Magnetochemical selective probe kit containing boric acid ester cracking sites as well as preparation method and application of magnetochemical selective probe kit

Abstract

The invention discloses a magnetic chemical selective probe kit containing boric acid ester cracking sites as well as a preparation method and application of the magnetic chemical selective probe kit, and belongs to the technical field of mass spectrum labeling probes. The kit disclosed by the invention consists of two parts, namely a magnetic probe and a magnet. The magnetic probe fixes a labeling reagent on the surfaces of the magnetic ferroferric oxide nanoparticles through a reversible borate reaction. Analyte metabolites are captured using labeled reactive groups. After the metabolite is captured and interferents are removed, the marked product is quickly released by regulating the pH value for subsequent mass spectrometry. The pH-regulated magnetic separation and labeling method has the remarkable advantages that the probe is low in cost and convenient to use, and a labeled product containing a boron element is convenient to identify. The probe can expand the analysis of different types of metabolites, such as carboxyl, amino, sulfydryl and the like, by changing labeled reaction groups. The kit has been successfully used for analyzing carboxyl compounds in complex samples such as serum, and has very important clinical application value and economic value.

Description

Technical Field

[0001] The 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 technology that combines mass spectrometry labeling probes with phase separation. Its characteristics are that on the basis of retaining the function and structure of traditional mass spectrometry probes (mass spectrometry sensitizing groups and reactive groups), two new parts, a linker arm containing a cleavage site and a solid phase matrix, are newly added, so that the probe itself not only has the function of labeling analytes, but also can separate and purify analytes in complex samples. The traditional CP usage process is as follows: First, CP is incubated with the sample extract, and the reactive group of the probe captures the analyte; then, the irrelevant excess liquid matrix in the extract is removed by physical separation; then, under the designed cleavage conditions, the labeled product is released; finally, the labeled product is collected for liquid chromatography-mass spectrometry (LC-MS) analysis.

[0003] Since CP has the functions of improving ionization efficiency, reducing matrix effects, and improving chromatographic separation, it is very suitable for mass spectrometry analysis of small molecule compounds in complex samples. At present, CP has been widely used in various research fields, such as intestinal bacteria-host co-metabolite analysis, disease marker discovery, metabolite rapid screening, metabolite network analysis, etc. However, although the current metabolomics research is very active, and chemical selective probes have many advantages in metabolite analysis.

[0004] For example, the article (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 the new progress in their application in natural product enrichment, metabolite derivatization and other fields, and their potential applications in metabolomics research. Another example is the article (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 product stability, and mild enzymatic or photolysis are time-consuming, so it is extremely urgent to find a suitable cleavage site. Secondly, stable isotope-coded probes are expensive to use, 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, a high-sensitivity magnetic CP with simple preparation, rapid labeling and release, low probe cost, and the ability to label various types of analytes has 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: (1) treating ferroferric oxide magnetic nanoparticles with a silane coupling agent, and coating the surface of the nanoparticles with silicon dioxide to obtain silicon-coated nanoparticles; (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; (3) mixing double-bond modified particles, functional monomers, porogens, crosslinking agents and initiators, wherein the molecules of the functional monomers include two characteristic structures: a terminal carbon-carbon double bond and a cis-dihydroxyl group; and subjecting the mixture to 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 react to form a boric acid ester, thereby obtaining a magnetic chemical selective probe; (5) The magnetic chemical selective probe is combined with the magnet component of the kit to obtain a magnetic chemical selective probe kit.

[0008] 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 the cis-dihydroxyl group after elution can be reused.

[0009] 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 solutions; 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.

[0010] 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 in the range of (20-50): 1; and the mass ratio of solution to reactant is 1: (1-3).

[0011] Preferably, in step (1), the reaction temperature is room temperature and the reaction time is 10-14 h.

[0012] 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, to form double-bond modified particles having carbon-carbon double bonds on the surface through reaction.

[0013] More preferably, the mass ratio of the silicon-coated particles, toluene and methacryloxypropyltrimethoxysilane is (2-4):1:(2-4).

[0014] Preferably, in step (2), the reaction temperature is room temperature and the reaction time is 20-28 h.

[0015] 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 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 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.

