Metal semiconductor material doped mass spectrum sample target plate and application thereof

Abstract

The invention relates to an application of a metal semiconductor material as a matrix in laser desorption ionization mass spectrometry detection, a method for detecting a sample by using the matrix, a kit containing the matrix, a mass spectrometry sample target plate doped with the matrix, a method for preparing the mass spectrometry sample target plate and a kit for detecting the sample by using the matrix. The invention also relates to a method for detecting a sample by using the mass spectrum sample target plate.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and relates to the application of a metal semiconductor material as a matrix in laser desorption ionization mass spectrometry detection, a method for detecting a sample using the matrix, a kit containing the matrix, a mass spectrometry sample target plate doped with the matrix, a method for preparing the mass spectrometry sample target plate, and a method for detecting a sample using the mass spectrometry sample target plate. Background Art

[0002] In recent years, laser desorption ionization mass spectrometry (LDI-MS) technology has gradually been successfully applied in small molecule detection. Compared with the traditional matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) technology, it uses a variety of nanomaterials (Nanomaterial, NM) to replace the organic small molecule weak acid matrix, which helps to overcome the low-mass background interference of the weak acid matrix itself. The sample does not need to be derivatized, the spectrum analysis is simple, the sensitivity is high, and it is fast and convenient.

[0003] The NM used in LDI-MS has the following characteristics: it has a high molar absorption coefficient in the laser radiation area, which can promote the "soft" desorption / ionization of the analyte; it has a low or no background in the low mass area, which helps to improve the sensitivity; it has a high specific surface area, thus ensuring the loading capacity of the analyte; it provides higher salt tolerance and more uniform sample points, which is more conducive to accurate quantification. In addition, it can also be functionalized, or it can be used as an extraction separation medium at the same time to achieve in-situ selective determination.

[0004] So far, NMs that have been used as LDI-MS matrices include carbon-based nanomaterials (C-NM), silicon-based nanomaterials (Si-NM), metal-organic frameworks (MOFs), covalent organic frameworks (COFs), metal or metal semiconductor-based nanomaterials (M-NM), etc., which have been successfully applied in the detection of drugs (poisons), amino acids, lipids, small peptides, sugars, etc. In terms of detection form, in most application examples, NM and samples are simply mixed or dripped layer by layer on the mass spectrometry sample target plate, and then measured after drying.

[0005] Based on appropriate photothermal, photoelectric, surface and interfacial physical and chemical behaviors, and their own conductive properties, metal semiconductor materials have great potential as matrices in LDI-MS detection. Summary of the invention

[0006] In the present invention, the inventor first screened and obtained a suitable metal semiconductor (metal oxide or metal sulfide) material as a matrix for LDI-MS, and further found that doping it in a sample target plate can achieve rapid real-time determination and accurate quantification of multiple analytes, and can also achieve non-biased, non-targeted mass spectrometry imaging. Based on the above findings, the present invention is completed. Specifically, the present invention relates to the following aspects.

[0007] The first aspect of the present invention relates to the use of metal semiconductor materials as a matrix in laser desorption ionization mass spectrometry detection, wherein the metal semiconductor material is selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets.

[0008] A second aspect of the present invention relates to a method for detecting a sample using laser desorption ionization mass spectrometry, comprising:

[0009] 1) Mix the sample to be tested with the matrix solution, drop it into the sample well of the mass spectrometry sample target plate, and dry it; or,

[0010] Applying a matrix to the bottom of the sample hole to form a matrix layer (for example, dropping a matrix solution to the bottom of the sample hole and drying to form a matrix layer), and then placing a sample to be tested on the matrix layer;

[0011] 2) Use MALDI-MS instrument to detect the samples,

[0012] The matrix is ​​selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets.

[0013] In certain embodiments, the matrix solution is a solution containing MoS2 powder, D-WO3 nanosheets, or MoS2 nanosheets.

[0014] In certain embodiments, the matrix solution is 0.1-1 mg / mL of MoS2 powder, D-WO3 nanosheets, or an aqueous solution of MoS2 nanosheets.

[0015] In certain embodiments, the matrix solution further comprises a proton transfer agent. In certain embodiments, the proton transfer agent is diammonium hydrogen citrate. In certain embodiments, the matrix solution further comprises 0.1-1 g / L (e.g., 0.2 g / L or 0.5 g / L) of diammonium hydrogen citrate.

[0016] In some embodiments, the matrix solution is an aqueous solution containing 0.1-1 mg / mL D-WO3 nanosheets and 0.1-1 g / L (e.g., 0.2 g / L or 0.5 g / L) diammonium hydrogen citrate. In some embodiments, the matrix is ​​an aqueous solution containing 0.1-1 mg / mL MoS2 powder or MoS2 nanosheets and 0.1-0.2 g / L diammonium hydrogen citrate.

[0017] In some embodiments, the sample contains organic small molecules and / or polypeptides. In some embodiments, the organic small molecules include alkaloids (e.g., physostigmine, magnocurine, strychnine, colchicine, neorin, aconitine), amino acids, etc.

[0018] A third aspect of the present invention relates to a kit for laser desorption ionization mass spectrometry, comprising a metal semiconductor material selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets. In certain embodiments, the kit further comprises a proton transfer agent, such as diammonium hydrogen citrate.

[0019] The fourth aspect of the present invention relates to a sample target plate for laser desorption ionization mass spectrometry, comprising a target plate and a sample hole located on the surface of the target plate, wherein at least the target plate at the bottom of the sample hole is doped with a metal semiconductor material, the metal semiconductor material being selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets. The sample hole is used to hold a sample to be tested.

[0020] In certain embodiments, in the sample target plate, at least the target plate at the bottom and wall of the sample hole is doped with a metal semiconductor material.

[0021] In certain embodiments, the doping amount of the metal semiconductor material is 0.1-1 wt‰ (eg, 0.1 wt‰, 0.2 wt‰, 0.5 wt‰, 1 wt‰) of the target plate weight at the site.

[0022] In certain embodiments, the metal semiconductor material is D-WO3 nanosheets, and the doping amount thereof is 0.2-1 wt‰, for example 0.5 wt‰, of the weight of the target plate at the site.

[0023] In certain embodiments, the metal semiconductor material is MoS2 nanosheets, and the doping amount thereof is 0.1-0.2 wt‰ of the target plate weight at the site.

[0024] In certain embodiments, the entire target plate is doped with metal semiconductor material, and the doping amount is 0.1-1 wt‰ (eg, 0.1 wt‰, 0.2 wt‰, 0.5 wt‰, 1 wt‰) of the weight of the target plate.

[0025] In some embodiments, the target plate is made of a high molecular polymer material (such as a photosensitive resin, a thermoplastic plastic, a rubber material) or a metal material (such as an alloy material) doped with a metal semiconductor material, and the doping amount of the metal semiconductor is 0.1-1wt‰ (such as 0.1wt‰, 0.2wt‰, 0.5wt‰, 1wt‰) of the weight of the high molecular polymer material or the metal material.

[0026] In certain embodiments, the sample target plate has an outer contour that matches the target holder of a MALDI-MS instrument.

[0027] In some embodiments, the sample target plate further has a fixing hole for fixedly connecting with the target holder of the MALDI-MS instrument, for example, the fixed connection can be achieved by a flange, screws, bolts or jackscrews.

[0028] In certain embodiments, when testing a sample, the target plate containing the sample to be tested is fixedly connected to the target holder of the matrix-assisted laser desorption / ionization mass spectrometer by means of a flange, screws, bolts or jackscrews.

[0029] In some embodiments, the surface of the target plate has a plurality of (e.g., 4, 8, 16, 24, 48, 96) sample holes arranged periodically (e.g., two-dimensionally periodically). In some embodiments, the plurality of sample holes are of equal size and shape. The shape of the sample hole can be any sample hole that can hold the sample to be tested, such as a rectangular hole, a hemispherical hole, a semi-ellipsoidal hole, a cylindrical hole, etc.

[0030] The fifth aspect of the present invention relates to a method for preparing the sample target plate described in the fourth aspect of the present invention, comprising:

[0031] 1) Mixing a metal semiconductor material with a polymer precursor material or a metal powder;

[0032] 2) preparing a sample target plate from the mixture obtained in step 1),

[0033] The metal semiconductor material is selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets.

[0034] In some embodiments, the mixture obtained in step 1) can be made into a sample target plate by 3D printing, precision molding, machining, hot and cold processing or manual molding. In some embodiments, the mixture obtained in step 1) can be made into a sample target plate by 3D printing.

[0035] The sixth aspect of the present invention relates to the use of the sample target plate described in the fourth aspect of the present invention in laser desorption ionization mass spectrometry detection of samples. In certain embodiments, the sample contains organic small molecules and / or polypeptides. In certain embodiments, the organic small molecules include alkaloids (e.g., physostigmine, magnocurine, strychnine, colchicine, neorin, aconitine), and amino acids.

[0036] A seventh aspect of the present invention relates to a method for detecting a sample using laser desorption ionization mass spectrometry, comprising:

[0037] 1) placing a sample to be tested in a sample well of the sample target plate described in the fourth aspect of the present invention;

[0038] 2) Use MALDI-MS instrument to detect the samples.

[0039] In certain embodiments, the sample contains small organic molecules and / or polypeptides. In certain embodiments, the small organic molecules include alkaloids (e.g., physostigmine, magnocurine, strychnine, colchicine, neorin, aconitine), amino acids. In certain embodiments, the sample is a slice of Chinese herbal medicine. In certain embodiments, the sample is a Chinese herbal medicine extract.

[0040] In the present invention, "doping" means adding a small amount of metal semiconductor into the substrate (main material) of the target plate to be doped, and then processing it into shape, so that the metal semiconductor material is doped into the target plate, wherein the doping amount can be 0.1-1wt‰.

[0041] In the present invention, the purity of the MoS2 powder is preferably greater than 98%, and more preferably 99%. The MoS2 powder can be synthesized according to known methods or commercially available.

[0042] In the present invention, the MoS2 nanosheets are prepared by a known chemical exfoliation method, for example, they can be prepared by referring to the method disclosed by Goki Eda et al. (Goki Eda, Hisato Yamaguchi, Damien Voiry, Takeshi Fujita, Mingwei Chen, and Manish Chhowalla, Photoluminescence from Chemically Exfoliated MoS2, Nano Lett. 2011, 11, 5111–5116. doi: 10.1021 / nl201874w), or by referring to the method described in Example 1 of the present invention.

[0043] In the present invention, the D-WO3 nanosheets (Defect-rich WO3 nanosheets) can be prepared by referring to the method disclosed by the research group of Professor Wang Qiang of Lanzhou University (Xin-Ping Zhai, Lin-Feng Gao, Hong Zhang, Yong Peng, Xiao-Dong Zhang, Qiang Wang, and Hao-Li Zhang, Defect Engineering of Ultrathin WO3 Nanosheets: Implications for Nonlinear Optoelectronic Devices, ACS Applied NanoMaterials, 2022 5(1), 1169-1177.DOI: 10.1021 / acsanm.1c03791).

[0044] In the present invention, the organic small molecules refer to organic compounds with a relatively small molecular weight, generally less than 1000, which mainly contain carbon and hydrogen elements, and may also contain other elements, such as oxygen, nitrogen, phosphorus, sulfur, etc. In the present invention, the organic small molecules include alkaloids, amino acids and other substances.

[0045] In the present invention, the amino acid is an organic compound containing a basic amino group and an acidic carboxyl group, including protein amino acids and non-protein amino acids. Protein amino acids refer to amino acids that can be found in proteins and are encoded in the standard genetic code, including glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, histidine, selenocysteine ​​and lysine, and pyrrolysine. Non-protein amino acids refer to other amino acids that are not found in proteins (such as ornithine, citrulline, carnitine, GABA and L-DOPA), or are not encoded by the standard genetic code (such as hydroxyproline and selenomethionine), the latter of which are usually derived from post-translational modification of proteins.

[0046] In the present invention, the polypeptide refers to a peptide consisting of 2-100 amino acid molecules.

[0047] In the present invention, the alkaloid is a class of nitrogen-containing organic compounds existing in nature (mainly plants, but some also exist in animals), has significant biological activity, and is one of the important effective ingredients in Chinese herbal medicine. The alkaloids mainly include: organic amines, such as ephedrine, leonurine, colchicine; pyrrolidines, such as codonopsis, senecioine, and crotonine; pyridines, such as niacin, arecoline, and lobeline; isoquinolines, such as berberine, morphine, and tetrandrine; indoles, such as reserpine, vincristine, and ergonovine; tropanes, such as atropine and scopolamine; imidazoles, such as pilocarpine; quinazolinones, such as febrifugine; purines, such as caffeine and theophylline; steroids, such as solanine, fritillary bulbine, and solanine; diterpenes, such as aconitine and delphinidin; others, such as galantamine and triptolide. In certain embodiments, the alkaloids include physostigmine, magnocurarine, strychnine, colchicine, neorin, and aconitine.

[0048] In the present invention, the polymer material refers to a high molecular weight (usually up to 104-106) compound material formed by repeated bond connection, especially those polymer materials that can be used as target materials for laser desorption ionization mass spectrometry, such materials include polyamide, polyester, polycarbonate, polyethylene, polypropylene and acrylonitrile-butadiene, etc. In the present invention, the polymer material includes photosensitive resin, thermoplastic plastic, rubber material, etc.

[0049] In the present invention, the photosensitive resin refers to a material used for photocuring rapid prototyping, which is a liquid photocurable resin, or liquid photosensitive resin, mainly composed of a photosensitive prepolymer, a photoinitiator or a photosensitizer, and a reactive diluent. The photosensitive prepolymer refers to a low molecular weight prepolymer that can be photocured, and its molecular weight is usually between 1,000 and 5,000, which is a determining factor for the final performance of the material. The commonly used photosensitive prepolymers in photosensitive resins mainly include epoxy resins (such as epoxy acrylate resins), unsaturated polyesters, polyurethanes, and polythiol / polyene photocurable resins. Both photoinitiators and photosensitizers play a role in promoting the initiation of polymerization during the polymerization process. Photoinitiators form some active substances such as free radicals or cations after absorbing light energy to initiate the reaction. The main photoinitiators include benzoin and its derivatives, acetophenone derivatives, triarylthiobell salts, etc. The mechanism of action of the photosensitizer mainly includes three types: energy conversion, hydrogen abstraction, and generation of charge transfer complexes. The main photosensitizers include benzophenone, Michler's ketone, thioxanthone, benzyl, etc. Diluents mainly refer to low molecular weight compounds containing reactive groups, which can not only reduce the viscosity of the system but also participate in the curing reaction and become part of the cured product. According to the number of reactive groups contained in each molecule, active diluents can be divided into monofunctional active diluents, bifunctional active diluents and multifunctional active diluents, such as monofunctional styrene (St), vinyl pyrrolidone (NVP), vinyl acetate (VA), butyl acrylate (BA), isooctyl acrylate (EHA), (meth) hydroxy acrylate (HEA, HEMA, HPA), etc.; bifunctional 1,6-hexanediol diacrylate (HDDA), tripropylene glycol diacrylate (TPGDA), neopentyl glycol diacrylate (NPGDA), etc.; multifunctional trimethylolpropane triacrylate (TMPTA), etc. According to the type of functional group, it can be divided into (meth) acrylates, vinyls, vinyl ethers, epoxies, etc. According to the curing mechanism, it can be divided into free radical type and cationic type. (Meth)acrylates are typical free radical reactive diluents, and the curing reaction is carried out by free radical photopolymerization. Epoxy is a cationic reactive diluent, and its curing reaction mechanism is cationic polymerization. Vinyl ethers can participate in both free radical polymerization and cationic polymerization, so they can be used as reactive diluents for two photocuring systems. Those skilled in the art can select various components of photosensitive resins according to the prior art, and assemble them to obtain the desired photosensitive resin. In one embodiment of the present invention, the photosensitive resin used is a photocurable transparent resin (model FLGPCL04), purchased from Formlab, which is composed of polymer monomers and prepolymers, to which ultraviolet light initiators (or photosensitizers) are added, which can immediately cause polymerization reactions and complete curing under the irradiation of ultraviolet light of a certain wavelength (250-300nm).

[0050] In the present invention, the thermoplastic plastic is a type of plastic that is plastic at a certain temperature and solidifies after cooling, including acrylonitrile-butadiene-styrene plastic (acrylonitrile-butadiene), polylactic acid (PLA), polyamide (nylon) (PA), polycarbonate (PC), polystyrene (PS), polycaprolactone (PCL), polyphenylsulfone (PPSF), thermoplastic polyurethane (elastic rubber), polyetheretherketone (PEEK), etc.

[0051] In the present invention, the rubber material includes silicone rubber and the like.

[0052] In the present invention, the metal material includes titanium, aluminum, and precious metal powder materials such as gold and silver.

[0053] In the present invention, the alloy material includes titanium alloy, cobalt-chromium alloy, stainless steel, aluminum alloy, nickel alloy and the like.

[0054] In the present invention, the polymer precursor material refers to a prepolymer formed by preliminary polymerization of monomers in the early stage of polymer material synthesis. The precursor material undergoes polymerization reaction under appropriate conditions (such as high temperature, light or other conditions) to form a polymer material.

[0055] Beneficial effects of the present invention

[0056] The present invention provides a metal semiconductor material suitable for use as a LDI-MS matrix, including MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets, which has one or more of the following advantages:

[0057] 1. The metal semiconductor material has good photothermal and photoelectric conversion efficiency, which can improve the dissociation and ionization efficiency of the analyte;

[0058] 2. The metal semiconductor material shows a strong absorption band in the range of 300-400nm, which matches the 355nm laser used in the MALDI-MS mass spectrometer, facilitating the absorption of sufficient laser energy for continuous transfer to the analyte;

[0059] 3. MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets themselves do not produce obvious interference in the low-mass region, which is conducive to obtaining highly sensitive and highly selective detection results;

[0060] 4. The metal semiconductor as a matrix also has high salt tolerance;

[0061] 5. Using this metal semiconductor material as an LDI-MS matrix, a variety of small molecule organic compounds and peptides can be detected.

[0062] The metal semiconductor material doped mass spectrometry sample target plate provided by the present invention has one or more of the following advantages:

[0063] 1. The main body of the target plate is made of materials doped with MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets. The target plate itself can efficiently absorb laser energy and transfer it to the analyte, thereby promoting the desorption and ionization of the analyte;

[0064] 2. By using the sample target plate of the present invention, it is not necessary to add a matrix when dropping the sample, and a highly sensitive mass spectrometry detection can be achieved, which can avoid the tedious operation of optimizing the matrix co-crystallization morphology that is often required during sample preparation, simplify the operation steps, save sample preparation time, and thus improve the detection efficiency;

[0065] 3. MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets themselves do not produce obvious interference in the low-mass region, which is conducive to obtaining highly sensitive and highly selective detection results;

[0066] 4. Metal semiconductor materials such as MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets have conductive properties. When coupled with commercial stainless steel target holders, no external conductive layer is required, which is portable and efficient.

[0067] 5. The target plate has an outer contour that matches the target holder in the MALDI-MS instrument and can match the target holder of a commercial mass spectrometer;

[0068] 6. The target plate has several sample holes on its surface for holding samples to be tested, which is compatible with commercial mass spectrometers and can achieve rapid and high-throughput sample detection;

[0069] 7. The target plate can be 3D printed, with a simple manufacturing process and stable strength;

[0070] 8. The material used for 3D printing the target plate is about 10 grams, and the cost of a single plate is less than RMB 20, which is low cost;

[0071] 9. The target plate is printed with photosensitive resin, which has the advantage of light weight;

[0072] 10. The target plate can be used for LDI-MS detection to achieve high-precision molecular weight determination of a wide range of organic small molecules (including but not limited to alkaloids, amino acids), peptides, etc.

[0073] 11. The target plate is used to test the sample, and the sample to be tested can be evenly distributed in the sample hole of the target plate, thereby reducing the "hot spots" generated during the co-crystallization process when using traditional organic matrices.

[0074] Therefore, it has better repeatability.

[0075] The method for detecting samples using the metal semiconductor material matrix or mass spectrometry sample target plate provided by the present invention does not require the addition of additional matrix during sample preparation, is easy to operate, has high detection efficiency, good repeatability, high sensitivity, and high selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The characterization results of the MoS2 nanosheets synthesized in Example 1 of the present invention are shown.

[0077] Figure 2 Shown are the characterization results of D-WO3 nanosheets.

[0078] Figure 3 The figure shows the results of LDI-MS detection of a mixed alkaloid sample using 1 g / L metal semiconductor material MoS2 powder as a matrix. The mixed alkaloid sample includes physostigmine, magnocurine, strychnine, colchicine, nepomrine, and aconitine, all with a concentration of 10 mg / L. The added volume of the mixed alkaloid sample is 1 μL. The red dots in the figure indicate the six alkaloids detected and a fragment signal of aconitine (m / z 646→586).

[0079] Figure 4 The figure shows the results of LDI-MS detection of a mixed alkaloid sample using MoS2 nanosheets, a metal semiconductor material, as a matrix at a concentration of 1 g / L. The mixed alkaloid sample includes physostigmine, magnocurine, strychnine, colchicine, neapolitanine, and aconitine, all at a concentration of 10 mg / L. The added volume of the mixed alkaloid sample is 1 μL. The red dots in the figure indicate the six alkaloids detected and a fragment signal of aconitine (m / z 646→586).

[0080] Figure 5 The figure shows the results of LDI-MS detection of a mixed alkaloid sample using D-WO3 nanosheets, a metal semiconductor material with a concentration of 1 g / L, as a matrix. The mixed alkaloid sample includes physostigmine, magnocurine, strychnine, colchicine, neorin, and aconitine, all with a concentration of 10 mg / L. The added volume of the mixed alkaloid sample is 1 μL. The red dots in the figure indicate the six alkaloids detected and a fragment signal of aconitine (m / z 646→586).

[0081] Figure 6 The effect of DHC concentration on the relative abundance of MS signal intensities of four alkaloids using D-WO3 nanosheets (A) or MoS2 nanosheets (B) as the matrix is ​​shown, and the error bars represent the standard deviation (σ).

[0082] Figure 7The titration fitting curves and linear fitting curves of three alkaloids (magnocurine, neapolitanine and aconitine) drawn using D-WO3 nanosheets (A) and MoS2 nanosheets (B) as matrices and detected by LDI-MS (n=6) are shown, in which benzylpyridinium chloride (10 μg / mL) was added as an internal standard.

[0083] Figure 8 Shown are the LDI-MS spectra measured using D-WO3 nanosheets (top) and MoS2 nanosheets (bottom) as matrices for four traditional Chinese medicine extracts (Aconiti Lateralis, Aconitum kusnezoffii, Radix Aconiti Kusnezoffii, and Magnolia officinalis), where red dots are used to mark the detected alkaloids.

[0084] Fig. 9 A top view and a physical picture of a mass spectrometry sample target plate doped with a metal semiconductor material prepared by 3D printing in Example 7 of the present invention are shown.

[0085] Fig.10 The results of LDI-MS detection by directly spotting the alkaloid mixture sample onto a mass spectrometry sample target plate doped with 1wt‰ MoS2 powder are shown.

[0086] Fig.11 The results of LDI-MS detection of the alkaloid mixture sample directly onto the mass spectrometry sample target plate doped with 0.2wt‰ MoS2 nanosheets are shown.

[0087] Fig.12 The results of LDI-MS detection of the alkaloid mixture sample directly onto the mass spectrometry sample target plate doped with 0.5wt‰ D-WO3 nanosheets are shown.

