• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Method and device (1) for determining a concentration (c) of a fluorescent substance in a medium, wherein the medium with the fluorescent substance is irradiated by an excitation radiation having an excitation wavelength (λex), so that the fluorescent substance emits fluorescence radiation with a Fluorescence wavelength (λem) is excited, whereby the intensity of the fluorescence radiation (Ifluor) is measured, from which the concentration (c) of the fluorescent substance in the medium is determined, wherein also the intensity (Itrans, ex) of a transmitted portion of the excitation radiation is measured, wherein the concentration (c) of the fluorescent substance from the intensity of the transmitted portion of the excitation radiation (I trans, ex) and the intensity of the fluorescence radiation (Ifluor) is determined.
    公开号:AT513863A1
    申请号:T50110/2013
    申请日:2013-02-15
    公开日:2014-08-15
    发明作者:
    申请人:Vwm Gmbh；
    IPC主号:
    专利说明:

    1
    The invention relates to a method for determining a concentration of a fluorescent substance in a medium, wherein the medium with the fluorescent substance is irradiated by an excitation radiation having an excitation wavelength, so that the fluorescent substance is excited to emit fluorescence radiation having a fluorescence wavelength, wherein the intensity the fluorescence radiation is measured, from which the concentration of the fluorescent substance in the medium is determined.
    The invention further relates to a device for determining a concentration of a fluorescent substance in a medium, comprising at least a first radiation source for emitting an excitation radiation having an excitation wavelength, a first sensor for measuring the intensity of a fluorescence radiation excited by the excitation radiation and having a fluorescence wavelength, and with a computing unit for determining the concentration of the fluorescent substance as a function of the intensity of the fluorescence radiation.
    From the prior art, a method for determining the presence of a fluorescent substance in a medium is known in which the fluorescent substance is excited by means of an excitation radiation targeted to fluorescence radiation. The intensity of the fluorescence radiation is measured in order to conclude, depending on the measurement result, on the presence of the fluorescent substance. However, the fluorescence measurement as such is generally not regarded as a reliable quantitative measurement method, since only in exceptional cases from the intensity of the fluorescence radiation on the concentration of the fluorescent substance can be deduced. In immunology, for example, antibodies are provided with a fluorescent chemical group so that the sites to which the antibodies bind are recognizable by fluorescence. The antigen concentration can thus be partly determined quantitatively.
    In any case, it has been observed in practice that the desired accuracy in concentration determination was not normally achieved by measuring the fluorescence. On the one hand, an influence of the geometry of the measuring chamber on the measuring accuracy was observed. Even with time-varying conditions no satisfactory results were achieved with the known method. This problem could not be solved so far.
    Accordingly, the object of the present invention is to provide a method and a device of the type mentioned, with which or with which a highly accurate determination of the concentration of a fluorescent substance is made possible. The aim is, in particular, to reduce or eliminate the influence of the geometry of the measuring chamber on the measuring accuracy. In addition, the concentration determination should be made precisely even with time-varying conditions.
    According to the invention, the intensity of a transmitted portion of the excitation radiation is also measured to solve this object, wherein the concentration of the fluorescent substance from the intensity of the transmitted portion of the excitation radiation and the intensity of the fluorescence radiation is determined.
    Advantageously, therefore, not only, as before, the fluorescence is measured, but it is also taken into account that the absorption of the excitation light in the sample with the fluorescent substance can affect the intensity of the measured fluorescence radiation. In particular, how strong the influence of the absorption is on the determination of the concentration may depend on the lengths which the light travels in the sample. Accordingly, the influence of the absorption depends in particular on the geometry of the measuring chamber. Since these effects were not taken into account in the previously used fluorescence measurement, the measured fluorescence intensity as such could not be used to deduce the concentration of the fluorescent substance in the sample. In contrast, the inventive method can be used as a highly accurate quantitative method for determining the concentration of the fluorescent substance. On the one hand, it has been shown that, when carrying out the method for determining the concentration of the fluorescent substance, the absorption increases in proportion to the fluorescence. This means that the excitation light necessary for the fluorescence measurement is attenuated with increasing depth of penetration into the sample medium, which leads to a reduction of the resulting fluorescence signal. In addition, in practice, in unknown samples, absorption of the excitation light in the sample often occurs, which is not causally related to the fluorescence or the concentration of the fluorescence-causing substance. Overall, therefore, in the case of pure fluorescence measurement results in a systemic error that can not be corrected by information from the fluorescence measurement. According to the invention, this error is eliminated by measuring the transmitted component of the excitation radiation, which provides information about the absorption of the excitation radiation as it passes through the measurement volume. The determination of the concentration of the fluorescent substance then takes place on the basis of both the fluorescence and the transmission of the excitation radiation. This method is particularly advantageous when turbidity of the sample or for other reasons increased absorption of the excitation radiation, for example by adding a reaction chemistry, for attenuation of the medium penetrating excitation radiation and subsequently also leads to a reduced fluorescence signal. In a pure fluorescence measurement, the change in the signal would not be distinguishable from a lower fluorescence, which would give an inaccurate or incorrect result in the concentration determination. In contrast, the simultaneous measurement of absorption and fluorescence on the one hand offers the advantage that the transmitted portion of the excitation radiation can be used to correct nonlinearities and false measurements due to the absorption of the excitation radiation in the sample. This is advantageous, for example, if different geometries of the sample chambers receiving the sample are to be considered. Furthermore, for example, from the relationship between absorption and fluorescence after metering in a reaction chemistry, in particular a substrate, a statement about the qualitative state of the added reaction chemistry can be made. A further advantage of the invention is that, in the case of a prolonged measurement process, changes in the concentration can be distinguished with high accuracy from variations in the permeability of the sample. In this case, it is particularly preferred if the intensity of the fluorescence and the intensity of the transmission of the excitation radiation are measured simultaneously on one and the same volume element of the medium with the fluorescent substance. This is particularly advantageous if the sample is inhomogeneous o its state changes rapidly in time. However, in the case of stable and homogeneous samples, which are also present in sufficient quantity, the fluorescence and the transmission of the excitation radiation can alternatively also be measured in the same device in succession or (even simultaneously) in separate measuring devices.
