Method for determining at least one analyte of interest - Patent Application 20070122997

Abstract

The present invention relates to a method for determining at least one analyte of interest and its use.The present invention further relates to a diagnostic system, a kit for determining at least one analyte of interest and their use.

Description

[Technical Field]

[0001] The present invention relates to a method for determining at least one analyte of interest and its use.The present invention further relates to a diagnostic system, a kit for determining at least one analyte of interest and their use. [Background technology]

[0002] Raman spectroscopy is a technique that specializes in measuring the frequency shift of inelastically scattered light from a sample when photons from an incident light strike a molecule and generate scattered photons. Raman spectra provide qualitative and quantitative information about the analyte, whether in a matrix, e.g., organic or inorganic, e.g., aqueous, matrix.

[0003] The main problem with (organic) matrices is that the components of the sample and / or matrix have a strong fluorescence effect alongside the Raman effect, where the parallel and more sensitive fluorescence effect limits this process, causing problems with the inability to detect analyte information from the Raman spectrum.

[0004] Therefore, fluorescence quenchers and the selective quenching of fluorescence are of great interest to analytical techniques.

[0005] Quenching of the fluorescent signal has been attempted in the past by adding KI to the solution to shorten the fluorescence lifetime, but molecular quenching has not been performed.

[0006] Therefore, there is an urgent need in the art to overcome the above-mentioned problems.

[0007] It is an object of the present invention to provide a method for determining at least one analyte of interest. It is also an object of the present invention to provide a detection system, a kit and their use for determining at least one analyte of interest.

[0008] This or these objects are solved by the subject matter of the independent claims. Further embodiments are subject to the dependent claims. Summary of the Invention

[0009] In the following, the present invention relates to the following aspects:

[0010] In a first aspect, the present invention provides a method for determining at least one analyte of interest, comprising: a) - maximum excitation wavelength λ max1 at least one analyte of interest capable of emitting scattered electromagnetic radiation when excited with monochromatic electromagnetic radiation having - maximum excitation wavelength λ max1 a fluorophore capable of emitting fluorescent electromagnetic radiation when excited with monochromatic electromagnetic radiation having and preparing a b) Maximum excitation wavelength λ max1 providing a quencher capable of quenching the fluorescent electromagnetic radiation of the fluorophore upon excitation with monochromatic electromagnetic radiation having c) mixing at least one analyte of interest, a fluorophore, and a quencher to form a sample; d) performing Raman spectroscopy; e) determining at least one analyte of interest via Raman spectroscopy; The present invention relates to a method, comprising:

[0011] In a second aspect, the present invention relates to the use of the method of the first aspect of the invention for determining at least one analyte of interest.

[0012] In a third aspect, the present invention relates to a diagnostic system for determining at least one analyte of interest in a sample.

[0013] In a fourth aspect, the present invention provides a kit suitable for carrying out the method of the first aspect of the invention, comprising: (A) at least one analyte of interest, preferably a deuterated analyte of interest as an internal standard; (B) Quencher and The present invention relates to a kit comprising or consisting of:

[0014] A fifth aspect is the use of the kit of the fourth aspect of the invention in the method of the first aspect of the invention. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows the Raman spectrum of acetonitrile as the analyte of interest. [Figure 2] 1 shows a quencher with an oligonucleotide as the hydrophilic group. [Figure 3A] 1 shows the Raman spectrum of acetonitrile as the analyte of interest. [Figure 3B] 1 shows the Raman spectrum of acetonitrile as the analyte of interest. [Figure 3C] 1 shows the Raman spectrum of acetonitrile as the analyte of interest. [Figure 4A] 1 shows Raman spectra of acetonitrile as the analyte of interest at different fluorophore dilutions. [Figure 4B] 1 shows Raman spectra of acetonitrile as the analyte of interest at different fluorophore dilutions. [Figure 5] The signal-to-noise ratio as a function of fluorophore concentration at different quencher concentrations (0 mg / l (blank)) is shown. [Figure 6] The signal-to-noise ratio as a function of fluorophore concentration at different quencher concentrations (0 mg / l (blank), 41.7 mg / l, 62.5 mg / l) is shown. [Figure 7] Figure 1 shows the signal-to-noise ratio as a function of fluorophore concentration at different quencher concentrations in biological matrix environments such as serum (0 mg / L (blank), 62.5 mg / L). DETAILED DESCRIPTION OF THE INVENTION

[0016] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific embodiments and examples described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0017] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the text of this specification shall control.

[0018] The elements of the present invention are described below. While these elements are listed with specific embodiments, it is understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless the context dictates otherwise.

[0019] definition The word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0020] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0021] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a "range" format. It is understood that such range formats are used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as boundaries of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of example, a numerical range of "4% to 20%" should be interpreted not only to include the explicitly recited value 4% to 20%, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle also applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the described characteristics.

[0022] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0023] In the context of this disclosure, the terms "analyte," "analyte molecule," or "analyte of interest" are used interchangeably to refer to chemical species analyzed by Raman spectroscopy. Chemical species, i.e., analytes, suitable for analysis by Raman spectroscopy can be any type of molecule present in a living organism, including, but not limited to, nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytosolic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characteristic of a particular modification of another molecule (e.g., a sugar moiety or phosphoryl residue on a protein, a methyl residue on genomic DNA), or substances internalized by an organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.), or metabolites of such substances. Such analytes can serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance within a living system that is used as an indicator of the biological state of that system.

[0024] The analyte or target analyte may be present in a sample, particularly a biological or clinical sample. The term "biological or clinical sample" is used interchangeably herein and refers to a portion or piece of a tissue, organ, or individual, usually smaller than the tissue, organ, or individual intended to represent the entire tissue, organ, or individual. Upon analysis, the biological or clinical sample provides information about the state of the tissue, or the health or disease state of the organ or individual. Examples of biological or clinical samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymphatic fluid, or solid biological or clinical samples such as dried blood spots and tissue extracts. Another example of a biological or clinical sample is a cell culture or tissue culture.

