A surface mesoporous matrix modified target plate, a preparation method and application thereof

Abstract

This invention relates to the field of mass spectrometry analysis technology, specifically to a target plate with a surface-modified mesoporous matrix, its preparation method, and its application. The method involves forming a precursor solution from a template agent, an acidic catalyst, and a matrix precursor, and then combining this with physical masking for confined film formation, aging and curing, and segmented heat treatment processes to construct an arrayed mesoporous matrix modification layer in situ on the surface of a conductive target plate. The resulting target plate improves the localization and uniformity of sample droplet distribution and crystallization, reduces background interference in low mass-to-charge ratio regions, and enhances the mass spectrometric response and detection sensitivity of small molecule analytes, making it suitable for the mass spectrometric detection of small biomolecule metabolites.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of mass spectrometry analysis and advanced nanomaterials, and particularly relates to a target plate with a surface mesoporous matrix modified, its preparation method and its application. Background Technology

[0002] Matrix-assisted laser desorption / ionization mass spectrometry (MALS) has become an important tool in life sciences and clinical metabolomics analysis due to its advantages such as high sensitivity, speed, and lack of complex pretreatment. However, when detecting small molecule metabolites with a mass-to-charge ratio of less than 500, traditional organic matrices, such as α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, exhibit fragment ion peaks under laser irradiation due to their poor matrix stability, leading to severe background interference. Furthermore, the co-crystallization process between the organic matrix and the sample is prone to producing coffee ring and sweet spot effects, resulting in uneven crystallization and significantly reducing the reproducibility of quantitative analysis.

[0003] To overcome the aforementioned shortcomings, surface-assisted laser desorption / ionization mass spectrometry (SALDS) has attracted widespread attention in recent years, with various high-performance inorganic nanomaterials being developed as novel background-free matrices. Through precise composition and structural design, the efficiency of electron-hole separation, photothermal conversion, and proton transport within the matrix can be significantly improved, enabling cascaded amplification of metabolite signals. In particular, functionalized mesoporous nanomaterials, with their large specific surface area, tunable pore size, and excellent light absorption capabilities, exhibit unique advantages in metabolite enrichment and energy transfer. Multidimensional body fluid metabolic fingerprints obtained using such nanomatrices have not only been validated in routine screening for major chronic diseases but have also demonstrated irreplaceable clinical potential in the early, precise diagnosis and targeted therapy evaluation of various complex degenerative diseases, cardiovascular diseases, and multicenter tumor cohorts.

[0004] However, despite significant breakthroughs in the desorption and ionization mechanisms of functional inorganic nanomatrices, existing technologies still face the following insurmountable process and platform technology bottlenecks when moving towards high-throughput, large-scale clinical multi-omics applications: First, uncontrolled evaporation of target droplets leads to poor reproducibility. Most existing nanomatrices are droplets added as suspensions onto a flat stainless steel target surface. During the evaporation and drying process, the sample and matrix droplets are highly susceptible to hydrodynamic influences, resulting in severe coffee ring and sweet spot effects. This leads to macroscopic migration and highly uneven distribution of metabolites within the target site. This random aggregation phenomenon results in high relative standard deviations (RSDs) between different batches and sites, making it difficult to meet the stringent quantitative standards of clinical mass spectrometry.

[0005] Second, there is a lack of universal matrix customization platforms for handling complex body fluids. Clinical metabolomics analysis targets various complex biological fluids, such as blood, urine, and saliva. Due to the heterogeneity of their endogenous components, their microenvironments exhibit significant differences in physicochemical properties such as ionic strength, pH, and polarity. This leads to drastically different matrix interferences and ion suppression effects for target small-molecule metabolites in different body fluids during mass spectrometry analysis. The synthesis of existing high-performance inorganic matrices often relies heavily on specific preparation routes, resulting in severe process silos. This incompatibility of synthetic routes prevents current technologies from flexibly adapting front-end matrix formulations within a single standard workflow, lacking plug-and-play platform customization capabilities, and severely limiting their high-throughput adaptation to massive amounts of diverse and complex clinical samples.