    According to a particularly preferred embodiment, the medium containing the fluorescent substance is further irradiated by a fluorescence absorption radiation having the fluorescence wavelength, the intensity of a transmitted portion of the fluorescence absorption radiation is measured and the concentration of the fluorescent substance is determined from the intensity of the transmitted portion of the excitation radiation, the intensity of the fluorescence radiation and the fluorescence Intensity of the transmitted portion of the fluorescence absorption radiation is determined. In this embodiment, therefore, the transmission or absorption of the sample when irradiated with the fluorescence wavelength is used as a further input variable for the determination of the concentration of the fluorescent substance. In this case, the measurement of the transmitted fraction of the fluorescence absorption radiation is carried out with the excitation radiation switched off, so that the transmission of the sample when irradiated with the fluorescence wavelength can be determined independently of the fluorescence excited by the excitation radiation. Accordingly, in this embodiment, the concentration of the fluorescent substance is determined from a measured triplet, which is composed of the fluorescence radiation on the one hand and the transmission of the excitation radiation and the fluorescence absorption radiation on the other. The transmission of excitation radiation or fluorescence absorption radiation when passing through the medium with the fluorescent substance are hereby applied as correction values to the fluorescence signal.
    In order to obtain even more accurate measurement results, it is preferred that the intensity of a scattered light having the excitation wavelength be measured and the concentration of the fluorescent substance be determined from the intensity of the transmitted fraction of the excitation radiation, the intensity of the fluorescence radiation and the intensity of the fluorescence Stray light, optionally (ie, if measured) and the intensity of the transmitted portion of the fluorescence absorption radiation, is determined. The measurement of the scattered radiation is accomplished, in particular, by a third radiation sensor, which is preferably sensitive to the excitation wavelength or to which a corresponding bandpass filter with central wavelength λΘΧ is connected upstream.
    For highly precise concentration measurement, it is preferably provided that the concentration of the fluorescent substance is determined from a characteristic field which determines the concentration of the fluorescent substance as a function of the intensity of the transmitted portion of the excitation radiation, the intensity of the fluorescence radiation and optionally (ie if measured) the intensity indicates the transmitted portion of the fluorescence absorption radiation and / or the intensity of the scattered light. The characteristic field can be obtained empirically. Alternatively, a calculation model may be used which relates the input quantities for the determination of the concentration of the fluorescent substance in relation to each other
    In this case, it is particularly favorable if the characteristic field is determined in a measurement method for determining the concentration of the fluorescent substance, in which the intensity of the fluorescence radiation, the intensity of the transmitted portion of the excitation radiation and optionally the intensity of the transmitted fluorescence are given for given calibration concentrations of the fluorescent substance Proportion of fluorescence absorption radiation and / or the intensity of the scattered light is measured. Advantageously, this can be used to determine a clear relationship between the concentration of the fluorescent substance and the input variables, which is used when determining an unknown concentration of the fluorescent substance. To increase the resolution, it is favorable if the measuring points of the characteristic field are interpolated in a manner known per se by an approximation curve. In the case of consideration of the scattered light, several measuring passes can be made for equal absorption and varying scattering. As an alternative to such a measurement or calibration method, the relationship between the concentration and the individual measured variables can also be obtained by a mathematical relationship.