[0025] The term "chromatography" refers to a process in which a liquid or gas-borne chemical mixture is separated into components as a result of differential distribution of the chemical components as they flow around or over a stationary liquid or solid phase. In embodiments of the present invention, the method or sample element or device or kit does not include a chromatographic step and a chromatographic unit, respectively.

[0026] The term "liquid chromatography" or "LC" refers to the process of selectively retarding one or more components of a fluid solution as the fluid permeates uniformly through a column or capillary passage of finely divided material. Retardation results from the distribution of mixture components between one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal phase liquid chromatography (NPLC), while methods in which the stationary phase is less polar than the mobile phase (e.g., a water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) are called reversed phase liquid chromatography (RPLC).

[0027] "High-performance liquid chromatography" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by passing a mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregular or spherical particles, a porous monolithic layer, or a porous membrane. HPLC has historically been divided into two distinct subclasses based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal-phase liquid chromatography (NPLC), while the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is called reverse-phase liquid chromatography (RPLC). Micro-LC refers to HPLC methods using columns with narrow internal diameters, typically less than 1 mm, e.g., about 0.5 mm. "Ultra-high performance liquid chromatography" or "UHPLC" refers to an HPLC method using a pressure of 120 MPa (17,405 lbf / in²), or approximately 1200 atmospheres. Rapid LC refers to an LC method using a short column with an internal diameter as described above and a length of less than 2 cm, e.g., 1 cm, at a flow rate as described above and a pressure as described above (micro LC, UHPLC). A short rapid LC protocol involves trapping / washing / elution steps using a single analytical column, achieving LC in a very short time of less than 1 minute.

[0028] Further well-known LC methods include hydrophilic interaction chromatography (HILIC), size-exclusion LC, ion-exchange LC, and affinity LC.

[0029] LC separations can be single-channel LC or multi-channel LC, which includes multiple LC channels arranged in parallel. In LC, analytes can be separated according to their polarity or log P value, size, or affinity, as commonly known to those skilled in the art.

[0030] The term "electromagnetic radiation" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to any special or customized meaning. This term can specifically, but is not limited to, refer to the form of energy waves as they travel through space (vacuum or matter). This consists of both electric and magnetic field components. Energy waves oscillate perpendicular to each other and in phase perpendicular to the direction of energy propagation. Electromagnetic radiation is classified into several types depending on the frequency of the wave. These types include (in order of decreasing frequency and increasing wavelength) cosmic radiation, gamma rays, X-ray radiation, ultraviolet radiation, visible radiation, IR radiation, terahertz radiation, microwave radiation, and radio waves. A small, variable window of frequencies is perceived by the eyes of various organisms and is known as the visible spectrum (λ 0.4-0.7 μm), or light.

[0031] The term "monochromatic electromagnetic radiation" is a broad term and should be given its ordinary and customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. The term may specifically refer to, but is not limited to, electromagnetic radiation of only one frequency or wavelength, particularly visible radiation. While completely monochromatic radiation cannot be produced, lasers produce radiation within a very narrow frequency band.

[0032] The term "scattered electromagnetic radiation" refers to electromagnetic radiation that is scattered.

[0033] As used herein, the term "patient sample" refers to a biological sample obtained for the purpose of in vitro evaluation. In the methods of the present invention, the sample or patient sample may preferably include any bodily fluid. Preferred samples are whole blood, serum, or plasma. As will be understood by those skilled in the art, any such evaluation is performed in vitro. The patient sample is then discarded. The patient sample is used only in the in vitro methods of the present invention, and no material from the patient sample is returned to the patient's body.

[0034] In the context of this disclosure, a sample may be derived from an "individual" or "subject." Typically, the subject is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0035] The term "hemolytic reagent" (HR) refers to a reagent that lyses cells present in a sample, and in the context of the present invention, refers to a hemolytic reagent, particularly a reagent that lyses cells present in a blood sample, including, but not limited to, red blood cells present in a whole blood sample. A well-known hemolytic reagent is water (HO). Further examples of hemolytic reagents include, but are not limited to, deionized water, liquids with high osmolality (e.g., 8 M urea), ionic liquids, and various detergents.

[0036] The term "fluorescent electromagnetic radiation" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to any special or customized meaning, and therefore will not be described in detail herein.

[0037] The term "quench" is a broad term and should be given its ordinary and customary meaning to those of ordinary skill in the art and should not be limited to any special or customized meaning, and therefore will not be described in detail herein.

[0038] The term "Förster resonance energy transfer" is a broad term and should be given its ordinary and customary meaning to those of skill in the art and should not be limited to any special or customized meaning, and therefore will not be described in detail herein.

[0039] The term "aqueous sample" can mean that the quencher has been modified to be water soluble up to 100% water content.

[0040] The term "hydrophobic" can mean that the compound is only minimally soluble or not soluble in polar solvents such as ethanol, methanol, or water.

[0041] A "clinical diagnostic system" is a laboratory automation device dedicated to the analysis of samples for in vitro diagnostics. Clinical diagnostic systems may have different configurations as needed and / or according to the desired laboratory workflow. Additional configurations can be achieved by coupling multiple devices and / or modules together. A "module" is a work cell, typically smaller in size than the entire clinical diagnostic system, with a dedicated function. This function may be an analytical function, a pre-analytical function, a post-analytical function, or a function supporting either a pre-analytical function, an analytical function, or a post-analytical function. In particular, a module can be configured to cooperate with one or more other modules to perform a dedicated task in a sample processing workflow, for example, by performing one or more pre-analytical and / or analytical and / or post-analytical steps. In particular, a clinical diagnostic system can include one or more analytical devices designed to perform respective workflows optimized for specific types of analysis, such as clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separations, mass spectrometry, Raman spectroscopy, etc. Thus, a clinical diagnostic system may include one analytical device or any combination of such analytical devices with their respective workflows, and pre-analytical and / or post-analytical modules may be coupled to individual analytical devices or shared by multiple analytical devices. Alternatively, pre-analytical and / or post-analytical functions may be performed by units integrated into the analytical devices. Clinical diagnostic systems may include functional units such as liquid handling units for pipetting, pumping, and / or mixing samples and / or reagents and / or system fluids, and may further include functional units for sorting, storage, transport, identification, separation, and detection. Clinical diagnostic systems may include a sample preparation station for automated preparation of samples containing analytes of interest, a liquid chromatography (LC) separation station optionally including multiple LC channels, and / or a sample preparation / LC interface for inputting the prepared sample into any one of the LC channels.The clinical diagnostic system may further include a controller programmed to assign samples to predefined sample preparation workflows, each of which includes a predefined sequence of sample preparation steps and requires a predefined time for completion depending on the analyte of interest. The clinical diagnostic system may further include a Raman spectrometer and an LC / Raman interface for connecting the LC separation station to the Raman analyzer. As used herein, the terms "automatically" and "automated" are broad terms and should be given their ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. The terms may specifically refer to, but are not limited to, a process performed entirely by at least one computer and / or computer network and / or machine, particularly without the need for human action and / or user interaction.

