Method and optode for determining the concentration of an analyte in a sample liquid

Abstract

A method and optode for determining a concentration of an analyte in a sample liquid is provided. The method comprises a radiation source, where excitation radiation is directed onto a carrier unit which is in contact with the sample liquid and has immobilized molecules of a sensor dye that is sensitive to the analyte. The excitation radiation induces luminescence radiation of the sensor dye. This radiation is detected by a radiation detector, which generates an output signal. The analyte concentration is ascertained from the detector output signal using an evaluation routine. This uses a property of the luminescence radiation on the interaction of the concentration of the analyte in the sample liquid used. The dependence of the examined property of the luminescence radiation on an indirect exchange interaction between the individual molecules of the sensor dye, which interact with each other over particles of the analyte.

Claims

1. A method for determining a concentration of an analyte in a sample liquid, in which, a) by means of a radiation source, excitation radiation is directed onto a carrier unit which is in contact with the sample liquid and which has immobilized molecules of a sensor dye that is sensitive to the analyte, wherein the mean distance R between immobilized molecules is smaller than the Förster radius r0, b) a portion of sensor dye luminescence radiation from which the sensor dye luminescence radiation is induced by the excitation radiation, is applied to a radiation detector in order to generate a detector output signal therefrom, and c) an evaluation routine ascertains the analyte concentration from the detector output signal, wherein a dependence of an examined property of the luminescence radiation on the concentration of the analyte in the sample liquid is used, characterized in that d) the dependence of the examined property of the luminescence radiation on an indirect exchange interaction between the individual molecules of the sensor dye is used to ascertain the analyte concentration, wherein the indirect exchange interaction takes place when the molecules of the sensor dye interact with the particles of the analyte, and characterized in that the evaluation routine evaluates a variable that is proportional to an exponential function $\exp \left(\frac{R}{r-r_0}\right)$ where R is a measure of a distance between the immobilized molecules of the sensor dye, r is a measure of the distance between the molecules of the analyte that interact with a molecule of the sensor dye, and r0 is the Förster radius.

2. The method according to claim 1, characterized in that the dependence of a luminescence radiation lifetime on the analyte concentration is used as the property of the luminescence radiation and the evaluation routine for ascertaining the analyte concentration is based on a known concentration of the sensor dye molecules immobilized in a polymer matrix and a known dependence of the lifetime of the luminescence radiation is based on the sensor dye molecule concentration and the analyte concentration.

3. The method according to claim 2, characterized in that, for the dependence of the lifetime of the luminescence radiation on the sensor dye molecule concentration and the analyte concentration, the proportionality $\tau \sim \exp \left(\frac{R}{r-r_0}\right)$ is used, where τ is the lifetime of the luminescence radiation of the immobilized molecules of the sensor dye which interact with the particles of the analyte, R is a measure of a distance between the immobilized molecules of the sensor dye, r is a measure of the distance between the molecules of the analyte that interact with a molecule of the sensor dye, and r0 is the Förster radius.

4. The method according to claim 2, characterized in that the lifetime of the luminescence radiation is ascertained by means of time-resolved measurement.

5. The method according to claim 2, characterized in that the lifetime of the luminescence radiation is ascertained by means of phase modulation.

6. The method according to claim 1, characterized in that a) the excitation radiation is directed onto the carrier unit in a polarized manner and b) intensities I∥ and I.sup.⊥ of sensor dye luminescence radiation, in which the sensor dye luminescence radiation is induced by the excitation radiation, and which intensities are given for two substantially mutually perpendicular polarization directions are determined and, as the property of the luminescence radiation, the degree of polarization $P=.Math.\frac{I_{\mathrm{II}}-I_{\perp }}{I_{\mathrm{II}}+I_{\perp }}.Math.$ on the concentration of the analyte is used, where P is polarization degree of the sensor dye luminescence radiation, I.sub.∥ corresponds to the intensity of the sensor dye luminescence radiation polarized in vertical direction or parallel to the polarization direction of the emission source (parallel), and I.sub.⊥ corresponds to the intensity of the sensor dye luminescence radiation polarized in horizontal direction or normal/perpendicular to the polarization direction of the emission source.