[0006] Therefore, there is an urgent need in this field to develop a novel universal mass spectrometry target plate with a universal preparation process that is compatible with various functionalized mesoporous materials. This plate would completely solve the problems of droplet anchoring and reproducibility through purely physical confinement, thereby meeting the high-efficiency screening needs of clinical metabolomics for multiple diseases with high sensitivity and high throughput. Summary of the Invention

[0007] To address the problems of sample droplet migration, uneven crystallization distribution, poor inter-spot reproducibility, and strong background interference in low mass-to-charge ratio regions in existing mass spectrometry target plates for small molecule metabolite detection, this invention provides a target plate modified with mesoporous titanium dioxide, its preparation method, and its applications. This method induces precursor assembly through block copolymer templates, combined with physical mask confinement film formation and segmented heat treatment processes, to construct an array of mesoporous titanium dioxide modification layers in situ on the surface of a conductive target plate. This improves the localized distribution and crystallization uniformity of the sample on the target plate surface, thereby enhancing the stability and repeatability of mass spectrometry detection.

[0008] Another objective of this invention is to provide a surface-modified mesoporous titanium dioxide target plate loaded with gold-palladium nanocrystals, based on the aforementioned mesoporous titanium dioxide modification system. By introducing gold-palladium alloy nanocrystals during precursor assembly, the energy transfer characteristics and analyte response performance of the target plate surface can be further tuned to meet the application requirements of different samples and different detection targets.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for preparing a target plate modified with mesoporous titanium dioxide on its surface, comprising the following steps: S1. Preparation of precursor micelle solution: The block copolymer template agent is dissolved in a volatile solvent, and an acidic catalyst and titanium precursor are added under stirring. Stirring is continued to obtain the precursor micelle solution. S2. Physical mask confined film formation: After pretreatment of the conductive target plate, a physical mask plate with array holes is attached to the surface of the conductive target plate. The precursor micelle solution is coated on the surface of the conductive target plate with the physical mask plate attached, and then aged and cured to form a precursor array on the surface of the target plate. S3. Mask Removal and Heat Treatment: After removing the physical mask, the resulting target plate is subjected to nitrogen atmosphere pre-calcination, air atmosphere calcination and final heat treatment in sequence to remove the template agent and complete the formation of the mesoporous skeleton, thereby obtaining a target plate with surface mesoporous titanium dioxide modification.

[0010] Furthermore, the block copolymer template agent is an amphiphilic block copolymer, preferably a polyethylene oxide-block-polystyrene diblock copolymer, or a Pluronic series polyethylene oxide-block-polypropylene oxide-block-polyethylene oxide triblock copolymer, such as at least one of P123, F127, and F108.

[0011] Furthermore, the volatile solvent is an ether solvent, a low-carbon alcohol solvent, a ketone solvent, a nitrile solvent, or a mixture thereof, preferably tetrahydrofuran.

[0012] Furthermore, the acidic catalyst includes hydrochloric acid and / or glacial acetic acid; The titanium precursor is a titanium alkoxide precursor, preferably at least one of tetrabutyl titanate, isopropyl titanate, and tetraethyl titanate, and more preferably tetrabutyl titanate.

[0013] Further, in step S1, the amount of the block copolymer template agent is 15-30 mg, the amount of the volatile solvent is 4-6 mL, the amount of concentrated hydrochloric acid added is 30-60 µL, the amount of glacial acetic acid added is 30-60 µL, the amount of the titanium precursor added is 0.15-0.30 mL, and the stirring time is 1.5-3 h. The concentrated hydrochloric acid is hydrochloric acid with a mass fraction of 35%-38%.

[0014] Furthermore, the conductive target plate is a metal conductive target plate, preferably a stainless steel target plate.

[0015] Furthermore, the physical mask is a solvent-resistant film or metal sheet, preferably one of polyimide film, polytetrafluoroethylene film or metal foil; the physical mask is provided with an array of holes, the aperture of which is 3.5-4.5 mm, the hole spacing is 0.8-1.2 mm, and the mask thickness is 0.02-0.05 mm.

[0016] Furthermore, in step S2, the conductive target plate is ultrasonically cleaned and pretreated before the physical mask is attached; the coating amount of the precursor micelle solution is 0.4-0.6 mL.

[0017] Further, in step S2, the aging and curing treatment includes: aging for 18-30 hours at 20-30°C and 35%-45% relative humidity, aging for 18-30 hours at 35-45°C, and curing for 12-24 hours at 90-110°C.

[0018] Further, in step S3, the nitrogen atmosphere pre-calcination temperature is 350-500℃, and the holding time is 1.5-3h; the air atmosphere calcination temperature is 380-420℃, and the holding time is 1.5-3h; the final heat treatment temperature is 380-420℃, and the holding time is 0.5-1.5h.

