Method for determining at least one target analyte

Abstract

The invention relates to a method for determining at least one target analyte and to the use thereof. The invention further relates to a diagnostic system, a kit and their use for determining at least one target analyte.

Description

Technical Field

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

[0002] Raman spectroscopy is a technique specifically for measuring the frequency shift of inelastically scattered light from a sample when photons from incident light strike molecules and generate scattered photons. Raman spectroscopy gives qualitative and quantitative information about analytes, even when in a matrix, such as an organic or inorganic (e.g., aqueous) matrix.

[0003] (Organic) matrices have the main problem that the sample and / or matrix components have a strong fluorescence effect in addition to the Raman effect, where the parallel and more sensitive effect of fluorescence limits this process. This leads to the problem that the analyte information from the Raman spectrum cannot be detected well.

[0004] Therefore, for analytical techniques, fluorescence quenchers and selective quenching of fluorescence are highly concerned.

[0005] In the past, attempts have been made to quench the fluorescence signal by reducing the fluorescence lifetime of adding KI to the solution. However, molecular quenching has never been carried out.

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

[0007] The object of the present invention is to provide a method for determining at least one analyte of interest. Further, the object of the present invention is to provide a diagnostic system, a kit, and their use for determining at least one target analyte.

[0008] This object 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] Hereinafter, the present invention relates to the following aspects:

[0010] In a first aspect, the present invention relates to a method for determining at least one analyte of interest, the method comprising the following steps:

[0011] a) Providing

[0012] - the at least one target analyte, which can emit scattered electromagnetic radiation when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1 and

[0013] - a fluorophore, which when excited by the maximum excitation wavelength λ max1that can emit fluorescent electromagnetic radiation when excited by the monochromatic electromagnetic radiation,

[0014] b) providing a quencher that can quench the fluorescent electromagnetic radiation of the fluorophore when excited by the monochromatic electromagnetic radiation having the maximum excitation wavelength λ max1 of the fluorophore when excited by the monochromatic electromagnetic radiation,

[0015] c) mixing the at least one target analyte, the fluorophore, and the quencher to form a sample,

[0016] d) performing Raman spectroscopy, and

[0017] e) determining the at least one target analyte via Raman spectroscopy.

[0018] In a second aspect, the present invention relates to the use of the method of the first aspect of the present invention for determining the at least one analyte of interest.

[0019] In a third aspect, the present invention relates to a diagnostic system for determining at least one target analyte in a sample.

[0020] In a fourth aspect, the present invention relates to a kit adapted to perform the method of the first aspect of the present invention, the kit comprising or consisting of the following items:

[0021] (A) at least one target analyte, preferably a target deuterated analyte as an internal standard, and

[0022] (B) a quencher.

[0023] In a fifth aspect, the use of the kit of the fourth aspect of the present invention in the method of the first aspect of the present invention. Description of the Drawings

[0024] Figure 1 Shows the Raman spectrum of acetonitrile as a target analyte.

[0025] Figure 2 Shows a quencher having an oligonucleotide as a hydrophilic group.

[0026] Figures 3A to 3C Shows the Raman spectrum of acetonitrile as a target analyte.

[0027] Figure 4A and 4B Shows the Raman spectrum of acetonitrile as a target analyte at different fluorophore dilutions.

[0028] Figure 5 and 6 Shows the signal-to-noise ratio varying with the fluorophore concentration at different quencher concentrations (0 mg / l (blank), 41.7 mg / l, 62.5 mg / l).

[0029] Figure 7 Shows the signal-to-noise ratio varying with the fluorophore concentration at different quencher concentrations (0 mg / l (blank), 62.5 mg / l) in a biological matrix environment such as serum. Detailed Description

[0030] 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 can vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise indicated, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0031] This specification text incorporates by reference several documents in their entirety. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.), whether cited above or below, is hereby incorporated by reference in its entirety. In the event of a conflict between the definitions or teachings of such incorporated references and the definitions or teachings cited in this specification text, the specification text shall prevail.

[0032] The elements of the present invention will be described below. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to form additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Additionally, unless the context otherwise requires, any arrangement and combination of all the described elements in this application shall be considered to be disclosed by the specification of this application.

[0033] Definition

[0034] The word "comprising" and variations such as "including" and "containing" should be understood to imply the inclusion of the 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.

[0035] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", "the", and "said" include plural referents.

[0036] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a "range" format. It should be understood that such range formats are used merely for convenience and brevity and should therefore be interpreted flexibly to include not only the explicitly recited values as the limits of the range, but also all the individual values or sub-ranges subsumed within that range as if each value and sub-range were explicitly recited. By way of illustration, the numerical range "4% to 20%" should be interpreted to include not only the explicitly recited values of 4% to 20%, but also the individual values and sub-ranges within the indicated range. Thus, this numerical range includes individual values such as 4, 5, 6, 7, 8, 9, 10, … 18, 19, 20% and sub-ranges such as 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges that refer to a minimum or maximum value. In addition, such interpretation applies regardless of the breadth of the range or feature described.

[0037] When used in connection with a numerical value, the term "about" means to encompass a numerical value within a range having a lower limit that is 5% less than the value indicated and an upper limit that is 5% greater than the value indicated.

[0038] In the context of the present disclosure, the terms "analyte", "analyte molecule", or "target analyte" are used interchangeably and refer to a chemical substance to be analyzed via Raman spectroscopy. A chemical substance suitable for analysis via Raman spectroscopy, i.e., an analyte, 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, cytoplasmic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules having a certain modification of another molecule (e.g., a sugar moiety or a phosphoryl residue on a protein, a methyl-residue on genomic DNA), or substances internalized by a living organism (e.g., therapeutic drugs, abused drugs, toxins, etc.) or metabolites of such substances. Such analytes can be used as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance within a biological system that serves as an indicator of the biological state of said system.

