A monatomic manganese-doped Ti3C2 nanomaterial self-supporting film, a preparation method and an assisted in-vivo curved surface mass spectrum imaging method

Abstract

The application discloses a monatomic manganese-doped Ti3C2 nanomaterial self-supporting film, a preparation method and an assisted in-vivo curved surface mass spectrum imaging method.The monatomic manganese-doped Ti3C2 (Mn@Ti3C2) has the advantages of low background interference, good salt resistance and protein resistance, high stability and accuracy, and lower detection limit and quantitative limit, as compared with traditional organic matrixes DHB and CHCA.Secondly, the Mn@Ti3C2 self-supporting film which is bendable and has high adsorption is used to assist LDI-MSI, so that the molecular coverage and resolution are improved, metabolites such as lactic acid, inositol, amino acid and coenzyme of the skin are in-situ detected, and the spatiotemporal dynamic change mechanism of metabolism is in-situ and non-invasively revealed from a microscale.The application has important significance for exploring biomarkers for disease diagnosis and provides theoretical guidance and technical support for the research on metabolic mechanisms of other in-vivo curved surface parts.

Description

TECHNICAL FIELD

[0001] The application relates to the field of mass spectrometry detection technology, in particular to a single-atom manganese-doped Ti3C2 nanomaterial self-supporting film, a preparation method thereof and a living body curved surface mass spectrometry imaging method assisted by the same. BACKGROUND

[0002] The sweat composition of the body surface skin is not as complex as serum, plasma or urine, is easy to collect, and can more sensitively provide metabolic marker information. Small molecule substances, such as amino acids and glucose, secreted by different skin parts can be used as metabolic markers of certain metabolic diseases of the human body, and have important research significance and are paid more and more attention. Due to the lack of in-situ high-sensitivity metabolite extraction methods and detection means, the current research on the metabolites on the skin surface still stays in the stage of invasive biopsy research by extracting tissues from the human body surface, which greatly limits the research progress.

[0003] Mass spectrometry (MS) has become the most commonly used analysis tool in metabolomics research due to its high sensitivity, good specificity, wide detection dynamic range and other advantages. At present, the most commonly used metabolomics research on metabolites is liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS), which combines the advantages of mass spectrometry and chromatography, has high selectivity and high sensitivity, and can provide the advantages of relative molecular mass and structure information, and has been widely used in many fields such as drug analysis, food analysis and environmental analysis. However, the sample pretreatment usually needs tissue homogenization, metabolite extraction and other processing procedures, which seriously destroys the actual spatial distribution of metabolites in the tissue. Mass spectrometry imaging (MSI) is a new type of molecular imaging technology combining mass spectrometry analysis and image visualization, which can locate various metabolites and spatial distribution information of various biological tissues at the microscopic level. Compared with other imaging methods, mass spectrometry imaging has the advantages of no need for specific labeling, no need for complex sample pretreatment and high molecular detection specificity, which brings new opportunities for in-situ characterization of trace chemical signals.

[0004] As the most mature mass spectrometry imaging technology at present, matrix-assisted laser desorption ionization mass spectrometry imaging technology (MALDI-MSI) can realize in-situ analysis of molecules in tissues at a spatial resolution of several microns, and has a very important role in the fields of biology and medicine. MALDI-MSI can show the location of target molecules in tissue sections, thereby providing a basis for medical diagnosis and treatment. However, most of the current MALDI-MSI research methods can only use optimized frozen tissue sections for research, and the experimental process cannot continuously detect in-situ distribution of metabolites in living organisms and curved positions, thereby having great application limitations in the research of skin metabolites. Therefore, it is crucial to develop a non-destructive sampling method and a high-sensitivity and high-throughput analysis method for in-situ research of metabolites on curved surfaces of living organisms. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to establish a new method for high-resolution in-vivo and curved surface mass spectrometry imaging spatial metabolomics analysis based on the excellent adsorption, flexibility and photoelectric performance of the new Mn@Ti3C2 nanometer two-dimensional material self-supporting film, and the visualization in-situ analysis advantage of mass spectrometry imaging technology. The in-vivo curved surface mass spectrometry imaging metabolomics research is carried out from human fingerprints and facial skin, and the spatiotemporal dynamic change rule is revealed.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] In a first aspect, the present application provides a new Mn@Ti3C2 nanometer material self-supporting film with high conductivity and good flexibility, which is in-situ doped with Mn when synthesizing Ti3AlC2; the prepared self-supporting film can be used for in-situ visual analysis of metabolites of skin.