[0019] In a preferred embodiment of the present invention, a gold-palladium alloy nanocrystal solution is further added in step S1 to prepare a surface-modified titanium dioxide target plate loaded with gold-palladium nanocrystals.

[0020] Furthermore, the amount of the gold-palladium alloy nanocrystal solution added is 0.3-0.7 mL, so that the theoretical loading of gold-palladium alloy nanocrystals in the obtained target plate is 3%-8 wt%.

[0021] Furthermore, the gold-palladium alloy nanocrystalline solution is prepared by the following method: S11. Add the gold precursor and palladium precursor to a mixed solution of oleylamine and toluene at a mass ratio of 0.8:1-1.2:1, wherein the gold precursor is a gold salt precursor, preferably chloroauric acid or its hydrate, and the palladium precursor is a palladium organic salt precursor, preferably palladium acetylacetonate; the volume ratio of oleylamine to toluene is 1:4-1:6, and stir to dissolve to obtain a precursor mixture. S12. Under nitrogen protection, the precursor mixture is first heated to 75-85℃ and reacted for 0.5-1.5h, then heated to 95-105℃ and reacted for another 0.5-1.5h to obtain a reaction solution containing gold-palladium alloy nanocrystals. S13. Add an ethanol aqueous solution with a volume fraction of 70%-80% to the reaction solution to allow the gold-palladium alloy nanocrystals to precipitate. Then, separate them by centrifugation at 9000-11000 rpm for 4-6 min, and wash them 2-4 times with an ethanol aqueous solution with a volume fraction of 70%-80%. Finally, redisperse them in cyclohexane to obtain the gold-palladium alloy nanocrystal solution.

[0022] The present invention also provides a target plate modified with mesoporous titanium dioxide on its surface, which is prepared by the above-described method.

[0023] The present invention also provides the application of the above-mentioned surface-mesoporous titanium dioxide modified target plate in mass spectrometry detection, preferably for the detection of small molecule metabolites, and especially suitable for the detection of small molecule analytes in the low mass-to-charge ratio region.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention combines block copolymer template-induced assembly, physical mask confinement film formation, and segmented heat treatment to form an arrayed mesoporous titanium dioxide modification layer in situ on the surface of a conductive target plate. This avoids the unevenness of the film layer caused by traditional powder dispersion followed by drop coating, and helps to improve the consistency and repeatability of the modification layer on the target plate surface.

[0025] This invention employs a physical mask confinement method to array and localize the precursor solution, which can improve the distribution of sample droplets on the target plate surface and reduce the edge migration tendency of the sample during the drying process. This helps to reduce the impact of the coffee ring effect and local crystallization inhomogeneity on the detection results and improve the reproducibility of intra-point and inter-point detection.

[0026] The mesoporous titanium dioxide modified layer constructed in this invention has a high specific surface area and good interfacial transport conditions, which is beneficial to the adsorption, enrichment and desorption / ionization response of analytes on the target plate surface. It can reduce the background interference caused by traditional organic matrices in the low mass-to-charge ratio region to a certain extent and improve the stability of small molecule metabolite detection.

[0027] The preparation method of the present invention has clear process steps, and the precursor system, mask parameters and heat treatment conditions can be controlled. It has good process repeatability and adaptability to array preparation, and is suitable for batch preparation on multi-target plates.

[0028] In a preferred embodiment, by introducing gold-palladium alloy nanocrystals into the mesoporous titanium dioxide system, the surface energy transfer characteristics of the target plate and the analyte response performance can be further controlled, thereby providing a more flexible material design scheme for different sample systems and different detection objects. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0030] Figure 1 This is an optical photograph of the precursor solution prepared in Example 1 of the present invention.

[0031] Figure 2 This is a scanning electron microscope image of the mesoporous titanium dioxide array prepared in Example 1 of the present invention.

[0032] Figure 3 This is an optical photograph of the composite precursor solution prepared in Example 2 of the present invention.

[0033] Figure 4 This is an optical micrograph of the product after co-crystallization with glutamic acid in Example 1 of the present invention.

[0034] Figure 5 This is a signal reproducibility diagram of the target plates prepared for the detection of glutamate in Test Example 1 and Test Example 2 of the present invention.