    In order to improve the accuracy in the measurement, it is favorable if the transmitted portion of the excitation radiation is deflected after passing through the medium with the fluorescent substance before the intensity of the transmitted portion of the excitation radiation is measured. This advantageously prevents interference effects which could occur when measuring the transmission of the excitation radiation in the direction of the radiation of the medium with the fluorescent substance.
    The device of the initially mentioned type is characterized in that a second sensor is provided for measuring the intensity of a transmitted portion of the excitation radiation, wherein the arithmetic unit for determining the concentration of the fluorescent substance in dependence on the intensity of the transmitted portion of the excitation radiation and the intensity of Fluorescence radiation is set up.
    Hereby, the same advantages and technical effects as in the previously explained method can be achieved, so that reference is made to the above statements. In this case, the arithmetic unit preferably has a memory in which a characteristic field is stored, which contains the relationship between the concentration of the fluorescent substance and the intensity of the transmitted portion of the excitation radiation, the intensity of the fluorescence radiation, preferably also the intensity of the transmitted portion of the fluorescence absorption radiation ,
    In order to further improve the accuracy of the concentration determination, it is favorable if a second radiation source is provided for irradiating the medium with the fluorescent substance with fluorescence absorption radiation having the fluorescence wavelength. The second radiation source is switched on or off independently of the first radiation source, so that the intensity of the transmitted portion of the fluorescence absorption radiation, i. the absorption of a radiation having the fluorescence wavelength when passing through the medium, in the switched-on state of the second radiation source and in the switched-off state of the first radiation source is determinable.
    Preferably, the first sensor is arranged to measure the intensity of the transmitted portion of the fluorescence absorption radiation. Alternatively, a separate, third sensor can be provided for measuring the transmitted portion of the fluorescence absorption radiation. The first sensor is connected to the arithmetic unit, which receives the intensity of the fluorescence and the intensity of the fluorescence absorption radiation as further input variables (in addition to the transmission of the excitation radiation) for determining the concentration of the fluorescent substance.
    Moreover, it is preferable if a third sensor is provided for measuring the intensity of a scattered light. The third sensor is connected to the arithmetic unit, which receives the intensity of the scattered light as a further input variable for determining the concentration of the fluorescent substance.
    According to a preferred embodiment, the first radiation source and / or the second radiation source are each assigned a reference sensor for calibrating the first or second radiation source. In a preferred embodiment, the reference sensor measures, for example by continuously decoupling a constant percentage of the excitation light from the beam path of the excitation light by means of a partially transmissive mirror and supplying it to the reference sensor, the intensity of the excitation radiation. This reference signal is compared at all times with the measured (transmission and fluorescence) signals. The measurement signals are therefore always related to the intensity of the excitation light measured by the reference sensor at the same time. This advantageously ensures that any fluctuations in the intensity of the excitation light have no influence on the measurement result.
    In a preferred application, a measuring chamber, in particular a cuvette, is provided for receiving the medium with the fluorescent substance. Preferably, the medium with the fluorescent substance is present as a liquid sample. Alternatively, the concentration of a fluorescent substance in a gaseous medium can be determined. In some embodiments, it is favorable if a plurality of measuring chambers are provided, which are each tuned to a different fluorescence or excitation wavelength, so that the concentrations of different fluorescent substances can be determined with the same device. In this case, it is favorable to use characteristics maps matched to the respective wavelengths, preferably with the aid of the measuring method described above.
    In accordance with a further preferred embodiment, it is provided that the first radiation source is set up to emit excitation radiation having a plurality of excitation wavelengths, in particular having a substantially continuous excitation spectrum. For this purpose, characteristic curves of the concentration are preferably used for an at least substantially continuous wavelength range of the excitation or fluorescence wavelength, whereby the method can be extended to the determination of the concentration of fluorescent substances in an at least substantially continuous wavelength range. For this purpose, the measuring method described above may be performed at narrow frequency intervals for a plurality of excitation and / or fluorescence wavelengths, whereby the characteristic fields are obtained for the wavelength range of interest. In this case, it is not necessary for the first radiation source to be configured to emit monochromatic excitation radiation (or the second radiation source to emit monochromatic fluorescence absorption radiation), as is preferably provided when examining a single, substantially discrete fluorescence or excitation wavelength. Instead, the first radiation source for emitting an excitation wavelength having several excitation wavelengths is 9/34 9
    Radiation, in particular with a substantially continuous excitation spectrum, set up .. As a first or second sensor, optionally as a reference sensor, a spectrometer is preferably provided in this embodiment. Moreover, it is advantageous in this embodiment, if the second radiation source for emitting the fluorescence absorption radiation, optionally also the sensor for the scattered light intensity, omitted, since the hereby representable information is already included in the absorption or fluorescence spectrum.
    In order to avoid disturbing effects which impair measuring accuracy, it is favorable if a deflecting mirror for deflecting the transmitted component of the excitation radiation is arranged between the measuring chamber and the second sensor.