[0039] An analyte or target analyte can be present in a sample, specifically a biological or clinical sample. The terms "biological or clinical sample" are used interchangeably herein and refer to a part or piece of a tissue, organ, or individual, typically smaller than such tissue, organ, or individual, and intended to represent the whole tissue, organ, or individual. When analyzed, the biological or clinical sample provides information about the state of the tissue or the health or diseased state of the organ or individual. Examples of biological or clinical samples include but are not limited to: fluid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph fluid; or solid samples such as dried blood spots and tissue extracts. Other examples of biological or clinical samples are cell cultures or tissue cultures.

[0040] The term "chromatography" refers to a process in which a chemical mixture, as it flows around or over a liquid or solid stationary phase carried by a liquid or gas, is separated into its various components as a result of the differential distribution of the chemical entities. In embodiments of the present invention, the method or sample element or device or kit respectively does not have a chromatography step and a chromatography unit.

[0041] The term "liquid chromatography" or "LC" refers to the process of selectively retarding one or more components in a fluid solution as the fluid uniformly percolates through a column of finely divided material or through a capillary channel. As this fluid moves relative to the stationary phase, the distribution of the mixture components between the one or more stationary phases and the bulk fluid (i.e., the mobile phase) causes this retardation. A method in which the stationary phase has a higher polarity than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) is called normal-phase liquid chromatography (NPLC), while a method in which the stationary phase has a lower polarity than the mobile phase (e.g., a water - methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is called reverse-phase liquid chromatography (RPLC).

[0042] "High performance liquid chromatography" or "HPLC" refers to a liquid chromatography method in which the separation degree is increased by forcing the mobile phase to pass through a stationary phase, usually a densely packed column, under pressure. Generally, the column is packed with a stationary phase composed of irregularly shaped or spherical particles, porous monoliths or porous membranes. Historically, HPLC has been divided into two different subclasses based on the polarity of the mobile phase and the stationary phase. The method in which the polarity of the stationary phase is higher than that of the mobile phase (for example, toluene as the mobile phase and silica as the stationary phase) is called normal phase liquid chromatography (NPLC), and vice versa (for example, a water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is called reverse phase liquid chromatography (RPLC). Microflow LC refers to an HPLC method using a column with a narrow inner column diameter (usually less than 1 mm, for example about 0.5 mm). "Ultra high performance liquid chromatography" or "UHPLC" refers to an HPLC method using 120 MPa (17,405 lbf / in2) or about 1200 atmospheres. Fast LC refers to an LC method using a column with the inner diameter as described above and a short length (<2 cm, for example 1 cm), which employs the flow rate as described above and uses the pressure as described above (microflow LC, UHPLC). The short fast LC protocol includes a capture / wash / elute step using a single analytical column and achieves LC in an extremely short time of <1 min.

[0043] Other well-known LC modes include hydrophilic interaction chromatography (HILIC), size exclusion LC, ion exchange LC, and affinity LC.

[0044] The LC separation can be single-channel LC or multi-channel LC including a plurality of LC channels arranged in parallel. In LC, the analyte can be separated according to the polarity or log P value, size or affinity of the analyte, as is commonly known to those skilled in the art.

[0045] The term "electromagnetic radiation" is a broad term and will be given the ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or custom meaning. Specifically, the term can refer to, but is not limited to, an energy wave form traveling through space (vacuum or matter). This energy wave form can consist of an electric field component and a magnetic field component. The energy waves are perpendicular to each other in phase and oscillate perpendicular to the energy propagation direction. Electromagnetic radiation is classified into several types according to the frequency of its waves. These types include (in the order of decreasing frequency and increasing wavelength) cosmic radiation, gamma radiation, X-ray radiation, ultraviolet radiation, visible radiation, IR radiation, terahertz radiation, microwave radiation, and radio waves. A small and variable frequency window is sensed by the eyes of various organisms, which is called the visible spectrum (λ 0.4 to 0.7 μm) or light.

[0046] The term "monochromatic electromagnetic radiation" is a broad term and will be given the ordinary and customary meaning to a person of ordinary skill in the art and is not limited to a special or custom meaning. Specifically, the term can refer to, but is not limited to, electromagnetic radiation of only one frequency or wavelength, especially visible radiation. It is not possible to produce completely monochromatic radiation, but lasers produce radiation within a very narrow frequency band.

[0047] The term "scattered electromagnetic radiation" refers to electromagnetic radiation that has been scattered.

[0048] 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 preferably can include any body fluid. Preferred samples are whole blood, serum or plasma. As will be understood by one skilled in the art, any such assessment is performed in vitro. The patient sample is then discarded. The patient sample is only used for the in vitro methods of the present invention and the material of the patient sample is not transferred back into the patient's body.

[0049] In the context of the present disclosure, a sample can be derived from an "individual" or a "subject". Generally, the subject is a mammal. Mammals include, but are not limited to, domesticated 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).

[0050] The term "hemolytic reagent (HR)" refers to a reagent that lyses the cells present in a sample. In the context of the present invention, the hemolytic reagent specifically refers to a reagent that lyses the 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 (H2O). Other examples of hemolytic reagents include, but are not limited to, deionized water, hypertonic liquids (e.g., 8M urea), ionic liquids and different detergents.

[0051] The term "fluorescent electromagnetic radiation" is a broad term and will be given the ordinary and customary meaning to a person of ordinary skill in the art and is not limited to a special or custom meaning. Therefore, it will not be explained in detail here.

[0052] The term "quenching" is a broad term and will be given the ordinary and customary meaning to a person of ordinary skill in the art and is not limited to a special or custom meaning. Therefore, it will not be explained in detail here.

