A method for imprinting mass spectrometry imaging based on titanium dioxide nanotubes

Abstract

The present invention discloses an imprinting mass spectrometry imaging method based on titanium dioxide nanotubes. The method comprises (1) preparing nanoparticle-hPDA-TDNT material and using it as a nanoimprinting substrate; (2) sample imprinting; (3) mass spectrometry imaging. Compared with traditional imprinting substrates, the nanoimprinting substrate preparation method of the present invention is simple and easy to use. The entire mass spectrometry imaging analysis process does not require complex sample pre-treatment steps such as frozen section sample processing or spraying the matrix after imprinting, which can ensure that the imaging results are true and reliable and avoid false positive results; the imprinting mass spectrometry imaging method of the present invention can simultaneously achieve the effects of in-situ imprinting and ion signal enhancement, and is applicable to both positive and negative ion imaging modes, and has wide universality and application prospects.

Description

Technical Field

[0001] The invention relates to the field of mass spectrometry imaging, and in particular to an imprinting mass spectrometry imaging method based on titanium dioxide nanotubes. Background Art

[0002] Mass spectrometry imaging (MSI) technology has been widely used in the study of the spatiotemporal distribution of small molecule metabolites, peptides and proteins in animal and plant tissues, and even single cells due to its advantages such as no need for chemical labeling, high sensitivity, spatiotemporal visualization, non-specific detection, and simultaneous acquisition of elements and molecules in one analysis (Anal. Chem., 2020, 92, 543-553; Science, 2020, 368, 283-290). Among them, matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) has become the most commonly used MSI method since its invention in the late 1980s due to its advantages such as high sensitivity, high salt tolerance, and low sample consumption (Chem. Rev., 2013, 113, 2309-2342). Despite this, the traditional MALDI-MSI method still has some shortcomings, such as the use of organic matrices, which can lead to serious spectral peak background interference in the low m / z range, hindering the detection and identification of small molecule metabolites. At the same time, matrix deposition and co-crystallization are also often uneven, which often lead to false positive results in mass spectrometry imaging (Mass Spectrom. Rev., 2022, 41, 373-420). Therefore, inorganic nanomaterials have gradually developed to solve the shortcomings of traditional matrices, such as carbon nanomaterials (Anal. Chem., 2010, 82, 6208), metal nanomaterials (ACS Nano, 2017, 11, 7424), metal organic framework materials (Angew. Chem. Int. Ed., 2020, 59, 10831; Small Methods, 2021, 5, 2001001), covalent organic framework materials (Adv. Funct. Mater., 2021, 31, 2105478), etc. Although the problems of spectral peak background interference and uneven matrix crystallization have been partially solved, time-consuming sample pretreatment processes such as matrix spraying are still required.

[0003] To this end, surface-assisted laser desorption / ionization mass spectrometry (SALDI-MS) based on nano-substrate materials has gradually developed. Thanks to the advantages of high specific surface area, high electrical conductivity and low thermal conductivity, and good photothermal effect, nano-substrate materials have shown unique analytical performance in the mass spectrometry analysis of small molecule compounds. Compared with the sample preparation method in MALDI-MS, which often requires matrix spraying or sublimation to the sample surface, the SALDI-MS method only needs to place the sample imprint or tissue section on the substrate to achieve enhanced laser desorption / ionization efficiency, greatly shortening the sample preparation time. At the same time, although the tissue section method has been widely used as a sample processing method for plant root tissue imaging (Front. Plant Sci., 2016, 7, 60), for biological samples such as petals and leaves that cannot be tissue sectioned, mass spectrometry imaging analysis can only be performed after imprint transfer. Recently, research groups have reported that porous TiO2 films and nanowire materials were prepared on the surface of indium tin oxide (ITO), which were used for imaging analysis of some metabolites in petals (ACS Appl. Mater. Interfaces, 2020, 12, 42567-42575) and leaves (Anal. Chem., 2022, 94, 952-959). However, ITO as a substrate itself is relatively expensive, and the number of endogenous metabolites identified is small. For fingerprint imaging analysis, existing substrate materials often require secondary nanoparticle deposition after imprinting to obtain a better signal response. Before the sample pretreatment time, it is difficult to meet the needs of high-throughput mass spectrometry imaging analysis. In addition to imprinting imaging, the sample preparation method of tissue sections often requires the use of matrix spraying after slicing to improve the ionization efficiency. If the matrix spraying step can be simplified, the scope of application of the mass spectrometry imaging method can be greatly improved. However, most SALDI materials currently on the market are usually single chemical compositions or surface modifications, which are difficult to simultaneously meet the mass spectrometry imaging analysis of complex biological tissues in daily life (such as plant / animal tissues and fingerprints, etc.). Therefore, it is very important to develop a composite nano-based material that integrates multiple analytical properties. It is not only required to have high photothermal conversion efficiency and low background interference, but also to have simple material preparation, wide universality, and can be used for mass spectrometry imaging analysis applications for tissue sections and imprinting samples. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an imprinting mass spectrometry imaging method based on titanium dioxide nanotubes.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] Application of nanoparticle-hPDA-TDNT material as imprinted mass spectrometry imaging substrate.

