of a direct bond, a lower alkylene having 1 to 8 carbon atoms optionally terminated with or including one or more divalent connecting groups selected from sulfonamide, amide, quaternary ammonium, pyridinium, ester, ether, sulfide, sulfone, phenylene, urea, thiourea, and urethane, or amine, wherein said quencher Q¹ or Q² is mixed at a molecular level with said fluorophore dye D¹ or D², and with the proviso that Q² when polyfunctional is linked to the matrix M² at one or two sites,

C. contacting a physiological fluid which contains analyte, with a dye and a quencher in vivo with an excitation light source coupled with a detector;

D. producing a detectable and quantifiable signal in the range of about 400 to 800 nm; and

E. determining the concentration of said polyhydroxyl-substituted analyte in said physiological fluid.

49. An optical device for the in vivo detection of polyhydroxyl-substituted organic molecules as the analyte between about 400 and 800 nm detection, which device comprises:

A. a fluorophore dye D, which is compatible with the analyte solution, wherein D is selected from;

(a) D¹ which is a fluorophore dye having the properties of

i. A fluorophore,

ii. An excitation in the range greater than 400 nm and less than 800 nm,

iii. Resistant to photobleaching under the conditions of analysis,

iv. A Stokes shift of about or greater than 30 nm,

v. Compatibility with said analyte solution, and wherein said

vi. Dye D¹ is quenched by methyl viologen to produce an experimentally determined apparent Stern-Volmer quenching constant (Ksv) greater than or equal to 50,

wherein the fluorophore dye D¹ which is neutral or negatively charged is;

(i) a discrete compound having a molecular weight of 1,000 daltons or greater, with the proviso that if the dye is substituted with negatively charged groups the molecular weight is 500 daltons or greater;

(ii) a pendant group or chain unit in a water-soluble or dispersible polymer having a molecular weight greater than about 10,000 daltons, and

optionally said polymer is non-covalently associated with a water-insoluble polymer matrix M¹ and is physically immobilized within said polymer matrix M¹

wherein

said polymer matrix M¹ is permeable to or in contact with said analyte solution; and

optionally where dye D¹ is negatively charged and the polymer is immobilized as a

complex with a cationic water-soluble polymer, said complex formed is permeable to or in contact with said analyte solution;

(b) D² is a fluorophore dye having the properties of

i. A fluorophore,

ii. An excitation in the range greater than 400 nm and less than 800,

iii. A Stokes shift of about or greater than 30 nm,

iv. Resistant to photobleaching under the conditions of analyses,

v. Compatibility in the analyte solution, and wherein

vi. Said Dye D² is quenched by methyl viologen to produce an apparent Stern-Volmer quenching constant (Ksv) greater than or equal to 50, wherein D² is covalently bonded to an insoluble polymer matrix M¹ wherein said polymer matrix M¹ is permeable to or in contact with said analyte;

wherein said fluorophore dye D² is a part of the structure;

M¹-L¹-D² with the proviso that D² which is polyfunctional is bonded to matrix M¹ at one, two or three sites;

L¹ is a hydrolytically stable covalent linking group selected from the group consisting of a direct bond, lower alkylene having 1 to 8 carbon atoms optionally terminated with or including one or more divalent connecting groups selected from sulfonamide, amide, ester, ether, sulfide, sulfone, phenylene, urethane, urea, and amine, and

B. a boronic acid-containing quencher moiety Q, wherein Q is comprised of a conjugated nitrogen-containing heterocyclic, aromatic bis-onium salt having the properties of compatibility in said analyte solution and produces a detectable change in the emission of the dye in the presence of said analyte, selected from;

(i) quencher Q¹ which is a discrete compound having a molecular weight of about 400 daltons or greater or is a pendant group or a chain unit in a water-soluble or water-dispersible polymer having a molecular weight greater than 10,000 daltons and said polymer optionally is non-covalently associated with the optional polymer matrix M¹ when present, and is physically immobilized in said polymer matrix, or optionally said polymer is immobilized as a complex with a negatively charged water-soluble polymer, or

(ii) quencher Q² which is covalently bonded by linking group L² to M¹ or to a second water insoluble polymer matrix M² producing M²-L²-Q² wherein L² is selected from the group consisting of a direct bond, a lower alkylene having 1 to 8 carbon atoms optionally terminated with or including one or more divalent connecting groups selected from sulfonamide, amide, quaternary ammonium, pyridinium, ester, ether, sulfide, sulfone, phenylene, urea, thiourea, and urethane, or amine, wherein said quencher Q¹ or Q² is mixed at a molecular level with said fluorophore dye D¹ or D², and with the proviso that Q² when polyfunctional is linked to the matrix M² at more than one site,

wherein when a dye and a quencher in contact with physiological fluid which contains an analyte in vivo is contact with an excitation light source coupled with a detector;

C. produces a detectable and quantifiable signal in the range of about 400 to 800 nm; and

D. determines the concentration of said polyhydroxyl-substituted analyte,

wherein the Dye D components and quencher Q components are immobilized in or attached to a polymer matrix M¹, M² or combinations thereof and

said device measures the concentration of polyhydroxyl-containing molecules periodically or continuously.

51. A composition of matter selected from:

52. The composition of matter of claim 51 selected from: