Patent Publication Number: US-2021164987-A1

Title: Method and Optode for Determining the Concentration of an Analyte in a Sample Liquid

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims benefit of and priority to German Patent Application No. DE10 2019 132 525.0, filed on Nov. 29, 2019 which is incorporated by reference in its entirety. 
     BACKGROUND 
     The invention relates to a method for determining the concentration of an analyte in a sample liquid according to the preamble of claim  1  and an optode according to the preamble of claim  9 . The sample liquid can be in particular a biological fluid, such as blood, serum intercellular fluid, cerebrospinal fluid, sweat or urine. 
     Various methods for the chemical analysis of such liquids are already known, for example flame spectroscopy, photometric methods, colorimetric methods, luminescence methods and methods using ion-selective electrodes or ion-selective optodes. The use of these methods, for example for the analysis of blood samples, requires the extraction of serum or plasma by means of centrifuge, the preparation of a precise volume of the serum sample and the dilution thereof with a precise volume of distilled water. 
     An optode of the type mentioned at the outset is known from EP 2805 151 B1, by means of which optode the pH or substance concentrations in a sample can be determined using a time-resolved spectroscopy method. It is proposed that a pH-sensitive dye be immobilized in a polymer matrix comprising sulfonated polyether ether ketone (SPEEK). This material allows the pH of the sample to be determined from the decay time of the luminescence of the sensor dye. In the case of materials used for the polymer matrix in the prior art, the interactions between the polymer matrix and the sensor dye led to changes in the optical properties of the sensor dye, which severely restricted its usability for pH determination. In addition to the pH value, other parameters, in particular substance concentrations, can also be determined. 
     U.S. Pat. No. 5,246,867 discloses a method for measuring the sugar concentration of a sample. For this purpose, a donor-acceptor pair is brought into contact with the sample, with either the molecules of the fluorescent donor or the molecules of the acceptor being immobilized on a carrier material. If a donor binds to an acceptor, this results in a measurable change in the fluorescence lifetime of the donor. The non-immobilized molecules of the donor-acceptor pair compete with the sugar present in the sample, such that, depending on the sugar concentration, the change in the fluorescence lifetime is eliminated or reduced. The change in the fluorescence lifetime is determined using phase-modulated fluorimetry or using time-resolved fluorimetry. 
     U.S. Pat. No. 6,395,556 B1 discloses a method for determining the concentration of an analyte in a sample liquid, wherein fluorescent sensor molecules are added to the sample liquid, the fluorescence properties of which molecules depend on the concentration of the analyte. Both the sample liquid and a reference liquid having fluorescent reference molecules are exposed to excitation radiation which excite both the reference molecules and the sensor molecules to fluorescence radiation. The fluorescence radiation of the reference liquid is polarized along a first polarization axis and the fluorescence radiation of the sample liquid is polarized along a second polarization axis which is substantially perpendicular to the first. One of the polarized emissions is weakened to such an extent that the intensities on both polarization axes are substantially the same. The analyte concentration can be deduced from the degree of weakening. 
     A method and an optode of the type mentioned at the outset are known from DE 10 2018 204 744 A1. According to this document, the concentration of analyte ions contained in an electrolyte is determined using a light-irradiated ion-selective membrane that has been immersed in the electrolyte. Reference measurements should not be necessary. A concentration-dependent shift of the spectral position of a luminescence-based absorption, reflection or transmission spectrum of an optode is measured. This method therefore requires the use of an expensive, high resolution spectrometer. The technical problem addressed by the invention is that of providing a method and an optode of the type mentioned at the outset, by means of which reference samples or reference measurements can be largely avoided in a cost-effective manner. 
     The technical problem is solved by means of a method of the type mentioned at the outset containing the characterizing features of claim  1  in an optode of the type mentioned at the outset containing the characterizing features of claim  9 . Advantageous embodiments are provided by the dependent claims. 
     SUMMARY OF THE INVENTION 
     The invention is thus based on a method in which, 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. 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. According to the invention, it is now proposed that 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 via particles of the analyte, is used to ascertain the analyte concentration. Particles of the analyte can be atoms, ions or molecules. 
     The exchange interaction, also called exchange energy, is a quantum mechanical phenomenon that occurs when identical particles interact with one another. The exchange interaction is a weak interaction, which in the present context is in the order of meV (millielectron volts) but has a high range. In the case of the indirect exchange interaction, this takes place via the influence of a further variable which is introduced by the particle of the analyte, for example, an electric field in the case of an ionized particle. A sensor dye molecule interacts with a particle of the analyte and this in turn with at least one further sensor dye molecule, which leads to an indirect exchange interaction between at least two molecules of the sensor dye that are involved. 
     In particular, evaluating a variable that is proportional to the exponential function 
     