[0007] Compared with the nanopaper previously reported by the inventors (CN111239238A), the nanoparticle-hPDA-TDNT material of the present application has the following beneficial effects: (i) Although the surface of the substrate is covered with nanoparticles, compared with the paper substrate, the TDNT metal material has better electrical conductivity and thermal conductivity, which is more conducive to exciting the plasma resonance effect between the laser and AuNPs, promoting local heating on the tissue imprint surface and generating high-density charge and carrier relaxation, thereby enhancing the desorption / ionization efficiency; (ii) The PDA coating has excellent ultraviolet light absorption and photothermal conversion efficiency, Therefore, it can also promote the thermal desorption of metabolites on the imprinted surface, thereby ionizing them; (iii) TNDT is an n-type semiconductor material with ultraviolet light-induced photocatalytic activity and electron-hole pairs. When it works synergistically with nanoparticles, especially metal nanoparticles, a Schottky barrier is generated after contact due to the different work functions between the nanoparticles and the n-type semiconductors. After absorbing light, the electrons are excited to cross the Schottky barrier and enter the nanoparticles, but they cannot cross the Schottky barrier again to recombine with the holes, so the holes are separated from the charges, which is conducive to the rapid transfer of a large amount of charges in the nanostructure, thereby improving the ionization efficiency of the analyte.

[0008] Therefore, the nanoparticle-hPDA-TDNT material can be applied to the imaging analysis of more types of biological tissue systems and metabolites, such as amino acids, sugars, flavonoids, alkaloids, peptides, phospholipids, triglycerides, etc.

[0009] The method for preparing the nanoparticle-hPDA-TDNT material of the present invention comprises the following steps:

[0010] S1. Preparation of TiO2 nanotube array TDNT: Anodizing the titanium sheet to obtain the TiO2 nanotube array TDNT;

[0011] S2. Preparation of polydopamine PDA coating on the surface of TiO2 nanotube array TDNT: The TiO2 nanotube array TDNT of S1. is placed in a Tris buffer containing dopamine hydrochloride and heated for reaction to prepare a PDA-TDNT material;

[0012] S3. Hydrophobic modification on the surface of the PDA-TDNT material: Spraying a hydrophobic silane solution on the surface of the PDA-TDNT material obtained in S2 to form a hydrophobic layer on the surface of the PDA-TDNT material to prepare a hPDA-TDNT material;

[0013] S4. Performing nanoparticle modification on the surface of the hPDA-TDNT material: spraying the nanoparticle solution evenly onto the hPDA-TDNT material to obtain the nanoparticle-hPDA-TDNT material.

[0014] In step S1, the TiO2 nanotube array TDNT can be prepared by referring to the existing method.

[0015] Preferably, in step S1., the TiO2 nanotube array is prepared by placing a titanium sheet in a mixed solution of NaF and Na2SO4 to perform an anodic oxidation reaction.

[0016] Preferably, in step S1., the concentration of NaF is 0.001-0.02 g / mL, and the concentration of Na2SO4 is 0.1-0.2 g / mL.

[0017] The solvent for preparing the mixed solution of NaF and Na2SO4 is preferably ultrapure water.

[0018] Preferably, in step S1., the voltage of the anodic oxidation reaction is 3 to 30 V. Most preferably, the voltage of the anodic oxidation reaction in step S1. is 20 V.

[0019] Preferably, in step S1., the time of the anodization reaction is 0.5 to 4 hours. Most preferably, the time of the anodization reaction in step S1. is 1 hour.