[0042] A "sample preparation station" can be a pre-analytical module coupled to one or more analytical devices or a unit within an analytical device designed to perform a series of sample processing steps aimed at removing or at least reducing interfering matrix components in the sample and / or concentrating the analytes of interest in the sample. Such processing steps can include any one or more of the following processing operations performed sequentially, in parallel, or alternating on a sample or multiple samples: pipetting (aspirating and / or dispensing) fluids, pumping fluids, mixing with reagents, incubation at a specific temperature, heating or cooling, centrifugation, separation, filtration, filtering, drying, washing, resuspension, dispensing, transfer, storage, etc.

[0043] Typically, an "internal standard" (ISTD) is a known quantity of a substance that exhibits similar properties to the analyte of interest when subjected to the Raman spectroscopic detection workflow (i.e., including any pretreatment, concentration, and actual detection steps). The ISTD exhibits similar properties to the analyte of interest, but is clearly distinguishable from it. For example, during a chromatographic separation, such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the Raman spectrometer simultaneously. However, the ISTD exhibits a different molecular weight than the analyte of interest from the sample. This allows ions from the ISTD and the analyte to be distinguished in Raman spectroscopic detection using their different mass-to-charge (m / z) ratios. Both are subjected to fragmentation to produce daughter ions. These daughter ions can be distinguished by their m / z ratios and their respective parent ions. As a result, the signals from the ISTD and the analyte can be determined and quantified separately. Because the ISTD is added in a known amount, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for relative comparison of the amount of analyte detected, allowing for unambiguous identification and quantification of the analyte of interest present in the sample when the analyte reaches the Raman spectrometer. Typically, but not necessarily, the ISTD is an isotopically labeled variant of the analyte of interest (e.g., 2 H, 13 C, or 15 (including labels such as N).

[0044] A "kit" is any article of manufacture (e.g., a package or container) comprising at least one reagent, such as a drug for treating a disorder or a probe for specifically detecting a biomarker gene or protein of the invention. The kit is preferably promoted, distributed, or sold as a unit for practicing the methods of the invention. Typically, the kit will further comprise carrier means compartmentalized to receive in tight confinement one or more container means, such as vials, tubes, etc. In particular, each of the container means contains one of the separate elements used in the method of the first aspect. The kit may further comprise one or more other reagents, including, but not limited to, a reaction catalyst. The kit may further comprise one or more other containers containing additional materials, including, but not limited to, buffers, internal standards, diluents, filters, needles, syringes, and a package insert with instructions for use. A label may be affixed to the container to indicate that the composition is to be used for a particular application, and may provide instructions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or may be provided directly on a computer or data processing device. Additionally, the kit may include standard amounts of biomarkers, as described elsewhere herein, for calibration purposes.

[0045] Embodiment In a first aspect, the present invention provides a method for determining at least one analyte of interest, comprising: a) - maximum excitation wavelength λ max1 providing at least one analyte of interest capable of emitting scattered electromagnetic radiation when excited with monochromatic electromagnetic radiation having - maximum excitation wavelength λ max1 providing a fluorophore capable of emitting fluorescent electromagnetic radiation when excited with monochromatic electromagnetic radiation having b) Maximum excitation wavelength λ max1providing a quencher capable of quenching the fluorescent electromagnetic radiation of the fluorophore upon excitation with monochromatic electromagnetic radiation having c) mixing at least one analyte of interest, a fluorophore, and a quencher to form a sample; d) performing Raman spectroscopy; e) determining at least one analyte of interest via Raman spectroscopy; The present invention relates to a method, comprising:

[0046] The inventors have surprisingly found that the subject matter of the present invention, in particular the method according to the first aspect of the invention, represents a simple and robust way to overcome the above-mentioned drawbacks: Fluorescence as a major problem in Raman spectroscopy is overcome by the described simple solution for fast and reliable Raman spectroscopy measurements, even in systems where fluorescence cannot be avoided or eliminated.

[0047] In particular, the method is carried out using fluorescence quenchers, e.g., oligonucleotides, that have hydrophilic groups to increase their solubility in water in order to reduce fluorescence by mixing the analyte sample with the quencher.

[0048] According to step a), at least one analyte of interest is provided, the at least one analyte having an excitation maximum wavelength λ max1 When excited with monochromatic electromagnetic radiation having a refractive index of 0.01, the material can emit scattered electromagnetic radiation.

[0049] In an embodiment of the first aspect of the present invention, the scattered electromagnetic radiation is inelastically scattered electromagnetic radiation.

[0050] In an embodiment of the first aspect of the present invention, the scattered electromagnetic radiation is Stokes scattering and / or anti-Stokes scattering. The scattered electromagnetic radiation can be measured by Raman spectroscopy. Raman spectroscopy is a technique that specializes in measuring the frequency shift of inelastically scattered light from a sample when photons from the incident light or electromagnetic radiation collide with molecules and generate scattered photons. The emerging scattered light or electromagnetic radiation can be photons of a lower frequency than the original photons, in which case it is known as Stokes Raman scattering, or at a higher frequency it is known as anti-Stokes Raman scattering.

[0051] In an embodiment of the first aspect of the present invention, the maximum excitation wavelength λ of the monochromatic electromagnetic radiation max1 is less than 1064 nm, for example 532 nm, 633 nm.

