Analyte detection by chip-based nanoESI detection system

Abstract

The present invention relates to a method, a diagnostic system, a kit and uses thereof for the efficient detection of analytes by a chip-based nanoESI detection system.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method, a diagnostic system, a kit and uses thereof for the efficient detection of analytes by a chip-based nanoESI detection system. [Background technology]

[0002] Background of the Invention Mass spectrometry is a widely used technique for the qualitative and quantitative analysis of chemicals ranging from small molecules to macromolecules. Generally, it is a very sensitive and specific method, even capable of analyzing complex biological samples, such as environmental or clinical samples. However, for some analytes, the sensitivity of the measurement remains an issue, especially when analyzed from complex biological matrices such as serum.

[0003] MS is often coupled with chromatographic techniques, particularly gas and liquid chromatography, such as HPLC. Here, the molecules of interest (analytes) are chromatographically separated and individually subjected to mass spectrometry. However, stand-alone mass spectrometers have significantly advanced the selectivity and sensitivity of direct detection methods by MS. Unlike traditional workflows categorized as sample preparation, chromatographic separation, and mass spectrometry detection, many sample preparation techniques can be directly coupled to stand-alone MS, offering superior performance.

[0004] To ensure reliable and sensitive mass spectrometric detection (avoiding matrix effects and interferences, and increasing sensitivity), target analytes must be chromatographically separated as closely as possible. Generally, this can be achieved using an isocratic or gradient system, such as a reversed-phase HPLC column and a gradient from aqueous to organic phase. Columns used for HPLC require flow rates of 0.1-1.0 ml / min. Under these optimal flow conditions, very narrow chromatographic peaks with very small peak volumes are produced.

[0005] Liquid extraction surface analysis (LESA) mass spectrometry is a direct surface sampling technique. Analytes are extracted from the surface via a liquid microjunction between the pipette tip and the sample surface. This approach allows for sampling of a wide variety of biological analytes, including drugs, lipids, and proteins, from various solid surfaces prior to electrospray ionization (ESI). Substrates analyzed with LESA include thin tissue sections, bacterial colonies grown on agar media, and dried blood spots on cards or polymer surfaces. Other direct analytical approaches that have been applied to the analysis of dried blood spots include desorption electrospray ionization (DESI), direct analysis in real time (DART), and paper spray.

[0006] Currently, several approaches combining bead-based solid supports with nanoESI-MS analysis have been described in the literature, most of which are based on microfluidic chips or devices.

[0007] It is well known that nanoESI mass spectrometry has been gaining importance over the past few years. However, combining direct surface sampling techniques with nanoESI-MS requires significant improvements in sample preparation techniques to achieve sensitive, adaptable, and rapid measurements from biological matrices. The application of solid support-based sample preparation techniques coupled with nanoESI-MS may overcome these hurdles.

[0008] However, current approaches are not applicable to efficient detection of analytes, e.g., high-throughput measurements, because the described microfluidic devices are difficult to mass-produce, leading to increased device complexity.

[0009] The coupling of solid support sample preparation with nanoESI mass spectrometry is limited by the complex design and fabrication of solid supports, including microfluidic devices, devices designed for specific applications lacking a broad analyte menu, the suitability of sample preparation affecting ionization, system requirements for fluidic or liquid chromatography components, the low throughput of most examples in the literature, and / or sample carryover affecting the measurement.

[0010] However, there remains a need to improve the efficiency of MS analytical methods, especially for methods that allow efficient detection of analytes from complex biological matrices. This is particularly important in random-access, high-throughput MS setups, where several different analytes exhibiting different chemical properties must be measured in a short period of time.

[0011] The present invention relates to a method for determining the presence or level of an analyte in a sample by a chip-based nanoESI detection system, which can efficiently detect at least one analyte in a biological sample, such as steroids, proteins, and other types of analytes.

[0012] It is an object of the present invention to provide a method, a diagnostic system, a kit and uses thereof for the efficient detection of an analyte by a chip-based nanoESI detection system.

[0013] 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

[0014] Summary of the Invention In the following, the present invention relates to the following aspects: In a first aspect, the present invention provides a method for determining the presence or level of an analyte in a sample by a chip-based nanoESI detection system, the chip-based nanoESI detection system comprising a conductive pipette tip and a nanoelectrospray nozzle, the method comprising the steps of: a) providing a sample comprising an analyte and a matrix, wherein the matrix is ​​non-magnetic; b) providing a microparticle, wherein the microparticle is magnetic; c) incubating the analyte with the microparticles to form an analyte-microparticle complex in the sample holder, wherein the analyte-microparticle complex is magnetic; d) separating the matrix and the analyte-microparticle complex by magnetic force; e) optionally washing the analyte-microparticle complexes in the sample holder; f) extracting the analyte from the analyte-microparticle complex by an extraction solvent and magnetic force, step (f) comprising: f1) supplying the extraction solvent by a conductive pipette tip; f2) contacting the extraction solvent with the analyte-microparticle complex in the sample holder; f3) extracting the analyte from the analyte-microparticle complex to form an extracted analyte, wherein the microparticles are held in the sample holder by magnetic force during the extraction step f3) and the conductive pipette tip contains the extracted analyte; g) directly contacting the extracted analyte with a conductive pipette tip comprising a nanoelectrospray nozzle of a chip-based nanoESI detection system to form a nanoelectrospray for ionizing the extracted analyte; direct contacting can mean directly contacting the respective object or surface, or contacting the respective object or surface with a liquid phase (e.g., the extracted analyte in an extraction solvent). h) determining the presence or level of an extracted analyte in a sample using a chip-based nanoESI detection system, wherein the chip-based nanoESI detection system uses mass spectrometry, ion mobility, and / or a combination thereof.

[0015] In a second aspect, the present invention relates to the use of the method of the first aspect for determining the presence or level of an analyte in a sample.

[0016] In a third aspect, the present invention relates to a diagnostic system for determining the presence or level of an analyte in a sample, comprising a chip-based nanoESI source, a conductive pipette tip, and a detector for carrying out the method according to the first aspect, wherein the chip-based nanoESI source comprises a nozzle and the detector uses mass spectrometry or ion mobility or a combination thereof.

[0017] In a fourth aspect, the present invention relates to the use of the diagnostic system of the third aspect in the method of the first aspect.

[0018] In a fifth aspect, the present invention provides a kit suitable for carrying out the method of the first aspect, comprising: (A) a microparticle for concentrating or purifying an analyte in a sample; (B) an extraction solvent for extracting the analyte from the microparticles; (C) optionally, an internal standard; and (D) Kits that optionally include a catalyst or other reagent, such as a derivatization reagent.

[0019] In a sixth aspect, the present invention relates to the use of the kit of the fifth aspect of the invention in the method of the first aspect of the invention. [Brief explanation of the drawings]

[0020] [Figure 1]1 illustrates a method for determining the presence or level of an analyte in a sample by a chip-based nanoESI detection system according to the present invention. [Figure 2] 1 shows a front view of a diagnostic system for carrying out the method according to the invention; [Figure 3] 1 shows a side view of a diagnostic system for carrying out the method according to the invention; [Figure 4] 1 shows the concentration of analytes with superparamagnetic beads as microparticles for subsequent extraction from a smooth surface and a chip-based nanoESI detection system. [Figure 5] 1 shows the concentration of analytes with superparamagnetic beads as microparticles for subsequent extraction from well plates and a chip-based nanoESI detection system. [Figure 6] Shown is a calibration set of testosterone-13C3 [M+H]+ detected after microparticle concentration, extraction, and ionization starting from analyte-spiked horse serum in the presence of internal standard (ISTD) aldosterone-13C3. [Figure 7] Shown is a calibration set of testosterone-13C3 [M+H]+ detected after microparticle concentration, extraction, and ionization starting from analyte-spiked horse serum in the presence of internal standard (ISTD) aldosterone-13C3. [Figure 8] 1 shows a calibration set of phenytoin-13C1-15N2[MH]- detected after microparticle concentration, extraction, and ionization starting from analyte-spiked horse serum in the presence of internal standard (ISTD) aldosterone-13C3. [Figure 9] In the range below 500 pg / mL, extracts from Figure 8 are shown with corresponding magnification. [Figure 10] Ion mobility separation of different analytes in a mixture is shown, applying microparticle-based sample enrichment with extraction and ionization in positive ion mode. [Figure 11]Shown is an overlay of five extracted ion mobilograms (drift times dt between 0 and 14 ms) detected after microparticle enrichment, extraction, and ionization from a single analyte mixture. [Figure 12] Microparticle-based sample enrichment / purification was applied to extraction and ionization in positive ion mode, and a comparison of the detection of testosterone-13C3 considering neat solution and horse serum matrix is ​​presented. [Figure 13] Ion mobility separation of different analytes in a mixture is demonstrated by applying microparticle-based sample enrichment with extraction and ionization in negative ion mode. [Figure 14] Ion mobility separation of different analytes in a mixture is demonstrated by applying microparticle-based sample enrichment with extraction and ionization in negative ion mode. [Figure 15] Microparticle-based sample enrichment / purification is applied to extraction and ionization in negative ion mode, and a comparison of the detection of estradiol-13C3 considering neat solution and horse serum matrix is ​​presented. [Figure 16] The application of microparticles, e.g., magnetic immunobeads, for the detection of estradiol-13C3 is demonstrated, applying microparticle sample enrichment and extraction and ionization in negative ion mode. [Figure 17] We demonstrate the application of microparticles, e.g., magnetic immunobeads, for the detection of testosterone-13C3, applying microparticle-based sample enrichment with extraction and ionization in positive ion mode. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description of the Invention 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.