[0016] Further preferably, the mass ratio of the double bond modified particles, the functional monomer, and the 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, the crosslinking agent, and the porogen.

[0017] The heating control is carried out in two steps. The first heating time is shorter, giving a certain time to prevent boiling and large-scale volatilization of the porogen. The second heating provides the necessary reaction time for the polymerization reaction and the porogenation process.

[0018] Further preferably, the time is 10 min-2 h, 1-4 h respectively.

[0019] 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.

[0020] In the art, the molecular design of probe reagents usually includes specific functional groups, such as labeling reaction groups and mass spectrometry sensitization groups, etc., to achieve its 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 radioactive isotope 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 and reducing the detection limit. These groups jointly 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 group contained in the analyte, correspondingly used are ethylaminophenylboronic acid, 4-carboxylphenylboronic acid, and 4-(maleimidohexanamide-N-methyl)phenylboronic acid as probe reagents, wherein the benzene ring is used to increase the hydrophobicity of the labeled product and increase the reverse phase chromatography retention; the nitrogen-containing part is a mass spectrometry sensitization group. The selection of labeling reaction groups and mass spectrometry sensitization groups is flexible based on practical applications, and those skilled in the art can select the appropriate type according to the type of analyte.

[0021] Preferably, in step (4), 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 succinimide; when the analyte contains a sulfhydryl group, the labeling reaction group is selected as maleimide or sulfhydryl group; when the analyte contains a carbonyl group, the labeling reaction group is selected as hydrazide or hydroxylamine; when the analyte contains an alkynyl group, the labeling reaction group is selected as azide.

[0022] The porogen is usually a volatile solvent, which is mixed into the polymer matrix and evaporates during the material forming or curing process, thereby leaving pores in the material. Any solvent with good volatility and good ability to disperse and dissolve the raw materials can be used as a porogen. In the present invention, acetonitrile is a particularly suitable porogen.

[0023] 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.

[0024] Preferably, in step (4), the incubation temperature is room temperature and the incubation time is 5 min-4 h.

[0025] Preferably, in step (4), the mass ratio of the hydroxyl-modified particles to the solution of the probe reagent is 1:(20-200); and the concentration of the solution of the probe reagent is 5 mg / mL.

[0026] In the art, solvents are used as media to promote dispersion of raw materials and to create suitable conditions for reaction. Those skilled in the art can select suitable solvent types according to the physicochemical properties of the optional raw materials involved in the method, in combination with actual conditions. Under the premise that the technicians know the above principles, the choice of solvent is not unique, such as toluene, acetonitrile, etc., all belong to suitable solvent types of the present invention.

[0027] In the second aspect of the present invention, a chemical selective probe kit is provided which has rapid labeling and release, low use cost, and the ability to label various types of analytes, and is prepared by the preparation method of the first aspect of the present invention.

[0028] In the third aspect of the present invention, there is provided an application of the chemical selective probe kit according to the second aspect of the present invention, specifically combining the chemical selective probe kit with a mass spectrometer for sample detection and identification.

[0029] In applications, the rapid identification strategy of screening using the abundance of natural isotopes of boron can quickly identify labeled products without stable isotope coding technology, significantly reducing the cost of using probes in large-scale sample analysis scenarios and facilitating the promotion of the method. In addition, the high-throughput analysis method of chemical selective labeling combined with mass spectrometry can simultaneously study different types of analytes in complex samples and obtain accurate and sensitive qualitative and quantitative results.

[0030] Preferably, the application comprises the following steps: 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 chemical selective probes, removal of supernatant and purification; S4, using an acidic solution to elute the magnetically fixed chemical selective probe to release the labeled product, and performing mass spectrometry detection and analysis; S5. Screen the labeled products according to the natural isotope abundance characteristics of boron.

[0031] The shortcomings of the preparation and use of traditional CP 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 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 enzyme or photolysis is time-consuming, so it is extremely urgent to find a suitable cleavage site. Secondly, the cost of using stable isotope-encoded 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 kit, simplifying the preparation steps, and reducing the cost of using the CP kit, which has high clinical application value and practical value.

