Method for traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring device with a calibration composition

Abstract

Method for traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring instrument with a calibration composition comprising at least one fluorescent calibration dye, characterized in that the method comprises the following steps: • Weighing out at least one calibration dye using a calibrated or verified precision balance; • Preparation of a stock solution of a calibration composition by adding a solvent or a polymer solution of known mass; • Prepare a dilution series with known concentrations of at least one calibration dye; • Measuring the absorption spectrum for each concentration of the dilution series of the calibration composition in an absorption spectrometer and calculating the molar extinction coefficient from the measured extinction for each concentration of the at least one calibration dye in the dilution series; • Measuring the emission spectrum for each concentration of the dilution series of the calibration composition in the single fluorescence measuring device; • Calculation of the integral fluorescence intensity for each concentration of at least one calibration dye in the dilution series from the emission spectra; • Assignment of the respective molar extinction coefficient and the respective integral fluorescence intensity to a concentration of the calibration composition.

Description

[0001] The invention relates to a method for the traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring instrument with a calibration composition comprising at least one fluorescent calibration dye. According to the invention, the traceability and thus reproducibility of the calibration chain is achieved by highly precise weighing of the calibration dye during the preparation of the calibration composition, thereby linking it to the SI units mass and mole. The invention further relates to a kit for the traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring instrument with a calibration composition comprising at least one fluorescent calibration dye, using the method according to the invention.

[0002] Fluorescence methods are widely used in the life sciences for the detection and quantification of analytes. Examples range from immunoassays to PCR methods, such as those used for the detection of viruses like COVID-19. Various fluorescent dyes (referred to here as fluorescent markers) are used to generate the signal. These dyes are, for example, covalently bound to specific antibodies or DNA sequences, or they intercalate into the DNA. Often, the fluorescence intensities measured at a specific emission wavelength range at a fixed excitation wavelength are used as readout parameters. These intensities are then correlated with the analyte concentration, for example, using a previously recorded calibration curve for analyte quantification. These measurements are frequently performed integrally, meaning that the fluorescence values ​​are combined to determine the analyte concentration.The wavelength selection for the emission signal is carried out using simple optical components such as filters.

[0003] The German patent application DE 10 2014 100 536 A1 discloses a test strip with sterically immobilized dyes for the internally referenced fluorometric determination of anions, in particular fluoride, from organic, mixed aqueous-organic, or organic-aqueous, and / or aqueous solutions, comprising a BODIPY core and an amidothiourea derivative residue. It should be noted that the integral fluorescence signal detected with inexpensive detectors such as photodiodes is influenced not only by the amount or concentration of the fluorescent marker to be detected, but also by the wavelength-dependent transmission profile of the filter and the spectral sensitivity of the detection system. Therefore, the measured integral fluorescence signal reflects a device-specific contribution.

[0004] Multiparametric fluorescence methods, which can utilize the fluorescence parameters relative or integral fluorescence intensity, emission spectrum, fluorescence excitation spectrum, fluorescence decay behavior or lifetime, and emission anisotropy, offer numerous advantages in their application and are therefore of great interest in a wide range of fields. They are characterized by • a relatively simple procedure; • diverse miniaturization options for the devices required for this; and • in particular, a high detection power that enables the detection of individual molecules or particles; from.

[0005] In addition to the device-specific contribution to the fluorescence signal already described, it must also be considered when performing fluorescence methods that the optical properties of fluorophores, such as the spectral position and intensity of their absorption and emission properties, are sensitive to the environment. Furthermore, the fluorescence intensity of the fluorophores depends on the excitation light intensity or excitation power density. This applies to all fluorescence methods and fluorescence instruments.

[0006] The dependence of fluorescence intensity on excitation light intensity is often determined by signal referencing (so-called ratiometric measurements, or measurements of the quotient of the analyte-dependent sample signal and an inert or constant reference signal). This reference signal can be generated using another reference fluorophore or by means of a reference detector such as a photodiode.

[0007] Instrument-specific contributions to measured "uncorrected" fluorescence signals are due to the wavelength-dependent optical and optoelectronic properties of the instrument components used, such as light sources, filters, and detectors. This means that even fluorescence signals measured with different fluorescence instruments of the same type can differ from one and the same sample.

[0008] Furthermore, the aging of device components can alter their signal contributions. Therefore, it is desirable to enable regular device calibration, functional testing, and the comparison of fluorescence measuring devices with one another. This is particularly relevant for comparable measurements and applications in legally regulated fields such as medicine or medical diagnostics. Moreover, this is also a requirement for the approval of fluorescence measurement methods and devices.

[0009] The integral fluorescence intensity of an analyte, measured with a fluorescence meter, is therefore not inherently quantifiable, but initially represents a relative integral fluorescence intensity. Quantification always requires calibration, which should take all the aforementioned points into account.

[0010] Measurements with fluorescence measuring instruments, particularly in medically relevant fields, must meet exceptionally high standards regarding reliability and measurement uncertainties. Calibration procedures that allow traceability of measurement results are suitable for this purpose. Traceable measurements are related to a recognized standard through an unbroken chain of comparative measurements with known measurement uncertainty. The requirements for traceability to SI units are defined in ISO / EN 17025.

[0011] Furthermore, the traceability of the measured values ​​is also of interest for the approval of new devices and, in principle, to create a high-quality possibility for the calibration or referencing of fluorescence signals in a fluorescence measuring device, where the measurement uncertainty is precisely known.

[0012] The MESF concept for calibrating fluorescence intensity is known from the prior art. Here, calibration is performed using fluorescent microparticles of known fluorescence intensity [1]. MESF stands for "molecular equivalent soluble fluorochrome" and is a measure of the number of fluorescence molecules that contribute to a measurement signal. This concept is primarily known from flow cytometry. However, it does not account for environmental changes in the signal-relevant properties of the fluorophores incorporated into the particles, particularly changes in the fluorescence quantum yield. Furthermore, the particles can differ in their dye loading density (particle-to-particle variation), which is not captured in ensemble measurements averaging over a large number of particles. This concept has also not yet been reduced to SI units.

[0013] Microchannel systems that can be filled with calibration solutions are also known. These systems are offered by various vendors and, as such, can at best serve as a component for a calibration concept in fluorescence spectroscopy. A calibration method that provides traceable signal referencing is not available.

[0014] It would therefore be desirable to have a method for calibrating or referencing a fluorescence measurement in a fluorescence measuring device that not only allows for simple calibration but also enables the measurement results to be traced back to SI units and is easy to reproduce.

[0015] For this purpose, the present invention provides a method for traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring instrument with a calibration composition comprising at least one fluorescent calibration dye, characterized in that the method comprises the steps • Weighing out at least one calibration dye using a calibrated or verified precision balance; • Preparation of a stock solution of a calibration composition by adding a solvent or a polymer solution of known mass; • Prepare a dilution series with known concentrations of at least one calibration dye; • Measuring the absorption spectrum for each concentration of the dilution series of the calibration composition in an absorption spectrometer and calculating the molar extinction coefficient from the measured extinction for each concentration of the at least one calibration dye in the dilution series; • Measuring the emission spectrum for each concentration of the dilution series of the calibration composition in the single fluorescence measuring device; • Calculation of the integral fluorescence intensity for each concentration of at least one calibration dye in the dilution series from the emission spectra; • Assignment of the respective molar extinction coefficient and the respective integral fluorescence intensity to a concentration of the calibration composition.

[0016] Furthermore, a kit for traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring instrument is provided, comprising a calibration composition comprising at least one fluorescent calibration dye and a method according to any one of claims 1 to 9, comprising • at least one calibration dye of known mass; • a work instruction. Detailed description

[0017] Calibration dyes can be used for signal referencing of fluorophore-labeled samples and for the comparability of fluorescence measuring instruments with each other (adjustment of instrument sensitivities and signal quantities, which are due to different spectral properties such as wavelength-dependent transmissions or spectral sensitivities of the optical and optoelectronic components used in the instrument setup and which can change over time due to aging of the instrument components) as well as for performance validation or functional testing (IPV).

[0018] In a first embodiment, the present invention is suitable for calibrating fluorescence measuring instruments, thereby enabling the comparison of different fluorescence measuring instruments. Furthermore, calibrating a fluorescence measuring instrument allows its functionality and instrument characteristics to be verified. Repeated calibrations over an extended period make it possible to immediately detect any changes in the instrument's functionality. Since the instrument characteristics of the fluorescence measuring instrument influence the measurement results, this method eliminates a significant source of error in fluorescence measurement.