    Preferably, the deflecting mirror causes a deflection of the trans-mediated portion of the excitation radiation by substantially 90 °. Advantageously, the accuracy in measuring the transmission of the excitation radiation can be significantly improved hereby.
    To decouple the excitation radiation, it is particularly advantageous if a beam splitter, in particular a frequency-selective beam splitter, is provided as deflecting mirror. Preferably, the frequency-selective beam splitter is designed as a long pass (i.e., in low-pass frequency space) which does not reflect radiation of the fluorescence wavelength, but transmits it.
    With this embodiment it can be achieved, in particular, that radiation of the fluorescence wavelength reflected by the second sensor does not pass back through the measuring chamber and subsequently to the first sensor. At the same time, this can prevent radiation of the fluorescence wavelength from reaching the second sensor from the measuring chamber. This is particularly important because the intensities of transmitted radiation and fluorescence radiation can be different orders of magnitude. The first sensor (fluorescence sensor) can therefore be much more sensitive than the second sensor (transmission sensor). Preferably, the deflection mirror is also associated with the second radiation source for emitting the fluorescence absorption radiation. In this case, it is also advantageous if the beam splitter 10/34 is frequency-selective. Preferably, the cut-off wavelength of the frequency-selective beam splitter is between the excitation wavelength and the fluorescence wavelength. This can be achieved, in particular, that the transmitted portion of the excitation radiation passes substantially entirely into the second sensor.
    The invention will be explained below with reference to preferred embodiments illustrated in the drawings, to which, however, it should not be restricted. In detail, in the drawing:
    Figure 1 is a view of a device according to the invention for determining a concentration of a fluorescent substance in a medium.
    Fig. 2 is a view of an alternative embodiment of the fluorescent substance concentration determination apparatus;
    Fig. 3 is a view of another embodiment of the fluorescent substance concentration determination apparatus;
    Fig. 4 is a view of another embodiment of the fluorescent substance concentration determining apparatus;
    5 shows a schematic view of a computing unit for determining the concentration of the fluorescent substance;
    6 shows a diagram to illustrate the relationship between the excited fluorescence and the transmission of the excitation radiation and the permeability of the medium, wherein different measurement curves for constant calibration concentrations are shown;
    7 shows a diagram with a characteristic field for determining the concentration of the fluorescent substance as a function of the fluorescence and the transmission of the excitation radiation; and
    8 is a diagram illustrating an increase of the fluorescence signal, which is not due to an increase in the concentration of the fluorescent substance, but to an increase in the transmission of the excitation radiation.
    1 shows a device 1 for determining a concentration c of a fluorescent substance in a medium which is accommodated in a measuring chamber 2. In the embodiment shown, a cuvette is provided as the measuring chamber 2, in which a liquid sample is taken up with the fluorescent substance. The device 1 has a first radiation source 3 for emitting an excitation radiation with an excitation wavelength λΘΧ. In addition, a first sensor 4 is provided for measuring the intensity Ifiuor of a fluorescence radiation excited by the excitation radiation (short: fluorescence), which has a larger fluorescence wavelength Xem compared to the excitation wavelength Xex. As is well known, the fluorescent light always has a longer wavelength, i. a lower frequency (or energy) than its excitation light.
    As can also be seen from FIG. 1, a second sensor 5 is additionally provided, with which the intensity of a transmitted portion of the excitation radiation Itrans, ex (in short: transmission of the excitation radiation) is measured. The transmission of the excitation radiation, which is indirectly proportional to the absorption of the excitation radiation when passing through the sample, is measured simultaneously with the measurement of the intensity of the fluorescence radiation Ifiuor.
    In addition, a computing unit 6 (see Fig. 5) is provided, with which the concentration c of the fluorescent substance is determined. In the embodiment shown, the concentration c is determined not only as a function of the fluorescence, but also depending on the transmission of the excitation radiation. Hereby, the accuracy of the concentration measurement compared to conventional methods, in which only the fluorescence signal is used, be substantially increased, if the fluorescence measurement as such could make possible a quantitative concentration determination.
    As can also be seen from FIG. 1, a second radiation source 7 is also provided for irradiating the sample with a radiation having the fluorescence wavelength, hereinafter referred to as fluorescence absorption radiation. The intensity I trans, em of the transmi¬ierten proportion (short: transmission) of the fluorescence absorption radiation is measured by means of the first sensor 3, while the first radiation source 3 is turned off.