[0088] Fig.13 Shown are the LDI-MS spectra of a mixture sample of four alkaloids obtained on a mass spectrometry sample target plate doped with 0.1wt‰, 0.2wt‰, 0.5wt‰ or 1wt‰ D-WO3 nanosheets (A) or MoS2 nanosheets (B).

[0089] Fig.14 The titration fitting curves and linear fitting curves of four alkaloids were drawn using LDI-MS (n=6) on a mass spectrometry sample target plate doped with 0.5wt‰ D-WO3 nanosheets or a mass spectrometry sample target plate doped with 0.2wt‰ MoS2 nanosheets, in which benzylpyridinium chloride (10μg / mL) was added as an internal standard.

[0090] Fig.15 The LDI-MS spectra of three traditional Chinese medicine extracts (Aconiti Kusnezoffii, Radix Aconiti Lateralis and Magnolia Bark) measured using two target plates: a mass spectrometry sample target plate doped with 0.5wt‰ D-WO3 nanosheets and a mass spectrometry sample target plate doped with 0.2wt‰ MoS2 nanosheets.

[0091] Fig.16 The results of mass spectrometry imaging analysis of Chinese herbal medicine slices using a mass spectrometry sample target plate doped with 0.2wt‰ MoS2 nanosheets are shown, wherein A is an optical photograph of a Magnolia bark slice; B is the MSI result of mesocurarine in the Magnolia bark slice; C is the result of staining the Magnolia bark slice with safranin fast green; D is the total average mass spectrum obtained by measuring the Magnolia bark slice using a mass spectrometry sample target plate doped with 0.2wt‰ MoS2 nanosheets. DETAILED DESCRIPTION

[0092] The substantive content of the present invention is further described below in conjunction with specific embodiments of the present invention. It should be understood that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to conventional conditions or manufacturer recommendations. If the manufacturer is not specified for the drugs or reagents used, they are all conventional products that can be obtained commercially.

[0093] Although many materials and operating methods used in the following examples are well known in the art, the present invention is still described in as much detail as possible. It is clear to those skilled in the art that the materials and operating methods used in the following examples are well known in the art unless otherwise specified.

[0094] See also Fig. 9 One embodiment of the present invention provides a sample target plate for laser desorption ionization mass spectrometry, comprising a target plate 1 and a sample hole 2 located on the surface of the target plate, wherein at least the target plate at the bottom of the sample hole 2 is doped with a metal semiconductor material, and the metal semiconductor material is selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets. The sample hole is used to hold a sample to be tested.

[0095] More preferably, at least the target plate at the bottom and the wall of the sample hole 2 is doped with a metal semiconductor material. More preferably, the entire target plate 1 is doped with a metal semiconductor material.

[0096] Preferably, the doping amount of the metal semiconductor material is 0.1-1wt‰ (eg, 0.1wt‰, 0.2wt‰, 0.5wt‰, 1wt‰) of the weight of the target plate at the site.

[0097] Preferably, the metal semiconductor material is D-WO3 nanosheets, and the doping amount thereof is 0.2-1wt‰, for example 0.5wt‰, of the weight of the target plate at the site.

[0098] Preferably, the metal semiconductor material is MoS2 nanosheets, and the doping amount thereof is 0.1-0.2wt‰ of the weight of the target plate at the position.

[0099] Preferably, the entire target plate 1 is doped with metal semiconductor material, and the doping amount is 0.1-1wt‰ (eg, 0.1wt‰, 0.2wt‰, 0.5wt‰, 1wt‰) of the weight of the target plate.

[0100] Preferably, the target plate is made of a polymer material (such as a photosensitive resin, a thermoplastic plastic, a rubber material) or a metal material (such as an alloy material) doped with a metal semiconductor material, and the doping amount of the metal semiconductor is 0.1-1wt‰ (such as 0.1wt‰, 0.2wt‰, 0.5wt‰, 1wt‰) of the weight of the polymer material or the metal material.

[0101] Preferably, the sample target plate has an outer contour that matches the target holder of the MALDI-MS instrument, so that when the analyte is detected, the target plate can fully cooperate with the target holder, so that the analyte is smoothly ionized, and flight acceleration and detection are achieved; it also ensures that the target plate can match and be compatible with the target holder of a commercial mass spectrometer, thereby achieving rapid and high-throughput sample detection.

[0102] Further preferably, the sample target plate also has a fixing hole for fixedly connecting with the target holder of the MALDI-MS instrument, for example, the fixed connection can be achieved by a flange, a screw, a bolt or a top screw. Further preferably, when the sample is tested, the target plate containing the sample to be tested is fixedly connected with the target holder of the matrix-assisted laser desorption ionization mass spectrometer by a flange, a screw, a bolt or a top screw.

[0103] Further preferably, the surface of the target plate has a plurality of (for example, 4, 8, 16, 24, 48, 96) periodically arranged sample holes, and the preferred arrangement is a two-dimensional periodic arrangement, so that a plurality of samples to be tested can be placed in a plurality of sample holes for testing, which makes the operation more convenient and the test results more accurate.

[0104] Further preferably, the above-mentioned plurality of sample holes are of equal size and same shape, and can be in the shape of rectangular hole, hemispherical hole, semi-ellipsoidal hole, cylindrical hole or any other sample hole capable of placing the sample to be tested.

[0105] In one embodiment, MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets can be added to a specific volume of commercial photosensitive resin and stirred and shaken thoroughly to make the MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets evenly dispersed in the photosensitive resin, and then processed and manufactured using 3D printing technology to obtain the sample target plate.

[0106] The sample target plate described in the present invention is doped with MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets, so the target plate itself can efficiently absorb laser energy and transfer energy to the analyte through a certain efficient transfer mechanism, thereby promoting effective desorption and ionization of the analyte from the surface of the target plate. Therefore, when using the sample target plate of the present invention, there is no need to add any additional matrix during sample preparation, and high-sensitivity mass spectrometry detection can be achieved, thereby simplifying the operating steps, saving sample preparation time and improving detection efficiency.

[0107] The doping amount of the MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets in the target plate is preferably 0.1wt‰~1wt‰. Too little will affect the performance of the LDI-MS sample target plate, while too much will increase the difficulty of processing and manufacturing and make it difficult to evenly disperse in the photosensitive resin. Therefore, the doping amount of MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets is preferably controlled within the above range.

[0108] Further preferably, the sample target plate of the present invention is prepared by 3D printing technology, thereby improving the manufacturing accuracy of the target plate, so that the target plate is better matched with the target holder to complete subsequent detection and analysis.

[0109] In some embodiments, the sample target plate of the present invention can use thermoplastics, various metals and alloys, rubber materials, light-hardening resin as a substrate, and dope MoS2 powder, D-WO3 nanosheets or MoS2 nanosheets in the substrate, and then process and shape. In some embodiments, a photosensitive resin can be used as the substrate of the target plate, and it is preferably a liquid photosensitive resin before molding, which is convenient for doping with MoS2 powder, D-WO3 nanosheets, and MoS2 nanosheets. The photosensitive resin will immediately cause a polymerization reaction under the irradiation of light of a certain wavelength, so that curing can be completed quickly.

[0110] The present invention is further described in detail below through specific examples.

[0111] Example 1 Preparation and characterization of 2D MoS2 nanosheets

[0112] The synthesis of MoS2 adopts chemical exfoliation method (Goki Eda, Hisato Yamaguchi, Damien Voiry, Takeshi Fujita, Mingwei Chen, and Manish Chhowalla, Photoluminescence from Chemically Exfoliated MoS2, Nano Lett. 2011, 11, 5111-5116. doi: 10.1021 / nl201874w). The specific steps are as follows: Under a nitrogen atmosphere, 5ml of 1.6M butyl lithium in n-hexane solution is added to 500mg MoS2 powder (purchased from Beijing Bailingwei Technology Co., Ltd., purity 99%), and then stirred at room temperature for 48 hours. After that, the unreacted butyl lithium is filtered out, and the semi-dried solid on the filter paper is rinsed 3 times with n-hexane to wash away the butyl lithium that is not completely filtered out. Then the solid on the filter paper is quickly placed in 150mL of ultrapure water, and water bath ultrasound is performed for 1 hour to complete the exfoliation of the MoS2 layer. The ultrasonicated liquid mixture containing MoS2 was then centrifuged at 3000 rpm for 10 min to remove the unpeeled MoS2 at the bottom layer, and the supernatant was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and then dialyzed in ultrapure water for 48 hours, with the ultrapure water being replaced every 8 hours. After the dialysis was completed, the solution of the MoS2 nanosheet layer (i.e., MoS2 nanosheet) was stored in a refrigerator at 4°C.