[0020] Preferably, in step S2., the concentration of dopamine hydrochloride in the Tris buffer containing dopamine hydrochloride is 0.5-5 mg / mL. When the concentration of dopamine hydrochloride is within this range, a better mass spectrometry signal can be obtained. More preferably, the concentration of dopamine hydrochloride in the Tris buffer containing dopamine hydrochloride is 1-4 mg / mL. Within this range, the signal intensity of most detected compounds can be further improved. Most preferably, the concentration of dopamine hydrochloride in the Tris buffer containing dopamine hydrochloride is 3 mg / mL.

[0021] Generally, the preparation method of the Tris buffer containing dopamine hydrochloride is to add dopamine hydrochloride of the target concentration into the Tris buffer. Generally, the concentration of the Tris buffer is 10 mM and the pH is 8.5.

[0022] Preferably, in step S2., the heating temperature is 80-99° C. Most preferably, in step S2., the heating temperature is 90° C.

[0023] Preferably, in step S2., the reaction time is 1 to 4 hours. Most preferably, in step S2., the reaction time is 3 hours.

[0024] Preferably, in step S2., the reaction is carried out under stirring.

[0025] In step S3., the thickness of the hydrophobic layer can be adjusted by controlling parameters such as the concentration of the hydrophobic silane solution, the spraying flow rate, and the number of spraying times.

[0026] Preferably, in step S3., the concentration of the hydrophobic silane solution is 0.001 to 0.01 mol / L; the flow rate of the spraying is 0.1 to 5 mL / min; and the number of spraying times is 1 to 20 times.

[0027] Most preferably, the concentration of the hydrophobic silane solution is 2.5 μL / mL; the flow rate of the spraying is 1 mL / min; and the number of spraying times is 10.

[0028] Preferably, in step S4., the nanoparticles are any one of Au, Ag, Pt, Pd, Co, Cu, C, Ge or Si nanoparticles.

[0029] More preferably, in step S4., the nanoparticles are metal nanoparticles, selected from any one of Au, Ag, Pt, Pd, Co, Cu or Ge nanoparticles.

[0030] Most preferably, in step S4., the nanoparticles are Au nanoparticles.

[0031] Preferably, in step S4., the shape of the nanoparticles is spherical, rod-shaped, star-shaped, linear, porous and / or sheet-shaped.

[0032] Preferably, the concentration of the nanoparticle dispersion in step S4. is 0.01 to 10 g / L. More preferably, the concentration of the nanoparticle dispersion in step S4. is 0.1 to 1 g / L.

[0033] Preferably, in step S4, the diameter distribution of the nanoparticles is 1-200 nm. Within this diameter range, the obtained nanoparticle-hPDA-TDNT material has a good resonance enhancement effect at 355 nm.

[0034] More preferably, in step S4, the diameter distribution of the nanoparticles is 60-80 nm. Within this diameter range, the obtained nanoparticle-hPDA-TDNT material has a better resonance enhancement effect at 355 nm.

[0035] Preferably, in step S4, the spraying method is ultrasonic spraying at a uniform speed.

[0036] Preferably, in step S4., the flow rate of the ultrasonic spray uniform spraying is 0.01 to 0.09 mL / min. Most preferably, the flow rate of the ultrasonic spray uniform spraying in step S4. is 0.03 mL / min.

[0037] Preferably, the number of spraying in step S4. is 50 to 500 times. Within this range, a better signal can be obtained. Most preferably, the number of spraying in step S4. is 250 times. Under this value, the time required for spraying can be reduced and a better mass spectrometry signal enhancement effect can be achieved.

[0038] A titanium dioxide nanotube-based imprinting mass spectrometry imaging method comprises the following steps:

[0039] S5. preparing the above-mentioned nanoparticle-hPDA-TDNT material and using it as a nanoimprinting substrate;

[0040] S6. Sample imprinting: imprinting the tissue sample on the nanoimprint substrate in S1., so that a tissue sample imprint is obtained on the nanoimprint substrate, and the tissue sample imprint includes at least chemical information of the distribution of endogenous substances in the sample;

[0041] S7. Mass spectrometry imaging: The tissue sample imprint is irradiated with a light source and detected by a mass analyzer to reconstruct the mass spectrometry signal intensity of the sampling two-dimensional dot matrix and the sample endogenous substances to obtain a two-dimensional mass spectrometry image with all the chemical information on the tissue sample imprint surface.