[0032] Based on the above technical scheme, the design concept and principle of the present invention is that first, silica is coated on the surface of magnetic ferroferric oxide nanoparticles that are easy to separate, and then carbon-carbon double bonds are introduced on the surface by methacryloxypropyltrimethoxysilane modification, and then double bonds are polymerized to form a connecting arm connected with a functional monomer, and a pore-forming agent is combined to form a pore at the organic polymer coating of the particle and the outer layer of silica. The effect of the micron-scale pore is to improve the reaction kinetics as a penetration hole, and 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-dihydroxyl group present in the connecting arm is incubated with the probe reagent containing the boric acid group, and connected by the borate ester group formed by the reaction. The boric acid group has affinity with the cis-dihydroxyl structure. For example, in the inventor's prior application (CN117147669A), the inventor designed functional monomers corresponding to nucleosides, carbohydrates, and glycoproteins based on this principle. In the present invention, the inventors further deepened the utilization of borate groups, and the functions of borate are mainly divided into two aspects: in the chemical selective probe, borate is connected to the ferroferric oxide nanoparticle part for easy separation and the probe reagent for capturing the analyte, and the probe reagent is extracted along with the separation after sampling; in the detection and identification of samples, the inventors use the characteristics of borate that it is stable in neutral and alkaline conditions and easy to cleave in acidic conditions, use it as a cleavage site and elute it in an acidic solution, so that the labeled product containing the analyte group can be easily separated and analyzed by mass spectrometry.

[0033] 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 the 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 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.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention provides a method for preparing a magnetic chemical selective probe kit. The magnetic chemical selective probe constructed based on borate ester reaction and ferroferric oxide nanoparticles can realize convenient preparation of the probe and separation of labeled products under mild conditions. Different types of chemical selective probes can use the same ferroferric oxide nanoparticles. The labeled objects of the probe can be expanded by simply replacing the labeling reaction group at the end of the probe reagent, and the kit has good versatility.

[0035] 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.

[0036] The 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

[0037] Figure 1 Schematic diagram of the preparation process of the magnetic chemical selective probe in Example 1; Figure 2 is a scanning electron microscope morphology image of the magnetic chemical selective probe in Example 2; Figure 3 This is the labeling optimization diagram of the carboxyl magnetic chemoselective probe in Example 3; Figure 4 This is a diagram showing the labeling results of the magnetic chemical selective probe on compounds with different structures in Example 4; Figure 5 This is the fragmentation characteristic diagram of the magnetic chemical selective probe labeled product in Example 5; Figure 6 This is a diagram of the serum targeted spike experiment of the carboxyl magnetic chemical selective probe in Example 6; Figure 7The non-targeted experimental process of carboxyl magnetic chemical selective probe serum and the logarithmic graph of the peaks screened in Example 7; Figure 8 Schematic diagram of the use of the magnetic chemical selective probe kit. DETAILED DESCRIPTION

