Detection of target analyte by chip-based nanoESI detection system

Abstract

The present invention relates to a method, a diagnostic system, a kit and their use for the efficient detection of a target analyte by a chip-based nanoESI detection system.

Description

Technical Field

[0001] The present invention relates to a method, a diagnostic system, a kit, and their use for efficiently detecting a target analyte by a chip-based nanoESI detection system. Background Art

[0002] Mass spectrometry (MS) is a technique widely used in the qualitative and quantitative analysis of chemical substances from small molecules to large molecules. Generally, it is a very sensitive and specific method, even allowing the analysis of complex biological samples (such as environmental samples or clinical samples). However, for several analytes, especially if analyzed from complex biological matrices such as serum, the sensitivity of the measurement remains a problem.

[0003] MS often is coupled with chromatographic techniques especially gas chromatography and liquid chromatography such as HPLC. Here, the target molecules (analytes) to be analyzed are chromatographically separated and mass spectrometrically analyzed separately. However, stand-alone mass spectrometry has made substantial progress in terms of selectivity and sensitivity of direct MS detection methods. Different from the traditional workflow divided into sample preparation, chromatographic separation, and mass spectrometric detection, many sample preparation techniques are directly associated with stand-alone MS that exhibits excellent performance.

[0004] To ensure reliable and sensitive mass spectrometric detection (avoiding matrix effects and interferences and improving sensitivity), it is necessary to separate the target analyte as well as possible by chromatography. Generally, this can be accomplished by an isocratic or gradient system (e.g., a reversed-phase HPLC column and a gradient from an aqueous phase to an organic phase). The columns for HPLC require a flow rate between 0.1 ml / min and 1.0 ml / min. Under these optimal flow conditions, very narrow chromatographic peaks are produced, which have very small peak volumes.

[0005] Liquid extraction surface analysis (LESA) mass spectrometry is a direct surface sampling technique. Analytes are extracted from the surface via a liquid microinterface between a pipette tip and the sample surface. This method is capable of sampling a variety of bioanalytes (such as drugs, lipids, and proteins) from a series of solid surfaces prior to electrospray ionization (ESI). Substrates analyzed by LESA include thin tissue sections, bacterial colonies grown on agar, dried blood spots on cards and polymer surfaces. Other direct analysis methods that have been applied to dried blood spot analysis include desorption electrospray ionization (DESI), direct analysis in real time (DART), and paperspray.

[0006] Currently, some methods combining bead-based solid supports with nano-ESI-MS analysis are described in the literature. Most of them are based on microfluidic chips or devices.

[0007] It is well known that the importance of nanoESI mass spectrometry has increased during the past few years. However, combining direct surface sampling techniques with nanoESI-MS requires significantly improved sample preparation techniques to achieve sensitive, adaptable, and rapid measurements of biological matrices. Combining solid support-based sample preparation techniques with nanoESI-MS can overcome these obstacles.

[0008] However, current methods are not suitable for the efficient detection of target analytes, such as for high-throughput measurements, because the microfluidic devices are difficult to mass-produce and do result in an increase in complexity itself.

[0009] Combining solid support-based sample preparation with nano-ESI mass spectrometry is limited by the following factors: the complex design and production of solid supports containing microfluidic devices, devices designed for specific applications lacking a broad menu of analytes, the adaptability of sample preparation affecting ionization, the system requirements of the fluid or liquid chromatography section, the low throughput of most examples in the literature, and / or sample residues affecting measurements.

[0010] However, there is still a need to increase the efficiency of MS analysis methods, especially methods that allow for the efficient detection of analytes from complex biological matrices. This is particularly important in random access high-throughput MS settings where several different analytes with different chemical properties have to be measured within a short period of time.

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

[0012] The object of the present invention is to provide a method, a diagnostic system, a kit, and their use for the efficient detection of target analytes by a chip-based nanoESI detection system.

[0013] This object or these objects are solved by the subject matter of the independent claims. Further embodiments are subject to the dependent claims. Summary of the Invention

[0014] Hereinafter, the present invention relates to the following aspects:

[0015] In a first aspect, the present invention relates to a method for determining the presence or level of a target 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 nanoliter electrospray nozzle, and the method comprises the following steps:

[0016] a) Provide a sample comprising a target analyte and a matrix, wherein the matrix is non-magnetic,

[0017] b) Provide microparticles, wherein the microparticles are magnetic,

[0018] c) Incubate the microparticles and the target analyte in a sample holder to form an analyte-microparticle complex, wherein the analyte-microparticle complex is magnetic,

[0019] d) Magnetically separate the matrix and the analyte-microparticle complex,

[0020] e) Optionally wash the analyte-microparticle complex in the sample holder,

[0021] f) Extract the analyte from the analyte-microparticle complex by an extraction solvent and magnetic force, step (f) comprising

[0022] f1) Provide the extraction solvent through a conductive pipette tip,

[0023] f2) Contact the extraction solvent with the analyte-microparticle complex in the sample holder,

[0024] f3) Extract the target analyte from the analyte-microparticle complex to form an extracted target analyte, wherein during extraction step f3), the microparticles are retained in the sample holder by magnetic force, wherein the conductive pipette tip comprises the extracted target analyte,

[0025] g) Bring the conductive pipette tip containing the extracted target analyte into direct contact with the nanoelectrospray nozzle of a chip-based nanoESI detection system to form a nanoelectrospray for ionizing the extracted target analyte. Direct contact can mean by directly touching the corresponding object or surface, or by contact of a liquid phase (e.g., the analyte extracted in the extraction solvent) with the corresponding object or surface.

[0026] h) Use a chip-based nanoESI detection system to determine the presence or level of the extracted target analyte in the sample, wherein the chip-based nanoESI detection system uses mass spectrometry, ion mobility, and / or a combination thereof.

[0027] 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 a target analyte in a sample.

[0028] In a third aspect, the present invention relates to a diagnostic system for determining the presence or level of a target analyte in a sample, the diagnostic system comprising a chip-based nanoESI source, a conductive pipette tip, and a detector for implementing the method according to the first aspect, wherein the chip-based nanoESI source comprises a nozzle, wherein the detector uses mass spectrometry or ion mobility or a combination thereof.

[0029] 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.

[0030] In a fifth aspect, the present invention relates to a kit adapted to perform the method of the first aspect, comprising:

[0031] (A) Particles for enriching or purifying a target analyte in a sample,

[0032] (B) An extraction solvent for extracting the target analyte from the particles,

[0033] (C) Optionally, an internal standard, and

[0034] (D) Optionally, a catalyst or other reagents. The other reagents are, for example, derivatization reagents.

[0035] In a sixth aspect, the present invention relates to the use of the kit of the fifth aspect of the present invention in the method of the first aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shows a method for determining the presence or level of a target analyte in a sample by a chip-based nanoESI detection system according to the present invention.

[0037] Figure 2 and Figure 3 Shows a front view of a diagnostic system for performing the method according to the present invention ( Figure 2 ) and a side view ( Figure 3 ).

[0038] Figure 4 a1) and Figure 4 b1) Show analyte enrichment on superparamagnetic beads as particles and subsequent extraction from a smooth surface and from a chip-based nanoESI detection system.

[0039] Figure 5 a2) and Figure 5 b2) Show analyte enrichment on superparamagnetic beads as particles and subsequent extraction from a microtiter plate and a chip-based nanoESI detection system.

[0040] Figure 6 and Figure 7 Show testosterone-13C3 [M+H] detected after particle enrichment, extraction, and ionization starting from analyte-spiked horse serum in the presence of the internal standard (ISTD) aldosterone-13C3 + calibration set.

[0041] Figure 8Shows phenytoin-13C1-15N2 [M-H] detected after particulate enrichment, extraction, and ionization starting from analyte-spiked horse serum in the presence of the internal standard (ISTD) aldosterone-13C3 - of a calibration set.

[0042] Figure 9 Shows an excerpt obtained by increasing the corresponding amplification in the range below 500 pg / mL Figure 8 thereof.

[0043] Figure 10 Shows ion mobility separation of different analytes in a mixture, applying particulate-based sample enrichment and extraction and ionization in positive ion mode.

[0044] Figure 11 A through Figure 11 E show the overlap of five extracted ion mobility plots (0 ms to 14 ms drift time dt) detected after particulate enrichment, extraction, and ionization in a single analyte mixture.

[0045] Figure 12 Shows a comparison of testosterone-13C3 detection, considering pure solution and horse serum matrix, applying particulate-based sample enrichment / purification and extraction and ionization in positive ion mode.

[0046] Figure 13 and Figure 14 A through Figure 14 E show ion mobility separation of different analytes in a mixture, applying particulate-based sample enrichment and extraction and ionization in negative ion mode.

[0047] Figure 15 Shows a comparison of estradiol-13C3 detection, considering pure solution and horse serum matrix, applying particulate-based sample enrichment / purification and extraction and ionization in negative ion mode.

[0048] Figure 16 Shows the application of particulates (e.g., magnetic immunobeads) for estradiol-13C3 detection, applying particulate sample enrichment and extraction and ionization in negative ion mode.

[0049] Figure 17 Shows the application of particulates (e.g., magnetic immunobeads) for testosterone-13C3 detection, applying particulate-based sample enrichment and extraction and ionization in positive ion mode. Detailed Description

[0050] Before the present invention is described in detail below, it should be understood that the 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 particular embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise indicated, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0051] The text of this specification incorporates by reference several documents in their entirety. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.), whether cited above or below, is hereby incorporated by reference in its entirety. In the event of a conflict between the definitions or teachings of such incorporated references and the definitions or teachings cited in this specification, the text of this specification shall control.