[0042] In step S6., the tissue sample may be microbial tissue, plant tissue (such as leaves, bulbs, petals, etc.), animal / insect tissue (such as brain, kidney, liver, muscle, etc.).

[0043] Most preferably, in step S6., the tissue sample is a tissue sample such as plant petals, plant leaves, strawberries, cherry tomatoes, human fingerprints, mouse brain slices, etc.

[0044] In step S7., the light source may be a continuous or pulsed light source. Preferably, the beam wavelength of the continuous light source is 46.9 to 2940 nm, the beam diameter is 0.01 to 10 mm, and the average power is not less than 0.1 mW. Preferably, the beam wavelength of the pulsed light source is 46.9 to 2940 nm, the pulse width is 1 fs to 1 ms, the beam diameter is 0.01 to 10 mm, the instantaneous pulse energy is 1 nJ to 100 mJ, and the pulse frequency is 0.05 to 100 MHz.

[0045] Preferably, in step S7., the light source is a 355nm pulsed laser.

[0046] Preferably, in step S7., the mass analyzer may be a quadrupole analyzer, an ion trap analyzer, a time-of-flight analyzer, a magnetic field analyzer, a Fourier transform analyzer, and the like.

[0047] The principle of the mass spectrometry imaging imprinting method of the present invention is similar to that of the conventional mass spectrometry imaging imprinting method. After the tissue sample imprint is prepared, the light beam emitted by the light source (including the light beam generated by the laser or other light source) is irradiated on the surface of the tissue sample imprint. Due to the coupling enhancement effect between the light beam and the nanoparticles, the desorption and ionization efficiency can be improved, so ions can be generated at a lower light beam energy. When the power density of the light beam enhanced by the nanoparticles is greater than or equal to the desorption (or atomization) threshold of the sample, the sample molecules (or atoms) within the irradiation range will be desorbed (or atomized) and ionized to generate ions due to the energy provided by the output light source. The ions are transmitted into the mass analyzer to be separated and detected, and the mass spectrum signal intensity of the sample endogenous substances in the sampling two-dimensional lattice and the imprint is reconstructed to obtain a mass spectrum containing molecular and atomic information at the sample sampling point.

[0048] Preferably, in step S7., the environment of the mass spectrometry imaging blotting method can be high pressure, normal pressure, low vacuum or high vacuum, and other gases (such as air, nitrogen, argon, helium, hydrogen or a mixture of the above gases, etc.) can be used as auxiliary gases, and the pressure range is 1×10 -7 ~1×10 8 Pa.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The present invention provides a new nanoparticle-hPDA-TDNT material, and an imprinting mass spectrometry imaging method based on the nanoparticle-hPDA-TDNT material (titanium dioxide nanotube). The novel titanium dioxide nanotube composite material has nanoscale surface uniformity, improved light-to-heat conversion efficiency, and higher desorption / ionization efficiency. The imprinting mass spectrometry imaging method based on the nanoparticle-hPDA-TDNT material as an imprinting substrate can be used for mass spectrometry imaging analysis of plant tissue (flowers, leaves, etc.), fingerprint imprinting, and animal tissue (mouse brain, etc.) slices. The substrate of the present invention is easy to use, and the entire mass spectrometry imaging analysis process does not require complex sample pre-treatment steps such as frozen section sample processing or spraying the matrix after imprinting, which can ensure that the imaging results are true and reliable and avoid false positive results; the imprinting mass spectrometry imaging method of the present invention can simultaneously achieve the effects of in-situ imprinting and ion signal enhancement, and is suitable for positive and negative ion imaging modes at the same time, and has wide universality and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1This is a schematic diagram of an imprinting mass spectrometry imaging method based on titanium dioxide nanotubes according to the present invention.

[0052] Figure 2 This is a scanning electron microscope image of the AuNP-hPDA-TDNT material described in Example 1 of the present invention, a) the substrate surface is magnified 35,000 times; b) the substrate surface is magnified 80,000 times.

[0053] Figure 3 This is an optical image of the imprinting of the petals of the lyre coral (J. integerrima) using AuNP-hPDA-TDNT material and the mass spectrometry imaging of its various endogenous metabolites.

