Solid-state derivatized hydrogel and its preparation method and application

Abstract

The present invention relates to the field of bioanalysis technology, and discloses a solid-state derivatized hydrogel, a preparation method, and an application thereof, and particularly relates to a solid-state derivatized hydrogel, a preparation method, and an application thereof, a biological tissue derivatization method, and a biological tissue metabolite characterization method. The solid-state derivatized hydrogel provided by the present invention comprises a hydrogel, a derivatization reagent, and water; the derivatization reagent is dispersed within and on the surface of a three-dimensional network structure of the solid-state derivatized hydrogel; the mass of the hydrogel accounts for 5 to 45% of the total mass of the hydrogel, the derivatization reagent, and water; the mass concentration of the derivatization reagent in a mixed solution formed by the hydrogel, the derivatization reagent, and water is 0.1 to 200 mg / mL; the solid-state derivatized hydrogel provided by the present invention derivatizes biological tissues with high derivatization efficiency, can significantly improve the detection sensitivity of endogenous metabolites in biological tissues, and effectively realizes mass spectrometry imaging analysis and in situ characterization of low-abundance, difficult-to-ionize metabolites in biological tissues.

Description

Technical Field

[0001] The present invention relates to the field of bioanalysis technology, and in particular to a solid-state derivatized hydrogel and a preparation method and application thereof, a biological tissue derivatization method, and a biological tissue metabolite characterization method. Background Art

[0002] Tissue derivatization is the chemical derivatization of metabolites containing reactive functional groups using derivatization reagents with highly mass spectrometrically responsive functional groups on biological tissue sections. The highly responsive derivative ions after derivatization are measured and visualized using mass spectrometry imaging technology, thereby achieving in situ characterization of low-abundance, difficult-to-ionize metabolites in biological tissues.

[0003] Mass spectrometry imaging technology usually uses probes to scan and detect multiple molecules contained in biological tissues or other samples point by point according to their spatial positions, obtaining a multidimensional data array of the relationship between their ion intensity and position. Then, data processing software is used to reconstruct and visualize ions of different mass-to-charge ratios (m / z) according to their intensity and spatial position, ultimately achieving simultaneous imaging analysis of multiple molecules.

[0004] Currently available tissue derivatization methods mostly rely on spray-coating methods for matrix-assisted laser desorption ionization mass spectrometry (MALDI). This method relies on a sophisticated spraying apparatus, which can easily overload the derivatization reagent. Furthermore, online derivatization methods, which add the derivatization reagent to the spray solvent, suffer from low efficiency and are only able to derivatize a very small number of individual metabolites, making it difficult to analyze a wide range of metabolites with different functional groups in biological tissues.

[0005] However, a large number of low-abundance, weakly ionized metabolites, such as neurotransmitters, steroid hormones, fatty acids, and fatty aldehydes, are present in biological tissues. Mass spectrometry detection of these metabolites has low sensitivity and is susceptible to interference from background ions during mass spectrometry imaging analysis. However, these metabolites have important biological functions and are involved in the development and progression of various diseases. They are biomarkers with great clinical potential. Understanding their spatial distribution and relative abundance in biological tissue samples is crucial for understanding their physiological and pathological mechanisms. Summary of the Invention

[0006] The technical problem solved by the present invention is to provide a solid-state derivatized hydrogel and its preparation method and application. The solid-state derivatized hydrogel provided by the present invention derivatizes biological tissues with high derivatization efficiency, can significantly improve the detection sensitivity of endogenous metabolites in biological tissues, and effectively realize mass spectrometry imaging analysis and in situ characterization of low-abundance and difficult-to-ionize metabolites in biological tissues.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The first aspect of the technical solution of the present invention is to provide a solid-state derivatized hydrogel, comprising a hydrogel, a derivatization agent, and water; the derivatization agent is dispersed inside and on the surface of the three-dimensional network structure of the solid-state derivatized hydrogel;

[0009] The mass of the hydrogel accounts for 5 to 45% of the total mass of the hydrogel, the derivatization reagent and water;

[0010] The mass concentration of the derivatization reagent in the mixed solution formed by the hydrogel, the derivatization reagent and water is 0.1-200 mg / mL.

[0011] Unless otherwise specified, the raw materials used in the present invention are commercially available products well known to those skilled in the art.

[0012] The solid-state derivatized hydrogel provided by the present invention comprises a hydrogel, a derivatization reagent and water; the derivatization reagent is uniformly dispersed inside and on the surface of the three-dimensional network structure of the solid-state derivatized hydrogel; in the present invention, the hydrogel preferably contains one or more of agar, agarose or gelatin, more preferably agar, agarose or gelatin, and most preferably agar or gelatin; in the present invention, the derivatization reagent preferably comprises one or more of Gilad's reagent P, dansyl chloride, dansyl hydrazide and benzoyl chloride, more preferably Gilad's reagent P, dansyl chloride, dansyl hydrazide or benzoyl chloride. In the present invention, when the derivatization reagent preferably comprises two or more of the above-mentioned specific substances, the present invention has no special requirements for the mass ratio of the specific substances, and any ratio can be used. In the present invention, the Gilad's reagent P is preferably d0-Girard's reagent P or d5-Girard's reagent P, and the d0-Girard's reagent P and d5-Girard's reagent P are isotopic to each other.