[0052] The elements of the present invention will be described below. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to form additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. In addition, unless the context otherwise requires, any arrangement and combination of all the elements described in this application shall be regarded as disclosed by the specification of this application.

[0053] Definition

[0054] The word "comprising" and variations such as "including" and "containing" should be understood to imply the inclusion of the stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The terms "comprising" and "including" may be used interchangeably.

[0055] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents.

[0056] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a "range" format. It should be understood that such range formats are used merely for convenience and brevity and should therefore be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also all the individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly recited. By way of illustration, the numerical range "4% to 20%" should be interpreted to include not only the values explicitly recited as 4% to 20% but also each and every value and sub-range within the indicated range. Thus, this numerical range includes individual values such as 4, 5, 6, 7, 8, 9, 10, … 18, 19, 20% and sub-ranges such as 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges reciting a minimum or a maximum value. In addition, such interpretation applies regardless of the breadth of the range or feature recited.

[0057] When used in connection with a numerical value, the term "about" means to cover a numerical value within a range having a lower limit that is 5% less than the indicated value and an upper limit that is 5% greater than the indicated value.

[0058] The term "mass spectrometry" ("Mass Spec" or "MS") or "mass spectrometric determination" or "mass spectrometric analysis" refers to an analytical technique for identifying a compound by its mass. MS is a method for filtering, detecting, and measuring ions based on their mass-to-charge ratio or "m / z". MS techniques generally include: (1) ionizing a compound to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating the mass-to-charge ratio. Compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Typically, one or more target molecules are ionized and subsequently introduced into a mass spectrometry instrument where, due to a combination of magnetic and electric fields, the ions follow a spatial path that depends on their mass ("m") and charge ("z"). The term "ionization" or "ionize" refers to the process of generating 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, while positive ions are those having a net positive charge of one or more units. MS methods may be performed in a "negative ion mode" in which negative ions are generated and detected or in a "positive ion mode" in which positive ions are generated and detected.

[0059] "Tandem mass spectrometry" or "MS / MS" involves multiple mass spectrometry selection steps in which fragmentation of the analyte occurs between stages. In a tandem mass spectrometer, ions are formed in the ion source and separated by mass-to-charge ratio in a first-stage mass spectrometry analysis (MS1). Ions of a specific mass-to-charge ratio (precursor ions or parent ions) are selected and fragmented ions (or product ions) are generated by collision-induced dissociation, ion-molecule reactions, or photodissociation. The resulting ions are then separated and detected in a second-stage mass spectrometry analysis (MS2).

[0060] Because a mass spectrometer separates and detects ions with slightly different masses, it is readily able to distinguish between different isotopes of a given element. Accordingly, mass spectrometry is an important method for the 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 global measurement of proteins in proteomics. Typically, de novo sequencing of peptides or proteins by mass spectrometry can be performed without prior knowledge of the amino acid sequence.

[0061] The term "electrospray ionization" or "ESI" refers to a method in which a solution travels along a short capillary tube to the end to which a high positive or high negative potential is applied. The solution reaching the end of the tube is evaporated (atomized) into a jet or spray in which there are very small solution droplets present in the solvent vapor. This droplet mist passes through an evaporation chamber, which is slightly heated to prevent condensation and to evaporate the solvent. As the droplets become smaller, the surface charge density increases until the natural repulsion between like charges causes ions as well as neutral molecules to be released.

[0062] The term "nanoelectrospray ionization" or "nanoESI" can refer to classical electrospray ionization at 10 nL / min or 20 nL / min. It can be a method that typically uses a flow rate below 1 μL / min in either a static or dynamic mode. Preferably, nanoESI uses a flow rate of 10 nl / min or 20 nl / min to 500 nl / min (e.g., 500 nl / min). 500 nl / min is equal to 0.5 μl / min.

[0063] The term "static nanoESI mass spectrometry" is used in the context of the present disclosure as a non-continuous flow nanoESI option. Analysis is typically defined by loading discrete samples into the emitter, and a nanoelectrospray is formed during the application of voltage together with a constant gas backpressure. In contrast, dynamic nanoESI mass spectrometry is characterized by a mobile phase that is pumped at a low flow rate through a small-diameter emitter when voltage is applied.

[0064] In the context of the present disclosure, the terms "analyte", "analyte molecule", or "target analyte" are used interchangeably and refer to a chemical substance to be analyzed via mass spectrometry, particularly nanoESI mass spectrometry. A chemical substance suitable for analysis via mass spectrometry, i.e., an analyte, can be any type of molecule present in a living organism, including but not limited to nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytoplasmic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, seco-steroids (e.g., vitamin D), molecules characterized by a certain modification of another molecule (e.g., sugar moiety or phosphoryl residue on a protein, methyl-residue on genomic DNA), substances internalized by an organism (e.g., therapeutic drugs, abused drugs, toxins, etc.) or metabolites of such substances. Such analytes can be used as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance within a biological system that serves as an indicator of the biological state of said system.

[0065] An analyte can be present in a target sample (e.g., a biological sample or a clinical sample). The terms "sample" or "target sample" are used interchangeably herein and refer to a part or section of a tissue, organ, or individual, typically smaller than such tissue, organ, or individual, and intended to represent the entire tissue, organ, or individual. At the time of analysis, the sample provides information about the state of the tissue or the health or diseased state of the organ or individual. Examples of samples include but are not limited to: fluid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph fluid; or solid samples such as dried blood spots and tissue extracts. Other examples of samples are cell cultures or tissue cultures.

[0066] In the context of the present disclosure, a sample can be derived from an "individual" or "subject". Generally, the subject is a mammal. Mammals include but are not limited to domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0067] As used herein, the term "serum" is the clear liquid portion of blood that can be separated from the clotted blood. As used herein, the term "plasma" is the clear liquid portion of blood that contains blood cells. Serum is different from plasma, which is the liquid portion of normal unclotted blood that contains red blood cells, white blood cells, and platelets. Coagulation causes the difference between serum and plasma. As used herein, the term "whole blood" contains all components of blood, e.g., white blood cells and red blood cells, platelets, and plasma.

[0068] In this context, "amount" or "quantity" encompasses absolute amounts, relative amounts or concentrations, and any value or parameter related to or derivable from them.

[0069] As used herein, the term "determining" the level of a target analyte refers to the quantification of the target analyte, e.g., to determine or measure the level of the target analyte in a pretreated sample. The level of the target analyte is determined by nanoESI mass spectrometry.

[0070] The term "hemolytic reagent" (HR) refers to a reagent that lyses the cells present in a sample. In the context of the present invention, the hemolytic reagent particularly refers to a reagent that lyses the cells present in a blood sample (including but not limited to, red blood cells present in a whole blood sample). A well-known hemolytic reagent is water (H2O). Other examples of hemolytic reagents include but are not limited to deionized water, hypertonic liquids (e.g., 8M urea), ionic liquids, and different detergents.

[0071] Generally, an "internal standard" (ISTD) is a substance of known amount that exhibits properties similar to those of the target analyte when undergoing a mass spectrometry detection workflow (i.e., including any pretreatment, enrichment, and actual detection steps). Although the ISTD exhibits properties similar to those of the target analyte, it can still be clearly distinguished from the target analyte. For example, during ion mobility separation, the ISTD has approximately the same drift time and ion mobility as the target analyte in the sample, respectively. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. However, the ISTD exhibits a different molecular mass from the target analyte from the sample. This allows for mass spectrometry discrimination between the ions from the ISTD and the ions from the analyte by their different mass-to-charge (m / z) ratios. Both undergo fragmentation and provide daughter ions. These daughter ions can be distinguished from each other and from their respective parent ions by their m / z ratios. Thus, independent determination and quantification of the signals from the ISTD and the analyte can be performed. Since the amount of the ISTD added is known, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for a relative comparison of the amounts of the detected analyte and enables unambiguous identification and quantification of the analyte when the target analyte present in the sample reaches the mass spectrometer. Generally but not necessarily, the ISTD is an isotopically labeled variant of the target analyte (including, for example 2 H, 13 C, or 15 N, etc. labels).

[0072] The term "in vitro method" is used to indicate that the method is performed outside a living organism, preferably on body fluids, isolated tissues, organs, or cells.

[0073] As used herein, the terms "automatically" or "automated" are broad terms and shall be given their ordinary and customary meaning to one of ordinary skill in the art and are not limited to a special or custom meaning. The term can specifically refer to, but is not limited to, a process that is carried out entirely by means of at least one computer and / or at least one computer network and / or at least one machine, in particular, without manual operation and / or interaction with a user.

[0074] A "kit" is any article (e.g., a package or container) that contains at least one reagent of the present invention, such as a medicament for treating a disease or a probe for specifically detecting a biomarker gene or protein. The kit is preferably marketed, distributed, or sold as a unit for performing the methods of the present invention. Generally, the kit may further include a carrier device that is compartmentalized to receive one or more container devices, such as vials, tubes, etc., in a confined space. In particular, each container is meant to contain one of the individual elements to be used in the methods of the first aspect. The kit may further include one or more other reagents, including but not limited to reaction catalysts. The kit may further contain one or more other containers that contain other materials, including but not limited to buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate the use of the composition for a specific application and may also indicate guidelines for in vivo or in vitro use. Computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly on a computer or data processing device. Additionally, the kit may contain a standard amount of a biomarker for calibration purposes as described elsewhere herein.

