U.S. patent application number 09/800667 was filed with the patent office on 2001-08-30 for fluorescent n-alkylated acrylamide copolymers and optical ph sensors.
Invention is credited to Barnard, Steven Mark, Berger, Joseph, Rouilly, Marizel, Waldner, Adrian.
Application Number | 20010018217 09/800667 |
Document ID | / |
Family ID | 4246288 |
Filed Date | 2001-08-30 |
United States Patent
Application |
20010018217 |
Kind Code |
A1 |
Barnard, Steven Mark ; et
al. |
August 30, 2001 |
Fluorescent N-alkylated acrylamide copolymers and optical pH
sensors
Abstract
The invention relates to a polymer composition comprising
pH-sensitive fluorescent dyes, to an ionic strength-independent
optical sensor for pH value determination that contains the
composition in the form of a membrane on a transparent support
material, and to an optical process, according to the fluorescence
method, that renders possible highly accurate pH value
determination independently of the ionic strength of the test
solution. The process is especially suitable for the determination
of the pH value of physiological solutions, especially for the
determination of the pH value of blood.
Inventors: |
Barnard, Steven Mark;
(Wellesley Hills, MA) ; Berger, Joseph; (Muttenz,
CH) ; Rouilly, Marizel; (Gipf-Oberfrick, CH) ;
Waldner, Adrian; (Allschwil, CH) |
Correspondence
Address: |
Michael R. Davis
WENDEROTH, LIND & PONACK
Suite 800
2033 "K" Street N.W.
Washington
DC
20006
US
|
Family ID: |
4246288 |
Appl. No.: |
09/800667 |
Filed: |
March 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09800667 |
Mar 8, 2001 |
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09051496 |
Nov 2, 1998 |
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09051496 |
Nov 2, 1998 |
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PCT/EP96/04426 |
Oct 11, 1996 |
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Current U.S.
Class: |
436/100 ;
340/630; 356/438 |
Current CPC
Class: |
G01N 33/84 20130101;
G01N 21/6428 20130101; Y10T 436/15 20150115; G01N 2021/7786
20130101; C08F 220/56 20130101 |
Class at
Publication: |
436/100 ;
340/630; 356/438 |
International
Class: |
G01N 033/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 23, 1995 |
CH |
2995/95 |
Claims
What is claimed is:
1. A water-insoluble copolymer which is composed of a) from 39.9 to
60% by weight of N,N-dimethylacrylamide or N,N-dimethyl
methacrylamide; b) from 60 to 39.9% by weight of a monomer of
formula Ia or Ib 13 wherein R.sub.a is hydrogen or
C.sub.1-C.sub.6alkyl and R.sub.b is C.sub.1-C.sub.12alkyl; with the
proviso that R.sub.a and Rb are not both methyl; c) from 0.1 to
0.7% by weight of a proton-sensitive fluorophore which is
covalently bonded to the polymer; and d) from 0 to 20% by weight of
a diolefinic crosslinking component, the sum of the percentage
weights of a) to d) being 100%.
2. A water-insoluble copolymer according to claim 1, wherein
N,N-dimethylacryl amide is used as monomer a).
3. A water-insoluble copolymer according to claim 1, wherein
R.sub.a is hydrogen and R.sub.b is a branched
C.sub.3-C.sub.8alkyl.
4. A water-insoluble copolymer according to claim 3, wherein
R.sub.a is hydrogen, R.sub.b is tert-butyl and the ratio of monomer
a) to monomer b) is 50 parts by weight to 50 parts by weight.
5. A water-insoluble copolymer according to claim 1, wherein
R.sub.a is methyl or ethyl and R.sub.b is linear
C.sub.3-C.sub.8alkyl.
6. A water-insoluble copolymer according to claim 5, wherein
R.sub.a is methyl and R.sub.b is n-butyl.
7. A water-insoluble copolymer according to claim 1, wherein the
proton-sensitive fluorophore is selected from the group consisting
of acridines, acridones, rhodamines, xanthenes, benzoxanthenes,
pyrenes and coumarins.
8. A water-insoluble copolymer according to claim 1, wherein the
fluorophore is covalently bonded to the polymer.
9. A water-insoluble copolymer according to claim 1, wherein the
fluorophore is a compound of formula II, III, IV, V or VI 14wherein
R.sub.1, R.sub.2, R.sub.5 and R.sub.6 are each independently of the
others hydrogen, --SO.sub.2--(C.sub.1-C.sub.6)alkylphenyl,
C.sub.1-C.sub.30alkyl, C.sub.1-C.sub.30alkyl--CO-- or a radical of
the formula --(C.sub.nH.sub.2n--O--).sub.m--R.sub.8; R.sub.3 is
hydrogen or --SO.sub.2--(C.sub.1-C.sub.6)alkylphenyl; R.sub.4 and
R.sub.7 are a C.sub.1-C.sub.30alkylene or a radical of the formula
--(C.sub.nH.sub.2n--O--).sub.m--R.sub.8; Z is a divalent radical
--NH--CO--; R.sub.8 is a direct bond or C.sub.1-C.sub.12alkylene; n
is an integer from 2 to 6 and m is an integer from 1 to 10, with
the proviso that the total number of carbon atoms is no more than
30; R.sub.9 and R.sub.10 are each independently of the other H,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4alkoxycarbonyl, C.sub.1-C.sub.4alkyl-SO.sub.2-- or
halogen, and either R.sub.11 is H and R.sub.12 is a divalent
radical --NH--CO--, --CO--NH--(C.sub.2-C.sub.12alk- ylene-O)--CO--,
-- CO--NH-- (C.sub.2-C.sub.12alkylene--NH)--CO-- or
--C(O)--NH--(CH.sub.2CH.sub.2--O).sub.1 to 6--CH.sub.2C(O)-- NH--,
or R.sub.11 is a divalent radical --NH--CO--,
--CO--NH--(C.sub.2-C.sub.12alk- ylene-O)--CO--,
--CO--NH--(C.sub.2-C.sub.12alkylene-NH)-- CO-- or
--C(O)--NH--(CH.sub.2CH.sub.2--O).sub.1 to 6--CH.sub.2C(O)--NH--,
and R.sub.12 is H; or wherein either R.sub.13 is H and R.sub.14 is
a divalent radical --NH--C(O)--,
--CO--NH--(C.sub.2C.sub.12alkylene-O)--CO--, -- CO--NH--
(C.sub.2-C.sub.12alkylene--NH)--CO-- or --C(O)--NH--(CH.sub.2CH.-
sub.2--O).sub.1 to 6--CH.sub.2C(O)-- NH--, or R.sub.13 is a
divalent radical --NH--C(O)--,
--CO--NH--(C.sub.2-C.sub.12alkylene-O)--CO--,
--CO--NH--(C.sub.2-C.sub.12alkylene-NH)-- CO-- or
--C(O)--NH--(CH.sub.2CH- .sub.2--O).sub.1 to 6--CH.sub.2C(O)--NH--,
and R.sub.14 is H, wherein the radical -- COOH is each in free form
or in salt form, or a C.sub.1-C.sub.20alkyl ester thereof.
