A magnetic chemical selective probe kit containing a boronate cleavage site, and its preparation method and application

Abstract

The present invention discloses a magnetic chemical selective probe kit containing a boronate cleavage site, a preparation method and an application thereof, and belongs to the technical field of mass spectrometry labeling probes. The kit of the present invention consists of two parts: a magnetic probe and a magnet. The magnetic probe fixes the labeling reagent on the surface of magnetic ferroferric oxide nanoparticles through a reversible boronate reaction. The analyte metabolites are captured using the labeling reaction group. After the metabolites are captured and the interferences are removed, the labeled products are quickly released for subsequent mass spectrometry analysis by regulating the pH. The significant advantages of this pH-regulated magnetic separation and labeling method are that the probe is low in cost, easy to use, and the labeled products containing boron elements are easy to identify. By changing the labeling reaction group, the probe can expand the analysis of different types of metabolites, such as carboxyl, amino and thiol groups. The kit has been successfully used to analyze carboxyl compounds in complex samples such as serum, and has very important clinical application value and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry labeling probes, and in particular to a magnetic chemical selective probe kit containing a boronate cleavage site, and a preparation method and application thereof. Background Art

[0002] Chemoselective probe (CP) is a sample pretreatment technique that combines mass spectrometry-labeled probes with phase separation. Its unique characteristic is that, while retaining the functionality and structure of traditional mass spectrometry probes (mass spectrometry sensitization groups and reactive groups), it adds two new components: a linker arm containing a cleavage site and a solid-phase matrix. This allows the probe to not only label analytes but also separate and purify analytes from complex samples. The traditional CP usage process is as follows: First, the CP is incubated with the sample extract, and the reactive groups of the probe capture the analyte. Then, extraneous liquid matrix is ​​removed from the extract through physical separation. Next, under designed cleavage conditions, the labeled product is released. Finally, the labeled product is collected for liquid chromatography-mass spectrometry (LC-MS) analysis.

[0003] Because CP can improve ionization efficiency, reduce matrix effects, and improve chromatographic separation, it is very suitable for mass spectrometry analysis of small molecules in complex samples. Currently, CP has been widely used in various research fields, such as gut bacteria-host co-metabolite analysis, disease marker discovery, rapid metabolite screening, and metabolite network analysis. However, despite the current active research in metabolomics, chemically selective probes have many advantages in metabolite analysis.

[0004] For example, the literature (Chen J, Tian Y, Zhang YX, et al. Chemoselective probes serving as promising derivatization tools in targeted metabolomics research [J]. Journal of Analysis and Testing, 2020, 4 (3): 175-182.) reviewed the workflow and design of chemical selective probes, introduced their new application progress in natural product enrichment, metabolite derivatization and other fields, as well as their potential applications in metabolomics research. For another example, the literature (Garg N, Conway LP, Ballet C, et al. Chemoselective probe containing a unique bioorthogonal cleavage site for investigation of gut microbiota metabolism [J]. Angewandte Chemie International Edition, 2018, 57 (42): 13805-13809.) designed and synthesized a unique multifunctional mass spectrometry chemical probe containing p-nitrocinnamoyloxycarbonyl as a new bioorthogonal cleavage site. Combined with magnetic beads, this chemical probe can extract metabolites directly from human samples and release them under mild conditions.

[0005] However, the problems of existing CPs are also very obvious. First, the preparation of probes is complicated, and the traditional step-by-step modification method can have as many as a dozen steps in the synthesis; moreover, the labeling efficiency of CP is poor, and the product release conditions are harsh and time-consuming. The release conditions of strong acid-base or strong redox reactions may affect the stability of the product, and mild enzymatic or photolysis takes a long time, so finding a suitable cleavage site is extremely urgent. Secondly, the cost of using stable isotope-coded probes is high, which limits their application and promotion. Finally, the probe coverage and throughput are insufficient. In the face of intertwined metabolic networks, simultaneous analysis of multiple types of sub-metabolomes is very necessary.

[0006] In view of this, the development and preparation of high-sensitivity magnetic CPs with simple preparation, rapid labeling and release, low probe cost, and the ability to label various types of analytes have very important practical application value and economic value. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, in a first aspect of the present invention, a method for preparing a magnetic chemical selective probe kit with convenient process is provided, comprising the following steps:

[0008] (1) Using a silane coupling agent to treat ferroferric oxide magnetic nanoparticles, the surface of the nanoparticles is coated with silicon dioxide to obtain silicon-coated particles;

[0009] (2) Modifying the silicon-coated particles using 3-Methacryloxypropyltrimethoxysilane (γ-MPS) to introduce carbon-carbon double bonds on the surface of the silicon-coated particles to obtain double-bond modified particles;

[0010] (3) mixing double-bond modified particles, a functional monomer, a porogen, a crosslinker, and an initiator, wherein the functional monomer molecule includes two characteristic structures: a terminal carbon-carbon double bond and a cis-dihydroxyl group; and performing a polymerization reaction to obtain hydroxyl-modified particles having cis-dihydroxyl groups on the surface;

[0011] (4) mixing the hydroxyl-modified particles with a solution of a probe reagent, wherein the molecular structure of the probe reagent includes a boronic acid group, a labeling reaction group, and a mass spectrometry sensitization group; incubating the cis-dihydroxyl group and the boronic acid group to form a borate ester, thereby obtaining a magnetic chemically selective probe;

[0012] (5) The magnetic chemical selective probe is combined with the magnet component of the kit to obtain a magnetic chemical selective probe kit.

