Covalently immobilized fluoroionophores as optical ion sensors

Details Covalently immobilized fluoroionophores as optical ion sensors Covalently immobilized fluoroionophores as optical ion sensors

1998-12-07

2002-07-09

Soderquist, Arlen (Department: 1743)

Chemistry: analytical and immunological testing

Metal or metal containing

C422S051000, C422S082050, C422S082060, C422S082070, C422S082080, C436S079000, C436S172000, C546S102000, C546S104000

Reexamination Certificate

active

06417005

ABSTRACT:

This application is a 371 application of PCT/EP97/01695 filed Apr. 4, 1997.
The present invention relates to fluoroionophores that are covalently bound to organic or inorganic materials either directly or via a bridging group, and to processes for their preparation. The invention relates also to a) a sensor for determining ions, polar substances or lipophilic substances, especially in aqueous solutions, which sensor comprises the immobilised fluoroionophores in an active layer; b) a method for the qualitative and quantitative determination of ions, polar substances or lipophilic substances, especially in aqueous solutions, using the optical sensor, and c) a composition, as a coating composition for sensors, of a polymer having covalently bound fluoroionophores or of a polymer in which there is incorporated an inorganic or organic carrier material to the surfaces of which fluoroionophores have been covalently bound.
The optical determination of ions has recently gained greater importance, the presence or concentration of ions being measured, for example, by means of a change in the absorption or fluorescence of a suitable dye. The sensors, also called optrodes, generally consist of a transparent support material and an active layer. The active layer normally comprises a transparent hydrophobic polymer and a lipophilic plasticiser for the purpose of obtaining adequate diffusion of the ions and adequate solubility of the active components. Active components are a specific ionophore as a complexing agent for ions, a counterion for maintaining electrical neutrality, and an indicator substance which, as a result of a chemical change or a physical change in the environment, emits a measurable optical signal. The disadvantages of many such optical sensors are that their response times are too long, they are not sufficiently stable, they are pH-dependent, and the active constituents are washed out.
The response times may be shortened by covalent linkage of ionophore and fluorophore to form the so-called fluoroionophores. Such fluoroionophores are known from WO 89/00997 and U.S. Pat. No. 4,367,072.
In
J. Mater. Chem
. 4(1), (1994), pp. 145-151, Perez-Jimenez et al describe two novel fluoroionophores that comprise 4 anthracene units covalently bound to calix[4]arene via an amide or ester bond. Disadvantageous fluoroescence-quenching effects may occur as a result of the close adjacency of the four anthracene units in the molecule.
EP 0 484 865 describes an optical potassium sensor that comprises a crown ether as ionophore covalently linked to two fluorophores. In order to disperse the fluoroionophore, the polymer gel is prepared in the presence of the functional-group-free fluoroionophore, the fluorophore being non-covalently bound to the polymer gel.
EP 0 578 630 describes a sensor membrane for an optical sensor having an indicator substance that is ionically bound to the polymer matrix of the sensor membrane. The indicator substance is likewise in the form of a pair of ions and is anchored in the polymer membrane by a counterion balancing the electrical charge of the dye molecule. The counterion is derived from compounds that comprise an ionic group and a) an oligomer radical of the monomer that forms the basis of the polymer matrix in question, b) long-chained alkyl or alkylene groups or c) silyl groups. When aqueous test samples, such as, for example, blood are used, even with that method it is not possible effectively to prevent the fluoroionophore from being washed out.
A substantial disadvantage of the methods described hitherto is that the fluoroionophores are in time washed out of the active layer of a sensor. The result is that the usable life of the sensors is too short and, even where their service life is relatively long, the sensors still have to be recalibrated.
It has now, surprisingly, been found that fluoroionophores having a functional group on the fluorophore can be covalently bound to the functional groups of an inorganic or organic material and still be suitable for fluorescence detection. It is in that manner possible virtually completely to prevent the fluoroionophore from being washed out of the active layer of the sensor. The usable life of the sensor is consequently considerably increased. Undesired error corrections of the measured values (drift) caused by inaccurate measurements resulting from the fluoroionophore being washed out can be avoided practically completely.
It has also, surprisingly, been found that the selective binding affinity of the ionophore for the ions is not significantly impaired, or is not impaired at all, by the covalent bonding or by the introduction only of a functional group.
It has also, surprisingly, been found that the bonding of ions to the ionophores of the covalently bound fluorojonophores results in an adequate perturbation of the fluorescing group, the complexing of ions by way of interactions between fluorophore and ionophore leading to a measurable signal change, with the result that measurements that are substantially independent of pH are possible. Consequently, direct analysis of ions in body fluids (blood, urine, serum), natural waters or waste water is possible.
The invention accordingly relates firstly to fluoroionophores, functionalised with reactive groups, of formula (I)
I—R
1
—F—R
2
—G  (I)
wherein
I is a monovalent residue of an ionophore,
F is a divalent residue of a fluorophore,
G is a functional group and
R
1 and R
2
are each independently of the other a direct bond or a bridging group.
Preferably, R
1
and R
2
are each independently of the other a bridging group.
Ionophores are natural or synthetic organic compounds that contain a plurality of mostly alternating electron-rich hetero atoms such as, for example, S, N and especially O, in an open-chained or cyclic carbon chain and that enable the ions to be measured to be selectively complexed. Such ionophores are described, for example, in US-A-4,645,744.
The monovalent ionophores from which I in formula (I) is derived may be substances that have an oligoether, polyether, oligoester, polyester, oligoamide or polyamide structure. Examples of such suitable substances may be crown ethers, coronandenes, cryptandenes, calixarenes, podandene or derivatives thereof, also cyclic peptides, for example valinomycin, nonactin, peptides such as gramicidin, and peptides which in the presence of the ion to be determined change their secondary, tertiary or quaternary structure for bonding the ion; it is also possible to use tetrahydrofuran-containing macrolides bonded via ester bridges, and analogous substances that are able to regulate transport in biological systems, or cyclic oligosaccharides, such as, for example, cyclodextrins or cyclophanes.
The functional group G may be a carboxy, sulfonic acid, acid halide, amide, thiol, amine alcohol, cyanate, isocyanate, oxime, aldehyde or ketone group or a polymerisable group.
The functional group G is preferably a hydroxyl, thiol or amine group.
The functional group G may also be a polymerisable group and in that case is preferably a vinyl group that is unsubstituted or substituted by C
1
-C
4
alkyl.
The polymerisable group is bonded to the fluorophore preferably via the bridging group R
2
. The polymerisable radical may be selected from the group —O—R
8
, —S—R
8
, —NR
7
R
8
, —NR
7
C(O)R
8
, —OC(O)R
8
, —C(O)OR
8
, —C(O)NR
7
R
8
, —CH═N—O—R
8
and —NH—C(O)—NR
7
R
8
, wherein R
7
is H or C
1
-C
4
alkyl and R
8
is an olefinic group having from 1 to 12 carbon atoms, preferably from 1 to 4 carbon atoms, it being possible for those groups to be bonded to the fluorophore directly or via a C
1
-C
20
alkylene radical. The polymerisable radicals are preferably —NR
7
C(O)R
8
wherein R
7
is preferably H and R
8
is an olefinic group.
R
8
is preferably an ethylenically unsaturated organic group of formula (V)
—CR
9
═CHR
10
  (V),
wherein
R
9
is H or C
1
-C
4
alkyl, especially methyl, and
R
10
is H, C
1
-C
12
alkyl, phenyl or benzyl.
R
9
is preferably H or methyl and R
10
is preferably H.
The

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