10. A water-insoluble copolymer according to claim 9, wherein
R.sub.1 and R.sub.2 of the fluorophore of formula II are each
independently of the other hydrogen or linear
C.sub.12-C.sub.24alkyl.
11. A water-insoluble copolymer according to claim 9, wherein
R.sub.4 of the fluorophore of formula II is a linear
C.sub.2-C.sub.16alkylene or a radical of the formula
--(C.sub.2H.sub.4--O--).sub.m--R.sub.8 wherein R.sub.8 and m are as
defined in claim 9.
12. A water-insoluble copolymer according to claim 9, wherein
R.sub.5 and R.sub.6 of the fluorophore of formula III are each
independently of the other linear C.sub.2-C.sub.12alkyl.
13. An optical sensor for an ionic strength-independent pH value
determination, consisting of A) a transparent support material B) a
layer of water-insoluble copolymers that are composed of a) from
39.9 to 60% by weight of N,N-dimethylacrylamide or
N,N-dimethylmethacrylamide; b) from 60 to 39.9% by weight of a
monomer of formula Ia or Ib 15 wherein R.sub.a is hydrogen or
C.sub.1-C.sub.6alkyl and R.sub.b is C.sub.1-C.sub.12alkyl; with the
proviso that R.sub.a and Rb are not both methyl; c) from 0.1 to
0.7% by weight of a proton-sensitive fluorophore which is
covalently bonded to the polymer; and d) from 0 to 20% by weight of
a diolefinic crosslinking component, the sum of the percentage
weights of a) to d) being 100%.
14. A sensor according to claim 13, wherein the fluorophore is
selected from the group consisting of acridines, acridones,
rhodamines, xanthenes and benzoxanthenes, pyrenes, coumarins and
fluoresceins.
15. A sensor according to claim 13, wherein the thickness of the
polymer layer B) is from 0.1 to 500 .mu.m.
16. A process for the ionic strength-independent, reversible
optical determination of the pH value of an aqueous sample
according to the fluorescence method, in which an optical sensor,
consisting of A) a transparent support material B) a layer of
water-insoluble copolymers that are composed of a) from 39.9 to 60%
by weight of N,N-dimethylacrylamide or N,N-dimethyl methacrylamide;
b) from 60 to 39.9% by weight of a monomer of formula Ia or Ib 16
wherein R.sub.a is hydrogen or C.sub.1-C.sub.6alkyl and R.sub.b is
C.sub.1-C.sub.12alkyl; with the proviso that R.sub.a and Rb are not
both methyl; c) from 0.1 to 0.7% by weight of a proton-sensitive
fluorophore which is covalently bonded to the polymer; and d) from
0 to 20% by weight of a diolefinic crosslinking component, the sum
of the percentage weights of a) to d) being 100%, is brought into
contact with an aqueous test sample and irradiated with excitation
light, the fluorescence is measured, and the pH value is calculated
from the measured fluorescence intensity taking calibration curves
into consideration.
17. A process according to claim 16, wherein the test solution has
a pH of from 6.5 to 8.5.
18. A process according to claim 16, wherein the ionic strength of
the test solution is from 0.05 to 5 mol/l.
19. A process according to claim 16, wherein the ionic strength is
provided essentially by 1,1- or 1,2-salts.
20. A process according to claim 16, wherein the test solution
consists partly or wholly of a body fluid.
21. The use of an optical sensor according to claim 13 for the
ionic strength-independent determination of the pH value of an
aqueous test solution according to the fluorescence method.
Description
[0001] The invention relates to a polymer composition comprising
pH-sensitive fluorescent dyes, to an ionic strength-independent
optical sensor for pH value determination that contains the
composition in the form of a membrane on a transparent support
material, and also to an optical process, according to the
fluorescence method, that renders possible highly accurate pH value
determination independently of the ionic strength of the test
solution. The process is especially suitable for the determination
of the pH value of physiological solutions, especially for the
determination of the pH value of blood.
[0002] It is known that the pK.sub.a value of an indicator varies
with the ionic strength of a solution and that that variation
depends on the level of the charge at the indicator. For example,
it has already been proposed in DE-A-3 430 935 to determine
computationally the ionic strength and the pH value from the
difference between the measured values of two sensors having
different ionic strength dependence of which one exhibits as low as
possible an ionic strength dependence, after calibration of said
sensors with known test solutions. The sensor described therein
that is almost independent of the ionic strength does not lie
exactly within the physiological pH range and has a low resolution.