[0019] When calibrating a fluorescence measurement with a fluorescence meter, various influencing factors must be considered. The fluorescence signal detected by a detector of a fluorescence measurement system, assuming otherwise constant instrument settings such as the slit width (spectral bandwidth) of the excitation and emission monochromators and the same optical components in the excitation and emission / detection beam paths, such as filters and polarizers (and polarizer position), depends on the following influencing factors: a.) The device-specific influencing factors - Spectral photon flux at excitation wavelength λ ex at the sampling location (also referred to as excitation intensity), - Spectral sensitivity s(λ) em ) of the detection system, which depends on the emission wavelength λ em depends on. b.) The sample-specific contributions - Absorption (A) of a fluorescent dye at the excitation wavelength λ ex (A(λ ex ) = 1-10 -E(λex) ; E: extinction; E = ε × c × d; ε(λ ex ): molar (decadic) extinction coefficient at wavelength λ ex ; c: concentration; d: layer thickness or optical path length, determined by the cuvette used), - Fluorescence quantum yield of the dye in the chosen solvent at the temperature used.

[0020] Therefore, the following parameters must be taken into account when calibrating a fluorescence measurement in a fluorescence measuring device: - the wavelength accuracy of the excitation and detection channels (when using dispersing elements), - the wavelength dependence of the (relative) spectral sensitivity of the detection system.

[0021] The invention provides a method in which a calibration composition is used for calibration or referencing, and thus for signal referencing, wherein the calibration composition comprises at least one fluorescent calibration dye. The calibration composition can therefore comprise one or more calibration dyes. In one embodiment, the calibration composition comprises between one and five calibration dyes, preferably between one and three. If the calibration composition comprises more than one calibration dye, any interaction between the calibration dyes must be ruled out, or the nature of the interaction must be well known, in order to exclude or account for any influence on measurement results in the fluorescence measurement.In a particularly preferred embodiment of the invention, the calibration composition comprises exactly one calibration dye.

[0022] Suitable calibration dyes within the meaning of the present invention are selected from a group comprising organic dyes, dye-doped or dye-loaded polymer systems and fluorescent nanoparticles.

[0023] Suitable fluorescent nanoparticles can be selected from a group comprising polymer nanoparticles and silica nanoparticles, each loaded with organic dyes or semiconductor quantum dots such as II / VI or III / V semiconductor quantum dots.

[0024] In one embodiment of the present invention, the at least one calibration dye is an organic dye, wherein the organic dye is selected from the group comprising ultraviolet dyes whose emission range lies in the spectral range between 200 nm and 425 nm, blue dyes whose emission range lies in the spectral range between 425 nm and 485 nm, green dyes whose emission range lies in the spectral range between 485 nm and 560 nm, yellow / orange dyes whose emission range lies in the spectral range between 560 nm and 600 nm, red dyes whose emission range lies in the spectral range between 600 nm and 780 nm and near-infrared dyes whose emission range lies in the spectral range between 780 nm and 1000 nm.

[0025] In one embodiment of the invention, the calibration dye is a red dye, preferably selected from the group of dyes whose emission lies in the spectral range of 600 nm to 780 nm.

[0026] In one embodiment, the red dye is selected from the group of acridines, arylmethane, azo, and cyanine dyes, carbopyronines, chlorines / bacteriochlorines, heterocycles, oxazines, phenanthridines, phthalocyanines, porphyrins, rhodamines, and similar substances.

[0027] In another embodiment, the red dye is selected from the group of dyes FEW S0387, Methylene Blue, DY 610, ATTO 610, Bodipy 610, Bodipy 650, ATTO 594, DY 480, ATTO 633, Alexa Fluor 647, Cyanine 5 (CY5), ATTO 680, Cyanine 5.5 [CY55], Quasar 670, Quasar 705, FAM alkyne 5-isomer, FEW S0536, FEW S0586, FEW S2038, FEW S2314, FEW S2405, FEW S2381, FEW S0247, FEW S0584, FEW S0223, FEW S2197, FEW S0436, FEW S0536, FEW S0586, FEW S2308, FEW S2314, FEW S2405, FEW S2381, DyLight 633, TO-PRO-3-DANN, DDAO pH 9.0, Nilblau, EtOH, TOTO-3-DANN, DyLight 649, Carboxynaphthofluorescein pH 10.0, Allophycocyanin, Alexa 633, Alexa Fluor 660, TruRed, Alexa Fluor 680, ATTO 620, ATTO Rho14, ATTO 643, ATTO 647, ATTO 655, ATTO Oxa12 and ATTO 665, 5 / 6 ROX. The dye names correspond to the common trade names of the respective companies that are the dye suppliers.

[0028] Table 1 lists some of the mentioned red dyes with their structural formulas. Table 1: Examples of red dyes Red dyes Structural formula 5 / 6 ROX FEW S0387 Methylene blue DY 610 ATTO 610 Bodipy 610 ATTO 633 Alexa Fluor 647 Cyanin5 (CY5) ATTO 680 5-isomer Alexa 633 ATTO 594 DY 480

[0029] In a further embodiment of the invention, the calibration dye is a blue dye, preferably selected from the group of dyes whose emission lies in the spectral range of 425 nm to 485 nm.

[0030] In one embodiment, the blue dye is selected from the group comprising aromatic hydrocarbons, biomolecules, coumarins, heterocycles, indoles, oligophenylenes, polycyclic aromatic hydrocarbons, stilbenes and similar compounds.

[0031] In another embodiment, the blue dye is selected from the group comprising Fluorescein 27, Fluorescein Isothiocyanate (FITC), FLU, ATTO 488, Oregon Green 488 (OG488), Oregon Green 514 (OG514), Rhodamine Green (RGR), DY 430, DY 431, DY 478, DY 488, DY 490, DY 495, DY 505, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, ATTO 495, ATTO 465, ATTO Rho110, ATTO 514, ATTO 520, FEW S2409, MitoTracker Green, calcein pH 9.0, Fluo 4, FDA, DTAF, CFDA, DyLight 488, Rhodamine 110, BCECF pH 5.5, PicoGreendsDNA, SYBR Green I, CyQuant GR-DANN, NeuroTrace 500 / 525, green, fluorescent Nissl stain-RNA, Rhodol Green antibody conjugate pH 8.0, Fluoro-Emerald, Nissl, 5-(and-6)-carboxy-2', 7'-dichlorofluorescein pH 9.0, DM-NERF pH 4.0, NBD-X, Cy2, JOE, TET, CAL FLUOR Gold 540, Quasar 570, Cy 3, TAMRA and FAM, FAM-alkyne 5 isomer.

[0032] Table 2 lists some of the mentioned blue dyes with their structural formulas. Table 2: Examples of blue dyes Blue dye Structural formula Fluorescein 27 (F27) FITC FAM FAM-alkyne, 5 isomers JOE TET CAL FLUOR Gold 540 Quasar 570 Cy 3 TAMRA FLU ATTO 488 Oregon Green 488 (OG 488) Oregon Green 514 (OG 514) Rhodamin Green (RGR)

[0033] In a further embodiment of the invention, the calibration dye is an ultraviolet dye, preferably selected from the group of dyes whose emission lies in the spectral range of 200 nm to 425 nm.

[0034] In one embodiment, the ultraviolet dye is selected from the group comprising aromatic hydrocarbons, biomolecules, coumarins, heterocycles, oligophenylenes, polycyclic aromatic hydrocarbons, polyenes / polyynes and similar compounds.

[0035] In one embodiment of the present invention, the ultraviolet dye is selected from the group comprising 7-mercapto-4-methylcoumarin, S-methyl-7-mercapto-4-methylcoumarin, S-3-oxobutyl-7-mercapto-4-methylcoumarin, benzene, toluene, o-xylene, m-xylene, p-xylene, mesitylene, phenol, anisole, benzoic acid, aniline, N,N-dimethylaniline, phenylboronic acid, terephthalic acid, biphenyl, p-terphenyl, p-quaterphenyl, p-quinquephenyl, 1,3,5-triphenylbenzene, benzidine, 2,5-diphenyloxazole, 1,4-bis(5-phenyl-2-oxazolyl)benzene, (R)-BINOL, naphthalene, anthracene, phenanthrene, pyrene, triphenylene, 9,10-diphenylanthracene, 2,3-diaminonaphthalene, 1,4,5,8-naphthalene tetra-carboxylic acid dianhydride, 1,2-diphenylacetylene, trans-stilbene, 1,4-diphenylbutadiine, 1,4-diphenylbutadiene, 1,6-diphenylhexatriene, 2-methylbenzoxazole, L-Phenylalanine, L-Tyrosine, L-Tryptophan, Adenine.