    1, the first radiation source 3 is assigned a reference sensor 9 and the second radiation source 7 is assigned a reference sensor 10 with which the first 3 or second radiation source 7 can be calibrated. For this purpose, a constant, comparatively small percentage of the excitation light is coupled out of the beam path of the excitation light by means of a partially transmissive mirror 25 and supplied to the reference sensor 9 in order to continuously detect the intensity of the excitation radiation. The transmission of the excitation radiation and the fluorescence (possibly also the scattered light intensity, see Fig. 3) is related to the reference signal at all times in order to carry out a calibration of the measurement signals. Correspondingly, the second radiation source 7 is provided with another partially transmissive mirror 26 with which a (comparatively small) proportion of the fluorescence absorption radiation is diverted into the reference sensor 10. Thus, a calibration can be made with regard to the fluorescence absorption radiation.
    As further shown in FIG. 1, the device 1 in the embodiment shown also has a deflecting mirror 11, which is arranged between the measuring chamber 2 and the second sensor 5. Hereby, the transmitted portion of the excitation radiation is deflected after leaving the measuring chamber 2 relative to the transmission direction. The deflection mirror 11 in this case causes a deflection of the transmitted portion of the excitation radiation by substantially 90 ° relative to the direction of the excitation radiation when passing through the measuring chamber 2. In the embodiment shown, a beam splitter is provided as deflecting mirror 11, which frequency selective with a cut-off wavelength between the excitation and the fluorescence wavelength is performed so that radiation of the fluorescence wavelength is not reflected but transmitted. This on the one hand 13/34 13 prevents reflected by the second sensor 5 radiation of the fluorescence wavelength passes through the measuring chamber to the first sensor 4 and radiation of the fluorescence wavelength from the measuring chamber 2 to the second sensor 5.
    In the embodiment shown, the reference sensor 10, the second radiation source 7 and the second sensor 5 are each preceded by an (optional) bandpass filter 12. A reference value for the intensity of the fluorescence absorption radiation is obtained at the reference sensor 10 in order to be able to take into account any fluctuations in the intensity of the fluorescence absorption radiation.
    As can further be seen from FIG. 1, a further deflecting mirror 13 is provided between the first radiation source 3 and the measuring chamber 2, with which the excitation radiation is coupled into the measuring chamber 2 under deflection by substantially 90 °. The deflecting mirror 13 is designed according to the deflecting mirror 11 as a frequency-selective beam splitter whose cutoff wavelength lies between the excitation λex and the fluorescence wavelength Xem. The first sensor 4 is arranged parallel to the transmission direction of the measuring chamber 2 on the side facing away from the measuring chamber 2 side of the deflecting mirror 13. In addition, the first sensor 4, as well as the reference sensor 9 and the first radiation source 3, optionally a bandpass filter 14 upstream. The optional use of the bandpass filters 12, 14 can be made in particular dependent on the characteristics of the sensors or radiation sources.
    Fig. 2 shows an alternative embodiment of the device 1 for determining the concentration c of the fluorescent substance, wherein only the differences from Fig. 1 will be discussed below.
    According to FIG. 2, a partially transmissive mirror 15 is arranged between the first radiation source 3 and the measuring chamber 2, with which in each case a portion of the excitation radiation is coupled into the measuring chamber 2 or fed to the reference sensor 9.
    At the reference sensor 9, a reference signal for the intensity of the excitation radiation is continuously detected. Optionally, the first radiation source 3 may be preceded by a filter 16 tuned to the excitation wavelength. A further filter 16 for the excitation wavelength Xex is connected upstream of the second sensor 5 for detecting the transmission of the excitation radiation. Corresponding, but matched to the fluorescence wavelength λem filters 17 are associated with the second radiation source 7 for emitting the fluorescence absorption radiation and the first sensor 4 for detecting the transmission of fluorescence absorption radiation or fluorescence.
    According to FIG. 2, a further semitransparent mirror 18 is arranged between the second radiation source 7 and the measuring chamber 2, by means of which in each case a portion of the fluorescence absorption radiation is coupled into the measuring chamber 2 or fed to the reference sensor 10. At the reference sensor 10, a reference signal for the intensity of the fluorescence absorption radiation is detected continuously, which allows the exact determination of the transmission of the fluorescence absorption radiation (and from this the absorption of the fluorescence radiation in the sample).
    According to FIG. 2, the fluorescence is measured at an angle of, in particular, 90 ° to the irradiation direction of the excitation radiation. In contrast to FIG. 1, no beam splitters are provided in the beam path between the measuring chamber 2 and the first sensor 4 or in the beam path between the measuring chamber 2 and the second sensor 5.
    As can be seen from FIG. 3, in this embodiment, in addition, the intensity Istreu, ex of a scattering radiation having the excitation wavelength Xex, is measured by means of a third sensor 27 and used to determine the concentration of the fluorescent substance. The third sensor 27 is preceded by (optionally) a bandpass filter 12. Otherwise, the arrangement according to FIG. 3 corresponds to the embodiment shown in FIG. 1, whereby of course a third sensor 27 for detecting the intensity of the scattered light can also be present in the embodiment according to FIG.