[0022] Throughout the text of this specification, several documents are cited. 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.

[0023] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that these elements can 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 to be disclosed by the description of this application unless the context indicates otherwise.

[0024] definition It will be understood that the word "comprise," and variations such as "comprises" and "comprising," 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. The terms "including" and "comprising" may be used interchangeably.

[0025] 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.

[0026] 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 ​​expressly 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 expressly 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.

[0027] 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.

[0028] The term "Mass Spectrometry" ("Mass Spec" or "MS"), or "mass spectrometric determination," or "mass spectrometric analysis," refers to an analytical technique used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally involve (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating their mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometry instrument, where a combination of magnetic and electric fields causes the ions to follow a path in space that depends on their mass ("m") and charge ("z"). The term "ionization" or "ionization" refers to the process of producing analyte ions having a net charge equal to one or more units. Negative ions are those having a net negative charge of one or more units, and positive ions are those having a net positive charge of one or more units. MS methods can be performed in either "negative ion mode," in which negative ions are generated and detected, or "positive ion mode," in which positive ions are generated and detected.

[0029] "Tandem mass spectrometry" or "MS / MS" involves multiple steps of selective mass analysis, with analyte fragmentation occurring between stages. In a tandem mass spectrometer, ions are generated in an ion source and separated by mass-to-charge ratio in the first stage of mass analysis (MS1). Ions of specific mass-to-charge ratios (precursor ions or parent ions) are selected, and fragment ions (daughter ions) are generated by collision-induced dissociation, ion-molecule reactions, or photodissociation. The resulting ions are then separated and detected in the second stage of mass analysis (MS2).

[0030] Mass spectrometer separates and detects ions with slightly different masses, so it can easily distinguish different isotopes of a given element.Therefore, mass spectrometry is an important method for accurate mass determination and characterization of analytes, including but not limited to low molecular weight analytes, peptides, polypeptides or proteins.Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, and the total measurement of proteins in proteomics.The de novo sequencing of peptides or proteins by mass spectrometry can usually be carried out without prior knowledge of amino acid sequence.

[0031] The term "electrospray ionization" or "ESI" refers to a method in which a solution is passed down a short length of capillary tube and a high positive or negative potential is applied to the end of the capillary tube. Upon reaching the end of the tube, the solution is vaporized (atomized) into a jet or spray of very small droplets in solvent vapor. This mist of droplets passes through an evaporation chamber, which is slightly heated to prevent condensation and evaporate the solvent. As the droplets become smaller, the electrical surface charge density increases until natural repulsion between like charges releases ions and neutral molecules.

[0032] The term "nanoelectrospray ionization" or "nanoESI" can refer to classical 10 or 20 nL / m electrospray ionization. It can be a method that typically uses flow rates of less than 1 μL / min in static or dynamic modes. Preferably, nanoESI uses flow rates of 10 or 20 nL / min to 500 nL / min, e.g., 500 nL / min. 500 nL / min is equivalent to 0.5 μL / min.

[0033] The term "static nanoESI mass spectrometry" is used in the context of this disclosure as an option for discontinuous flow nanoESI. Analysis is typically defined by loading a sample into an emitter and forming a nanoelectrospray while a voltage is applied with a constant gas backpressure. In contrast, dynamic nanoESI mass spectrometry is characterized by a mobile phase pumped at a low flow rate through a small diameter emitter while a voltage is applied.

[0034] 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 mass spectrometry, particularly nanoESI mass spectrometry. Chemical species, i.e., analytes, suitable for analysis by mass spectrometry 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 may serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance in a living system that is used as an indicator of the biological state of that system.

[0035] The analyte may be present in a sample of interest, for example, a biological sample or a clinical sample. The terms "sample" or "subject sample" are used interchangeably herein and refer to a portion or part of a tissue, organ, or individual, typically smaller than the tissue, organ, or individual intended to represent the entire tissue, organ, or individual. Upon analysis, the sample provides information about the state of the tissue, or the health or disease state of the organ or individual. Examples of 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 samples such as dried blood spots and tissue extracts. Another example of a sample is a cell culture or tissue culture.

[0036] 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).

[0037] As used herein, the term "serum" refers to the clear liquid portion of blood that can be separated from clotted blood. As used herein, the term "plasma" refers to the clear liquid portion of blood that contains blood cells. Serum differs from plasma, which contains the liquid portion of normal, unclotted blood, which contains red and white blood cells and platelets. It is the blood clot that differentiates serum from plasma. As used herein, the term "whole blood" refers to all components of blood, such as red and white blood cells, platelets, and plasma.

[0038] In this context, "level" or "level value" encompasses absolute amount, relative amount or concentration, as well as any value or parameter that correlates thereto or can be derived therefrom.

[0039] As used herein, the term "determining" the level of an analyte refers to quantifying the analyte, e.g., determining or measuring the level of the analyte in a pre-treated sample. The level of the analyte is determined by nanoESI mass spectrometry.

[0040] 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.

[0041] Typically, an "internal standard" (ISTD) is a known quantity of a substance that exhibits similar properties to the analyte when subjected to a mass spectrometric detection workflow (i.e., including any pretreatment, enrichment, and actual detection steps). The ISTD exhibits similar properties to the analyte but is clearly distinguishable from it. For example, during ion mobility separation, the ISTD has approximately the same drift time and ion mobility as the analyte from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. However, the ISTD exhibits a different molecular weight than the analyte from the sample. This allows ions from the ISTD and the analyte to be distinguished in mass spectrometry using their different mass-to-charge (m / z) ratios. Both are then 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 an analyte from a 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(s) present in the sample when the analyte(s) reach the mass spectrometer. Typically, although not necessarily, the ISTD is an isotopically labeled variant of the analyte (e.g., 2 H, 13 C, or 15 (including labels such as N).

[0042] The term "in vitro method" is used to indicate that the method is performed outside an organism, preferably on a body fluid, isolated tissue, organ or cell.

[0043] As used herein, the terms "automatically" or "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 any special or customized meaning. The terms may specifically refer to, but are not limited to, a process that is performed entirely by at least one computer and / or computer network and / or machine, especially without the need for human action and / or user interaction.

[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 present invention. The kit is preferably advertised, distributed, or sold as a unit for practicing the methods of the present 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, diluents, filters, needles, syringes, and package inserts 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] As used herein, the term "microparticle" is a broad term and should be given its common and ordinary meaning to those skilled in the art, without being limited to any specific or special meaning. This term may specifically, but is not limited to, refer to any particulate material of microscopic size. Microparticles may have an average diameter ranging from 100 nm to 100 μm, specifically from 500 to 200 nm to 50 μm. Microparticles are sometimes referred to as beads. Microparticles may be spherical or globular in shape. However, slight variations from the spherical or globular shape may be possible. In particular, microparticles have at least one surface to which an analyte can be attached, for example, by covalent or van der Waals forces. As used herein, the term "surface" is a broad term and should be given its common and ordinary meaning to those skilled in the art, without being limited to any specific or special meaning. This term may specifically, but is not limited to, refer to the entire area that separates any object from the outside. Thus, a body may have multiple surfaces. Specifically, a microparticle may have a core surrounded by a surface. The surface and the core may comprise different materials. Furthermore, the surface and the core may have different properties. For example, the core may be magnetic. The surface may be configured to capture molecules, for example, non-polar molecules from a wide range of polar molecules, when the microparticles are incubated with a sample containing such molecules. The terms microparticle and bead may be used interchangeably.

[0046] In particular, the microparticles may be selected from the group consisting of magnetic microparticles, specifically magnetic microparticles having a magnetic core and a modified surface; silica microparticles, specifically silica microparticles having a silica core and a modified surface; melamine resin microparticles, specifically melamine resin microparticles having a melamine resin core and a modified surface; poly(styrene)-based microparticles, specifically poly(styrene)-based microparticles having a poly(styrene) core and a modified surface; and poly(methyl methacrylate) microparticles, specifically poly(methyl methacrylate) microparticles having a poly(methyl methacrylate) core and a modified surface. However, other particles may also be feasible. The melamine resin microparticles may have an average diameter of 500 nm to 20 μm, preferably 2 μm to 4 μm, and most preferably 3 μm. The poly(styrene)-based microparticles may have an average diameter of 500 nm to 50 μm, preferably 2 μm to 4 μm, and most preferably 3 μm. The poly(methyl methacrylate) microparticles may have an average diameter of 500 nm to 50 μm, preferably 2 μm to 4 μm, and most preferably 3 μm. The modified surface of the magnetic microparticles may be a modified poly(styrene) surface, and the magnetic microparticles may have an average diameter of 5 μm to 50 μm, preferably 10 μm to 30 μm, and most preferably 20 μm. The modified surface of the magnetic microparticles may be a silica surface, and the magnetic microparticles may have an average diameter of 100 nm to 1000 nm, preferably 200 nm to 500 nm, and most preferably 300 nm. The modified surface of the silica microparticles may be a cyanopropylsilane-functionalized surface, and the silica microparticles may have an average diameter of 5 μm to 100 μm, preferably 20 μm to 80 μm, and most preferably 40 μm. Other dimensions may also be feasible.