[0036] Table 3 lists some of the ultraviolet dyes mentioned, along with their structural formulas. Table 3: Examples of ultraviolet dyes Ultraviolet dye Structural formula phenol Biphenyl naphthalene 1,4-Diphenylbutadiyne 2-Methylbenzoxazol L-Phenylalanine

[0037] In a further embodiment of the invention, the calibration dye is a green dye, preferably selected from the group of dyes whose emission lies in the spectral range of 485 nm to 560 nm.

[0038] In one embodiment, the green dye is selected from the group comprising acridines, arylmethane dyes, biomolecules, BODIPY dyes, chlorines / bacteriochlorines, coumarins, dipyrins, fluoresceins, heterocycles, oligophenylenes, perylenes, polycyclic aromatic hydrocarbons, polyenes / polyynes, pyrenes, rhodamines, xanthenes, stilbenes and similar substances.

[0039] In einer weiteren Ausführungsform ist der grüne Farbstoff ausgewählt aus der Gruppe aufweisend ATTO 425, 8-Amino-1,3,6-tris-(3-hydroxypropyl)-pyren sulfone, 8-Amino-[N,N',N''-tris(2-hydroxyethyl)-N,N',N''-trimethyl]pyren-1,3,6-trisulfonamid, 8-Methylamino-N,N',N''-tris(2-hydroxyethyl)-N,N',N''-trimethylpyren-1,3,6-trisulfonamide, ATTO 465, ATTO 495, 1,3,5,7,8-Pentamethyl-pyrromethin Fluoroborat, 1,3,5,7-Tetramethyl-pyrromethin Fluoroborat, 2-Chloro-1,3,5,7,8-pentamethyl-pyrromethin Fluoroborat, 8-Hydroxymethyl-2-cyano-1,3,5,7-tetramethyl-pyrromethen fluoroborat, 2-Chloro-1,3,5,7-tetramethyl-pyrromethen fluoroborat, 8-Hydroxymethyl-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 8-Hydroxymethyl-2,6-dicyano-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 8-Acetoxymethyl-2-cyano-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 2,6-Diethyl-1,3,5,7,8-pentamethyl-pyrromethin Fluoroborat, 2,6-Dichloro-1,3,5,7,8-pentamethyl-pyrromethin Fluoroborat, 2,6-Diethyl-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 8-Acetoxymethyl-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 8-Acetoxymethyl-2,6-dicyano-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 2,6-Dichloro-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 8-Hydroxymethyl-2-chloro-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 8-Acetoxymethyl-2-chloro-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, 8-Hydroxymethyl-2,6-dichloro-1,3,5,7-tetramethyl-pyrromethin Fluoroborat, Fluorescein, Rhodamin 110, ATTO 488, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 430LS, rac-BINAP, Tetracen, Rubrene, 8-Anilino-1-naphthalenesulfonsäure, Curcumin, Thiazol-Orange, 7-Benzylamino-4-nitrobenz-2-oxa-1,3-diazoe, Riboflavin, Coumarin 6, Acridin-Orange, Acridingelb G, Acridone, Auramin O, Perylen, Fluorescein, Eosin Y, Rhodamin 123, Rhodamin 6G, Lucifer yellow CH, N,N'-Difluoroboryl-1,9-dimethyl-5-phenydipyrrin, N,N'-Difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)dipyrrin, N,N'-Difluoroboryl-1,9-dimethyl-5-[(4-(2-trimethylsilylethynyl)pheny]dipyrrine, bis(5-phenyldipyrrinato)zinc.,

[0040] Table 4 lists some of the mentioned green dyes with their structural formulas. Table 4: Examples of green dyes Green dye Structural formula Curcumin 7-Benzylamino-4-nitrobenz-2-oxa-1,3-diazole Acridine yellow Perylen Eosin Y N, N'-Difluoroboryl-1,9-dimethyl-5-phenydipyrrine CuC-1

[0041] In a further embodiment of the invention, the calibration dye is a yellow / orange dye preferably selected from the group of dyes whose emission lies in the spectral range of 560 nm to 600 nm.

[0042] In one embodiment, the yellow / orange dye is selected from the group comprising acridines, arylmethane dyes, BODIPY dyes, cyanine dyes, phthalocyanines, polyenes / polyynes, porphyrins, pyrenes, quinones, rhodamines, xanthenes, stilbenes and similar substances.

[0043] In a further embodiment, the yellow / orange dye is selected from the group comprising 8-acetoxymethyl-2,6-dichloro-1,3,5,7-tetramethyl-pyrromethine fluoroborate, PM597, Alexa Fluor 532, Janelia Fluor 549, tetramethylrhodamine, ATTO 550, 2-(3,6-di(pyrrolidin-1-yl)xanthylium-9-yl)benzoate, 2-(3,6-di(azepane-1-yl)xanthylium-9-yl)benzoate, 2-(3,6-di(piperidin-1-yl)xanthylium-9-yl)benzoate, ATTO Rho3B, ATTO 565, ATTO Rho11, ATTO 542, 4-dimethylamino-4'-nitrostilbene, Alizarin red S, Neutral red, Nile red, and Merocyanine. 540, p-fuchsin, rose bengal, rhodamine B, sulforhodamine B, rhodamine 101 inner salt, sulforhodamine 101, MgOEP, ZnOEP, boron subphthalocyanine chloride.

[0044] Table 5 lists some of the mentioned yellow / orange dyes with their structural formulas. Table 5: Examples of yellow / orange dyes Yellow / orange dye Structural formula 4-Dimethylamino-4'-nitrostilbene Alizarin red S Neutral red Merocyanin 540 Rose Bengal Borone subphthalocyanine chloride

[0045] In a further embodiment of the invention, the calibration dye is an infrared dye, preferably selected from the group of dyes whose emission lies in the spectral range of 780 nm to 900 nm.

[0046] In one embodiment, the infrared dye is selected from the group comprising cyanine dyes, phthalocyanines and similar dyes.

[0047] In another embodiment, the infrared dye is selected from the group comprising Alexa Fluor 750, 1,1'-Diethyl-3,3,3',3'-tetramethylindotricarbocyanine Iodide, Indocyanine green, H2Nc(tBu).

[0048] Table 6 lists some of the infrared dyes mentioned, along with their structural formulas. Table 6: Examples of infrared dyes Infrared dye Structural formula 1,1'-Diethyl-3,3,3',3'-tetramethylindotricarbocyanine iodide Indocyanine green H2Nc(tBu)

[0049] In one process step, the at least one calibration dye is weighed out using a calibrated or verified precision balance. The use of a calibrated or verified precision balance ensures highly accurate weighing of the at least one calibration dye. Furthermore, the measurement uncertainty is precisely known.

[0050] Subsequently, a stock solution of the calibration composition is prepared by adding a solvent or a polymer solution of known mass.

[0051] If the calibration composition includes a solvent, the solvent is preferably an organic or an inorganic solvent. The choice of the appropriate solvent is subject to various criteria.

[0052] The solvent must be selected based, among other things, on the type of sample container used in the fluorescence spectroscopy. Some polymer chips used for this purpose are not resistant to organic solvents, so an inorganic solvent must be used in this case. Suitable inorganic solvents include, for example, aqueous buffer solutions. If, however, sample containers made of materials such as glass or polymers that are resistant to organic solvents are used, organic solvents can also be used. In one embodiment of the present invention, ethanol or methanol is used as the organic solvent. Therefore, the criterion of water solubility of the calibration dyes is not strictly necessary according to the invention.

[0053] Suitable water-soluble solvents can be, for example, aqueous buffer solutions or water.

[0054] The solvent is preferably added to the at least one calibration dye using a calibrated pipette, so that the mass of the added solvent is precisely known via its volume. Likewise, the measurement uncertainty regarding the volume or mass of the added solvent is known.

[0055] In a further embodiment of the invention, a polymer solution can be added to the at least one calibration dye instead of a solvent. Suitable polymers include, for example, silicone and epoxy resin. The polymers are generally dissolved in an organic solvent to provide a polymer solution. To prepare such a polymer solution, the sample is weighed using a calibrated precision balance, and the solvent is added using a calibrated pipette. In this way, both the mass of the polymer and the mass of the solvent, along with the associated measurement uncertainty, are precisely known.

[0056] In another embodiment, the calibration dye is present as a fluorescent nanoparticle, which is preferably dispersed in a solvent or in a polymer solution.

[0057] If the calibration composition is in liquid form, this has the advantage that photochemically decomposed molecules can diffuse back in during exposure, thus avoiding local bleaching effects.

[0058] In one embodiment of the present invention, the calibration composition comprises a curable polymer solution. An advantage of calibration compositions comprising a curable polymer solution is that they can be filled into the measuring containers used for the sample, such as cuvettes, microtiter plates, chips, microchannel systems, or other vessels, and then cured.