[0053] The term "Förster resonance energy transfer" is a broad term and will be given the ordinary and customary meaning to a person of ordinary skill in the art and is not limited to a special or custom meaning. Therefore, it will not be explained in detail here.

[0054] The term "aqueous sample" can mean a water content in which the quencher is modified to be up to 100% water-soluble.

[0055] The term "hydrophobic" can mean that a compound is only sparingly or not soluble in polar solvents such as ethanol, methanol or water.

[0056] A "clinical diagnostic system" is laboratory automation equipment specifically designed to analyze samples for in vitro diagnosis. Depending on requirements and / or the desired laboratory workflow, the clinical diagnostic system can have different configurations. Additional configurations can be obtained by coupling together multiple devices and / or modules. A "module" is a working unit with a dedicated function, typically smaller than the entire clinical diagnostic system. This function can be an analytical function, but can also be a pre-analytical function or a post-analytical function, or can be an auxiliary function for any one of the pre-analytical, analytical, or post-analytical functions. 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 steps and / or analytical steps and / or post-analytical steps. In particular, the clinical diagnostic system can include one or more analytical devices designed to perform corresponding workflows optimized for certain types of analysis (e.g., clinical chemistry, immuno-chemistry, coagulation, hematology, liquid chromatography, mass spectrometry, Raman spectroscopy, etc.). Thus, the clinical diagnostic system can include one analytical device with a corresponding workflow or any combination of such analytical devices, where pre-analytical modules and / or post-analytical modules can be coupled to individual analytical devices or shared by multiple analytical devices. In an alternative, pre-analytical functions and / or post-analytical functions can be performed by units integrated within the analytical device. The clinical diagnostic system can include functional units such as a liquid handling unit for aspirating and / or pumping and / or mixing samples and / or reagents and / or system fluids, and functional units for sorting, storing, transporting, identifying, separating, detecting. The clinical diagnostic system can include a sample preparation station for automatically preparing a sample containing an analyte of interest, a liquid chromatography (LC) separation station optionally including multiple LC channels, and / or a sample preparation / LC interface for optionally inputting the prepared sample into any one of the LC channels. The clinical diagnostic system can further include a controller programmed to allocate samples to predefined sample preparation workflows, each workflow including a predefined sequence of sample preparation steps and requiring a predefined completion time (depending on the target analyte). The clinical diagnostic system can further include a Raman spectrometer and an LC / Raman interface for connecting the LC separation station to the Raman spectrometer. As used herein, the term "automatically" or "automatic" is a broad term and is given the ordinary and customary meaning to a person of ordinary skill in the art and is not limited to a special or custom meaning. The term can specifically refer to, but is not limited to, a process that is carried out entirely by means of at least one computer and / or at least one computer network and / or at least one machine, and in particular, does not require manual operation and / or interaction with a user.

[0057] A "sample preparation station" can be a pre - analytical module coupled to one or more analytical devices or units within an analytical device, designed to perform a series of sample - handling steps that are intended to remove or at least reduce interfering matrix components in a sample and / or enrich the analyte of interest in the sample. Such handling steps can include any one or more of the following handling operations performed sequentially, in parallel, or in an interleaved manner on one or more samples: aspirating (suctioning and / or dispensing) fluids, pumping fluids, mixing with reagents, incubating at a certain temperature, heating or cooling, centrifuging, separating, filtering, sieving, drying, washing, resuspending, aliquoting, transferring, storing, etc.).

[0058] Typically, an "internal standard" (ISTD) is a known amount of a substance that exhibits properties similar to the target analyte when undergoing a Raman spectroscopy detection workflow (i.e., including any pre - treatment, enrichment, and actual detection steps). Although the ISTD exhibits properties similar to the target analyte, it can still be clearly distinguished from the target analyte. For example, during chromatographic separations such as gas chromatography and 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 mass from the target analyte from the sample. This enables Raman spectroscopic discrimination between the ions from the ISTD and the ions from the analyte by their different mass - to - charge (m / z) ratios. Both undergo fragmentation and provide daughter ions. These daughter ions can be distinguished from each other and from their respective parent ions by their m / z ratios. Thus, independent determination and quantification of the signals from the ISTD and the analyte can be performed. Since the amount of the ISTD added is known, 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 a relative comparison of the amount of the detected analyte and enables unambiguous identification and quantification of the analyte when the target analyte present in the sample reaches the Raman spectrometer. Typically but not necessarily, the ISTD is an isotopically labeled variant of the target analyte (containing, for example 2 H, 13 C, or 15 N, etc. labels).

[0059] "Kit" refers to any article (e.g., a package or container) that contains at least one reagent of the present invention, such as a pharmaceutical for treating a disease or a probe for specifically detecting a biomarker gene or protein. The kit is preferably promoted, distributed, or sold as a unit for performing the method of the present invention. Generally, the kit may further include a separated carrier device for receiving one or more container devices, such as vials, tubes, etc., in a tightly defined space. In particular, each container is meant to contain one of the individual elements to be used in the method of the first aspect. The kit may further include one or more other reagents, including but not limited to reaction catalysts. The kit may further contain one or more other containers containing other materials, including but not limited to buffers, internal standards, diluents, filters, needles, syringes, and package inserts with instructions for use. Labels may be present on the containers to indicate the use of the composition for a specific application and may also indicate guidelines for in vivo or in vitro use. 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 directly on a computer or data processing device. Additionally, the kit may contain a standard amount of a biomarker for calibration purposes as described elsewhere herein.