       
         
           
             exp 
              
             
               ( 
               
                 R 
                 
                   r 
                   - 
                   
                     r 
                     0 
                   
                 
               
               ) 
             
           
         
       
     
     can be advantageous for the evaluation routine. This dependence is a consequence of the indirect exchange interaction, where R is a measure of the distance between the immobilized molecules of the sensor dye and r is a measure of the distance between the particles of the analyte, which interact with the molecules of the sensor dye. The indirect exchange interaction results when the distance between an involved immobilized molecule of the sensor dye and an analyte particle mediating the exchange interaction is at most r0. r0 is what is known as the Förster radius (Theodor Förster, “ Experimentelle and theoretische Untersuchung des zwischenmolekularen Übergangs von Elektronenanregungsenergie ” [Experimental and theoretical investigation of the intermolecular transition of electron excitation energy],  Zeitschrift für Naturforschung  [Journal for Nature Research], 4a, pp. 321-327 [1949]; T. Förster,  Zwischenmolekulare Energiewanderung and Fluoreszenz  [Intermolecular Energy Migration and Fluorescence],  Annalen der Physik, Volume  437, Issue 1-2, [1948], pp. 55-75). The indirect exchange interaction becomes relevant if both R&lt;r0 and r&lt;r0 apply. 
     The aforementioned variables R and r result from the concentration of the immobilized molecules of the sensor dye or the analyte concentration sought. 
     For a small mass fraction of the immobilized molecules of the sensor dye in the total mass of the carrier unit and sensor dye, the following applies in good approximation: 
     
       
         
           
             R 
             ≈ 
             
               1 
               
                 
                   ( 
                   
                     NMN 
                     a 
                   
                   ) 
                 
                 
                   1 
                   2 
                 
               
             
           
         
       
     
     where N is the molality of the immobilized molecules of the sensor dye based on the carrier unit, M is the density of the carrier unit and Na is the Avogadro number (≈6.022 10 23  mol −1 ). 
     One can find values below 6 mmol/kg may be given for N. For determining salt ions Na+, a tetramethylammonium salt (e.g. Sodium Green™) can be used as sensor dye, for example. 
     R is a measure of the average distance between the individual immobilized molecules of the sensor dye and is thus a parameter that is known by the optode, specifically by the specified molality of the molecules of the sensor dye, which molality is based on the mass of the carrier unit. This molality can be specified when the carrier unit is being manufactured. 
     r0 depends on the type of sensor dye and can be ascertained experimentally or by numerical calculation, for example, and can reach up to 10 nm. 
     The variable r represents the average distance between the particles of the analyte and, in turn, it applies in good approximation with negligible mass of the analyte in the sample liquid: 
     
       
         
           
             r 
             ≈ 
             
               1 
               
                 
                   ( 
                   
                     nmN 
                     a 
                   
                   ) 
                 
                 
                   1 
                   2 
                 
               
             
           
         
       
     
     where n is the molality of the analyte in the sample liquid and m is the density of the sample liquid. The molality n is a direct measure of the analyte concentration sought. 
     The method according to the invention can be performed in such a way that luminescence radiation lifetime dependence on 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 the polymer matrix and a known dependence of the lifetime of the luminescence radiation is based on the sensor dye molecule concentration as well as the analyte concentration. 
     The method according to the invention can be performed in such a way that proportionality is used for the dependence of the lifetime of the luminescence radiation on the sensor dye molecule concentration and the analyte concentration as follows 
     
       
         
           
             τ 
             ∼ 
             
               exp 
                
               
                 ( 
                 
                   R 
                   
                     r 
                     - 
                     
                       r 
                       0 
                     
                   
                 
                 ) 
               
             
           
         
       
     
     where τ is the lifetime of the sensor dye luminescence radiation of the immobilized molecules which interact with the analyte. 
     Luminescence radiation lifetime can be ascertained using time-resolved measurement or phase modulation. 
     As an alternative to ascertaining the lifetime of the luminescence radiation, analyte concentration can also be determined using the degree of polarization of the luminescence radiation. For this purpose, the method according to the invention is performed in such a way that the excitation radiation is directed onto the carrier unit in a polarized manner and, from a luminescence radiation of the sensor dye induced by the excitation radiation, intensities III and I ⊥  of two polarization directions that are substantially mutually perpendicularly are determined and, as the property of the luminescence radiation, the dependence of the degree of polarization 
     
       
         