[0059] In a further embodiment, the calibration dye is a dye-doped or dye-loaded polymer system, to which, in a preferred embodiment, a solvent is added according to the inventive method. In a further preferred embodiment, this solution is filled into a measuring container and cured or polymerized therein.

[0060] In one embodiment, the method according to the invention therefore additionally includes the step of hardening the calibration composition.

[0061] In the inventive method, a dilution series with known concentrations of the at least one calibration dye is prepared for the calibration composition. For this purpose, the stock solution of the calibration composition is preferably diluted stepwise by adding a solvent. The addition of the solvent is again carried out using a calibrated pipette, so that the mass of the added solvent and the associated measurement uncertainty are precisely known. For high-quality calibration, the dilution series should consist of calibration compositions with at least two different concentrations of the at least one calibration dye. In principle, the dilution series can consist of any number of different concentrations of the at least one calibration dye.In one embodiment of the present invention, the dilution series consists of 2 to 10 different concentrations of the at least one calibration dye, preferably of 2 to 8 different concentrations of the at least one calibration dye, and particularly preferably of 3 different concentrations of the at least one calibration dye.

[0062] According to the invention, a defined volume of the calibration composition is filled into a measuring container using a calibrated pipette and optionally solidified. Suitable measuring containers are, as already mentioned, cuvettes, microtiter plates, chips, microchannel systems, or other vessels.

[0063] The emission and absorption spectrum of the calibration composition are measured for each concentration of the dilution series of the calibration composition in the fluorescence meter and in an absorption meter.

[0064] From the measured absorption spectra, the absorbance A of the at least one calibration dye is determined for each concentration of the at least one calibration dye in the dilution series, and the absorbance E is calculated from this according to formula (1). From the absorbance E, the molar extinction coefficient ε of the at least one calibration dye is calculated according to formula (2). Preferably, the absorption spectra are corrected for solvent and baseline during the evaluation. A(λex)=1−10−E(λex) ε=Ec d

[0065] The molar extinction coefficient ε is calculated according to formula (2) from the extinction E, the concentration of the calibration dye c, and the layer thickness d of the measuring vessel. E is the dimensionless extinction, i.e., the reduction in the intensity of the light measured in the fluorescence meter, and is determined from the absorption spectrum. This procedure is familiar to those skilled in the art.

[0066] Furthermore, the integral fluorescence intensity for each concentration of at least one calibration dye in the dilution series is determined from the emission spectra. For this purpose, the integral under the emission spectrum is calculated. This procedure is also familiar to those skilled in the art.

[0067] At higher concentrations of calibration dyes, emitted photons can be reabsorbed. This leads to an underestimation of the fluorescence intensity or the number of emitted photons in the spectral region of the short-wavelength edge of the emission spectrum. This also results in a redshift of the emission spectra. To minimize the contribution of reabsorption effects to the specification of the integral fluorescence intensity, a preferred embodiment of the method only considers the area integral of the emission band in a spectral region where there is no overlap between absorption and emission, or where the overlap between absorption and emission is minimal.

[0068] The wavelength range in which the at least one calibration dye of the calibration composition emits should overlap with the detection range of the filters and photodiodes used in the fluorescence measuring device. Ideally, the overlap should be as large as possible to ensure good detection of the emission spectrum of the at least one calibration dye.

[0069] It is particularly preferred if the photoluminescence quantum yield of the at least one calibration dye is as high as possible in order to achieve a good signal / noise ratio at the photodetector in the fluorescence measuring device.

[0070] Preferably, the at least one calibration dye exhibits very good thermal and photochemical stability and very low temperature dependence of the optical properties, in particular the fluorescence quantum yield, in the temperature range used for the sample.

[0071] It is further preferred if the at least one calibration dye exhibits low, and especially preferably no, aggregation in the solvent or polymer solution of the calibration composition.

[0072] Furthermore, adsorption on the walls of the measuring vessel used for measurement is undesirable. In one embodiment of the present invention, the at least one calibration dye exhibits no or only negligible adsorption on the walls of the measuring vessel used. Negligible in this context means that the effect has no measurable influence on the measurement results.

[0073] The method according to the invention is carried out within the linearity range of the detection system of the fluorescence measuring device. This range must therefore be known beforehand. In one embodiment of the present invention, the linearity range of the detection system is thus determined, and the method according to the invention is applied within this linearity range. As is known to those skilled in the art, the linearity range is that region of the detector in which it displays linear fluorescence intensities.

[0074] Fluctuations in excitation power intensity, e.g., due to fluctuations in the light source of the fluorescence meter, are detected either by a reference channel (output of the measurement signal as the quotient of the signals from the emission detection system and the reference detector) or, in devices without a separate reference detector, by referencing the signal of a dye determined under constant measurement conditions. This referencing can be performed using a reference dye that has a known and / or constant signal intensity. The reference dye can, for example, be contained in a PCR plate or microtiter plate. Alternatively, a scattering solution or a scatterer can be used whose measurement signal depends on the excitation intensity. Both variants are known from the prior art.

[0075] According to the invention, a molar extinction coefficient ε and an integral fluorescence intensity can be assigned to each concentration of the calibration composition. Due to the weighing of the at least one calibration dye and optionally a polymer using a calibrated or verified precision balance and the use of calibrated pipettes when adding solvents, the concentration of the calibration dye in the measuring vessel is precisely known. If the volume in the measuring vessel is known, the integral fluorescence intensity can be traced back to how many grams or moles of the calibration dye produce the measured integral fluorescence intensity in the fluorescence measuring instrument, thus providing a traceable calibration of the fluorescence measuring instrument.

[0076] If the inventive method is carried out at time intervals with the same calibration composition and the same fluorescence measuring device under comparable measurement conditions, the measured integral fluorescence intensity should always remain the same for a concentration of the calibration composition within the measurement tolerance. Comparable measurement conditions mean that all measurement conditions that can influence the measurement signal are the same. These are, in particular, the temperature, the measurement geometry, the optical components of the fluorescence measuring device, and the excitation wavelength. Device-specific influencing factors such as the spectral photon flux at the excitation wavelength λ are also considered. ex at the sample location and the spectral sensitivity s(λ) emThe values ​​of the detection system should ideally remain unchanged from previous measurements. If deviations occur during the calibration measurement that lie outside the measurement tolerance, this indicates that the device-specific properties of the fluorescence measuring instrument used have changed.

[0077] The method according to the invention can therefore be advantageously used to test the functionality of a fluorescence measuring device and to monitor it at regular intervals. This is particularly important for quality assurance of subsequent measurements with other samples. A malfunctioning fluorescence measuring device and the associated altered device-specific influences on the measurement signal can thus be ruled out.

[0078] A further advantage is that the method according to the invention can be used to reference different fluorescence measuring instruments in a traceable manner. By applying the method according to the invention with identical calibration compositions and concentrations in different fluorescence measuring instruments, the respective integral fluorescence intensities determined can be compared and the instruments referenced to one another. This referencing always inherently incorporates the instrument-specific properties of the respective fluorescence measuring instruments used. Therefore, the method according to the invention makes it possible to compare measurements in different fluorescence measuring instruments.

[0079] Another area of ​​application is the production control of fluorescence spectrometers. Instrument manufacturers aim to ensure that their manufactured fluorescence measuring devices consistently exhibit similar performance. However, since even the optical components of a fluorescence spectrometer do not exhibit exactly the same optical properties, variations in the device-specific characteristics of different fluorescence spectrometers can occur. The method according to the invention allows the influence of these device-specific properties on the fluorescence signal to be determined after the manufacturing of the fluorescence measuring devices. This makes it possible to assess the quality of the fluorescence measuring devices and thus ensure the highest possible quality in production.

[0080] The method according to the invention establishes a traceable and reproducible calibration chain. It utilizes the fact that weighing out samples, for example of the at least one calibration dye, even in the range of a few milligrams, can be carried out with very high precision and accuracy using a correspondingly precise calibrated or verified precision balance. The traceability and reproducibility of the calibration chain according to the invention are thus achieved by linking it to the SI units mass or mole. When using solvents, the uncertainties of defined dilution series prepared with calibrated pipettes can be easily determined via gravimetric or photometric control. For gravimetric control, the solutions and dilution series are prepared directly on a calibrated balance.For photometric control, a calibrated absorption spectrometer is used, and verification is carried out by measuring absorption spectra (also in cuvettes of different and precisely known layer thicknesses) and by previously determined extinction coefficients. Defined sample weights are used for determining the extinction coefficients. Using a calibrated or verified precision balance and calibrated pipettes, solutions are prepared via gravimetric control as described above. Thus, all prerequisites for establishing a complete measurement uncertainty balance, as required for traceability from SI units according to ISO / EN 17025, are fulfilled in the inventive method.