[0047] Therefore, the term "chip-based nanoESI detection system" as used herein 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 specific or particular meaning.

[0048] In particular, the chip-based nanoESI detection system includes a conductive pipette tip and a nanoelectrospray nozzle.

[0049] The term "conductive pipette tip" as used herein 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 material of the conductive pipette tip may at least partially comprise a conductive material selected from the group consisting of graphene, carbon nanotubes, carbon black, carbon fiber, stainless steel, aluminum, titanium, chromium, conductive metals, and alloys thereof. The conductive pipette tip may be a disposable conductive pipette tip.

[0050] As used herein, the term "nanoelectrospray nozzle" is a broad term and should be given its common and ordinary meaning to those skilled in the art, and should not be limited to a specific or special meaning. The nanoelectrospray nozzle can be a single-use nozzle and / or a multi-use nozzle having an inner diameter of less than 1 mm. The nanoelectrospray nozzle can be disposed on a consumable tip containing a fixed amount of nanoelectrospray nozzle. The nanoelectrospray nozzle can be a disposable nanoelectrospray nozzle.

[0051] As used herein, the term "incubation" is a broad term and should be given its ordinary and customary meaning to those skilled in the art, without being limited to any specific or special meaning. This term may specifically refer, but is not limited to, the mixing of at least two substances and / or the addition of at least one substance to another. Specifically, a solid or particulate substance may be added to and / or mixed with a liquid sample. Apart from the process of addition and / or mixing, incubation may further include a period called the incubation time. During the incubation time, one of the two substances may be adsorbed onto the surface of the other of the two substances. During the incubation time, additional conditions, such as temperature and / or other conditions, may be selected to promote the desired adsorption. Thus, in step b), microparticles may be added to the sample and, optionally, mixed with the sample. In step b), the sample may be incubated with the microparticles for an incubation time of 1 second to 60 minutes, preferably 1 minute to 30 minutes, and most preferably 3 minutes to 12 minutes. However, other durations may also be feasible.

[0052] As used herein, the term "analyte-particle complex" 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 may specifically, but not exclusively, refer to an assembly comprising at least one microparticle and at least one analyte, specifically one microparticle and multiple analytes. The microparticles and the analyte, specifically the analyte, that form the complex may reversibly associate. Thus, components of the complex may either remain or dissociate from the complex, at least under certain conditions. The analyte-microparticle complex may be formed based on at least one attractive force between the microparticle and the analyte. In particular, the attractive force may act between the surface of the microparticle and the analyte. Thus, the analyte, which may initially be distributed in the sample, specifically in the liquid phase of the sample, may accumulate during adsorption on the surface of the microparticle. The attractive force may include van der Waals forces and electrostatic forces. Other attractive forces are also possible. Specifically, at least one chemical bond may be formed between the microparticle and the analyte, specifically between the surface of the microparticle and the analyte, as part of the formation of the analyte-microparticle complex. The analyte-microparticle complex is sometimes referred to as an analyte-loaded microparticle.

[0053] In step c), the analyte can be incubated with the microparticles, which can cause the analyte to be adsorbed onto the surface of the microparticles and form analyte-microparticle complexes. In this context, the expression can be understood to mean the formation of multiple analyte-microparticle complexes. This means that in step c), the sample can be incubated with the microparticles, which can cause the analyte to be adsorbed onto the surface of the microparticles and form analyte-microparticle complexes.

[0054] The term "sample holder" as used herein 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 intended use of a sample holder is to present a sample for further analysis. Sample holder options include well plates, glass plates, plain or structured surfaces, etc.

[0055] As used herein, the term "magnetic force" is a broad term and should be given its common and ordinary meaning to those skilled in the art and should not be limited to any particular or special meaning. Magnetic force can result from the application of a magnetic field provided by a permanent magnet or an electromagnet.

[0056] As used herein, the term "extract" is a broad term and should be given its ordinary and accustomed meaning to those skilled in the art and should not be limited to any specific or particular meaning. Extraction is a separation process that consists of the separation of a substance, e.g., an analyte, from a matrix.

[0057] As used herein, the term "contacting" is a broad term and should be given its ordinary and accustomed meaning to those of ordinary skill in the art and should not be limited to a specific or particular meaning. Generally, this can be described by coming together or touching, such as with objects or surfaces.

[0058] As used herein, the term "direct contacting" is a broad term and should be given its ordinary and accustomed meaning to those skilled in the art, and should not be limited to any specific or particular meaning. Direct contacting can mean either directly contacting the respective object or surface, or contacting the respective object or surface with a liquid phase (e.g., the analyte to be extracted in an extraction solvent).

[0059] As used herein, the term "random-access" is a broad term and should be given its common and ordinary meaning to those skilled in the art, and should not be limited to any specific or particular meaning. In general, a process can refer to the ability to perform analysis or transfer information randomly and directly, rather than being accessed in a fixed order.

[0060] 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 are obtained 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, but may also be a pre-analytical or post-analytical function, or a function supporting either the pre-analytical, analytical, or post-analytical function. In particular, a module may be configured to cooperate with one or more other modules to perform a dedicated task of 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 may be composed of one or more analytical devices designed to perform respective workflows optimized for a specific type of analysis, such as clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separations, mass spectrometry, etc. Thus, a clinical diagnostic system may consist of one analytical device or any combination of such analytical devices for 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. A clinical diagnostic system may comprise functional units such as liquid handling units for pipetting, pumping, and / or mixing of samples and / or reagents and / or system fluids, and may further comprise functional units for sorting, storage, transport, identification, separation, and detection.

[0061] The clinical diagnostic system may include a sample preparation station for automatically preparing a sample containing an analyte, and optionally a separation station. In particular, the clinical diagnostic system does not include a separation station, such as an LC-HPLC unit or an HPLC unit.

[0062] The clinical diagnostic system can 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. The clinical diagnostic system can further include a mass spectrometer (MS).

[0063] 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 analyte 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, sieving, drying, washing, resuspension, aliquoting, transport, storage, etc.

[0064] The clinical diagnostic system, e.g., a sample preparation station, may also include a buffer unit for receiving multiple samples before a new sample preparation initiation sequence is initiated, and the samples may be individually randomly accessible, and their individual preparation may be initiated according to the sample preparation initiation sequence.

[0065] Embodiment In a first aspect, the present invention provides a method for determining the presence or level of an analyte in a sample by a chip-based nanoESI detection system, the chip-based nanoESI detection system comprising a conductive pipette tip and a nanoelectrospray nozzle, the method comprising the steps of: a) providing a sample comprising an analyte and a matrix, wherein the matrix is ​​non-magnetic; b) providing a microparticle, wherein the microparticle is magnetic; c) incubating the analyte with the microparticles to form an analyte-microparticle complex in the sample holder, wherein the analyte-microparticle complex is magnetic; d) separating the matrix and the analyte-microparticle complex by magnetic force; e) optionally washing the analyte-microparticle complexes in the sample holder; f) extracting the analyte from the analyte-microparticle complex by an extraction solvent and magnetic force, step (f) comprising: f1) supplying the extraction solvent by a conductive pipette tip; f2) contacting the extraction solvent with the analyte-microparticle complex in the sample holder; f3) extracting the analyte from the analyte-microparticle complex to form an extracted analyte, wherein the microparticles are held in the sample holder by magnetic force during the extraction step f3) and the conductive pipette tip contains the extracted analyte; g) directly contacting the extracted analyte with a conductive pipette tip comprising a nanoelectrospray nozzle of a chip-based nanoESI detection system to form a nanoelectrospray ESI spray for ionizing the extracted analyte; h) determining the presence or level of an extracted analyte in a sample using a chip-based nanoESI detection system, wherein the chip-based nanoESI detection system uses mass spectrometry, ion mobility, and / or a combination thereof.

[0066] In this way, for example, the performance and throughput of standalone MS can be significantly improved when combined with efficient sample processing strategies. Miniaturization and integration into the final analysis, as well as full automation of the entire analytical process, can increase throughput and reduce the complexity and separate sample preparation. The capabilities of direct MS can be further enhanced by highly selective gas-phase separation techniques such as high-resolution MS and ion mobility MS.