[0035] Figure 6 This is a signal reproducibility diagram of the target plates prepared for the detection of histidine in Test Example 1 and Test Example 2 of the present invention.

[0036] Figure 7 This is a mass spectrum of the target plate prepared in Example 1 of the present invention for detecting a mixed standard solution. The mixed solution includes histidine, valine, glucose, glutamic acid, aspartic acid, arginine, taurine, and phenylalanine.

[0037] Figure 8 This is a mass spectrum of the target plate prepared in Example 2 of the present invention for detecting a mixed standard solution. The mixed solution includes histidine, valine, glucose, glutamic acid, aspartic acid, arginine, taurine, and phenylalanine.

[0038] Figure 9 This is a mass spectrum of a mixed standard solution detected using 2,5-dihydroxybenzoic acid as the matrix material. The mixed solution includes histidine, valine, glucose, glutamic acid, aspartic acid, arginine, taurine, and phenylalanine.

[0039] Figure 10 This is a mass spectrum of α-cyano-4-hydroxycinnamic acid detected in a mixed standard solution. The mixed solution includes histidine, valine, glucose, glutamic acid, aspartic acid, arginine, taurine, and phenylalanine. Detailed Implementation

[0040] The technical problems, technical solutions, and advantages of the present invention will be explained in detail below with reference to exemplary embodiments. The exemplary embodiments described below are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] Polyethylene oxide-block polystyrene, using ethylene oxide-styrene (PEO-b-PS) supplied by Xi'an Qiyue Biotechnology, item number Q-0159337. Example 1

[0042] This embodiment provides a method for in-situ construction of mesoporous titanium dioxide composite array target plates based on a combination of physical plate masking and evaporation-induced self-assembly technology.

[0043] The specific preparation steps are as follows: Step 1: Preparation of precursor solution: Weigh 20 mg of polyethylene oxide-block polystyrene and add it to 5 mL of tetrahydrofuran. Sonicate the solution at room temperature for 5 minutes to obtain solution A. Then, while stirring, add 40 μL of concentrated hydrochloric acid (36% by mass) and 40 μL of glacial acetic acid (≥99.5% by mass) dropwise to solution A. Next, add 0.2 mL of tetrabutyl titanate and stir the mixture continuously at room temperature for 2 hours to obtain the precursor solution. Figure 1 A homogeneous and stable general-purpose evaporation-induced self-assembly precursor solution was demonstrated.

[0044] Step 2: Physical localization mask and in-situ assembly: 1. Target pretreatment: Select a stainless steel target plate with 384 holes (Bruker MTP 384 polishing target plate), with a hole diameter of 4mm and a hole spacing of 1mm, and place it in isopropanol and TA30 (a mixed solution containing 30% volume of acetonitrile and 0.1% trifluoroacetic acid) for ultrasonic cleaning. 2. Utilizing a physical confinement mechanism, a 384-well high-temperature resistant polyimide film pre-fabricated with an array of micropores (pore diameter of 4 mm and pore spacing of 1 mm) was selected as a physical mask. Through physical pressing, the mask was seamlessly and tightly bonded to the target plate surface, thereby constructing a region of micropores with a depth of 0.03 mm on the target plate to be coated. This micron-level thickness, combined with a fixed pore diameter, tightly confines the maximum wet film volume within a single array of pores to approximately 377 nL. 3. The precursor solution obtained in step one is uniformly applied to the surface of a target plate with a mask at a total coating volume of 0.5 mL, and the solution is filled into each array well by directional scraping. During the scraping process, excess sol is carried away from the target plate surface by the scraper, and only the solution retained in the array wells serves as an effective precursor for subsequent film formation. Based on a well diameter of 4 mm and a mask thickness of 0.03 mm, the theoretical maximum wet film volume per well is approximately 377 nL. With subsequent solvent evaporation and inorganic framework shrinkage, the precursor solution in a single well is eventually assembled in situ to form a cured film with a surface matrix packing density of approximately 0.23 μg / mm², weighing approximately 2.88 μg. 2 The coated target plate was then placed in an evaporation chamber at 25°C and 40% relative humidity for 24 hours to age; then transferred to a 40°C constant temperature chamber for 24 hours to age; and finally transferred to a 100°C oven for further constant temperature curing for 24 hours. Step 3: Removal of physical mask and template remover: Peel off the polyimide film on the surface, leaving a uniformly distributed precursor array on the target plate; place the target plate in a tube furnace and heat it to 350°C at a heating rate of 1°C / min under a nitrogen atmosphere, and hold it at the same temperature for 2 hours, then heat it to 400°C at a heating rate of 1°C / min under an air atmosphere, and hold it at the same temperature for 2 hours; then place it in a muffle furnace and heat it to 400°C at a heating rate of 2°C / min, and hold it at the same temperature for 1 hour, thus obtaining a surface mesoporous titanium dioxide modified target plate; Figure 2 The mesoporous structure of the thin film is shown. Example 2