[0075] As used herein, the term "particle" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or custom meaning. The term can specifically refer to, but is not limited to, any particulate matter of microscopic size. Particles can have an average diameter in the range of 100 nm to 100 μm, specifically 500200 nm to 50 μm. Particles can also be referred to as beads. Particles can be spherical or globular in shape. However, slight derivations from the spherical or globular shape may be possible. In particular, particles have at least one surface that can attach to a target analyte, for example, by covalent means or van der Waals forces. The term "surface" as used herein is a broad term and is given its ordinary and customary meaning to one of ordinary skill in the art and is not limited to a special or custom meaning. The term can specifically refer to, but is not limited to, the entire region that externally delimits any object. Thus, an object can have multiple surfaces. Specifically, a particle can have a core surrounded by a surface. The surface and the core can include different materials. In addition, the surface and the core can have different properties. Exemplarily, the core can be magnetic. When the particle is incubated with a sample containing such molecules, the surface can be configured to capture molecules, such as a wide range of polar to non-polar molecules. The terms particle and bead can be used interchangeably.

[0076] In particular, the microparticles can 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; poly(methyl methacrylate) microparticles, specifically poly(methyl methacrylate) microparticles having a poly(methyl methacrylate) core and a modified surface. However, other particles are also feasible. The melamine resin microparticles can have an average diameter of from 500 nm to 20 μm, preferably from 2 μm to 4 μm, more preferably 3 μm. The poly(styrene)-based microparticles can have an average diameter of from 500 nm to 50 μm, preferably from 2 μm to 4 μm, more preferably 3 μm. The poly(methyl methacrylate) microparticles can have an average diameter of from 500 nm to 50 μm, preferably from 2 μm to 4 μm, more preferably 3 μm. The modified surface of the magnetic microparticles can be a modified poly(styrene) surface, and the magnetic microparticles can have an average diameter of from 5 μm to 50 μm, preferably from 10 μm to 30 μm, most preferably 20 μm. The modified surface of the magnetic microparticles can be a silica surface, and the magnetic microparticles can have an average diameter of from 100 nm to 1000 nm, preferably from 200 nm to 500 nm, most preferably 300 nm. The modified surface of the silica microparticles can be a cyanopropylsilane-functionalized surface, and the silica microparticles can have an average diameter of from 5 μm to 100 μm, preferably from 20 μm to 80 μm, most preferably 40 μm. Other sizes can also be feasible.

[0077] As used herein, the term "chip-based nanoESI detection system" is a broad term and should be given its ordinary and customary meaning to a person of ordinary skill in the art and should not be limited to a special or custom meaning.

[0078] In particular, the chip-based nanoESI detection system includes a conductive pipette tip and a nanoliter electrospray nozzle.

[0079] As used herein, the term "conductive pipette tip" is a broad term and is given its ordinary and customary meaning to a person of ordinary skill in the art and is not limited to a special or custom meaning. The conductive pipette tip can comprise a conductive material selected from the group consisting of at least partially graphene, carbon nanotubes, carbon black, carbon fibers, stainless steel, aluminum, titanium, chromium, conductive metals, and alloys thereof. The conductive pipette tip can be a disposable conductive pipette tip.

[0080] As used herein, the term "nanoelectrospray nozzle" is a broad term and is given the ordinary and customary meaning to one of ordinary skill in the art, and is not limited to a special or custom meaning. A nanoelectrospray nozzle can be a single-use and / or multi-use nozzle with an inner diameter less than 1 mm. The nanoelectrospray nozzle can be disposed on a disposable chip that contains a specific number of nanoelectrospray nozzles. The nanoelectrospray nozzle can be a disposable nanoelectrospray nozzle.

[0081] As used herein, the term "incubation" is a broad term and should be given the ordinary and customary meaning to one of ordinary skill in the art, and should not be limited to a special or custom meaning. The term can specifically refer to, but is not limited to, the mixing of at least two substances and / or the addition of at least one substance to another substance. Specifically, a solid or particulate substance can be added to and / or mixed with a liquid sample. In addition to the process of addition and / or mixing, incubation can further include a period of time called the incubation time. During the incubation time, one of the two substances can be adsorbed on the surface of the other of the two substances. During the incubation time, other conditions, such as temperature and / or other conditions, can be selected to facilitate the desired adsorption. Thus, in step b), the microparticles can be added to the sample and can optionally be mixed with the sample. In step b), the sample can be incubated with the microparticles for a period of 1 second to 60 minutes, preferably 1 minute to 30 minutes, and most preferably 3 minutes to 12 minutes. However, other durations are also possible.

[0082] As used herein, the term "analyte-microparticle complex" is a broad term and should be given the ordinary and customary meaning to one of ordinary skill in the art, and should not be limited to a special or custom meaning. The term can specifically refer to, but is not limited to, an entity that contains at least one microparticle and at least one analyte, specifically one microparticle and multiple analytes. The microparticle and analyte that form the complex, specifically the analyte, can bind reversibly. Thus, at least under certain conditions, the components of the complex can leave the complex or dissociate from the complex. The analyte-microparticle complex can form based on at least one attraction between the microparticle and the analyte. In particular, the attraction can 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 the adsorption process on the surface of the microparticle. The attraction can include van der Waals forces and electrostatic attraction. Other attractions are also possible. Specifically, as part of the formation of the analyte-microparticle complex, at least one chemical bond can be formed between the microparticle and the analyte, specifically between the surface of the microparticle and the analyte. The analyte-microparticle complex can also be referred to as a microparticle carrying an analyte.

[0083] In step c), the target analyte can be incubated with the microparticles, where the analyte can adsorb on the surface of the microparticles and an analyte-microparticle complex can be formed. In this context, this expression can be understood as forming multiple analyte-microparticle complexes. In step c), this sample can be incubated with the microparticles, where the analyte can adsorb on the surface of the microparticles and an analyte-microparticle complex can be formed.

[0084] As used herein, the term "sample holder" is a broad term and is given its ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or custom meaning. The intended use of the sample holder can provide a sample for further analysis. Sample holder options are, for example, multiwell plates, glass plates, and flat or structured surfaces.

[0085] As used herein, the term "magnetic force" is a broad term and will be given its ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or custom meaning. The magnetic force can be generated by the application of a magnetic field introduced by a permanent magnet or an electromagnet.

[0086] As used herein, the term "extraction" is a broad term and will be given its ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or custom meaning. Extraction is a separation process consisting of separating a substance, such as an analyte, from a matrix.

[0087] As used herein, the term "contact" is a broad term and will be given its ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or custom meaning. Generally, it can be described by aggregation or touching starting from an object or a surface.

[0088] As used herein, the term "direct contact" is a broad term and will be given its ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or custom meaning. Direct contact can mean contacting the corresponding object or surface by directly touching it, or by a liquid phase (e.g., an analyte extracted in an extraction solvent) with the corresponding object or surface.

[0089] As used herein, the term "random access" is a broad term and will be given its ordinary and customary meaning to those of ordinary skill in the art and is not limited to a special or custom meaning. Generally, this process can describe the ability to perform an analysis or directly transfer information randomly, rather than being accessed in a fixed order.

[0090] A "clinical diagnostic system" is a laboratory automation device specifically designed to analyze samples for in vitro diagnosis. Depending on the needs and / or the desired laboratory workflow, the clinical diagnostic system can have different configurations. By coupling together multiple devices and / or modules, additional configurations can be obtained. A "module" is a working unit with a dedicated function, usually smaller than the entire clinical diagnostic system. This function can be an analytical function, but it can also be a pre-analytical function or a post-analytical function, or it can be an auxiliary function for any one of the pre-analytical, analytical, or post-analytical functions. In particular, a module can be configured to cooperate with one or more other modules to perform a dedicated task of a sample processing workflow, for example, by performing one or more pre-analytical steps and / or analytical steps and / or post-analytical steps. In particular, the clinical diagnostic system can include one or more analytical devices designed to perform corresponding workflows optimized for certain types of analysis (e.g., clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography, mass spectrometry, etc.). Thus, the clinical diagnostic system can include one analytical device or any combination of such analytical devices with corresponding workflows, where pre-analytical modules and / or post-analytical modules can be coupled to individual analytical devices or shared by multiple analytical devices. In an alternative, the pre-analytical function and / or the post-analytical function can be performed by units integrated in the analytical device. The clinical diagnostic system can include functional units such as a liquid handling unit for aspirating and / or pumping and / or mixing samples and / or reagents and / or system fluids, and functional units for sorting, storing, transporting, identifying, separating, detecting.

[0091] The clinical diagnostic system can include a sample preparation station, optionally a separation station, for the automated preparation of samples containing the target analyte. In particular, the clinical diagnostic system does not include a separation station, such as an LC-HPLC unit or an HPLC unit.

[0092] The clinical diagnostic system can also include a controller programmed to allocate samples to predefined sample preparation workflows, each workflow including a predefined sequence of sample preparation steps and requiring a predefined completion time (depending on the target analyte). The clinical diagnostic system can also include a mass spectrometer (MS).

[0093] "Sample preparation station" can be a pre - analysis module coupled to one or more analytical devices or units 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 enriching the analyte of interest in the sample. Such processing steps can include any one or more of the following processing operations performed sequentially, in parallel, or interleaved on one or more samples: aspirating (suctioning and / or dispensing) fluids, pumping fluids, mixing with reagents, incubating at a certain temperature, heating or cooling, centrifuging, separating, filtering, sieving, drying, washing, resuspending, aliquoting, transferring, storing, etc.).

