Fluorescent N-alkylated acrylamide copolymers and optical pH...

Details Fluorescent N-alkylated acrylamide copolymers and optical pH... Fluorescent N-alkylated acrylamide copolymers and optical pH...

2001-03-08

2001-10-16

Henderson, Christopher (Department: 1713)

Chemistry: analytical and immunological testing

Including titration or ph determination

C422S051000, C422S082080

Reexamination Certificate

active

06303385

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
2. Description of Related Art
It is known that the pK
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.
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.
BRIEF SUMMARY OF THE INVENTION
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.
DETAILED DESCRIPTION OF THE INVENTION
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.
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.
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.
The invention relates to 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
 wherein R
a
is hydrogen or C
1
-C
6
alkyl and R
b
is C
1
-C
12
alkyl; with the proviso that R
a
and R
b
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%.
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.
The alkyl radicals may be linear or branched. Examples of C
1
-C
12
alkyl 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.
The monomer preferably used as monomer a) is N,N-dimethylacrylamide. Preferred water-insoluble copolymers are obtained when R
a
is hydrogen and R
b
is a branched C
3
-C
8
alkyl. Especially preferred are water-insoluble copolymers in which R
a
is hydrogen, R
b
is tertiary butyl and the ratio of monomer a) to monomer b) is 50 parts by weight to 50 parts by weight.
Another group of preferred water-insoluble copolymers is obtained when R
a
is methyl or ethyl and R
b
is linear C
3
-C
8
alkyl. Especially preferably, R
a
is methyl and R
b
is n-butyl.
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.
Preferably, the fluorophores are selected from the group consisting of acridines, acridones, rhodamines, xanthenes, benzoxanthenes, pyrenes and coumarins, which are either admixed with or covalenty bonded to the polymer.
Preferred are water-insoluble copolymers in which the flourophore is covalently bonded to the polymer.
Especially preferred are water-insoluble copolymers in which the fluorophore is a compound of formula II, III, IV, V or VI
wherein
R
1
, R
2
, R
5
and R
6
are each independently of the others hydrogen, —SO
2
—(C
1
-C
6
)alkylphenyl, C
1
-C
30
alkyl, C
1
-C
30
alkyl-CO— or a radical of the formula —(C
n
H
2n
—O—)
m
—R
8
;
R
3
is hydrogen or —SO
2
—(C
1
-C
6
)alkylphenyl;
R
4
and R
7
are a C
1
-C
30
alkylene or a radical of the formula —(C
n
H
2n
—O—)
m
—R
8
;
Z is a divalent radical —NH—CO—;
R

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