[0060] Embodiment

[0061] In a first aspect, the present invention relates to a method for determining at least one analyte of interest, the method comprising the steps of:

[0062] a)

[0063] - providing the at least one target analyte that is capable of emitting scattered electromagnetic radiation when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1 and

[0064] - providing a fluorophore that is capable of emitting fluorescent electromagnetic radiation when excited by the monochromatic electromagnetic radiation having the maximum excitation wavelength λ max1 ;

[0065] b) providing a quencher that is capable of quenching the fluorescent electromagnetic radiation of the fluorophore when excited by the monochromatic electromagnetic radiation having the maximum excitation wavelength λ max1 ;

[0066] c) mixing the at least one target analyte, the fluorophore, and the quencher to form a sample,

[0067] d) performing Raman spectroscopy, and

[0068] e) determining the at least one target analyte via Raman spectroscopy.

[0069] The inventors have surprisingly found that the subject matter of the present invention, in particular the method according to the first aspect of the present invention, shows a simple and robust way to overcome the above disadvantages. By the simple solution described, fluorescence (as the main problem in Raman spectroscopy) is overcome so that fast and reliable Raman spectra can be measured even in systems where fluorescence cannot be avoided or removed.

[0070] In particular, the method is carried out by a fluorescence quencher having a hydrophilic group (such as an oligonucleotide) to enhance the solubility of the fluorescence quencher in water so as to reduce fluorescence by mixing the analyte sample with the quencher.

[0071] According to step a), at least one target analyte is provided. The at least one target analyte is capable of emitting scattered electromagnetic radiation when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1 .

[0072] In an embodiment of the first aspect of the present invention, the scattered electromagnetic radiation is inelastic scattered electromagnetic radiation.

[0073] 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 specifically for measuring the frequency shift of inelastically scattered light from a sample when photons from incident light or electromagnetic radiation hit molecules and generate scattered photons. The emitted scattered light or electromagnetic radiation can be: photons having a lower frequency than the original photons, and in this case, it is called Stokes Raman scattering; or photons having a higher frequency than the original photons, and is called anti-Stokes Raman scattering.

[0074] In an embodiment of the first aspect of the present invention, the maximum excitation wavelength λ max1 of the monochromatic electromagnetic radiation is less than 1064 nm, such as 532 nm, 633 nm.

[0075] In an embodiment of the first aspect of the present invention, the monochromatic electromagnetic radiation is generated by a krypton ion laser (530.9 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 and 514.5 nm), or a diode laser (630 and 780 nm).

[0076] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, seco-steroids, molecules having some modified properties of another molecule, substances that have been internalized by an organism, metabolites of such substances, and combinations thereof.

[0077] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: 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-hydroxy pregnenolone, 17-hydroxyprogesterone, androsterone, epiandrosterone, Δ4-androstenedione, 11-deoxycortisol, corticosterone, 21-deoxycortisol, 11-deoxycorticosterone, allopregnanolone and aldosterone; Δ8-tetrahydrocannabinolic acid, benzoylecgonin, salicylic acid, 2-hydroxybenzoic acid, gabapentin, pregabalin, valproic acid, vancomycin, methotrexate, mycophenolic acid, montelukast, repaglinide, furosemide, telmisartan, gemfibrozil, diclofenac, ibuprofen, indomethacin, zomepirac, isoxepac, and penicillin. In an embodiment of the first aspect of the present invention, the analyte molecules containing one or more carboxyl groups are: amino acids 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, acetylcholine, amcinonide, amyl oxamate, amylocaine, anileridine, arecoline, artesunate, and pethidine; cantharidin, succinic anhydride, trimellitic anhydride, and maleic anhydride; cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcidiol, calcitriol, tachysterol, lumisterol, and tacalcitol; 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 (calcidiol), 3-epi-25-hydroxyvitamin D2, 3-epi-25-hydroxyvitamin 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 mononitrate, guaifenesin, clavulanic acid, miglitol, zalcitabine, isoproterenol, acyclovir, methocarbamol, tramadol, venlafaxine, atropine, clofedanol, α-hydroxyalprazolam, α-hydroxytriazolam, lorazepam, oxazepam, temazepam, ethyl glucuronide, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-demethyltramadol, desmetramadol, dihydroquinidine, and quinidine. In an embodiment of the first aspect of the present invention, where the analyte molecule contains 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, canaglifozin, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyl digitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine,Capecitabine, adenosine arabinoside, 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, thiamine tetrahydrofurfuryl disulfide, octreotide, desmopressin, vapreotide, terlipressin, linaclotide, and peginesatide. 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, scopolamine methylbromide, eplerenone, mupirocin, natamycin, and troleandomycin; estrogen; estrogen-like compounds, estrone (El), estradiol (E2), 17α-estradiol, 17β-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol; and / or their metabolites. In the examples, the metabolites are selected from the group consisting of: estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-ketoE2), 16α-hydroxyestrone (16α-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-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (El), estrone sulfate (Els), 17α-estradiol (E2a), 17β-estradiol (E2B),Estradiol Sulfate (E2S), Equilin (EQ), 17α-Dihydroequilin (EQα), 17β-Dihydroequilin (EQβ), Equilenin (EN), 17-Dihydroequilenin (ENα), 17α-Dihydroequilenin, 17β-Dihydroequilenin (ENβ), Δ8,9-Dehydroestrone (dEl), Δ8,9-Dehydroestrone Sulfate (dEls), Δ9-Tetrahydrocannabinol, Mycophenolic Acid (β or b can be used interchangeably, α and a can be used interchangeably); 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, Amfepramone, Butylone, 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-benzodioxolylbutylamine, 2-amino-1-(3,4-methylenedioxyphenyl)butane, 1,3-benzodioxolylbutylamine, normeperidine, O-Destramadol, nortramadol, tramadol, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin, sisomicin, and 5-methylcytosine; ribose, deoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, N-acetylneuraminic acid, etc. In the examples, the analyte molecules are oligosaccharides, particularly selected from the group consisting of: disaccharides, trisaccharides, tetrasaccharides, polysaccharides. In an embodiment of the first aspect of the present invention, the disaccharides are 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 above-mentioned monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide moiety, zidovudine, and azidocillin.