[0054] Figure 4 This is an optical image of AuNP-hPDA-TDNT material used for imprinting of Ginkgo biloba leaves and mass spectrometry imaging of its various endogenous metabolites.

[0055] Figure 5 This is an optical image of the imprinting of strawberry pulp using AuNP-hPDA-TDNT material and mass spectrometry imaging of its various endogenous metabolites.

[0056] Figure 6 This is the imprinted optical image of cherry tomato pulp using AuNP-hPDA-TDNT material and the mass spectrometry imaging of its various endogenous metabolites.

[0057] Figure 7 AuNP-hPDA-TDNT material is used for optical images of human fingerprints and mass spectrometry images of illicit drugs and various endogenous metabolites on fingerprints (step size is 45μm).

[0058] Figure 8 AuNP-hPDA-TDNT material is used for high-resolution mass spectrometry imaging analysis of human fingerprints (step size is 10μm), and the fingerprint detail distribution map with individual specificity such as pores, bifurcations, and interruption areas in the fingerprint can be obtained.

[0059] Fig. 9 This is the mass spectrum of AuNP-hPDA-TDNT material used for endogenous metabolite analysis of human fingerprints.

[0060] Fig.10 AuNP-hPDA-TDNT material was used to imprint optical images of mouse brain slices (5μm) and mass spectrometry images of their various endogenous lipid metabolites.

[0061] Fig.11 Comparison of mass spectra of lipid components in mouse brain slices using traditional DHB matrix and AuNP-hPDA-TDNT material.

[0062] Fig.12 The electron microscope images of the surface micro-nano structure of TDNT after treatment at 5V, 10V and 20V (Example 1), wherein (a), (b) and (c) correspond to the electron microscope images at 5V and magnifications of 35,000×, 80,000× and 200,000×, respectively; (d), (e) and (f) correspond to the electron microscope images at 10V and magnifications of 35,000×, 80,000× and 200,000×, respectively; (g), (h) and (i) correspond to the electron microscope images at 10V and magnifications of 35,000×, 80,000× and 200,000×, respectively.

[0063] Fig.13 The electron microscope images of the surface micro-nanostructure of PDA-TDNT after stirring for 1.5h, 2h and 3h (Example 1), wherein (a), (b) and (c) correspond to the electron microscope images at magnifications of 35,000×, 80,000× and 200,000× at 1.5h, respectively; (d), (e) and (f) correspond to the electron microscope images at magnifications of 35,000×, 80,000× and 200,000× at 2h, respectively; (g), (h) and (i) correspond to the electron microscope images at magnifications of 35,000×, 80,000× and 200,000× at 3h, respectively.

[0064] Fig.14 The electron microscope images of the surface micro-nanostructure characterization of the AuNP-hPDA-TDNT material after spraying 50 times, 100 times, 150 times, 200 times (Example 1), and 250 times, wherein (a), (b), and (c) correspond to the electron microscope images at magnifications of 35,000×, 80,000×, and 200,000× at 50 times, respectively, and (d), (e), and (f) correspond to the electron microscope images at magnifications of 35,000×, 80,000×, and 200,000× at 100 times, respectively. Electron micrographs of , (g), (h), and (i) correspond to electron micrographs taken 150 times at magnifications of 35,000×, 80,000×, and 200,000×, respectively; (j), (k), and (l) correspond to electron micrographs taken 200 times at magnifications of 35,000×, 80,000×, and 200,000×, respectively; (m), (n), and (o) correspond to electron micrographs taken 250 times at magnifications of 35,000×, 80,000×, and 200,000×, respectively. DETAILED DESCRIPTION

[0065] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are provided for illustrative purposes only and are not intended to limit the present invention. The test methods for which specific conditions are not specified in the following examples are usually carried out under conventional conditions, and the instruments involved are all commercially available products. Unless otherwise stated, percentages and parts are calculated by weight.

[0066] Example 1

[0067] A method for preparing AuNP-hPDA-TDNT material is as follows:

[0068] S1. The titanium sheet was placed in an aqueous solution containing 0.005 g / mL NaF and 0.142 g / mL Na2SO4 and subjected to an anodic oxidation reaction at a DC voltage of 20 V for 1 h to obtain a TiO2 nanotube array (TDNT) sheet.