7. The method according to claim 6, characterized in that the function $P\sim \exp \left(\frac{R}{r-r_0}\right),$ is used, where P is polarization degree of the sensor dye luminescence radiation, R is a measure of a distance between the immobilized molecules of the sensor dye, r is a measure of the distance between the molecules of the analyte that interact with a molecule of the sensor dye, and r0 is the Förster radius.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the drawings:

(2) FIG. 1A-1D show four exemplary variants of a sensor device,

(3) FIG. 2 is a graph showing laser pulse power over time,

(4) FIG. 3 is a graph showing normalized radiation intensity as ascertained by a photon counter,

(5) FIG. 4 is a graph showing the luminescent decay rate as a function of the concentration of sodium ions in a tested sample liquid,

(6) FIG. 5 shows a fifth sensor device with a continuous light source,

(7) FIG. 6 is a graph showing the degree of polarization of the luminescence radiation as a function of the concentration of the analyte, and

(8) FIG. 7 shows a multi-core optical fiber which has ten core strands.

(9) FIG. 1A-1D show four exemplary variants of a sensor device, also called an optode, which is suitable for carrying out a time-resolved method for determining the concentration of a chemical substance (analyte) in a sample liquid. The sample liquid can be in the form of a single drop or a plurality of drops. This is preferably a biological sample liquid, such as blood, serum, cerebrospinal fluid, intercellular fluid, sweat, or urine.

DETAILED DESCRIPTION

(10) Insofar as the following description of the figures refers to a sensor element or a sensor sub-element, this element has a carrier unit having immobilized molecules of a sensor dye sensitive to the analyte or is formed from such a carrier unit.

(11) A first sensor device 1 has a sample container 2, a pulsed light source 3, e.g. for laser radiation or LED radiation, and a photon counter 4. In the lower region of the sample container 2, preferably inside the sample container 2, a first sensor element 5 is located, the surface of which is intended to come into direct contact with the sample liquid to be analyzed (not shown here). The sensor element 5 has a carrier unit made of a functionalized polymer in which a sensor dye sensitive to the analyte is immobilized and which is hydrogenated when the sample liquid to be analyzed is fed in. The analyte particles penetrate the sensor element 5 such that the sensor dye's immobilized molecules can interact with the analyte particles. The sensor dye immobilized molecules generate a luminescence response to the incident pulsed light, with photons of the luminescence response being guided toward the photon counter 4.

(12) In the case of the first sensor device 1, radiation travels via a beam splitter 6 and an optical fiber 7, which can be a glass fiber, for example. The light originating from the light source 3 is guided via the beam splitter 6 through the optical fiber 7 toward the sensor element 5. Photons originating from the luminescence response arrive at the photon counter 4 via the beam splitter 6. Before entering the photon counter 4, the photons originating from the luminescence response can optionally pass through an optical filter 8, for example a high-pass filter, which is intended to prevent the entry of excitation radiation.

(13) The first pulsed light source 3 used emits in the spectral range of the excitation radiation for the molecules of the sensor dye, for example 405 nm or 488 nm. FIG. 2 shows preferred properties of a pulsed light source, as can be used in the first sensor device 1, for example. The spectral range of the excitation radiation is determined by the optical properties of the sensor dye selective places. The decay time tfal of the pulsed light source should be 0.1 ns or shorter. The repetition rate of the pulsed light source should preferably be in the range of megahertz (MHz) or kilohertz (kHz). With a time period trr between two pulses of the light source 3 of 20 ns, the repetition rate 1/trr of the pulsed light source is 50 MHz. A repetition rate of, for example, 50 MHz makes it possible to measure luminescence decay times of down to 20 ns.

(14) The photon counter 4 detects the incoming photons as a function of time. The time resolution of the photon counter 4 should be in the range of 100 ps (0.1 ns) or better. In order to increase the signal-to-noise ratio and to eliminate measurement noise, the measurement can be performed over multiple pulses of the light source 3.