[0081] In a further embodiment of the invention, the method additionally comprises the steps • Provide a sample containing at least one fluorescent marker; • Measure at least one absorption spectrum of the sample in an absorption spectrometer and calculate the molar extinction coefficient from the measured extinction; • Measure at least one emission spectrum of the sample in the fluorescence measuring device under the same measurement conditions as the emission spectra of each concentration of the dilution series of the calibration composition were measured; • Calculation of the integral fluorescence intensity of the sample from the emission spectrum; • Mapping of the integral fluorescence intensity of the at least one emission spectrum of the sample to the integral fluorescence intensity of a concentration of the calibration composition; wherein the spectral properties of the calibration composition are adapted to the at least one fluorescence marker of the sample.

[0082] In this embodiment, the method according to the invention is suitable for traceably referencing the fluorescence measurement of a sample that has at least one fluorescent marker.

[0083] In this embodiment, a sample is provided that contains at least one fluorescent marker. Suitable samples within the meaning of the present invention are fluorescence assays and / or other calibration objects. According to the invention, the samples contain at least one fluorescent marker.

[0084] Fluorescence assays are based on fluorescence measurements and are well known to experts. Examples include DNA- or PCR-based COVID-19 fluorescence assays and fluorescence assays for other disease-specific biomarkers. Disease-based biomarkers can include, for example, cancer-related biomarkers such as HER2 / Neu, inflammatory biomarkers such as CRP, biomarkers for autoimmune diseases, and similar biomarkers.

[0085] Other calibration objects can, for example, be further calibration solutions. In one embodiment, a calibration solution corresponding to a calibration composition as described in the present invention is suitable for this purpose. In another embodiment of the invention, the other calibration objects can be selected from the group comprising doped polymer systems, dye-containing polymer systems, and fluorescent glasses.

[0086] According to the invention, the sample comprises at least one fluorescent marker. In one embodiment of the present invention, the at least one fluorescent marker of the sample is selected from the group consisting of blue dyes whose emission range lies in the spectral range between 425 nm and 485 nm and red dyes whose emission range lies in the spectral range between 600 nm and 780 nm. Dyes that meet these conditions have already been described with respect to calibration dyes. In another embodiment, the at least one fluorescent marker in the sample is selected from the group consisting of FAM and ATTO 647N, whose structural formulas are given in Table 7. These can advantageously be used, for example, in a DNA- or PCR-based COVID-19 fluorescence assay. Table 7: Examples of fluorescent markers Fluorescence markers Structural formula FAM ATTO 647N

[0087] In a further embodiment of the invention, the sample can also have several fluorescent markers. In one embodiment of the invention, the sample has 2 to 5 fluorescent markers, preferably 2 to 3 fluorescent markers, wherein the individual fluorescent markers must be spectrally distinguishable. They can be spectrally distinguished if their measurement signals can be separated from one another.

[0088] According to the invention, at least one absorption spectrum of the sample is measured in an absorption spectrometer and the molar extinction coefficient is calculated from the measured absorption according to formulas (1) and (2).

[0089] Furthermore, at least one emission spectrum of the sample is measured in the fluorescence analyzer under the same measurement conditions as those under which the emission spectra of each concentration in the dilution series of the calibration composition were measured. Identical measurement conditions mean that all measurement conditions that can influence the measurement signal are the same. These include, in particular, the temperature, as well as the measurement geometry, the optical components of the fluorescence analyzer, and the excitation wavelength. The measurement geometry refers to the configuration of the optical beam path within the analyzer.

[0090] All measurements of the sample and the calibration composition are performed in the same absorption spectrometer and fluorescence measuring device.

[0091] For the most accurate and reliable reference and instrument calibration, functional testing, and adjustment possible, calibration, verification, and control measurements using the inventive method should be performed under conditions as similar as possible to those used with the samples. This includes the instrument parameters used in the excitation and detection channels, the measurement geometry employed, and ideally, the measuring containers used as well.

[0092] The measurement of the absorption and emission spectra of the sample and the calibration composition is therefore preferably carried out with the same instrument parameters, the same measurement geometry, and optionally the same type of measuring container. In a particularly preferred embodiment, the same type of measuring container is used.

[0093] All device-specific influencing factors, in particular the spectral photon flux at the excitation wavelength λ exat the sample location and the spectral sensitivity s(λ) em The parameters of the detection system are therefore the same for all measurements.

[0094] Fluorescence measurements using reader systems are often performed in a front-face geometry. Here, the meniscus of the measured sample or calibration solution in the measuring container can influence the measurement. The meniscus depends on the material of the measuring container (polymer systems, glass, etc.) and its wetting properties, as well as on the composition of the solvent or polymer used. For the best possible match between sample and calibration solution, either the same solvent or polymer solution should be used in both the sample and the calibration solution, or the solvent or polymer solution of the calibration solution must be selected so that the menisci of the sample and the calibration solution are as similar as possible.Organic solvents such as ethanol often have the advantage of providing a smoother meniscus than some aqueous solvents, such as some bioanalytically relevant aqueous buffers.

[0095] In one embodiment of the invention, the sample contains the same solvent or polymer solution as the calibration composition.

[0096] By adding surfactants, the meniscus in the measuring vessel can be modified to match the meniscus of the calibration composition to that of the sample. It is important to ensure that the at least one surfactant does not significantly alter the calibration-relevant spectral properties of the at least one calibration dye. Suitable surfactants include anionic surfactants such as SDS (sodium dodecyl sulfate), cationic surfactants such as CTAB (cetyltrimethylammonium bromide), neutral surfactants, PEG systems, proteins such as BSA, and micelle formers. The at least one surfactant is selected to minimize the appearance of the meniscus and thus eliminate any noticeable edge effects. The surface should appear as planar as possible.

[0097] In one embodiment of the present invention, at least one surfactant of known mass is added during the preparation of the calibration composition.

[0098] When a surfactant is added, this is done using a calibrated pipette, so that the added volume, and therefore the mass of the surfactant, is precisely known. Likewise, the measurement uncertainty of the volume, and thus the mass, is known.

[0099] In a further embodiment of the invention, the edge effect caused by the meniscus is hidden or covered by a mask.

[0100] Essential for the present procedure and the quality of the associated calibration is that the spectral properties of the calibration composition are adapted to the at least one fluorescence marker of the sample.

[0101] In particular, according to the invention, the position of the emission spectrum of the at least one calibration dye in the calibration composition should be adapted to the emission spectrum of the at least one fluorescent marker used in the sample. This means that, according to the invention, the emission spectrum of the at least one calibration dye in the calibration composition overlaps with the emission spectrum of at least one fluorescent marker in the sample. Preferably, the overlap of the two emission spectra should be as large as possible. The greatest possible overlap contributes to a higher quality of calibration.

[0102] It should be noted that the optical properties of calibration dyes, such as the spectral position and shape of the absorption and emission spectra, the intensity parameters such as the molar extinction coefficient, and the fluorescence quantum yields, depend on their environment. The solvent or polymer solution in the calibration composition therefore influences the spectral properties of the at least one calibration dye in the calibration composition. Therefore, according to the invention, the spectral properties of the at least one calibration dye must be considered as a function of the other components of the calibration composition. This also offers the possibility of favorably influencing the spectral properties of the at least one calibration dye in the calibration composition by adding specific solvents or polymer solutions.The spectral properties of the calibration composition and also of the sample can be determined by measuring the absorption spectrum, as already described.

[0103] In one embodiment of the invention, a solvent or a polymer solution and optionally a surfactant are selected for the preparation of the calibration composition such that the spectral properties of the at least one calibration dye in the calibration composition, in particular the position of the emission spectrum and / or the intensity parameters, are favorably influenced. Favorably, in this context, means that the spectral properties, i.e., the position and shape of the emission spectra, of the sample and the standard should be as similar as possible and ideally identical.

[0104] Furthermore, the fluorescence of the at least one calibration dye must be excitable at the same wavelength as the fluorescence of the at least one fluorescent marker of the sample. Additionally, the excitation wavelength for a channel of the fluorescence spectrometer must lie within the absorption spectrum of the at least one calibration dye and should also be spectrally separated from the detection window.

[0105] In one embodiment of the method according to the invention, the fluorescence of the at least one calibration dye is excited at the same wavelength as the fluorescence of at least one fluorescent marker of the sample, and the emission spectrum of the at least one calibration dye overlaps with the emission spectrum of at least one fluorescent marker of the sample.