[0013] The probe uses ferroferric oxide magnetic nanoparticles, which have the advantages of low cost, easy preparation, convenient operation, efficient separation, and stable physicochemical properties. The magnetic nanoparticles that expose cis-dihydroxyl groups after elution can be reused.

[0014] Preferably, the specific operation of step (1) is as follows: using ferrosoferric oxide nanoparticles and tetraethoxysilane (TEOS) as reactants, and using water, ammonia water, and ethanol as a solution; mixing the reactants with the solution and reacting them, and purifying after the reaction is completed to obtain silicon-coated particles containing silicon dioxide on the surface.

[0015] Further preferably, the volume ratio of ammonia water, ethanol and water is 1: (5-10): (20-40); the mass ratio of ferrosoferric oxide nanoparticles to tetraethoxysilane is (20-50): 1; and the mass ratio of solution to reactant is 1: (1-3).

[0016] Preferably, in step (1), the reaction temperature is room temperature and the reaction time is 10-14 h.

[0017] Preferably, the specific operation of step (2) is as follows: adding the silicon-coated particles to a solvent, mixing to make them uniformly dispersed, and then adding methacryloxypropyltrimethoxysilane for modification, and reacting to form double-bond modified particles containing carbon-carbon double bonds on the surface.

[0018] More preferably, the mass ratio of the silicon-coated particles, toluene, and methacryloxypropyltrimethoxysilane is (2-4):1:(2-4).

[0019] Preferably, in step (2), the reaction temperature is room temperature and the reaction time is 20-28 h.

[0020] Preferably, the specific operation of step (3) is as follows: double-bond modified particles, functional monomers, porogens, crosslinking agents, and initiators are mixed, and the porogen uses a low-boiling point solvent; within a certain period of time, the resulting mixture is heated until the porogen boils, and the heating temperature reaches the polymerization reaction requirement; the boiling state is maintained, and half of the amount of the porogen is evaporated within a certain period of time; then, the product is cooled, recovered, and purified to obtain hydroxyl-modified particles containing cis-dihydroxy groups on the surface.

[0021] Further preferably, the mass ratio of the double bond modified particles, functional monomer, and crosslinking agent is (1-3):1:(1-3); the added amount of the initiator is 0.2 wt.%-5 wt.% of the total mass of the functional monomer, crosslinking agent, and porogen.

[0022] The heating control is carried out in two steps. The first heating time is relatively short, giving a certain amount of time to prevent boiling and large-scale volatilization of the porogen. The second heating provides the necessary reaction time for the polymerization reaction and porogenation process.

[0023] More preferably, the time is 10 min-2 h, and 1-4 h, respectively.

[0024] Preferably, in step (3), the functional monomer includes one of 3-methacrylamide dopamine (N-Methacryldopamine, MDA), N-(3,4-dihydroxybutyl) acrylamide, and N-(4,5-dihydroxypentyl) acrylamide.

[0025] In the art, the molecular design of probe reagents usually contains specific functional groups, such as labeling reaction groups and mass spectrometry sensitization groups, to achieve their application in analytical chemistry. The labeling reaction group is the part of the probe molecule used to bind to the target analyte. It can be an antibody antigen, a fluorophore, an enzyme label, a radioisotope or a stable isotope, etc., which is used to improve the detection sensitivity and specificity of the probe. The mass spectrometry sensitization group is a structure used to improve the ionization efficiency of the analyte in the mass spectrometer, which is crucial for improving the sensitivity of mass spectrometry analysis and reducing the detection limit. These groups together ensure the effectiveness and sensitivity of the probe in analytical chemistry. For example, in an embodiment of the present invention, based on the type of groups contained in the analyte, corresponding probe reagents such as p-ethylaminophenylboronic acid, 4-carboxylphenylboronic acid, and 4-(maleimidohexanamide-N-methyl)phenylboronic acid are used, wherein the benzene ring is used to increase the hydrophobicity of the labeled product and increase reverse phase chromatography retention; the nitrogen-containing part is the mass spectrometry sensitization group. The selection of labeling reaction groups and mass spectrometry sensitization groups is flexible based on actual application, and those skilled in the art can select the appropriate type according to the type of analyte.

[0026] Preferably, in step (4), the labeling reaction group is selected based on the type of analyte of the chemical selective probe kit; when the analyte contains a carboxyl group, the labeling reaction group is selected as an amino group; when the analyte contains an amino group, the labeling reaction group is selected as a carboxyl group or a succinimide group; when the analyte contains a sulfhydryl group, the labeling reaction group is selected as a maleimide or a sulfhydryl group; when the analyte contains a carbonyl group, the labeling reaction group is selected as a hydrazide or a hydroxylamine group; when the analyte contains an alkynyl group, the labeling reaction group is selected as an azide group.

[0027] Porogens are typically volatile solvents that, after being mixed into the polymer matrix, evaporate during the material's molding or curing process, leaving pores within the material. Any solvent with good volatility and the ability to disperse and dissolve the raw materials can be used as a porogen. In the present invention, acetonitrile is a particularly suitable porogen.

[0028] Preferably, in step (4), the porogen includes at least one of water, methanol, acetonitrile, cyclohexane, toluene, 1,4-butanediol, dodecanol, and n-propanol; the crosslinker includes one of N,N'-methylenebisacrylamide (MBA) and ethylene glycol dimethacrylate (EDMA); and the initiator includes one of 2,2'-azobisisobutyronitrile (AIBN), ammonium persulfate, and benzoyl peroxide.