(15) FIG. 3 shows the electrical signal generated by the photon counter 4 after amplification and conversion into digital form. The individual measuring points each represent the radiation intensity determined by the photon counter at a particular point in time. For example, laser pulses with a wavelength of 488 nm were produced with the first pulsed light source 3, which leads to a time-dependent fluorescence response.

(16) The graph in FIG. 3, adapted to the measuring points, is a straight line in the logarithmic representation of the graph and shows the time-resolved fluorescence decay, the sensor element 5 of the first sensor device 1 being selective for sodium ions (Na+) as the analyte. The measurement was performed with a known sodium concentration of 15 mmol per liter. The graph was normalized to one million counting pulses and the range of the decay phase was adapted to an exponential decay function.

(17) For the graph, the normalized intensity

(18) $I=I_{\max }\exp \left(-\frac{t-t_{\mathrm{delay}}}{\tau }\right)$

(19) where I.sub.max=1E6, t.sub.delay=12.2 ns, and τ=0.75 ns, is in the range of the decay phase, where t is the time, τ is the experimentally ascertained luminescence lifetime, and tdelay is the time delay, dependent on the length of the signal transmission path, between the trigger signal of the power supply for the light source 3 and the signal of the photon counter 4. When the tdelay is known, the luminescence lifetime can therefore be ascertained from the graph adapted to the measuring points.

(20) On the basis of the proportionality

(21) $\tau \approx {\tau }^{*}\exp \left(\frac{R}{r-r_0}\right)$

(22) and the known variables R and r0, the average distance r of the particles of the analyte in the sample liquid and thus the analyte concentration sought can be ascertained.

(23) τ corresponds to the luminescence lifetime, which is determined from the measured decay of the luminescence according to the formula

(24) $I=I_{\max }\exp \left(-\frac{t-t_{\mathrm{delay}}}{\tau }\right).$

(25) R is the average distance between the immobilized molecules of the sensor dye and is specified by the design of the carrier unit, e.g. membrane, which design is provided when the optode is manufactured. r0 is the theoretically calculable or experimentally determinable maximum distance between a particle of the analyte and each participating sensor dye molecule, within which an interaction between the particle of the analyte and the molecules of the sensor dye can occur and an indirect exchange interaction between the involved sensor dye molecules occurs (Förster radius). τ* is the increase in the lifetime of the luminescence of the sensor dye caused by the indirect exchange interaction. This parameter can also be calculated theoretically or determined experimentally for each sensor dye (Zwischenmolekulare Energiewanderung and Fluoreszenz [Intermolecular energy migration and fluorescence], Annalen der Physik, Volume 437, Issue 1-2, [1948], pp. 55-75). A value of τ*=0.6 ns is obtained for the dependence shown in FIG. 4.

(26) The concentration can thus be determined without a reference measurement.

(27) FIG. 4 shows the dependence of the concentration of sodium ions in the examined sample liquid on the decay rate of the luminescence. The decay rate is to be understood as an equivalent to the term “lifetime”. Using this graph, after a plurality of measurements have been performed in a given sensor device, the concentration of the sodium ions can be ascertained directly from the luminescence lifetime using further measurements.

(28) FIG. 1B shows a second sensor device 9, in which a plurality of light beams, generated by a pulsed light source 10, travel via a dichroic mirror 11 and then each via a core of a multi-core optical fiber 12 to a second sensor element 13 arranged in a sample container 15, which element has a plurality of sensor sub-elements. Each sensor sub-element can be provided for a specific analyte, such that the concentrations of multiple analytes can be determined in a sequence of multiple measurements.

(29) The luminescence radiation emanating from the sensor element 13 is guided via the multi-core optical fiber 12 and the dichroic mirror 11 onto a photon counter 14. The dichroic mirror 11 reflects the radiation from the light source, e.g. laser light with a wavelength of 400 to 500 nm, toward the sample and, additionally, allows the luminescence radiation emanating from the sensor element 13 to pass to the photon counter 14.