[0094] A clinical diagnostic system (such as a sample preparation station) can also include a buffer unit for receiving a plurality of samples before starting a new sample preparation start sequence, where the samples can be accessed individually and randomly, and individual preparation can be initiated according to the sample preparation start sequence.

[0095] Embodiment

[0096] In a first aspect, the present invention relates to a method for determining the presence or level of a target analyte in a sample by a chip - based nanoESI detection system, where the chip - based nanoESI detection system includes a conductive pipette tip and a nano - electrospray nozzle, and the method includes the following steps:

[0097] a) Providing a sample comprising a target analyte and a matrix, where the matrix is non - magnetic,

[0098] b) Providing microparticles, where the microparticles are magnetic,

[0099] c) Incubating the microparticles and the target analyte in a sample holder to form an analyte - microparticle complex, where the analyte - microparticle complex is magnetic,

[0100] d) Separating the matrix and the analyte - microparticle complex by magnetic force,

[0101] e) Optionally washing the analyte - microparticle complex in the sample holder,

[0102] f) Extracting the analyte from the analyte - microparticle complex by an extraction solvent and magnetic force, step (f) includes

[0103] f1) Providing the extraction solvent through the conductive pipette tip,

[0104] f2) Contacting the extraction solvent with the analyte - microparticle complex in the sample holder,

[0105] f3) Extract the target analyte from the analyte-particle complex to form the extracted target analyte, wherein during the extraction step f3), the particles are retained in the sample holder by magnetic force, and wherein the conductive pipette tip comprises the extracted target analyte.

[0106] g) Bring the conductive pipette tip containing the extracted target analyte into direct contact with the nanoelectrospray ionization (nanoESI) nozzle of a chip-based nanoESI detection system to form a nanoelectrospray ESI spray for ionizing the extracted target analyte.

[0107] h) Determine the presence or level of the extracted target analyte in the 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.

[0108] By combining with an efficient sample handling strategy, this method can significantly enhance the performance and throughput of, for example, stand-alone mass spectrometry (MS). Miniaturization and integration into the final analysis, as well as full automation of the entire analytical process, can increase throughput and reduce complexity and separately performed sample preparation. The performance of direct MS can be further enhanced by high-selectivity gas separation techniques such as high-resolution MS and ion mobility MS.

[0109] The method according to the present invention can exhibit the following advantages:

[0110] Reduced complexity and robustness

[0111] a. Extremely low sample / eluent / extraction solution consumption

[0112] b. Efficient extraction

[0113] c. Significantly reduced sample injection and thus reduced background / matrix in MS

[0114] d. Disposable tips and spray nozzles

[0115] e. Modular components

[0116] Simplified workflow

[0117] f. Sample preparation via a particle workflow is directly related to ionization for MS

[0118] g. The particle workflow can be simply adapted to specific target analytes without changing ionization

[0119] h. No chromatography column

[0120] i. No HPLC gradient / eluent system

[0121] j. Isobaric separation via ion mobility or immunofunctionalized particles

[0122] k. Capable of being extended for high throughput and low throughput

[0123] Physical fitness

[0124] l. Enhanced sensitivity by nanoESI

[0125] m. Analyte concentration in the particle extraction step

[0126] n. Increased S / N level

[0127] o. Spray and multiple MS experiments

[0128] In an embodiment of the first aspect of the present invention, the conductive pipette tip containing the extracted target analyte is free of particles.

[0129] In an embodiment of the first aspect of the present invention, the material of the conductive pipette tip includes a conductive material selected from the group consisting of at least partially graphene, carbon nanotubes, carbon black, carbon fiber, stainless steel, aluminum, titanium, chromium, conductive metals, and their alloys. Preferably, the conductive material is selected from the group consisting of graphene, carbon nanotubes, carbon black, carbon fiber, and combinations thereof.

[0130] In an embodiment of the first aspect of the present invention, the conductive pipette tip contains a particle content of less than 20%, 15%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, or 0.01% relative to the total content of particles.

[0131] 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 mechanical contact.

[0132] 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 electrical contact. This may mean that a bridge can be formed between the target analyte and the nozzle.

[0133] 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 the direct contact between the extracted target analyte and the nozzle of the chip-based nanoESI detection system.

[0134] 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 the direct contact between the extracted target analyte and the nozzle of the chip-based nanoESI detection system and the conductive pipette tip.

[0135] In an embodiment of the first aspect of the present invention, the ratio of the microparticles to the 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. This ratio means weight percentage (w / w).

[0136] In an embodiment of the first aspect of the present invention, the microparticles are superparamagnetic or paramagnetic.

[0137] In an embodiment of the first aspect of the present invention, the magnetic force is induced by a permanent magnet or an electromagnet.

[0138] In an embodiment of the first aspect of the present invention, the sample is a biological sample from an individual, preferably a human.

[0139] In an embodiment of the first aspect of the present invention, the sample is a biological or clinical sample selected from the group consisting of blood, serum, plasma, urine, saliva, spinal fluid, and dried blood spots.

[0140] In an embodiment of the first aspect of the present invention, the sample is a hemolyzed whole blood sample, especially a hemolyzed human whole blood sample. Hemolysis can be induced by using a hemolysis reagent.

[0141] In an embodiment of the first aspect of the present invention, the matrix includes analyte interference components derived from the biological sample, microparticles, sample preparation solution, their mixtures, or combinations.

[0142] In an embodiment of the first aspect of the present invention, the matrix is a solution.

[0143] In an embodiment of the first aspect of the present invention, the matrix contains an internal standard.

[0144] In an embodiment of the first aspect of the present invention, step e) includes

[0145] e1) adding a washing solution, and

[0146] e2) removing the washing supernatant after magnetic separation.

[0147] In an embodiment of the first aspect of the present invention, before step f) and preferably after step e), the method includes the following steps:

[0148] i) drying the analyte-microparticle complex, and / or

[0149] ii) storing the analyte-microparticle complex.

[0150] In an embodiment of the first aspect of the present invention, the method is automated.

[0151] In an embodiment of the first aspect of the present invention, the method is performed in a random access mode.

[0152] In an embodiment of the first aspect of the present invention, the method is an in vitro diagnostic method.

[0153] In an embodiment of the first aspect of the present invention, the method is performed continuously.

[0154] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, seco-steroids, molecules characterized by a certain modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0155] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: testosterone, epitestosterone, dihydrotestosterone (DHT), desoxymethyltestosterone (DMT), tetrahydrogestrinone (THG), aldosterone, estrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone, 16-ketoestradiol, 16-α-hydroxyestrone, 2-hydroxyestrone-3-methyl ether, prednisone, prednisolone, pregnenolone, progesterone, dehydroepiandrosterone (DHEA), 17-hydroxypregnenolone, 17-hydroxyprogesterone, androsterone, epiandrosterone, Δ4-androstenedione, 11-deoxycortisol, corticosterone, 21-deoxycortisol, 11-deoxycorticosterone, allopregnanolone, and aldosterone.

[0156] In an embodiment of the first aspect of the present invention, the target 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, isoxepac, and penicillin. In an embodiment of the first aspect of the present 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.

[0157] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: pyridoxal, N-acetyl-D-glucosamine, acrivastine, streptomycin, and josamycin.

[0158] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: cocaine, heroin, Ritalin, aceclofenac, acetylcholine, amcinonide, amiloride, amilocaine, ampipridine, arecoline, artesunate, and pethidine.

[0159] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: cantharidin, succinic anhydride, trimellitic anhydride, and maleic anhydride.

[0160] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol, and tacalcitol. In particular, the secosteroid compound is vitamin D, especially vitamin D2 or D3 or a derivative thereof. In a specific embodiment, the secosteroid compound 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, 24,25-dihydroxyvitamin D3. In an embodiment of the first aspect of the present invention, the analyte molecule containing 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.

[0161] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: benzyl alcohol, menthol, L-carnitine, pyridoxine, metronidazole, isosorbide mononitrate, guaifenesin, clavulanate, Miglitol, zalcitabine, isoproterenol, acyclovir, methocarbamol, tramadol, venlafaxine, atropine, clofedanol, α-hydroxyalprazolam, α-hydroxytriazolam, lorazepam, nordazepam, temazepam, ethyl glucuronide, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-demethyltramadol, dihydroquinidine, and quinidine. In an embodiment of the first aspect of the present invention, where the analyte molecule contains more than one hydroxyl group, the analyte is selected from the group consisting of: vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacitidine, ribavirin, floxuridine, gemcitabine, streptozotocin, adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenoson, lincomycin, clindamycin, Canaglifozin, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyl digitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, and plicamycin.

[0162] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: thiomandelic acid, DL-captopril, DL-thioprofen, N-acetylcysteine, D-penicillamine, glutathione, L-cysteine, zefenoprilat, tiopronin, dimercaprol, and succimer.

[0163] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: glutathione disulfide, disulfiram, selenium disulfide, disulfiram, lipoic acid, L-cystine, fursultiamine, octreotide, desmopressin, vapreotide, terlipressin, linaclotide, and peginesatide. The selenium disulfide can be selenium disulfide SeS2 or selenium hexasulfide Se2S6.

[0164] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: carbamazepine-10,11-epoxide, carfilzomib, furosemide epoxide, fosfomycin, sevelamer hydrochloride, cerulenin, scopolamine, tiotropium, tiotropium bromide, methscopolamine bromide, eplerenone, mupirocin, natamycin, and oleandomycin.