[0029] Preferably, in step (4), the incubation temperature is room temperature and the incubation time is 5 min-4 h.

[0030] Preferably, in step (4), the mass ratio of the hydroxyl-modified particles to the probe reagent solution is 1:(20-200); and the concentration of the probe reagent solution is 5 mg / mL.

[0031] In this area, solvents are used as media to promote dispersion of raw materials and to create suitable reaction conditions. Those skilled in the art can select suitable solvent types based on the physicochemical properties of the optional raw materials involved in this method, in combination with actual conditions. Under the premise that the technicians know the above principles, the choice of solvent is not unique, and for example, toluene, acetonitrile, etc., all belong to suitable solvent types of the present invention.

[0032] In the second aspect of the present invention, a chemical selective probe kit is provided which has rapid labeling and releasing, low use cost, and the ability to label various types of analytes. The kit is prepared using the preparation method of the first aspect of the present invention.

[0033] In a third aspect of the present invention, an application of the chemical selective probe kit according to the second aspect of the present invention is provided, specifically combining the chemical selective probe kit with a mass spectrometer for sample detection and identification.

[0034] In this application, a rapid identification strategy using the abundance of natural boron isotopes for screening can rapidly identify labeled products without stable isotope coding technology, significantly reducing the cost of using probes in large-scale sample analysis scenarios and facilitating method promotion. Furthermore, high-throughput analysis methods combining chemical selective labeling with mass spectrometry can simultaneously study different types of analytes in complex samples, obtaining accurate and sensitive qualitative and quantitative results.

[0035] Preferably, the application comprises the following steps:

[0036] S1. extracting the sample using a solvent to obtain an extract;

[0037] S2. taking a predetermined amount of the extract and adding it to the chemical selective probe kit, and incubating to capture the analyte in the sample;

[0038] S3, magnetic separation of the chemically selective probe, removal of the supernatant and purification;

[0039] S4, eluting the magnetically immobilized chemically selective probe with an acidic solution to release the labeled product for mass spectrometry analysis;

[0040] S5. Screen the labeled products based on the natural isotope abundance characteristics of boron.

[0041] The shortcomings of the preparation and use of traditional CP test kits are very obvious. First, the preparation of probes is complicated, and the synthesis steps of the traditional step-by-step modification method can be as many as more than a dozen steps; moreover, the labeling efficiency of CP is poor, and the product release conditions are harsh and time-consuming. The release conditions of strong acid-base or strong redox reactions may affect the stability of the product, and mild enzymatic or photolysis are time-consuming, so it is extremely urgent to find a suitable cleavage site. Secondly, the cost of using stable isotope-coded probes is high, which limits their application and promotion. Finally, the probe coverage and flux are insufficient. In the face of intertwined metabolic networks, simultaneous analysis of multiple types of sub-metabolomes is very necessary. To this end, the present invention focuses on modifying the construction strategy of the CP test kit, simplifying the preparation steps, and reducing the cost of using the CP test kit, which has high clinical application value and practical value.

[0042] Based on the above technical solution, the design concept and principle of the present invention lies in first coating the surface of easily separable magnetic ferroferric oxide nanoparticles with silica. Then, using methacryloxypropyltrimethoxysilane modification, carbon-carbon double bonds are introduced onto the surface. Subsequently, through double bond polymerization, these double bonds are linked to functional monomers to form linkers. A porogen is then incorporated into the organic polymer coating on the particles and the outer silica layer to form pores. These micron-sized pores act as penetrating pores to enhance reaction kinetics, while the mesoporous effect primarily provides a confined reaction site. Furthermore, the present invention introduces a boronic acid group beyond the conventional molecular structure of the probe reagent. The cis-dihydroxyl groups present in the linker are incubated with the probe reagent containing the boronic acid group, and the resulting boronate ester groups are bonded. Boronic acid groups have an affinity for cis-dihydroxyl structures. For example, in the inventors' prior application (CN117147669A), the inventors used this principle to design functional monomers corresponding to nucleosides, carbohydrates, and glycoproteins. In the present invention, the inventors have further deepened the utilization of borate groups. The functions of borate are mainly divided into two aspects: in the chemically selective probe, borate is connected to the ferroferric oxide nanoparticle part for easy separation and the probe reagent for capturing the analyte. After sampling, the probe reagent is extracted along with the separation. In the detection and identification of samples, the inventors use the characteristics of borate that is stable in neutral and alkaline conditions and easily cleaved in acidic conditions. It is used as a cleavage site and eluted in an acidic solution, so that the labeled product containing the analyte group can be easily separated and analyzed by mass spectrometry.

[0043] The effects of the above designs and improvements are mainly reflected in the following aspects. The density of the labeling reagent of the present invention is higher than that of the reported technical solutions, and the collision probability of the reaction is higher, which is conducive to the reaction; secondly, the porous structure on the surface of the chemically selective probe test material utilizes through-holes to improve the reaction kinetics, and the mesopores provide a confinement effect, thereby improving the labeling efficiency. Moreover, the magnetic separation mode is simple and efficient, allowing for simultaneous pre-treatment of multiple independent samples. In the face of the simultaneous analysis of intertwined metabolic networks and multiple types of sub-metabolomes, it is only necessary to label multiple types of chemically selective probes with labeled reaction groups with the same sample at the same time, so that multiple types of metabolites can be analyzed simultaneously, solving the technical defects of insufficient probe coverage and flux.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] The present invention provides a method for preparing a magnetic chemoselective probe kit. The magnetic chemoselective probe constructed based on borate reaction and ferroferric oxide nanoparticles can realize convenient preparation of the probe and separation of the labeled product under mild conditions. Moreover, different types of chemoselective probes can use the same ferroferric oxide nanoparticles. The labeling objects of the probe can be expanded by simply replacing the labeling reaction group at the end of the probe reagent, which has good versatility.