[0013] In the present invention, the mass of the hydrogel accounts for 5-45% of the total mass of the hydrogel, the derivatization reagent and water, preferably 10-35%, and more preferably 12.5-30%; the mass concentration of the derivatization reagent in the mixed solution comprising the hydrogel, the derivatization reagent and water is 0.1-200 mg / mL, preferably 10-150 mg / mL, and most preferably 25-100 mg / mL.

[0014] In the present invention, the solid-state derivatized hydrogel further includes a pH regulator. The amount of the pH regulator is preferably used to adjust the pH value of the solid-state derivatized hydrogel to the pH value required for the derivatization reaction between the derivatization reagent and the metabolites in the biological tissue section. In the present invention, the pH regulator preferably includes a weak acid solution or a weak base solution. In the present invention, the weak acid solution preferably includes a formic acid solution or an acetic acid solution, and the weak base solution preferably includes ammonia water. In the present invention, the mass concentration of the weak acid solution is preferably 0.5-3%, more preferably 1-2%; the mass concentration of the weak base solution is preferably 0.5-3%, more preferably 1-2%.

[0015] The second aspect of the technical solution of the present invention provides a method for preparing the solid-state derivatized hydrogel according to the first aspect of the technical solution of the present invention, comprising the following steps:

[0016] mixing the hydrogel, the derivatization reagent, and water to obtain a mixed solution;

[0017] allowing the mixed solution to stand to obtain a solid-state derivatized hydrogel;

[0018] The mass percentage of the hydrogel in the mixed solution is 5 to 45%;

[0019] The mass concentration of the derivatization reagent in the mixed solution is 0.1-200 mg / mL.

[0020] The present invention provides a method for preparing the solid-state derivatized hydrogel described in the above technical solution, comprising the following steps:

[0021] mixing the hydrogel, the derivatization reagent, and water to obtain a mixed solution;

[0022] allowing the mixed solution to stand to obtain a solid-state derivatized hydrogel;

[0023] The mass percentage of the hydrogel in the mixed solution is 5 to 45%;

[0024] The mass concentration of the derivatization reagent in the mixed solution is 0.1-200 mg / mL.

[0025] The present invention mixes hydrogel, a derivatization reagent and water to obtain a mixed solution.

[0026] The present invention has no special requirements for the mixing order of the hydrogel, the derivatization agent, and water. In a specific embodiment of the present invention, the mixing is preferably carried out by preparing the hydrogel and water into a hydrogel solution, and then mixing with the derivatization agent. In the present invention, the mixing temperature is preferably 70° C. In the present invention, a pH adjuster is preferably added after the mixing. In the present invention, the type and range of the pH adjuster are the same as above and will not be repeated here.

[0027] After obtaining the mixed solution, the present invention allows the mixed solution to stand to obtain a solid-state derivatized hydrogel.

[0028] In the present invention, the resting temperature is preferably -20 to 4°C, more preferably -15 to 2°C; the resting time is preferably 4 to 10 hours, more preferably 5 to 8 hours, and most preferably 6 hours. In the present invention, the resting is preferably performed in a shaped mold, and the present invention has no particular requirements for the shape and structure of the shaped mold. By controlling the resting temperature to -20 to 4°C, the present invention allows the mixed solution to more effectively solidify into a solid derivatized hydrogel with a certain degree of elasticity and mechanical strength.

[0029] The present invention prepares a solid-state derivatized hydrogel by mixing a hydrogel, a derivatization reagent and water and then allowing the mixture to stand. The derivatization reagent is dispersed inside and on the surface of a three-dimensional network structure of the solid-state derivatized hydrogel. The derivatization reagent dispersed on the surface of the solid-state derivatized hydrogel forms a water molecule layer containing the derivatization reagent on the surface of the solid-state derivatized hydrogel through the physical adsorption of the solid-state derivatized hydrogel, thereby providing a good liquid microenvironment for the derivatization reaction.

[0030] The third aspect of the technical solution of the present invention provides the use of the solid-state derivatized hydrogel described in the first aspect of the technical solution or the solid-state derivatized hydrogel obtained by the preparation method described in the second aspect of the technical solution in the preparation of derivatized biological tissue.

[0031] The present invention has no special requirements for the application of the solid-state derivatized hydrogel in biological tissue derivatization. The solid-state derivatized hydrogel can be brought into contact with biological tissue to carry out the derivatization reaction.

[0032] A fourth aspect of the technical solution of the present invention provides a biological tissue derivatization method, comprising the following steps:

[0033] Provide biological tissue sections;

[0034] attaching the solid-state derivatized hydrogel to the surface of the biological tissue slice and performing a derivatization reaction to prepare a derivatized biological tissue slice;

[0035] The solid-state derivatized hydrogel is the solid-state derivatized hydrogel described in the above technical solution or the solid-state derivatized hydrogel obtained by the preparation method described in the above technical solution.

[0036] The present invention provides biological tissue slices.

[0037] In the present invention, the biological tissue slices preferably include heart tissue slices, liver tissue slices, spleen tissue slices, lung tissue slices, kidney tissue slices, brain tissue slices, muscle tissue slices, gonad tissue slices or tumor tissue slices.