[0045] The specific steps for preparing a target plate modified with mesoporous titanium dioxide supported on gold-palladium nanocrystals are as follows: Step 1: Preparation of gold-palladium nanocrystals: 65 mg of chloroauric acid and 65 mg of palladium acetylacetonate were dissolved in a mixed solution of 7.5 mL of oleylamine and 37.5 mL of toluene. After stirring for 1 hour under a nitrogen atmosphere, the solution was transferred to an oil bath at 80 °C and heated and stirred for 1 hour. Then, it was stirred at 100 °C for 1 hour. Subsequently, it was added to a 75% ethanol aqueous solution for precipitation. After centrifugation at 10,000 rpm for 5 minutes, it was washed three times with a 75% ethanol aqueous solution. Finally, it was redispersed in cyclohexane and the volume was adjusted to 10 mL to obtain a gold-palladium alloy nanocrystal solution. Preparation of a composite precursor solution containing gold-palladium nanocrystals: Weigh 20 mg of polyethylene oxide-block polystyrene and add it to 5 mL of tetrahydrofuran. Then, add 0.5 mL of the above gold-palladium alloy nanocrystal solution dropwise to the system and sonicate at room temperature for 5 minutes. While continuously stirring, add 40 μL of concentrated hydrochloric acid (36% by mass) and 40 μL of glacial acetic acid (≥99.5% by mass) to the above mixed solution. Next, add 0.2 mL of tetrabutyl titanate and stir at room temperature for 2 hours to obtain a mesoporous titanium dioxide precursor solution with a theoretical gold-palladium alloy loading of 5.8 wt%. Figure 3 As shown, the precursor solution is reddish-brown; Step 2: Physical Masking and Coating. The specific operations and parameters for this step are the same as those in Step 2 of Example 1.

[0046] Step 3: Step-by-step aging and in-situ firing molding. The specific operation and parameters of this step are the same as those in Step 3 of Example 1.

[0047] Test Example 1: This test example provides a method for detecting glutamic acid using a mesoporous titanium dioxide composite array target plate.

[0048] Step 1: Add 1 mg of glutamic acid to 1 mL of deionized water to obtain a 1 mg / mL glutamic acid solution. Then, on the target plate prepared in Example 1, add 1 μL of the glutamic acid solution to each of the target sites A1-A8, dry at room temperature, and then perform the test. Figure 4 The dried deposition morphology of glutamic acid solution on the modified target plate surface is shown; Step 2: Mass spectrometry analysis was performed on an UlrafleXtreme MALDI-TOF / TOF MS system equipped with a 355 nm Nd:YAG laser; mass spectra were acquired in positive ion reflectance mode, with a mass scan range of 100-1000 m / z and an accelerating voltage of 20 kV; the laser intensity and frequency were set to 80% and 2000 Hz, respectively, and all MALDI-MS analyses used 2000 sampling times; mass calibration was performed before data acquisition to obtain accurate mass measurements; Figure 5 The mass spectrometry results show [glutamate + Na] + The intensity of the addition peak.

[0049] Test Example 2: This test example provides a method for detecting histidine using a mesoporous titanium dioxide composite array target plate.

[0050] Step 1: Add 1 mg of histidine to 1 mL of deionized water to obtain a 1 mg / mL histidine solution; then, on the target plate prepared in Example 1, take 1 μL of histidine solution and drop it onto the target sites B1-B8 respectively, dry at room temperature, and then perform the test.

[0051] Step 2: Mass spectrometry analysis was performed on an UlrafleXtreme MALDI-TOF / TOF MS system equipped with a 355 nm Nd:YAG laser; mass spectra were acquired in positive ion reflectance mode, with a mass scan range of 100-1000 m / z and an accelerating voltage of 20 kV. The laser intensity and frequency were set to 80% and 2000 Hz, respectively, and all MALDI-MS analyses used 2000 samplings; mass calibration was performed before data acquisition to obtain accurate mass measurements. Figure 6 The mass spectrometry results show [histidine + Na] + The intensity of the addition peak.

