US2004028612A1PendingUtilityA1
Optical determination of glucose utilizing boronic acid adducts
Priority: Dec 5, 2000Filed: Jun 5, 2003Published: Feb 12, 2004
Est. expiryDec 5, 2020(expired)· nominal 20-yr term from priority
G01N 33/52
49
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Claims
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. 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
exact text as granted — not AI-modifiedWe claim:
1 . An optical method for the in vivo detection of polyhydroxyl-substituted organic molecules as the analyte between about 430 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) D 1 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 rim,
v. Compatibility with said analyte solution, and wherein said
vi. Dye D 1 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 D 1 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 M 1 and is physically immobilized within said polymer matrix M 1 wherein said polymer matrix M 1 is permeable to or in contact with said analyte solution; and
optionally where D 1 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) D 2 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 D 2 is quenched by methyl viologen to produce an apparent Stern-Volmer quenching constant (Ksv) greater than or equal to 50, wherein D 2 is covalently bonded to an insoluble polymer matrix M 1 wherein said polymer matrix M 1 is permeable to or in contact with said analyte; wherein said fluorophore dye D 2 is a part of the structure: M 1 -L 1 -D 2 with the proviso that D 2 which is polyfunctional is bonded to matrix M 1 at one, two or three sites;
L 1 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 ten-ninated 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 dye in the presence of said analyte, selected from: (i) quencher Q 1 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 M 1 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 Q 2 which is covalently bonded by linking group L 2 to M 1 or to a second water insoluble polymer matrix M 2 producing M 2 -L 2 -Q 2 wherein L 2 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 Q 1 or Q 2 is mixed at a molecular level with said fluorophore dye D 1 or D 2 , and with the proviso that Q 2 when polyfunctional is linked to the matrix M 2 at one or two sites,
C. contacting a physiological fluid which contains analyte, a dye and a quenched 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 where the Dye D 1 is selected form the group consisting of pyranine derivatives having the structures of:
where R 1 , R 2 and R 3 are each —NHR 4 is —CH 2 —CH 2 (—O—CH 2 —CH 2 ) n —X 1 ;
wherein X 1 is selected from —OH, —OCH 3 —CO 2 H, —CONH 2 , —SO 3 H, or —NH 2 ; and
n is between about 70 and 10,000.
3 . The method of claim 1 wherein the dye D 1 is selected from the group consisting of pyranine derivatives having the structure of
where R 1 , R 2 and R 3 are each —NH—CH 2 —CH 2 (—O—CH 2 —CH 2 ,) n —X 1 , and X 1 is selected from —OH, —OCH 3 , —CO 2 H, —CONH 2 , —SO 3 H, or —NH 2 , n is about 100 to 10,000.
4 . The method of claim 1 wherein the Dye D 1 or D 2 is prepared from pyramine derivatives having the structure:
or from a dye monomer selected from the group consisting of:
where
R 4 —H and
R 5 is selected from —R 6 —NH—(C═O)—(C═CH 2 )—R 7 , —R 6 —O—(C==O)—(C—R 7 ═CH 2 ) or —CH 2 —C 6 H 4 —CH—CH 2 or —CH 2 —CH═CH 2 , and where R 6 is lower alkylene having 2 to 6 carbon atoms and
where R 7 is —H or; —CH 3 and
Z is a blocking group that can be removed by hydrolysis selected from: —(C═O)—R 8 —Y
where R 8 is a lower alkylene having 1 to 4 carbon atoms and Y is selected from —H, —OH, —CO 2 H, —SO 3 H, —(C═O)—NH—R 9 , or —CO 2 —R 9 ,
where R 9 is a lower alkyl having 1 to 4 carbon atoms.
5 . The method of claim 1 wherein the precursors of quenchers Q 1 and Q 2 are 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 Z 1 or Z 2 is either a polymerizable ethylenically unsaturated group selected from:
(i) —R 10 —CO 2 —C(R 11 )═CH 2 , —R 10 —NH—(C═O)—C(R 12 )═CH 2 , or —CH 2 —C 6 H 4 —CH═CH 2 , where R 10 is a lower alkylene or hydroxyalkylene of 2 to 6 carbon atoms and where R”=-H or —CH 3 ; or
(ii) a coupling group selected from: —R 12 -Z 3 —
where R 12 is —CH 2 C 6 H 4 — or alkylene of 2 to 6 carbon atoms and
Z 3 is —OH, —SH, —CO 2 H, or —NH 2 .
6 . The method of claim 5 where the precursors are selected from:
wherein X is chloride, bromide or combinations thereof.