[0067] The method according to the invention can exhibit the following advantages: Reduced complexity and robustness a. Extremely low sample / eluate / extract consumption b. Highly efficient extraction c. Significantly reduced sample injection volume, and therefore less background / matrix in MS d. Disposable tips and spray nozzles e. Modular assembly Simplified Workflow f. Sample preparation with a particulate workflow directly correlates with ionization for MS g. The particulate workflow can be easily adapted to specific analytes without changing the ionization h. No chromatography column i. No HPLC gradient / eluent system j. Isobaric separation using ion mobility or immunofunctionalized microparticles k. Scalable for both high and low throughput performance l.Improved sensitivity with nanoESI m. Analyte concentration in the microparticle extraction step nImproved S / N level o. Spray and multiple MS experiments

[0068] In an embodiment of the first aspect of the present invention, the conductive pipette tip containing the extracted analyte is free of particulates.

[0069] In an embodiment of the first aspect of the present invention, the material of the conductive pipette tip at least partially comprises a conductive material selected from the group consisting of graphene, carbon nanotubes, carbon black, carbon fiber, stainless steel, aluminum, titanium, chromium, conductive metals, and alloys thereof. Preferably, the conductive material is selected from the group consisting of graphene, carbon nanotubes, carbon black, carbon fiber, and combinations thereof.

[0070] In an embodiment of the first aspect of the present invention, the conductive pipette tip comprises a particulate content relative to the total particulate content that is less than 20%, 15%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1% or 0.01%.

[0071] In an embodiment of the first aspect of the present invention, the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is a direct mechanical contact.

[0072] In an embodiment of the first aspect of the present invention, the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is a direct electrical contact, which may mean that a kind of bridge may be formed between the analyte and the nozzle.

[0073] In an embodiment of the first aspect of the present invention, the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is direct contact between the extracted analyte and the nozzle of the chip-based nanoESI detection system.

[0074] In an embodiment of the first aspect of the present invention, the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is direct contact between the extracted analyte and the nozzle and conductive pipette tip of the chip-based nanoESI detection system.

[0075] In an embodiment of the first aspect of the present invention, the ratio of microparticles to extraction solvent in step f) is in the range of 0.1:1 to 50:1, preferably 0.5:1 to 25:1, more preferably 1:1 to 10:1, where the ratio means % by mass (w / w).

[0076] In an embodiment of the first aspect of the present invention, the microparticles are superparamagnetic or paramagnetic.

[0077] In an embodiment of the first aspect of the present invention, the magnetic force is induced by a permanent magnet or an electromagnet.

[0078] In an embodiment of the first aspect of the invention, the sample is a biological sample and is derived from an individual, preferably a human.

[0079] In an embodiment of the first aspect of the present invention, the analyte molecule is present in a biological or clinical sample selected from the group consisting of blood, serum, plasma, urine, saliva, spinal fluid, and dried blood spots.

[0080] In an embodiment of the first aspect of the present invention, the sample is a hemolyzed whole blood sample, in particular a hemolyzed human whole blood sample. Hemolysis can be induced using a hemolysis reagent.

[0081] In an embodiment of the first aspect of the present invention, the matrix comprises analyte-interfering components derived from a biological sample, a microparticle, a sample preparation solution, a mixture, or a combination thereof.

[0082] In an embodiment of the first aspect of the present invention, the matrix is ​​a solution.

[0083] In an embodiment of the first aspect of the present invention, the matrix is ​​an internal standard.

[0084] In an embodiment of the first aspect of the present invention, step e) comprises: e1) Addition of cleaning solution, and e2) Removal of the washing supernatant after magnetic separation.

[0085] In an embodiment of the first aspect of the invention, before step f), preferably after step e), the method comprises: i) drying the analyte-microparticle complex, and / or ii) washing the analyte-microparticle complex.

[0086] In an embodiment of the first aspect of the invention, the method is automated.

[0087] In an embodiment of the first aspect of the present invention, the method is performed in a random access mode.

[0088] In an embodiment of the first aspect of the invention, the method is an in vitro diagnostic method.

[0089] In an embodiment of the first aspect of the invention, the method is carried out continuously.

[0090] In an embodiment of the first aspect of the present invention, the 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 specific modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0091] In an embodiment of the first aspect of the present invention, the 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-alpha-hydroxyestrone, 2-hydroxyestrone-3-methyl ether, prednisone, prednisolone, pregnenolone, progesterone, dehydroepiandrosterone (DHEA), 17-hydroxypregnenolone, 17-hydroxyprogesterone, androsterone, epiandrosterone, Δ4-androstenedione, 11-deoxycortisol, corticosterone, 21-deoxycortisol, 11-deoxycorticosterone, allopregnanolone, and aldosterone.

[0092] In an embodiment of the first aspect of the invention, the analyte is selected from the group consisting of 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 invention, the analyte molecule containing one or more carboxyl groups is an amino acid 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.

[0093] In an embodiment of the first aspect of the present invention, the analyte is selected from the group consisting of pyridoxal, N-acetyl-D-glucosamine, alcaftadine, streptomycin, and josamycin.

[0094] In an embodiment of the first aspect of the present invention, the analyte is selected from the group consisting of cocaine, heroin, Ritalin, aceclofenac, acetylcholine, amcinonide, amiloxate, amylocaine, anileridine, aranidipine, artesunate, and pethidine.

[0095] In an embodiment of the first aspect of the present invention, the analyte is selected from the group consisting of cantharidin, succinic anhydride, trimellitic anhydride, and maleic anhydride.

[0096] In embodiments of the first aspect of the invention, the analyte is selected from the group consisting of cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol, and tacalcitol. In particular, the secosteroid is vitamin D, particularly vitamin D2 or D3, or a derivative thereof. In certain embodiments, the secosteroid is selected from the group consisting of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 (calcifediol), 3-epi-25-hydroxyvitamin D2, 3-epi-25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 (calcitriol), 24,25-dihydroxyvitamin D2, and 24,25-dihydroxyvitamin D3. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more diene groups is selected from the group consisting of vitamin A, tretinoin, isotretinoin, alitretinoin, natamycin, sirolimus, amphotericin B, nystatin, everolimus, temsirolimus, and fidaxomicin.

[0097] In an embodiment of the first aspect of the present invention, the analyte is selected from the group consisting of benzyl alcohol, menthol, L-carnitine, pyridoxine, metronidazole, isosorbide mononitrate, guaifenesin, clavulanic acid, miglitol, zalcitabine, isoprenaline, acyclovir, methocarbamol, tramadol, venlafaxine, atropine, clofedanol, alpha-hydroxyalprazolam, alpha-hydroxytriazolam, lorazepam, oxazepam, temazepam, ethyl glucuronide, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-desmethyltramadol, desmetramadol, dihydroquinidine, and quinidine. 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, azacytidine, ribavirin, floxuridine, gemcitabine, streptozotocin, adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenoson, lincomycin, clindamycin, canaglyph Selected from the group consisting of rhodin, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, and plicamycin.

[0098] In an embodiment of the first aspect of the present invention, the analyte is selected from the group consisting of thiomandelic acid, DL-captopril, DL-thiorphan, N-acetylcysteine, D-penicillamine, glutathione, L-cysteine, zofenoprilat, tiopronin, dimercaprol, and succimer.

[0099] In an embodiment of the first aspect of the present invention, the analyte is selected from the group consisting of glutathione disulfide, dipyrithione, selenium sulfide, disulfiram, lipoic acid, L-cystine, fursultiamine, octreotide, desmopressin, vapreotide, terlipressin, linaclotide, and peginesatide. The selenium sulfide can be selenium disulfide, SeS2, or selenium hexasulfide, Se2S6.

[0100] In an embodiment of the first aspect of the present invention, the analyte is selected from the group consisting of carbamazepine-10,11-epoxide, carfilzomib, furosemide epoxide, fosfomycin, sevelamer hydrochloride, cerulenin, scopolamine, tiotropium, tiotropium bromide, methylscopolamine bromide, eplerenone, mupirocin, natamycin, and troleandomycin.

[0101] In an embodiment of the first aspect of the present invention, the analyte is selected from the group consisting of estrogen, estrogenic compounds, estrone (E1), estradiol (E2), 17a-estradiol, 17b-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, 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-ketoE2), 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-OHE1), 2-hydroxyestradiol. 17α-dihydroequilenin (ENa), 17β-dihydroequilenin (ENb), Δ8,9-dihydroestrone (dEl), Δ8,9-dihydroestrone sulfate (dEls), Δ9-tetrahydrocannabinol, and mycophenolic acid, wherein β or b may be used interchangeably, and α and a may be used interchangeably.

[0102] In an embodiment of the first aspect of the invention, the analyte is 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, butyrone, etcathinone, elefehedron, 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-destramadol, desmetamadol, tramadol, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin, sisomicin, and 5-methylcytosine.

[0103] In an embodiment of the first aspect of the invention, the analyte is selected from the group consisting of ribose, desoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, N-acetylneuraminic acid, etc. In an embodiment, the analyte molecule is an oligosaccharide, in particular selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and polysaccharides. In an embodiment of the first aspect of the invention, the disaccharide is selected from the group consisting of sucrose, maltose, and lactose. In an embodiment of the first aspect of the invention, the analyte molecule is a substance comprising the above-mentioned monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide moiety.

[0104] In an embodiment of the first aspect of the present invention, the analyte is zidovudine or azidocillin.