[0113] Example 2 Characterization of MoS2 Nanosheets and D-WO3 Nanosheets

[0114] The MoS2 nanosheets and D-WO3 nanosheets (Defect-rich WO3 nanosheets, donated by Professor Wang Qiang's research group at Lanzhou University) synthesized in Example 1 were characterized.

[0115] The instruments used for characterization include: Thermo Scientific Nicolet 6700 Fourier transform infrared spectrometer purchased from Thermo Scientific (USA), Varian Cary 300 UV-visible spectrophotometer purchased from Varian (USA), Zetasizer Nano-ZS90 dynamic light scattering instrument purchased from Malvern Instruments (USA), 5410 high-speed centrifuge purchased from Eppendorf (Germany), and magnetic stirrer purchased from SCILOGEX (USA).

[0116] About 1 mg of NM (MoS2 nanosheets or D-WO3 nanosheets) was dissolved in 1 mL of ultrapure water and ultrasonically treated for 1 hour before use to produce a uniform dispersed solution, thereby preparing NM matrix solutions, respectively, and collecting UV-visible absorption spectra, Fourier transform infrared spectroscopy (KBr pellet method), and dynamic light scattering (DLS) particle size data.

[0117] The NM matrix solution was diluted to 0.1 mg / mL, and 200 μL was taken to obtain transmission electron microscopy images and energy dispersive X-ray spectroscopy (EDX) images using the STEM and EDX modules of a FEI Tecnai G2 F20 transmission electron microscope (FEI Company, USA).

[0118] The characterization results of MoS2 nanosheets are shown in Figure 1 , where A is the transmission electron microscope (TEM) image; B is the energy spectrum (EDX) image; C is the ultraviolet-visible absorption spectrum (UV-vis); and D is the Fourier transform infrared (FT-IR) spectrum. The characterization results of D-WO3 nanosheets are shown in Figure 2 , where A is the transmission electron microscope (TEM) image; B is the energy spectrum (EDX) image; C is the ultraviolet-visible absorption spectrum (UV-vis) image; and D is the Fourier transform infrared (FT-IR) spectrum.

[0119] In general, NMs in LDI-MS can act as effective laser radiation absorbers and LDI promoters, enabling the direct analysis of a variety of analytes. TEM and EDX images show that both MoS2 nanosheets and D-WO3 nanosheets have sufficient and uniform elemental distribution. UV-Vis spectra show that both MoS2 nanosheets and D-WO3 nanosheets show stronger absorption bands in the range of 300-400 nm, which matches the 355 nm laser used in the Autoflex III Smartbeam MALDI-MS mass spectrometer (Bruker, Germany), which will be beneficial for NMs to absorb sufficient laser energy for continuous transfer to analytes. In fact, this is also a prerequisite for selecting NMs as LDI-MS matrices. The characterization results show that MoS2 nanosheets and D-WO3 nanosheets have good photothermal and photoelectric conversion efficiencies and can be used as candidate substances for LDI-MS matrices.

[0120] Example 3 MoS2 powder, MoS2 nanosheets or D-WO3 nanosheets as matrix for alkaloid mixture determination

[0121] About 1 mg of metal semiconductor material (MoS2 powder, MoS2 nanosheets or D-WO3 nanosheets) was dissolved in 1 mL of ultrapure water and ultrasonically treated for 1 hour before use to produce a uniform dispersed solution, thereby preparing matrix solutions of three metal semiconductor materials. PEG 600 was diluted 1000 times with water (concentration after dilution was about 1.12 mg / mL) and dissolved in an aqueous solution containing 0.1% NaCl as a mass axis calibration solution in positive ion mode.

[0122] Dissolve various alkaloids in ultrapure water to obtain a 1 mg / mL stock solution of alkaloids. Prepare a mixed solution of six alkaloids (physostigmine, magnocurine, strychnine, colchicine, neorin, and aconitine) by mixing different alkaloid stock solutions in equal proportions. Dilute the mixed solution to the desired concentration before use.

[0123] The thin layer spotting method (take 1 μL NM matrix solution, first spot the matrix layer, and after natural drying, take 1 μL sample (where the concentration of each alkaloid is 10 μg / mL), and spot the sample layer on the matrix layer) was used for spotting on the Polished steel MALDI target plate. Mass spectral data were acquired on an Autoflex III Smartbeam MALDI-MS mass spectrometer, and the mass spectrum of each point was collected 1000 times in the m / z range of 100 to 1000 Da in positive ion reflectance mode or negative ion reflectance mode (ion source 1 voltage = 19 kV, ion source 2 voltage = 16.65 kV, detector voltage = 1.796 kV). Mass axis calibration was performed by the external standard method, using PEG 600 with m / z 300-1000 Da as a low molecular weight calibration substance. MS data were collected and analyzed by FlexControl and FlexAnalysis software. In positive ion reflectron mode, the total average mass spectrum was collected by total ion counts under the average signal of 1000 shots in the mass range of 100-1000 Da. The Gubbs test was used to remove outliers from the collected data and perform graphical analysis in Origin 2021 (OriginLab, USA).

[0124] For the six alkaloids investigated, when MoS2 powder, MoS2 nanosheets or D-WO3 nanosheets were used as the matrix for LDI-MS detection, serious background interference occurred in the negative ion mode. When the concentration of the alkaloids was at the level of 10 μg / mL, no spectral information of any small molecules could be obtained.

[0125] Considering the molecular nature of alkaloids as tertiary amines, the detection performance of various metal semiconductor materials for alkaloids in positive ion mode at a concentration of 1g / L was tested. Figure 3 , Figure 4 and Figure 5 As shown, the red dots indicate the detected six alkaloids and a fragment signal of aconitine (m / z 646→586). The results show that the detection of alkaloid samples with a concentration of 10 μg / mL using MoS2 powder, MoS2 nanosheets or D-WO3 nanosheets as LDI-MS matrices can display the response spectra of the six alkaloids, and the background response generated at the same time has relatively little background interference in the full range of m / z 200-700.

[0126] The matrix concentration is an important factor affecting its detection performance. This embodiment also investigates the effects of other concentrations of D-WO3 nanosheets and MoS2 nanosheets on the MS signal intensity of six alkaloids. The results show that the concentration of D-WO3 nanosheets or MoS2 nanosheets between 0.1-1 mg / mL does not significantly affect the MS signal intensity of six alkaloids. However, considering the solubility of these two metal semiconductor materials, 1 mg / mL D-WO3 nanosheets and 0.2 mg / mL MoS2 nanosheets are preferably used as matrices for LDI-MS detection.

[0127] Example 4: Effect of DHC on relative abundance of MS signal intensity using D-WO3 nanosheets or MoS2 nanosheets as matrix for alkaloid mixture detection

[0128] Diammonium Hydrogen Citrate (DHC, purity 99%) was purchased from Sigma-Aldrich (USA).

[0129] About 1 mg of D-WO3 nanosheets or 0.2 mg of MoS2 nanosheets were dissolved in 1 mL of ultrapure water and ultrasonically treated for 1 hour before use to produce a uniform dispersed solution, thereby preparing the matrix solution of the metal semiconductor material. Different concentrations of DHC were added to the matrix solution as a proton transfer agent to investigate the effect of different concentrations of DHC on the performance of NM matrix in detecting alkaloids. PEG 600 (concentration of about 1.12 g / mL) was diluted 1000 times and dissolved in an aqueous solution containing 0.1% NaCl as a mass axis correction solution in the positive ion mode.