[0052] In an embodiment of the first aspect of the present invention, the monochromatic electromagnetic radiation is produced by a krypton ion laser (530.9 nm and 647.1 nm), a He:Ne laser (632.8 nm), a Nd:YAG laser (1064 nm and 532 nm), an argon ion laser (488.0 nm and 514.5 nm), or a diode laser (630 nm and 780 nm).

[0053] In an embodiment of the first aspect of the present invention, the analyte of interest is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characteristic of a particular modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0054] In an embodiment of the first aspect of the present invention, the analyte of interest is testosterone, epitestosterone, dihydrotestosterone (DHT), desoxymethyltestosterone (DMT), tetrahydrogestrinone (THG), aldosterone, estrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone, 16-ketoestradiol, 16-α-hydroxyestrone, 2-hydroxyestrone-3-methyl ether, prednisone, prednisolone, pregnenolone, progesterone, dehydroepiandrosterone (DHEA), 17-hydroxypregnenolone, 17-hydroxyprogesterone, androsterone. and aldosterone, Δ8-tetrahydrocannabinolic acid, benzoylecgonine, salicylic acid, 2-hydroxybenzoic acid, gabapentin, pregabalin, valproic acid, vancomycin, methotrexate, mycophenolic acid, montelukast, repaglinide, furosemide, telmisartan, gemfibrozil, diclofenac, ibuprofen, indomethacin, zomepirac, isoxepak, and penicillin.In an embodiment of the first aspect of the present invention, the analyte molecule containing one or more carboxyl groups is selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, tryptophan, alanine, isoleucine, leucine, methionine, phenylalanine, valine, proline and glycine, pyridoxal, N-acetyl-D-glucosamine, alcaftadine, streptomycin and josamycin, cocaine, heroin, Ritalin, aceclofenac, acetylcholinesterase inhibitor ... , amcinonide, amiloxate, amylocaine, anileridine, aranidipine, artesunate and pethidine, cantharidin, succinic anhydride, trimellitic anhydride and maleic anhydride, cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol and tacalcitol, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 (calcifediol), 3-epi-25-hydroxyvitamin D2, 3-epi-25-hydroxyvitamin D Vitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 (calcitriol), 24,25-dihydroxyvitamin D2, 24,25-dihydroxyvitamin D3, vitamin A, tretinoin, isotretinoin, alitretinoin, natamycin, sirolimus, amphotericin B, nystatin, everolimus, temsirolimus and fidaxomicin, benzyl alcohol, menthol, L-carnitine, pyridoxine, metronidazole, isosorbide dinitrate, guaifenesin, clavulanic acid, miglioretin and an amino acid selected from the group consisting of benzodiazepine, benzocaine, benzophenone, benzocaine, benzoyl peroxidase ...In an embodiment of the first aspect of the invention, wherein the analyte molecule comprises more than one hydroxyl group, the analyte is selected from the group consisting of vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacitidine, ribavirin, floxuridine, gemcitabine, streptozotocin, adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenoson, lincomycin, clindamycin, canagliflozin, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, framycetin, paromomycin, epinephrine, erythromycin ... Selected from the group consisting of rithromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, plicamycin, thiomandelic acid, DL-captopril, DL-thiorphan, N-acetylcysteine, D-penicillamine, glutathione, L-cysteine, zofenoprilat, tiopronin, dimercaprol, succimer, glutathione disulfide, dipyrithione, selenium sulfide, disulfiram, lipoic acid, L-cystine, fursultiamine, octreotide, desmopressin, vapreotide, terlipressin, linaclotide, peginesatide, and plicamycin.The selenium sulfide can be selenium disulfide, SeS2, or selenium hexasulfide, Se2S6, carbamazepine-10,11-epoxide, carfilzomib, furosemide epoxide, fosfomycin, sevelamer hydrochloride, cerulenin, scopolamine, tiotropium, tiotropium bromide, methylscopolamine bromide, eplerenone, mupirocin, natamycin, and troleandomycin, estrogen, estrogenic compounds, estrone (EL), estradiol (E2), 17a-estradiol, 17b-estradiol, estriol (E3), 16-epiestriol, 17-estriol, and 16,17-epiestriol, and / or metabolites thereof. In embodiments, the metabolite is estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-keto-E2), 16a-hydroxyestrone (16a-OHEl), 2-methoxyestrone (2-MeOEl), 4-methoxyestrone (4-MeOEl), 2-hydroxyestrone-3-methyl ether (3-MeOEl), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4 17α-dihydroequilenin (ENa), 17β-dihydroequilenin (ENb), Δ8,9-dehydroestrone (dE1), Δ8,9-dehydroestrone sulfate (dE1s), Δ9-tetrahydrocannabinol, and mycophenolic acid.α and a can be used interchangeably and include 3,4-methylenedioxyamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 3,4-methylenedioxymethamphetamine, amphetamine, methamphetamine, N-methyl-1,3-benzodioxolylbutanamine, 7-aminoclonazepam, 7-aminoflunitrazepam, 3,4-dimethylmethcathinone, 3-fluoromethcathinone, 4-methoxymethcathinone, 4-methylethcathinone, 4-methylmethcathinone Non, amfepramone, butyrone, ethcathinone, elephedrone, methcathinone, methylone, methylenedioxypyrovalerone, benzoylecgonine, dehydronorketamine, ketamine, norketamine, methadone, normethadone, 6-acetylmorphine, diacetylmorphine, morphine, norhydrocodone, oxycodone, oxymorphone, phencyclidine, norpropoxyphene, amitriptyline, clomipramine, dothiepin, doxepin, imipramine , nortriptyline, trimipramine, fentanyl, glycylxylidide, lidocaine, monoethylglycylxylidide, N-acetylprocainamide, procainamide, pregabalin, 2-methylamino-1-(3,4-methylenedioxyphenyl)butane, N-methyl-1,3-benzodioxolylbutanamine, 2-amino-1-(3,4-methylenedioxyphenyl)butane, 1,3-benzodioxolylbutanamine, normeperidine, O-destramad Examples of suitable analyte molecules include 5-methylcytosine, ribose, deoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, and N-acetylneurominic acid. In embodiments, the analyte molecule is an oligosaccharide, particularly selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and polysaccharides. In embodiments of the first aspect of the present invention, the disaccharide is selected from the group consisting of sucrose, maltose, and lactose.In an embodiment of the first aspect of the present invention, the analyte molecule is a substance comprising the monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide moieties described above, zidovudine, and azidocillin.