    According to FIG. 4, a first radiation source 3 with a broad, continuous excitation spectrum is used. The first 15/34 15
    Sensor 4, second sensor 5 and the reference sensor 9 are hereby designed as spectrometers 28, 28 ', 28' '. In this embodiment, the second radiation source 7 for emitting the fluorescence absorption radiation and the third sensor 27 for the scattered light intensity can be dispensed with.
    As can be seen from FIG. 5, the arithmetic unit 6 receives the fluorescence, the transmission of the excitation radiation and, optionally, the transmission of the fluorescence absorption radiation and the intensity of the scattered light as input variables. The arithmetic unit 6 has a memory 8, in which a characteristic field for determining the concentration c of the fluorescent substance from the input variables is stored. As the output of the arithmetic unit 6, the arithmetic unit 6 supplies the sought concentration c of the fluorescent substance.
    As already mentioned, the concentration of the fluorescent substance is determined with the aid of the arithmetic unit 6 from the measured variables, which are linked to one another by a characteristic field stored in the memory 8. The family of characteristics can be determined in a measurement procedure upstream of the concentration determination, which is explained below.
    According to FIG. 6, in a first step of the measurement or calibration process, individual measurement curves 19 are applied for defined concentrations c1, c2 of the fluorescent substance, on the one hand the fluorescence (measurement curves 19 ') and on the other hand the transmission of the excitation radiation (measurement curves 19' '). depending on a permeability D of the sample is determined. The permeability D of the sample can be varied stepwise, for example, by metering in an opaque substance. This produces the measurement curves 19 for the fluorescence (measurement curves 19 ') and transmission (measurement curves 19' ') as a function of the permeability or opacity of the sample at a constant concentration of the fluorescent substance.
    As can be seen from FIG. 7, in the next step the fluorescence is plotted against the transmission of the excitation radiation (measurement curves 20), each measurement curve 20 corresponding to a specific calibration concentration c1 to c3. This creates a set of curves 16/34 16 in two-dimensional space. Depending on the design, the transmission of the fluorescence absorption radiation or the intensity of the scattered light can additionally be measured as a function of the permeability of the sample. In this case, instead of the two-dimensional diagram of FIG. 7, a flock in three-dimensional space is obtained (not shown). Each point in the diagram (two- or three-dimensional, depending on the design) corresponds to a specific concentration of the fluorescent substance, so that a clear relationship between the measured quantities and the concentration c is obtained.
    Alternatively, both scattered light and fluorescence absorbing radiation may be considered, thus obtaining a four-dimensional state matrix in which each combination of the four parameters (i.e., fluorescence, excitation radiation, fluorescence absorption radiation, and scattered radiation) is uniquely associated with a fluorescent substance concentration c.
    Depending on the number of measurement curves 20, the accuracy of the concentration determination can be increased. In addition, a mathematical or numerical interpolation can also be carried out in order to enable a direct conversion of a measured value doublet (or, depending on the model, a measured value triplet or measured value quartet) into the sought concentration c.
    The advantages of this method can be seen, for example, from FIG. 8, which schematically illustrates the time course of the fluorescence (signal 21) during a measuring process. A liquid sample is investigated, to which a buffer is added at time 22. Later, a substrate is dosed, which causes the formation of the fluorescent substance (arrow 23). After metering in the substrate, the fluorescence signal first increases abruptly due to the intrinsic fluorescence of the substrate. Thereafter, a further increase in fluorescence over time is observed, giving the appearance of an increasing concentration of the fluorescent substance. By measuring the transmission of the excitation radiation (signal 24), however, it can be ascertained that the transmission initially decreases abruptly when the buffer is introduced, since the buffer in the 17/34 17
    Sample leads to that as good as no light gets into the second sensor 5. Subsequently, the permeability of the sample increases again over time, so that the transmission of the excitation radiation increases. This also increases the intensity of the excitation signal for the fluorescence and thus the fluorescence signal. Accordingly, the supposed increase in fluorescence is not due to a change in the concentration of the fluorescent substance but to an increase in the transmission of the excitation radiation.
    The method described above can be used to determine the concentration of different types of fluorescent substances.
    The intensity of the fluorescence of a substance may be dependent both on the pH value and on the temperature at the time of the measurement. The invention described allows an improvement in the accuracy of fluorescence measurement even without a defined pH value or without a defined temperature. However, to be able to determine the concentration of a substance in the sample, the measured sample should have the same pH and temperature as in the calibration. This is ensured by adding a defined amount of buffer and a precise temperature control of the measuring device.
    In the following, as an example of the method described above, the determination of the concentration of sodium fluorescein in waters is determined.