[0130] Dissolve various alkaloids in ultrapure water to obtain a 1 mg / mL stock solution of alkaloids. Prepare a mixed solution of four alkaloids (magnocurine, strychnine, colchicine, and aconitine) by mixing different alkaloid stock solutions in equal proportions. Dilute the mixed solution to the desired concentration before use.

[0131] The sample was spotted on a Polished steel MALDI target plate using a thin layer spotting method, and the alkaloid mixture sample (wherein the concentration of each alkaloid was 10 mg / L) was subjected to LDI-MS detection using an Autoflex III Smartbeam MALDI-MS mass spectrometer. The spotting and detection methods were the same as those in Example 3.

[0132] Test results such as Figure 6 As shown. Diammonium hydrogen citrate (DHC) is a commonly used proton transfer agent in MALDI-MS. With the increase of DHC concentration, the peak intensity of magnocurine, strychnine, colchicine and aconitine basically increases first and then remains flat (or decreases), and can be increased by up to 3 to 4 times (D-WO3 nanosheet matrix) or 2 to 4 times (MoS2 nanosheet matrix). The results show that when D-WO3 nanosheets are used as a matrix, adding 0.1-1g / L (preferably 0.5g / L) of DHC can significantly enhance the relative abundance of MS signal intensity. When MoS2 nanosheets are used as a matrix, adding 0.1-0.2g / L (preferably 0.1g / L) of DHC can significantly enhance the relative abundance of MS signal intensity.

[0133] Example 5: Methodological verification of LDI-MS detection of alkaloid mixture using D-WO3 nanosheets and MoS2 nanosheets as matrices

[0134] About 1 mg of D-WO3 nanosheets or 0.2 mg of MoS2 nanosheets were dissolved in 1 mL of ultrapure water and ultrasonically treated for 1 hour before use to produce a uniform dispersed solution, thereby preparing a matrix solution of metal semiconductor materials. PEG 600 (concentration of about 1.12 g / mL) was diluted 1000 times and dissolved in an aqueous solution containing 0.1% NaCl as a mass axis calibration solution in the positive ion mode.

[0135] Various alkaloids were dissolved in ultrapure water to obtain a 1 mg / mL stock solution of the alkaloids. A mixed solution of three alkaloids (magnocurine, neorin and aconitine) was prepared by mixing different alkaloid stock solutions in equal proportions. Before use, the mixed solution was diluted to obtain a series of standard samples, and benzylpyridinium chloride (10 μg / mL) was added as an internal standard.

[0136] The sample was spotted on a Polished steel MALDI target plate using a thin layer spotting method, and the alkaloid mixture sample (wherein the concentration of each alkaloid was 10 mg / L) was subjected to LDI-MS detection using an Autoflex III Smartbeam MALDI-MS mass spectrometer. The spotting and detection methods were the same as those in Example 3. A titration curve and a linear standard curve were drawn based on the detection results, as shown in FIG. Figure 7 shown.

[0137] This example carried out a preliminary method validation, using the internal standard (IS) addition method to add benzyl pyridinium ion ([BP] + ) established a calibration curve for IS. When LDI-MS is used to detect alkaloids, the internal standard method can effectively correct the response value fluctuation caused by different NMs. This example selected three representative alkaloids (magnocurine, neorin and aconitine), and plotted their titration curves and linear standard curves ( Figure 7 The results showed that the three alkaloids followed an S-shaped curve in the range of 0.5 to 64 μg / mL, and D-WO3 nanosheets as the matrix (R 2 >0.995) than MoS2 nanosheets as matrix (R 2 The linear range of the three alkaloids in both matrices was 0.5-8 μg / mL.

[0138] Example 6 D-WO3 nanosheets and MoS2 nanosheets as matrices for actual sample analysis

[0139] Weigh each of Aconite, Radix Aconiti Kusnezoffii, Radix Aconiti Chuanwu, and Cortex Magnoliae Officinalis, soak them, and reflux extract them for 1 hour with 10 times the amount of ultrapure water. After filtering with 8 layers of gauze, reflux extract the residue again for 1 hour by adding 8 times the amount of ultrapure water. After filtering with 3 layers of gauze, combine the filtrates and concentrate them to 0.5 g / mL to obtain the aqueous extracts of Aconite, Radix Aconiti Kusnezoffii, Radix Aconiti Chuanwu, and Cortex Magnoliae Officinalis, which were stored at 4°C before use.

[0140] About 1 mg of D-WO3 nanosheets or 0.2 mg of MoS2 nanosheets were dissolved in 1 mL of ultrapure water and ultrasonically treated for 1 hour before use to produce a uniform dispersed solution, thereby preparing a matrix solution of metal semiconductor materials. PEG 600 (concentration of about 1.12 g / mL) was diluted 1000 times and dissolved in an aqueous solution containing 0.1% NaCl as a mass axis calibration solution in the positive ion mode.

[0141] The sample was spotted on a Polished steel MALDI target plate using a thin layer spotting method, and the water extracts of Aconite, Aconitum kusnezoffii, Aconitum carmichaelii and Magnolia officinalis obtained above were subjected to LDI-MS detection using an Autoflex III Smartbeam MALDI-MS mass spectrometer. The spotting and detection methods were the same as those in Example 3.

[0142] The LDI-MS spectra of four traditional Chinese medicine extracts (Aconite, Aconitum, Radix Aconiti Kusnezoffii, and Magnolia Bark) measured using D-WO3 nanosheets and MoS2 nanosheets as matrices are shown in Figure 2. Figure 8The related alkaloids detected were annotated with reference to the literature, including 14-acetylcaraquinone (m / z 420.2745), neoline (m / z 438.2852), aconitine (m / z 454.2801), benzoylhypaconitine (m / z 574.301), benzoylhypaconitine (m / z 588.317), benzoylmeaconitine (m / z 590.296), benzoylmeaconitine (m / z 604.312), hypoaconitine (m / z 616.312), 3-deoxyaconitine (m / z 630.327), meaconitine (m / z 632.307), aconitine (m / z 646.322) and magnocurarine (m / z 314.1737). Among them, magnocurine is the main toxic alkaloid component of Magnolia officinalis, and the other alkaloids are the main toxic components of three Aconitum Chinese medicinal materials (Aconite, Aconitum kusnezoffii and Aconitum carmichaelii), and belong to the Aconitum alkaloids.

[0143] Example 7 3D Printing Metal Semiconductor Material Impurity Spectrum Sample Target Plate

[0144] Photopolymer transparent resin (model: FLGPCL04) was purchased from Formlabs (USA). 3ds Max 2020 (Autodesk) software was used to design the 3D model. Form 3 printer was purchased from Formlabs (USA) and equipped with 405nm UV light with a power of 250mW. FlexImaging4.1 software was used for MSI data acquisition and visualization, and SCiLS Lab 2020b software was used for imaging data analysis.

[0145] Metal semiconductor materials (i.e., MoS2 powder, D-WO3 nanosheets, or MoS2 nanosheets) were mixed with a photocurable transparent resin (model FLGPCL04) to prepare a 3D printable metal semiconductor material-doped resin. The appropriate concentration of metal semiconductor material was gently added to the glass beaker to which the resin had been added, and then the mixture was vigorously stirred until the NM was evenly dispersed. Before entering the 3D printer to start printing, the resin was ultrasonically treated for 60 minutes and stirred again to be uniform.

[0146] Referring to the size of the commercial stainless steel 96-well MALDI target plate, a 3D model of a 3D metal semiconductor material doped target plate was designed in 3ds Max 2020 (Autodesk) and exported as a slice file format (STL), which can be applied to the steelMTP NALDI adapter (Bruker Daltonik, Germany). The digital model was imported into the Form 3 printer (Formlabs, Somerville, MA, USA) and printed using the original transparent resin or the resin doped with the metal semiconductor material, with a layer thickness accuracy of 25 μm. After printing, the target plate was immersed in ethanol and gently stirred for a short time to remove the uncured resin to obtain a mass spectrometry sample target plate doped with MoS2 powder, D-WO3 nanosheets or MoS2 nanosheets.