[0008] In a second aspect, the present application provides a preparation method of the single-atom manganese-doped Ti3C2 nanometer material self-supporting film, which comprises the following steps:

[0009] (1) Ti, Al, C and Mn elements are used as raw materials, mixed uniformly by ball milling, heated under the action of Ar protection to obtain Mn@Ti3AlC2 powder;

[0010] (2) HF is used to etch the Mn@Ti3AlC2 to remove Al atoms, and DMSO is used to strip the multi-layer Mn@Ti3C2 solution to make it become single-layer Mn@Ti3C2;

[0011] (3) The single-layer Mn@Ti3C2 solution is added into a suction filter bottle, a vacuum pump is opened for suction filtration, and after the filter paper is sucked out, the Mn@Ti3C2 self-supporting film is dried.

[0012] Further, the molar ratio of Ti, Al, C, and Mn in step (1) is 3:1.2:0.1:2, the temperature rising rate is 10℃ / min, the temperature is raised to 1500℃, and the temperature is kept for 5h.

[0013] Further, the etching step in step (2) is that the Mn@Ti3AlC2 powder is slowly added into a 50% HF solution, continuously stirred at room temperature for 24h, after the reaction is completed, the obtained suspension is washed with ultrapure water, put into a centrifuge and centrifuged for 8 times, each time for 5min, the rotating speed is set to 4000rpm, until the pH of the final solution is greater than or equal to 6; the precipitate after centrifugation is dissolved in DMSO, continuously stirred for 24h under the protection of argon, and then the solution is centrifuged for 1h, the rotating speed is set to 5000rpm, and the supernatant is collected.

[0014] Further, the volume of the Mn@Ti3C2 solution subjected to suction filtration in step (3) is 5mL, the concentration is 10mg / mL, and the suction filtration time is 20-30min.

[0015] In a third aspect, the application provides an application of the monatomic manganese-doped Ti3C2 nanomaterial self-supporting film in detection of small molecule compounds.

[0016] The application provides a new substrate of the Mn@Ti3C2 self-supporting film for mass spectrometry imaging spatial metabolomics analysis of fingerprints and facial skin, which comprises the following steps:

[0017] (1) Preparation of a fingerprint sample: the right index finger is coated with procaine standard powder, and the left index finger is blank control, then the molecules of the two groups of fingerprints are simultaneously pressed on the Mn@Ti3C2 film by using a direct pressing method, and the Mn@Ti3C2 film is directly detected by LDI-MSI technology;

[0018] (2) Preparation of chin and cheek skin samples: two rectangles are randomly selected on the cheeks and chin of a volunteer, and two pieces of Mn@Ti3C2 film are tightly pasted on the two selected areas;

[0019] (3) Preparation of forehead skin samples: a rectangle is selected on the forehead of a volunteer, 2% dihydro myricetin solution is uniformly sprayed on 1 / 2 area of the rectangle by using a spray bottle, and the other 1 / 2 area is blank control, and a piece of Mn@Ti3C2 film with the same area as the rectangle is pasted on the selected area;

[0020] (4) MALDI mass spectrometry imaging analysis and detection, the mass-to-charge ratio detected is 100-1000, and the sample scanning resolution in MALDI mass spectrometry imaging analysis is 100μm.

[0021] Further, the imprinting time in step (1) is 10min.

[0022] Further, the chin and cheek skin sampling time in step (2) is 10 min.