The construction of those sensors is effected without embedding
into a polymer matrix and consequently has the disadvantage that
the dye is in direct contact with the test solution. The
fluorescent dye of the sensors, which is the same in each case, is
in that arrangement immobilised directly on the surface of glass
supports by way of bridging groups, one of the sensors containing
additional charges for achieving a high polarity and ionic strength
dependence and the other sensor being so modified that it is
essentially non-polar, hydrophobic and independent of the ionic
strength. A quite considerable disadvantage of those sensors is
that the fluorescent dye is exposed directly to external influences
of the test solutions, and both physical influences (for example
dissolution of the dye, deposits on the surface) and chemical
influences (decomposition of the dye) quickly make the sensors
unusable. In addition, in the case of excitations in an evanescent
field, interference between the evanescent measuring field and the
fluorescence of the test sample cannot be completely avoided, which
reduces the accuracy of the measurement. The response time of those
sensors is on the other hand short, since the fluorescent dye
bonded to the surface immediately comes into contact with the test
solution. The sensitivity is regarded as adequate.
[0003] The method of optical pH determination using two sensors
that respond to different extents to the ionic strength of a test
solution is expensive in respect of apparatus and a subsequent,
additional calculation step has to be carried out.
[0004] It has now been found that, by selection of quite specific
copolymers of acrylamides and methacrylamides in conjunction with
the selection of a narrow concentration range of a fluorescent dye,
which is embedded in the polymer matrix, it is possible to produce
an optical pH sensor that allows highly accurate optical pH
measurement that is independent of ionic strength in the
physiological pH range of from 6.5 to 8.2. By that means, a second
measurement and the calculation step for eliminating the ionic
strength are dispensed with. The high degree of accuracy of the pH
value measurement is of great importance especially in the analysis
of human blood, since the measurement can be used, for example, for
monitoring the therapy of metabolic diseases. For a quick and
inexpensive test it is therefore especially advantageous if only
one sensor has to be used. The analytical apparatus can
consequently also be miniaturised more easily.
[0005] The shelf life and working life of those sensors is high
since the fluorescent dye is effectively protected by the polymer
matrix against damaging or interfering influences of the test
medium. The sensitivity is not reduced in such sensors and the
response times are surprisingly short.
[0006] By means of the polymer compositions it is possible to set
very accurately, for example, the hydrophilic property, hydrophobic
property, polarity and/or dielectric constant of the matrix, which,
combined with the selected concentration range of the fluorophore,
results in a measurement that is independent of ionic strength
within a particular pH value range.
[0007] The response times and the conditioning times correspond to
the short periods of time required of optical measuring systems
despite embedding of the fluorophore, those parameters being
dependent essentially on the membrane thickness.
[0008] The invention relates to water-insoluble copolymers that are
composed of
[0009] a) from 39.9 to 60% by weight of N,N-dimethylacrylamide or
N,N-dimethylmethacrylamide;
[0010] b) from 60 to 39.9% by weight of a monomer of formula Ia or
Ib 1
[0011] wherein R.sub.a is hydrogen or C.sub.1-C.sub.6alkyl and
R.sub.b is C.sub.1-C.sub.12alkyl; with the proviso that R.sub.a and
Rb are not both methyl;
[0012] c) from 0.1 to 0.7% by weight of a proton-sensitive
fluorophore which is covalently bonded to the polymer; and
[0013] d) from 0 to 20% by weight of a diolefinic crosslinking
component, the sum of the percentage weights of a) to d) being
100%.
[0014] Within the scope of the present invention, "water-insoluble"
denotes that at most traces of less than 0.1% are able to dissolve.
In order, on the other hand, to be able to produce a good contact
with the test medium, the copolymer must, however, be
swellable.
[0015] The alkyl radicals may be linear or branched. Examples of
C.sub.1-C.sub.12alkyl are the linear or branched radicals: methyl,
ethyl and the various position isomers of propyl, butyl, pentyl,
hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl.
[0016] The monomer preferably used as monomer a) is
N,N-dimethylacrylamide. Preferred water-insoluble copolymers are
obtained when R.sub.a is hydrogen and R.sub.b is a branched
C.sub.3-C8alkyl. Especially preferred are water-insoluble
copolymers in which R.sub.a is hydrogen, R.sub.b is tertiary butyl
and the ratio of monomer a) to monomer b) is 50 parts by weight to
50 parts by weight.
[0017] Another group of preferred water-insoluble copolymers is
obtained when R.sub.a is methyl or ethyl and R.sub.b is linear
C.sub.3-C.sub.8alkyl. Especially preferably, R.sub.a is methyl and
R.sub.b is n-butyl.
[0018] Suitable proton-sensitive fluorescent dyes are, for example,
those from the group of the xanthenes and benzoxanthenes, for
example fluorescein, halogenated fluoresceins,
seminaphthofluoresceins, seminaphthorhodafluors, 2,3-benzo
fluorescein, 3,4-benzofluorescein, the isomers of benzorhodamine
and substituted derivatives, the isomers of benzochromogen and
substituted derivatives; acridines, for example acridine,
9-amino-6-chloroacridine; acridones, for example 7-hydroxyacridone
and 7-hydroxybenz acridone; pyrenes, for example
8-hydroxypyrene-1,3,6-trisulfonic acid; cyanine dyes; and
coumarins, for example 7-hydroxycoumarin and
4-chloromethyl-7-hydroxycoumarin. The fluorescent dyes may be
functionalised with olefinically unsaturated groups in order to
bind to the polymer backbone.
[0019] Preferably, the fluorophores are selected from the group
consisting of acridines, acridones, rhodamines, xanthenes,
benzoxanthenes, pyrenes and coumarins, which are either admixed
with or covalently bonded to the polymer.
[0020] Preferred are water-insoluble copolymers in which the
flourophore is covalently bonded to the polymer.