[0105] In an embodiment of the first aspect of the present invention, the method does not include a chromatography step, but includes at least one method selected from the following group: chromatography, high performance liquid chromatography (HPLC), liquid chromatography-high performance liquid chromatography (LC-HPLC), gas chromatography (GC), gel permeation chromatography (GPC), flash chromatography, etc. The chromatography is, for example, size exclusion chromatography.

[0106] In an embodiment of the first aspect of the invention, the method is carried out in the order a, then b, then c, then d, then optionally e, then f, then g, then h.

[0107] In a second aspect, the present invention relates to the use of the method of the first aspect for determining the presence or level of an analyte in a sample. All embodiments mentioned for the first aspect of the invention apply to the second aspect of the invention and vice versa.

[0108] In a third aspect, the present invention relates to a diagnostic system for determining the presence or level of an analyte in a sample, comprising a chip-based nanoESI source for carrying out the method according to the first aspect, a conductive pipette tip, and a detector, wherein the chip-based nanoESI source comprises a conductive pipette tip and a nozzle, and the detector uses mass spectrometry or ion mobility or a combination thereof. 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.

[0109] In an embodiment of the third aspect of the present invention, the diagnostic system is a stand-alone system.

[0110] In an embodiment of the third aspect of the invention, the system is integrated into other systems that are capable of determining the presence or level of an analyte based on (electro)chemiluminescence or clinical chemistry.

[0111] In an embodiment of the third aspect of the present invention, the diagnostic system is a clinical diagnostic system.

[0112] In an embodiment of the third aspect of the present invention, the nanoESI source may be, for example, Advion's chip-based electrospray ionization technology, which combines the advantages of liquid chromatography, mass spectrometry, chip-based injection, fraction collection, and direct surface analysis into one integrated ion source platform. Other known nanoESI sources are also possible. NanoESI sources are known to those skilled in the art and therefore will not be described in detail.

[0113] In an embodiment of the third aspect of the present invention, the mass spectrometer may be, for example, a triple quadrupole mass spectrometer or a linear ion trap mass spectrometer. Mass spectrometers are known to those skilled in the art and therefore will not be described in detail.

[0114] In a fourth aspect, the present invention relates to the use of the diagnostic system of the third aspect in the method of the first aspect. 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 apply to the fourth aspect of the invention and vice versa.

[0115] In a fifth aspect, the present invention provides a kit suitable for carrying out the method of any one of the preceding aspects, comprising: (A) a microparticle for concentrating or purifying an analyte in a sample; (B) an extraction solvent for extracting the analyte from the microparticles; (C) optionally an internal standard, and (D) Kits, optionally including a catalyst or other reagent, such as a derivatization reagent. 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.

[0116] In a sixth aspect, the present invention relates to the use of the kit of the fifth aspect in the method of the first aspect. 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 and / or the fourth aspect of the invention and / or the fifth aspect of the invention apply to the sixth aspect of the invention and vice versa.

[0117] In summary, without excluding further embodiments, the following embodiments can be envisaged:

[0118] Embodiment 1. A method for determining the presence or level of an analyte in a sample by a chip-based nanoESI detection system, wherein the chip-based nanoESI detection system includes a conductive pipette tip and a nanoelectrospray nozzle, and the method comprises the following steps: a) providing a sample comprising an analyte and a matrix, wherein the matrix is ​​non-magnetic; b) providing a microparticle, wherein the microparticle is magnetic; c) incubating the analyte with the microparticles to form an analyte-microparticle complex in the sample holder, wherein the analyte-microparticle complex is magnetic; d) separating the matrix and the analyte-microparticle complex by magnetic force; e) optionally washing the analyte-microparticle complexes in the sample holder; f) extracting the analyte from the analyte-microparticle complex by an extraction solvent and magnetic force, step (f) comprising: f1) supplying the extraction solvent by a conductive pipette tip; f2) contacting the extraction solvent with the analyte-microparticle complex in the sample holder; f3) extracting the analyte from the analyte-microparticle complex to form an extracted analyte, wherein the microparticles are held in the sample holder by magnetic force during the extraction step f3) and the conductive pipette tip contains the extracted analyte; g) directly contacting the extracted analyte with a conductive pipette tip comprising a nanoelectrospray nozzle of a chip-based nanoESI detection system to form a nanoelectrospray for ionizing the extracted analyte; direct contacting can mean directly contacting the respective object or surface, or contacting the respective object or surface with a liquid phase (e.g., the extracted analyte in an extraction solvent). h) determining the presence or level of an extracted analyte in a sample using a chip-based nanoESI detection system, wherein the chip-based nanoESI detection system uses mass spectrometry, ion mobility, and / or a combination thereof.

[0119] Embodiment 2 The method of embodiment 1, wherein the conductive pipette tip containing the extracted analyte is free of particulates.

[0120] Embodiment 3. The method of any of the preceding aspects, wherein the material of the conductive pipette tip at least partially comprises a conductive material selected from the group consisting of graphene, carbon nanotubes, carbon black, carbon fiber, stainless steel, aluminum, titanium, chromium, conductive metals, and alloys thereof.

[0121] Embodiment 4. The method of any preceding aspect, wherein the conductive pipette tip comprises a particulate content relative to the total particulate content that is less than 20%, 15%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, or 0.01%.

[0122] Embodiment 5. The method of any preceding aspect, wherein the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is direct mechanical contact.

[0123] Embodiment 6 The method of any preceding aspect, wherein the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is direct electrical contact.

[0124] Embodiment 7. The method of any preceding aspect, wherein the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is direct contact between the extracted analyte and the nozzle of the chip-based nanoESI detection system.

[0125] Embodiment 8. The method of any preceding aspect, wherein the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is direct contact between the extracted analyte and the nozzle of the chip-based nanoESI detection system and the conductive pipette tip.

[0126] Embodiment 9. The method of any preceding aspect, wherein the ratio of microparticles to extraction solvent in step f) is in the range of 0.1:1 to 20:1, preferably 0.5:1 to 15:1, more preferably 1:1 to 1:10, or wherein the ratio of microparticles to extraction solvent in step f) is in the range of 0.1:1 to 50:1, preferably 0.5:1 to 25:1, more preferably 1:1 to 10:1. Ratios mean % by weight (w / w).

[0127] Embodiment 10 The method of any preceding aspect, wherein the microparticle is superparamagnetic or paramagnetic.

[0128] Embodiment 11. The method of any preceding aspect, wherein the magnetic force is induced by a permanent magnet or an electromagnet.

[0129] Embodiment 12 The method of any of the preceding aspects, wherein the sample is a biological sample and is from an individual, preferably a human.

[0130] Embodiment 13 The method of any preceding aspect, wherein the analyte molecule is present in a biological or clinical sample selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, and dried blood spots.

[0131] Embodiment 14 The method of any of the preceding aspects, wherein the sample is a hemolyzed whole blood sample, particularly a hemolyzed human whole blood sample.

[0132] 15. The method of any of the preceding aspects, wherein the matrix comprises analyte-interfering components derived from a biological sample, a particulate, a sample preparation solution, a mixture, or a combination thereof.

[0133] Embodiment 16 The method of any preceding aspect, wherein the matrix is ​​a solution.

[0134] Embodiment 17 The method of any preceding aspect, wherein the matrix comprises an internal standard.

[0135] Embodiment 18. Step e) is e1) Addition of cleaning solution, and e2) removal of wash supernatant after magnetic separation.

[0136] Embodiment 19. Before step f), preferably after step e), the method comprises: i) drying the analyte-microparticle complex, and / or

[0023] Aspect 10. The method of any preceding aspect, comprising the step of: ii) washing the analyte-microparticle complex.

[0137] Embodiment 20. The method of any preceding aspect, wherein the method is automated.

[0138] Embodiment 21. The method of any preceding aspect, wherein the method is performed in a random-access mode.

[0139] Embodiment 22 The method of any preceding aspect, wherein the method is an in vitro diagnostic method.

[0140] Embodiment 23 The method of any preceding aspect, wherein the method is performed sequentially.

[0141] Embodiment 24. The method of any of the preceding aspects, wherein the 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 specific modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0142] Embodiment 25. The method of any preceding aspect, wherein the method does not include a chromatography step, but includes at least one or more methods selected from the following group: chromatography, high performance liquid chromatography (HPLC), liquid chromatography-high performance liquid chromatography (LC-HPLC), gas chromatography (GC), gel permeation chromatography (GPC), flash chromatography. The chromatography is, for example, size exclusion chromatography.

[0143] Embodiment 26. The method of any preceding aspect, wherein the method is performed in the order a, then b, then c, then d, then optionally e, then f, then g, then h.

[0144] Embodiment 27. Use of the method of any one of the preceding aspects 1 to 26 for determining the presence or level of an analyte in a sample.

[0145] Embodiment 28. A diagnostic system for determining the presence or level of an analyte in a sample, comprising a chip-based nanoESI source, a conductive pipette tip, and a detector for performing the method of any one of the preceding aspects, wherein the chip-based nanoESI source includes a nozzle and the detector uses mass spectrometry or ion mobility or a combination thereof.