[0106] The components of the calibration composition (calibration dye, solvent, polymer solution, surfactant) are therefore always specifically tailored to the at least one fluorescent marker of the sample in order to meet the described conditions.

[0107] In this embodiment, the integral fluorescence intensity of the sample is determined from the emission spectrum. The integral fluorescence intensity of the at least one emission spectrum of the sample is then assigned an integral fluorescence intensity of a concentration of the calibration composition.

[0108] This means that, according to the present method, at least one emission spectrum of a sample is measured, and the integral fluorescence intensity of the sample is calculated from this under the measurement conditions used. Furthermore, emission spectra are also measured for calibration compositions with different concentrations of calibration dye, and the respective integral fluorescence intensity is determined from these. The integral fluorescence intensity of the sample can then be referenced using the measurement of the calibration composition. For this purpose, a comparison is made to determine at which concentration of calibration dye in the calibration composition the integral fluorescence intensity is equal to the integral fluorescence intensity of the sample.The reference then indicates that the measured integral fluorescence intensity of the sample corresponds to the integral fluorescence intensity of a specific calibration dye at a precisely defined concentration of the calibration dye. Since the weights of the calibration dye and the amount of solvent or polymer solution in the calibration composition, as well as their measurement uncertainties, are precisely known, the reference is advantageously traceable to the SI units of grams or moles.

[0109] Furthermore, the present invention comprises a kit for traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring instrument with a calibration composition comprising at least one fluorescent calibration dye, comprising the method according to the invention. • at least one calibration dye of known mass; • a work instruction.

[0110] All characteristics already described for the process apply to the kit and vice versa. To avoid repetition, please refer to the previously described characteristics of the process.

[0111] The kit includes at least one calibration dye of known mass. In one embodiment, the calibration dye can be weighed into a suitable transport container using a calibrated or verified precision balance. Preferably, a mass of the calibration dye is weighed that is suitable for preparing a stock solution by adding a solvent or a polymer solution.

[0112] In a preferred embodiment of the present invention, a defined mass of a calibration dye is weighed out using a calibrated or verified precision balance, and a stock solution is prepared in a suitable transport container by adding a defined quantity of a solvent or a polymer solution using a calibrated pipette. The solvent is then evaporated, leaving only the calibration dye in the transport container.

[0113] The kit is particularly suitable for shipping to a user. In this respect, it is especially advantageous that the calibration dye in the kit is initially in solid form. Transporting calibration dyes in solution causes various problems that can be avoided by this method. Among other things, it avoids stability problems of solutions that can arise due to thermal influences. This is particularly true for very dilute dye concentrations below 10 -5 Wall absorption effects can occur, which alter the concentration of the solutions. This effect is also prevented by the kit according to the invention.

[0114] The kit also includes operating instructions. These instructions specify the required weight of the calibration dye and the associated measurement uncertainty. Furthermore, the instructions detail the step-by-step procedures to be performed sequentially. A) to prepare a stock solution of the calibration composition and B) to carry out the inventive method for referencing or calibrating a fluorescence measurement in a fluorescence measuring device.

[0115] The invention is explained in more detail below with reference to 7 figures and 2 exemplary embodiments. Fig. 1 (A) and (B) represent absorption and emission spectra of two fluorescent markers; Fig. Figures 2 (A) to (H) represent absorption and emission spectra of blue dyes that can be used as calibration dyes; Fig.Figures 3 (A) to (I) represent absorption and emission spectra of red dyes that can be used as calibration dyes; Fig. Figure 4 schematically represents the position of a cuvette relative to a light source and a detector; Fig. Figure 5 presents the absorption spectra of fluorescein 27 in 0.1M NaOH for different dilutions of two stock solutions; Fig. Figures 6 (A) to (F) represent the emission spectra for different dilutions of two stock solutions; Fig. Figures 7 (A) and (B) represent emission spectra of Fluorescein 27 in 0.1M NaOH, (A) normalized at 540 nm and (B) normalized at maximum.

[0116] Fig. 1(A) represents the absorption and emission spectrum of the fluorescent marker FAM and Fig.Figure 1(B) shows the absorption and emission spectrum of the fluorescent marker ATTO647N. Both fluorescent markers are particularly suitable for use in a COVID-19 fluorescence assay.

[0117] Fig. Figures 2 (A) to (H) represent absorption and emission spectra of blue dyes that can be used as calibration dyes. The graphs show the spectra of the following dyes: figure dye figure dye 2(A) Fluorescein 27® 2(E) Oregon Green 488 (OG488) 2(B) Fluorescein isothiocyanate (FITC) 2(F) Oregon Green 514 (®14) 2(C) FLU 2(G) Rhodamin Green (RGR) 2(D) ATTO488 2(H) ATTO594

[0118] The structural formulas of the respective dyes can be found in Table 2. The fluorescence properties of the dye Fluorescein 27 dissolved in 0.1 M NaOH were investigated using absorption and emission spectrometry in an absorption and fluorescence spectrometer. The following data summary provides an overview of the results: solvents 0.1 M NaOH Absorption range 400 nm - 500 nm Absorption maximum 474 nm Emission area 490 nm - 700 nm; excitation wavelength 474 nm Emission peak 527 nm Photoluminescence quantum yield 88% (0.1 M NaOH) A(λ ex ) the measuring solution 0.11 (Extinction) Extinction coefficient measuring solution 280.000 M -1 cm -1 (Absorptionsmaximum 503 nm, 0,1 M NaOH)

[0119] Fig.Figures 3 (A) to (I) represent absorption and emission spectra of red dyes that can be used as calibration dyes. The graphs show the spectra of the following dyes: figure dye figure dye 3(A) W S0387 3(E) ATTO633 3(B) Methylene blue (MB) 3(F) Alexa Fluor 647 3(C) ATTO610 3(G) Cyanine5 (Cy5) 3(D) Bodipy 610 3(H) ATTO680 3(I) DY 480

[0120] The structural formulas of the respective dyes can be found in Table 1. The fluorescence properties of the dye FEW S0387 dissolved in PBS and 0.05% SDS were investigated using absorption and emission spectrometry in an absorption and fluorescence spectrometer. The following data summary provides an overview of the measurement results: solvents PBS & 0.05% SDS Absorption range 500 nm - 700 nm Absorption maximum 644 nm Emission area 620 nm - 850 nm; excitation wavelength 608 nm Emission peak 663 nm Photoluminescence quantum yield 19% (PBS & 0.05% SDS) A(λ ex ) the measuring solution 0,025 Extinction coefficient measuring solution 176.600 M -1 cm -1 (Absorptionsmaximum 644 nm, PBS & 0,05 % SDS)

[0121] The fluorescence properties of the dye methylene blue (MB) dissolved in PBS and 0.05% SDS are also shown in Fig. 3(B) was investigated using absorption and emission spectrometry in an absorption and fluorescence spectrometer. The following data compilation provides an overview of the results and measurement conditions: solvents PBS & 0.05% SDS Absorption range 500 nm - 720 nm Absorption maximum 665 nm Emission area 625 nm - 900 nm; excitation wavelength 620 nm Emission peak 685 nm Photoluminescence quantum yield 3% (PBS & 0.05% SDS) A(λ ex ) the measuring solution 0,018 Extinction coefficient measuring solution 157.200 M -1 cm -1 (Absorptionsmaximum 664 nm, PBS & 0,05 % SDS)

[0122] Fig. Figure 4 schematically illustrates the position of a cuvette relative to a light source and a detector. The right-hand figure shows the positioning in absorption measurements, and the left-hand figure shows the positioning in emission measurements.

[0123] Fig. Figure 5 presents the absorption spectra of fluorescein 27 in 0.1 M NaOH for different dilutions of two stock solutions. Starting with two stock solutions with a dye concentration of 2 × 10 -4 Moles of each stock solution were diluted with a dye concentration of 1*10- 4 Mol, 1*10 -5 Mole and 1*10 -6 Mole was prepared and the absorption spectrum was measured.

[0124] Fig. Figures 6 (A) to (F) represent the emission spectra for the dilutions of the two stock solutions already described.

