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

• US 6,627,177 B2
• Filed: 12/05/2000
• Issued: 09/30/2003
• Est. Priority Date: 12/05/2000
• Status: Expired due to Term

• 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.
• Primary Examiner(s): Padmanabhan, Sreeni
• Assistant Examiner(s): Wells, Lauren Q.
• Application Number:
  US09/731,323
• Publication Number:
  US 20020106326A1
• Time in Patent Office: 1,029 Days
• Field of Search: 424/9.6, 514/646, 514/741, 514/765
• 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 polyhydroxyl-substituted organic molecules as the analyte between about 430 and 800 nm detection in physiological media having a p11 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 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 Stern-Volmer quenching constant (Ksv) greater than or equal to 50,

wherein the fluorophore dye D¹ is selected from the group consisting of

(i) a discrete compound having a molecular weight of 1,000 daltons or greater and

(ii) a pendant group or chain unit which is also attached to a water soluble or dispersible polymer which has a molecular weight greater than about 10,000 daltons, wherein optionally said water soluble or dispersible polymer is non-covalently associated with water insoluble polymer matrix M¹ and is thus 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 nm,

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 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;

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

where R¹, R² and R³ are each —

NHR⁴

wherein R⁴ is —

CH₂—

CH₂(—

O—

CH₂—

CH₂)_n—

X¹;

wherein X¹ is selected from —

OH, —

OCH₃, —

CO₂H, —

CONH₂, —

SO₃H, or —

NH₂; and

n is between about 100 and 10,000.

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

where R¹, R² and R³ are each —

NH—

CH₂—

CH₂(—

O—

CH₂—

CH₂)_n—

X¹, and X¹ selected from —

OH, —

OCH₃, —

CO₂H, —

CONH₂, —

SO₃H, or —

NH₂, n is about 70 to 10,000.

4. The method of claim 1 wherein the Dye D²is prepared from pyranine derivatives and comprises a group which is a reaction product of a compound of the structure:

or a dye monomer

 

where R⁴=—

H and

R⁵ is selected from —

R⁶—

NH—

(C═

O)—

(C═

CH₂)—

R⁷, —

R⁶—

O—

(C═

O)—

(C═

CH₂)—

R⁷,

or —

CH₂—

C₆H₄—

CH═

CH₂ or —

CH₂ CH═

CH₂, and where R⁶ is lower alkylene having 2 to 6 carbon atoms and

where R⁷ is —

H, or;

—

CH₃ and

Z is a blocking group that can be removed by hydrolysis) selected from;

5. The method of claim 1 wherein the quencher Q² comprises a group which is a reaction product of a compound of a structure selected from the group consisting of

where (V)²⁺ is a nitrogen containing conjugated heterocyclic aromatic group selected from isomers of dipyridyls dipyridyl ethylenes, dipyridyl phenylenes, phenanthrolines, or diazafluorenes;

and where Z¹ or Z² is either a polymerizable ethylenically unsaturated group selected from;

(i) —

R¹⁰—

CO₂—

C(R¹¹)═

CH₂, —

R¹⁰—

NH—

(C═

O)—

C(R²)═

CH₂, or —

CH₂—

C₆H₄—

CH═

CH₂,

here R¹⁰ is a lower alkylene or hydroxyalkylene of 2 to 6 carbon atoms and where R¹¹═

—

H or —

CH₃;

or

(ii) a coupling group selected from;

—

R¹²—

Y¹

where R¹² is —

CH₂C₆H₄—

or alkylene of 2 to 6 carbon atoms and

Y¹ is —

OH, —

SH, —

CO₂H, or —

NH₂.

6. The method of claim 5 where the quenchers comprise a reaction product of a compound of the structure:

wherein X is chloride or bromide.

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 D² comprises a group which is a reaction product of a compound of the structure:

10. The method of claim 1 wherein in substep B , quencher Q² comprises a group which is a reaction product of a compound of the structure:

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:

wherein n is between about 70 and 200.

13. The method of claim 1 wherein in substep A the polymeric dye D² comprises a group which is a reaction product of a compound of the structure:

and in step B the quencher comprises a group which is a reaction product of a monomer selected from the group consisting of

 

wherein X is bromide or chloride.

14. The method of claim 1 wherein the polyhydroxyl-substituted organic molecules which are detected are sugars selected from the group consisting of glucose and fructose.

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

wherein n is about 70 to 200.

16. The method of claim 14 wherein the quencher Q² comprises a structure which is a reaction product of a monomer selected from the group consisting of

wherein X is bromide or chloride.

17. An in vivo optical sensing device wherein the Dye D components and quencher Q components are immobilized within or covalently 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 within an in vivo biological medium periodically or continuously.

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