[0146] Embodiment 29. The diagnostic system of the preceding aspect 28, wherein the system is a stand-alone system.

[0147] Embodiment 30. The diagnostic system of any of the preceding aspects 28 to 29, wherein the system is integrated into another system capable of determining the presence or level of an analyte based on (electro)chemiluminescence or clinical chemistry.

[0148] Embodiment 31 Use of the diagnostic system of any one of the preceding aspects 28 to 30 in a method according to any one of the preceding aspects 1 to 26.

[0149] Embodiment 32. A kit suitable for carrying out the method of any one of the preceding aspects 1 to 26, comprising: (A) a microparticle for concentrating or purifying an analyte in a sample; (B) an extraction solvent for extracting the analyte from the microparticles; (C) optionally an internal standard, and (D) A kit, optionally including a catalyst or other reagent, such as a derivatization reagent.

[0150] Embodiment 33. Use of the kit of the preceding claim 32 in a method according to any one of the preceding aspects 1 to 26. [Example]

[0151] The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0152] Figure 1 illustrates a method for determining the presence or level of an analyte in a sample using a chip-based nanoESI detection system according to the present invention. A solid support bead purification / enrichment workflow is shown, combining direct extraction and chip-based nanoESI ionization.

[0153] In Figure 1, I), a sample is prepared containing an analyte and a matrix. The matrix is ​​non-magnetic. The matrix may contain analyte-interfering components from the biological sample, microparticles, sample preparation solutions, and / or mixtures. Optionally, the matrix contains an internal standard.

[0154] In Figure 1 II) the addition of magnetic microparticles is shown. The microparticles can be a microparticle suspension. The microparticles and the analyte are then incubated to form analyte-microparticle complexes, for example in a sample holder. The analyte-microparticle complexes are magnetic.

[0155] In Figure 1 III), the supernatant matrix is ​​removed after magnetic separation, and the matrix and analyte-particle complexes are separated by magnetic force.

[0156] 1A and 1B show optional washing steps of the analyte-microparticle complexes, for example in a sample holder, where 1B shows the addition of a washing solution and 1B shows the removal of the washing supernatant after magnetic separation.

[0157] After the extraction steps Va) and Vb), an extraction solvent is delivered by a conductive pipette tip, and the extraction solvent and the analyte-microparticle complex are brought into contact in the sample holder (Va). The analyte is extracted from the analyte-microparticle complex to form an extracted analyte. In Vb) of FIG. 1, the extracted analyte can be captured after magnetic separation. Optionally, during step Va), the magnet can also be separated while adding the extraction solvent, which can facilitate mechanical stirring.

[0158] As shown in Figure 1(VI), the conductive pipette tip containing the extracted analyte is brought into direct contact with the nanoelectrospray nozzle of the chip-based nanoESI detection system to form a nanoelectrospray ESI spray for ionizing the extracted analyte.

[0159] After direct contact with the nozzle of the nanoESI system (VI) and application of a voltage, a continuous nanoESI spray can be formed. The analyte ion species generated here can be analyzed in an analytical system (VII) that considers a mass spectrometry device, an ion mobility separation device, or a combination thereof.

[0160] Optionally, between steps IVb) and Va), the analyte-microparticle complexes can be dried and stored prior to extraction and analysis, which is considered a further advantage of the method.

[0161] Particularly in the presence of biological matrices such as blood or serum, a defined concentration of the desired analyte may allow for direct ionization without the use of expensive chromatography systems or fluidic devices. Directly combining sample preparation, analyte extraction, and ionization has the advantage of reducing the need for large amounts of solvents, fluid components, and disposable materials. All steps of the magnetic bead-based analytical workflow, from sample addition to the final extraction immediately prior to ionization, can be performed in a single assay cup.

[0162] 2 and 3 show a front view (FIG. 2) and a side view (FIG. 3) of a diagnostic system for carrying out a method according to the present invention. The diagnostic system includes an analytical module 1, a sample solution 2 supplied to an assay cup, a pipetting unit 3, an analyte solution 4 containing a microparticle suspension, residual analytes adsorbed to the microparticles 5, an analytical module 6, a microparticle extraction module 7, a microparticle separation module 8, a conductive pipette tip 9 (a microparticle extraction tip), a microparticle extraction solvent reservoir 10, a microparticle capture plate 11, a nanospray tip (a chip-based nanoESI source) 12, and an analytical module inlet 13. The nozzle and detector are not shown in FIGS. 2 and 3.

[0163] To demonstrate the breadth of applicability using the method described herein, a variety of analytes, not just microparticles, were tested. Additionally, the use of this method starting from biological matrices was explored in more detail. By combining this sample preparation and ionization method with ion mobility, the enrichment and separation of multiple analytes in a mixture was demonstrated. Furthermore, the ability to quantify analytes was successfully demonstrated.

[0164] Figures 4a1) and 4b1) show the subsequent extraction from a smooth surface and enrichment of the analyte with superparamagnetic beads as microparticles for a chip-based nanoESI detection system. The respective enlargements of the detected full-scan mass spectra (mass-to-charge ratio m / z 553-562) around the protonated analyte leucine enkephalin signal [M+H]+ at m / z 556.3 are shown (b1) and can be compared to the extraction of a blank bead sample (a1)—both normalized to the same counts (relative abundance, ra). The microparticle workflow began with 100 μL of 100 ng / mL leucine enkephalin prepared in a single well of a twin.tech® PCR Plate 96, skirted, 150 μL volume, Eppendorf AG, while a separate well contained 100 μL of blank deionized water for comparison. Fifteen microparticle suspensions (10 mg / mL, Bead A = superparamagnetic polystyrene-coated carboxylic acid-modified beads) were added to both samples and incubated for a total of 3 min. After magnetic separation and removal of the supernatant, the analyte-microparticle complexes were washed twice with 100 μL of deionized water. The remaining analyte-microparticle complexes were transferred to microscope slides using a pipette tip. Analytes were then extracted from the microparticles on the slides using 10 μL of extraction solvent containing 80% acetonitrile (ACN) and 0.1% formic acid (FA). Subsequently, nanoESI ionization was performed using a chip-based device (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed using a Synapt G2-Si mass spectrometer (Waters Corp.) in time-of-flight (ToF) positive ion mode with an acquisition time of 60 s. The analyte concentration, extraction, and subsequent ionization of leucine enkephalin in combination with microparticles from a microscope slide were successful, as shown in Figure 4b1), where protonated analyte ions were clearly detected. The blank experiment in Figure 4a1) showed only a negligible background signal, demonstrating that the peptide leucine enkephalin can be selectively analyzed directly from a smooth surface without significant background interference.

[0165] Figures 5a2) and 5b2) show the subsequent extraction of an analyte with microparticles, e.g., superparamagnetic beads, from a well plate and subsequent enrichment with a chip-based nanoESI detection system. A magnification of the detected full-scan mass spectrum (m / z 553–562) around the protonated analyte leucine enkephalin signal [M+H]+ at m / z 556.3 is shown (b2) and can be compared to the extraction of a blank bead sample (a2)—both normalized to the same counts. The workflow began with 100 ng / mL leucine enkephalin (100 μL) prepared in a single well of a well plate (twin.tech® PCR Plate 96, skirted, 150 μL volume, Eppendorf AG), while a separate well contained 100 μL of blank deionized water for comparison. Fifteen microparticle suspensions (10 mg / mL, Bead A = superparamagnetic polystyrene-coated carboxylic acid-modified beads) were added to both samples and incubated for a total of 3 min. After magnetic separation and removal of the supernatant, the analyte-microparticle complexes were washed twice with 100 μL of deionized water. The remaining analyte-microparticle complexes were then extracted with 10 μL of 80% ACN + 0.1% FA and subsequently ionized by nanoESI using a chip-based device (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed on a Synapt G2-Si mass spectrometer (Waters Corp.) in ToF positive ion mode with an acquisition time of 60 s. The protonated analyte ions were clearly detected in Figure 5a2, confirming the successful enrichment, extraction, and subsequent ionization of leucine enkephalin analytes using microparticles. No significant overlap was observed in the blank experiment, demonstrating that the peptide leucine enkephalin could be selectively analyzed directly from the sample wells without interference from background signals.

[0166] This highlights the enormous potential of combining microparticle-based sample preparation techniques with subsequent extraction and chip-based nanoESI ionization directly from one sample well. After adsorption and concentration of the desired analytes on the microparticles, the analytes can be extracted and ionized directly in a single pipetting tip, and further processing or transfer in different analytical vessels is no longer necessary. Thus, additional materials and time-consuming steps can be avoided.

[0167] Figures 6 and 7 show the testosterone-13C3 [M+H] detected after microparticle concentration, extraction, and ionization starting from analyte-spiked horse serum in the presence of the internal standard (ISTD) aldosterone-13C3. + The following abbreviations are used: ar = area ratio (= area(analyte) / area(ISTD)); c = concentration).