(30) A third sensor device 16 according to FIG. 1C has a similar design to the second sensor device 9 according to FIG. 1B, having a pulsed light source 17, a dichroic mirror 18, a sample container 19, and a sensor element 20 consisting of sensor sub-elements. The multi-core optical fiber 12 of the second sensor device 9 is, however, dispensed with. Instead, the beam is freely guided to the sensor element 20 and, from there, guided to a photon counter 21.

(31) In the variant of a fourth sensor device 22, a first optical fiber 23 guides the radiation generated by a pulsed light source 25 to a fourth sensor element 26 arranged in a sample container 27, and a second optical fiber 24 guides luminescence radiation from the fourth sensor element 26 toward a photon counter 28. Before hitting the fourth photon counter 28, the luminescence radiation passes through an optical filter 29.

(32) In the following, a method utilizing light polarization is presented as an alternative to the time-resolving method.

(33) FIG. 5 shows a fifth sensor device 30 (optode) with a non-pulsed but continuous light source 31 (e.g. laser or LED), a sensor element 33 arranged in a sample container 32, a first photodetector 34, and a second photodetector 35. After leaving the light source 38, the radiation passes through a linear polarizer 36 to produce linear polarization of the radiation. A first optical fiber 37 then guides the linearly polarized excitation light 40 to the sensor element 33, where it is used for optical excitation. In the sensor element 33 there is a luminescence response to the linearly polarized optical excitation, which response is dependent on the concentration of the analyte.

(34) A captured portion of the luminescence radiation is guided by a second optical fiber 38 toward an optical filter 39, e.g. a high-pass filter, which in particular filters out scattered excitation light. By means of a polarizing beam splitter 41, the incident luminescence radiation is split into two partial beams 42 and 43, which exhibit mutually perpendicular polarizations. The polarization directions are each symbolized by double arrows. The first partial beam 42 strikes the first photodetector 34, and the second partial beam 43 strikes the second photodetector 35. Photodetectors 34 and 35 ascertain the degree of polarization

(35) 0$P=.Math.\frac{I_{\mathrm{II}}-I_{\perp }}{I_{\mathrm{II}}+I_{\perp }}.Math.$

(36) of the luminescence radiation, where I∥ and I.sup.⊥ represent the intensities of the partial beams having mutually perpendicular polarization directions. The degree of polarization has the proportionality

(37) $P\sim \exp \left(\frac{R}{r-r_0}\right),$

(38) which is why, from the known variables R and r0 and the relationship between r and the analyte concentration sought (see explanations in the introduction to the description), said concentration can be ascertained.

(39) FIG. 6 is a graph showing a dependence of the degree of polarization of the luminescence radiation on the concentration of the analyte, the dependence having been ascertained in the aforementioned manner.

(40) FIG. 7 shows an example of a multi-core optical fiber 44 having ten core strands 45, each of the strands 45 acting as an independent optical fiber and each ending in a separate sensor element.

(41) TABLE-US-00001 List of reference numerals 1 first sensor device 2 first sample container 3 first pulsed light source 4 photon counter 5 sensor element 6 beam splitter 7 optical fiber 8 first optical filter 9 second sensor device 10 second pulsed light source 12 multi-core optical fiber 13 second sensor element 14 photon counter 15 sample container 16 third sensor device 17 third pulsed light source 18 dichroic mirror 19 sample container 20 sensor element 21 third photon counter 22 fourth sensor device 23 optical fiber 24 optical fiber 25 fourth pulsed light source 26 fourth sensor element 27 sample container 28 fourth photon counter 29 optical filter 30 fifth sensor device 31 continuous light source 32 sample container 33 sensor element 34 first photodetector 35 second photodetector 36 linear polarizer 37 first optical fiber 38 second optical fiber 39 optical filter 40 linearly polarized excitation light 41 polarizing beam splitter 42 first partial beam 43 second partial beam 44 multi-core optical fiber 45 core strand