[0038] In an embodiment of the present invention, the method for preparing a biological tissue slice preferably comprises the following steps:

[0039] The biological tissue is thawed, embedded, sliced and dried in sequence to obtain the biological tissue slices.

[0040] In the present invention, the temperature of the biological tissue is preferably -80°C, and the thawing temperature is preferably -20°C; in the present invention, the embedding agent is preferably Leica Cryo-Gel, and the present invention has no special requirements for the specific implementation process of the embedding. In the present invention, the slicing is preferably performed in a microtome, and the present invention has no special requirements for the specific implementation process of the slicing. In the present invention, the thickness of the biological tissue slice is preferably 12μm. In the present invention, the drying temperature is preferably 25°C, and the drying time is preferably 6h. The present invention has no special requirements for the source of the biological tissue. In a specific embodiment of the present invention, the biological tissue is rat tissue.

[0041] After obtaining the solid-state derivatized hydrogel and the biological tissue slice, the present invention adheres the solid-state derivatized hydrogel to the surface of either side of the biological tissue slice to perform a derivatization reaction to obtain the derivatized biological tissue slice.

[0042] In the present invention, the temperature of the derivatization reaction is preferably 37° C.; the time is preferably 15 min to 5 h, more preferably 30 min to 3 h, and most preferably 1 h to 2 h.

[0043] Prior to attachment, the present invention preferably prepares the solid-state derivatized hydrogel into a solid-state derivatized hydrogel sheet. In the present invention, the preparation method is preferably cutting. In the present invention, the size of the solid-state derivatized hydrogel sheet is preferably 20 mm × 10 mm × 10 mm. In the present invention, the size of the solid-state derivatized hydrogel sheet is preferably fine-tuned based on the size of the biological tissue slice, and the size of the solid-state derivatized hydrogel sheet is preferably the same as the size of the biological tissue slice.

[0044] The solid-state derivatized hydrogel prepared by the present invention forms a water molecule layer containing a derivatization reagent on the surface of the solid-state derivatized hydrogel by physical adsorption, providing a good liquid microenvironment; the water molecule layer on the surface of the solid-state derivatized hydrogel is fully in contact with the surface of a biological tissue slice, so that the derivatization reagent in the water molecule layer and the metabolites in the biological tissue undergo a full derivatization reaction; and the solid-state derivatized hydrogel is used to remove inorganic salts, quaternary ammonium salts, and water-soluble metabolites with high mass spectrometry response on the surface of the derivatized biological tissue.

[0045] A fifth aspect of the technical solution of the present invention provides a method for characterizing biological tissue metabolites for non-diagnostic and non-therapeutic purposes, comprising the following steps:

[0046] Obtain a derivatized biological tissue section according to the derivatization method described in the fourth aspect of the technical solution;

[0047] The metabolites in the derivatized biological tissue sections are characterized by mass spectrometry imaging technology.

[0048] The present invention obtains derivatized biological tissue slices according to the derivatization method described in the above technical solution.

[0049] In the present invention, the derivatized biological tissue sections are preferably pretreated before characterization. In the present invention, the pretreatment is preferably drying, the drying temperature is preferably 25° C., the drying time is preselected to be 6 hours, and the drying is preferably vacuum drying. The present invention has no special requirements for the vacuum degree of the vacuum drying.

[0050] The present invention adopts mass spectrometry imaging technology to characterize the metabolites in the derivatized biological tissue slices.

[0051] In the present invention, the method for characterizing metabolites in the derivatized biological tissue slice using mass spectrometry imaging technology comprises the following steps:

[0052] Using mass spectrometry imaging technology, a multidimensional data array of the relationship between the mass-to-charge ratio, ion intensity, and position of derivatized metabolite ions in derivatized biological tissue sections is obtained;

[0053] The accurate mass of the derivatized metabolite ions generated after the derivatization of the reference metabolites was determined, with a mass error of 5 ppm. The derivatized metabolite ions were extracted and identified, and the molecular structure of the metabolites was further inferred by secondary mass spectrometry analysis. The derivatized metabolites in the derivatized biological tissue sections were reconstructed and visualized according to the ion intensity and position relationship using the data processing software in mass spectrometry imaging technology.

[0054] The present invention adopts mass spectrometry imaging technology to obtain a multidimensional data array of the relationship between the mass-to-charge ratio, ion intensity and position of derivatized metabolite ions in derivatized biological tissue slices.

[0055] The present invention has no special requirements for the mass spectrometry imaging technology, and can use mass spectrometry imaging technology well known in the art; in a specific embodiment of the present invention, the mass spectrometry imaging technology is air flow assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) independently developed by the inventors.

[0056] In the present invention, the specific implementation process of the mass spectrometry imaging technology is as follows: preparing a spray solution, setting the voltage to 7KV, the spray flow rate to 5μL / min, the spray gas pressure to 0.8MPa, the X-axis scanning speed to 2mm / s, and the Y-axis step distance to 2mm / s. The spray solution is preferably a mixed solution of acetonitrile and water, and the volume ratio of acetonitrile to water is preferably 8:2; setting the acquisition mode to positive ion mode, scanning and detecting the metabolites in the derivatized biological tissue section point by point according to the spatial position, and obtaining a multidimensional data array of the mass-to-charge ratio, ion intensity, and position relationship of the derivatized metabolite ions in the derivatized biological tissue section.