[0055] Such analyte molecules may be present in a sample, for example, a biological or clinical sample, such as a body fluid, eg, blood, serum, plasma, urine, saliva, spinal fluid, etc., a tissue or cell extract.

[0056] In embodiments of the first aspect of the invention, the sample is selected from the group consisting of blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva, and lymphatic fluid, cell culture, tissue culture, and solid samples such as dried blood spots or tissue extracts. In some embodiments of the first aspect of the invention, the analyte molecule may be present in a purified or partially purified sample, for example, a sample that is a purified or partially purified protein mixture or extract.

[0057] In an embodiment of the first aspect of the invention, the sample is obtained from a patient sample selected from the group consisting of a serum, plasma and whole blood sample from an individual.

[0058] In an embodiment of the first aspect of the present invention, the sample is a human sample, preferably a hemolyzed whole blood sample, in particular a hemolyzed human whole blood sample. The hemolyzed whole blood sample can be hemolyzed with a hemolysis reagent.

[0059] According to step (a), a fluorophore is prepared. The fluorophore has an excitation maximum wavelength λ max1 When excited with monochromatic electromagnetic radiation having the formula:

[0060] In an embodiment of the first aspect of the present invention, the fluorophore is selected from the group consisting of chemicals containing several combined aromatic groups or planar or cyclic molecules with several π bonds. Xanthene derivatives: fluorescein, rhodamine, Oregon green, eosin, Texas red Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine Squalene derivatives and ring-substituted squaraines, e.g., Seta and Square dyes Squalene rotaxane derivatives: see tau dyes Naphthalene derivatives (dansyl and prodan derivatives) Coumarin derivatives Oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole Anthracene derivatives: anthraquinones, such as DRAQ5, DRAQ7, CyTRAK Orange Pyrene derivatives: Cascade Blue, etc. Oxazine derivatives: Nile Red, Nile Blue, Cresyl Violet, Oxazine 170, etc. Acridine derivatives: proflavine, acridine orange, acridine yellow, etc. Arylmethine derivatives: auramine, crystal violet, malachite green Tetrapyrol derivatives: porphine, phthalocyanine, bilirubin Dipyrromethene derivatives: BODIPY, aza-BODIPY

[0061] According to method step b), a quencher is provided, which has an excitation maximum wavelength λ max1 The fluorescent electromagnetic radiation of the fluorophore can be quenched when excited with monochromatic electromagnetic radiation having

[0062] In an embodiment of the first aspect of the present invention, the quencher is SO4 2- , PO4 3- , an oligonucleotide, a quaternary amine, a PEG group, an alcohol, or a hydrophilic group selected from COOH.

[0063] In an embodiment of the first aspect of the invention, the oligonucleotides are oligomers having different numbers of linked nucleotides, namely adenine, cytosine, guanine and / or thymine.

[0064] In an embodiment of the first aspect of the present invention, the quaternary amine is (CH3)4NCl or (CH3CH2)4NCl.

[0065] In an embodiment of the first aspect of the invention, the PEG groups are polyethylene glycols having different numbers of ethylene glycol units, for example from 1 to 100, for example 25 or 50 PEG units.

[0066] In an embodiment of the first aspect of the present invention, the alcohol is OH, CH3OH, C2H5OH, C3H7OH, C4H9OH.

[0067] In an embodiment of the first aspect of the present invention, the hydrophilic group is an oligonucleotide.

[0068] In an embodiment of the first aspect of the present invention, the quencher comprises a polyaromatic azo backbone.

[0069] In an embodiment of the first aspect of the present invention, the quencher molecules and the fluorophore molecules have a distance of less than 6 nm, 5 nm, or 3 nm, so that the quenching process can be easily carried out.

[0070] In an embodiment of the first aspect of the present invention, the excitation wavelength of the quencher is in the range of 530 nm to 540 nm or 570 nm to 590 nm.

[0071] In an embodiment of the first aspect of the invention, the excitation wavelength of the quencher is λ with a tolerance of + / - 20 nm, preferably + / - 15 nm, + / - 10 nm or + / - 5 nm. max1 The range is.

[0072] In an embodiment of the first aspect of the present invention, the quenching range (absorption range) of the quencher is in the range of 470 nm to 660 nm, preferably 480 nm to 580 nm (inclusive), or 550 nm to 650 nm (inclusive).

[0073] In an embodiment of the first aspect of the present invention, the quenching is Förster resonance energy transfer.

[0074] In an embodiment of the first aspect of the present invention, the quencher is a BHQ1 quencher or a BHQ2 quencher.

[0075] In an embodiment of the first aspect of the present invention, the quencher is a BHQ1 quencher or a BHQ2 quencher, and the BHQ1 quencher is modified with an oligonucleotide, or the BHQ2 quencher is modified with an oligonucleotide.

[0076] In an embodiment of the first aspect of the invention, the quencher comprises the formula: TIFF2025538582000001.tif34104 TIFF2025538582000002.tif31100

[0077] The structures of BHQ1 and BHQ2 are modified with oligonucleotides (*=5'-TTx-3', X=BHQ1 or BHQ2).

[0078] According to method step c), at least one analyte of interest, a fluorophore and a quencher are mixed to form a sample.

[0079] In an embodiment of the first aspect of the invention, mixing can be performed by combining all components, fluorophore, quencher, analyte, and solvent, by shaking or stirring in a selected vessel.

[0080] In an embodiment of the first aspect of the present invention, the sample is an aqueous sample.

[0081] In an embodiment of the first aspect of the present invention, the ratio of fluorophore to quencher is in the range of 4 / 1 to 1 / 25.

[0082] In an embodiment of the first aspect of the present invention, the sample is a solvent-based sample and the quencher is hydrophobic.

[0083] According to method step d), Raman spectroscopy is carried out, in particular the sample is measured by Raman spectroscopy, in particular the frequency shift of inelastically scattered electromagnetic radiation from the sample is measured when a photon from the incident electromagnetic radiation strikes a molecule and generates a scattered photon.