           
             P 
             = 
             
                
               
                 
                   
                     I 
                     II 
                   
                   - 
                   
                     I 
                     ⊥ 
                   
                 
                 
                   
                     I 
                     II 
                   
                   + 
                   
                     I 
                     ⊥ 
                   
                 
               
                
             
           
         
       
     
     on the concentration of the analyte is used. 
     Advantageously, proportionality dependence 
     
       
         
           
             P 
             ∼ 
             
               exp 
                
               
                 ( 
                 
                   R 
                   
                     r 
                     - 
                     
                       r 
                       0 
                     
                   
                 
                 ) 
               
             
           
         
       
     
     can be used. 
     This proportionality of the degree of polarization of the aforementioned natural exponential function (e function) results from the above-described indirect exchange interaction of immobilized molecules of the sensor dye via particles of the analyte, which provides an additional effect in the electric field. 
     Exemplary embodiments of the method according to the invention and of the optode according to the invention are illustrated below with reference to figures. For the sake of simpler linguistic representation, some of the exemplary embodiments listed below concern the determination of the concentration of an analyte of a single type, e.g. cooking salt (NaCl), using a specific sensor dye, e.g. a tetramethylammonium salt (e.g. Sodium Green™). However, it is conceivable for the concentration of a plurality of analytes to be determined simultaneously or in succession. In addition, more than one sensor dye may be used. 
    
    
     
       BREIF DESCRIPTION OF THE DRAWINGS 
       In the drawings: 
         FIG. 1A-1D  show four exemplary variants of a sensor device, 
         FIG. 2  is a graph showing laser pulse power over time, 
         FIG. 3  is a graph showing normalized radiation intensity as ascertained by a photon counter, 
         FIG. 4  is a graph showing the luminescent decay rate as a function of the concentration of sodium ions in a tested sample liquid, 
         FIG. 5  shows a fifth sensor device with a continuous light source, 
         FIG. 6  is a graph showing the degree of polarization of the luminescence radiation as a function of the concentration of the analyte, and 
         FIG. 7  shows a multi-core optical fiber which has ten core strands. 
     
    
    
       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 
     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. 
     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&#39;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 . 
     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. 
     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. 
     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 . 
       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. 
     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. 
     For the graph, the normalized intensity 
     
       
         
           
             I 
             = 
             
               
                 I 
                 max 
               
                
               
                 exp 
                  
                 
                   ( 
                   
                     - 
                     
                       
                         t 
                         - 
                         
                           t 
                           delay 
                         
                       
                       τ 
                     
                   
                   ) 
                 
               
             
           
         
       
     
     where I max =1E6, t 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. 
     On the basis of the proportionality 
     
       
         
           
             τ 
             = 
             
               
                 τ 
                 2 
               
                
               
                 exp 
                  
                 
                   ( 
                   
                     R 
                     
                       r 
                       - 
                       
                         r 
                         0 
                       
                     
                   
                   ) 
                 
               
             
           
         
       
     
     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. 
     τ corresponds to the luminescence lifetime, which is determined from the measured decay of the luminescence according to the formula 
     
       
         
           
             I 
             = 
             
               
                 I 
                 max 
               
                
               
                   
               
                
               
                 
                   exp 
                    
                   
                     ( 
                     
                       - 
                       
                         
                           
                             - 
                             t 
                           
                           - 
                           
                             t 
                             delay 
                           
                         
                         τ 
                       
                     
                     ) 
                   
                 
                 . 
               
             
           
         
       
     
     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 . 
     The concentration can thus be determined without a reference measurement. 
       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. 
       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. 
     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 . 
     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 . 
     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 . 
     In the following, a method utilizing light polarization is presented as an alternative to the time-resolving method. 
       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. 
     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 
     
       
         
           
             P 
             = 
             
                
               
                 
                   
                     I 
                     II 
                   
                   - 
                   
                     I 
                     ⊥ 
                   
                 
                 
                   
                     I 
                     II 
                   
                   + 
                   
                     I 
                     ⊥ 
                   
                 
               
                
             
           
         
       
     
     of the luminescence radiation, where I ∥ and I ⊥  represent the intensities of the partial beams having mutually perpendicular polarization directions. The degree of polarization has the proportionality 
     
       
         
           
             
               P 
               ∼ 
               
                 exp 
                  
                 
                   ( 
                   
                     R 
                     
                       r 
                       - 
                       
                         r 
                         0 
                       
                     
                   
                   ) 
                 
               
             
             , 
           
         
       
     
     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. 
       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. 
       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. 
     
       
         
           
               
             
               
                   
               
               
                 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