[0057] After obtaining a multidimensional data array of the mass-to-charge ratio, ion intensity, and positional relationship of the derivatized metabolite ions in the derivatized biological tissue sections, the accurate mass of the derivatized metabolite ions generated after the reference metabolite of the present invention is derivatized is set to 5 ppm, the derivatized metabolite ions are extracted and identified, and the molecular structure of the metabolite is further inferred by secondary mass spectrometry analysis. The derivatized metabolites in the derivatized biological tissue sections are reconstructed and visualized according to the ion intensity and positional relationship using data processing software in mass spectrometry imaging technology.

[0058] In the present invention, the method for obtaining the accurate mass of the derivatized metabolite ion generated after the metabolite is derivatized includes data stored in a known database or data obtained by laboratory measurement of derivatized spiked biological tissue pulp strips. In the present invention, the known database includes the Metlin database, the HMDB database, or the Lipid Maps database.

[0059] In the present invention, the method for obtaining data by laboratory measurement of derivatized spiked biological tissue homogenate strips is the same as the method for obtaining the multidimensional data array of the mass-to-charge ratio, ion intensity and position relationship of the derivatized metabolite ions in the derivatized biological tissue sections, and will not be repeated here.

[0060] In the present invention, the method for preparing the derivatized spiked biological tissue homogenate strips comprises the following steps:

[0061] The biological tissue homogenate is mixed with a standard stock solution containing a metabolite standard to obtain a spiked biological tissue homogenate;

[0062] solidifying the spiked biological tissue homogenate to obtain a spiked biological tissue homogenate strip;

[0063] The spiked biological tissue homogenate strip is derivatized according to the derivatization method described in the above technical solution to obtain a derivatized spiked biological tissue homogenate strip.

[0064] The invention mixes biological tissue homogenate with a standard stock solution containing a metabolite standard substance to obtain a spiked biological tissue homogenate.

[0065] In the present invention, the biological tissue homogenate is preferably a mixed solution of biological tissue and physiological saline. In the present invention, the volume ratio of the mass of the biological tissue to the physiological saline is preferably 3:4; in the present invention, the metabolite standards in the standard stock solution containing metabolite standards preferably include one or more of carbonyl metabolites, hydroxyl metabolites, carboxyl metabolites and thiol metabolites, more preferably carbonyl metabolites, and most preferably pregnenolone, testosterone, androsterone and fatty aldehydes; in the present invention, the mass concentration of the metabolite standards in the spiked biological tissue homogenate is preferably 100 μg / mL.

[0066] After obtaining the spiked biological tissue homogenate, the present invention solidifies the spiked biological tissue homogenate to obtain a spiked biological tissue homogenate strip.

[0067] The present invention has no special requirements for the specific implementation scheme of the curing. In a specific embodiment of the present invention, the curing is as follows: using a hole puncher to make continuous 25 mm rectangular holes on the PVC self-adhesive sticker; adhering the punched PVC self-adhesive sticker to a positively charged anti-shedding slide; using a micropipette to draw 5 μL of the spiked biological tissue homogenate into the rectangular holes of the slide; and placing the slide in a vacuum desiccator to dry for 6 hours.

[0068] After obtaining the spiked biological tissue homogenate strip, the present invention derivatizes the spiked biological tissue homogenate strip according to the derivatization method described in the above technical solution to obtain a derivatized spiked biological tissue homogenate strip.

[0069] In the present invention, the range of the metabolites is the same as the range of standard metabolite types, which will not be repeated here.

[0070] The present invention provides a solid-state derivatization hydrogel, comprising a hydrogel, a derivatization reagent, and water; the derivatization reagent is dispersed inside and on the surface of the three-dimensional network structure of the solid-state derivatization hydrogel; the mass of the hydrogel accounts for 5 to 45% of the total mass of the hydrogel, the derivatization reagent, and water; the mass concentration of the derivatization reagent in the mixed solution formed by the hydrogel, the derivatization reagent, and water is 0.1 to 200 mg / mL. The solid-state derivatization hydrogel provided by the present invention uniformly disperses the derivatization reagent inside and on the surface of the three-dimensional network structure of the solid-state derivatization hydrogel; the derivatization reagent dispersed on the surface of the solid-state derivatization hydrogel forms a water molecule layer containing the derivatization reagent on the surface of the solid-state derivatization hydrogel through the physical adsorption effect of the solid-state derivatization hydrogel, providing a good liquid microenvironment for the derivatization reaction; the water molecule layer on the surface of the solid-state derivatization hydrogel is in full contact with the surface of the biological tissue, so that the derivatization reagent in the water molecule layer and the metabolite undergo a full derivatization reaction, and when the concentration of the derivatization reagent in the water molecule layer is When the reaction is reduced, the derivatization reagent dispersed in the three-dimensional network structure of the hydrogel continuously diffuses into the water molecule layer under the action of the concentration difference, thereby improving the derivatization efficiency of metabolites in biological tissues; at the same time, the water molecule layer on the surface of the solid-state derivatized hydrogel is used to dissolve the water-soluble inorganic salts, quaternary ammonium salts, and metabolites with high mass spectrometry response that are easily soluble in water on the surface of the biological tissue after derivatization, and diffuses into the three-dimensional network structure of the solid-state derivatized hydrogel under the action of the concentration difference, thereby achieving the removal of water-soluble inorganic salts, quaternary ammonium salts, and metabolites with high mass spectrometry response that are easily soluble in water, and reducing background interference. When the solid-state derivatization hydrogel provided by the present invention derivatizes biological tissues, the derivatization efficiency is high, and the detection sensitivity of endogenous metabolites in biological tissues can be significantly improved, and mass spectrometry imaging analysis and in situ characterization of low-abundance, difficult-to-ionize metabolites in biological tissues can be effectively realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Schematic diagram of the method for derivatizing biological tissue using the solid-state derivatized hydrogel provided in Example 1 of the present invention;