[0165] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: estrogen, estrogen-like compounds, estrone (El), estradiol (E2), 17α-estradiol, 17β-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol and / or their metabolites. In an embodiment, the metabolites are selected from the group consisting of: estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-ketoE2), 16α-hydroxyestrone (16α-OHEl), 2-methoxyestrone (2-MeOEl), 4-methoxyestrone (4-MeOEl), 2-hydroxyestrone-3-methyl ether (3-MeOEl), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (El), estrone sulfate (Els), 17α-estradiol (E2a), 17β-estradiol (E2B), estradiol sulfate (E2S), equilin (EQ), 17α-dihydroequilin (EQa), 17β-dihydroequilin (EQb), equilenin (EN), 17-dihydroequilenin (ENa), 17α-dihydroequilenin, 17β-dihydroequilenin (ENb), Δ8,9-dehydroestrone (dEl), Δ8,9-dehydroestrone sulfate (dEls), Δ9-tetrahydrocannabinol, and mycophenolic acid. β or b can be used interchangeably. α and a can be used interchangeably.

[0166] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: 3,4-methylenedioxyamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 3,4-methylenedioxymethamphetamine, amphetamine, methamphetamine, N-methyl-1,3-benzodioxolylbutanamine, 7-aminonitrazepam, 7-aminoflunitrazepam, 3,4-dimethylmethcathinone, 3-fluoromethcathinone, 4-methoxymethcathinone, 4-methylethcathinone, 4-methylmethcathinone, phentermine, butylone, ethylcathinone, elephedrone, methcathinone, methylone, methylenedioxypyrovalerone, benzoylecgonine, dehydrodeschloroketamine, ketamine, norketamine, methadone, normethadone, 6-acetylmorphine, diacetylmorphine, morphine, norhydrocodone, oxycodone, oxymorphone, phencyclidine, norpropoxyphene, amitriptyline, clomipramine, dosulepin, 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, norpethidine, O-demethyltramadol, desmethyltramadol, tramadol, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin, sisomicin, and 5-methylcytosine.

[0167] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of: ribose, deoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and N-acetylneuraminic acid, etc. In an embodiment, the analyte molecule is an oligosaccharide, particularly selected from the group consisting of: disaccharide, trisaccharide, tetrasaccharide, and polysaccharide. In an embodiment of the first aspect of the present invention, the disaccharide is selected from the group consisting of: sucrose, maltose, and lactose. In an embodiment of the first aspect of the present invention, the analyte molecule is a substance containing the above monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide moiety.

[0168] In an embodiment of the first aspect of the present invention, the target analyte is zidovudine or aztreonam.

[0169] In an embodiment of the first aspect of the present invention, the method does not comprise a chromatography step, which chromatography step comprises at least one or more methods selected from the group consisting of: chromatography, high performance liquid chromatography (HPLC), liquid chromatography - high performance liquid chromatography (LC - HPLC), gas chromatography (GC), gel permeation chromatography (GPC), flash chromatography. Chromatography is, for example, size exclusion chromatography.

[0170] In an embodiment of the first aspect of the present invention, the methods are carried out in the following order: a, then b, then c, then d, then optionally e, then f, then g, and then h.

[0171] 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 a target analyte in a sample. All embodiments mentioned for the first aspect of the present invention are applicable to the second aspect of the present invention, and vice versa.

[0172] In a third aspect, the present invention relates to a diagnostic system for determining the presence or level of a target analyte in a sample, the diagnostic system comprising a chip - based nanoESI source, a conductive pipette tip, and a detector for implementing the method according to the first aspect, wherein the chip - based nanoESI source comprises a conductive pipette and a nozzle, and wherein the detector uses mass spectrometry or ion mobility or a combination thereof. All embodiments mentioned for the first aspect and / or the second aspect of the present invention are applicable to the third aspect of the present invention, and vice versa.

[0173] In an embodiment of the third aspect of the present invention, the system is a stand - alone system.

[0174] In an embodiment of the third aspect of the present invention, the system is integrated in other systems capable of determining the presence or level of a target analyte based on (electro)chemiluminescence or clinical chemistry.

[0175] In an embodiment of the third aspect of the present invention, the diagnostic system is a clinical diagnostic system.

[0176] In an embodiment of the third aspect of the present invention, the nanoESI source can be, for example, a chip - based electrospray ionization technology from Advion. It combines the advantages of liquid chromatography, mass spectrometry, chip - based infusion, fraction collection, and direct surface analysis into an integrated ion source platform. Other known nanoESI sources are also possible. The nanoESI source is known to the person skilled in the art and is therefore not explained in detail.

[0177] In an embodiment of the third aspect of the present invention, the mass spectrometer can be, for example, a triple quadrupole mass spectrometer or a linear ion trap mass spectrometer. The mass spectrometer is known to the person skilled in the art and is therefore not explained in detail.

[0178] 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 mentioned for the first aspect and / or the second aspect and / or the third aspect of the present invention are applicable to the fourth aspect of the present invention, and vice versa.

[0179] In a fifth aspect, the present invention relates to a kit adapted to perform the method of any one of the foregoing aspects, comprising:

[0180] (A) Particles for enriching or purifying a target analyte in a sample,

[0181] (B) An extraction solvent for extracting the target analyte from the particles,

[0182] (C) Optionally, an internal standard, and

[0183] (D) Optionally, a catalyst or other reagent, such as a derivatization reagent. All embodiments mentioned for the first aspect and / or the second aspect and / or the third aspect and / or the fourth aspect of the present invention are applicable to the fifth aspect of the present invention, and vice versa.

[0184] 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 and / or the second aspect and / or the third aspect and / or the fourth aspect and / or the fifth aspect of the present invention are applicable to the sixth aspect of the present invention, and vice versa.

[0185] Summarizing and without excluding other possible embodiments, the following embodiments can be envisaged:

[0186] Example 1. A method for determining the presence or level of a target analyte in a sample by a chip-based nanoESI detection system, wherein the chip-based nanoESI detection system comprises a conductive pipette tip and a nanoliter electrospray nozzle, the method comprising the steps of:

[0187] a) Providing a sample comprising a target analyte and a matrix, wherein the matrix is non-magnetic,

[0188] b) Providing particles, wherein the particles are magnetic,

[0189] c) Incubating the particles and the target analyte in a sample holder to form an analyte-particle complex, wherein the analyte-particle complex is magnetic,

[0190] d) Separating the matrix and the analyte-particle complex by magnetic force,

[0191] e) Optionally washing the analyte-particle complex in the sample holder,

[0192] f) Extracting the analyte from the analyte - particle complex by extraction solvent and magnetic force, step (f) comprising

[0193] f1) Providing the extraction solvent through a conductive pipette tip,

[0194] f2) Contacting the extraction solvent with the analyte - particle complex in a sample holder,

[0195] f3) Extracting the target analyte from the analyte - particle complex to form the extracted target analyte, wherein during extraction step f3), the particles are retained in the sample holder by magnetic force, and wherein the conductive pipette tip comprises the extracted target analyte,

[0196] g) Directly contacting the conductive pipette tip containing the extracted target analyte with the nano - ESI detection nozzle of a chip - based nano - ESI detection system to form a nano - electrospray for ionizing the extracted target analyte. The direct contact can mean by directly touching the corresponding object or surface, or by contact of a liquid phase (e.g., the analyte extracted in the extraction solvent) with the corresponding object or surface.

[0197] h) Determining the presence or level of the extracted target analyte in the sample using a chip - based nano - ESI detection system, wherein the chip - based nano - ESI detection system uses mass spectrometry, ion mobility, and / or a combination thereof.

[0198] Example 2. The method according to aspect 1, wherein the conductive pipette tip containing the extracted target analyte is free of particles.

[0199] Example 3. The method according to any one of the foregoing aspects, wherein the material of the conductive pipette tip comprises a conductive material selected from the group consisting of at least partially graphene, carbon nanotubes, carbon black, carbon fibers, stainless steel, aluminum, titanium, chromium, conductive metals, and their alloys.

[0200] Example 4. The method according to any one of the foregoing aspects, wherein the conductive pipette tip contains a particle content of less than 20%, 15%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, or 0.01% relative to the total content of the particles.

[0201] Example 5. The method according to any one of the foregoing aspects, wherein the direct contact between the conductive pipette tip and the nozzle of the chip - based nano - ESI detection system is direct mechanical contact.

[0202] Example 6. The method according to any one of the foregoing aspects, wherein the direct contact between the conductive pipette tip and the nozzle of the chip - based nano - ESI detection system is direct electrical contact.

[0203] Example 7. The method according to any one of the preceding aspects, wherein the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is the direct contact between the extracted target analyte and the nozzle of the chip-based nanoESI detection system.

[0204] Example 8. The method according to any one of the preceding aspects, wherein the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is the direct contact between the extracted target analyte and the nozzle of the chip-based nanoESI detection system and the conductive pipette tip.

[0205] Example 9. The method according to any one of the preceding aspects, wherein the ratio of particulate matter: 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 the ratio of particulate matter: 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. This ratio means mass percentage (w / w).

[0206] Example 10. The method according to any one of the preceding aspects, wherein the particulate matter is superparamagnetic or paramagnetic.

[0207] Example 11. The method according to any one of the preceding aspects, wherein the magnetic force is induced by a permanent magnet or an electromagnet.

[0208] Example 12. The method according to any one of the preceding aspects, wherein the sample is a biological sample from an individual, preferably a human.