[0084] In an embodiment of the first aspect of the present invention, Raman spectroscopy may be performed by a Raman spectrometer.

[0085] In an embodiment of the first aspect of the present invention, the Raman spectrometer comprises a radiation source, a monochromator, a sample holder, and a detector. Dispersive Raman spectroscopy and Fourier transform Raman spectroscopy can be performed, which differ in the laser source and the method of detecting Raman scattering.

[0086] In an embodiment of the first aspect of the present invention, step (d) is carried out in the liquid phase.

[0087] According to method step e), at least one analyte of interest is determined by Raman spectroscopy. To obtain a signal-to-noise ratio from the spectrum, the maximum intensity of the fluorescent signal of the fluorophore and the maximum intensity of the strong signal of the analyte can be used.

[0088] In a second aspect, the present invention relates to the use of the method of the first aspect of the invention for determining at least one analyte of interest. All embodiments mentioned for the first aspect of the invention apply to the second aspect of the invention and vice versa.

[0089] In an embodiment of the second aspect of the present invention, the presence or level of at least one analyte of interest in a sample is determined.

[0090] In a third aspect, the present invention provides a diagnostic system for determining at least one analyte of interest in a sample, comprising: a radiation source; - sample holder and a wavelength selector; -Detector and and a diagnostic system for carrying out the method according to the first aspect of the invention, comprising a spectrometer having: All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention apply to the third aspect of the invention and vice versa.

[0091] In an embodiment of the third aspect of the present invention, the radiation source can emit monochromatic electromagnetic radiation. In particular, the radiation source is a laser. Several types of lasers can be used as radiation sources or excitation sources.

[0092] In an embodiment of the third aspect of the present invention, the radiation source can be selected from the group consisting of krypton ion (530.9 nm and 647.1 nm), He:Ne (632.8 nm), Nd:YAG (1064 nm and 532 nm), argon ion (488.0 nm and 514.5 nm), and diode lasers (630 nm and 780 nm). The use of a 1064 nm near-infrared (NIR) excitation laser can cause lower fluorescence effects than visible wavelength lasers.

[0093] In an embodiment of the third aspect of the present invention, Raman spectroscopy has a significant advantage for remote sensing when it is combined with optical fibers. The optical fiber serves to transmit the Raman signal by collecting scattered photons. The optical fiber system includes fibers in which the laser excitation can be transmitted along one fiber and the scattered radiation can be transmitted to a detector along a different fiber.

[0094] In the embodiment of the third aspect of the present invention, Raman spectroscopy can be performed by a Raman spectrometer.There are several commercially available handheld Raman spectrometers known and available, such as SciAps Reporter (formerly DeltaNu, Inc.), Snowy Range Instrument CBEX, Thermo Scientific FirstDefender (formerly Ahura, Inc.) and B&W TEK NanoRam, for example, see Driscoll, AJ, Harpster, MH, Johnson, PA (2013).The development of surface-enhanced Raman scattering as a detection modality for portable in vitro diagnostics: progress and challenges.Physical Chemistry Chemical Physics, 15(47), 20415-20433.

[0095] In an embodiment of the third aspect of the present invention, the Raman spectroscopy is surface-enhanced Raman scattering (SRS), coherent anti-Stokes Raman scattering (CARS), tip-enhanced Raman scattering (TERS), and / or stimulated and resonance Raman spectroscopy.

[0096] In an embodiment of the third aspect of the present invention, the sample holder may be a cuvette or a well plate capable of holding a liquid sample.

[0097] In an embodiment of the third aspect of the invention, the wavelength selector comprises software-implemented wavelength selection and automatic switching of mirrors towards the desired radiation source.

[0098] In an embodiment of the third aspect of the invention, the detector comprises a CCD camera detector that converts incident photons, for example Raman signals, into electrical signals from which wavelength dependent intensity functions are obtained.

[0099] In an embodiment of the third aspect of the present invention, the diagnostic system is a clinical diagnostic system.

[0100] In an embodiment of the fifth aspect of the present invention, the clinical diagnostic system comprises a sample preparation station.

[0101] In an embodiment of the fifth aspect of the present invention, a clinical diagnostic system, for example a sample preparation station, comprises a buffer unit for receiving a plurality of samples before a new sample preparation initiation sequence is initiated, the samples may be individually randomly accessible and their individual preparation may be initiated according to the sample preparation initiation sequence.

[0102] Clinical diagnostic systems utilize Raman spectroscopy, which is simpler, more reliable, and therefore more suitable for clinical diagnosis. In particular, high throughput or even higher can be achieved using random-access sample preparation and LC separation, while enabling online coupling to Raman spectroscopy. Furthermore, the process can be fully automated, improving yield and reducing the skill level required.

[0103] In a fourth aspect, the present invention provides a kit suitable for carrying out a method according to the first aspect of the invention, comprising: (A) at least one analyte of interest, preferably a deuterated analyte of interest as an internal standard; (B) Quencher and The present invention relates to a kit comprising or consisting of:

[0104] All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention apply to the fourth aspect of the invention and vice versa.

[0105] In a fifth aspect, the present invention relates to the use of a kit according to the fourth aspect of the invention in a method according to the first aspect of the invention.

[0106] All embodiments described for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention apply to the fifth aspect of the invention and vice versa.

[0107] In further embodiments, the present invention relates to the following aspects: 1. A method for determining at least one analyte of interest, comprising: a) - maximum excitation wavelength λ max1 at least one analyte of interest capable of emitting scattered electromagnetic radiation when excited with monochromatic electromagnetic radiation having - maximum excitation wavelength λ max1 a fluorophore capable of emitting fluorescent electromagnetic radiation when excited with monochromatic electromagnetic radiation having and preparing a b) Maximum excitation wavelength λ max1 providing a quencher capable of quenching the fluorescent electromagnetic radiation of the fluorophore upon excitation with monochromatic electromagnetic radiation having c) mixing at least one analyte of interest, a fluorophore, and a quencher to form a sample; d) performing Raman spectroscopy; e) determining at least one analyte of interest via Raman spectroscopy; A method comprising:

[0108] 2. The method of embodiment 1, wherein the sample is selected from the group consisting of blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva, and lymphatic fluid, cell culture, tissue culture, and solid samples such as dried blood spots or tissue extracts.

