Method for quantitatively determining a chemical parameter of a sample

(42) Date of commencement of the patent: 15.12.1990 (45) Date of issue: 25. 7.1991

<td>(56) Documents:</td><td>(73) Patent owner:</td>

<td>DE-OS 3346810 EP-A2-0214768 EP-A2-0242527</td><td>AVL COMPANY FOR INTERNAL COMBUSTION ENGINES UM) MESSTECHNIK MBH. PROF.DR.DR.HC HANS LIST A-8020 GRAZ, STYRIA (AT).</td>

<td></td><td>(72) Inventor:</td>

<td></td><td>WOLFBEIS OTTO S. DR. GRAZ, STYRIA (AT). LEINER MARCO JEAN-PIERRE DR. GRAZ, STYRIA (AT).</td>

(54) METHOD FOR THE QUANTITATIVE DETERMINATION OF AT LEAST ONE CHEMICAL PARAMETER OF A SAMPLING MEDIUM (57) In a method for the quantitative determination of at least one chemical parameter of a sample medium, in which two substances in close physical contact are used, as first substance a fluorophore with an excitation and an emission spectrum, which does not respond to the parameter to be determined, and as a second substance a substance which reacts to the parameter of the sample medium to be determined by changing its absorption spectrum, wherein the emission spectrum of the fluorophore overlaps at least partially with the absorption spectrum of the second substance, is proposed to improve long-term stability and avoid frequent calibrations, the second substance used is a chromophore influencing the fluorescence of the fluorophore, whose absorption maximum is shifted depending on the parameter to be determined and that the reduction of the fluorescence decay time t caused by energy transfer between the chromophore and the fluorophore is used for the quantitative determination of the chemical parameter.

AT 393 035 ufflseraaifl

AT 393 035 Β

The invention relates to a method for the quantitative determination of at least one chemical parameter of a sample medium, in which two substances in close physical contact are used, as first substance a fluorophore with an excitation and an emission spectrum, which does not respond to the parameter to be determined, and as a second substance a substance which reacts to the parameter of the sample medium to be determined by changing its absorption spectrum, wherein the emission spectrum of the fluorophore overlaps at least partially with the absorption spectrum of the second substance.

The determination of physical or chemical parameters, in particular the determination of substances contained in a sample medium by means of optical sensors, has made significant progress in recent years. Such optical sensors (optodes) usually consist of an indicator, which is applied to a solid support and is in contact with the sample. The change, caused by the parameter to be determined, of at least one optical property of the indicator is measured in an optical system consisting of a light source, optical filters, possibly fiber-optic light guides, and photodetectors.

A distinction is made between absorption, reflection and fluorescence-optical sensors. In the hitherto known embodiments, the fluorescence intensity is predominantly used as the measurement parameter, the concentration [Q] of a substance Q which quenches the fluorescence of the indicator being determined according to the following equation:

F₍/ F = 1 + K_SV[Q] (1)

Here mean F_O and F the fluorescence intensities of the indicator quenched by Q in the absence or in the presence of the quenching gas in a concentration [Ql · Kg_V is an indicator-specific constant.

In the aforementioned method according to EP-A 214 768, a method is now described for the determination of certain substances, in which a fluorescent substance (fluorophore) and an absorber substance are immobilized in close spatial contact on a support. The emission spectrum of the fluorescent substance after excitation overlaps with the absorption spectrum of the absorber substance. While the fluorophore does not react to the substance to be determined, a reaction with the absorber substance occurs upon sample contact, through which the degree of overlap of the emission and absorption spectrum varies depending on the concentration of the substance to be determined. The resulting change in fluorescence intensity is measured and used as a measure of the parameter to be determined.

The disadvantages of the intensity measurement consist primarily in the fact that the fluorescence intensity F is influenced by fluctuations in the intensity of the excitation light source. Furthermore, F is dependent on the concentration of the fluorophore, so that fading of the same drift occurs. Finally, photodetectors can also vary in their sensitivity, which in turn affects the measured intensity F.

In those cases where the analytical measure acts as a dynamic quencher on the fluorescence of an indicator, one can also use the change in fluorescence fading as a parameter since, according to Stem-Vomer, between the fluorescence decay time in the presence (t) and in the absence (ίθ) of a quencher Concentration [Q] the following relation holds:

ιθ / ι = ι + κ_8ν[θ] (2)

Dynamic quenchers are those quenchers that quench the fluorescence of a molecule by deactivating the excited state of a molecule as a result of a dynamic collision process. This is in contrast to the static quencher, which exerts its effect in that it forms a loose complex with the fluorescence even in the ground state, which does not fluoresce. The two extinguishing mechanisms have different effects on the cooldown. * The dynamic extinguisher reduces the cooldown after (2), while the static one does not

Typical sensors based on the measurement of cooldown have been described in DE-OS 3 346 810. The advantage of measuring the fluorescence decay time is, above all, that the method is significantly less susceptible to errors than if the concentration of the quencher Q is determined by measuring the fluorescence intensity, since the lifetime t of a fluorophore is independent of the intensity of the light source, the concentration of the fluorescence Dye and the sensitivity of the photodetector. Thus, lifetime measurements are considerably superior to the intensity measurements despite the higher metrological effort required.

