In known devices and methods for the quantitative determination of the concentration of fluorophores of at least one substance in a sample, the sample is irradiated with light of an excitation wavelength emitted from an excitation light source. The intensity of the fluorescent light of an emission wavelength emitted from the sample is measured by means of a detection element. Such a device and such a method are, for example, known from DE 10 2008 057 115 A1.
In order to guarantee exact measurement results, a reference object, in particular a so-called fluorescence standard or a suitable optical element, is used to calibrate a measured intensity value of the fluorescent light. When irradiated with excitation light of a predetermined wavelength and intensity, the reference object emits light having a known wavelength distribution and/or intensity. The long-term stability of fluorescence standards is often insufficient, whereas the use of an optical element as a reference object, as known from DE 10 2008 057 115 A1, guarantees the required long-term stability. Reference objects can be used as a calibration standard as well as additionally or alternatively as a quality assurance standard or as a reference standard for the direct referencing of performed measurements. Reference standards are often used in so-called scanning systems in which the reference standard and at least one sample are successively irradiated with light, and the light that is incident on a detection element and comes from the reference standard is detected as a first measurement value and the light that is incident on the detection element and comes from the sample is detected as a second measurement value, wherein by means of the ratio of the first measurement value to the second measurement value the concentration of fluorophores in the sample is determined. Preferably, the invention, too, can be realized as a scanning system.
From EP 0 237 363 A2, a method and a device for determining the fluorescence of a test sample are known. From U.S. Pat. No. 6,242,114 an optical composite having fluorescent properties is known, which comprises a support layer with fluorescent material, which is optically coupled to a selective spectral filter.
In the fluorescence-based analysis of samples, in particular of body fluids, for example in the fluorescence-based analysis of sample fluids applied to test strips, in known devices absolute values are determined by a referencing with an internal reference standard. The excitation efficiency of the fluorophore used for analyzing the sample is generally wavelength-dependent so that the sample or the test strip with the sample is irradiated with a narrow-band light having a preset intensity in order to be able to draw conclusions on the number of fluorophores and thus on the amount of a substance in the sample to which the fluorophores have been attached based on the light emitted from the sample or, respectively, the test strip. A change in the spectrum of the light source generating the excitation light, in particular a shift in the wavelength of the light emitted from the light source, partially results in considerable distortions of the measurement result, in particular a change in wavelength has a very different effect or has an even opposite effect on the light emitted from the fluorophores and on the light emitted from the reference object. The wavelength or, respectively, the wavelength spectrum of the light source can, for example, change due to temperature influences. Further, such a change in wavelength is also possible as a result of so-called spontaneous jumps which may occur in the course of the lifetime of the light source. Such spontaneous jumps, for example, occur in the case of lasers used as a light source. In the case of a change in wavelength as a result of temperature influences, a compensation can be accomplished with a suitable algorithm by way of a temperature measurement in that the temperature influences are appropriately taken into account in the evaluation of the measurement values. In the case of the mentioned spontaneous wavelength jumps, such a compensation is not possible so that there will be faulty measurements.
It is the object of the invention to specify devices and methods for determining the concentration of fluorophores in a sample, by which the influence on a measurement result due to a changing wavelength of the light source used for illuminating the sample is reduced.
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