[0209] Example 13. The method according to any one of the preceding aspects, wherein the sample is a biological or clinical sample selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, and dried blood spots.

[0210] Example 14. The method according to any one of the preceding aspects, wherein the sample is a hemolyzed whole blood sample, especially a hemolyzed human whole blood sample.

[0211] 15. The method according to any one of the preceding aspects, wherein the matrix comprises analyte interfering components derived from the biological sample, particulate matter, sample preparation solution, their mixtures or combinations.

[0212] Example 16. The method according to any one of the preceding aspects, wherein the matrix is a solution.

[0213] Example 17. The method according to any one of the preceding aspects, wherein the matrix comprises an internal standard.

[0214] Example 18. The method according to any one of the preceding aspects, wherein step e) comprises e1) adding a washing solution, and

[0215] e2) removing the washing supernatant after magnetic separation.

[0216] Example 19. The method according to any one of the preceding aspects, wherein before step f) and preferably after step e), the method comprises the steps of:

[0217] i) drying the analyte - particle complex, and / or

[0218] ii) storing the analyte - particle complex.

[0219] Example 20. The method according to any one of the preceding aspects, wherein the method is automated.

[0220] Example 21. The method according to any one of the preceding aspects, wherein the method is performed in a random access mode.

[0221] Example 22. The method according to any one of the preceding aspects, wherein the method is an in vitro diagnostic method.

[0222] Example 23. The method according to any one of the preceding aspects, wherein the method is performed continuously.

[0223] Example 24. The method according to any one of the preceding aspects, wherein the target analyte is selected from the group consisting of: nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, seco - steroids, molecules characterized by some modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0224] Example 25. The method according to any one of the preceding aspects, wherein the method does not contain a chromatography step, and the chromatography step comprises at least one or more methods selected from the group consisting of: chromatography, high - performance liquid chromatography (HPLC), liquid chromatography - high - performance liquid chromatography (LC - HPLC), gas chromatography (GC), gel permeation chromatography (GPC), flash chromatography. Chromatography is, for example, size - exclusion chromatography.

[0225] Example 26. The methods are performed in the following order: a, then b, then c, then d, then optionally e, then f, then g, and then h.

[0226] Example 27. Use of the method according to any one of the preceding aspects 1 to 26 for determining the presence or level of a target analyte in a sample.

[0227] Example 28. A diagnostic system for determining the presence or level of a target analyte in a sample, the diagnostic system comprising a chip-based nanoESI source, a conductive pipette tip, and a detector for performing the method according to any one of the foregoing aspects, wherein the chip-based nanoESI source comprises a nozzle, and wherein the detector uses mass spectrometry or ion mobility or a combination thereof.

[0228] Example 29. The diagnostic system according to aspect 28 foregoing, wherein the system is a stand-alone system.

[0229] Example 30. The diagnostic system according to any one of aspects 28 to 29 foregoing, wherein the system is integrated in other systems capable of determining the presence or level of the target analyte based on (electro)chemiluminescence or clinical chemistry.

[0230] Example 31. Use of the diagnostic system according to any one of aspects 28 to 30 foregoing in the method according to any one of aspects 1 to 26 foregoing.

[0231] Example 32. A kit adapted to perform the method according to any one of aspects 1 to 26 foregoing, the kit comprising

[0232] (A) Particles for enriching or purifying the target analyte in the sample,

[0233] (B) An extraction solvent for extracting the target analyte from the particles,

[0234] (C) Optionally, an internal standard, and

[0235] (D) Optionally, a catalyst or other reagent, such as a derivatization reagent.

[0236] Example 33. Use of the kit according to aspect 32 foregoing in the method according to any one of aspects 1 to 26 foregoing.

[0237] Example

[0238] The following examples are provided to illustrate, but not limit, the invention claimed herein.

[0239] Figure 1 A method for determining the presence or level of a target analyte in a sample by a chip-based nanoESI detection system according to the present invention is shown. A solid support bead purification / enrichment workflow combined with direct extraction and chip-based nano-ESI ionization is shown.

[0240] In Figure 1In I), a sample including a target analyte and a matrix is provided. The matrix is non-magnetic. The matrix may include analyte interfering components derived from a biological sample, microparticles, sample preparation solutions, and / or mixtures. Optionally, the matrix includes an internal standard.

[0241] In Figure 1 II), the addition of magnetic microparticles is shown. The microparticles can be a microparticle suspension. Then, the microparticles and the target analyte are incubated to form an analyte-microparticle complex, for example, in a sample holder. The analyte-microparticle complex is magnetic.

[0242] In Figure 1 III), the supernatant matrix is removed after magnetic separation. The matrix and the analyte-microparticle complex are separated by magnetic force.

[0243] Figure 1 IVa) and IVb) optionally show a washing step of the analyte-microparticle complex, for example, in a sample holder. Figure 1 IVa) shows the addition of a washing solution, and Figure 1 IVb) shows the removal of the washed supernatant after magnetic separation.

[0244] After the extraction procedure in Va) and Vb), an extraction solvent is provided by a conductive pipette tip, and the extraction solvent contacts the analyte-microparticle complex in the sample holder (Va). The target analyte is extracted from the analyte-microparticle complex to form an extracted target analyte. In Figure 1 Vb), the extracted target analyte can be adsorbed after magnetic separation. Optionally, during step Va), the magnet can also be removed while adding the extraction solvent, which can help with mechanical stirring.

[0245] As Figure 1 shown in VI), it shows the direct contact of the conductive pipette tip containing the extracted target analyte with the nanoESI detection system's nanoliter electrospray nozzle on a chip to form a nanoliter electrospray ESI spray for ionizing the extracted target analyte.

[0246] After direct contact with the nozzle of the nano-ESI system in VI) and applying a voltage, a continuous nan-ESI spray can be formed. The resulting ionic species of the target analyte can be analyzed with an analytical system VII), considering a mass spectrometry device, an ion mobility separation device, or a combination thereof.

[0247] Optionally, between step IVb) and Va), the analyte-microparticle complex can be dried and stored before extraction and analysis, which is regarded as an additional benefit of this method.

[0248] In particular, in the presence of a biological matrix such as blood or serum, the defined enrichment and concentration of the analyte of interest enables direct ionization without the use of expensive chromatographic systems or fluidic devices. The direct combination of sample preparation, analyte extraction, and ionization has the advantage of reducing large amounts of solvents, fluid components, and disposable materials. All magnetic bead-based analyte workflow steps can be performed in a single assay cup starting from sample addition until final extraction and prior to ionization.

[0249] Figure 2 and Figure 3 shows a front view ( Figure 2 ) and a side view ( Figure 3 ) of a diagnostic system for performing the method according to the invention. The diagnostic system includes an analysis module 1, a sample solution 2 provided in an assay cup; a pipetting unit 3, an analyte solution 4 having a particulate suspension, a residual analyte 5 adsorbed on the particles, an analysis module 6, a particle extraction module 7, a particle separation module 8; a conductive pipette tip 9 (particle extraction tip), a particle extraction solvent reservoir 10, a particle capture plate 11, a nano-spray chip (chip-based nanoESI source) 12; an analysis module inlet 13. Figure 2 and Figure 3 The nozzle and detector are not shown in

[0250] To demonstrate the broad applicability of the method described herein, different analytes as well as particles were tested. In addition, the use of this method starting from a biological matrix was tested in more detail. The combination of such sample preparation and ionization methods together with ion mobility has been verified in the enrichment and separation of multiple analytes in a mixture. In addition, the ability to quantify the target analyte has also been successfully demonstrated.

[0251] Figure 4 a1) and Figure 4 b1) show analyte enrichment on superparamagnetic beads as particles and subsequent extraction from a smooth surface and detection from a chip-based nanoESI detection system. The corresponding enlarged view (b1)) of the full-scan mass spectrum (mass-to-charge ratio m / z 553 to 562) detected around the protonated analyte leucine enkephalin signal [M+H]+ at m / z 556.3 is shown and can be compared with the extraction of the blank bead sample (a1)) (both normalized to the same number of counts (relative abundance ra)). The particle workflow starts with 100 ng / mL leucine enkephalin (100 μL) in a well plate ( The 96-well PCR plate, skirted, with a volume of 150 μL, was provided in a single well of an Eppendorf AG), and 100 μL of blank deionized water was placed in different wells for comparison. 15 μL of the particle suspension (10 mg / mL, Bead A = superparamagnetic polystyrene-coated carboxylic acid-modified beads) was added to both samples, and the total incubation was for 3 minutes. After magnetic separation and removal of the supernatant, the analyte-particle complex was washed twice with deionized water (100 μL). The remaining analyte-particle complex was transferred to a microscope slide using a pipette tip. Subsequently, the analyte was extracted from the particles spread on the slide using an extraction solvent (10 μL) containing 80% acetonitrile (ACN) + 0.1% formic acid (FA), and then ionized by nanoESI using a chip-based instrument (Triversa NanoMate, Advion Inc.). Mass analysis of the resulting ions was performed in the time-of-flight (ToF) positive ion mode using a Synapt G2-Si mass spectrometer (Waters Corp.) for a collection time of 60 seconds. The enrichment, extraction, and subsequent ionization of the analyte combined with the particles from the microscope slide were successful because the protonated analyte ions were clearly detected in Figure 4 b1). Figure 4 The blank experiment in a1) showed only a small background signal, demonstrating the ability to selectively analyze the peptide leucine enkephalin without significant interference from the background signal directly from the smooth surface.