[0078] Such analyte molecules can be present in a sample, such as a biological or clinical sample, such as a body fluid (e.g., blood, serum, plasma, urine, saliva, cerebrospinal fluid, etc.), a tissue or cell extract, etc.

[0079] In an embodiment of the first aspect of the present invention, the samples are selected from the group consisting of: blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph fluid, cell cultures, tissue cultures, and solid samples, such as dried blood spots or tissue extracts. In some embodiments of the first aspect of the present invention, the analyte molecule can be present in a sample that is a purified or partially purified sample, e.g., a purified or partially purified protein mixture or extract.

[0080] In an embodiment of the first aspect of the present invention, the sample is obtained from a patient sample, which is selected from the group consisting of: serum samples, plasma samples, and whole blood samples from an individual.

[0081] In an embodiment of the first aspect of the present invention, the sample is a human sample, preferably a hemolyzed whole blood sample, particularly a hemolyzed human whole blood sample. A hemolyzing agent can be used to hemolyze the hemolyzed whole blood sample.

[0082] According to step a), a fluorophore is provided. The fluorophore is capable of emitting fluorescent electromagnetic radiation when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1 .

[0083] In an embodiment of the first aspect of the present invention, the fluorophore is selected from the group consisting of: a chemical substance containing a number of combined aromatic groups, or a planar or cyclic molecule having a number of π bonds. As examples, the following can be listed

[0084] Xanthene derivatives: fluorescein, rhodamine, Oregon Green, eosin and Texas Red cyanine derivatives: cyanine, indocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine squaraine derivatives and ring-substituted squaraines, including Seta and Square dyes

[0085] Squaraine rotaxane derivatives: see τ dyes

[0086] Naphthalene derivatives (dansyl and Prodan derivatives)

[0087] Coumarin derivatives

[0088] Oxadiazole derivatives: pyridyl oxazole, nitrobenzoxadiazole and benzoxadiazole

[0089] Anthracene derivatives: anthraquinone, including DRAQ5, DRAQ7 and CyTRAK Orange

[0090] Pyrene derivatives: cascade blue, etc.

[0091] Oxazine derivatives: Nile Red, Nile Blue, cresyl violet, oxazine 170, etc.

[0092] Acridine derivatives: proflavine, acridine orange, acridine yellow, etc.

[0093] Arylmethine derivatives: auramine, crystal violet, malachite green

[0094] Tetrapyrrole derivatives: porphin, phthalocyanine, bilirubin

[0095] Dipyrromethene derivatives: BODIPY, azabodipy

[0096] According to step b), a quencher is provided. The quencher is capable of quenching the fluorescent electromagnetic radiation of the fluorophore when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1 .

[0097] In an embodiment of the first aspect of the present invention, the quencher contains a hydrophilic group selected from the following: SO4 2- , PO4 3-, oligonucleotide, quaternary amine, PEG group, alcohol, COOH.

[0098] In an embodiment of the first aspect of the present invention, the oligonucleotide is an oligomer having a varying number of nucleotides attached with adenine, cytosine, guanine, and / or thymine.

[0099] In an embodiment of the first aspect of the present invention, the quaternary amine is (CH3)4NCl or (CH3CH2)4NCl.

[0100] In an embodiment of the first aspect of the present invention, the PEG group is polyethylene glycol having a varying number of ethylene glycol units (e.g., between 1 and 100 (e.g., 25 or 50) PEG units).

[0101] In an embodiment of the first aspect of the present invention, the alcohol is OH, CH3OH, C2H5OH, C3H7OH, C4H9OH.

[0102] In an embodiment of the first aspect of the present invention, the hydrophilic group is an oligonucleotide.

[0103] In an embodiment of the first aspect of the present invention, the quencher comprises a polycyclic aromatic - azo backbone.

[0104] In an embodiment of the first aspect of the present invention, the distance between the molecule of the quencher and the molecule of the fluorophore is less than 6 nm or 5 nm or 3 nm. Thus, the quenching process can be easily carried out.

[0105] 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.

[0106] In an embodiment of the first aspect of the present invention, the excitation wavelength of the quencher is in the range of λ max1 with a tolerance of + / - 20 nm, preferably + / - 15 nm, + / - 10 nm, or + / - 5 nm.

[0107] 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 (including the boundary values), or 550 nm to 650 nm (including the boundary values).

[0108] In an embodiment of the first aspect of the present invention, the quenching is Förster resonance energy transfer.

[0109] In an embodiment of the first aspect of the present invention, the quencher is a BHQ1 quencher or a BHQ2 quencher.

[0110] In an embodiment of the first aspect of the present invention, the quencher is a BHQ1 quencher or a BHQ2 quencher, wherein the BHQ1 quencher is modified with an oligonucleotide, or wherein the BHQ2 quencher is modified with an oligonucleotide.

[0111] In an embodiment of the first aspect of the present invention, the quencher comprises the following formula:

[0112]

[0113]

[0114] The structures of BHQ1 and BHQ2 are modified with an oligonucleotide (* = 5'-TTx-3', X = BHQ1 or BHQ2).

[0115] According to method step c), at least one target analyte, a fluorophore, and a quencher are mixed to form a sample.

[0116] In an embodiment of the first aspect of the present invention, the mixing can be carried out by combining all components of the fluorophore, quencher, analyte, and solvent by shaking or stirring in a selected container.

[0117] In an embodiment of the first aspect of the present invention, the sample is an aqueous-based sample.

[0118] In an embodiment of the first aspect of the present invention, the ratio of the fluorophore to the quencher is in the range of 4 / 1 to 1 / 25.

[0119] In an embodiment of the first aspect of the present invention, the sample is a solvent-based sample and the quencher is hydrophobic.