Previously known optical methods based on the measurement of the cooldown are only for dynamic

Löscher the fluorescence suitable, ie z. For example, oxygen, SO₂ or halothane. For example

-2AT393 035B of the pH or other analytical parameters, which result in color changes or envelopes with known per se indicators, lifetime measurements are unsuitable since the color-forming reactions take place in the electrical ground state.

In this connection, reference should be made to EP-A-0 242 527, which describes a method for the determination of chemical analytes with the aid of antigens or antibodies, ie certain proteins which are capable of specifically binding the analyte. This is a so-called homogeneous immunoassay, thus a method in which an antigen binds a specific antibody. By the incoming binding, energy transfer from one donor fluorescent moiety bound to a binding partner to one acceptor fluorescent moiety bound to the other binding partner is possible as long as there is a spectral overlap and donor and acceptor are within the FörsterRadius.

The object of the present invention is to refine the method mentioned above so that parameters which can not be detected by means of the advantageous method of measuring the decay time can be supplied to quantitative determination.

This object is achieved according to the present invention in that as the second substance, a fluorescence of the fluorophore influencing chromophore is used, the Absorptionsmaxium is shifted depending on the parameter to be determined and that caused by energy transfer between the chromophore and fluorophore reduction of fluorescence decay time t for the quantitative determination of the chemical parameter. It has surprisingly been found that the energy transfer between the chromophore and the fluorophore causes a change in the fluorescence decay time known hitherto only in the case of dynamic fluorescence quenching, which effect is used according to the invention for determining the parameters.

Depending on the width of the absorption band of the nonfluorescent chromophore, the emission of the fluorophore is influenced, which leads to a reduction of its decay time. As a result, surprisingly, it is also possible to detect substances via the measurement of the cooldown which do not occur as dynamic quenchers of the fluorescence radiation of the fluorophore.

The method described thus has a considerably improved long-term stability compared to conventional methods, which allows the use of corresponding measuring arrangements in measuring stations, whereby the frequently required frequent calibrations are unnecessary.

The theoretical basis for this effect is the so-called energy transfer (ET). Thereafter, electronic energy can be transferred from a donor (here the fluorophore) to an acceptor (here the anaiytic sensitive chromophore). Free photons do not occur in this process. The ET obeys the Förster equation k_et= r₀⁶/ (A)

P) where Kgy is the rate constant for the ET and R_Q or r the so-called critical distance or the current distance of donor and acceptor mean. R_Q (Förster radius) is the distance at which the probability of a Förster ET is the same as that of a spontaneous ET.

The efficiency of the energy transfer thus depends on the quantum efficiency of the donor, the overlap of the emission spectrum of the acceptor, and their relative orientation and distance. Typical transfer distances are between 0.5 and 10 nm. The distance between donor and acceptor has a great influence on the energy transfer since it depends on its sixth power.

An application of the invention provides that used to determine the hydrogen ion concentration as a fluorophore 7-diethylaminocoumarin-3-carboxylic acid and as a chromophore Methylorange wild, or that for their determination as a fluorophore 8-aminopyrene-l, 3,6-trisulfonate and as a chromophore phenol red is used.

Advantageously, the fluorophore (donor) and chromophore (acceptor) are covalently linked together.

In the table below examples of different chemical parameters are given for examples of Fhiorophore / chromophore combinations, care being taken that the fluorescence decay time of the fluorophores used is measurable.

-3AT393 035B

<td>fluorophore (Donor)</td><td>chromophore (Acceptor)</td><td>Measuring wavelength (nm)</td><td>Chem. Parameters</td>

<td>7-Diethylaminocouma- rin-3-carbonsüure</td><td>methyl orange</td><td>480</td><td>H⁺</td>

<td>8-aminopyrene-l, 3,6- trisulfonate</td><td>phenol</td><td>490</td><td>H⁺</td>

<td>Sulfrhodamine 101</td><td>Alizarin complexone</td><td>590</td><td>Ba²⁺</td>

<td>Rhodamine 6G</td><td>Beryllon</td><td>570</td><td>mg²*</td>

<td>2,7-Dichlorflou-</td><td>catechol-</td><td>540</td><td>CD²⁺</td>

<td>rescein</td><td>violet</td><td></td><td></td>

<td>fluorescein</td><td>Chromazurol S</td><td>520</td><td>al³⁺</td>

<td>Coumarin 7</td><td>Eriochrom Black T</td><td>485</td><td>Ca²⁺, Pb²⁺</td>

<td>Coumarin 343</td><td>murexid</td><td>450</td><td>Cu²⁺</td>

<td>Coumarin 334</td><td>zincon</td><td>440</td><td>Zn²⁺</td>