[0023] Further, in step (3), dihydromyricetin is sprayed every 2 min, 5 times, 3 mL in total, and the interval between spraying dihydromyricetin and resampling is 2 h.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] (1) The application designs a single-atom doped Ti3C2, prepares a new type of Mn@Ti3C2 nanomaterial, and first uses it for LDI-MSI.

[0026] (2) Compared with the traditional matrix, the LDI-MS method established by the application using the new type of Mn@Ti3C2 nanomaterial has higher peak intensity and lower background noise, can be used for detecting endogenous small molecule metabolites and exogenous antiepileptic drugs, has good repeatability of detection results and good linear relationship, and can be used for quantitative analysis.

[0027] (3) The application breaks the limitations of biopsy and traditional frozen tissue sections, explores the in-situ spatial distribution of metabolites on human fingerprints and facial skin, and develops a method of live curved surface live body mass spectrometry imaging. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0029] Figure 1 It is a schematic diagram of the preparation process of the Mn@Ti3C2 self-supporting film of the application.

[0030] Figure 2are Transmission Electron Microscopy (TEM) of Ti3C2, TEM of Mn@Ti3C2, High Resolution TEM of Mn@Ti3C2, High Resolution Aberration-corrected Electron Microscopy of Mn@Ti3C2, Selected Area Electron Diffraction (SAED) image of Mn@Ti3C2, High Angle Annular Dark Field Scanning Transmission Electron Microscopy (HAADF-STEM) of Mn@Ti3C2, and Energy Dispersive X-ray element mapping (EDX mapping) image of Mn@Ti3C2, wherein (a) TEM of Ti3C2; (b) TEM of Mn@Ti3C2; (c) High Resolution TEM of Mn@Ti3C2; (d) High Resolution Aberration-corrected Electron Microscopy of Mn@Ti3C2; (e) SAED of Mn@Ti3C2; (f) HAADF-STEM of Mn@Ti3C2; (g) EDS mapping (Mn); (h) EDS mapping (Ti); (i) EDS mapping (C);

[0031] Figure 3 are X-ray Photoelectron Spectroscopy (XPS) of Mn@Ti3C2, wherein (a) XPS survey spectrum of Mn@Ti3C2; (b-c) XPS (Mn 2p); (d) XPS (Ti 2p); (e) XPS (C 1s); (f) XPS (O 1s);

[0032] Figure 4 are Scanning Electron Microscopy images (SEM) of Mn@Ti3C2, wherein (a) planar morphology of Mn@Ti3C2 thin film; (b-c) cross-sectional morphology of Mn@Ti3C2 thin film;

[0033] Figure 5 are Atomic Force Microscopy images (AFM) of Mn@Ti3C2, X-ray Diffraction images (XRD) of Ti3C2 and Mn@Ti3C2, and Ultraviolet Absorption Spectra (UV-vis) of Ti3C2 and Mn@Ti3C2, wherein (a) AFM; (b) XRD; (c) UV-vis;

[0034] Figure 6 are intensity plots of detecting four standard mixtures (Glu, Gln, Asn and Pro) using Mn@Ti3C2, Ti3C2, DHB, CHCA matrix, respectively;

[0035] Figure 7 are intensity plots of detecting CBZ and CBZ-E using Mn@Ti3C2, Ti3C2, DHB, CHCA matrix, respectively;

[0036] Figure 8 are mass spectra of two standard (CBZ and CBZ-E) mixed with different salts or proteins, wherein (a) CBZ with 0.5 M NaCl; (b) CBZ with 0.5 M KCl; (c) CBZ with 5 mg·mL-1 CBZ of BSA; (d) CBZ-E with 0.5 M NaCl; (e) CBZ-E with 0.5 M KCl; (f) CBZ-E with 5 mg-mL -1 CBZ-E of BSA;

[0037] Figure 9 LDI-MS signal stability plots of CBZ and CBZ-E at two concentrations of 10 pg / mL and 50 pg / mL, where (a) CBZ sample at 10 measurement locations within the same spot stability; (b) CBZ sample between 10 different spots signal stability; (c) CBZ-E sample at 10 measurement locations within the same spot stability; (d) CBZ-E sample between 10 different spots signal stability.