[0052] Test Example 3: This test example provides a method for detecting glutamic acid using a gold-palladium nanocrystal modified mesoporous titanium dioxide composite array target plate.

[0053] Step 1: Add 1 mg of glutamic acid to 1 mL of deionized water to obtain a 1 mg / mL glutamic acid solution. Then, on the target plate prepared in Example 2, add 1 μL of the glutamic acid solution to the target sites A1-A8, dry at room temperature, and then perform the test.

[0054] Step 2: Mass spectrometry analysis was performed on an UlrafleXtreme MALDI-TOF / TOF MS system equipped with a 355 nm Nd:YAG laser; mass spectra were acquired in positive ion reflectance mode, with a mass scan range of 100-1000 m / z and an accelerating voltage of 20 kV; the laser intensity and frequency were set to 80% and 2000 Hz, respectively, and all MALDI-MS analyses used 2000 sampling times; mass calibration was performed before data acquisition to obtain accurate mass measurements; Figure 5 The mass spectrometry results show [glutamate + Na] + The intensity of the addition peak.

[0055] Test Example 4: This test example provides a method for detecting histidine using a gold-palladium nanocrystal modified mesoporous titanium dioxide composite array target plate.

[0056] Step 1: Add 1 mg of histidine to 1 mL of deionized water to obtain a 1 mg / mL histidine solution; then, on the target plate prepared in Example 2, take 1 μL of histidine solution and drop it onto the target points B1-B8, dry it at room temperature, and then perform the test.

[0057] Step 2: Mass spectrometry analysis was performed on an UlrafleXtreme MALDI-TOF / TOF MS system equipped with a 355 nm Nd:YAG laser. Mass spectra were acquired in positive ion reflectance mode, with a mass scan range of 100–1000 m / z and an accelerating voltage of 20 kV. The laser intensity and frequency were set to 80% and 2000 Hz, respectively. All MALDI-MS analyses used 2000 sampling times. Mass calibration was performed before data acquisition to obtain accurate mass measurements. Figure 6 The mass spectrometry results show [histidine + Na] + The intensity of the addition peak.

[0058] Test Example 5: This test example provides a method for detecting mixed standard solutions using a mesoporous titanium dioxide composite array target plate. Step 1: Prepare a 1 mg / mL solution of histidine, valine, glucose, glutamic acid, aspartic acid, arginine, taurine, and phenylalanine, taking 50 μL of each to prepare a mixed standard solution. Then, on the target plate prepared in Example 1, add 1 μL of the mixed standard solution to the target site, dry at room temperature, and then perform the test.

[0059] Step 2: Mass spectrometry analysis was performed on an UlrafleXtreme MALDI-TOF / TOF MS system equipped with a 355 nm Nd:YAG laser. Mass spectra were acquired in positive ion reflectance mode, with a mass scan range of 100–1000 m / z and an accelerating voltage of 20 kV. The laser intensity and frequency were set to 80% and 2000 Hz, respectively. All MALDI-MS analyses used 2000 sampling times. Mass calibration was performed before data acquisition to obtain accurate mass measurements. Figure 7 The mass spectrometry peaks of each standard analyte are displayed.

[0060] Test Example 6: This test example provides a method for detecting mixed standard solutions using a gold-palladium nanocrystal modified mesoporous titanium dioxide composite array target plate. Step 1: Prepare a 1 mg / mL solution of histidine, valine, glucose, glutamic acid, aspartic acid, arginine, taurine, and phenylalanine, taking 50 μL of each to prepare a mixed standard solution. Then, on the target plate prepared in Example 2, add 1 μL of the mixed standard solution to the target site, dry at room temperature, and then perform the test.

[0061] Step 2: Mass spectrometry analysis was performed on an UlrafleXtreme MALDI-TOF / TOF MS system equipped with a 355 nm Nd:YAG laser; mass spectra were acquired in positive ion reflectance mode, with a mass scan range of 100-1000 m / z and an accelerating voltage of 20 kV. The laser intensity and frequency were set to 80% and 2000 Hz, respectively, and all MALDI-MS analyses used 2000 samplings; mass calibration was performed before data acquisition to obtain accurate mass measurements. Figure 8 The mass spectrometry peaks of each standard analyte are displayed.