[0125] Fig.Figures 7 (A) and (B) represent emission spectra of fluorescein 27 in 0.1M NaOH, where in Fig. 7 (B) the emission spectra are normalized at the maximum and in Fig. 7 (A) at 540 nm. Example 1 - Calibration of a fluorescence measuring device

[0126] Two sample weights of the dye Fluorescein 27 were prepared using a calibrated ultramicrobalance, and the weighed amounts were dissolved in a defined volume of 0.1 M NaOH solvent. The resulting stock solution and the measurement solutions prepared from it by defined dilution steps were photometrically measured using a calibrated spectrophotometer (determination of the absorbance at the excitation wavelength and the molar extinction coefficient at the dye's absorption maximum) and fluorometrically characterized using a calibrated fluorescence spectrometer. All spectroscopic measurements were performed with identical instrument settings.The spectrophotometer was calibrated with respect to intensity and wavelength accuracy. Intensity was calibrated using commercial calibration solutions, such as those offered by Merck KGaA, and wavelength accuracy was calibrated using certified filters, such as those offered by Hellma GmbH & Co. KG. The fluorescence spectrometer was calibrated with respect to wavelength, spectral sensitivity, and the linearity range of the detection system. Measuring instruments used

[0127] For the absorption spectra measurements, a calibrated Cary 5000 absorption spectrometer from Varian, with a calibrated wavelength and intensity scale or axis, was used, as well as cuvettes of different layer thicknesses (0.2 and 1 cm).

[0128] For the emission spectra measurements, a calibrated Edinburgh Instruments FSP20 fluorescence spectrometer with a calibrated wavelength and intensity scale, a calibrated spectral sensitivity, and a known linearity range of the emission detection system was used. All measurements with solutions of the dye Fluorescein 27 in 0.1 M NaOH of varying concentrations were performed with the same fluorescence spectrometer settings, utilizing the linear range of the emission detector.

[0129] The following pipettes were used for the preparation of the stock solutions and dilutions: - EP1: calibrated Eppendorf pipette with a pipetting range of 500 µl - 2500 µl - EP2: calibrated Eppendorf pipette with a pipetting range of 100 µl - 1000 µl

[0130] A calibrated precision balance was used for weighing the solids. Description of the procedure 1. Two samples F27_01 and F27_02 of the dye Fluorescein 27 were prepared in a comparable mass range using the same calibrated microbalance. 2. One stock solution each, SL01 and SL02, of the calibration composition was prepared, with a target dye concentration of 2*10 -4 Mole was prepared by dissolving the weighed mass of dye with a volume of solvent 0.1 M NaOH solution (see Table 8) adjusted to the weighed mass. Pipette EP1 was used to add the solvent. Table 8: Composition of the stock solutions SL01 and SL02 of the calibration compositions SL01 SL02 Fluorescein weighing 27 (µg) 554,0 574,3 Solvent (ml) 6,904 (=3 * 2 + 1,904) 7,157 (=3* 2 + 1,157) 3. Two diluted measuring solutions, SL01_V1 and SL02_V1, with a dye concentration of 1*10 -4M was prepared from the previously prepared stock solutions (1:1 dilution) using pipette EP1 by adding 2 ml of solvent 0.1 M NaOH to 2 ml of the respective dye stock solution in a sample vial of 7 ml total volume. 4. Two 0.2 cm x 1.0 cm cuvettes, A and B, were filled with the measuring solutions SL01_V1 and SL02_V1. The absorption and emission spectra were then measured. - The Cary 5000 absorption spectrometer was used to measure the absorption spectra. Each measurement was performed once (single determination). The measurement was carried out in the upper measuring range of the absorption spectrometer, with the orientation of the cuvette as shown in the diagram. Fig. The arrangement corresponded to the schematic shown in Figure 4 on the left. The 1.0 cm long side of the cuvette was perpendicular to the light source, meaning the optical path length or the relevant layer thickness in the beam path was 0.2 cm. - The FSP920 emission spectrometer was used to measure the emission spectra with excitation at a wavelength of 475 nm. Each emission spectrum was measured three times. The cuvette was prepared according to the instructions in Fig. The cuvette was positioned according to the schematic arrangement shown on the right. The 1.0 cm long side of the cuvette was perpendicular to the light source, and the 0.2 cm long side of the cuvette pointed towards the detector. The optical path length was therefore 0.2 cm during excitation and 0.5 cm during detection with a 0° / 90° measurement geometry. 5. Two diluted measurement solutions SL01_V2 and SL02_V2 of the calibration composition were prepared from the measurement solutions SL0x_V1 (from step 3) with a dye concentration of 1*10 -5M was prepared by a further dilution step (dilution 1 to 10). For this purpose, 600 µl of the respective measuring solution_V1 were added using pipette EP2 to 5.4 ml (= 2 * 2 ml + 1.4 ml) of solvent using pipette EP1 in a sample vial with a total volume of 7 ml. 6. Two 1.0 cm * 1.0 cm cuvettes A and B were filled with the measuring solutions SL01_V2 and SL02_V2 and the absorption and emission spectra were measured as described in step 4. 7. Two further measurement solutions SL01_V3 and SL02_V3 were prepared from the measurement solutions SL01_V2 and SL02_V2 (from step 5) with dye concentrations of 1*10 -6 M was prepared by a further dilution step (dilution factor 1 to 10) by adding 600 µl of the respective stock solution using pipette EP2 to 5.4 ml (= 2 * 2 ml + 1.4 ml) of solvent using pipette EP1 in a sample vial of 7 ml total volume. 8. Two 1.0 cm*1.0 cm cuvettes A and B were filled with the dilution solutions SL01_V3 and SL02_V3 and the absorption and emission spectra were measured as described in step 4. Results of the absorption measurements:

[0131] The molar extinction coefficients ε given in Tables 9 to 11 are derived from the respective measured absorbance A at the absorption maximum, the dye concentration c of the sample solution, and the path length d of the cuvette of 0.2 cm or 1 cm according to formulas (1) and (2). The underlying absorption spectra are solvent- and baseline-corrected. The measured absorption spectra are shown in the Fig. 5 shown. Table 9: Molar extinction coefficients determined for measuring solutions SL01 and SL02, dilution 1 (1*10-4 M, solvent 0.1 M NaOH), determined at the absorption maximum at 491 nm; 0.2 cm cuvettes (d = 0.2 cm) sample measured extinction e(l max ) / (L M -1 cm -1 ) SL01_V1 SL01_V2 SL02_V1 SL02_V2 A 1,456 1,462 72.800 73.100 B 1,441 1,459 72.050 72.950 mean 1,4545 72.725 Standard deviation 0,0093 466,4 coefficient of variation 0,0064 0,0064 Table 10: Determined molar extinction coefficients for measuring solutions SL01 and SL02, dilution 2 (1*10-5 M, solvent 0.1 M NaOH), determined at the absorption maximum at 491 nm; 1 cm cuvette sample measured absorption e(l max ) / (LM -1 cm -1 ) SL01_V1 SL01_V2 SL02_V1 SL02_V2 A 0,7521 0,7451 75.210 74.510 B 0,7480 0,7435 74.800 74.350 mean 0,7472 74.717,5 Standard deviation 0,0038 377,5 coefficient of variation 0,0051 0,0051 Table 11: Determined molar extinction coefficients for measuring solutions SL01 and SL02, dilution 3 (1×10-6 M, solvent 0.1 M NaOH), determined at the absorption maximum at 491 nm, 1 cm cuvette sample measured absorption e(l max ) / (LM -1 cm -1 ) SL01_V1 SL01_V2 SL02_V1 SL02_V2 A 0,0715 0,0718 71.500 71.800 B 0,0717 0,0701 71.700 70.100 mean 0,0713 71.275,0 Standard deviation 0,0008 793,2 coefficient of variation 0,0111 0,0111 Results of the emission measurements

[0132] The dye fluorescein 27 exhibits an overlap of its absorption and emission spectra. At higher concentrations of the dye, this leads to a reabsorption of emitted photons and thus to an underestimation of the fluorescence intensity or the number of emitted photons in the spectral region of the short-wavelength edge of the emission spectrum. This also results in a redshift of the emission spectra. To minimize the contribution of reabsorption effects to the integral fluorescence intensity reported in Table 12, the area integral of the emission band was determined only in the wavelength range between 540 nm and 650 nm, i.e., in a spectral range where the overlap of absorption and emission is as small as possible.

[0133] The Fig. Figures 6 (A) to (F) represent the emission spectra of the individual calibration compositions with different concentrations of calibration dye.

[0134] The ones in Table 12 and the ones in Fig. The emission spectra and fluorescence intensities shown in Figures 7 (A) and (B) are dimensionless and refer to the number of photons (photon counts) measured by the detector of the fluorescence spectrometer used.