[0038] Figure 10 Standard curve plots of CBZ and CBZ-E at 1-45 ng-pL -1

[0039] Figure 11 Fingerprint MSI map of procaine, where (a) procaine MS map and fingerprint procaine MSI image using Mn@Ti3C2 self-supporting membrane matrix; (b) three-dimensional PCA score plot of fingerprint metabolites; (c) fingerprint MSI image using Mn@Ti3C2 self-supporting membrane matrix in positive ion mode;

[0040] Figure 12 MSI map of small molecule metabolites of chin and cheek skin, where (a) three-dimensional PCA score plot of cheek and chin; (b-i) mass spectra of cheek and chin metabolite distribution comparison;

[0041] Figure 13 MSI map of small molecule metabolites of forehead skin, where (a) LDI-MS map of dihydromyricetin; (b) three-dimensional PCA score plot of metabolites of forehead sprayed dihydromyricetin region versus unsprayed region; (c-i) mass spectra imaging images of metabolite distribution comparison of forehead sprayed dihydromyricetin region versus unsprayed region;

[0042] Figure 14 Application map of Mn@Ti3C2 self-supporting membrane of the present application. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. Such description, however, is to be considered in all aspects as illustrative and not restrictive, understood to be based upon certain examples of aspects, features and embodiments of the application.

[0044] ​It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for a range of values of, for example, concentrations, solvent amounts, and the like, there are multiple values within the range that also are specifically disclosed. Every intermediate value of the ranges is incorporated herein.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials that are described in the documents. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0046] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0047] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0048] Sweat of the skin is directly involved in the regulation of body temperature and metabolism. Metabolomics, as a branch of systems biology, can explore the metabolites produced by the life activities of organisms in a whole system approach. MALDI-MSI technology can simultaneously in situ visualize and analyze the spatial distribution and spatiotemporal dynamic change mechanism of various small molecule metabolites such as amino acids, sugars, lipids, organic acids and other exogenous compounds in the metabolic activities of living organisms. Compared with other imaging methods, it has the outstanding advantages of not needing specific labeling, not needing complex sample pretreatment, and large detection throughput. In addition, compared with the previously developed metabolomics analysis methods (HPLC-MS, GC-MS), the metabolite analysis by mass spectrometry imaging technology can effectively avoid the loss of spatial information caused by the pretreatment process such as tissue homogenization and metabolite extraction. The present application uses a new substrate of Mn@Ti3C2 self-supporting film with good flexibility, high conductivity and good adsorption. Based on the in situ visualization analysis advantage of MALDI mass spectrometry imaging technology, a new method of in vivo and curved surface mass spectrometry imaging spatial metabolomics analysis will be established, realizing the in situ, visual and non-invasive research of complex living body curved surface metabolic mechanism from the microscopic scale, and revealing the spatiotemporal dynamic change mechanism of human fingerprint and facial skin metabolism.

[0049] Example 1 Preparation and characterization of Mn@Ti3C2 and freestanding films

[0050] (1) Preparation of Mn@Ti3C2 and freestanding films

[0051] 1) In-situ doping of manganese element on Ti3AlC2 structure, Al element and Mn element are combined with Ti element and C element in the process of synthesizing MAX phase, this method does not damage the layered MAX structure and lattice parameters. The specific method is as follows: Ti, Al, C, Mn elements are used as raw materials, the molar ratio is 3: 1.2: 0.1: 2, ball milling is carried out, and the temperature is raised to 1500℃ at a rate of 10℃ / min under the action of Ar protection, and then the sintering is carried out for 5h, and then Mn@Ti3AlC2 powder is obtained. 2g of Mn@Ti3AlC2 powder is slowly added to 40mL of 50% HF solution, and continuously stirred at room temperature for 24h. After the reaction is completed, the obtained suspension is washed with ultrapure water, and then centrifuged for 8 times, each time for 5 minutes, and the speed is set to 4000rpm, until the pH of the final solution is greater than or equal to 6. 1g of the precipitate after centrifugation is dissolved in 26mL of DMSO, continuously stirred for 24h under the protection of argon, and then the solution is centrifuged for 1h at a speed of 5000rpm, and the supernatant is collected.