[0062] Test Example 7: Detection method for mixed standard solutions of commercial matrix 2,5-dihydroxybenzoic acid (DHB).

[0063] Step 1: Prepare 50 μL of each of the following 1 mg / mL solutions: histidine, valine, glucose, glutamic acid, aspartic acid, arginine, taurine, and phenylalanine. Add 1 μL of 10 mg / mL 2,5-dihydroxybenzoic acid (DHB) onto a stainless steel target plate. Then, add 1 μL of the mixed standard solution when the matrix droplets are about to dry, and dry at room temperature for subsequent mass spectrometry analysis.

[0064] Step 2: Mass spectrometry analysis was performed on an UlrafleXtreme MALDI-TOF / TOF MS system equipped with a 355 nm Nd:YAG laser. Mass spectra were acquired in positive ion reflectance mode, with a mass scan range of 100–1000 m / z and an accelerating voltage of 20 kV. The laser intensity and frequency were set to 80% and 2000 Hz, respectively. All MALDI-MS analyses used 2000 sampling times. Mass calibration was performed before data acquisition to obtain accurate mass measurements. Figure 9 The study showed that not all small metabolite molecules were detected.

[0065] Test Example 8: Detection method for mixed standard solutions of commercial matrix α-cyano-4-hydroxycinnamic acid (CHCA).

[0066] Step 1: Prepare 50 μL solutions of 1 mg / mL histidine, valine, glucose, glutamic acid, aspartic acid, arginine, taurine, and phenylalanine to form a mixed standard solution. Add 1 μL of 10 mg / mL α-cyano-4-hydroxycinnamic acid (CHCA) to a stainless steel target plate. Then, add 1 μL of the mixed standard solution just before the matrix droplet dries, and allow to dry at room temperature for subsequent mass spectrometry analysis.

[0067] Step 2: Mass spectrometry analysis was performed on an UlrafleXtreme MALDI-TOF / TOF MS system equipped with a 355 nm Nd:YAG laser; mass spectra were acquired in positive ion reflectance mode, with a mass scan range of 100-1000 m / z and an accelerating voltage of 20 kV; the laser intensity and frequency were set to 80% and 2000 Hz, respectively, and all MALDI-MS analyses used 2000 sampling times; mass calibration was performed before data acquisition to obtain accurate mass measurements. Figure 10 The study showed that not all small metabolite molecules were detected.

[0068] Depend on Figure 7-10It can be seen that, under the same mixed standard detection conditions, the target plates prepared in Examples 1 and 2 can both produce mass spectrometry responses to multiple small molecule metabolites in the mixed standard solution; however, when using traditional commercial organic matrices DHB and CHCA for detection, not all target small molecules were detected. This result indicates that the surface-modified mesoporous titanium dioxide target plate constructed in this invention has better detection applicability in mixed small molecule systems, effectively reducing background interference from traditional organic matrices in the low mass-to-charge ratio region and the ion suppression effect caused by the coexistence of multiple components, thereby improving the detection capability of target small molecules. This may be because the mesoporous titanium dioxide modification layer has a high specific surface area and pore confinement effect, which is beneficial for the adsorption, enrichment, and uniform distribution of analytes on the target plate surface; simultaneously, the introduction of gold-palladium alloy nanocrystals in the preferred embodiment further enhances the target plate's utilization efficiency of laser energy and interfacial charge transfer capability, thereby helping to improve the desorption and ionization response of mixed small molecule analytes. Therefore, the target plate of the present invention is not only suitable for the detection of single small molecule analytes, but also exhibits good detection performance in a multi-component competitive environment, and has good application potential for mass spectrometry analysis of small molecule metabolites in complex biological samples.

[0069] Table 1 shows the glutamate signal intensity at different target sites of the target plates prepared in Examples 1 and 2 of this invention. Mesoporous titanium dioxide is the target plate prepared in Example 1; gold-palladium-mesoporous titanium dioxide is the target plate prepared in Example 2.

[0070] Table 1 1 7238 16321 2 7208 16138 3 7168 15771 4 7023 15565 5 6876 15467 6 6864 15338 7 6787 14967 8 6779 14929 Table 2 shows the histidine signal intensity at different target sites of the target plates prepared in Examples 1 and 2 of this invention.