[0120] According to method step d), Raman spectroscopy is carried out, particularly by measuring the sample by Raman spectroscopy. In particular, the frequency shift of the inelastically scattered electromagnetic radiation from the sample is measured when photons from the incident electromagnetic radiation strike the molecules and generate scattered photons.

[0121] In an embodiment of the first aspect of the present invention, Raman spectroscopy can be carried out by a Raman spectrometer.

[0122] 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 carried out, which differ in their laser sources and the methods for detecting Raman scattering.

[0123] In an embodiment of the first aspect of the present invention, step (d) is carried out in the liquid phase.

[0124] According to method step e), at least one target analyte is determined via Raman spectroscopy. To obtain the signal-to-noise ratio from the spectrum, the maximum intensity of the fluorescence signal of the fluorophore and the maximum intensity of the strong signal of the analyte can be used.

[0125] In a second aspect, the invention relates to the use of the method of the first aspect of the invention for determining the at least one analyte of interest. All the embodiments mentioned for the first aspect of the invention are applicable to the second aspect of the invention, and vice versa.

[0126] In an embodiment of the second aspect of the invention, the presence or level of at least one target analyte in a sample is determined.

[0127] In a third aspect, the invention relates to a diagnostic system for determining at least one target analyte in a sample, the diagnostic system comprising a spectrometer having

[0128] - a radiation source,

[0129] - a sample holder,

[0130] - a wavelength selector, and

[0131] - a detector for performing the method according to the first aspect of the invention. All the embodiments mentioned for the first aspect and / or the second aspect of the invention are applicable to the third aspect of the invention, and vice versa.

[0132] In an embodiment of the third aspect of the invention, the radiation source is capable of emitting monochromatic electromagnetic radiation. In particular, the radiation source is a laser. Several types of lasers can be used as the radiation source or excitation source.

[0133] In an embodiment of the third aspect of the invention, the radiation source can be selected from the group consisting of: krypton ion (530.9 and 647.1 nm), He:Ne (632.8 nm), Nd:YAG (1064 nm and 532 nm), argon ion (488.0 and 514.5 nm), and diode laser (630 and 780 nm). Using a 1064 nm near-infrared (NIR) excitation laser can produce a lower fluorescence effect than a visible wavelength laser.

[0134] In an embodiment of the third aspect of the invention, Raman spectroscopy has great remote sensing advantages when associated with an optical fiber. The optical fiber is responsible for transporting the Raman signal by collecting scattered photons. The fiber optic system includes an optical fiber in which laser excitation can be transmitted along one fiber, while the scattered radiation can be transmitted along a different fiber to the detector.

[0135] In an embodiment of the third aspect of the present invention, Raman spectroscopy can be carried out by a Raman spectrometer. It is known that there are several commercially available handheld Raman spectrometers that can be used, such as SciAps ReporteRt (formerly known as DeltaNu, Inc.), Snowy Range Instrument CBEXt, Thermo Scientific FirstDefendert (formerly known as Ahura, Inc.), and B&WTEK NanoRamt, see for example Driscoll, A.J., Harpster, M.H., Johnson, P.A. (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.

[0136] 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.

[0137] In an embodiment of the third aspect of the present invention, the sample holder can be a cuvette or a well plate that can hold a liquid sample.

[0138] In an embodiment of the third aspect of the present invention, the wavelength selector includes software-related selection of wavelengths and automated switching of the mirror towards the desired radiation source.

[0139] In an embodiment of the third aspect of the present invention, the detector includes a CCD camera detector that converts incident photons (such as Raman signals) into electrical signals, and the electrical signals give a wavelength-dependent intensity function.

[0140] In an embodiment of the third aspect of the present invention, the diagnostic system is a clinical diagnostic system.

[0141] In an embodiment of the fifth aspect of the present invention, the clinical diagnostic system includes a sample preparation station.

[0142] In an embodiment of the fifth aspect of the present invention, a clinical diagnostic system (such as a sample preparation station) includes a buffer unit for receiving a plurality of samples before initiating a new sample preparation start sequence, where the samples can be individually randomly accessible, and the individual preparation of the samples can be initiated according to the sample preparation start sequence.

[0143] The clinical diagnostic system makes the use of Raman spectroscopy more convenient and reliable, and is thus suitable for clinical diagnosis. In particular, in the case of random access sample preparation and LC separation, high throughput or more can be achieved while being able to be online coupled to Raman spectroscopy. In addition, the process can be fully automated, increasing the turn-around time and reducing the required skill level.

[0144] In a fourth aspect, the present invention relates to a kit adapted to perform the method according to the first aspect of the present invention, the kit comprising or consisting of the following:

[0145] (A) at least one target analyte, preferably a target deuterated analyte as an internal standard, and

[0146] (B) a quencher.

[0147] All embodiments mentioned for the first aspect and / or the second aspect and / or the third aspect of the present invention are applicable to the fourth aspect of the present invention, and vice versa.

[0148] In a fifth aspect, the present invention relates to the use of the kit according to the fourth aspect of the present invention in the method according to the first aspect of the present invention.

[0149] All embodiments mentioned for the first aspect and / or the second aspect and / or the third aspect and / or the fourth aspect of the present invention are applicable to the fifth aspect of the present invention, and vice versa.

[0150] In a further embodiment, the present invention relates to the following aspects:

[0151] 1. A method for determining at least one analyte of interest, the method comprising the steps of:

[0152] a) providing

[0153] - the at least one target analyte, which is capable of emitting scattered electromagnetic radiation when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1 and

[0154] - a fluorophore, which is capable of emitting fluorescent electromagnetic radiation when excited by the monochromatic electromagnetic radiation having the maximum excitation wavelength λ max1

[0155] ​b) Provide a quencher that can quench the fluorescent electromagnetic radiation of the fluorophore when excited by the monochromatic electromagnetic radiation having the maximum excitation wavelength λ max1 of the fluorophore,

[0156] c) Mix the at least one target analyte, the fluorophore, and the quencher to form a sample,

[0157] d) Perform Raman spectroscopy, and

[0158] e) Determine the at least one target analyte via Raman spectroscopy.