[0038] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0039] In the following embodiments: Benzotriazole-N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) with a purity of 98%, CAS number: 94790-37-1, was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; 1-Hydroxybenzotriazole (HOBT), purity 98%, CAS number: 2592-95-2, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; N,N-Diisopropylethylamine (DIEA), purity 99%, CAS number: 7087-68-5, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; 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.; Triethylamine (TEA), purity 99.5%, CAS number: 121-44-8, purchased from Shanghai Merril Biochemical Technology Co., Ltd.; 2-Chloro-1-methylpyridinium iodide (CMPI), purity 98%, CAS number: 14338-32-0, was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0040] Example 1 The preparation method of the magnetic chemical selective probe kit is as follows Figure 1As shown, the steps are as follows: (1) Using ferroferric oxide magnetic nanoparticles as raw materials, 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; then 20 mL of TEOS, 25 mL of ammonia water, 221.67 mL of pure water and 778.33 mL of ethanol were 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 at 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; (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 at a speed of 300 rmp for 24 h. After the reaction, the mixture was washed alternately with methanol and water, and dried with nitrogen for later use to obtain double bond-modified particles with carbon-carbon double bonds on the surface. (3) First, 500 mg of double-bond modified particles were ultrasonically dispersed in 192 mL of acetonitrile, and then 504 mg of 3-MDA, 583 mg of EDMA and 7.5 mg of AIBN were added to the solution in sequence; then the three-necked flask was placed in a 50 °C water bath, and the water bath was heated to boiling acetonitrile within 30 min, and the boiling state was maintained, and half of the acetonitrile was evaporated within 2 h; after the reaction was completed, methanol and water were alternately washed, and then dried with nitrogen to obtain hydroxyl-modified particles with cis-dihydroxy groups on the surface, which were recorded as Fe3O4@SiO2@MPS@MDA; (4) Synthesis of thiol labeling reagent: Dissolve 200 mg of 6-maleimidocaproic acid, 432 mg of HBTU, 154 mg of HOBT and 213 mg of 4-aminomethylphenylboronic acid hydrochloride in 10 mL of DMF; slowly add 785 mL of DIEA; react at room temperature for 12 h; Synthesis of amino labeling reagent: 200 mg 4-carboxyphenylboronic acid, 340 mg EDCI, and 320 mg HOBT were dissolved in 10 mL DMF; 360 mg TEA was added, mixed at room temperature for 10 min, and then 207 mg N-hydroxysuccinimide was added; the reaction was allowed to react at room temperature for 12 h; Carboxyl labeling reagent: 4-((2-aminoethyl)phenyl)borate hydrochloride is a commercial product; The washed and dried Fe3O4@SiO2@MPS@MDA was divided into three groups of samples, each with 20 mg, and then 1 mL of 5 mg / mL carboxyl labeling reagent, amino labeling reagent and thiol labeling reagent were added respectively; incubated at room temperature for 3 hours; after the end, the supernatant was removed with the assistance of a magnet, the probe was washed twice with acetonitrile, and dried with nitrogen to obtain a magnetic chemical selective probe; (5) The prepared magnetic chemical selective probe is combined with the universal components of the kit to obtain a magnetic chemical selective probe kit.

[0041] Example 2 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 synthesis process of the magnetic chemical selective probe (hereinafter referred to as the magnetic probe) was investigated by scanning electron microscopy. The results are as follows Figure 2 shown.

[0042] Figure 2 (a) The morphology of commercial bare Fe3O4 nanoparticles and modified nanomagnetic beads was characterized by scanning electron microscopy. The morphology of commercial bare Fe3O4 nanoparticles varies, including spherical and cubic, and the average diameter is about 150 nm. Figure 2 (b) Fe3O4 nanoparticles coated with SiO2, whose size grows to about 250 nm and whose morphology is still 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 For the carboxyl magnetic chemical selective probe, the particle size did not increase much, about 370 nm, at this time the number of cubic particles was relatively reduced, and more particles were irregularly spherical. From the SEM image, we can see the translucent modification outside the magnetic core, indicating that the modification was successful.

[0043] Example 3 This Example 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.

[0044] First, prepare 1 ppm and 10 ppm mixed standards. The carboxyl mixed standard contains pentanoic acid, cyclohexanoic acid, octanoic acid, deoxycholic acid, 8-aminooctanoic acid and deuterated pentanoic acid-d9; the amino mixed standard contains glycine, L-alanine, methionine, cyclohexylamine and deuterated glycine-d2; the thiol 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 the carboxyl magnetic probe, the naming 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 times optimization diagram of the carboxyl magnetic probe.

[0045] (1) Optimization of probe dosage A series of different masses of carboxyl, amino and thiol magnetic chemoselective probes were taken, 2, 5, 10, 20, 30, 40 mg respectively, in EP tubes, and then 500 mL of 1 ppm carboxyl mixed standard, 20 mL TEA and 20 mL CMPI (20 mmol / mL) were added to the carboxyl probe; 500 mL of 1 ppm amino mixed standard and 20 mL TEA (20mmol / mL) were added to the amino probe; 500 mL of 1 ppm thiol mixed standard was added to the thiol probe, and the reaction was carried out at 40 ℃, room temperature and room temperature for 30 min respectively. After the reaction, acetonitrile was used to wash twice, and then 200 mL of eluent (0.1M HCl:ACN=1:9) was added to it and eluted twice. Finally, the eluent was dried under nitrogen atmosphere, re-dissolved with 100 mL of re-solvent (ACN:H2O=1:9), and injected into LC-MS. Since the types and concentrations of metabolites in non-targeted metabolomics analysis are unknown, the amount of probe used cannot be too small to prevent the failure to capture more metabolite information. At the same time, it can be concluded from the experimental results that the change in the amount of probe after 20 mg is basically flat, and a slight increase or decrease is related to the nonspecific adsorption or labeling efficiency of the probe. Therefore, the optimal amount of magnetic probe is 20 mg.