[0071] Table 2 1 11862 16867 2 11552 16846 3 11250 16828 4 10887 16453 5 10878 15442 6 10585 16249 7 10507 16125 8 10329 16037 Table 2 As shown in Tables 1 and 2, compared with Example 1, the surface-modified mesoporous titanium dioxide target plate with gold-palladium nanocrystals prepared in Example 2 exhibited higher mass spectrometry response intensity for small molecule analytes. Specifically, the average signal intensity of glutamic acid increased from 6992.9 to 15562.0, an increase of approximately 122.5%; the average signal intensity of histidine increased from 10981.3 to 16355.9, an increase of approximately 48.9%. This indicates that the introduction of gold-palladium nanocrystals can effectively enhance the mass spectrometry response of the target plate to target small molecules and improve detection sensitivity. This may be because the composite interface formed between the gold-palladium alloy nanocrystals and mesoporous titanium dioxide improves the absorption and utilization efficiency of laser energy by the target plate and enhances the interfacial charge transfer capability, thereby promoting the desorption and ionization of target small molecules, ultimately manifesting as an increase in mass spectrometry signal intensity.

[0072] The above description represents preferred embodiments of the present invention. It should be noted that the present invention is not limited to the exemplary embodiments disclosed above. The specification is merely intended to help those skilled in the art to comprehensively understand the specific details of the present invention. For those skilled in the art, any improvements, modifications, easily conceivable variations, or substitutions made within the scope of the technology disclosed in the present invention without departing from the principles described herein should be included within the protection scope of the present invention.

Claims

1. A method for preparing a surface mesoporous titanium dioxide modified target plate, characterized in that, Includes the following steps: S1. Preparation of precursor micelle solution: The block copolymer template agent is dissolved in a volatile solvent, and an acidic catalyst and titanium precursor are added to prepare the precursor micelle solution. S2. Physical mask confined film formation: The precursor micelle solution is coated on the surface of a conductive target plate with a physical mask attached, forming a precursor array on the target plate surface; S3. Mask removal and heat treatment forming: After removing the physical mask, the obtained target plate is subjected to heat treatment in sequence to remove the template agent and complete the formation of the mesoporous skeleton, thereby obtaining the target plate modified with mesoporous titanium dioxide on the surface.

2. The method for preparing a target plate modified with surface mesoporous titanium dioxide as described in claim 1, characterized in that, The block copolymer template agent is an amphiphilic block copolymer.

3. The method for preparing a target plate modified with surface mesoporous titanium dioxide as described in claim 1, characterized in that, The volatile solvent is an ether solvent, a low-carbon alcohol solvent, a ketone solvent, a nitrile solvent, or a mixture thereof.

4. The method for preparing a target plate modified with surface mesoporous titanium dioxide as described in claim 1, characterized in that, The acidic catalyst includes hydrochloric acid and / or glacial acetic acid; the titanium precursor is a titanium alkoxide precursor.

5. The method for preparing a target plate modified with surface mesoporous titanium dioxide as described in claim 1, characterized in that, In step S1, the amount of the block copolymer template agent is 15-30 mg, the amount of the volatile solvent is 4-6 mL, the amount of concentrated hydrochloric acid added is 30-60 µL, the amount of glacial acetic acid added is 30-60 µL, the amount of titanium precursor added is 0.15-0.30 mL, and the stirring time is 1.5-3 h.

6. The method for preparing a target plate modified with surface mesoporous titanium dioxide as described in claim 1, characterized in that, The conductive target plate is a metal conductive target plate.

7. The method for preparing a target plate modified with surface mesoporous titanium dioxide as described in claim 1, characterized in that, The physical mask is one of polyimide film, polytetrafluoroethylene film, or metal foil; the physical mask has an array of holes with a hole diameter of 3.5-4.5 mm, a hole spacing of 0.8-1.2 mm, and a mask thickness of 0.02-0.05 mm.

8. A target plate with a surface modified with mesoporous titanium dioxide, characterized in that, It is prepared by the preparation method described in any one of claims 1-7.

9. The application of the surface-mesoporous titanium dioxide-modified target plate according to claim 8 in the analysis of small biomolecule metabolites.

10. The application of the surface-mesoporous titanium dioxide-modified target plate as described in claim 9 in the analysis of small biomolecule metabolites, characterized in that, The biological small molecule metabolites include amino acids, lipids, organic acids, or sugars; the applications include high-throughput quantitative detection or mass spectrometry imaging analysis.

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