[0147] The target plate is printed by a commercial desktop light-curing 3D printer, and the printing material is a photosensitive resin doped with 1wt‰ MoS2 powder, 0.2wt‰ MoS2 nanosheets or 0.5wt‰ D-WO3 nanosheets. The MoS2 powder, D-WO3 nanosheets or MoS2 nanosheets in the target plate are uniformly dispersed, can efficiently absorb laser energy, and transfer part of the laser energy to the analyte through a certain mechanism (including thermally driven or non-thermal desorption, charge transfer, phase change, nanophoton ionization and other desorption / ionization mechanisms) to promote the desorption and ionization of the analysis; therefore, in the preparation and detection process of the sample to be tested, high-sensitivity mass spectrometry detection can be achieved without the need for the addition of traditional organic matrix, which is fast and high-throughput.

[0148] In addition, this embodiment also prepares mass spectrometry sample target plates with doping concentrations of 0.1wt‰, 0.2wt‰, 0.5wt‰ or 1wt‰ D-WO3 nanosheets or MoS2 nanosheets.

[0149] Fig. 9 The metal semiconductor material doped mass spectrometry sample target plate prepared in this embodiment is shown. The main material of the sample target plate is a photosensitive resin doped with MoS2, D-WO3 nanosheets, and MoS2 nanosheets. The sample target plate includes a target plate 1 and a sample hole 2 located on the surface of the target plate. The entire target plate 1 is doped with metal semiconductor materials.

[0150] The sample target plate as a whole can be regarded as a convex font structure, which can be divided into two rectangular blocks, wherein the upper surface of the large rectangular block has 96 circular sample holes arranged in 8 rows and 12 columns, which are used to hold the samples to be tested by the MALDI-MS instrument; wherein the small rectangular block has four positioning holes, which are used to be fixedly connected with the target holder of the MALDI-MS instrument by screws. The sample target plate is printed by a commercial desktop light-curing 3D printer, and the printing and manufacturing process is simple and low-cost.

[0151] Example 8 Detection of alkaloid mixture based on metal semiconductor material doped mass spectrometry sample target plate

[0152] The alkaloid mixture sample was subjected to LDI-MS determination using the metal semiconductor material doped mass spectrometry sample target plate prepared in Example 7, wherein the alkaloid mixture sample contained six alkaloids, namely, physostigmine, magnocurine, strychine, colchicine, neorin, and aconitine, and the concentration of each alkaloid was 10 mg / L, and the preparation method was the same as that in Example 3. First, 1 μL of the alkaloid mixture sample was transferred and directly spotted onto the metal semiconductor material doped mass spectrometry sample target plate prepared in Example 7; after the sample was naturally dried, the target plate was placed in a MALDI-MS mass spectrometer for detection, and the detection method was the same as that in Example 3. The detection results are shown in FIG. Fig.10 , 11 , 12, where the spectral peaks of the alkaloids are marked with red dots. It can be seen that without adding additional traditional organic matrices, directly spotting the sample onto the mass spectrometry sample target plate doped with 1wt‰ MoS2 powder, 0.2wt‰ MoS2 nanosheets or 0.5wt‰ D-WO3 nanosheets can display the response spectra of all six alkaloids, and the background response generated at the same time, among which aconitine is measured in the form of fragment signals (m / z 646→586). Relatively less background interference is generated in the full range of m / z 200-700, and the alkaloid mixture has a higher spectral peak intensity.

[0153] Example 9 Effect of doping concentration in metal semiconductor material doped mass spectrometry sample target on detection results

[0154] A mixture of four alkaloids (magnocurine, strychnine, colchicine and neorin) was prepared, each alkaloid concentration was 10 mg / L, and the preparation method was the same as in Example 3. 1 μL of the alkaloid mixture sample was pipetted and directly spotted onto the mass spectrometry sample target plate doped with 0.1 wt‰, 0.2 wt‰, 0.5 wt‰ or 1 wt‰ D-WO3 nanosheets or MoS2 nanosheets prepared in Example 7; after the sample was naturally dried, the target plate was placed in a MALDI-MS mass spectrometer for detection, and the detection method was the same as in Example 3.

[0155] Test results such as Fig.13 The results show that the mass spectrometry sample target plates doped with 0.1-1wt‰ D-WO3 nanosheets or MoS2 nanosheets can display the corresponding spectra of the four alkaloids, wherein for D-WO3 nanosheets, the preferred doping concentration is 0.2-1wt‰, such as 0.5wt‰; for MoS2 nanosheets, the preferred doping concentration is 0.1-0.2wt‰.

[0156] Example 10 Methodological verification of LDI-MS detection using a mass spectrometry sample target plate doped with metal semiconductor materials

[0157] Various alkaloids were dissolved in ultrapure water to obtain a 1 mg / mL stock solution of alkaloids. A mixed solution of four alkaloids (colchicine, magnocurine, strychnine, and neorin) was prepared by mixing different alkaloid stock solutions in equal proportions. Before use, the mixed solution was diluted to obtain a series of standard samples, and benzylpyridinium chloride (10 μg / mL) was added as an internal standard.

[0158] 1 μL of the standard sample was directly applied to the mass spectrometry sample target plate doped with 0.5 wt‰ D-WO3 nanosheets or the mass spectrometry sample target plate doped with 0.2 wt‰ MoS2 nanosheets prepared in Example 7; after the sample was dried naturally, the target plate was placed in a MALDI-MS mass spectrometer for detection, and the detection method was the same as that in Example 3. A titration curve and a linear standard curve were drawn according to the detection results, as shown in FIG. Fig.14 shown.

[0159] This example uses the internal standard (IS) addition method to establish a calibration curve using benzylpyridinium ion ([BP]+) as IS. When LDI-MS is used to detect alkaloids, the internal standard method can effectively correct the response value fluctuation caused by different NMs.

[0160] The results showed that colchicine fit the S-shaped curve in the range of 4-64μg / mL (mass spectrometry sample target plate doped with 0.5wt‰D-WO3 nanosheets) and 16-64μg / mL (mass spectrometry sample target plate doped with 0.2wt‰MoS2 nanosheets), and the other three alkaloids fit the S-shaped curve in the range of 2-64μg / mL. The mass spectrometry sample target plate doped with 0.5wt‰D-WO3 nanosheets (R2>0.823) provided better fitting performance than the mass spectrometry sample target plate doped with 0.2wt‰MoS2 nanosheets (R2>0.754). In the two target plates, the linear ranges of the four alkaloids were 8-64μg / mL (mass spectrometry sample target plate doped with 0.5wt‰D-WO3 nanosheets) and 16-64μg / mL (mass spectrometry sample target plate doped with 0.2wt‰MoS2 nanosheets), and the linear ranges of the other three alkaloids were 2-64μg / mL (such as Fig.14Compared with the metal semiconductor material used as the matrix solution for LDI-MS detection in Example 5, after the metal semiconductor material is doped into the mass spectrometry sample target plate, the LOD of the detection method is significantly increased. It is speculated that the possible reason is that when the metal semiconductor material is used as the LDI-MS matrix, the NM and the analyte can fully contact and play, which is more conducive to the desorption and ionization of the analyte. After it is doped into the mass spectrometry sample target plate, the contact between the NM and the analyte on the target plate surface is not sufficient, so the desorption and ionization efficiency is slightly reduced.

[0161] Example 11: Analyzing actual samples using a mass spectrometry sample target plate doped with metal semiconductor materials

[0162] Weigh each of Radix Aconiti Kusnezoffii, Radix Aconiti Chuanwu, and Cortex Magnoliae Officinalis, soak them, and reflux extract them for 1 hour with 10 times the amount of ultrapure water. After filtering with 8 layers of gauze, add 8 times the amount of ultrapure water to reflux extract the residue for another 1 hour. After filtering with 3 layers of gauze, combine the filtrates and concentrate them to 0.5 g / mL to obtain the aqueous extracts of Radix Aconiti Kusnezoffii, Radix Aconiti Chuanwu, and Cortex Magnoliae Officinalis, respectively, and store them at 4°C before use.