[0168] The horizontal axis shows the spike concentration (c, ng / mL) of testosterone-13C3 in horse serum (100 µL) before bead workflow sample preparation. The vertical axis represents the area ratio, normalized by the internal standard aldosterone-13C3 (m / z 364.2 → 346.0, collision energy 16 eV) from the testosterone-13C3 multiple reaction monitoring (MRM) transition m / z 292.1 → 100.0 (collision energy 18 eV), over a 60-second measurement period. Figure 7 shows the corresponding magnification of Figure 6 for the range below 0.5 ng / mL.

[0169] Eight samples were prepared, ranging in concentration from 46 ng / mL to 4.6 pg / mL of testosterone-13C3, along with a blank horse serum sample. All samples contained the same concentration of aldosterone-13C3 (18 ng / mL) as an internal standard. Sample preparation involved adding 15 μL of microparticle suspension (10 mg / mL, Bead A) to all samples in the well plate and incubating them for a total of 3 minutes. After magnetic separation and removal of the supernatant, the analyte-microparticle complexes were washed twice with water (100 μL). The remaining analyte-microparticle complexes were then extracted with 80% ACN + 0.1% FA (10 μL) and subsequently ionized by nanoESI using a chip-based instrument (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed in positive ion mode using a Xevo TQ-XS mass spectrometer (Waters Corp.). The total analysis time was set to 60 seconds, and the MRM transition of the analyte testosterone-13C3 (m / z 292.1 → 100.0, collision energy 18 eV) was selected and referenced to the MRM transition of aldosterone-13C3 (m / z 364.2 → 346.0, collision energy 16 eV). Calculation of the detection area ratio was supported using the TargetLynx software tool (Waters Corp.). Considering the data from this single dilution series, the lowest detectable value was estimated to be approximately 35 pg / mL.

[0170] Figure 8 shows the detected phenytoin-13C1-15N2[MH] after bead concentration, extraction, and ionization starting from spiked horse serum in the presence of the internal standard (ISTD) aldosterone-13C3. - The calibration set is shown.

[0171] The horizontal axis shows the spike concentration (c, ng / mL) of phenytoin-13C1-15N2 in horse serum (100 µL) before microparticle workflow sample preparation. The vertical axis shows the area ratio, obtained by normalizing the phenytoin-13C1-15N2 MRM transition m / z 254.0 → 103.0 (collision energy 20 eV) to the internal standard aldosterone-13C3 (m / z 362.2 → 334.2, collision energy 16 eV) over a 60-second measurement period. Figure 9 shows the extraction by adding the corresponding magnification to Figure 8 in the range below 0.5 ng / mL.

[0172] Eight samples containing phenytoin-13C1-15N2 at concentrations ranging from 46 ng / mL to 4.6 pg / mL were prepared, along with a blank horse serum sample. All samples contained the same concentration of aldosterone-13C3 (18 ng / mL) as an internal standard. Sample preparation involved adding 15 μL of Bead A suspension (10 mg / mL) to all samples in the well plate and incubating them for a total of 3 minutes. After magnetic separation and removal of the supernatant, the analyte-particle complexes were washed twice with water (100 μL). The remaining analyte-particle complexes were then extracted with 10 μL of 80% ACN + 0.08 mM NHF + NH4OH pH = 9.0, followed by nanoESI ionization using a chip-based device (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed in negative ion mode using a Xevo TQ-XS mass spectrometer (Waters Corp.). The total analysis time was set to 60 seconds, and the MRM transition of the analyte phenytoin-13C1-15N2 (m / z 254.0 → 103.0, collision energy 20 eV) was selected and referenced to the MRM transition of aldosterone-13C3 (m / z 362.2 → 334.2, collision energy 16 eV). Calculation of the detection area ratio was supported using the TargetLynx software tool (Waters Corp.). Considering the data from this single dilution series, the lowest detectable value was estimated to be approximately 12 pg / mL.

[0173] Both the results for the testosterone-13C3 and phenytoin-13C1-15N2 dilution series highlight the high sensitivity of the method in negative and positive ion modes, even in the presence of difficult biological matrices.

[0174] Figure 10 shows the ion mobility separation of different analytes in a mixture applying microparticle-based sample enrichment with extraction and ionization in positive ion mode.

[0175] Figures 11A-11E show overlaid five extracted ion mobilograms (drift times dt of 0-14 ms) detected after microparticle enrichment, extraction, and ionization from a single analyte mixture. All five substances, designated carbamazepine-13C6, testosterone-13C3, linezolid-13C6, 24,25-dihydroxyvitamin D3-13C5, and cyclosporine A-D10, represent important diagnostic and therapeutic analytes and are spiked to a final concentration of 100 ng / mL (100 μL, neat solution). 15 μL of Bead A suspension (10 mg / mL) was added to the analyte-mixture solution in the well plate and incubated for 3 min. After magnetic separation and removal of the supernatant, the analyte-microparticle complexes were washed twice with water (100 μL). The remaining analyte-microparticle complexes were then extracted with 80% ACN + 0.1% FA (10 μL) and subsequently ionized by nanoESI using a chip-based device (Triversa NanoMate, Advion Inc.). Separation and mass analysis of the resulting ions were performed using a Synapt G2-Si mass spectrometer (Waters Corp.) in IMS-ToF positive ion mode with an acquisition time of 60 seconds, coupled with ion mobility separation (IMS; wave speed 650 m / s, pulse height 40 V). All spiked analytes were enriched and successfully extracted from one single analyte mixture. For 24,25-dihydroxyvitamin D3-13C5, [M + H - 2H2O] was used. + An ion species was observed at m / z 386.2, while all other analytes were the corresponding protonated adducts [M+H] + appeared in.

[0176] Comparison of the detection of testosterone-13C3 applying bead-based sample enrichment / purification in extraction and ionization in positive ion mode considering neat solution and horse serum matrix: Figure 12 shows the peak of testosterone-13C3 [M+H] at m / z 292.3 detected after microparticle enrichment, extraction, and ionization starting from a neat solution a3) and then supplemented with horse serum b3). + The extracted ion mobilograms (0.5–4.0 ms, drift time dt) of the mass signals are overlaid. The starting concentration was 10 ng / mL. To the total analyte solution (100 μL) in the well plate, 15 μL of Bead A suspension (10 mg / mL) was added and incubated for 3 min. After magnetic separation and removal of the supernatant, the analyte-particle complexes were washed twice with water (100 μL). The remaining analyte-bead complexes were then extracted with 80% ACN + 0.1% FA (10 μL) and subsequently ionized by nanoESI using a chip-based device (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed using a Synapt G2-Si mass spectrometer (Waters Corp.) in IMS-ToF positive ion mode with an acquisition time of 60 s, coupled with ion mobility separation (IMS; wave speed 650 m / s, pulse height 40 V). Despite the complex matrix, starting with the analyte and spiking it with horse serum resulted in similar counts of detected testosterone-13C3 compared to the same experiment from the neat analyte solution. This highlights the excellent ability to concentrate and purify analytes from complex matrices that make direct injection and measurement from the initial sample impossible. Microparticle-based sample concentration / purification was applied to extraction and ionization in positive ion mode, and a comparison of the detection of testosterone-13C3 considering neat solution and horse serum matrix is ​​shown.

[0177] Figures 13 and 14A-14E show ion mobility separation of different analytes in a mixture applying microparticle-based sample enrichment with extraction and ionization in negative ion mode.

[0178] Figures 14A-14E show overlaid five extracted ion mobilograms (drift time dt, 0-14 ms) detected after microparticle enrichment, extraction, and ionization from a single analyte mixture. All five substances, designated phenytoin-13C1-15N2, estradiol-13C3, aldosterone-13C3, 24,25-dihydroxyvitamin D3-13C5, and cyclosporine A-D10, represent important diagnostic and therapeutic analytes. All five were spiked to a final concentration of 10 ng / mL (100 μL, neat solution), except for aldosterone-13C3, which was included at 20 ng / mL. 15 μL of Bead A suspension (10 mg / mL) was added to the analyte-mixture solution in the well plate and incubated for 3 min. After magnetic separation and removal of the supernatant, the analyte-microparticle complexes were washed twice with water (100 μL). The remaining analyte-microparticle complexes were then extracted with 80% ACN + 0.08 mM NHF + NHOH pH = 9.0 (10 μL) and subsequently ionized by nanoESI using a chip-based device (Triversa NanoMate, Advion Inc.). Separation and mass analysis of the resulting ions were performed using a Synapt G2-Si mass spectrometer (Waters Corp.) in IMS-ToF positive ion mode, coupled with ion mobility separation (IMS; wave speed 650 m / s, pulse height 40 V), with an acquisition time of 60 s. All spiked analytes were enriched and successfully extracted from one single analyte mixture. The corresponding proton loss [MH] of the analyte was calculated. - appeared in.