[0069] S2. The TDNT sheet was then removed and gently ultrasonically cleaned in ultrapure water for 1 min, and then placed in a 10M Tris buffer containing 3 mg / mL dopamine hydrochloride and stirred at 90°C for 3 h to obtain a polydopamine-modified titanium dioxide nanotube (PDA-TDNT) material.

[0070] S3. The PDA-TDNT was ultrasonically cleaned in anhydrous ethanol and ultrapure water for 0.5 min each, and then a 2.5 μL / mL anhydrous ethanol solution of perfluorooctyltrichlorosilane was evenly sprayed onto the surface of the PDA-TDNT 10 times at a flow rate of 1 mL / min using an ultrasonic sprayer to obtain hydrophobic treated polydopamine-modified titanium dioxide nanotubes (hPDA-TDNT).

[0071] S4. Prepare a dispersion of gold nanoparticles (AuNPs) with a diameter of 60 to 80 nm and a concentration of 0.1 mg / mL and spray it evenly onto the surface of hPDA-TDNT at a flow rate of 0.03 mL / min 200 times to obtain AuNP-hPDA-TDNT material.

[0072] Figure 2 The characterization results of AuNP-hPDA-TDNT materials reflect that the diameter of TiO2 nanotubes is about 64.1±12.4nm, and the AuNPs are evenly distributed on the surface, avoiding false positive results of mass spectrometry imaging.

[0073] After the AuNP-hPDA-TDNT material is prepared as a substrate, the tissue sample is directly pressed to leave the imprint on the substrate, or the slice is placed on the substrate, and then the substrate is attached to the target plate and sent to the mass spectrometer for detection. The light source is used to irradiate different types of sample tissues, and the ions generated by ionization are detected one by one by the mass analyzer, and the sampling two-dimensional lattice and the mass spectrometry signal intensity of different substances are reconstructed to obtain the mass spectrometry imaging of all chemical information on the tissue surface ( Figure 1 ).

[0074] Test results of different tissue samples

[0075] Figure 3 The results of mass spectrometry imaging of the petals of the lyre coral after being imprinted on the AuNP-hPDA-TDNT material are shown in the figure. It can be clearly seen in the figure that the petal outline and texture can be completely imprinted on the AuNP-hPDA-TDNT nano-based material. In addition, the in-situ spatial distribution of endogenous metabolites (such as jasmonic acid, apigenin, cycas reticulin, astragalus glycoside, anthocyanins, etc.) inside the petal tissue can be obtained at the same time, indicating that the imprinting mass spectrometry imaging method based on titanium dioxide nanotubes described in the present invention is suitable for mass spectrometry imaging analysis of plant petal tissue imprinting.

[0076] Figure 4 The results of mass spectrometry imaging of ginkgo leaf after imprinting on AuNP-hPDA-TDNT material are shown in the figure. It can be clearly seen that the outline and texture of the leaf can be completely imprinted on the AuNP-hPDA-TDNT material. In addition, the in-situ spatial distribution of endogenous metabolites (such as monosaccharides, disaccharides, ginkgolide, isoscopolamine, ginkgo biloba flavonoids, etc.) in the ginkgo leaf can be obtained at the same time, indicating that the imprinting mass spectrometry imaging method based on titanium dioxide nanotubes described in the present invention is suitable for mass spectrometry imaging analysis of plant leaf tissue imprinting.

[0077] Figure 5 The results of mass spectrometry imaging after strawberry pulp was imprinted on AuNP-hPDA-TDNT material are shown in the figure. It can be clearly seen in the figure that the outline and texture of the strawberry can be completely imprinted on the AuNP-hPDA-TDNT material. And the in-situ spatial distribution of endogenous metabolites in strawberries (such as monosaccharides, disaccharides, abscisic acid, glutamine, anthocyanins, etc.) can be obtained at the same time, indicating that the imprinting mass spectrometry imaging method based on titanium dioxide nanotubes described in the present invention is suitable for mass spectrometry imaging analysis of imprinting of fruit tissues such as strawberries.