[0109] 3. The method of embodiment 1 or 2, wherein the sample is obtained from a patient sample selected from the group consisting of a serum, plasma, and whole blood sample from the individual.

[0110] 4. The method according to any one of aspects 1 to 3, wherein the sample is a human sample, preferably a hemolyzed whole blood sample, in particular a hemolyzed human whole blood sample.

[0111] 5. The method of any one of aspects 1 to 4, wherein the sample is an aqueous sample.

[0112] 6. The quencher is SO4 2- , PO4 3- 6. The method of any one of aspects 1 to 5, wherein the hydrophilic group is selected from an oligonucleotide, a quaternary amine, a PEG group, an alcohol, and COOH.

[0113] 7. The method of any one of aspects 1 to 6, wherein the hydrophilic group is an oligonucleotide.

[0114] 8. The method of any one of embodiments 1 to 7, wherein the quencher comprises a polyaromatic-azo backbone.

[0115] 9. The method of any one of embodiments 1 to 8, wherein the quencher molecules and the fluorophore molecules have a distance of less than 6 nm, or 5 nm, or 3 nm.

[0116] 10. The maximum excitation wavelength of the quencher is λ max1 The method of any one of embodiments 1 to 9, wherein the ΔT is + / −20 nm.

[0117] 11. The method according to any one of aspects 1 to 10, wherein the quenching range (absorption range) of the quencher is within the range of 470 nm to 660 nm, preferably 480 nm to 580 nm (inclusive) or 550 nm to 650 nm (inclusive).

[0118] 12. The method of any one of aspects 1-11, wherein the quench is Förster resonance energy transfer.

[0119] 13. The method of any one of aspects 1 to 12, wherein the quencher is a BHQ1 quencher or a BHQ2 quencher, preferably a BHQ1 quencher modified with an oligonucleotide or a BHQ2 quencher modified with an oligonucleotide.

[0120] 14. The method of any one of aspects 1-13, wherein the analyte of interest is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characteristic of a particular modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0121] 15. The method of any one of aspects 1-14, wherein step (d) is carried out in the liquid phase.

[0122] 16. The method of any one of aspects 1-15, wherein the scattered electromagnetic radiation is inelastically scattered electromagnetic radiation.

[0123] 17. The method of any one of aspects 1-16, wherein the scattered electromagnetic radiation is Stokes scattered and / or anti-Stokes scattered.

[0124] 18. Maximum excitation wavelength λ of monochromatic electromagnetic radiation max1 is less than 1064 nm.

[0125] 19. The method of any one of aspects 1-18, wherein the monochromatic electromagnetic radiation is generated by a krypton ion laser (530.9 nm and 647.1 nm), a He:Ne laser (632.8 nm), a Nd:YAG laser (1064 nm and 532 nm), an argon ion laser (488.0 nm and 514.5 nm), or a diode laser (630 nm and 780 nm).

[0126] 20. The method of any one of aspects 1-19, wherein the fluorophore is selected from the group consisting of chemicals containing several combined aromatic groups, or planar or cyclic molecules with several π bonds.

[0127] 21. The method of any one of embodiments 1 to 20, wherein the concentration of fluorophore is varied between 2.5 mg / l and 250 mg / l, with a concentration of quencher of 0, 51.7 or 62.5 mg / l.

[0128] 22. The method of any one of aspects 1-21, wherein the sample is a solvent-based sample and the quencher is hydrophobic.

[0129] 23. Use of a method according to any one of aspects 1 to 22 for determining at least one analyte of interest in a sample.

[0130] 24. The use according to embodiment 23, wherein the presence or level of at least one analyte of interest in a sample is determined.

[0131] 25. A diagnostic system for determining said at least one analyte of interest in a sample, comprising: a radiation source; - sample holder and a wavelength selector; -Detector and 23. A system for performing the method of any one of aspects 1-22, comprising a spectrometer having:

[0132] 26. A kit suitable for carrying out the method according to any one of aspects 1 to 22, comprising: (A) at least one analyte of interest, preferably a deuterated analyte of interest as an internal standard; (B) Quencher and A kit comprising or consisting of:

[0133] 27. Use of the kit according to aspect 26 in a method according to any one of aspects 1 to 22. [Example]

[0134] The following examples are offered to illustrate, but not to limit, the presently claimed invention.

[0135] Figure 1 shows the Raman spectrum of acetonitrile as the analyte of interest. -1 The intensity (counts) as a function of Raman shift is shown. A strong signal from acetonitrile is visible, but the background shows a broad signal exceeding the intensity of the acetonitrile signal. Samples contain or consist of 100 μl of phenol red solution as the fluorophore, 50 μl of acetonitrile as the analyte of interest, and 150 μl of water and quencher. Samples can be prepared using a concentrated solution of phenol red, mixed with acetonitrile as the analyte of interest, and combined with a quencher solution. The quencher solution can be prepared by adding water to the quencher solid to reach a concentration of 62.5 mg / L. To obtain a dilution series, the fluorophore is diluted to reach concentrations such as 250 mg / L, 125 mg / L, 50 mg / L, 25 mg / L, 12.5 mg / L, 5 mg / L, and 2.5 mg / L of fluorophore in the final sample.

[0136] Qualitative and / or quantitative determination of acetonitrile as the analyte of interest can be performed as follows. a) - maximum excitation wavelength λ max1 acetonitrile, which is capable of emitting scattered electromagnetic radiation when excited with monochromatic electromagnetic radiation having, for example, 532 nm, and - maximum excitation wavelength λ max1 A phenol red solution fluorophore capable of emitting fluorescent electromagnetic radiation when excited with monochromatic electromagnetic radiation having and preparing a b) Maximum excitation wavelength λmax1providing a quencher, e.g., hydrophilic BHQ1 and BHQ2, capable of quenching the fluorescent electromagnetic radiation of the fluorophore upon excitation with monochromatic electromagnetic radiation having c) mixing acetonitrile, a phenol red solution and a quencher to form a sample; d) performing Raman spectroscopy; e) Measuring acetonitrile by Raman spectroscopy.

