Method for quantitatively determining a chemical parameter of a sample.

0001The 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 the first substance a fluorophore with an excitation and an emission spectrum that does not respond to the parameter to be determined, and as a second substance a substance reacting to the parameter of the sample medium to be determined by changing its absorption spectrum, wherein the emission spectrum of the fluorophore at least partially overlaps with the absorption spectrum of the second substance.

0002The determination of physical or chemical parameters, in particular the determination of substances contained in a sample medium with the aid 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 in at least one optical property of the indicator caused by the parameter to be determined is measured in an optical system consisting of a light source, optical filters, possibly fiber-optic light guides, and photodetectors.

0003A distinction is made between absorption, reflection and fluorescence optical sensors. In the previously 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 using the following equation:<maths id="math0001" num=""><math display="inline"><mrow><msub><mrow><mtext>F</mtext></mrow><mrow><mtext>O</mtext></mrow></msub><msub><mrow><mtext>/ F = 1 + K</mtext></mrow><mrow><mtext>sv</mtext></mrow></msub><mtext>[Q] (1)</mtext></mrow></math><img file="EP0397641B1_D0001.tif" /></maths> Here F mean_O and F the fluorescence intensities of the indicator quenched by Q in the absence or presence of the quencher in a concentration [Q]. K_sv is an indicator-specific constant.

0004In the method mentioned at the outset in accordance with EP-A 214 768, a method for determining certain substances is now described which is based on a reduction in fluorescence due to an energy transfer between a fluorescent substance (fluorophore) and an absorber substance which are immobilized on a support in close physical contact. 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, there is a reaction with the absorber substance when the sample comes into contact, by means of which the degree of overlap of the emission and absorption spectrum varies depending on the concentration of the substance to be determined. The change in fluorescence intensity that can be achieved in this way is measured and used as the Mab for the parameter to be determined.

0005The 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 the same drift will develop. Finally, the sensitivity of photodetectors can also vary, which in turn affects the measured intensity F.

0006In those cases where the analytical parameter acts as a dynamic quencher for the fluorescence of an indicator, the change in the fluorescence decay can also be used as a parameter, since according to Stern-Volmer between the fluorescence decay time in the presence (t) and in the absence (t_O) of an extinguisher of concentration [Q] the following relationship applies:<maths id="math0002" num=""><math display="inline"><mrow><msub><mrow><mtext>t</mtext></mrow><mrow><mtext>O</mtext></mrow></msub><msub><mrow><mtext>/ t = 1 + K</mtext></mrow><mrow><mtext>sv</mtext></mrow></msub><mtext>[Q] (2)</mtext></mrow></math><img file="EP0397641B1_D0002.tif" /></maths> Dynamic quenchers are those fluorescent quenchers which quench the fluorescence of a molecule by deactivating the excited state of a molecule without radiation as a result of a dynamic collision process. This is in contrast to the static quencher, which exerts its effect by forming a loose complex with the fluorescent in its basic state, which does not fluoresce. The two extinguishing mechanisms have different effects on the cooldown: The dynamic quencher reduces the cooldown according to (2), the static one does not.

0007Typical sensors, which are based on the measurement of the decay time, have been described in DE-OS 3 346 810. The main advantage of measuring the fluorescence decay time is 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 Dye and the sensitivity of the photodetector is. Thus, lifetime measurements are considerably superior to the intensity measurements, despite the higher technical expenditure required.

0008Previously known optical methods based on the measurement of the decay time are only suitable for dynamic quenchers of fluorescence, for example oxygen, SO₂ or halothanes. Lifetime measurements are unsuitable for determining, for example, the pH value or other analytical parameters which result in color changes or changes with indicators known per se, since the color-forming reactions take place in the electrical ground state.

0009In this connection, reference is also made to EP-A-0 242 527, which describes a method for the determination of chemical analytes with the aid of antigens or antibodies, that is to say certain proteins which are able to bind the analyte specifically. This is a so-called homogeneous immunoassay, a method in which an antigen binds a specific antibody. The binding that occurs enables energy transfer from a donor fluorescent, which is bound to one binding partner, to an acceptor fluorescent, which is bound to the other binding partner, as long as there is a spectral overlap and the donor and acceptor are within the Förster radius.

0010The object of the present invention is to develop the method mentioned at the outset in such a way that parameters which have hitherto not been ascertainable using the advantageous method of measuring the decay time can be fed to the quantitative determination.

0011This object is achieved according to the present invention in that a chromophore influencing the fluorescence of the fluorophore is used as the second substance, the absorption maximum of which is shifted depending on the parameter to be determined, and in that the reduction in the fluorescence decay time t caused by energy transfer between the chromophore and fluorophore is used for quantitative determination of the chemical parameter is used. Surprisingly, it has been shown that the energy transfer between the chromophore and the fluorophore causes a change in the fluorescence decay time that has hitherto only been known in dynamic fluorescence quenching, which effect is used according to the invention to determine the parameters.