[0078] Figure 6It reflects the mass spectrometry imaging results of cherry tomato pulp after being imprinted on AuNP-hPDA-TDNT material. It can be clearly seen in the figure that the outline and texture of the strawberry can be completely imprinted on the AuNP-hPDA-TDNT material. And the in-situ spatial distribution of endogenous metabolites (such as glutamine, glutamic acid, monosaccharides, disaccharides, phenylalanine, 4-hydroxyisoleucine, 5-hydroxytryptamine, etc.) in the cherry tomato pulp can be obtained at the same time, indicating that the imprinting mass spectrometry imaging method based on titanium dioxide nanotubes described in the present invention is suitable for mass spectrometry imaging analysis of imprinting of fruit tissues such as cherry tomatoes.

[0079] Figure 7 The results of mass spectrometry imaging of human fingerprints on AuNP-hPDA-TDNT materials (step size of 45 μm) are shown. The in-situ distribution of endogenous metabolites (such as FA (16:0), WE (32:1), WE (34:1), TG (43:1), TG (46:1), TG (47:1), TG (48:1), etc.) and exogenous contraband in fingerprints can be clearly seen in the figure, indicating that the imprinting mass spectrometry imaging method based on titanium dioxide nanotubes described in the present invention is suitable for mass spectrometry imaging analysis of invisible fingerprints.

[0080] Figure 8 The high-resolution mass spectrometry imaging results (step size of 10 μm) of human fingerprints imprinted on AuNP-hPDA-TDNT materials are shown in the figure. The distribution of fingerprint details with individual specificity, such as pores, bifurcations, and interruption areas in the fingerprint, can be clearly seen in the figure, indicating that the imprint mass spectrometry imaging method based on titanium dioxide nanotubes described in the present invention has ultra-high spatial resolution.

[0081] Fig. 9 It is reflected that the mass spectrum of human fingerprints imprinted on AuNP-hPDA-TDNT material can clearly obtain the distribution map of fingerprint details with individual specificity such as pores, bifurcations, and interruption areas in the fingerprint, indicating that the imprint mass spectrometry imaging method based on titanium dioxide nanotubes described in the present invention has ultra-high spatial resolution.

[0082] Fig.10 The results of mass spectrometry imaging of rat brain slices (5 μm thick) on AuNP-hPDA-TDNT materials are shown in the figure. The imprint of rat brain slices and the in-situ distribution of various endogenous lipid metabolites in the rat brain (such as PC (32:0), PC (34:2), PC (36:4), PE (36:4), PE (38:4), PE (38:2), etc.) can be clearly seen in the figure, indicating that the imprinting mass spectrometry imaging method based on titanium dioxide nanotubes described in the present invention is suitable for mass spectrometry imaging analysis of animal tissue slices.

[0083] Fig.11The results of mass spectrometry analysis of rat brain using traditional organic matrix CHCA and AuNP-hPDA-TDNT materials were compared. The results showed that compared with the traditional MALDI method, the imprinting mass spectrometry imaging method of the present invention can identify more endogenous lipid metabolites (such as PC, PE, etc.) in rat brain tissue.

[0084] Effect of different anodic oxidation voltages in Examples 2 to 5

[0085] According to the method and parameters of Example 1, only the DC voltage in S1. was changed to 5V, 10V, 15V, and 25V respectively; Examples 2 to 5 were prepared. Among them, the surface micro-nanostructure characterization electron microscope images of TDNT after treatment at 5V, 10V, and 20V (Example 1) are as follows Fig.12 shown.

[0086] Effect of different dopamine reaction times in Examples 6 to 9

[0087] According to the methods and parameters of Examples 6 to 9, only the stirring time in S2. was modified to 1.5h, 2h, 2.5h, and 3h; Examples 6 to 9 were prepared. Among them, the surface micro-nanostructure characterization electron microscope images of PDA-TDNT after 1.5h, 2h, and 3h treatment (Example 1) are as follows Fig.13 shown.

[0088] Effect of different AuNP spraying times in Examples 10-13

[0089] According to the method and parameters of Example 1, only the number of times of spraying nanogold onto the surface of hPDA-TDNT in S4. was modified to 50 times, 100 times, 150 times, and 250 times respectively; wherein, the surface micro-nanostructure characterization electron microscope images of AuNP-hPDA-TDNT after 50 times, 100 times, 150 times, 200 times (Example 1), and 250 times are as follows Fig.14 shown.

[0090] The AuNP-hPDA-TDNT obtained in Examples 2 to 13 was used for detection of coral leaves, ginkgo leaves, strawberry pulp, cherry tomato pulp, human fingerprints, and mouse brain slices, with similar results to Example 1.