    Sodium fluorescein is used as a fluorescence tracer to study groundwater flows. The optimum excitation wavelength for sodium fluorescein is 491 nm, the maximum of the fluorescence radiation is 516 nm. To determine the fluorescein concentration in the sample, the measuring device is first calibrated by, as described above, a characteristic field is created. For this purpose, reference solutions are measured one after the other, each of which has a defined fluorescein concentration and a constant pH, for example pH 7, set by a buffer solution. These reference solutions are measured at constant temperature, for example 18/34 18 at 25 ° C. In the course of the calibration, each reference solution is repeatedly measured with the successive addition of an increasing concentration of an opaque substance, for example formazine. The respective combinations of the measured values of the fluorescence intensity, transmission intensity, if appropriate also for scattered light intensity and intensity of the fluorescence absorption radiation, with constant fluorescence concentration and simultaneous increase of formazine concentration are recorded. This process is repeated for different fluorescein concentrations, so that a sufficiently dense characteristic field arises, which makes it possible to represent the entire concentration range to be covered sufficiently by interpolation or mathematical calculation.
    After completion of the calibration, the measuring device is ready for use. Now any sample, provided with a defined amount of buffer for adjusting the pH, the measuring device is supplied, the intensity values for fluorescence, Tranmission, possibly also for scattered light and fluorescence absorption radiation, are determined. With the help of this measurement data set, the actual fluorescence concentration is determined from the characteristic field. 19/34
    权利要求:
    Claims (16)
    [1]
    19. A method for determining a concentration (c) of a fluorescent substance in a medium, wherein the medium with the fluorescent substance is irradiated by an excitation radiation having an excitation wavelength (Xex), so that the fluorescent substance for emitting fluorescence radiation with a Fluorescence wavelength (Xem) is excited, whereby the intensity of the fluorescence radiation (IfiUOr) is measured, from which the concentration (c) of the fluorescent substance in the medium is determined, characterized in that in addition the intensity (Itrans, ex) of a transmitted portion of the Excitation radiation is measured, wherein the concentration (c) of the fluorescent substance from the intensity of the transmitted portion of the excitation radiation (Itrans, ex) and the intensity of the fluorescence radiation (Ifiuor) is determined.
    [2]
    2. The method according to claim 1, characterized in that the medium with the fluorescent substance further irradiated by a fluorescence wavelength (Äem) having fluorescence absorption radiation, the intensity of a transmitted portion of the fluorescence absorption radiation (Itrans, em) measured and the concentration (c) of the fluorescent Substance from the intensity of the transmitted portion of the excitation radiation (Itrans, ex) / the intensity of the fluorescence radiation (Ifiuor) and the intensity of the transmitted portion of the fluorescence absorption radiation (Itrans, em) is determined.
    [3]
    3. The method according to claim 1 or 2, characterized in that in addition the intensity of the excitation wavelength (Xex) having scattered light (Istreu, ex) measured and the concentration (c) of the fluorescent substance from the intensity of the transmitted portion of the excitation radiation (Itrans, ex) r the intensity of the fluorescence radiation (Ifiuor) and the intensity of the scattered light (Istreu, ex) f and possibly also the intensity of the transmitted portion of the fluorescence absorption radiation (Itrans, em) / is determined.
    [4]
    4. The method according to any one of claims 1 to 3, characterized in that the concentration (c) of the fluorescent sub- 20/34 20 punch is determined from a family of characteristics, the concentration (c) of the fluorescent substance as a function of the intensity of transmitted intensity of the excitation radiation (Itrans, ex) r indicates the intensity of the fluorescence radiation (Ifiuor) and optionally on the intensity of the transmitted portion of the fluorescence absorption radiation (Itrans, em) and / or the intensity of the scattered light.
    [5]
    5. The method according to claim 4, characterized in that the characteristic field is determined in a determination of the concentration (c) of the fluorescent substance upstream measuring method in which for predetermined calibration concentrations (c1-c3) of the fluorescent substance in each case the intensity of fluorescence radiation (Ifiuor ), the intensity of the transmitted portion of the excitation radiation (Itrans, ex) and optionally the intensity of the transmitted portion of the fluorescence absorption radiation (Ifrans, em) and / or the intensity of the scattered light is measured.
    [6]
    6. The method according to any one of claims 1 to 5, characterized in that the transmitted portion of the excitation radiation is deflected after passing through the medium with the fluorescent substance before the intensity of the transmitted portion of the excitation radiation (Itrans, ex) is measured.