0012Depending on the width of the absorption band of the non-fluorescent chromophore, the emission of the fluorophore is influenced, which leads to a reduction in its decay time. As a result, substances that do not appear as dynamic quenchers of the fluorescent radiation of the fluorophore can surprisingly also be detected by measuring the decay time.

0013The described method thus has a considerably improved long-term stability compared to conventional methods, which allows the use of appropriate measuring arrangements in measuring stations, whereby the frequent calibrations previously required are unnecessary.

0014The theoretical basis for this effect is the so-called energy transfer (ET). Electronic energy can then be transferred from a donor (here the fluorophore) to an acceptor (here the analytically sensitive chromophore). Free photons do not occur in this process. The ET obeys the Förster equation<maths id="math0003" num=""><math display="inline"><mrow><msub><mrow><mtext>K</mtext></mrow><mrow><mtext>ET</mtext></mrow></msub><msub><mrow><mtext> = R</mtext></mrow><mrow><mtext>O</mtext></mrow></msub><mtext>⁶ / (r⁶t) (3)</mtext></mrow></math><img file="EP0397641B1_D0003.tif" /></maths> where k_ET is the rate constant for the ET and R_O or r mean the so-called critical distance or the current distance between donor and acceptor. R_O (Ranger radius) is the distance at which the probability of a Ranger ET is the same as that of a spontaneous ET.

0015The efficiency of the energy transfer thus depends on the quantum yield of the donor, the overlap of the emission spectrum of the acceptor, and their relative orientation and distance. Typical transmission distances are between 0.5 - 10 nm. The distance between donor and acceptor has a great influence on the energy transfer since it depends on its 6th power.

0016An application according to the invention provides that 7-diethylaminocoumarin-3-carboxylic acid is used as the fluorophore and methyl orange as the chromophore for the determination of the hydrogen ion concentration, or 8-aminopyrene-1,3,6-trisulfonate and as the chromophore phenol red for the determination thereof as the fluorophore is used.

0017The fluorophore (donor) and chromophore (acceptor) are advantageously covalently linked to one another.

0018Examples of fluorophore / chromophore combinations are given in the table below for determining different chemical parameters, care being taken to ensure that the fluorescence decay time of the fluorophores used can be measured. <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Fluorophore (donor)</entry><entry namest="col2" nameend="col2" align="center">Chromophore (acceptor)</entry><entry namest="col3" nameend="col3" align="center">Measuring wave length (nm)</entry><entry namest="col4" nameend="col4" align="center">Chemical parameters</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">7-diethylaminocoumarin-3-carboxylic acid</entry><entry namest="col2" nameend="col2" align="left">Methyl orange</entry><entry namest="col3" nameend="col3" align="right">480</entry><entry namest="col4" nameend="col4" align="left">H⁺</entry></row><row><entry namest="col1" nameend="col1" align="left">8-aminopyrene-1,3,6-trisulfonate</entry><entry namest="col2" nameend="col2" align="left">Phenol red</entry><entry namest="col3" nameend="col3" align="right">490</entry><entry namest="col4" nameend="col4" align="left">H⁺</entry></row><row><entry namest="col1" nameend="col1" align="left">Sulfrhodamine 101</entry><entry namest="col2" nameend="col2" align="left">Alizarin complexon</entry><entry namest="col3" nameend="col3" align="right">590</entry><entry namest="col4" nameend="col4" align="left">Ba²⁺</entry></row><row><entry namest="col1" nameend="col1" align="left">Rhodamine 6G</entry><entry namest="col2" nameend="col2" align="left">Beryllon</entry><entry namest="col3" nameend="col3" align="right">570</entry><entry namest="col4" nameend="col4" align="left">Mg²⁺</entry></row><row><entry namest="col1" nameend="col1" align="left">2,7-dichlorofluorescein</entry><entry namest="col2" nameend="col2" align="left">Catechol violet</entry><entry namest="col3" nameend="col3" align="right">540</entry><entry namest="col4" nameend="col4" align="left">Cd²⁺</entry></row><row><entry namest="col1" nameend="col1" align="left">Fluorescein</entry><entry namest="col2" nameend="col2" align="left">Chromazurol S</entry><entry namest="col3" nameend="col3" align="right">520</entry><entry namest="col4" nameend="col4" align="left">Al³⁺</entry></row><row><entry namest="col1" nameend="col1" align="left">Coumarin 7</entry><entry namest="col2" nameend="col2" align="left">Eriochromblack T</entry><entry namest="col3" nameend="col3" align="right">485</entry><entry namest="col4" nameend="col4" align="left">Ca²⁺, Pb²⁺</entry></row><row><entry namest="col1" nameend="col1" align="left">Coumarin 343</entry><entry namest="col2" nameend="col2" align="left">Murexid</entry><entry namest="col3" nameend="col3" align="right">450</entry><entry namest="col4" nameend="col4" align="left">Cu²⁺</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Coumarin 334</entry><entry namest="col2" nameend="col2" align="left">Zincon</entry><entry namest="col3" nameend="col3" align="right">440</entry><entry namest="col4" nameend="col4" align="left">Zn²⁺</entry></row></tbody></tgroup></table></tables>