[0091] The above embodiment is only one of the implementation modes of the present invention. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the premise and principle of the present concept are all equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of a nanoparticle-hPDA-TDNT material as an imprinting mass spectrometry imaging substrate, wherein the nanoparticle-hPDA-TDNT material is prepared by a method comprising the following steps: S1. Preparation of TiO2 nanotube array TDNT: Anodizing a titanium sheet to obtain the TiO2 nanotube array TDNT; S2. Preparation of polydopamine PDA coating on the surface of TiO2 nanotube array TDNT: The TiO2 nanotube array TDNT of S1. is placed in a Tris buffer containing dopamine hydrochloride and heated for reaction to prepare a PDA-TDNT material; S3. Hydrophobic modification on the surface of the PDA-TDNT material: Spraying a hydrophobic silane solution on the surface of the PDA-TDNT material obtained in S2 to form a hydrophobic layer on the surface of the PDA-TDNT material to prepare a hPDA-TDNT material; S4. Modifying the surface of the hPDA-TDNT material with nanoparticles: spraying the nanoparticle dispersion evenly onto the hPDA-TDNT material to obtain the nanoparticle-hPDA-TDNT material; In step S4., the nanoparticles are any one of Au, Ag, Pt, Pd, Co, Cu, C, Ge or Si nanoparticles; The printing is to directly imprint the tissue sample so that the imprint is left on the nanoparticle-hPDA-TDNT material.

2. The application according to claim 1, characterized in that: In step S2., the concentration of dopamine hydrochloride in the Tris buffer containing dopamine hydrochloride is 0.5-5 mg / mL.

3. The application according to claim 1, characterized in that: In step S2., the heating temperature is 80-99°C.

4. The use according to claim 1, characterized in that: In step S3., the concentration of the hydrophobic silane solution is 0.001-0.01 mol / L; the flow rate of the spraying is 0.1-5 mL / min; and the number of spraying times is 1-20 times.

5. The use according to claim 1, characterized in that: In step S4., the shape of the nanoparticles is spherical, rod-shaped, star-shaped, linear, porous and / or sheet-shaped.

6. The use according to claim 1, characterized in that: In step S4., the diameter distribution of the nanoparticles is 1-200 nm.

7. The use according to claim 1, characterized in that: In step S4, the spraying method is ultrasonic spraying at a uniform speed.

8. A method for imprinting mass spectrometry imaging based on titanium dioxide nanotubes, characterized in that: The steps include: S5. Preparation of nanoparticle-hPDA-TDNT material as a nanoimprinting substrate; S6. Sample imprinting: imprinting the tissue sample on the nanoimprint substrate in S5, so that a tissue sample imprint is obtained on the nanoimprint substrate, wherein the tissue sample imprint includes at least chemical information of the distribution of endogenous substances in the sample; S7. Mass spectrometry imaging: The tissue sample imprint is irradiated with a light source and detected by a mass analyzer, and the sampling two-dimensional dot matrix and the mass spectrometry signal intensity of the sample endogenous substance are reconstructed to obtain a two-dimensional mass spectrometry image with all the chemical information on the tissue sample imprint surface; The nanoparticle-hPDA-TDNT material is prepared by a method comprising the following steps: S1. Preparation of TiO2 nanotube array TDNT: Anodizing a titanium sheet to obtain the TiO2 nanotube array TDNT; S2. Preparation of polydopamine PDA coating on the surface of TiO2 nanotube array TDNT: The TiO2 nanotube array TDNT of S1. is placed in a Tris buffer containing dopamine hydrochloride and heated for reaction to prepare a PDA-TDNT material; S3. Hydrophobic modification on the surface of the PDA-TDNT material: Spraying a hydrophobic silane solution on the surface of the PDA-TDNT material obtained in S2 to form a hydrophobic layer on the surface of the PDA-TDNT material to prepare a hPDA-TDNT material; S4. Modifying the surface of the hPDA-TDNT material with nanoparticles: spraying the nanoparticle dispersion evenly onto the hPDA-TDNT material to obtain the nanoparticle-hPDA-TDNT material; In step S4., the nanoparticles are any one of Au, Ag, Pt, Pd, Co, Cu, C, Ge or Si nanoparticles.

Images