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

• US 20020106810A1
• 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 6,653,141 B2
• Time in Patent Office: Days
• US Class Current: 436/172
• 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;

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 4 carbon atoms, sulfonamide, amide, ester, ether, sulfide, sulfone, phenylene, urethane, urea, thiourea and amine, and

D² is said fluorophore dye which is covalently bonded to said polymer matrix M¹, with the proviso that D² as a poly-functional moiety is covalently bonded to said 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 aromatic nitrogen-containing heterocyclic bis-onium salt selected from;

(i) Q¹ which is a discrete soluble compound or is a pendant group or chain unit in a water soluble or dispersible polymer or organic liquid soluble or dispersible polymer and said polymer is optionally non-covalently associated with an optional polymer matrix M¹ when present and immobilized within said polymer matrix M¹, 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, and

wherein Q² is covalently bonded by linking group L² to M¹ or to a second 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 4 carbon atoms, sulfonamide, amide, ester, ether, sulfide, sulfone, phenylene, urethane, urea, thiourea and amine, with the proviso that Q² as a poly-functional moiety is covalently bonded to said matrix M¹ or M² at one or two sites;

wherein said quencher Q¹ or Q² is mixed at a molecular level with said fluorophore dye D¹ or D², and

C. contacting a test solution of analyte, a dye and a quencher in vitro with an excitation light source coupled with a detector;

D. producing a detectable and quantifiable signal in the range of about 300 to 800 nm; and

E. determining the concentration of said polyhydroxyl-substituted analyte in said aqueous liquid, organic liquid or combinations thereof.

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, R²and R³ are same or different and wherein R^{1 2}and IV 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-sindacene-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:

4. The method of claim 1 wherein Q¹ is selcted 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:

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

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:

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

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

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

18. A in vitro 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 matter of claim 19 wherein R⁴ is —

H, R⁵is —

CH₂CH₂—

(OCH₂CH₂)—

X¹;

X is —

OH, —

OCH₃; and

n is between about 2 and 200.

19. A composition of matter selected from the group consisting of pyranine derivatives having the structure of:

21. A composition of matter selected from the group consisting of

22. The composition of matter of claim 21 wherein R⁷ is —

H;

R⁸ is R⁹ —

NH (C═

O)—

C(═

CH₂)—

CH₃; and

Z is —

(C═

O)CH₃.