[0021] Especially preferred are water-insoluble copolymers in which
the fluorophore is a compound of formula II, III, IV, V or VI 2
[0022] wherein
[0023] R.sub.1, R.sub.2, R.sub.5 and R.sub.6 are each independently
of the others hydrogen, --SO.sub.2--(C.sub.1-C.sub.6)alkylphenyl,
C.sub.1-C.sub.30alkyl, C.sub.1-C.sub.30alkyl-CO-- or a radical of
the formula --(C.sub.nH.sub.2n--O--).sub.m--R.sub.8;
[0024] R.sub.3 is hydrogen or
--SO.sub.2--(C.sub.1-C.sub.6)alkylphenyl;
[0025] R.sub.4and R.sub.7 are a C.sub.1-C.sub.30alkylene or a
radical of the formula --(C.sub.nH.sub.2n--O--).sub.m--R.sub.8;
[0026] Z is a divalent radical --NH--CO--;
[0027] R.sub.8 is a direct bond or C.sub.1-C.sub.12alkylene;
[0028] n is an integer from 2 to 6 and m is an integer from 1 to
10, with the proviso that the total number of carbon atoms is no
more than 30;
[0029] R.sub.9 and R.sub.10 are each independently of the other H,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4alkoxycarbony- l, C.sub.1-C.sub.4alkyl-SO.sub.2-- or
halogen, and either
[0030] R.sub.11 is H and R.sub.12 is a divalent radical --NH--CO--,
--CO--NH--(C.sub.2-C.sub.12alkylene-O)--CO--, -- CO--NH--
(C.sub.2-C.sub.12alkylene-NH)--CO-- or
--C(O)--NH--(CH.sub.2CH.sub.2--O).- sub.1 to 6--CH.sub.2C(O)--
NH--, or R.sub.11 is a divalent radical --NH--CO--,
--CO--NH--(C.sub.2-C.sub.12alkylene-O)--CO--,
--CO--NH--(C.sub.2-C.sub.12alkylene-NH)-- CO-- or
--C(O)--NH--(CH.sub.2CH- .sub.2--O).sub.1 to 6--CH.sub.2C(O)--NH--,
and R.sub.12 is H; or wherein either
[0031] R.sub.13 is H and R.sub.14 is a divalent radical
--NH--C(O)--, --CO--NH--(C.sub.2-C.sub.12alkylene-O)--CO--, --
CO--NH-- (C.sub.2-C.sub.12alkylene-NH)--CO-- or
--C(O)--NH--(CH.sub.2CH.sub.2--O).- sub.1 to 6--CH.sub.2C(O)--
NH--, or R.sub.13 is a divalent radical --NH--C(O)--,
--CO--NH--(C.sub.2-C.sub.12alkylene-O)--CO--,
--CO--NH--(C.sub.2-C.sub.12alkylene-- NH)--CO-- or
--C(O)--NH--(CH.sub.2CH.sub.2--O).sub.1 to 6--CH.sub.2C(O)--NH--,
and R.sub.14 is H, wherein the radical -- COOH is each in free form
or in salt form, or a C.sub.1-C.sub.20alkyl ester thereof.
[0032] Alkyl as such or as a structural element of other groups,
such as, for example, of alkoxy and alkoxycarbonyl is, with
appropriate consideration given in each case to the number of
carbon atoms respectively included in the corresponding group or
compound, either straight-chain, that is to say methyl, ethyl,
propyl or butyl, or branched, e.g. isopropyl, isobutyl, sec-butyl
or tert-butyl.
[0033] Halogen is fluorine, chlorine, bromine or iodine, especially
fluorine, chlorine or bromine, more especially chlorine or
bromine.
[0034] Examples from which the divalent radicals Z, R.sub.11,
R.sub.12, R.sub.13 and R.sub.14 may arise are the acryloylamine
group --NHCOCH.dbd.CH.sub.2, the methacryloylamine group
--NHCOC(CH.sub.3).dbd.CH.sub.2, and the
2-(methacryloyloxy)-ethylaminocar- bonyl group
--CONHCH.sub.2CH.sub.2OCOC(CH.sub.3).dbd.CH.sub.2.
[0035] Preferably, R.sub.1 and R.sub.2 of the fluorophore of
formula II are each independently of the other hydrogen or linear
C.sub.12-C.sub.24alkyl.
[0036] Also preferably, R.sub.4 of the fluorophore of formula II is
a linear C.sub.2-C.sub.16alkylene or a radical of the formula
--(C.sub.2H.sub.4--O--).sub.m--R.sub.8 wherein R.sub.8 and m are as
defined hereinbefore.
[0037] Another group of preferred water-insoluble copolymers is
formed by those in which R.sub.5 and R.sub.6 of the fluorophore of
formula III are each independently of the other linear
C.sub.2-C.sub.12alkyl.
[0038] Copolymerisable fluorescent dyes contain, for example, an
ethylenically unsaturated group (vinyl, crotonyl, methallyl) that
is bonded directly or via a bridging group to the fluorescent dye.
The monomers a) and b) are known. A known copolymerisable
fluorescent dye is, for example, 3-oder
4-acryloylaminofluorescein.
[0039] Polymers with fluorescent dyes comprising the bridging
groups --O--C(O)-- and --C(O)--O--
C.sub.2-C.sub.12alkylene--O--C(O)-- are obtainable, for example, by
esterification with fluorescent dyes that contain carboxyl or
hydroxyl groups. Polymers with fluorescent dyes comprising the
bridging groups --NH--C(O)--O-- and
--NH--C(O)--O--C.sub.2-C.sub.12alkylene--O--C(O)-- are accessible,
for example, by way of isocyanate-functionalised fluorescent dyes
and hydroxyl group-containing polymers.
[0040] The reactions described above may be carried out in a manner
known per se, for example in the absence or presence of a suitable
solvent, as required with cooling, at room temperature or with
heating, e.g. in a temperature range of from approximately
5.degree. C. to approximately 200.degree. C., preferably
approximately from 20.degree. C. to 120.degree. C., and, if
necessary, in a closed vessel, under pressure, in an inert gas
atmosphere and/or under anhydrous conditions.
[0041] The preparation of the polymers may be carried out according
to methods known per se.