[0046] The present invention provides a magnetic chemical selective probe kit, which has the advantages of rapid separation and release of labeled products, low use cost, and the ability to label various types of analytes.

[0047] The present invention provides an application of a magnetic chemical selective probe kit, which can accurately and sensitively obtain qualitative and quantitative results and has the advantage of being easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the preparation process of the magnetic chemical selective probe in Example 1;

[0049] Figure 2 This is a scanning electron microscope image of the magnetic chemical selective probe in Example 2;

[0050] Figure 3 This is the labeling optimization diagram of the carboxyl magnetic chemoselective probe in Example 3;

[0051] Figure 4 This is a diagram showing the labeling results of magnetic chemical selective probes on compounds with different structures in Example 4;

[0052] Figure 5 This is the fragmentation characteristic diagram of the magnetic chemical selective probe labeled product in Example 5;

[0053] Figure 6 This is a diagram of the serum targeted spike experiment of the carboxyl magnetic chemoselective probe in Example 6;

[0054] Figure 7 The non-targeted experimental process of the carboxyl magnetic chemical selective probe serum and the logarithmic graph of the screened peaks in Example 7;

[0055] Figure 8 Schematic diagram of the use of the magnetic chemical selective probe kit. DETAILED DESCRIPTION

[0056] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0057] In the following embodiments:

[0058] Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) with a purity of 98%, CAS number: 94790-37-1, was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0059] 1-Hydroxybenzotriazole (HOBT), purity 98%, CAS number: 2592-95-2, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0060] N,N-Diisopropylethylamine (DIEA), purity 99%, CAS number: 7087-68-5, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0061] N1-((Ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (EDCI), purity 99%, CAS number: 7084-11-9, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.;

[0062] Triethylamine (TEA), purity 99.5%, CAS number: 121-44-8, purchased from Shanghai Mairui Biochemical Technology Co., Ltd.

[0063] 2-Chloro-1-methylpyridinium iodide (CMPI), purity 98%, CAS number: 14338-32-0, was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0064] Example 1

[0065] Preparation method of magnetic chemical selective probe kit, such as Figure 1 As shown, the steps are as follows:

[0066] (1) Using ferroferric oxide magnetic nanoparticles as raw material, the magnetic particles were coated with silicon using the Stober method; 500 mg of commercial ferroferric oxide nanoparticles were ultrasonically dispersed in a flask containing 25 mL of pure water; 20 mL of TEOS, 25 mL of ammonia water, 221.67 mL of pure water and 778.33 mL of ethanol were then evenly dispersed in a 2 L three-necked flask, and then the ultrasonically dispersed ferroferric oxide nanoparticles were added; the reaction was carried out at room temperature and a speed of 500 rpm for 12 h; after the reaction, the particles were washed alternately with methanol and water, and then dried with nitrogen for later use to obtain silicon-coated particles with silicon dioxide on the surface;

[0067] (2) 500 mg of the silicon-coated magnetic material was ultrasonically dispersed in 205 mL of toluene solution, and then 450 mg of γ-MPS was added. The mixture was reacted at room temperature and 300 rpm for 24 h. After the reaction, the mixture was washed alternately with methanol and water, and dried with nitrogen to obtain double-bond modified particles with carbon-carbon double bonds on the surface.

[0068] (3) First, 500 mg of double-bond modified particles were ultrasonically dispersed in 192 mL of acetonitrile. Then, 504 mg of 3-MDA, 583 mg of EDMA, and 7.5 mg of AIBN were added to the solution in sequence. The three-necked flask was then placed in a 50 °C water bath. Within 30 min, the water bath was heated to boiling acetonitrile and kept boiling. Half of the acetonitrile was evaporated within 2 h. After the reaction, the particles were washed alternately with methanol and water and dried with nitrogen to obtain hydroxyl-modified particles containing cis-dihydroxy groups on the surface, which were recorded as Fe3O4@SiO2@MPS@MDA.

[0069] (4) Synthesis of thiol labeling reagent: 200 mg of 6-maleimidocaproic acid, 432 mg of HBTU, 154 mg of HOBT, and 213 mg of 4-aminomethylphenylboronic acid hydrochloride were dissolved in 10 mL of DMF; 785 mL of DIEA was slowly added dropwise; the reaction was carried out at room temperature for 12 h.

[0070] Synthesis of amino-labeling reagent: Dissolve 200 mg of 4-carboxyphenylboronic acid, 340 mg of EDCI, and 320 mg of HOBT in 10 mL of DMF; add 360 mg of TEA, mix at room temperature for 10 min, and then add 207 mg of N-hydroxysuccinimide; react at room temperature for 12 h.

[0071] Carboxyl labeling reagent: 4-((2-aminoethyl)phenyl)borate hydrochloride is a commercial product;

[0072] The cleaned and dried Fe3O4@SiO2@MPS@MDA was divided into three groups of samples, each with 20 mg. 1 mL of 5 mg / mL carboxyl labeling reagent, amino labeling reagent, and thiol labeling reagent were added to each sample. The samples were incubated at room temperature for 3 hours. After incubation, the supernatant was removed with the aid of a magnet. The probe was washed twice with acetonitrile and dried with nitrogen to obtain a magnetic chemoselective probe.

