Polyhydroxyl-substituted organic molecule sensing optical in vivo method utilizing a boronic acid adduct and the device thereof

• US 20020106326A1
• Filed: 12/05/2000
• Published: 08/08/2002
• 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 6,627,177 B2
• Time in Patent Office: Days
• US Class Current: 424/9.6
• 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

Abstract

The present invention concerns an improved optical method and optical sensing device for determining the levels of polyhydroxyl-substituted organic molecules in vivo in aqueous media. 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. An optical method for the in vivo detection of polyhdroxyl-substituted organic molecules as the analyte between about 430 and 600 nm detection in physiological media having a pH of about 7.3-7.5, which method comprises:

A. obtaining a flurorophore 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 430 nm and less than 600 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 is a discrete compound having a molecular weight of 1,000 daltons or greater or a pendant group or chain unit attached to a water soluble or dispersible polymer having a molecular weight greater than about 10,000 daltons, or optionally said polymer is non-covalently associated with water insoluble polymer matrix M¹ and is physically immobilized within said polymer matrix M¹; and

wherein said polymer matrix M¹ 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 430 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 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

wherein;

M¹ is said polymer matrix,

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

D² is said fluorophore dye which is covalently bonded to said polymer matrix M¹;

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

B. Combining with a analyte solution-compatible boronic acid-containing quencher molecule Q, wherein Q is a conjugated nitrogen-containing heterocyclic, aromatic bis-onium salt selected from;

(i) Q¹ which is a pendant group or a chain unit in a water-soluble or water-dispersable 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 immobilized within said polymer matrix wherein Q¹ is a compound having the properties of;

compatibility in said analyte solution,

produces a detectable change in the emission of the dye in the presence of said analyte, or

(ii) Q² which is a structure having the properties of;

compatibility in said analyte solution

produces a detectable change in the emission of the dye in the presence of said analyte,

wherein Q² 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, 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 being polyfunctional is linked to the matrix M² at one or two sites,

C. contacting a physiological fluid which contains analyte, 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 430 to 600 nm; and

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

2. The method of claim 1 wherein the Dye D¹ is selected from the group consisting of pyranine derivatives having the structure of:

3. The method of claim 1 wherein the Dye D¹ is selected from the group consisting of pyranine derivatives having the structure of:

4. The method of claim 1 wherein the Dye D² is prepared from pyramine derivatives having the structure:

5. The method of claim 1 wherein the precursors of quenchers Q¹ and Q² are selected from the group consisting of:

6. The method of claim 5 where the precursors are:

7. The method of claim 1 wherein in substep A,

the fluorophore is D¹.

8. The method of claim 1 wherein in substep A,

the fluorophore is D².

9. The method of claim 1 wherein in substep B, quencher Q is Q¹.

10. The method of claim 1 wherein in substep B, quencher Q is Q².

11. The method of claim 1 wherein in substep A, D is D¹ and in substep B, Q is Q¹.

12. The method of claim 1 wherein in substep A the fluorophore D¹ is selected from pyranine derivatives having the structure of:

13. The method of claim 1 wherein in substep A the precursor to the polymeric dye D² is:

14. The method of claim 1 wherein the polyhydroxyl-substituted organic molecules are sugars selected from glucose or fructose.

15. The method of claim 14 wherein the Dye D¹ is selected from the group consisting of

16. The method of claim 14 wherein the quencher Q² is prepared from a quencher precursor from the group consisting of

17. An in vivo optical device wherein the Dye D components and quencher Q components are immobilized in or attached to a polymer matrix M¹, M² or combinations thereof as defined in claim 1 and

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

20. The composition of matter of claim 19 where the fluorophore is N,N′

,N″

-tris-(1-aminoethyl-2-polyethylene glycol (n˜

125)-methoxy)-8-hydroxy-pyrene-1,3,6-tris-sulfonamide or a polymer of N,N′

,N″

-tris-(1-aminopropyl-3-methacrylamide)-8-acetoxy-pyrene-1,3,6-tris-sulfonamide.

21. The composition of matter of claim 20 wherein the viologen including a boronic acid functional group is a polymer of 4-N′

-(benzyl-4-boronic acid)-4′

-N′

-(benzyl-(3, or

     4)-ethenyl)-dipyridinium bromide chloride (m-, or p-SBBV).

22. The composition of matter of claim 21 wherein the polymer matrix is a hydrogel comprised of 2-hyroxyethylmethacrylate polymers, polyethyleneglycol polymers, and combinations thereof.

18. A composition of matter selected from the group consisting of compounds of the structure:

19. A composition of matter which comprises, a glucose responsive polymer assembly, itself comprising a fluorophore which is excited by light of 430-800 nm, which is susceptible to quenching by a viologen, a viologen including at least one boronic acid functional group as a quencher, and a glucose permeable polymer matrix.

23. The composition of matter of Q¹ or Q² precursors are selected from