[0072] Figure 2 This is a comparison of the mass spectrometry imaging results of androsterone and progesterone before and after derivatization of spiked rat liver tissue homogenate strips with the solid-state derivatization hydrogel provided in Application Example 1 of the present invention;

[0073] Figure 3 This is a comparison chart of the mass spectrometry response results of androsterone and progesterone before and after derivatization of spiked rat liver tissue homogenate strips with the solid-state derivatization hydrogel provided in Application Example 1 of the present invention;

[0074] Figure 4 This is a comparison chart of the mass spectrometry imaging results of unknown metabolites before and after derivatization of rat kidney tissue slices with the solid-state derivatization hydrogel provided in Application Example 2 of the present invention;

[0075] Figure 5 This is a comparison chart of the mass spectrometry response results of unknown metabolites before and after derivatization of rat kidney tissue slices with the solid-state derivatization hydrogel provided in Application Example 2 of the present invention;

[0076] Figure 6 Comparison of mass spectrometry imaging results of L-carnitine C18:0, C18:1, C18:2, and C16:0 before and after derivatization of rat kidney tissue slices with the solid-state derivatization hydrogel provided in Application Example 2 of the present invention;

[0077] Figure 7 Comparison of mass spectrometry imaging results of lysophosphatidylcholine C18:0, C18:1, C18:2, and C16:0 before and after derivatization of rat kidney tissue slices with the solid-state derivatization hydrogel provided in Application Example 2 of the present invention;

[0078] Figure 8 Comparison of mass spectrometry imaging results of phosphatidylcholine C34:1 and C34:2 before and after derivatization of rat kidney tissue slices with the solid-state derivatization hydrogel provided in Application Example 2 of the present invention;

[0079] Figure 9 Comparison of the mass spectrometry response results of L-carnitine C18:0, C18:1, C18:2, C16:0, lysophosphatidylcholine C18:0, C18:1, C18:2, C16:0, and phosphatidylcholine C34:1 and C34:2 before and after derivatization of rat kidney tissue slices with the solid-state derivatization hydrogel provided in Application Example 2 of the present invention;

[0080] Figure 10 A photo of the derivatization of rat brain tissue slices by the solid-state derivatized hydrogel provided in Application Example 3 of the present invention;

[0081] Figure 11 Spatial distribution diagrams of mass spectrometry imaging of short-chain fatty aldehyde metabolites FAL3:0, FAL4:0, FAL5:0, FAL6:0, FAL7:0, FAL8:0, FAL9:0, and FAL10:0 after derivatization of rat kidney tissue slices, brain tissue slices, and liver tissue slices with the solid-state derivatization hydrogel provided in Application Examples 2 to 4 of the present invention;

[0082] Figure 12 Spatial distribution diagrams of mass spectrometry imaging of long-chain fatty aldehyde metabolites FAL11:0, FAL12:0, FAL13:0, FAL14:0, FAL15:0, FAL16:0, FAL17:0, and FAL18:0 after derivatization of rat kidney tissue slices, brain tissue slices, and liver tissue slices with the solid-state derivatization hydrogel provided in Application Examples 2 to 4 of the present invention;

[0083] Figure 13Spatial distribution diagrams of mass spectrometry imaging of short carbon-chain oxygenated fatty acid metabolites FA5:1;O, FA7:1;O, FA8:1;O, FA9:1;O, and FA10:1;O after derivatization of rat kidney tissue slices, brain tissue slices, and liver tissue slices with the solid-state derivatization hydrogel provided in Application Examples 2 to 4 of the present invention;

[0084] Figure 14 Spatial distribution diagrams of long-chain oxygenated fatty acid metabolites FA11:1;O, FA12:1;O, FA13:1;O, and FA14:1;O after derivatization of rat kidney tissue slices, brain tissue slices, and liver tissue slices with the solid-state derivatization hydrogel provided in Application Examples 2 to 4 of the present invention;

[0085] Figure 15 Spatial distribution diagrams of mass spectrometry imaging of lipid metabolites FAL16:4;O and FA17:5;O2 after derivatization of rat kidney tissue slices, brain tissue slices and liver tissue slices with the solid-state derivatization hydrogel provided in Application Examples 2 to 4 of the present invention;

[0086] Figure 16 Spatial distribution diagrams of mass spectrometry imaging of lipid metabolites FAL6:2 and FA9:4 after derivatization of rat kidney tissue slices, brain tissue slices, and liver tissue slices with the solid-state derivatization hydrogel provided in Application Examples 2 to 4 of the present invention;

