Polyhydroxyl-substituted organic molecule sensing method and device

• US 6,653,141 B2
• Filed: 12/05/2000
• Issued: 11/25/2003
• 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): SNAY, JEFFREY R
• Application Number:
  US09/731,325
• Publication Number:
  US 20020106810A1
• Time in Patent Office: 1,085 Days
• Field of Search: 436/95, 436/131, 436/172, 422/82.07, 422/82.08, 422/82.11, 250/458.1, 250/459.1
• US Class Current: 436/95
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• 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 in aqueous or organic media. Specifically, a dye is combined with an conjugated nitrogen-containing aromatic 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 fermentation and process streams.

Claims

1. An optical method for the in vitro detection between about 300 and 800 nm of polyhydroxyl-substituted organic molecules as the analyte in an analyte solution selected from aqueous liquid, an organic liquid or combinations thereof at pH of about 5 to 9, 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 300 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 is a discrete soluble compound, or is a pendant group or chain unit in a water-soluble or dispersible or organic liquid soluble or dispersible polymer, and said polymer optionally is non-covalently associated with an 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 300 nm and less than 800,

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

iv. Resident to photobleaching under the conditions of analysis,

v. Compatibility in the analyte solution, and wherein said

vi. 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;

2. The method of claim 1 wherein the Dye D¹ is selected from the group consisting of

pyranine;

pyranine derivatives having the structure of;

 

where

R¹, R² and R³ are same or different and wherein R¹, R² and R³ are each selected from;

—

OH, —

N(R⁴)R⁵, wherein R⁴ is selected from —

H, —

CH₃, and —

CH₂CH₂OH, and

R⁵ is selected from —

CH₂—

CH₂(—

O—

CH₂—

CH₂)_n—

X¹ or R⁶X¹, wherein X¹ is selected from —

OH, —

OCH₃, —

CO₂H, —

CONH₂, —

SO₃H, or —

NH₂, and R⁶ is a lower alkylene or hydroxyalkylene having 2 to 6 carbon atoms;

n is about 2 to 10,000, coumarin 343;

eosin Y;

fluorescein;

4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene-2,6-disulfonic acid, disodium salt (Molecular Probes D-3238);

Lucifer Yellow Iodoacetamide dipotassium salt (Molecular Probes L-1338); and

fluorescein-5-(and-6)-sulfonic acid, trisodium salt (Molecular Probes F-1130).

3. The method of claim 1 wherein the Dye D² is prepared from:

or from a dye monomer selected from the group consisting of;

 

where

R⁷=—

H, —

CH₂—

CH═

CH₂ and when R⁷=—

H then

R⁸ is selected from —

R⁹—

NH—

(C═

O)—

(C═

CH₂)—

R¹⁰, —

R⁹—

O—

(C═

O)—

(C═

CH₂)—

R¹¹, or —

CH₂—

C₆H₄—

CH═

CH₂, and where R⁹ is lower alkylene having 2-6 carbon atoms and

where R¹⁰ and R¹¹ are each —

H, —

CH₃ and when R⁷ is —

CH₂—

CH═

CH₂ then R⁸ is —

H, —

CH₂—

CH═

CH₂,

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

4. The method of claim 1 wherein Q¹ is selected from the group consisting of nitrogen containing conjugated heterocyclic aromatic bis-onium salts wherein the heterocyclic aromatic core is selected from the isomers of dipyridyl, phenanthroline, dipyridylethylene, dipyridylphenylene, and diazafluorene wherein the two nitrogen atoms are each in different aromatic rings and the nitrogen atoms are in all positions of the ring capable of forming an onium salt, and at least one of the substituents on the nitrogens is selected from ortho-, meta-, or para-benzyl boronic acids, preferably where both substituents are benzyl boronic acids.

5. The method of claim 1 wherein the quencher Q¹ is selected from the group consisting of:

wherein X is chloro or bromo.

6. A method of claim 1 where the quenchers Q¹ and Q² are derived from precursors selected from:

wherein (V)²⁺ is a nitrogen containing conjugated heterocyclic aromatic group selected from isomers of dipyridyls, dipyridyl ethylenes, dipyridyl phenylenes, phenanthrolines, or diazafluorenes wherein the two nitrogen atoms are each in a different aromatic ring and the nitrogen atoms are in all positions of the ring capable of forming an onium salt; and

Z¹ or Z² each are either a polymerizable ethylenically unsaturated group selected from;

7. The method of claim 1 wherein the quenchers Q¹ and Q² are prepared from a quencher precursor selected from the group consisting of:

wherein X is chloride, bromide or combinations thereof.

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

the fluorophore is D¹.

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

the fluorophore is D².

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

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

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

13. 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 2 and 200.

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

and in step B the quencher is selected from the group consisting of

 

wherein X is bromide, chloride or combinations thereof.

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

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

wherein n is about 2 to 200.

17. The method of claim 15 wherein the quencher Q¹ is selected from the group consisting of

wherein X is bromide, chloride, or combinations thereof.

18. An optical device for the in vitro detection between about 300 and 800 nm of polyhydroxyl-substituted organic molecules as the analyte in an analyte solution selected from aqueous liquid, an organic liquid or combinations thereof at pH of about 5 to 9, which device comprises:

a fluorophore Dye D,

a boronic acid—

containing quencher molecule Q,

a polymer matrix M or M¹,

an excitation light source, and a detector, wherein

A. said 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 300 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 is a discrete soluble compound, or is a pendant group or chain unit in a water-soluble or dispersible or organic liquid solid or dispersible polymer, and said polymer optionally is non-covalently associated with an 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 300 nm and less than 800,

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

iv. Resident to photobleaching under the conditions of analysis,

v. Compatibility in the analyte solution, and wherein said

vi. Dye D² is quenched by methyl viologen to produce an apparent Stem-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;