[0135] Fig. Figure 7 shows the emission spectra of fluorescein 27 in 0.1 M NaOH, where in Fig. 7(A) plot the curves on the fluorescence intensity at a wavelength of 540 nm and in Fig. 7(B) are normalized to the maximum. Dye solutions with a concentration of 1×10 -6 M show the shortest wavelength emission spectra, the emission spectra of the dye solutions with concentrations of 1×10 -5 M and 1×10 -4M are shifted towards longer wavelengths. This redshift with increasing dye concentration and the relative decrease in intensity of the emission spectra normalized at 540 nm with increasing dye concentration, as shown in the right-hand figure, are due to internal filtering effects or reabsorption. Table 12: Integral fluorescence intensity of the unnormalized emission spectra of fluorescein 27 in 0.1 M NaOH in the nearly reabsorption-free range of 540 nm - 650 nm (hatched area in Figures 6 (A) to (F)) Integral fluorescence intensity I1 (relative units) sample Dilution of stock solutions SL01 / SL02 SL01_V1* SL02_V1* SL01_V2 SL02_V2 SL01_V3 SL02_V3 A_M01 24.801.400,0 24.962.100,0 8.247.129,9 8.274.617,4 1.228.456,4 1.270.908,9 A_M02 24.864.800,0 25.002.900,0 8.287.354,5 8.303.095,9 1.230.963,6 1.268.294,8 A_M03 24.679.900,0 25.037.600,0 8.263.666,8 8.337.678,3 1.232.161,3 1.270.047,0 B_M01 25.058.400,0 25.311.300,0 8.430.505,1 8.610.678,8 1.237.574,9 1.267.185,1 B_M02 24.986.300,0 25.342.800,0 8.425.188,4 8.608.907,8 1.235.397,1 1.264.735,1 B_M03 24.905.100,0 25.248.100,0 8.396.373,0 8.613.142,1 1.238.084,2 1.262.351,9 mean 24.882.650,0 25.150.800,0 8.341.702,9 8.458.020,1 1.233.772,9 1.267.253,8 StAbw. 134.285,62 168.753,00 84.654,50 168.674,13 3.858,37 3.244,16 VarKoeff 0,0054 0,0067 0,0101 0,0199 0,0031 0,0026 mean 25.016.725,0* 8.399.861,5 1.250.513,3 StAbw. 201.869,51 14.0995,08 17.812,08 VarKoeff 0,0081 0,0168 0,0142 * determined in a 0.2 × 1.0 cm cuvette (see Fig. ) Table 13: Correlation of the concentration of fluorescein 27 with the measured integral fluorescence intensity I1 (in photon numbers or counts = cts) I1 (cts) 8.399.861,5 1.250.513,3 Dye concentration c (in M = mol / l) 1×10 -5 1×10 -6 Abs (l) ex ) 0,414 0,036 I1 / c 8,3999×10 11 1,2505×10 12

[0136] This example illustrates how a traceable calibration of a fluorescence measurement can be performed in a fluorescence measuring instrument according to the present invention. The values ​​determined in the exemplary embodiment can now serve as a reference for a subsequent instrument verification. In this verification, the procedure is repeated on the same instrument exactly as described in the exemplary embodiment. Changes in instrument-specific influencing factors would be reflected in a change in the integral fluorescence intensity I1 at the measured concentrations of the calibration composition. Example 2 - Referencing a sample

[0137] Exemplary embodiment 1 can also be applied analogously to an assay (for the detection of targets and target structures such as disease-relevant or disease-specific biomolecules) and the dye used therein to generate the fluorescence signal (assay fluorescence marker), for example with the following steps: i.) Selection of the calibration dye: must be matched to the spectral absorption and emission properties of the fluorescent marker used in the assay in the solvent used. This means that the absorption and emission spectra of the calibration dye and the assay fluorescent marker must be measured with one and the same absorption and fluorescence meter in the solvents selected for the calibration dye and the fluorescent marker, respectively, and preferably under identical measurement conditions. ii.) Signal matching for the calibration and assay fluorescence markers: either comparative determination of the molar extinction coefficients and the fluorescence quantum yields of the calibration dye and the assay fluorescence marker to determine the signal-determining brightness of both dye systems, or comparison of the intensities of the fluorescence signals of a solution of the calibration dye and the assay fluorescence marker of known concentration. The concentration-based signal comparison involves determining the fluorescence emission spectra for the calibration dye and the assay fluorescence marker using the same fluorescence meter in the solvents selected for the calibration dye and the fluorescence marker, respectively, under identical measurement conditions. This comparison, or the fluorescence factor determined in this way for matching the fluorescence intensities, can then later be used for the calibration and referencing of the assay.In the first case, a traceability to spectroscopic measurements is performed, and in the second case, a traceability to mass is performed. iii.) By comparing the fluorescence intensities of the calibration dye and the assay fluorescence marker, the fluorescence intensity measured in the assay for the fluorescence marker (or a biomolecule labeled with this fluorescence marker, such as a specific DNA sequence, protein, peptide, or other analyte) is assigned an intensity of the calibration dye X with a concentration Y. Bibliography [1] Schädel et al. PTB-Mitteilungen 118 (2008), Issue 4, 248-254 [2] D. Cavazos-Elizondo, A. Aguirre-Soto, Anal. Sens. 2022, 2, e202200004. [3] Taniguchi, M. and Lindsey, JS (2018), Photochem Photobiol, 94: 290-327.

Claims

[1] Method for the traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring device with a calibration composition comprising at least one fluorescent calibration dye characterized by that the procedure comprises the steps: • Weighing of at least one calibration dye using a calibrated or verified precision balance; • Preparing a stock solution of a calibration composition by adding a solvent or a polymer solution of known mass; • Preparing a dilution series with known concentrations of at least one calibration dye; • Measuring the absorption spectrum for each concentration of the dilution series of the calibration composition in an absorption spectrometer and calculating the molar extinction coefficient from the measured absorbance for each concentration of the at least one calibration dye of the dilution series; • Measuring the emission spectrum for each concentration of the dilution series of the calibration composition in one fluorescence measuring instrument; • Calculation of the integral fluorescence intensity for each concentration of at least one calibration dye in the dilution series from the emission spectra; • Assignment of the respective molar extinction coefficient and the respective integral fluorescence intensity to a concentration of the calibration composition. [2] Method according to claim 1, characterized by that the procedure continues to include the steps: • Providing a sample comprising at least one fluorescent marker; • Measuring at least one absorption spectrum of the sample in an absorption spectrometer and calculating the molar extinction coefficient from the measured absorbance; • Measuring at least one emission spectrum of the sample in the fluorescence measuring instrument under the same measurement conditions as the emission spectra of each concentration of the dilution series of the calibration composition were measured; • Calculation of the integral fluorescence intensity of the sample from the emission spectrum; • Assigning the integral fluorescence intensity of the at least one emission spectrum of the sample to the integral fluorescence intensity of a concentration of the calibration composition; wherein the spectral properties of the calibration composition are adapted to the at least one fluorescence marker of the sample. [3] Method according to one of the preceding claims, characterized bythat the same instrument parameters, the same measuring geometry and, optionally, the same type of measuring container are used as measurement conditions when measuring the emission spectra of the sample as when measuring the emission and / or absorption spectrum of the calibration solution. [4] Method according to one of the preceding claims, characterized by that the spectral properties of the calibration composition are adapted to the at least one fluorescent marker of the sample in that the fluorescence of the at least one calibration dye can be excited at the same wavelength as the fluorescence of at least one fluorescent marker of the sample, and that the emission spectrum of the at least one calibration dye overlaps with the emission spectrum of at least one fluorescent marker of the sample. [5] Method according to one of the preceding claims, characterized by that the calibration composition comprises a solvent or a polymer solution. [6] Method according to one of the preceding claims, characterized by that during the preparation of the calibration composition, at least one surfactant of known mass is added to it. [7] Method according to one of the preceding claims, characterized by that the at least one calibration dye is selected from the group comprising organic dyes, dye-doped or dye-loaded polymer systems and fluorescent nanoparticles. [8] Method according to claim 7, characterized bythat the organic dye is selected from the group comprising ultraviolet dyes whose emission range lies in the spectral range between 200 nm and 425 nm, blue dyes whose emission range lies in the spectral range between 425 nm and 485 nm, green dyes whose emission range lies in the spectral range between 485 nm and 560 nm, yellow / orange dyes whose emission range lies in the spectral range between 560 nm and 600 nm, red dyes whose emission range lies in the spectral range between 600 nm and 780 nm, and infrared dyes whose emission range lies in the spectral range between 780 nm and 900 nm. [9] Method according to one of the preceding claims, characterized bythat the at least one fluorescent marker of the sample is selected from the group comprising blue dyes whose emission range lies in the spectral range between 425 nm and 485 nm, and red dyes whose emission range lies in the spectral range between 600 nm and 780 nm. [10] Kit for traceable calibration or referencing of a fluorescence measurement in a fluorescence measuring device with a calibration composition comprising at least one fluorescent calibration dye with a method according to one of claims 1 to 9, comprising • at least one calibration dye of known mass; • a work instruction.

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