[0073] (5) The prepared magnetic chemical selective probe is combined with the universal components of the kit to obtain a magnetic chemical selective probe kit.

[0074] Example 2

[0075] This example characterizes the morphology of the magnetic chemical selective probe in the magnetic chemical selective probe kit prepared in Example 1. The physical morphology of the magnetic chemical selective probe (hereinafter referred to as the magnetic probe) during its synthesis was investigated using scanning electron microscopy. Figure 2 shown.

[0076] Figure 2 (a) Scanning electron microscopy analysis of the morphology of commercial bare Fe3O4 nanoparticles and modified nanomagnetic beads. The morphology of commercial bare Fe3O4 nanoparticles varies, including spherical and cubic shapes, with an average diameter of approximately 150 nm. Figure 2 (b) Fe3O4 nanoparticles after coating with SiO2, whose size grows to about 250 nm and the morphology remains spherical and cubic. Figure 2 (c) γ-MPS and MDA-modified ferroferric oxide nanoparticles. The average diameter of the magnetic nanoparticles increased to about 300 nm, and the morphology was spherical and cubic. Figure 2 This is a carboxyl magnetic chemoselective probe. The particle size increases slightly, reaching approximately 370 nm. At this point, the number of cubic particles decreases, while more particles exhibit irregular spherical shapes. The SEM image shows a translucent modification outside the magnetic core, indicating successful modification.

[0077] Example 3

[0078] In this embodiment, the labeling conditions of the magnetic chemical selective probe kit in Example 1 of the present invention were optimized to study its performance in practical applications.

[0079] First, prepare 1 ppm and 10 ppm mixed standards. The carboxyl mixed standard contains n-pentanoic acid, cyclohexanoic acid, octanoic acid, deoxycholic acid, 8-aminooctanoic acid and deuterated n-pentanoic acid-d9; the amino mixed standard contains glycine, L-alanine, methionine, cyclohexylamine and deuterated glycine-d2; the sulfhydryl mixed standard contains L-cysteine, DL-homocysteine, N-acetylcysteamine, N-acetyl-L-cysteine, cysteinylglycine and 2-naphthol. The data here only takes the carboxyl magnetic chemical selective probe (hereinafter referred to as carboxyl magnetic probe, the nomenclature of other labeling reaction groups is the same) as an example, and the results are as follows. Figure 3 shown. Figure 3 In the figure, (a) is the quality optimization diagram of the carboxyl magnetic probe; (b) is the catalyst dosage optimization diagram of the carboxyl magnetic probe; (c) is the labeling time optimization diagram of the carboxyl magnetic probe; (d) is the elution number optimization diagram of the carboxyl magnetic probe.

[0080] (1) Optimization of probe dosage

[0081] A series of carboxyl, amino, and thiol magnetic chemoselective probes (2, 5, 10, 20, 30, and 40 mg, respectively) were placed in EP tubes. To the carboxyl probe, 500 mL of a 1 ppm carboxyl standard mix, 20 mL of TEA, and 20 mL of CMPI (20 mmol / mL) were added. To the amino probe, 500 mL of a 1 ppm amino standard mix and 20 mL of TEA (20 mmol / mL) were added. To the thiol probe, 500 mL of a 1 ppm thiol standard mix was added. The reaction was carried out at 40°C, room temperature, and room temperature for 30 min, respectively. After the reaction, the sample was washed twice with acetonitrile and eluted twice with 200 mL of eluent (0.1 M HCl:ACN = 1:9). The eluent was evaporated to dryness under nitrogen and reconstituted with 100 mL of resolvent (ACN:H₂O = 1:9) for LC-MS injection. Because the types and concentrations of metabolites are unknown in non-targeted metabolomics analysis, the probe dosage must be kept to a minimum to prevent failure to capture more metabolite information. Furthermore, experimental results indicate that probe dosages above 20 mg generally flatten out, with slight increases or decreases related to the probe's nonspecific adsorption or labeling efficiency. Therefore, the optimal magnetic probe dosage is 20 mg.

[0082] (2) Optimization of the catalyst dosage of the probe

[0083] 20 mg of each carboxyl and amino magnetic chemoselective probe was placed in an EP tube. 500 mL of the corresponding 1 ppm mixed standard was then added to each probe. 20 mmol / mL CMPI and 20 mmol / mL LTEA were then added to the carboxyl probe as catalysts, while 20 mmol / mL TEA was added to the amino probe as catalyst. Catalyst dosages of 10, 20, 40, 60, 100, and 200 mL were used, respectively, and the reaction was carried out at 40°C and room temperature for 30 min. After the reaction, the tube was washed twice with acetonitrile and then eluted twice with 200 mL of eluent (0.1 M HCl:ACN = 1:9). The eluent was then dried under nitrogen and reconstituted with 100 mL of reconstitution solvent (ACN:H2O = 1:9) for LC-MS injection. The overall trend of the catalyst dosage did not change much, and 40 mL was the optimal dosage. When the catalyst dosage continued to increase, the peak area no longer changed significantly, and a large amount of catalyst would also bring about the problem of matrix effect.