[0252] Figure 5 a2) and Figure 5 b2) show the enrichment of the analyte on the particles (e.g., superparamagnetic beads), and subsequent extraction from the well plate and chip-based nano-ESI detection system. The corresponding enlarged view (b2)) of the full scan mass spectrum (m / z 553 to 562) detected around the protonated analyte leucine enkephalin signal [M+H]+ at m / z 556.3 is shown, and it can be compared with the extraction of the blank bead sample (a2)) (both normalized to the same number of counts). The workflow started with 100 ng / mL leucine enkephalin (100 μL), which was in the well plate ( Provided in a single well of a 96-well PCR plate, skirted, 150 μL volume, Eppendorf AG), and 100 μL of blank deionized water was placed in a different well for comparison. 15 μL of a particulate suspension (10 mg / mL, BeadA = superparamagnetic polystyrene-coated carboxylic acid-modified beads) was added to both samples and incubated for a total of 3 minutes. After magnetic separation and removal of the supernatant, the analyte-particulate complex was washed twice with deionized water (100 μL). The remaining analyte-particulate complex was 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 using a Synapt G2-Si mass spectrometer (Waters Corp.) in ToF positive ion mode with a collection time of 60 seconds. Enrichment, extraction, and subsequent ionization of leucine enkephalin bound to the particulates and the analyte were successful as protonated analyte ions were clearly detected in Figure 5 a2). The blank experiment showed no significant overlap, demonstrating the ability to selectively analyze leucine enkephalin directly from the sample well without background signal interference.

[0253] This highlights the great potential of combining particulate-based sample preparation techniques with subsequent extraction and chip-based nano-ESI ionization directly from a sample well. After adsorption and enrichment of the desired analyte on the particulates, no further processing or transfer in different analytical containers is required as the analyte can be directly extracted and ionized with a single pipette tip. Thus, additional materials and time-consuming steps can be avoided.

[0254] Figure 6 and Figure 7 shows the calibration set of testosterone-13C3 [M+H] detected after particulate enrichment, extraction, and ionization starting from analyte-spiked horse serum in the presence of the internal standard (ISTD) aldosterone-13C3. + The following abbreviations were used: ar = area ratio (= area(analyte) / area(ISTD)); c = concentration).

[0255] The horizontal axis represents the spiked concentration (c, in ng / mL) of testosterone-13C3 in horse serum (100 μL) before bead workflow sample preparation. The vertical axis represents the area ratio derived by normalizing the multiple reaction monitoring (MRM) transition m / z 292.1→100.0 (collision energy 18 eV) of testosterone-13C3 to the internal standard aldosterone-13C3 (m / z 364.2→346.0, collision energy 16 eV) over a 60-second measurement time. Figure 7Shows the obtained by adding the corresponding amplification in the range below 0.5 ng / mL Figure 6 extract.

[0256] As the concentration range, eight different samples of testosterone-13C3 between 46 ng / mL and 4.6 pg / mL and a blank horse serum sample were prepared. Each sample contained the same concentration of aldosterone-13C3 (18 ng / mL) and was carried out as an internal standard. Sample preparation included adding 15 μL of the particulate suspension (10 mg / mL, BeadA) to each sample in a well plate and incubating them for a total of 3 minutes. After magnetic separation and removal of the supernatant, the analyte-particulate complex was washed twice with water (100 μL). Then the remaining analyte-particulate complex was 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 an Xevo TQ-XS mass spectrometer (WatersCorp.). The total analysis time was set to 60 seconds. The analyte testosterone-13C3 MRM transition m / z 292.1 → 100.0 (collision energy 18 eV) was selected and referenced to the aldosterone-13C3 MRM transition (m / z 364.2 → 346.0, collision energy 16 eV). The TargetLynx software tool (Waters Corp.) was used to support the calculation of the detected area ratio. Considering the data of this single dilution series, the lowest possible detection value was estimated to be approximately 35 pg / mL.

[0257] Figure 8 Shows the calibration set of phenytoin-13C1-15N2 [M-H] detected after bead enrichment, extraction, and ionization starting from analyte-spiked horse serum in the presence of the internal standard (ISTD) aldosterone-13C3 - set.

[0258] The horizontal axis represents the spiked concentration (c, in ng / mL) of phenytoin-13C1-15N2 in horse serum (100 μL) before the particulate workflow sample preparation. The vertical axis represents the area ratio, derived 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 measurement time of 60 seconds. Figure 9 Shows the obtained by adding the corresponding amplification in the range below 0.5 ng / mL Figure 8 extract.

[0259] As concentration ranges, eight different samples of phenytoin-13C1-15N2 between 46 ng / mL and 4.6 pg / mL and blank horse serum samples were prepared. Each sample contained the same concentration of aldosterone-13C3 (18 ng / mL), which was used as an internal standard for implementation. Sample preparation included adding 15 μL of BeadA suspension (10 mg / mL) to each sample in a well plate and incubating them for a total of 3 minutes. After magnetic separation and removal of the supernatant, the analyte-microparticle complex was washed twice with water (100 μL). Then the remaining analyte-microparticle complex was extracted with 80% ACN + 0.08 mM NH4F + NH4OH pH = 9.0 (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 negative ion mode using an Xevo TQ-XS mass spectrometer (Waters Corp.). The total analysis time was set to 60 seconds, the analyte phenytoin-13C1-15N2 MRM transition m / z 254.0 → 103.0 (collision energy 20 eV) was selected, and the aldosterone-13C3 MRM transition (m / z 362.2 → 334.2, collision energy 16 eV) was used as a reference. The TargetLynx software tool (Waters Corp.) was used to support the calculation of the detected area ratio. Considering the data from this single dilution series, the lowest possible detection value was estimated to be approximately 12 pg / mL.

[0260] Both results for the dilution series of testosterone-13C3 and phenytoin-13C1-15N2 highlight the high sensitivity of the method in both negative and positive ion modes, even in the presence of challenging biological matrices.

[0261] Figure 10 Ion mobility separation of different analytes in a mixture is shown, applying particle-based sample enrichment and extraction and ionization in positive ion mode.

[0262] Figure 11 A to Figure 11Panel E shows the overlap of five extracted ion mobility plots (drift times dt from 0 ms to 14 ms) detected after particle enrichment, extraction, and ionization in a single analyte mixture. These five substances represent important diagnostic and therapeutic analytes, namely carbamazepine-13C6, testosterone-13C3, linezolid-13C6, 24,25-dihydroxyvitamin D3-13C5, and cyclosporine A-D10, respectively, and were spiked to a final concentration of 100 ng / mL (100 μL, pure solution). 15 μL of BeadA suspension (10 mg / mL) was added to the analyte mixture solution in the well plate and incubated for 3 minutes. After magnetic separation and removal of the supernatant, the analyte-particle complex was washed twice with water (100 μL). Then the remaining analyte-particle complex was extracted with 80% ACN + 0.1% FA (10 μL), and subsequently ionized by nanoESI using a chip-based instrument (Triversa NanoMate, Advion Inc.). Separation and mass analysis of the resulting ions were performed in IMS-ToF positive ion mode using a Synapt G2-Si mass spectrometer (Waters Corp.) in combination with ion mobility separation (IMS; wave velocity 650 m / s, wave height 40 V) with a collection time of 60 seconds. All spiked analytes were enriched and successfully extracted from the single analyte mixture. For 24,25-dihydroxyvitamin D3-13C5, the [M+H-2H2O]+ ion was observed at m / z 386.2, while all other analytes appeared at their corresponding protonated adducts [M+H]+. + ions, while all other analytes appeared in their corresponding protonated adducts [M+H]+. + in.

[0263] Comparison of testosterone-13C3 detection, considering pure solution versus horse serum matrix, applying bead-based sample enrichment / purification and extraction and ionization in positive ion mode:

[0264] Figure 12 shows testosterone-13C3 [M+H]+ at m / z 292.3 detected after particle enrichment, extraction, and ionization starting from a pure solution a3) and additionally spiked in horse serum b3). +Overlap of ion mobility plots (drift time dt from 0.5 ms to 4.0 ms) for extraction of mass signals. Starting concentrations were both 10 ng / mL. To each analyte mixture solution (100 μL) in a microplate was added 15 μL of BeadA suspension (10 mg / mL) and incubated for 3 minutes. After magnetic separation and removal of the supernatant, the analyte - particle complex was washed twice with water (100 μL). The remaining analyte - bead complex was then extracted with 80% ACN + 0.1% FA (10 μL) and subsequently ionized by nanoESI using a chip - based instrument (Triversa NanoMate, Advion Inc.). The resulting ions were mass - analyzed in IMS - ToF positive ion mode using a Synapt G2 - Si mass spectrometer (Waters Corp.) with ion mobility separation (IMS; wave velocity 650 m / s, wave height 40 V) for 60 seconds of acquisition time. Despite the complex matrix, similar testosterone - 13C3 counts were detected starting from analyte - spiked horse serum compared to the same experiment from a pure analyte solution. This highlights the excellent ability to enrich and purify the target analyte from a complex matrix, which would not be possible to measure by direct injection from the initial sample. A comparison of testosterone - 13C3 detection is shown, considering pure solution versus horse serum matrix, applying particle - based sample enrichment / purification and extraction and ionization in positive ion mode.

[0265] Figure 13 and Figure 14 A through Figure 14 E show the ion mobility separation of different analytes in the mixture, applying particle - based sample enrichment and extraction and ionization in negative ion mode.