7 . The method of claim 1 wherein in substep B, Q 1 or Q 2 is prepared from a precursor selected from:
where V 3 and Z 4 or Z 5 are 2, 3 or 4-(CH═CH 2 )-pyridinium; —N—(CH 2 ) w —O(C═O)C(CH 3 )═CH 2 ); —O—(CH 2 ) w , —O—CH 2 —(CH═CH 2 ); —O—(CH 2 ) w —O—(C═O)CH(═CH 2 ); and —O—(CH 2 ) n —O—(C═O) C(CH 3 )═CH 2 ; and w is a integer from 2 to 6, or Z 4 and Z 5 have the same definitions as above for Z 1 and Z 2 .
8 . The method of claim 1 wherein in substep A, the fluorophore is D 1 .
9 . The method of claim 1 wherein in substep A, the fluorophore is D 2 .
10 . The method of claim 1 wherein in substep B, quencher Q is Q 1 .
11 . The method of claim 1 wherein in substep A, D is D 1 and in substep B, Q is Q 1 .
12 . The method of claim 1 wherein in substep A the fluorophore D 1 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 precursor to the polymeric dye D 2 is:
and in step B the quencher is prepared from the group consisting of
wherein X is bromide or chloride.
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 D 1 is selected from the group consisting of
wherein n is about 70 to 200.
16 . The method of claim 14 wherein the quencher Q 2 is prepared from a quencher precursor from the group consisting of
wherein X is bromide or chloride.
17 . An optical device for the in vivo detection of polyhydroxyl-substituted organic molecules as the analyte between about 430 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) D 1 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 D 1 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 D 1 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 M 1 and is physically immobilized within said polymer matrix M 1 wherein said polymer matrix M 1 is permeable to or in contact with said analyte solution; and
optionally where D 1 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) D 2 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 D 2 is quenched by methyl viologen to produce an apparent Stern-Volmer quenching constant (Ksv) greater than or equal to 50, wherein D 2 is covalently bonded to an insoluble polymer matrix M 1 wherein said polymer matrix M 1 is permeable to or in contact with said analyte; wherein said fluorophore dye D 2 is a part of the structure: M 1 -L 1 -D 2 with the proviso that D 2 which is polyfunctional is bonded to matrix M 1 at one, two or three sites;
L 1 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 ten-ninated 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 Q 1 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 M 1 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 Q 2 which is covalently bonded by linking group L 2 to M 1 or to a second water insoluble polymer matrix M 2 producing M 2 -L 2 -Q 2 wherein L 2 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 Q 1 or Q 2 is mixed at a molecular level with said fluorophore dye D 1 or D 2 , and with the proviso that Q 2 when polyfunctional is linked to the matrix M 2 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 430 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 M 1 , M 2 or combinations thereof and said device measures the concentration of polyhydroxyl-containing molecules periodically or continuously.
18 . The device of claim 17 wherein the dye is selected from the group described in claim 2 .
19 . The device of claim 17 wherein the quencher is selected from the group described in claim 16 .
20 . The device of claim 17 wherein the polymer matrix is prepared from monomers selected from the group consisting of HPTS-MA and HPTS-CO 2 -MA.
21 . The device of claim 17 wherein the dye and the quencher is selected from the group described in claim 16 .
wherein the polymer is prepared from monomers selected from the group consisting of HPTS-CO 2 -MA and HPTS-MA.
22 . The device of claim 17 wherein the dye is described in claim 2 .
wherein the quencher described in claim 16 is selected from
wherein the polymer is HPTS-MA
23 . A composition of matter selected from the group consisting of compounds of the structure:
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 in all positions of the ring are capable of forming a quaternary salt; and
Z 1 or Z 2 is either a polymerizable ethylenically unsaturated group selected from: —R 15 —CO 2 —C(R 16 )═CH 2 , —R 15 —NH—(C═O)—C(R 16 )═CH 2 , —CH 2 —C 6 H 4 —CH═CH 2 ;
R 15 is a lower alkylene or hydroxyalkylene of 2 to 6 carbon atoms;
R 16 =—H, —CH 3 or a coupling group selected from —R 17 -Z 3 , wherein R 17 is —CH 2 C 6 H 4 — or alkylene of 2 to 6 carbon atoms, and
Z 3 is selected from OH—, —SH, —CO 2 H, or —NH 2 —.
24 . 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.
25 . The composition of matter of claim 24 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-methacrylamidopropyl)-8-acetoxy-pyrene-1,3,6-tris-sulfonamide.
26 . The composition of matter of claim 25 wherein the viologen including a boronic acid functional group is a polymer of 4-N-(benzyl-3 or 4-boronic acid)-4′-N′-(benzyl-4-ethenyl)-dipyridinium bromide chloride (m-, or p-SBBV).
27 . The composition of matter of claim 26 wherein the polymer matrix is a hydrogel comprised of 2-hyroxyethylmethacrylate polymers, polyethyleneglycol polymers, and combinations thereof.
28 . The composition of matter of Q 1 or Q 2 precursors are selected from
where X is bromide or chloride.
29 . A composition of matter of the structure
30 . A composition of matter as a polymer including the structure:
31 . A composition of matter of the structure:Join the waitlist — get patent alerts
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