[0084] (3) Optimization of probe labeling time

[0085] 20 mg of each carboxyl, amino, and thiol magnetic chemoselective probe was placed in an EP tube. To the carboxyl probe, 500 mL of a 1 ppm carboxyl standard mix, 20 mL of TEA, and 20 mL of CMPI (20 mmol / mL) were added. To the amino probe, 500 mL of a 1 ppm amino standard mix and 20 mL of TEA (20 mmol / mL) were added. To the thiol probe, 500 mL of a 1 ppm thiol standard mix was added. The reaction was carried out at 40°C, room temperature, and room temperature for 5, 10, 20, 30, and 60 min, respectively. After completion of the reaction, the tube was washed twice with acetonitrile and eluted twice with 200 mL of eluent (0.1 M HCl:ACN = 1:9). The eluent was then dried under nitrogen and reconstituted with 100 mL of resolvent (ACN:H₂O = 1:9) for LC-MS injection. The probe reached its peak response at 20 min.

[0086] (4) Optimization of probe elution times

[0087] 20 mg of each carboxyl, amino, and thiol magnetic chemoselective probe was placed in an EP tube. To the carboxyl probe, 500 mL of a 10 ppm carboxyl standard mix, 20 mL of TEA, and 20 mL of CMPI (20 mmol / mL) were added; to the amino probe, 500 mL of a 10 ppm amino standard mix and 20 mL of TEA (20 mmol / mL); and to the thiol probe, 500 mL of a 10 ppm thiol standard mix. The reaction was then incubated at 40°C, room temperature, and room temperature for 30 min, respectively. After completion of the reaction, the sample was washed twice with acetonitrile and then eluted with 200 mL of eluent (0.1 M HCl:ACN = 1:9) for 2, 3, 4, 5, and 6 cycles, respectively. The eluent was then dried under nitrogen and reconstituted with 100 mL of reconstitution solvent (ACN:H₂O = 1:9) for LC-MS injection. The probe response gradually increased with increasing elution times, but the trend was not significant.

[0088] Example 4

[0089] This example studies the universality of the magnetic chemical selective probe kit in Example 1 for labeling different types of analytes. Figure 4 shown. Figure 4 In the figure, (a)-(d) are the labeling results of carboxyl magnetic probes on compounds with different structures; (e)-(h) are the labeling results of amino magnetic probes on compounds with different structures; (i)-(l) are the labeling results of thiol magnetic probes on compounds with different structures.

[0090] 20 mg of each carboxyl, amino, and thiol magnetic chemoselective probe was placed in an EP tube. To the carboxyl probe, 500 mL of a 10 ppm carboxyl standard mix, 20 mL of TEA, and 20 mL of CMPI (20 mmol / mL) were added. To the amino probe, 500 mL of a 10 ppm amino standard mix and 20 mL of TEA (20 mmol / mL) were added. To the thiol probe, 500 mL of a 10 ppm thiol standard mix was added. The reaction was then incubated at 40°C, room temperature, and room temperature for 30 min, respectively. After completion of the reaction, the sample was washed twice with acetonitrile and eluted twice with 200 mL of eluent (0.1 M HCl:ACN = 1:9). The eluent was then dried under nitrogen and reconstituted with 100 mL of resolvent (ACN:H₂O = 1:9) for LC-MS injection.

[0091] Carboxyl, amino, and thiol magnetic chemoselective probes are used to specifically label carboxyl, amino, and thiol metabolites through CMPI / TEA coupling reaction, NHS active ester reaction, and thiol-maleimide click chemistry reaction, respectively. Four structurally different compounds were selected for each type of analyte (carboxyl compounds: n-pentanoic acid, o-methylbenzoic acid, deoxycholic acid, and cyclohexanecarboxylic acid; amino compounds: glycine, butylamine, aniline, and cyclohexylamine; thiol compounds: cysteamine, cysteine, naphthiophenol, and N-acetylcysteine) to investigate the labeling of different types of analytes by the probes, such as Figure 4 The experimental results show that the three probes can successfully detect compounds of different structural types and also exhibit boron-specific isotope peaks. Therefore, this type of magnetic probe kit can be used to label different types of metabolites in complex samples.

[0092] Example 5

[0093] This example studies the mass spectrometry fragmentation characteristics of the product labeled by the magnetic chemical selective probe kit in Example 1. Figure 5 shown. Figure 5 Among them, (a)-(b) are the secondary fragmentation characteristic diagrams of carboxyl-labeled products; (c)-(d) are the secondary fragmentation characteristic diagrams of amino-labeled products; (e)-(f) are the secondary fragmentation characteristic diagrams of sulfhydryl-labeled products.

[0094] The mass spectrometric fragmentation characteristics of the labeled products were investigated in liquid-liquid reactions. 20 mL of each carboxyl, amino, and thiol labeling reagent was placed in an EP tube. To the carboxyl labeling reagent, 500 mL of a 10 ppm carboxyl standard mix, 20 mL of TEA, and 20 mL of CMPI (20 mmol / mL) were added. To the amino labeling reagent, 500 mL of a 10 ppm amino standard mix and 20 mL of TEA (20 mmol / mL) were added. To the thiol labeling reagent, 500 mL of a 10 ppm thiol standard mix was added. The reactions were then incubated at 40°C, room temperature, and room temperature for 30 min, respectively. After the reaction, the eluent was dried under nitrogen and reconstituted with 100 mL of a reconstitution solvent (ACN:H₂O = 1:9) for LC-MS injection.