[0046] (2) Optimization of the catalyst dosage of the probe Take 20 mg of carboxyl and amino magnetic chemical selective probes in EP tubes, and then add 500 mL of 1 ppm corresponding mixed standards to the probes. Then add 20 mmol / mL CMPI and 20 mmol / mL LTEA as catalysts to the carboxyl probe; add 20 mmol / mL TEA as catalyst to the amino probe, use 10, 20, 40, 60, 100 and 200 mL of catalyst, respectively, and react at 40 ℃ and room temperature for 30 min. After the reaction, wash with acetonitrile twice, add 200 mL of eluent (0.1M HCl:ACN=1:9) to it, and elute twice. Finally, the eluent was dried under nitrogen atmosphere, re-dissolved with 100 mL of resolvent (ACN:H2O =1:9), and injected by LC-MS. The overall trend of changing the amount of catalyst did not change much, and 40 mL was the optimal amount. When the amount of catalyst 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.

[0047] (3) Optimization of probe labeling time 20 mg of carboxyl, amino and thiol magnetic chemoselective probes were placed in EP tubes respectively. Then, 500 mL of 1 ppm carboxyl mixed standard, 20 mL TEA and 20 mL CMPI (20 mmol / mL) were added to the carboxyl probe; 500 mL of 1 ppm amino mixed standard and 20 mL TEA (20 mmol / mL) were added to the amino probe; 500 mL of 1 ppm thiol mixed standard was added to the thiol probe, and the reaction was carried out at 40 ℃, room temperature and room temperature for 5, 10, 20, 30 and 60 min, respectively. After the reaction, acetonitrile was used to wash twice, and then 200 mL of eluent (0.1M HCl:ACN=1:9) was added to it and eluted twice. Finally, the eluent was dried under nitrogen atmosphere, and then re-dissolved with 100 mL of re-solvent (ACN:H2O=1:9) and injected into LC-MS. The probe reached the best response at 20 min.

[0048] (4) Optimization of probe elution times 20 mg of carboxyl, amino and thiol magnetic chemical selective probes were taken in EP tubes respectively, and then 500 mL of 10 ppm carboxyl mixed standard, 20 mL TEA and 20 mL CMPI (20 mmol / mL) were added to the carboxyl probe; 500 mL of 10 ppm amino mixed standard and 20 mL TEA (20 mmol / mL) were added to the amino probe; 500 mL of 10 ppm thiol mixed standard was added to the thiol probe, and the reaction was carried out at 40 ℃, room temperature and room temperature for 30 min respectively. After the reaction, acetonitrile was used to wash twice, and then 200 mL of eluent (0.1M HCl:ACN=1:9) was added to it, and eluted 2, 3, 4, 5, and 6 times in sequence. Finally, the eluent was dried under nitrogen atmosphere, and then re-dissolved with 100 mL of re-solvent (ACN:H2O=1:9) and injected by LC-MS. The probe response gradually increased with the increase of elution times, but the change trend was not very significant.

[0049] Example 4 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 for compounds with different structures; (e)-(h) are the labeling results of amino magnetic probes for compounds with different structures; (i)-(l) are the labeling results of thiol magnetic probes for compounds with different structures.

[0050] 20 mg of carboxyl, amino and thiol magnetic chemoselective probes were placed in EP tubes respectively. Then, 500 mL of 10 ppm carboxyl mixed standard, 20 mL TEA and 20 mL CMPI (20 mmol / mL) were added to the carboxyl probe; 500 mL of 10 ppm amino mixed standard and 20 mL TEA (20 mmol / mL) were added to the amino probe; 500 mL of 10 ppm thiol mixed standard was added to the thiol probe, and the reaction was carried out at 40 ℃, room temperature and room temperature for 30 min respectively. After the reaction, acetonitrile was used to wash twice, and then 200 mL of eluent (0.1M HCl:ACN=1:9) was added to it and eluted twice. Finally, the eluent was dried under nitrogen atmosphere, and then re-dissolved with 100 mL of resolvent (ACN:H2O=1:9) and injected into LC-MS.