[0042] The reactants may be reacted with one another as they are,
that is to say without the addition of a solvent or diluent, e.g.
in the melt. Generally, however, the addition of a solvent or
diluent or of a mixture of solvents is advantageous. There may be
mentioned as examples of such solvents and diluents: water; esters,
such as ethyl acetate; ethers, such as diethyl ether, dipropyl
ether, diisopropyl ether, dibutyl ether, tert-butyl methyl ether,
ethylene glycol monomethyl ether, ethylene glycol monoethyl ether,
ethylene glycol dimethyl ether, dimethoxy diethyl ether,
tetrahydrofuran or dioxane; ketones, such as acetone, methyl ethyl
ketone or methyl isobutyl ketone; alcohols, such as methanol,
ethanol, propanol, isopropanol, butanol, ethylene glycol or
glycerol; amides, such as N,N-dimethylformamide,
N,N-diethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone or
hexamethylphosphoric acid triamide; nitrites, such as acetonitrile
or propionitrile; and sulfoxides, such as dimethyl sulfoxide.
[0043] The copolymerisable fluorescent dyes may be prepared
according to processes known per se, and the starting materials are
either available commercially or can be prepared according to
analogous processes.
[0044] One possible method of preparing compounds of formula II or
III comprises
[0045] a) in compounds of formula IIc or IIIc 3
[0046] removing the phthalimide group under acidic conditions and,
where appropriate, in a second step
[0047] b) further reacting the reaction products with acrylic acid
chloride or methacrylic acid chloride or
[0048] c) where appropriate, removing the para-toluenesulfonyl
group from the reaction products of the starting materials of
formula IIc under acidic conditions,
[0049] the radicals R.sub.1, R.sub.2, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 being as defined hereinbefore.
[0050] The methods for the removal of the protecting groups are
known per se and may be used in an analogous manner in the
preparation of the compounds of formulae II and III.
[0051] The compounds of formula IIc can be prepared in a manner
known per se by stepwise alkylation with different alkylating
agents, or alkylation with an alkylating agent or acylating agent
of commercially available 3,6-diaminoacridine. Suitable alkylating
agents are, for example, dialkyl sulfates or monohaloalkanes,
especially chloro-, bromo- and iodo-alkanes. Suitable acylating
agents are, for example, carboxylic acid anhydrides and,
especially, carboxylic acid halides, such as, for example,
carboxylic acid chlorides. That reaction may be carried out in the
presence of inert polar and aprotic solvents, for example ethers,
alkylated acid amides and lactams or sulfones, and at elevated
temperatures, for example from 50 to 150.degree. C. Expediently, a
hydrogen halide acceptor is added, for example an alkali metal
carbonate or a tertiary amine, especially a sterically hindered
tertiary amine.
[0052] The compounds of formula IIIc are obtainable, for example,
by the reaction of phthalic acid anhydride with 2 molar equivalents
of 3-monoalkylaminophenol. Another possible method of preparation
is the reaction of 3-monoalkylaminophenol with one molar equivalent
of 2-hydroxy-4-dialkylamino-2'-carboxyl-benzophenone. Those
reactions are described, for example, in U.S. Pat. No.
4,622,400.
[0053] Compounds of formulae IV, V and VI may be prepared in an
analogous manner.
[0054] Conditions for the reactions are known per se. They may be
carried out, for example, in the presence of a suitable solvent or
diluent or a mixture thereof, as required with cooling, at room
temperature or with heating, e.g. in a temperature range of from
approximately -10.degree. C. to the boiling temperature of the
reaction mixture, preferably from approximately 0.degree. C. to
approximately 25.degree. C., and, if necessary, in a closed vessel,
under pressure, in an inert gas atmosphere and/or under anhydrous
conditions. Especially advantageous reaction conditions are
disclosed in the Examples.
[0055] Preferably, the copolymers have a mean molecular weight of
from 2 000 to 500 000, especially from 10 000 to 350 000 daltons,
determined according to the gel permeation method using standard
polymers of known molecular weight.
[0056] The water-insoluble copolymers may be crosslinked in the
form of a layer, for example with from 0.01 to 20%, preferably from
0.1 to 10%, and especially preferably from 0.5 to 5%, by weight of
a crosslinking agent based on the polymer. Suitable crosslinking
agents are, for example, acrylic acid or methacrylic acid esters or
amides of polyols, preferably diols to tetrols, or polyamines,
preferably diamines to tetramines. Such crosslinking agents are
known and widely described in the literature. Some examples of
polyols are ethylene glycol, 1,3-propanediol, 1,4-butanediol,
1,6-hexanediol, diethylene glycol, 1,1,1-trihydroxymethyl ethane or
propane, pentaerythritol and dipentaerythritol. Some examples of
polyamines are ethylenediamine, 1,3-propanediamine,
1,4-butanediamine, 1,6-hexanediamine, diethylenetriamine and
triethylenetetramine. Another known crosslinking agent is, for
example, divinylbenzene. Alkylene-bis-dialkylmaleimidyl compounds,
for example, are also suitable, for example
ethylene-bis-(dimethyl)maleimidyl.
[0057] The invention relates also to a composition and to an
optical sensor for an ionic strength-independent determination of
pH value, consisting of
[0058] A) a transparent support material
[0059] B) a layer of water-insoluble copolymers that are composed
of
[0060] a) from 39.9 to 60% by weight of N,N-dimethylacrylamide or
N,N-dimethylmethacrylamide;
[0061] b) from 60 to 39.9% by weight of a monomer of formula Ia or
Ib 4
[0062] wherein R.sub.a is hydrogen or C.sub.1-C.sub.6alkyl and
R.sub.b is C.sub.1-C.sub.12alkyl; with the proviso that R.sub.a and
Rb are not both methyl;
[0063] c) from 0.1 to 0.7% by weight of a proton-sensitive
fluorophore which is covalently bonded to the polymer; and
[0064] d) from 0 to 20% by weight of a diolefinic crosslinking
component,
[0065] the sum of the percentage weights of a) to d) being
100%.
[0066] The copolymer and fluorescent dye preferences given above
apply likewise to the sensor.
[0067] Sensors in which the fluorophore is admixed with the polymer
are suitable principally for once-only use. If the polymer membrane
is provided with a permeable and hydrophilic protective layer, then
both those sensors and also, in general, sensors with
polymer-bonded fluorophores, which may also contain a protective
layer on the membrane, may be used repeatedly or for continuous
measurements.