[0095] The labeled metabolites will show certain mass spectrometric characteristics, such as characteristic fragments, specific fracture modes, natural isotope abundance, in-source cleavage, etc., which are of great help in qualitative and quantitative analysis. Figure 5 As shown, the characteristic fragments of the carboxyl-labeled product are derived from the characteristic fragments of the labeling reagent ( m / z 166.1034, 149.0768, 131.0663 or 105.0699); the characteristic fragments of the amino-labeled products are derived from the characteristic fragments of the labeling reagent ( m / z166.0670, 149.0405 or 105.0335); the characteristic fragments of the thiol-labeled product are derived from the characteristic fragments of the labeling reagent ( m / z 152.0877, 135.0612 or 91.0542). The three labeled products all break in the same way, resulting in [M+H-44] + The secondary mass spectrometry fragments lose one water and produce [M+H-18] + By identifying characteristic fragments, different types of compounds can be classified even with a single injection.

[0096] Example 6

[0097] This example studies the targeted screening of different types of metabolites in human serum by the magnetic chemical selective probe kit in Example 1. Figure 6 shown. Figure 6 In the figure, (a)-(c) are the chromatograms and mass spectra of the cyclohexane acid-labeled product in serum; (d)-(f) are the chromatograms and mass spectra of the o-methylbenzoic acid-labeled product in serum; (g)-(h) are the chromatograms and mass spectra of the n-pentanoic acid-labeled product in serum.

[0098] The carboxyl magnetic chemoselective probe kit of Example 1 of the present invention was used for targeted metabolome analysis in human serum samples. 3-aminophenylboronic acid was used as the labeling reagent for carboxyl groups. In this example, the working solution was prepared similarly to that of Example 3, but at a concentration of 10 ppm. The serum sample preparation process was as follows: 120 mL of serum sample was extracted with 650 mL of a 10 ppm carboxylic acid mixed standard solution prepared with ACN. After sonication on ice for 30 minutes, the sample was centrifuged at 10,000 rpm at 4°C, and the supernatant was collected. A control group used ACN in place of the carboxylic acid mixed standard solution, with all other parameters remaining unchanged. Serum labeling: 20 mL of CMPI (20 mmol / mL) and 20 mL of TEA (20 mmol / mL) were added to 100 mL of serum sample (spike group, control group, and blank group). Then, 20 mg of the carboxyl magnetic probe (labeling reagent: 3-aminophenylboronic acid) was added. Labeling was incubated at 40°C for 30 minutes.

[0099] like Figure 6 Figure 2 shows the chromatograms and mass spectra of a control group and a spiked group of three carboxylic acid-labeled products. The mass spectra of the control and spiked groups show distinct mass spectral responses, and the labeled products can be accurately identified based on the boron isotope peak clusters. These examples demonstrate the potential of magnetic chemoselective probe kits for targeted metabolomics analysis of trace targets in complex samples.

[0100] Example 7

[0101] This example studies the non-targeted screening and qualitative analysis of different types of metabolites in human serum by the magnetic chemical selective probe kit in Example 1. Figure 7 shown. Figure 7 In the figure, (a) is the data processing flow chart for non-targeted screening of carboxyl metabolites in serum; (b) is the logarithm of peaks screened after extraction.

[0102] In this example, the serum labeling process is similar to that of Example 6. However, the data processing method is as follows. Figure 7 As shown in (a), after MZmine data extraction, a total of 22,243 peak pairs were obtained. After MATLAB condition screening, 78 peaks containing characteristic isotopic peaks were found. Thermo Xcalibur software generated 19 predicted molecular formulas from these characteristic peaks. After subtracting the elements introduced by the labeling reagent, further matching was performed against the public database HMDB (https: / / hmdb.ca / ), ultimately identifying five molecules that are likely carboxylic acid metabolites. In summary, this demonstrates that magnetic chemoselective probes based on boron element signatures are a convenient and accurate metabolite screening strategy.

[0103] The usage process of the above magnetic chemical selective probe kit is as follows Figure 8 shown.

[0104] In summary, the present invention first coats the surface of ferrosoferric oxide nanoparticles for easy separation with silica, then uses methacryloxypropyltrimethoxysilane modification to introduce carbon-carbon double bonds on the surface, which are then connected to functional monomers through double bond polymerization to form connecting arms, and combines with porogens to form channels at the organic polymer coating on the outer layer of the particles and silica. The effect of the micron-scale channels is to improve the reaction kinetics as penetrating holes, while the mesoporous effect is mainly to provide a confined effect as a reaction site. The present invention introduces a boric acid group outside the conventional probe reagent molecular structure design, and the cis-dihydroxy group present in the connecting arm is incubated with the probe reagent containing the boric acid group, and connected through the borate ester group formed by the reaction. Boronic acid groups have an affinity for cis-dihydroxy structures. The inventors have further deepened the utilization of borate ester groups. The functions of borate esters are mainly divided into two aspects: in chemically selective probes, borate esters are connected to the easy-to-separate ferroferric oxide nanoparticle part and the probe reagent used to capture the analyte. After sampling, the probe reagent is extracted along with the separation. In the detection and identification of samples, the inventors use the characteristics of borate esters that are stable in neutral and alkaline conditions and easily cleaved in acidic conditions. They use them as cleavage sites and elute them in acidic solutions, so that the labeled products containing analyte groups can be easily separated and analyzed by mass spectrometry.

[0105] Compared with traditional methods, this method offers significant advantages in rapid and cost-effective probe preparation, rapid labeling reaction, and easy identification of boron-containing labeled products. By varying the labeling reactive group, the probe can be expanded to analyze different types of metabolites, such as carboxyl, amino, and sulfhydryl groups. This kit has been successfully used to analyze carboxyl compounds in complex samples such as serum, demonstrating significant clinical and economic value.