[0163] 1 μL of the water extract of Aconitum kusnezoffii, Aconitum carmichaelii and Magnolia officinalis obtained above was transferred and directly spotted onto the metal semiconductor material doped mass spectrometry sample target plate prepared in Example 7; after the sample was naturally dried, the target plate was placed in a MALDI-MS mass spectrometer for detection, and the detection method was the same as in Example 3.

[0164] In this example, two target plates, a mass spectrometry sample target plate doped with 0.5wt‰ D-WO3 nanosheets and a mass spectrometry sample target plate doped with 0.2wt‰ MoS2 nanosheets, were used to test actual samples to evaluate their applicability in screening alkaloids in traditional Chinese medicine extraction. The results are shown in Figure 2. Fig.15 As shown, the mass spectrum provided by the same metal semiconductor material matrix in a conventional steel target plate ( Figure 8 ), the number of alkaloid signals obtained by the 3D NM target plate was slightly less, the signal intensity was slightly lower, but the background interference was also reduced. The relevant alkaloids detected were annotated with reference to the literature, including aconitine (m / z 454.2801), benzoylhypaconitine (m / z 574.301), benzoylhypaconitine (m / z 588.317), benzoylaconitine (m / z604.312), 3-deoxyaconitine (m / z 630.327) and magnocurarine (m / z314.1737). Among them, magnocurarine is the main toxic alkaloid component of Magnolia officinalis, and the other alkaloids are the main toxic components of Aconitum genus Chinese medicine (Aconiti Kusnezoffii and Aconiti Lateralis), belonging to the aconitum alkaloids (such as Fig.15 shown).

[0165] Example 12: Mass spectrometry imaging analysis of Chinese herbal medicine slices using a metal semiconductor material-doped mass spectrometry sample target plate

[0166] The plant tissue of Magnolia officinalis was cut into 10 μm thick using a freezing microtome. The tissue slice was gently removed using forceps and transferred to the mass spectrometry sample target plate doped with 0.2 wt‰ MoS2 nanosheets prepared in Example 7. The target plate was placed in a vacuum dryer for a few minutes. After the sample became dry, the target plate was placed in a MALDI-MS mass spectrometer for detection. The detection method was the same as in Example 3. The detection results are shown in FIG. Fig.16 As shown, A is the optical photograph of Magnolia bark slices; B is the MSI result of magnocurine in Magnolia bark slices; C is the result of Magnolia bark slices stained with safranin fast green; D is the total average mass spectrum measured by the mass spectrometry sample target plate doped with 0.2wt‰ MoS2 nanosheets on Magnolia bark slices.

[0167] The results showed that the target alkaloid, magnocurarine, was successfully observed in the tissue sections of Magnolia officinalis using the mass spectrometry sample target plate doped with 0.2wt‰ MoS2 nanosheets. The spatial resolution of MSI was 200μm. The distribution of magnocurarine in the sections of Magnolia officinalis can be clearly seen from the figure, and based on the abundance in the mass spectrum, it can be preliminarily judged that its content is at a low level (such as Fig.16 shown).

[0168] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of metal semiconductor materials as matrix in laser desorption ionization mass spectrometry detection, wherein the metal semiconductor materials are selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets.

2. A method for detecting a sample using laser desorption ionization mass spectrometry, comprising: 1) Mix the sample to be tested with the matrix solution, drop it into the sample well of the mass spectrometry sample target plate, and dry it; or, Applying a matrix to the bottom of the sample hole to form a matrix layer (for example, dropping a matrix solution to the bottom of the sample hole and drying to form a matrix layer), and then placing a sample to be tested on the matrix layer; 2) Use MALDI-MS instrument to detect the samples, wherein the matrix is ​​selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets; Preferably, the matrix solution is a solution containing MoS2 powder, D-WO3 nanosheets or MoS2 nanosheets. Preferably, the matrix solution is 0.1-1 mg / mL of MoS2 powder, D-WO3 nanosheets or MoS2 nanosheets aqueous solution; Optionally, the matrix solution further contains a proton transfer agent. Preferably, the proton transfer agent is diammonium hydrogen citrate, Preferably, the matrix solution further contains 0.1-1 g / L (e.g., 0.2 g / L or 0.5 g / L) of diammonium hydrogen citrate; Preferably, the matrix solution is an aqueous solution containing 0.1-1 mg / mL D-WO3 nanosheets and 0.1-1 g / L (e.g., 0.2 g / L or 0.5 g / L) diammonium hydrogen citrate, or the matrix is ​​an aqueous solution containing 0.1-1 mg / mL MoS2 powder or MoS2 nanosheets and 0.1-0.2 g / L diammonium hydrogen citrate.

3. The method according to claim 2, wherein the sample contains small organic molecules and / or polypeptides, Preferably, the organic small molecules include alkaloids and amino acids.

4. A kit for laser desorption ionization mass spectrometry, comprising a metal semiconductor material, wherein the metal semiconductor material is selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets, Preferably, a proton transfer agent is also included, such as diammonium hydrogen citrate.

5. A sample target plate for laser desorption ionization mass spectrometry, comprising a target plate (1) and a sample hole (2) located on the surface of the target plate, wherein at least the target plate at the bottom of the sample hole (2) is doped with a metal semiconductor material, wherein the metal semiconductor material is selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets, Preferably, at least the target plate at the bottom and the wall of the sample hole (2) is doped with a metal semiconductor material; Preferably, the doping amount of the metal semiconductor material is 0.1-1wt‰ (e.g., 0.1wt‰, 0.2wt‰, 0.5wt‰, 1wt‰) of the weight of the target plate at the site; Preferably, the metal semiconductor material is D-WO3 nanosheets, and the doping amount thereof is 0.2-1wt‰ of the weight of the target plate at the site, for example 0.5wt‰; Preferably, the metal semiconductor material is MoS2 nanosheets, and the doping amount thereof is 0.1-0.2wt‰ of the weight of the target plate at the position.

6. The sample target plate of claim 5, wherein the entire target plate is doped with a metal semiconductor material, and the doping amount is 0.1-1wt‰ (e.g., 0.1wt‰, 0.2wt‰, 0.5wt‰, 1wt‰) of the weight of the target plate; Preferably, the target plate is made of a polymer material (such as a photosensitive resin, a thermoplastic plastic, a rubber material) or a metal material (such as an alloy material) doped with a metal semiconductor material, and the doping amount of the metal semiconductor is 0.1-1wt‰ (such as 0.1wt‰, 0.2wt‰, 0.5wt‰, 1wt‰) of the weight of the polymer material or the metal material.

7. The sample target plate of claim 5 or 6, wherein the sample target plate has an outer contour matching a target holder of a MALDI-MS instrument, Preferably, the sample target plate also has a fixing hole for fixedly connecting with the target holder of the MALDI-MS instrument.

8. A method for preparing the sample target plate according to claims 5-7, comprising: 1) Mixing a metal semiconductor material with a polymer precursor material or a metal powder; 2) preparing a sample target plate from the mixture obtained in step 1), wherein the metal semiconductor material is selected from MoS2 powder, D-WO3 nanosheets and MoS2 nanosheets, Preferably, the mixture obtained in step 1) is made into a sample target plate by 3D printing, precision molding, machining, hot and cold processing or manual molding; Preferably, the mixture obtained in step 1) is made into a sample target plate by a 3D printing method.

9. Use of the sample target plate according to claims 5-7 in laser desorption ionization mass spectrometry detection of samples, Preferably, the sample contains small organic molecules and / or polypeptides. Preferably, the organic small molecules include alkaloids and amino acids.

10. A method for detecting a sample using laser desorption ionization mass spectrometry, comprising: 1) placing the sample to be tested in the sample well of the sample target plate described in claims 5-7; 2) Use MALDI-MS instrument to detect the samples, Preferably, the sample contains small organic molecules and / or polypeptides. Preferably, the organic small molecules include alkaloids and amino acids.

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