[0052] 2) The filter bottle is tightly connected with the sand core filter, the upper side of the sand core filter is soaked with ultrapure water, the filter paper is put into the sand core filter, and the cylindrical glass funnel and the safety bottle are fixed with the clamp. 5mL of Mn@Ti3C2 solution with a concentration of 10mg / mL is taken and added into the funnel, the vacuum pump is opened to filter for 20-30min, when no water drops down, the filter paper is taken out and dried in a vacuum dryer until the water in the film is completely volatilized.

[0053] (2) Characterization of Mn@Ti3C2 and freestanding films

[0054] ① Figure 2 TEM results of Ti3C2 and Mn@Ti3C2 are shown. Ti3C2 is etched to show single-layer or small-layer structure (a), and the flaky structure of Mn@Ti3C2 is not changed after doping of single-atom Mn (b). In addition, Figure 2 c shows that Mn@Ti3C2 has obvious lattice fringes, and the interplanar spacing of Mn@Ti3C2 is 0.3225nm. According to the high-angle annular dark-field image (HAADF) of Mn@Ti3C2, the Mn single atom is marked with a red circle, which shows that the Mn single atom is successfully added to Ti3C2 (d). The EDS image shows that Mn@Ti3C2 contains Mn, Ti and C elements (e). Figure 2 Figure 2 Figure 2 Figure 2 ​​​f-i). These results show that the new single-atom in-situ doping strategy successfully replaces the traditional composite doping method to dope Mn atoms in Ti3C2.

[0055] ②The element composition and valence of Mn@Ti3C2 nanocomposites were determined by X-ray photoelectron spectroscopy (XPS), and the results are shown in Figure 3 a-f.

[0056] ③The morphology of the Mn@Ti3C2 self-supporting film was studied from both the plane and the cross-section. As can be seen from the SEM images, the surface of the Mn@Ti3C2 self-supporting film is relatively flat ( Figure 4 a), and the cross-section is neat ( Figure 4 b), which means that the Mn@Ti3C2 self-supporting film will be more suitable for the curved surface of the biological body, which will help to detect more small molecules in situ. In addition, the thickness of the Mn@Ti3C2 self-supporting film is about 22.72 μm ( Figure 4 c).

[0057] ④The AFM results show that the thickness of the sheet structure of 1 mg / mL Mn@Ti3C2 solution is about 17.5 nm under a scale of 2 μm ( Figure 5 a). The nanometer thin film not only saves the cost of work, but also ensures a high ionization efficiency, meeting the needs of small molecule analysis.

[0058] ⑤The XRD patterns of Ti3C2 and Mn@Ti3C2 show that Figure 5 b), with the addition of Mn, the (002) peak shifts from 6.64° to 5.91°, which indicates that the addition of Mn single atoms increases the interlayer spacing, and the (002) peak of Mn@Ti3C2 is higher than that of Ti3C2, which indicates that the doping of Mn atoms improves the crystallinity and stability of Ti3C2.

[0059] ⑥ Figure 5 c is the UV-vis spectrum of Ti3C2 and Mn@Ti3C2. The results show that both Ti3C2 and Mn@Ti3C2 solutions have absorbance at 335 nm (the optimal wavelength for MALDI-MS ionization), and after the addition of Mn atoms, the absorbance of Mn@Ti3C2 at 335 nm is significantly higher than that of Ti3C2, which is due to the heterogeneous structure of Ti3C2 after the doping of Mn atoms. Therefore, the above results can prove that Mn@Ti3C2 has potential application value in LDI-MS analysis.