[0106] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a magnetic chemical selective probe kit, characterized in that: The steps include: (1) Using a silane coupling agent to treat ferroferric oxide nanoparticles, the surface of the nanoparticles is coated with silicon dioxide to obtain silicon-coated particles; (2) Modifying the silicon-coated particles with methacryloxypropyltrimethoxysilane to introduce carbon-carbon double bonds on the surface of the silicon-coated particles to obtain double-bond modified particles; (3) mixing double-bond modified particles, a functional monomer, a porogen, a crosslinker, and an initiator, wherein the functional monomer molecule includes two characteristic structures: a terminal carbon-carbon double bond and a cis-dihydroxyl group; and performing a polymerization reaction to obtain hydroxyl-modified particles having cis-dihydroxyl groups on the surface; (4) mixing the hydroxyl-modified particles with a solution of a probe reagent, wherein the molecular structure of the probe reagent includes a boronic acid group, a labeling reaction group, and a mass spectrometry sensitization group; incubating the cis-dihydroxyl group and the boronic acid group to form a borate ester, thereby obtaining a magnetic chemically selective probe; The labeling reaction group is selected based on the type of analyte in the chemical selective probe kit; when the analyte contains a carboxyl group, the labeling reaction group is selected as an amino group; when the analyte contains an amino group, the labeling reaction group is selected as a carboxyl group or a succinimide group; when the analyte contains a sulfhydryl group, the labeling reaction group is selected as a maleimide or a sulfhydryl group; when the analyte contains a carbonyl group, the labeling reaction group is selected as a hydrazide group; when the analyte contains an alkyne group, the labeling reaction group is selected as an azide group; (5) The magnetic chemical selective probe is combined with the magnet component of the kit to obtain a magnetic chemical selective probe kit.

2. The method for preparing the chemical selective probe kit according to claim 1, wherein The specific operation of step (1) is as follows: using ferrosoferric oxide nanoparticles and tetraethoxysilane as reactants, and using water, ammonia water, and ethanol as solution; mixing the reactants with the solution and reacting, and after the reaction is completed, purifying to obtain silicon-coated particles containing silicon dioxide on the surface; wherein the volume ratio of ammonia water, ethanol, and water is 1: (5-10): (20-40).

3. The method for preparing the chemical selective probe kit according to claim 1, wherein: In the step (1), the mass ratio of ferrosoferric oxide nanoparticles to tetraethoxysilane is (20-50):1; the mass ratio of the solution to the reactant is 1:(1-3); the reaction temperature is room temperature, and the reaction time is 10-14 h.

4. The method for preparing the magnetic chemical selective probe kit according to claim 1, wherein The specific operation of step (2) is as follows: adding the silicon-coated particles to the solvent, mixing to make them uniformly dispersed, and then adding methacryloxypropyltrimethoxysilane for modification, to form double-bond modified particles with carbon-carbon double bonds on the surface through reaction; the mass ratio of the silicon-coated particles, toluene, and methacryloxypropyltrimethoxysilane is (2-4):1:(2-4); the reaction temperature is room temperature, and the reaction time is 20-28 h.

5. The method for preparing the magnetic chemical selective probe kit according to claim 1, wherein The specific operation of step (3) is as follows: double bond modified particles, functional monomers, porogens, crosslinking agents, and initiators are mixed, and the porogen uses a low boiling point solvent; within a certain period of time, the obtained mixture is heated until the porogen boils, and the heating temperature reaches the polymerization reaction requirement; the boiling state is maintained, and half of the amount of porogen is evaporated within a certain period of time; then the product is cooled, recovered, and purified to obtain hydroxyl modified particles containing cis-dihydroxy groups on the surface; wherein the mass ratio of double bond modified particles, functional monomers, and crosslinking agents is (1-3):1:(1-3); the amount of initiator added is 0.2 wt.%-5wt.% of the total mass of functional monomers, crosslinking agents, and porogens; and the heating time is 10 min-2 h and 1-4 h, respectively; the functional monomer includes one of 3-methylacrylamidopamine, N-(3,4-dihydroxybutyl)acrylamide, and N-(4,5-dihydroxypentyl)acrylamide.

6. The method for preparing the magnetic chemical selective probe kit according to claim 1, wherein: The porogen includes at least one of water, methanol, acetonitrile, cyclohexane, toluene, 1,4-butanediol, dodecanol, and n-propanol; the crosslinker includes one of N,N'-methylenebisacrylamide and ethylene glycol dimethacrylate; the initiator includes one of 2,2'-azobisisobutyronitrile, ammonium persulfate, and benzoyl peroxide; the incubation temperature is room temperature, and the incubation time is 5 min-4 h; the mass ratio of the hydroxyl-modified particles and the probe reagent solution is 1:(20-200); and the concentration of the probe reagent solution is 5 mg / mL.

7. A magnetic chemical selective probe kit, characterized in that: The method is as described in any one of claims 1 to 6.

8. A use of the magnetic chemical selective probe kit according to claim 7, characterized in that: The magnetic chemical selective probe kit is combined with mass spectrometry for sample detection and identification.

9. Use of the chemical selective probe kit according to claim 8, characterized in that: The steps include: S1. extracting the sample using a solvent to obtain an extract; S2. taking a predetermined amount of the extract and adding it to the chemical selective probe kit, and incubating to capture the analyte in the sample; S3, magnetic separation of chemically selective probes, rapid removal of supernatant and purification; S4, eluting the magnetically immobilized chemically selective probe with an acidic solution to release the labeled product for mass spectrometry analysis; S5. Screen the labeled products based on the natural isotope abundance characteristics of boron.

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