[0068] The geometric form of the support material may vary widely;
it may be, for example, fibres, cylinders, spheres, cuboids or
cubes. Furthermore, flow systems in which continuous measurements
or successive measurements may be carried out are possible. Planar
sensors are preferred. The support material is transparent. It may
be, for example, inorganic glass or transparent plastics, such as a
polycarbonate, a polyester (for example polyethylene
terephthalate), a polyamide, or a polyacrylate or
polymethacrylate.
[0069] The planar sensor may be of any external shape, for example
square, rectangular or round. It may have a surface area of from
0.01 to approximately 50 cm.sup.2, more advantageously from 0.02 to
10 cm.sup.2. The measuring zone of the sensor may have a surface
area of less than 5 mm.sup.2, preferably less than or equal to 2
mm.sup.2. The measuring zone may be correspond exactly to one
completely coated surface of the sensor. Advantageously, a coating
that is on both sides but that is locally separated may be
used.
[0070] The sensor may comprise one or more locally separated
membrane layers; in the latter case parallel measurements may be
carried out with identical or different test samples.
[0071] Preferably, the thickness of the polymer layer B) is from
0.1 to 500 .mu.m, especially preferably from 1 to 100 .mu.m.
[0072] The production of such layers can be carried out in a manner
known per se, for example by dissolving the composition in an
organic solvent, then casting to form a film and finally removing
the solvent.
[0073] Also possible for the production of the layers are processes
known from coating technology. Examples are spin-coating, spraying
or knife application processes, with spin-casting processes being
preferred.
[0074] Suitable solvents include alcohols, ethers, esters, acid
amides and ketones. Especially suitable are readily volatile
solvents, especially tetrahydrofuran.
[0075] In addition to those processes, in which the composition is
first of all dissolved, moulded and the solvent subsequently
evaporated again, hot-moulding processes are also possible, since
the composition is a thermoplastic material. Suitable processes
include extrusion, injection moulding, pressing or blowing
processes as known from thermoplastic plastics processing.
[0076] The layer may be transparent or slightly opaque. Preferably
it is transparent.
[0077] In order to improve the adhesion, the support materials may
be treated beforehand with adhesion promoters. For the same
purpose, a plasma treatment of the support material in order to
produce functional groups on the surface is also possible. The
surface may also be provided with copolymerisable groups in order
to achieve an especially high level of adhesion. Known adhesion
promoters for glasses are, for example,
triethoxy-glycidyloxy-silane, 3-azidopropyl-triethoxysilane and
3-aminopropyl-triethoxysilane. The thus treated surfaces may be
further modified, for example with
O--(N-succinimidyl)-6-(4'-azido-2'-nitrophenyl-
amino)-hexanoate.
[0078] The invention relates also to a process for the ionic
strength-independent, reversible optical determination of the pH
value of an aqueous sample according to the fluorescence method, in
which process an optical sensor, consisting of
[0079] A) a transparent support material
[0080] B) a layer of water-insoluble copolymers that are composed
of
[0081] a) from 39.9 to 60% by weight of N,N-dimethylacrylamide or
N,N-dimethylmethacrylamide;
[0082] b) from 60 to 39.9% by weight of a monomer of formula Ia or
Ib 5
[0083] wherein R.sub.a is hydrogen or C.sub.1-C.sub.6alkyl and
R.sub.b is C.sub.1-C.sub.12alkyl; with the proviso that R.sub.a and
Rb are not both methyl;
[0084] c) from 0.1 to 0.7% by weight of a proton-sensitive
fluorophore which is covalently bonded to the polymer; and
[0085] d) from 0 to 20% by weight of a diolefinic crosslinking
component,
[0086] the sum of the percentage weights of a) to d) being 100%, is
brought into contact with an aqueous test sample and irradiated
with excitation light, the fluorescence is measured, and the pH
value is calculated from the measured fluorescence intensity taking
calibration curves into consideration.
[0087] The above-described preferences in respect of the copolymers
and fluorescent dyes apply likewise to the sensor.
[0088] In detail, the procedure may be as follows: after
calibration with samples of known pH, a measurement of the
fluorescence intensity in contact with a test solution of unknown
composition is carried out and the pH with respect to the measured
fluorescence intensity is determined directly from the
calibration.
[0089] The sensors are brought into contact with the calibrating
solutions and with the test samples. This may be effected by hand
(for example by means of pipetting) or using a suitable automatic
flow system, the sensors being mounted in fixed position in a flow
cell. Such flow cells are known to the person skilled in the art
and may be adapted in a simple manner to the purpose in
question.
[0090] UV lamps (for example mercury vapour lamps, halogen lamps),
lasers, diode lasers and light-emitting diodes may be used as light
sources for the excitation of the fluorescence. It may be expedient
to filter out, by means of filters, light of the wavelength at
which the fluorescent dye has an absorption maximum. The
fluorescent light emitted by the sensors can be collected, for
example using a lens system, and then directed to a detector, for
example a secondary electron multiplier or a photodiode. The lens
system may be so arranged that the fluorescence radiation is
measured through the transparent support, via the edges of the
support, or via the analytical sample. Advantageously, the
radiation is deflected in a manner known per se by means of a
dichroic mirror. The fluorescence of the sensors is measured
preferably during contact with the calibrating solutions or sample
solutions.
[0091] The measurement may be effected under photostationary
conditions with continuous illumination, but can, if required,
alternatively be time-resolved. This can be achieved, for example,
by a laser pulse of limited duration or by modulation of the
intensity of a light source.
[0092] The response times may be less than 30 seconds and a first
measurement is already possible after less than about 5 minutes.
The sensors are furthermore distinguished by a high storage
stability.
[0093] Preferably, the process is used for test solutions that have
a pH of from 6.5 to 8.5, especially preferably a pH value of from
6.7 to 7.8.
[0094] The ionic strength of the test solution is preferably from
0.05 to 5 mol/l, especially preferably from 0.05 to 1 mol/l.
[0095] The test solution may comprise salts of inorganic or organic
acids. Examples are salts of citric acid, lactic acid or acetic
acid or also salts of phosphoric acid, hydrochloric acid and
sulfuric acid, or carbonate.