[0051] 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. Four compounds with different structures 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 probe, such as Figure 4 The experimental results show that the three probes can successfully detect compounds of different structural types and also show the unique isotope peak of boron element. Therefore, this type of magnetic probe kit can be used to label different types of metabolites in complex samples.

[0052] Example 5 This example studies the mass spectrometry fragmentation characteristics of the product labeled by the magnetic chemical selective probe kit in Example 1. The results are as follows: Figure 5 shown. Figure 5 In the figure, (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 thiol-labeled products.

[0053] The mass spectrometry fragmentation characteristics of the labeled products were investigated in liquid-liquid reactions. Take 20 mL of carboxyl, amino, and thiol labeling reagents in EP tubes, then add 500 mL of 10 ppm carboxyl mixed standard, 20 mL TEA, and 20 mL CMPI (20 mmol / mL) to the carboxyl labeling reagent; add 500 mL of 10 ppm amino mixed standard and 20 mL TEA (20 mmol / mL) to the amino labeling reagent; add 500 mL of 10 ppm thiol mixed standard to the thiol labeling reagent, and react at 40 °C, room temperature, and room temperature for 30 min, respectively. After the reaction, the eluent was dried under nitrogen atmosphere, and then re-dissolved with 100 mL of redissolved solvent (ACN:H2O=1:9) and injected by LC-MS.

[0054] 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 for qualitative and quantitative analysis. Figure 5 As shown, the characteristic fragments of the carboxyl-labeled product include characteristic fragments derived from the labeling reagent ( m / z 166.1034, 149.0768, 131.0663 or 105.0699); the characteristic fragments of the amino-labeled product 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 types of labeled products all have carbon-boron bond cleavage, producing [M+H-44] + The secondary mass spectrometry fragments lose one water and produce [M+H-18] + By identifying the characteristic fragments, different types of compounds can be classified even with a single injection.

[0055] Example 6 This example studies the targeted screening of different types of metabolites in human serum by the magnetic chemical selective probe kit in Example 1. The results are as follows: 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-toluic acid labeled product in serum; (g)-(h) are the chromatograms and mass spectra of the n-pentanoic acid labeled product in serum.

[0056] The carboxyl magnetic chemical selective probe kit of Example 1 of the present invention was studied for targeted metabolomics analysis in human serum samples, and 3-aminophenylboronic acid was selected as the labeling reagent for the carboxyl group. In this embodiment, the preparation of the working solution is similar to that of Example 3, but the concentration is increased to 10 ppm. The serum sample preparation process is as follows: 120 mL of serum sample was taken, and 650 mL of a 10 ppm carboxylic acid mixed standard solution prepared with ACN was added for one-step extraction. After ultrasonication for 30 min under ice bath, centrifugation was performed at 10000 rmp and 4 ° C, and the supernatant was collected. The control group only used ACN instead of the extract containing the carboxylic acid mixed standard, and the others remained unchanged. Serum labeling: First, 20 mL CMPI (20 mmol / mL) and 20 mL TEA (20 mmol / mL) were added to 100 mL serum samples (divided into spiked group, control group and blank group), and then 20 mg of carboxyl magnetic probe (labeling reagent is 3-aminophenylboronic acid) was added thereto, and labeled at 40 ° C for 30 min.

[0057] like Figure 6 As shown in the figure, the chromatograms and mass spectra of the control group and the spiked group of three carboxylic acid labeled products are listed. The mass spectra of the control group and the spiked group have significantly changed mass spectrometric responses, and the labeled products can be accurately identified based on the isotope peak clusters of the boron element. The above examples fully demonstrate that the magnetic chemical selective probe kit can be used for targeted metabolomics analysis of trace targets in complex samples.

[0058] Example 7 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. The results are as follows Figure 7 shown. Figure 7 In the figure, (a) is the data processing flow chart of non-targeted screening of carboxyl metabolites in serum; (b) is the peak logarithm screened after extraction.