[0266] Figure 14 A through Figure 14Panel E shows the overlap of five extracted ion mobility maps (drift time dt from 0 ms to 14 ms) detected after particle enrichment, extraction, and ionization in a single analyte mixture. These five substances represent important diagnostic and therapeutic analytes, namely phenytoin-13C1-15N2, estradiol-13C3, aldosterone-13C3, 24,25-dihydroxyvitamin D3-13C5, and cyclosporin A-D10, and were spiked to a final concentration of 10 ng / mL (100 μL, pure solution), except for aldosterone-13C3, which had a concentration of 20 ng / mL. 15 μL of BeadA suspension (10 mg / mL) was added to the analyte mixture solution in a well plate and incubated for 3 minutes. After magnetic separation and removal of the supernatant, the analyte-particle complex was washed twice with water (100 μL). The remaining analyte-particle complex was then extracted with 80% ACN + 0.08 mM NH4F + NH4OH pH = 9.0 (10 μL), followed by ionization by nanoESI using a chip-based instrument (Triversa NanoMate, Advion Inc.). Separation and mass analysis of the resulting ions were performed in IMS-ToF positive ion mode using a Synapt G2-Si mass spectrometer (Waters Corp.) with ion mobility separation (IMS; wave velocity 650 m / s, wave height 40 V), and the acquisition time was 60 seconds. All spiked analytes were enriched and successfully extracted from the single analyte mixture. The analytes appeared at their corresponding proton losses [M-H] - .

[0267] Figure 15 Shows a comparison of estradiol-13C3 detection, considering pure solution versus horse serum matrix, applying particle-based sample enrichment / purification and extraction and ionization in negative ion mode. Figure 15 Shows estradiol-13C3 [M-H] at m / z 274.3 detected after particle enrichment, extraction, and ionization starting from pure solution a4) and additionally spiked in horse serum b4). -Overlap of the ion mobility plots (drift times from 0.5 ms to 4.0 ms) for the extraction of the quality signal. The starting concentration for both was 10 ng / mL. To each analyte mixture solution (100 μL) in the well plate, 15 μL of BeadA suspension (10 mg / mL) was added and incubated for 3 minutes. After magnetic separation and removal of the supernatant, the analyte - particle complex was washed twice with water (100 μL). Then the remaining analyte - particle complex was extracted with 80% ACN + 0.08 mM NH4F + NH4OH pH = 9.0 (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 the IMS - ToF negative ion mode using a SynaptG2 - Si mass spectrometer (Waters Corp.) with ion mobility separation (IMS; wave velocity 650 m / s, wave height 40 V) for a collection time of 60 seconds. Despite the complex matrix, similar estradiol - 13C3 counts were detected starting from spiked horse serum compared to the same experiment from a pure analyte solution. This highlights the excellent ability to enrich and purify the target analyte from a complex matrix, which would not be possible to measure by direct injection from the initial sample.

[0268] Figure 16 Shows the application of particles (e.g., magnetic immunobeads) for the detection of estradiol - 13C3, with particle sample enrichment and extraction and ionization in the negative ion mode. Estradiol - 13C3 from a pure analyte mixture; particle workflow for superparamagnetic immunobeads conjugated with anti - estradiol antibody (“iBead(E2)”). Analyte concentrations were 42 ng / mL, 8 ng / mL, 4 ng / mL estradiol - 13C3 and compared to blank water. To each analyte mixture 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 - particle complex was washed twice with water (100 μL). Then the remaining analyte - particle complex was extracted with 80% ACN + 0.08 mM NH4F + NH4OH pH = 9.0 (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 the IMS - ToF negative ion mode using a Synapt G2 - Si mass spectrometer (Waters Corp.) with ion mobility separation (IMS; wave velocity 950 m / s and wave height 40 V) for a collection time of 60 seconds. Figure 16 Shows the relevant [M - H] of estradiol - 13C3 extracted at a drift time of 3.34 ms- Overlap of the full-scan mass spectra of the signals. For the test concentrations of 42 ng / mL, 8 ng / mL, and 4 ng / mL, the extraction and ionization of estradiol-13C3 directly from the immunobeads were successful, while the blank spectra showed little background signal in the observed mass region.

[0269] Figure 17 Illustrated is the application of microparticles (e.g., magnetic immunobeads) for the detection of testosterone-13C3, which applies microparticle-based sample enrichment and extraction and ionization in the positive ion mode. Testosterone-13C3 from a pure analyte mixture; the microparticle workflow of superparamagnetic immunobeads conjugated with anti-testosterone antibody ("iBead(Te)"). The analyte concentrations were 833 pg / mL, 417 pg / mL, and 83 pg / mL testosterone-13C3, and were compared with blank water. To each analyte mixture solution (100 μL) in a well plate, 15 μL of the iBead(Te) suspension (4 mg / mL) was added and incubated for 10 minutes. After magnetic separation and removal of the supernatant, the analyte-microparticle complex was washed 2 times with water (100 μL). Then the remaining analyte-microparticle complex was 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 the IMS-ToF positive ion mode using a Synapt G2-Si mass spectrometer (Waters Corp.) combined with ion mobility separation (IMS; wave velocity 850 m / s and wave height 40 V), with an acquisition time of 60 seconds. Figure 17 Illustrated is the relevant [M+H] of testosterone-13C3 extracted at a drift time of 2.98 ms + Overlap of the full-scan mass spectra of the signals. For the test concentrations of 833 pg / mL and 417 pg / mL, the extraction and ionization of testosterone-13C3 directly from the microparticles (e.g., immunobeads) were successful. Even at a concentration of 83 pg / mL, the signal was still high compared to the blank HS iBead(Te) extract.

[0270] The method of the present invention shows a valuable tool for measuring clinically important analytes at low concentrations. Successful examples of the application using specific microparticles (e.g., immunobeads) highlight the modularity and broad applicability, as customized microparticles (e.g., immunobeads) can be targeted for the detection of target analytes that would otherwise be impossible to achieve. In addition, different microparticle materials for different target analytes can be easily implemented on one instrument without changing the sample preparation and ionization processes themselves.

[0271] This patent application claims the priority of European Patent Application No. 22211164.3, the content of which is incorporated herein by reference.

[0272] List of references

[0273] 1 – Analysis module

[0274] 2 – Sample solution provided in the measurement cup

[0275] 3 – Pipetting unit

[0276] 4 – Analyte solution containing particulate suspension

[0277] 5 – Residual analyte adsorbed on the particles

[0278] 6 – Analysis module

[0279] 7 – Particle extraction module

[0280] 8 – Particle separation module for bead workflow

[0281] 9 – Particle extraction tip

[0282] 10 – Particle extraction solvent reservoir

[0283] 11 – Particle capture plate

[0284] 12 – Nanoliter spray chip

[0285] 13 – Analysis module inlet

Claims

1. A method for determining the presence or level of a target analyte in a sample by a chip-based nanoESI detection system, wherein the chip-based nanoESI detection system comprises a conductive pipette tip and a nanoelectrospray nozzle, the method comprising the steps of: a) providing the sample comprising the target analyte and a matrix, wherein the matrix is non-magnetic, b) providing microparticles, wherein the microparticles are magnetic, c) incubating the microparticles and the target analyte in a sample holder to form an analyte-microparticle complex, 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 complex in the sample holder, f) extracting the analyte from the analyte-microparticle complex by an extraction solvent and magnetic force, the step (f) comprising f1) providing the extraction solvent through the conductive pipette tip, f2) contacting the extraction solvent with the analyte-microparticle complex in the sample holder, f3) extracting the target analyte from the analyte-microparticle complex to form an extracted target analyte, wherein during the extraction step f3), the microparticles are retained in the sample holder by magnetic force, wherein the conductive pipette tip contains the extracted target analyte, g) directly contacting the conductive pipette tip containing the extracted target analyte with the nanoelectrospray nozzle of the chip-based nanoESI detection system to form a nanoelectrospray for ionizing the extracted target analyte, h) using the chip-based nanoESI detection system to determine the presence or level of the extracted target analyte in the sample, 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 target analyte is free of microparticles.

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

4. The method according to any one of claims 1 or 3, wherein the conductive pipette tip contains a microparticle content of less than 20%, 15%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, or 0.01% relative to the total content of the microparticles.

5. The method according to any one of the preceding claims, wherein the direct contact between the conductive pipette tip and the nozzle of the chip-based nanoESI detection system is a direct electrical contact.

6. The method according to any one of the preceding claims, wherein the microparticles are superparamagnetic or paramagnetic.

7. The method according to any one of the preceding claims, wherein the matrix comprises an analyte interference component derived from a biological sample, microparticles, a sample preparation solution, a mixture or combination thereof.

8. The method according to any one of the preceding claims, wherein the matrix is a solution.

9. The method according to any one of the preceding claims, wherein the method is automated and / or is performed in a random access mode.

10. The method according to any one of the preceding claims, wherein the method does not contain a chromatography step, and the chromatography step comprises 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.

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

12. A diagnostic system for determining the presence or level of a target analyte in a sample, the diagnostic system comprising a chip-based nanoESI source, a conductive pipette tip, and a detector for implementing the method according to any one of claims 1 to 10 preceding, wherein the chip-based nanoESI source comprises a nozzle, and wherein the detector uses mass spectrometry or 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 preceding.

14. A kit adapted to perform the method according to any one of claims 1 to 10 preceding, the kit comprising (A) microparticles for enriching or purifying a target analyte in a sample, (B) an extraction solvent for extracting the target analyte from the microparticles, (C) an optional internal standard, and (D) an optional catalyst or other reagent, such as a derivatization reagent.

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

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