[0179] Figure 15 shows a comparison of the detection of estradiol-13C3 considering a neat solution and a horse serum matrix when microparticle-based sample enrichment / purification is applied to extraction and ionization in negative ion mode. Figure 15 shows the detection of estradiol-13C3 [MH] at m / z 274.3 after microparticle enrichment, extraction, and ionization starting from a neat solution a4) and then supplemented with horse serum b4). -The extracted ion mobilograms (0.5–4.0 ms, drift time) of the mass signals are overlaid. The starting concentration was 10 ng / mL. To the total analyte solution (100 μL) in the well plate, 15 μL of Bead A suspension (10 mg / mL) was added and incubated for 3 min. After magnetic separation and removal of the supernatant, the analyte-particle complexes were washed twice with water (100 μL). The remaining analyte-particle complexes were then extracted with 80% ACN + 0.08 mM NHF + NH4OH pH = 9.0 (10 μL) and subsequently ionized by nanoESI using a chip-based device (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed using a Synapt G2-Si mass spectrometer (Waters Corp.) in IMS-ToF negative ion mode with an acquisition time of 60 seconds, coupled with ion mobility separation (IMS; wave speed 650 m / s, pulse height 40 V). Despite the complex matrix, spiked horse serum starting from the analyte resulted in similar counts of detected estradiol-13C3 compared to the same experiment from the neat analyte solution. This highlights the excellent ability to enrich and purify analytes from complex matrices that would otherwise be impossible to measure by direct injection from the initial sample.

[0180] Figure 16 shows an example of the application of microparticles, such as magnetic immunobeads, for the detection of estradiol-13C3. This involves the concentration and extraction of the microparticle sample and ionization in negative ion mode. The estradiol-13C3 mixture from the neat analyte mixture was prepared using a microparticle workflow in which anti-estradiol antibodies were conjugated to superparamagnetic immunobeads (iBead(E2)). The analyte concentrations were 42 ng / mL, 8 ng / mL, and 4 ng / mL estradiol-13C3, compared to a water blank. To the total analyte solution (100 μL) in the well plate, 10 μL of iBead(E2) suspension (11 mg / mL) was added and incubated for 10 minutes. After magnetic separation and removal of the supernatant, the analyte-microparticle complex was washed twice with water (100 μL). The remaining analyte-microparticle complexes were then extracted with 80% ACN + 0.08 mM NHF + NHOH pH = 9.0 (10 μL) and subsequently ionized by nanoESI using a chip-based device (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed using a Synapt G2-Si mass spectrometer (Waters Corp.) in IMS-ToF negative ion mode coupled with ion mobility separation (IMS; wave speed 950 m / s, pulse height 40 V) with an acquisition time of 60 s. Figure 16 shows the relative [M−H] of estradiol-13C3 at a drift time of 3.34 ms. - An overlay of the extracted full-scan mass spectra is shown. Direct extraction and ionization of estradiol-13C3 from immunobeads was successful at test concentrations of 42 ng / mL, 8 ng / mL, and 4 ng / mL, with blank spectra showing little background signal in the observed mass region.

[0181] Figure 17 shows the application of microparticles, e.g., magnetic immunobeads, to detect testosterone-13C3. This microparticle-based sample enrichment involves extraction and ionization in positive ion mode. The microparticle workflow involved extracting testosterone-13C3 from a neat analyte mixture and then ionizing it with anti-testosterone antibody-conjugated superparamagnetic immunobeads ("iBead(Te)"). Analyte concentrations were 833 pg / mL, 417 pg / mL, and 83 pg / mL, respectively, compared with a water blank. To the total analyte solution (100 μL) in a well plate, 15 μL of iBead(Te) suspension (4 mg / mL) was added and incubated for 10 min. After magnetic separation and removal of the supernatant, the analyte-microparticle complexes were washed twice with water (100 μL). The remaining analyte-particle complexes were then extracted with 80% ACN + 0.1% FA (10 μL) and subsequently ionized by nanoESI using a chip-based device (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed using a Synapt G2-Si mass spectrometer (Waters Corp.) in IMS-ToF positive ion mode with an acquisition time of 60 seconds, coupled with ion mobility separation (IMS; wave speed 850 m / s, pulse height 40 V). Figure 17 shows the relevant [M+H] peaks of testosterone-13C3 at a drift time of 2.98 ms. - An overlay of the extracted full-scan mass spectra is shown. Direct extraction and ionization of testosterone-13C3 from microparticles such as immunobeads was successful at test concentrations of 833 pg / mL and 417 pg / mL. Even at the 83 pg / mL concentration, the signal is even higher compared to the blank HS iBead(Te) extract.

[0182] The method of the present invention is a valuable tool for measuring clinically important analytes at low concentrations. Successful applications using specific microparticles, such as immunobeads, highlight the modularity and breadth of applicability, as tailored microparticles, such as immunobeads, can be targeted to detect analytes that would not otherwise be possible. Furthermore, different microparticle materials targeting different analytes can be easily introduced into a single device without modifying the sample preparation or ionization process itself.

[0183] This patent application claims priority from European Patent Application No. 22211164.3, the contents of which are incorporated herein by reference. [Explanation of symbols]

[0184] 1. Analysis Module 2 Sample solution supplied to the assay cup 3 Pipetting Unit 4. Analyte solution containing a microparticle suspension 5. Residual analytes adsorbed onto microparticles 6 Analysis Module 7. Particle Extraction Module 8. Particle Separation Module for Bead Workflow 9. Microparticle Extraction Tip 10 Microparticle Extraction Solvent Reservoir 11. Particle Capture Plate 12 Nanospray Tip 13 Analytical Module Inlet

Claims

1. A method for determining the presence or level of an analyte in a sample using a tip-based nanoESI detection system, wherein the tip-based nanoESI detection system includes a conductive pipette tip and a nanoelectrospray nozzle, and the method comprises the following steps: a) Preparing the sample including the analyte and the matrix, wherein the matrix is ​​nonmagnetic. b) Preparing fine particles, wherein the fine particles are magnetic, c) Incubating the microparticles and the analyte in a sample holder to form an analyte-microparticle composite, wherein the analyte-microparticle composite is magnetic. d) Separating the matrix and the analyte-microparticle composite by magnetic force, e) Optionally, wash the analyte-particulate composite in the sample holder. f) Extracting the analyte from the analyte-particulate composite by means of an extraction solvent and magnetic force, step (f) is, f1) Supplying the extraction solvent using the conductive pipette tip, f2) Bringing the extraction solvent and the analyte-microparticle composite into contact with the sample holder, f3) Extracting the analyte from the analyte-microparticle composite to form the extracted analyte, wherein the microparticles are held in the sample holder by magnetic force during the extraction step f3), and the conductive pipette tip contains the extracted analyte. g) Directly contacting the extracted analyte with the conductive pipette tip, which includes the nanoelectrospray nozzle of the tip-based nanoESI detection system, in order to form a nanoelectrospray for ionizing the extracted analyte. h) A method for determining the presence or level of the extracted analyte in a sample using the chip-based nanoESI detection system, wherein the chip-based nanoESI detection system uses mass spectrometry, ion mobility, and / or a combination thereof.

2. The method according to claim 1, wherein the conductive pipette tip containing the extracted analyte does not contain fine particles.

3. The method according to claim 1, wherein the material of the conductive pipette tip comprises at least partially a conductive material selected from the group consisting of graphene, carbon nanotubes, carbon black, carbon fiber, stainless steel, aluminum, titanium, chromium, conductive metals, and alloys thereof.

4. The method according to claim 1, wherein the conductive pipette tip comprises a particulate content relative to the total content of the particulates, wherein the conductive pipette tip is 20%, 15%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, or less than 0.01%.

5. The method according to claim 1, wherein the direct contact between the conductive pipette tip and the nozzle of the tip-based nanoESI detection system is direct electrical contact.

6. The method according to claim 1, wherein the fine particles are superparamagnetic or paramagnetic.

7. The method according to claim 1, wherein the matrix comprises analyte interfering components derived from a biological sample, fine particles, sample preparation solution, mixture, or combination thereof.

8. The method according to claim 1, wherein the matrix is ​​a solution.

9. The method according to claim 1, wherein the method is automated and / or performed in random-access mode.

10. The method according to claim 1, wherein the method does not include a chromatography step and comprises at least one method selected from the following group: chromatography, high-performance liquid chromatography (HPLC), liquid chromatography-high-performance liquid chromatography (LC-HPLC), gas chromatography (GC), gel permeation chromatography (GPC), and flash chromatography.

11. Use of the method according to any one of claims 1 to 10 for determining the presence or level of an analyte in a sample.

12. A diagnostic system for determining the presence or level of an analyte in a sample, comprising a tip-based nanoESI source, a conductive pipette tip, and a detector for carrying out the method according to any one of claims 1 to 10, wherein the tip-based nanoESI source includes a nozzle, and the detector uses mass spectrometry, ion mobility, or a combination thereof.

13. Use of the diagnostic system according to claim 12 in the method according to any one of claims 1 to 10.

14. A kit suitable for carrying out the method described in any one of claims 1 to 10, (A) Fine particles for concentrating or purifying the analyte in the sample, (B) Extraction solvent for extracting the analyte from the fine particles, (C) Optionally, an internal standard, and (D) Optionally, a kit comprising a catalyst or other reagent, such as a derivatizing reagent.

15. Use of the kit according to claim 14 in the method according to any one of claims 1 to 10.

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