[0059] 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, there were 78 peaks containing isotopic characteristic peaks. The obtained characteristic peaks were generated by Thermo Xcalibur software to generate 19 expected molecular formulas. After deducting the elements introduced by the labeling reagents, further matching was performed in the public database HMDB (https: / / hmdb.ca / ), and finally 5 molecules that may be carboxylic acid metabolites were obtained. In summary, the above shows that the magnetic chemical selective probe based on the characteristics of the boron element is a convenient and accurate metabolite screening strategy.

[0060] The use process of the above magnetic chemical selective probe kit is as follows Figure 8 shown.

[0061] In summary, the present invention first coats silica on the surface of ferroferric oxide nanoparticles that are easy to separate, then uses methacryloxypropyltrimethoxysilane to modify the surface to introduce carbon-carbon double bonds, and then connects with functional monomers through double bond polymerization to form a connecting arm, and combines with a porogen to form a channel at the organic polymer coating of the particles and the outer layer of silica. The effect of the micron-scale channel is to improve the reaction kinetics as a penetration hole, and 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-dihydroxyl 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. Boric acid groups have affinity for cis-dihydroxy structures, and the inventors have further deepened the use of borate ester groups. The functions of borate esters are mainly divided into two aspects: in chemically selective probes, borate esters connect the ferric oxide nanoparticle part that is easy to separate 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 easy to cleave 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.

[0062] Compared with the traditional method, the significant advantages of the present invention are rapid and low-cost probe preparation, rapid labeling reaction, and convenient identification of boron-containing labeled products. By changing the labeling reaction group, the probe can expand the analysis of different types of metabolites, such as carboxyl, amino and thiol. 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.

[0063] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for preparing a magnetic chemical selective probe kit, characterized in that: The steps include: (1) treating ferroferric oxide nanoparticles with a silane coupling agent, and coating the surface of the nanoparticles with silicon dioxide to obtain silicon-coated particles; (2) Modifying the silicon-coated particles using 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, functional monomers, porogens, crosslinking agents and initiators, wherein the molecules of the functional monomers include two characteristic structures: a terminal carbon-carbon double bond and a cis-dihydroxyl group; and subjecting the mixture to 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 react to form a boric acid ester, thereby obtaining a magnetic chemical selective probe; (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, characterized in that: 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 solutions; mixing the reactants with the solution and reacting them, and purifying them after the reaction 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, characterized in that: In the step (1), the mass ratio of ferrosoferric oxide nanoparticles to tetraethoxysilane is in the range of (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, characterized in that: The specific operation of step (2) is as follows: adding silicon-coated particles to a solvent, mixing to make them uniformly dispersed, and then adding methacryloxypropyltrimethoxysilane for modification, to form double-bond modified particles having carbon-carbon double bonds on the surface through reaction; the mass ratio of 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, characterized in that: The specific operation of step (3) is as follows: double bond modified particles, functional monomers, porogens, crosslinking agents, and initiators are mixed, and the porogens use low boiling point solvents; 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 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; the heating time is 10 min-2 h and 1-4 h, respectively; the functional monomer comprises one of 3-methylacrylamide dopamine, 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, characterized in that: In the step (4), 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 succinimide; when the analyte contains a sulfhydryl group, the labeling reaction group is selected as maleimide or sulfhydryl group; when the analyte contains a carbonyl group, the labeling reaction group is selected as hydrazide or hydroxylamine; when the analyte contains an alkynyl group, the labeling reaction group is selected as azide.

7. The method for preparing the magnetic chemical selective probe kit according to claim 1, characterized in that: 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 solution of hydroxyl-modified particles and the probe reagent is 1:(20-200); and the solution concentration of the probe reagent is 5 mg / mL.

8. A magnetic chemical selective probe kit, characterized in that: The method is prepared according to any one of claims 1 to 7.

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

10. Use of the chemical selective probe kit according to claim 9, 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, using an acidic solution to elute the magnetically fixed chemical selective probe to release the labeled product, and performing mass spectrometry detection and analysis; S5. Screen the labeled products according to the natural isotope abundance characteristics of boron.

Images