[0137] The fluorophore emits fluorescence upon 532 nm laser excitation. Raman spectroscopy can be performed using a Raman spectrometer such as the HORIBA LabRAM HR Evolution. The spectrum in Figure 1 provides information on what a fluorescence-affected spectrum may look like.

[0138] Figure 2 shows a quencher with an oligonucleotide as the hydrophilic group. 2- ;PO4 3- The quenchers may contain other or additional hydrophilic groups selected from oligonucleotides, quaternary amines, PEG groups, alcohols, COOH, etc. The quenchers have excitation maxima λ of 534 nm (hydrophilic BHQ-1) and 573 nm (hydrophilic BHQ-2), respectively. max1 The quenchers have a quenching range of 400-500 nm (hydrophilic 5'-TTx-3'X=BHQ1) and 550 nm-650 nm (hydrophilic 5'-TTx-3'X=BHQ2), respectively.

[0139] Figures 3A-3C show the Raman spectrum of acetonitrile as the target analyte. Measurements were performed via the HORIBA LabRAM HR Evolution high-throughput screening system. Photographs 3A-3C show the high-throughput screening in LaSpec6 software. 3A shows an overlay of all spectra with different fluorophore concentrations, 3B shows a single spectrum view, and 3C shows a 96-well plate view for performing well selection to show the specific spectrum of each well.

[0140] Figures 4A and 4B show Raman spectra of acetonitrile as the analyte of interest at different fluorophore dilutions. Samples can be prepared as described in Figure 1. Figure 4A shows a 1 / 1 dye, 250 mg / L fluorophore dilution, and Figure 4B shows a 1 / 100 dye, 2.5 mg / L fluorophore dilution. The figures demonstrate the effect of dilution on the signal-to-noise ratio. For 4A, the spectrum shows fluorescence, while the spectrum for 4B shows almost no fluorescence due to the high dilution factor. Dilutions are performed by adding water to the fluorophore solution to reach the desired concentration.

[0141] Figure 5 shows the signal-to-noise ratio as a function of fluorophore dilution in the absence of any quencher. Fluorophore dilution allows for the detection of acetonitrile. This signal-to-noise ratio is calculated between the maximum intensity of the analyte signal and the maximum signal of the quencher fluorescence (described as noise). The increase in signal-to-noise ratio with fluorophore dilution provides a benchmark for identifying the effect of the quencher on fluorescence.

[0142] Figure 6 shows the signal-to-noise ratio as a function of fluorophore dilution at different quencher concentrations (0 mg / L (blank), 41.7 mg / L, 62.5 mg / L). The quencher results in an increase in signal-to-noise ratio at higher fluorophore concentrations compared to the blank sample.

[0143] This patent application claims priority from European Patent Application No. 22209071.4, the contents of which are incorporated herein by reference.

Claims

1. A method for determining at least one target analyte, a) - Maximum excitation wavelength λ max1 The at least one target analyte, which can emit scattered electromagnetic radiation when excited with monochromatic electromagnetic radiation having, - The maximum excitation wavelength λ max1 A fluorophore that can emit fluorescent electromagnetic radiation when excited by the monochromatic electromagnetic radiation having the above Steps to prepare, b) the maximum excitation wavelength λ max1 The steps include: preparing a quencher capable of quenching the fluorescence electromagnetic radiation of the fluorophore when excited with the monochromatic electromagnetic radiation having the c) The step of mixing the at least one target analyte, the fluorophore, and the quencher to form a sample, d) Steps to perform Raman spectroscopy, e) A step of determining the at least one target analyte by Raman spectroscopy. Methods that include...

2. The method according to claim 1, wherein the sample is selected from the group consisting of blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph, cell cultures, tissue cultures, and solid samples such as dried blood spots or tissue extracts, preferably the sample is obtained from a patient sample selected from the group consisting of serum, plasma, and whole blood samples from an individual, and / or the target analyte is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characteristic of specific modifications of another molecule, substances internalized by the organism, metabolites of such substances, and combinations thereof.

3. The method according to claim 1, wherein the sample is an aqueous sample.

4. The aforementioned quencher is SO 4 2- , PO 4 3- The method according to claim 1, comprising a hydrophilic group selected from oligonucleotides, quaternary amines, PEG groups, alcohols, and COOH groups.

5. The method according to claim 4, wherein the hydrophilic group is an oligonucleotide and the quencher comprises a polyaromatic-azo skeleton.

6. The maximum excitation wavelength of the quencher is λ max1 The method according to claim 1, wherein the wavelength is ±20 nm.

7. The method according to claim 1, wherein the quench range (absorption range) of the quencher is in the range of 470 nm to 660 nm, preferably 480 nm to 580 nm (including the boundary) or 550 nm to 650 nm (including the boundary).

8. The method according to claim 1, wherein the quencher is a BHQ1 quencher or a BHQ2 quencher.

9. The method according to claim 1, wherein step (d) is performed in the liquid phase.

10. The method according to claim 1, wherein the concentration of the fluorophore varies between 2.5 mg / l and 250 mg / l, compared to a quencher concentration of 0, 51.7, or 62.5 mg / l.

11. The method according to claim 1, wherein the sample is a solvent-based sample and the quencher is hydrophobic.

12. Use of the method according to any one of claims 1 to 11 for determining the at least one target analyte in a sample.

13. A diagnostic system for determining at least one target analyte in a sample, - Radiation source, - Sample holder and, - Wavelength selector, - Detector and A diagnostic system for performing the method according to any one of claims 1 to 11, comprising a spectrometer having a spectrometer.

14. A kit suitable for carrying out the method according to any one of claims 1 to 11, (A) At least one target analyte, preferably a deuterated target analyte as an internal standard, (B) Quencher and A kit that includes or consists of.

15. The use of a kit suitable for carrying out the method according to any one of claims 1 to 11, wherein the kit is (A) At least one target analyte, preferably a deuterated target analyte as an internal standard, (B) Quencher and Use including or consisting of.

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