[0087] Figure 17 Spatial distribution diagrams of mass spectrometry imaging of lipid metabolites FAL17:4;O and FA19:1;O2 after derivatization of rat kidney tissue slices, brain tissue slices and liver tissue slices with the solid-state derivatization hydrogel provided in Application Examples 2 to 4 of the present invention;

[0088] Figure 18 Application Example 5 of the present invention provides a comparison of the spatial distribution of mass spectrometry imaging of metabolites FAL10:2; O, FAL6:0; O, FAL14:3 and FAL11:0 after derivatization of rat kidney tissue slices and brain tissue slices with solid-state derivatization hydrogels prepared using isotope d0-Girard reagent and d5-Girard reagent. DETAILED DESCRIPTION

[0089] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0090] Example 1

[0091] A mixed aqueous solution of gelatin hydrogel containing 1% formic acid and 50 mg / mL d0-Girard reagent P at a concentration of 15% was prepared at 70°C, transferred to room temperature and cooled for 1 hour, and then placed in a 4°C refrigerator for 6 hours to obtain a solid derivatized hydrogel containing a derivatization reagent with a certain mechanical strength.

[0092] Application Example 1

[0093] A spiked rat liver tissue homogenate containing the above standard metabolites at a concentration of 100 μg / mL was prepared by taking 0.1 mL of the standard stock solution containing pregnenolone, testosterone, androsterone, and fatty aldehydes (FAL 6:0, FAL 7:0, and FAL 8:0), 0.4 mL of normal saline, and 0.3 g of rat liver tissue homogenate;

[0094] The spiked rat liver tissue homogenate prepared above was used to create continuous 2 mm × 5 mm rectangular holes on a PVC adhesive sheet using a hole punch. The PVC adhesive sheet was then adhered to a positively charged, anti-shedding glass slide. Using a micropipette, 5 μL of the spiked rat liver tissue homogenate was accurately pipetted into the rectangular hole on the glass slide. The slide was then placed in a vacuum desiccator and evacuated for 6 h to prepare spiked rat liver tissue homogenate strips.

[0095] The solid derivatized hydrogel prepared in Example 1 was cut into a series of complete solid derivatized hydrogel sheets in a size of 20 mm × 10 mm × 10 mm, and the size was fine-tuned according to the spiked rat liver tissue homogenate strips. The prepared solid derivatized hydrogel sheet was attached to one side of the spiked rat liver tissue homogenate strips and placed at 37° C. for 2 hours to complete the derivatization reaction between the metabolites in the spiked rat liver tissue homogenate strips and the derivatization reagent, thereby obtaining the derivatized spiked rat liver tissue homogenate strips. The strips were then dried in a vacuum for 6 hours.

[0096] A spray solution with a volume ratio of acetonitrile to water of 8:2 was prepared, and derivatized and spiked rat liver tissue homogenate strips were subjected to AFADESI-MSI testing. The voltage was set to 7 kV, the spray needle flow rate was 5 μL / min, the spray gas pressure was 0.8 MPa, the X-axis scanning speed was 2 mm / s, the Y-axis step distance was 2 mm / s, and the acquisition mode was positive ion mode. Multidimensional data arrays of the relationship between the mass-to-charge ratio, ion intensity, and position of the derivatized metabolite ions in the derivatized and spiked rat liver tissue homogenate strips were obtained.

[0097] The same method was used to determine the multidimensional data array of the relationship between the mass-to-charge ratio, ion intensity, and position of the metabolite ions of the standard in the underivatized spiked rat liver tissue homogenate strips;

[0098] The comparison charts of mass spectrometry imaging results and mass spectrometry response results of androsterone and progesterone in the derivatized and spiked rat liver tissue homogenate strips and the non-derivatized and spiked rat liver tissue homogenate strips were obtained, as shown in FIG. Figure 2 、 Figure 3 As shown in Table 1 , the mass spectrometric responses of the derivatized androsterone and progesterone were increased by 124.72 times and 30.39 times, respectively.

[0099] Application Example 2

[0100] Frozen rat kidney tissue was transferred from -80°C to -20°C, embedded with Lecia Cryo-Gel embedding gel and fixed in a microtome. Serial 12 μm sections were made to obtain rat kidney tissue sections, which were then dried at 25°C for 6 hours before use.

[0101] The solid derivatized hydrogel prepared in Example 1 was cut into a series of complete solid derivatized hydrogel sheets in a size of 20 mm × 10 mm × 10 mm, and the size of the sheets was finely adjusted according to the rat kidney tissue slices. The prepared solid derivatized hydrogel sheets were attached to one surface of the rat kidney tissue slices and placed at 37° C. for 2 hours to complete the derivatization reaction between the metabolites in the rat tissue and the derivatization reagent, thereby obtaining derivatized rat kidney tissue slices. The slices were then dried in a vacuum for 6 hours.