[0096] Preferably, the test solution comprises essentially 1,1- or
1,2-salts. Examples of 1,1-salts are LiCl, NaCl, KCl and
NH.sub.4Cl. Examples of 1,2-salts are CaCl.sub.2, MgCl.sub.2 and
K.sub.2SO.sub.4 as described, for example, in G. Kortum, Lehrbuch
der Elektrochemie, 4th edition, Verlag Chemie 1966, page 156.
[0097] Preferably, the test solution consists partly or wholly of a
body fluid. Especially preferably it consists partly or wholly of
blood.
[0098] The process can be performed as a single measurement or can
be performed continuously.
[0099] The invention relates also to the use of an optical sensor
described above for the ionic strength-independent optical
determination of the pH value of an aqueous test solution according
to the fluorescence method.
[0100] The following Examples illustrate the invention.
EXAMPLE A
Preparation of the Functionalised Fluorescent Dyes
Example A1
Preparation of 4-acryloylamidofluorescein (101)
[0101] 6
[0102] 5 g of 4-aminofluorescein are suspended in 200 ml of acetone
and, at 0.degree. C., 1.4 ml of acryloyl chloride in 2 ml of
acetone are added dropwise in the course of 10 min. The suspension
is stirred for 3 hours at room temperature. The crystals are
filtered off, washed with acetone and ether and dried. 5.7 g of
compound (101) having a melting point of > 200.degree. C., are
obtained. MS-FD: 402.
Example A2
Preparation of Compound (102)
[0103] a) 7
[0104] 29 ml of triethylene glycol monochlorohydrin and 20 g of
sodium azide are stirred overnight at 110.degree. C. without
solvent. The reaction mixture is diluted with ether and filtered
off. The solvent is evaporated and the filtrate is concentrated
under a high vacuum at 110-115.degree. C. Compound 103 is obtained
in a yield of 86%.
[0105] b) 8
[0106] 8.2 g of NaH (washed with pentane) are suspended in 150 ml
of dry tetrahydrofuran. 30 g of compound (103) are added dropwise
at 5.degree. C. The mixture is stirred for a further 30 min. and
subsequently reacted with 38 ml of .alpha.-bromoacetic acid
tert-butyl ester in 60 ml of tetrahydrofuran. The mixture is
stirred overnight, the ether is evaporated and the organic phase is
washed three times with water and once with salt solution and then
dried. The oil which remains is distilled under a high vacuum at
from 140 to 150.degree. C. Compound (104) is obtained. FAB-MS: 290
[M+H].sup.+. .sup.1H-NMR (CDCl.sub.3): 1.45 ppm (9H, s, t-Bu); 4.03
(2H, s, OCH.sub.2COO).
[0107] c) 9
[0108] The azide group of compound (104) is quantitatively reduced
with hydrogen, 5% Pd/C being used as catalyst and 1,4-dioxane being
used as solvent. Compound (105) is obtained. .sup.1H-NMR
(CDCl.sub.3): 1.45 ppm (9H, s, t-Bu); 2.2 (2H, broad, NH.sub.2);
2.9 (2H, t, J=6 Hz, CH.sub.2N); 4.03 (2H, s, OCH.sub.2COO).
[0109] d) 10
[0110] Compound (105) is dissolved in CH.sub.2Cl.sub.2 and treated
with 1.5 equiv. of NEt.sub.3 and 1.5 equiv. of acryloyl chloride at
0.degree. C. The clear solution is stirred for 5 hours, and then
washed with water, salt solution and water. The organic phase is
dried. The oil which remains is purified by chromatography on
silica gel with CH.sub.2Cl.sub.2 as eluant. The yield is 74% of
compound (106). .sup.1H-NMR (CDCl.sub.3): 1.45 ppm (9H, s, t-Bu);
4.03 (2H, s, OCH.sub.2COO); 5.10 (1H, m, CH.dbd.C); 6.05-6.35 (2H,
m, C.dbd.CH.sub.2).
[0111] e) 11
[0112] The tert-butyl ester of compound (106) is removed with a 1:1
mixture of trifluoroacetic acid and CH.sub.2Cl.sub.2 at room
temperature in the course of 6 hours. The reaction product (107) is
used directly for the next step without being purified.
[0113] f) The acid (107) is treated with one equivalent of
carbonyldiimidazole in tetrahydrofuran for 3 hours at room
temperature. 0.9 equivalent of 4-aminofluorescein in
tetrahydrofuran is added to that solution and the mixture is
stirred for 72 hours at room temperature. The reaction mixture is
dried and the product is purified by chromatography on silica gel
using MeOH/CH.sub.2Cl.sub.2 as eluant. Orange crystals of compound
(102) are obtained in a yield of 37% 12
[0114] with a melting point of 205.degree. C. (decomposition).
FAB-MS: 591 [M+H].sup.+, 613 [M+Na].sup.+, 629 [M+K].sup.+.
B) Preparation of the Copolymers
Example B1
[0115] In an ampoule provided with a 3-way tap, which is connected
to a vacuum and nitrogen, 2.19 g (22.1 mmol) of N,N
dimethylacrylamide, 2.81 g (22.1 mmol) of N-tert-butylacrylamide,
200 mg of 4-acryloylamidofluoresce- in (compound 101 from Example
A1) and 25 mg of azobisisobutyronitrile are dissolved in 15 ml of
dimethyl sulfoxide. The atmosphere in the ampoule is replaced with
nitrogen by a freeze/thaw cycle carried out three times. The
ampoule is maintained at 60.degree. C. for 2 days in a water bath.
The viscous contents of the ampoule are diluted with 100 ml of warm
methanol and the copolymer is precipitated by cautiously pouring
dropwise into 2 l of water with stirring. The copolymer is
filtered, and roughly dried. The precipitation is repeated twice
more. The thus purified end product is dried under a high vacuum at
60.degree..