[0159] 2. The method according to aspect 1, wherein the sample is selected from the group consisting of: blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph fluid, cell cultures, tissue cultures, and solid samples such as dried blood spots or tissue extracts.

[0160] 3. The method according to any one of the foregoing aspects, wherein the sample is obtained from a patient sample, and the patient sample is selected from the group consisting of: serum, plasma, and whole blood samples from an individual.

[0161] 4. The method according to any one of the foregoing aspects, wherein the sample is a human sample, preferably a hemolyzed whole blood sample, particularly a hemolyzed human whole blood sample.

[0162] 5. The method according to any one of the foregoing aspects, wherein the sample is an aqueous-based sample.

[0163] 6. The method according to any one of the foregoing aspects, wherein the quencher comprises a hydrophilic group selected from the group consisting of: SO4 2- , PO4 3- , oligonucleotides, quaternary amines, PEG groups, alcohols, COOH.

[0164] 7. The method according to any one of the foregoing aspects, wherein the hydrophilic group is an oligonucleotide.

[0165] 8. The method according to any one of the foregoing aspects, wherein the quencher comprises a polycyclic aromatic - azo backbone.

[0166] 9. The method according to any one of the foregoing aspects, wherein the distance between the molecule of the quencher and the molecule of the fluorophore is less than 6 nm or 5 nm or 3 nm.

[0167] 10. The method according to any one of the foregoing aspects, wherein the maximum excitation wavelength of the quencher is λ max1 ±20 nm.

[0168] 11. The method according to any one of the foregoing aspects, wherein the quenching 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 values), or 550 nm to 650 nm (including the boundary values).

[0169] 12. The method according to any one of the foregoing aspects, wherein the quenching is Förster resonance energy transfer.

[0170] 13. The method according to any one of the foregoing aspects, wherein the quencher is a BHQ1 quencher or a BHQ2 quencher, preferably wherein the BHQ1 quencher is modified with an oligonucleotide, or wherein the BHQ2 quencher is modified with an oligonucleotide.

[0171] 14. The method according to any one of the foregoing aspects, wherein 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 characterized by a certain modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0172] 15. The method according to any one of the foregoing aspects, wherein step (d) is carried out in a liquid phase.

[0173] 16. The method according to any one of the foregoing aspects, wherein the scattered electromagnetic radiation is inelastic scattered electromagnetic radiation.

[0174] 17. The method according to any one of the foregoing aspects, wherein the scattered electromagnetic radiation is Stokes scattering and / or anti-Stokes scattering.

[0175] 18. The method according to any one of the foregoing aspects, wherein the maximum excitation wavelength λ of the monochromatic electromagnetic radiation max1 is less than 1064 nm.

[0176] 19. The method according to any one of the foregoing aspects, wherein the monochromatic electromagnetic radiation is generated by a krypton ion laser (530.9 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 and 514.5 nm), or a diode laser (630 and 780 nm).

[0177] 20. The method according to any one of the foregoing aspects, wherein the fluorophore is selected from the group consisting of chemical substances containing a number of combined aromatic groups, or planar molecules or cyclic molecules having a number of π bonds.

[0178] 21. The method according to any one of the foregoing aspects, wherein at a quencher concentration of 0, 51.7 or 62.5 mg / l, the concentration of the fluorophore varies between 2.5 mg / l up to 250 mg / l.

[0179] 22. The method according to any one of the foregoing aspects, wherein the sample is a solvent-based sample and the quencher is hydrophobic.

[0180] 23. Use of the method according to any one of aspects 1 to 22 for determining the at least one target analyte in a sample.

[0181] 24. The use according to aspect 23, wherein the presence or level of the at least one target analyte in the sample is determined.

[0182] 25. A diagnostic system for determining at least one target analyte in a sample, the diagnostic system comprising a spectrometer having

[0183] - a radiation source,

[0184] - a sample holder,

[0185] - a wavelength selector, and

[0186] - a detector for performing the method according to any one of aspects 1 to 22.

[0187] 26. A kit adapted to perform the method according to any one of the foregoing aspects 1 to 22, the kit comprising or consisting of:

[0188] (A) at least one target analyte, preferably a target deuterated analyte as an internal standard, and

[0189] (B) a quencher.

[0190] 27. Use of the kit according to aspect 26 in the method according to any one of the foregoing aspects 1 to 22.

[0191] Example

[0192] The following examples are provided to illustrate but not limit the invention claimed herein.

[0193] Figure 1 The Raman spectrum of acetonitrile as a target analyte is shown. Shown as a function of cm -1The intensity (count) of the Raman shift variation. The strong signal of acetonitrile is visible, but the background shows a broad signal that exceeds the intensity of the acetonitrile signal. The sample contains or consists of the following: 100 μl of a phenol red solution as a fluorophore, 50 μl of acetonitrile as the target analyte, and 150 μl of water and a quencher. The sample can be prepared by using a concentrated solution of phenol red, mixing it with acetonitrile as the target analyte, and combining it with the 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 of 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 the fluorophore in the final sample.

[0194] The qualitative and / or quantitative determination of acetonitrile as the target analyte can be carried out as follows:

[0195] a) Provide

[0196] - Acetonitrile, which is capable of emitting scattered electromagnetic radiation when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1 (e.g., 532 nm), and

[0197] - A phenol red solution fluorophore, which is capable of emitting fluorescent electromagnetic radiation when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1

[0198] b) Provide a quencher, such as hydrophilic BHQ1 or BHQ2, which is capable of quenching the fluorescent electromagnetic radiation of the fluorophore when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ λmax1

[0199] c) Mix the acetonitrile, the phenol red solution, and the quencher to form a sample,

[0200] d) Perform Raman spectroscopy, and

[0201] e) Determine acetonitrile via Raman spectroscopy.