[0102] A spray solution with a volume ratio of acetonitrile to water of 8:2 was prepared, and derivatized rat kidney tissue sections were subjected to AFADESI-MSI testing. The voltage was set to 7 kV, the spray needle flow rate was 5 μL / min, the spray gas pressure was 0.8 MPa, the X-axis scanning speed was 2 mm / s, the Y-axis step distance was 2 mm / s, and the acquisition mode was positive ion mode. Multidimensional data arrays of the relationship between the mass-to-charge ratio, ion intensity, and position of the derivatized metabolite ions in the derivatized rat kidney tissue sections were obtained.

[0103] The mass error was set to 5 ppm, and the multidimensional data array of the mass-to-charge ratio, ion intensity, and position relationship of metabolites in the derivatized rat kidney tissue sections was extracted using the AFADESI-MSI data processing software to obtain the mass spectrometry imaging of the metabolites in the derivatized rat kidney tissue sections.

[0104] Comparison of the mass spectrometry imaging results and mass spectrometry response results of unknown metabolites in rat kidney tissue sections after derivatization and without derivatization, such as Figure 4 and Figure 5 As shown in Table 1, the mass spectrometric response of the derivatized unknown metabolite increased by 33.58 times; Figure 6 、 Figure 7 、 Figure 8 and Figure 9 As shown, after the rat kidney tissue sections were derivatized with solid-state derivatization hydrogel, the mass spectrometry responses of the underivatized L-carnitine C18:0, C18:1, C18:2 and C16:0, hemolytic phosphatidylcholine C18:0, C18:1, C18:2 and C16:0, and phosphatidylcholine C34:1 and C34:2 metabolites were all improved by more than 4.5 times, indicating that the derivatization method provided by the present invention using solid-state derivatization hydrogel effectively removes high concentrations of salt components, reduces matrix effects, and improves the mass spectrometry response of weakly polar metabolites.

[0105] Table 1 Mass spectrometry response results of androsterone and progesterone in rat liver tissue homogenates before and after derivatization, and unknown metabolites in rat kidney tissue sections before and after derivatization

[0106]

[0107] Table 2 Mass spectrometric response results of L-carnitine C18:0, C18:1, C18:2 and C16:0, lysophosphatidylcholine C18:0, C18:1 and C16:0, and phosphatidylcholine C34:1 and C34:2 metabolites in rat kidney tissue sections before and after derivatization

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[0109]

[0110] Application Example 3

[0111] The frozen rat brain tissue was transferred from -80°C to -20°C, embedded with Lecia Cryo-Gel embedding glue and fixed in a microtome. Serial 12 μm sections were made to obtain rat brain tissue sections, which were then dried at 25°C for 6 hours before use.

[0112] The solid derivatized hydrogel was cut into a series of complete solid derivatized hydrogel sheets in the size of 20 mm × 10 mm × 10 mm, and the size was fine-tuned according to the rat brain tissue slices. The prepared solid derivatized hydrogel sheets were attached to the surface of the rat brain tissue slices (such as Figure 10 The sections were placed at 37°C for 2 hours to complete the derivatization reaction between the metabolites in the rat tissue and the derivatization reagent, thereby obtaining derivatized rat brain tissue sections; and the sections were placed in a vacuum dryer for 6 hours.

[0113] A spray solution with a volume ratio of acetonitrile to water of 8:2 was prepared, and derivatized rat brain tissue sections were subjected to AFADESI-MSI testing. The voltage was set to 7 kV, the spray needle flow rate was 5 μL / min, the spray gas pressure was 0.8 MPa, the X-axis scanning speed was 2 mm / s, the Y-axis step distance was 2 mm / s, and the acquisition mode was positive ion mode. Multidimensional data arrays of the relationship between the mass-to-charge ratio, ion intensity, and position of the derivatized metabolite ions in the derivatized rat brain tissue sections were obtained.

[0114] The mass error was set to 5 ppm, and the multidimensional data array of the mass-to-charge ratio, ion intensity, and position relationship of metabolites in the derivatized rat brain tissue slices was extracted using the AFADESI-MSI data processing software to obtain the mass spectrometry imaging of the metabolites in the derivatized rat brain tissue slices.

[0115] Application Example 4

[0116] Frozen rat liver tissue was transferred from -80°C to -20°C, embedded with Lecia Cryo-Gel embedding gel and fixed in a microtome. Serial 12 μm sections were made to obtain rat liver tissue sections, which were then dried at 25°C for 6 hours before use.

[0117] The solid derivatized hydrogel prepared in Example 1 was cut into a series of complete solid derivatized hydrogel sheets in a size of 20 mm × 10 mm × 10 mm, and the size was finely adjusted according to the rat liver tissue slices. The prepared solid derivatized hydrogel sheets were attached to one surface of the rat liver tissue slices and placed at 37° C. for 2 hours to complete the derivatization reaction between the metabolites in the rat liver tissue and the derivatization reagent, thereby obtaining derivatized rat liver tissue slices. The slices were then dried in a vacuum for 6 hours.

[0118] A spray solution with a volume ratio of acetonitrile to water of 8:2 was prepared, and the derivatized rat liver tissue sections were subjected to AFADESI-MSI testing. The voltage was set to 7 kV, the spray needle flow rate was 5 μL / min, the spray gas pressure was 0.8 MPa, the X-axis scanning speed was 2 mm / s, the Y-axis step distance was 2 mm / s, and the acquisition mode was positive ion mode. Multidimensional data arrays of the relationship between the mass-to-charge ratio, ion intensity, and position of the derivatized metabolite ions in the derivatized rat liver tissue sections were obtained.