[0116] Yield: 3.6 g or 69% of the theoretical yield, glass
transition temperature T.sub.g=156.degree. C., content of
N-tert-butylacrylamide=45.- 7% by weight (determined by
IR-spectroscopy), inherent viscosity of a 0.5% solution in
chloroform at 25.degree. C. .eta..sub.inh=1.07 dl/g.
Example B2
[0117] The procedure is as in Example B1 except that 125 mg of the
fluorescent dye (101) from Example A1 are added. A copolymer having
the following characteristics is obtained. Yield 4.4 g or 86% of
the theoretical yield, T.sub.g=152.degree. C., content of
N-tert-butylacrylamide= 54.2% by weight (determined by
IR-spectroscopy), inherent viscosity of a 0.5% solution in
chloroform at 25.degree. C. .eta..sub.inh=1.39 dl/g, dye content
determined by UV-spectroscopy= 2.2% by weight.
Example B3
[0118] The procedure is as in Example B1 except that 15 mg of the
fluorescent dye (101) from Example A1 are added. A copolymer having
the following characteristics is obtained. Yield 3.4 g or 68% of
the theoretical yield, T.sub.g=149.degree. C., content of
N-tert-butylacrylamide= 58.7% by weight (determined by
IR-spectroscopy), inherent viscosity of a 0.5% solution in
chloroform at 25.degree. C. .eta..sub.inh=1.68 dl/g, dye content
determined by UV-spectroscopy= 0.26% by weight.
Example B4
[0119] In an ampoule provided with a 3-way tap, which is connected
to a vacuum and nitrogen, 2.19 g (22.1 mmol) of
N,N-dimethylacrylamide, 2.81 g (22.1 mmol) of
N-tert-butylacrylamide, 35 mg of compound 102 from Example A2 and
25 mg of azobisisobutyronitrile are dissolved in 15 ml of dimethyl
sulfoxide. The atmosphere in the ampoule is replaced with nitrogen
by a freeze/thaw cycle carried out three times. The ampoule is
maintained at 60.degree. C. for 5 days in a water bath. The viscous
contents of the ampoule are diluted with 25 ml of warm methanol and
the copolymer is precipitated by cautiously pouring dropwise into
1.5 l of ether with stirring. The copolymer is filtered, and
roughly dried. The precipitation is repeated twice more. The thus
purified end product is dried under a high vacuum at 50.degree. for
2 days.
[0120] Yield: 4.05 g or 81% of the theoretical yield, glass
transition temperature T.sub.g=151.degree. C., content of
N-tert-butylacrylamide=51.- 9% by weight (determined by
IR-spectroscopy), inherent viscosity of a 0.5% solution in
tetrahydrofuran at 25.degree. C. .eta..sub.inh=1.34 dl/g, dye
content determined by UV-spectroscopy=0.44%.
Example B5
[0121] The procedure is as in Example B4 except that 2.31 g (23.29
mmol) of N,N-dimethylacrylamide, 2.69 g (19.06 mmol) of
N-methyl-N-butylacrylam- ide and 35 mg of the fluorescent dye (101)
from Example A1 are added.
[0122] A copolymer having the following characteristics is
obtained. Yield 3.72 g or 74% of the theoretical yield,
T.sub.g=90.degree. C., content of N,N-dimethylacrylamide=55.7 mol %
(determined by IR-spectroscopy), inherent viscosity of a 0.5%
solution in chloroform at 25.degree. C. .eta..sub.inh=1.17 dl/g,
dye content determined by UV-spectroscopy=0.62% by weight.
C
Production of the Sensors
Example C1
[0123] Glass substrates (platelets of 18 mm diameter) are first of
all cleaned in 30% sodium hydroxide solution and then activated in
65% nitric acid. The activated platelets are then silanised with
3-aminopropyltrimethoxysilane. The silanised platelets are left to
react for 1 hour at room temperature in a solution of
O-(N-succinimidyl)-6-(4'-- azido-2'-nitrophenylamino)-hexanoate in
dimethylformamide/borax buffer (5:1). The polymer of Example B1
(5%) is dissolved in methanol at from 20.degree. to 25.degree. and
applied in the form of a thin film, by spin-coating at a speed of
500 revs/min for 20 seconds, onto the platelets functionalised with
azido groups, irradiated for 15 min. and then dried for 12 h at
60.degree. under nitrogen. The layer thicknesses of the membranes
are approximately 1 .mu.m.
Examples C2 to C5
[0124] Procedure is as in Example C1 and the corresponding polymers
of Examples B2 to B5 are used
D
Application Examples
[0125] General Method
[0126] The sensors are mounted in a flow cell. The calibration and
sample solutions are metered by pumps and conveyed through the
cell. The measuring arrangement is thermostatically controlled. The
light of a halogen lamp (white light, excitation wavelength 480 nm)
is conducted through an excitation filter and reflected at a
dichroic mirror and focussed by lenses onto the planar sensors. The
fluorescent light emitted by the sensors (at 520 nm) is collected
by the same lens system and directed by an emission filter and the
dichroic mirror to a photodiode. The fluorescence of the sensors is
recorded while they are being acted upon by the calibration and
sample solutions. The pH value can be determined directly from the
measured value.
[0127] The following Table 1 illustrates the dependence of the pH
on the ionic strength of the electrolyte in the pH range of from
6.7 to 8.0 that results on the basis of the different membrane
compositions.
1TABLE 1 pK.sub.a Ionic strength Sensor from at ionic strength
dependence at 0.1 Example Amount of dye 0.1 mol/l and 0.3 mol/l C1
Comparison 4% by wt. not determinable very high (>1).sup.1 test.
C2 Comparison 2.2% by wt. 7.3 high (1).sup.1 test C3 0.26% by wt.
7.3 independent (0).sup.1 C4 0.44% by wt. 7.3 independent (0).sup.1
C5 0.62% by wt. 7.5 independent (0).sup.1 .sup.1The numerical
values relate to the difference between the pK.sub.a values, which
were measured once at an ionic strength of 0.1 mol/l and once at an
ionic strength of 0.3 mol/l.
* * * * *