[0202] The fluorophore produces fluorescence under 532 nm laser excitation. Raman spectroscopy can be carried out using a Raman spectrometer, such as a HORIBA LabRAM HR Evolution. Figure 1 The spectrum in

[0203] Figure 2 shows a quencher with an oligonucleotide as a hydrophilic group. The quencher can contain other or additional hydrophilic groups selected from the following: SO4 2- 、PO4 3-, oligonucleotides, quaternary amines, PEG groups, alcohols, COOH. The quencher has maximum excitation wavelengths λ of 534 nm (hydrophilic BHQ-1) and 573 nm (hydrophilic BHQ-2) respectively max1 . The quencher has quenching ranges of 400 to 500 nm (hydrophilic 5'-TTx-3' X = BHQ1) and 550 nm to 650 nm (hydrophilic 5'-TTx-3' X = BHQ2) respectively.

[0204] Figures 3A to 3C The Raman spectrum of acetonitrile as the target analyte is shown. The measurement is carried out via the high-throughput screening HORIBA LabRAM HR Evolution. Figures 3A to 3C The high-throughput screening in LaSpec6 software is shown, where 3A is the coverage of all spectra with different fluorophore concentrations, 3B is the single-spectrum view and 3C is the 96-well plate view for well selection to show the specific spectrum for each well.

[0205] Figure 4A and 4B The Raman spectrum of acetonitrile as the target analyte is shown at different fluorophore dilutions. The sample can be prepared as described for Figure 1 above. Figure 4A "1 / 1 dye, concentration 250 mg / l fluorophore" is shown, and Figure 4B "1 / 100 dye, concentration 2.5 mg / l fluorophore" is shown. Each figure shows the effect of dilution on the signal-to-noise ratio. For 4A, the spectrum shows fluorescence, and due to the high dilution factor, the spectrum in 4B shows almost no fluorescence. The dilution has been done by adding water to the fluorophore solution to reach the desired concentration.

[0206] Figure 5 The signal-to-noise ratio varying with fluorophore dilution is shown, where there is no quencher. Acetonitrile can be detected by the dilution of the fluorophore. 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 increased signal-to-noise ratio due to the dilution of the fluorophore gives a benchmark for identifying the effect of the quencher on fluorescence.

[0207] Figure 6 The signal-to-noise ratio varying with fluorophore dilution at different quencher concentrations (0 mg / l (blank), 41.7 mg / l, 62.5 mg / l) is shown. Compared with the blank sample, the quencher produces an increased signal-to-noise ratio at higher fluorophore concentrations.

[0208] This patent application claims the priority of European patent application 22209071.4, the content of which is incorporated herein by reference.

Claims

1. A method for determining at least one target analyte, the method comprising the following steps: a) Providing - the at least one target analyte, which is capable of emitting scattered electromagnetic radiation when excited by monochromatic electromagnetic radiation having a maximum excitation wavelength λ max1 and - A fluorophore that is capable of emitting fluorescent electromagnetic radiation when excited by the monochromatic electromagnetic radiation having the maximum excitation wavelength λ max1 thereof, b) Provide a quencher that is capable of quenching the fluorescent electromagnetic radiation of the fluorophore when excited by the monochromatic electromagnetic radiation having the maximum excitation wavelength λ max1 thereof, c) Mixing the at least one target analyte, the fluorophore, and the quencher to form a sample, d) Performing Raman spectroscopy, and e) Determining the at least one target analyte via Raman spectroscopy.

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 lymphatic fluid, cell cultures, tissue cultures, and solid samples such as dried blood spots or tissue extracts, preferably wherein the sample is obtained from a patient sample, the patient sample being selected from the group consisting of serum, plasma, and whole blood samples from an individual, and / or wherein the target analyte is selected from the group consisting of: nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, seco-steroids, molecules having the properties of a certain modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

3. The method according to any one of the preceding claims, wherein the sample is an aqueous-based sample.

4. The method according to any one of the preceding claims, wherein the quencher comprises a hydrophilic group selected from the following: SO4 2- , PO4 3- , oligonucleotide, quaternary amine, PEG group, alcohol, COOH.

5. The method according to any one of the preceding claims, wherein the hydrophilic group is an oligonucleotide and the quencher comprises a polycyclic aromatic-azo backbone.

6. The method according to any one of the preceding claims, wherein the maximum excitation wavelength of the quencher is λ max1 ±20 nm.

7. The method according to any one of the preceding claims, wherein the quenching 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 values), or 550 nm to 650 nm (including the boundary values).

8. The method according to any one of the preceding claims, wherein the quencher is a BHQ1 quencher or a BHQ2 quencher.

9. The method according to any one of the preceding claims, wherein step (d) is performed in the liquid phase.

10. The method according to any one of the preceding claims, wherein the concentration of the fluorophore varies between 2.5 mg / l up to 250 mg / l compared to a quencher concentration of 0, 51.7, or 62.5 mg / l.

11. The method according to any one of the preceding claims, 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 12 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, the diagnostic system comprising a spectrometer having - A radiation source, - A sample holder, - A wavelength selector, and - A detector for performing the method according to any one of claims 1 to 12.

14. A kit suitable for performing the method according to any one of the preceding claims 1 to 12, the kit comprising or consisting of: (A) At least one target analyte, preferably a target deuterated analyte as an internal standard, and (B) A quencher.

15. Use of the kit according to claim 14 in the method according to any one of the preceding claims 1 to 12.

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