[0119] The mass error was set to 5 ppm, and the multidimensional data array of the mass-to-charge ratio, ion intensity, and position relationship of metabolites in the derivatized rat liver tissue sections was extracted using the AFADESI-MSI data processing software to obtain the mass spectrometry imaging of the metabolites in the derivatized rat liver tissue sections.

[0120] The mass spectrometry data of rat kidney, brain, and liver obtained in Application Examples 2 to 4 were compared with the Metlin, HMDB, and Lipid Maps databases, and a total of 266 fatty aldehyde and oxygenated fatty acid metabolites were obtained according to the rules of increasing lipid carbon chain number and increasing unsaturation, as shown in Table 3.

[0121] According to the accurate mass of the derivatized metabolites in Table 3, some metabolite data in rat brain, kidney and liver tissues were extracted, and the spatial distribution imaging of short-chain and long-chain fatty aldehydes and oxygenated fatty acids was characterized, such as Figure 11 、 Figure 12 、 Figure 13 、 Figure 14 、 Figure 15 、 Figure 16 and Figure 17The results show that the biological tissue derivatization method provided by the present invention can achieve the derivatization of different types of metabolites in different biological tissue samples, effectively improving the sensitivity of mass spectrometry imaging analysis.

[0122] Table 3 Application Examples 2-4 Accurate identification of 266 fatty aldehydes and oxygenated fatty acid metabolites in biological tissues by derivatization and mass spectrometry

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[0132]

[0133] Example 2

[0134] The preparation method is the same as that of Example 1, except that in this example, d0-Girard reagent P and d5-Girard reagent P are used to prepare the solid derivatized hydrogel using two derivatization reagents.

[0135] Application Example 5

[0136] The two solid-state derivatized hydrogels prepared in Example 2 were used to perform derivatization and mass spectrometry imaging analysis on adjacent rat kidney tissue slices and brain tissue slices, respectively. Figure 18As shown, adjacent rat kidney tissue sections and brain sagittal tissue sections were derivatized using d0-Girard reagent P solid-state derivatization hydrogel and d5-Girard reagent P solid-state derivatization hydrogel, respectively. The metabolites FAL10:2;O, FAL6:0;O, FAL14:3, and FAL11:0 were all derivatized by the two derivatization reagents, producing ions with a mass difference of 5 neutrons. The same metabolite derivatized by the two derivatization reagents showed the same fine tissue micro-region distribution characteristics in the two adjacent tissue sections, which further demonstrated that the target metabolite and the derivatization reagents had indeed undergone a derivatization reaction. The co-localization of the target metabolites by the two derivatization reagents, d0-Girard reagent P and d5-Girard reagent P, further eliminated false positive results in the identification of derivatized metabolites.

[0137] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A biological tissue derivatization method, characterized in that: The following steps are involved: Provide biological tissue sections; A solid-state derivatized hydrogel is attached to the surface of the biological tissue slice to perform a derivatization reaction; the solid-state derivatized hydrogel includes gelatin, a derivatization reagent, and water; the derivatization reagent is dispersed inside and on the surface of the three-dimensional network structure of the solid-state derivatized hydrogel; the derivatization reagent includes one or more of Girard reagent P, dansyl chloride, dansyl hydrazide, and benzoyl chloride; the mass of the gelatin accounts for 5 to 45% of the total mass of the gelatin, the derivatization reagent, and water; and the mass concentration of the derivatization reagent in the mixed solution formed by the gelatin, the derivatization reagent, and water is 0.1 to 200 mg / mL.

2. The biological tissue derivatization method according to claim 1, characterized in that: The preparation method of the solid-state derivatized hydrogel comprises the following steps: mixing gelatin, a derivatization reagent, and water to obtain a mixed solution; allowing the mixed solution to stand to obtain a solid-state derivatized hydrogel; The mass percentage of gelatin in the mixed solution is 5 to 45%; The mass concentration of the derivatization reagent in the mixed solution is 0.1-200 mg / mL.

3. The biological tissue derivatization method according to claim 2, characterized in that: The temperature of the standing state is -20 to 4° C., and the time is 4 to 10 hours.

4. The biological tissue derivatization method according to claim 1, characterized in that: The temperature of the derivatization reaction is 25-40° C., and the time is 15 minutes to 5 hours.

5. The biological tissue derivatization method according to claim 1, characterized in that: The biological tissue slices include heart tissue slices, liver tissue slices, spleen tissue slices, lung tissue slices, kidney tissue slices, brain tissue slices, muscle tissue slices, gonad tissue slices or tumor tissue slices.

6. A method for characterizing biological tissue metabolites for non-diagnostic and non-therapeutic purposes, characterized in that: The following steps are involved: Obtaining a derivatized biological tissue section according to the derivatization method according to any one of claims 1 to 5; The metabolites in the derivatized biological tissue sections are characterized by mass spectrometry imaging technology.

7. The characterization method according to claim 6, characterized in that The metabolites include one or more of carbonyl metabolites, hydroxyl metabolites, carboxyl metabolites and sulfhydryl metabolites.

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