    [7]
    7. Apparatus (1) for determining a concentration of a fluorescent substance in a medium, comprising at least a first radiation source (3) for emitting an excitation radiation having an excitation wavelength (Xex), with a first sensor (4) for measuring the intensity of a through the Excitation radiation excited, a fluorescence wavelength (Xem) having fluorescent radiation, and with a computing unit (6) for determining the concentration of the fluorescent substance from the intensity of fluorescence radiation, characterized in that a second sensor (5) for measuring the intensity of a transmitted portion of the excitation radiation is provided, wherein the computing unit (6) for determining the concentration of the fluorescent substance in dependence on the intensity of the transmitted portion of the excitation radiation (Itrans, ex) and the intensity of the fluorescence radiation (Ifiuor) is set up. 21/34 21
    [8]
    8. Device (1) according to claim 7, characterized in that a second radiation source (7) for irradiating the medium with the fluorescent substance with a fluorescence wavelength (Xem) having fluorescence absorption radiation is provided.
    [9]
    9. Device (1) according to claim 8, characterized in that the first sensor (4) for measuring the intensity of a transmit-tierten proportion of fluorescence absorption radiation (Itrans, em) is set up.
    [10]
    10. Device (1) according to one of claims 7 to 9, characterized in that a third sensor (27) is provided for measuring the intensity of a scattered light.
    [11]
    11. Device (1) according to one of claims 7 to 10, characterized in that the first radiation source (3) and / or the second radiation source (7) each have a reference sensor (9, 10) for calibrating the first (3) or associated with the second radiation source (7).
    [12]
    12. Device (1) according to any one of claims 7 to 11, characterized in that a measuring chamber (2), in particular a cuvette, is provided for receiving the medium with the fluorescent substance.
    [13]
    13. Device (1) according to claim 12, characterized in that between the measuring chamber (2) and the second sensor (5) a deflecting mirror (11) for deflecting the transmitted portion of the excitation radiation is arranged.
    [14]
    14. Device (1) according to claim 13, characterized in that the deflecting mirror (11) causes a deflection of the transmitted portion of the excitation radiation by substantially 90 °.
    [15]
    15. Device (1) according to claim 14, characterized in that as deflecting mirror (11) a beam splitter, in particular a frequency-selective beam splitter, is provided. 22/34 22
    [16]
    16. Device according to one of claims 7 to 15, characterized in that the first radiation source (3) for emitting a plurality of excitation wavelengths (λex) having excitation radiation, in particular with a substantially continuous excitation spectrum, is set up, wherein as the first sensor (4) and as a second sensor (5), optionally also as a reference sensor (9), in each case a spectrometer (28, 28 ', 28' ') is or are provided. 23/34
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    同族专利:
    公开号 | 公开日
    CN105074434A|2015-11-18|
    IL240383A|2020-04-30|
    RU2015132842A|2017-03-21|
    AT513863B1|2014-12-15|
    RU2664786C2|2018-08-22|
    JP6441238B2|2018-12-19|
    WO2014124476A3|2014-10-23|
    EP2956758B1|2019-11-27|
    JP2016510412A|2016-04-07|
    WO2014124476A2|2014-08-21|
    US9423350B2|2016-08-23|
    EP2956758A2|2015-12-23|
    CN105074434B|2019-05-21|
    US20150362430A1|2015-12-17|
    IL240383D0|2015-09-24|
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    WO2009079629A2|2007-12-18|2009-06-25|Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California|A cardiac output monitor probe and calibrator|RU2715934C1|2019-06-28|2020-03-04|Общество с ограниченной ответственностью "Люмисенсис Лаборатория"|Analyzer for selective determination of volatile aromatic hydrocarbons|
    RU192708U1|2019-06-28|2019-09-26|Общество с ограниченной ответственностью "Люмисенсис Лаборатория"|ANALYZER FOR THE SELECTIVE DETERMINATION OF VOLATILE AROMATIC HYDROCARBONS|
    法律状态:
    2021-07-15| PC| Change of the owner|Owner name: VWMS INVENTIONS GMBH, AT Effective date: 20210525 |
    优先权:
    申请号 | 申请日 | 专利标题
    ATA50110/2013A|AT513863B1|2013-02-15|2013-02-15|Method and device for determining a concentration of a fluorescent substance in a medium|ATA50110/2013A| AT513863B1|2013-02-15|2013-02-15|Method and device for determining a concentration of a fluorescent substance in a medium|
    RU2015132842A| RU2664786C2|2013-02-15|2014-02-10|Method and device for determining concentration|
    EP14708792.8A| EP2956758B1|2013-02-15|2014-02-10|Method and device for determining the concentration of a fluorescent substance in a medium|
    JP2015557287A| JP6441238B2|2013-02-15|2014-02-10|Method and apparatus for determining concentration|
    CN201480009058.0A| CN105074434B|2013-02-15|2014-02-10|Method and apparatus for determining the fluorescent material concentration in medium|
    US14/765,921| US9423350B2|2013-02-15|2014-02-10|Method and device for determining a concentration|
    PCT/AT2014/050036| WO2014124476A2|2013-02-15|2014-02-10|Method and device for determining a concentration|
    IL240383A| IL240383A|2013-02-15|2015-08-05|Method and device for determining a concentration|
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