[0060] Example 2 Performance evaluation of Mn@Ti3C2 assisted LDI-MS

[0061] ① A small molecule mixture composed of asparagine (Asn), glutamine (Gln), proline (Pro), and glutamic acid (Glu), as well as carbamazepine and 10,11-epoxycarbamazepine, were detected by LDI-MS. Figure 6 As shown in b, the [M+H] content of proline was detected using Ti3C2. + Peak (m / z 116.1) [M+Na] + Peak (m / z 138.1) and [M+K] + Peak (m / z 154.1); [M+H] of asparagine + Peak (m / z 133.1), [M+Na] + Peak (m / z 155.1), [M+K] + Peak (m / z 171.1); [M+H] of glutamate + Peak (m / z 148.1) and [M+K] + Peak (m / z 186.1); [M+H] of glutamine + Peak (m / z 147.1), [M+Na] + Peak (m / z 169.1) and [M+K] + Peak (m / z 185.1). Proline [M+H] was detected using 2,5-dihydroxybenzoic acid (DHB) as a matrix. + Peak (m / z 116.1), [M+Na] + Peak (m / z 138.1), [M+K] + Peak (m / z 154.1); Asparagine [M+K] + Peak (m / z 171.1); glutamic acid [M+H] + Peak (m / z 148.1), [M+Na] + Peak (m / z 170.1), [M+K] + Peak (m / z 186.1); glutamine [M+H] + Peak (m / z 147.1), [M+Na] + Peak (m / z 169.1), [M+K] + Peak (m / z 185.1)( Figure 6 c). Using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix, three adducts of four small molecules can be detected. However, CHCA exhibits numerous impurity peaks in the small molecule mass range of m / z 115-300, leading to false positive results when detecting small molecules. Figure 6 d). For Mn@Ti3C2, [M+H] can be observed in all small molecules. + Peak, [M+Na] +Peaks and [M+K] + Peaks were observed, and the adducts of small molecules exhibited higher intensities compared to DHB and Ti3C2 matrices. Figure 6 a).

[0062] ② LDI-MS was used to detect the exogenous antiepileptic compound carbamazepine (CBZ) and its metabolite 10,11-epoxycarbamazepine (CBZ-E). From... Figure 7 Figures b and f show that [M+Na] in CBZ was detected using a Ti3C2 matrix. + Peak (m / z 259.2) and [M+K] + Peak (m / z 275.2); [M+Na] of CBZ-E + Peak (m / z 275.2) and [M+K] + Peak (m / z 291.2). CBZ[M+H] was detected using DHB and CHCA as matrices. + Peak (m / z 237.2), [M+Na] + Peak (m / z 259.2), [M+K] + Peak (m / z 275.2); CBZ-E[M+H] + Peak (m / z 253.2), [M+Na] + Peak (m / z 275.2), [M+K] + Peak (m / z 291.2)( Figure 7 (c, d, g, and h). [M+H] ions of CBZ and CBZ-E were detected using Mn@Ti3C2 as the matrix. + Peak, [M+Na] + Peaks and [M+K] + The peak, whose adduct intensity is higher than that of DHB and Ti3C2 matrices ( Figure 7 a and e).

[0063] ② NaCl (1M), KCl (1M), and BSA (10 mg / mL) solutions were mixed with CBZ (500 μg / mL) and CBZ-E (500 μg / mL) in equal proportions to evaluate their effects on Mn@Ti3C2. Figure 8 As shown in a and d, [M+Na] of CBZ and CBZ-E can still be detected in the spectra after the addition of 1M NaCl. + In the spectrum containing 1M KCl, [M+K] CBZ and CBZ-E can be detected at high levels. + ( Figure 8 (b and e). Additionally, when BSA is added, the [M+Na] of CBZ and CBZ-E... + Peaks and [M+K] +Peaks can be detected simultaneously Figure 8 c and f). These results demonstrate that Mn@Ti3C2 as a matrix exhibits excellent tolerance to salts and proteins, indicating that Mn@Ti3C2 has high selectivity and sensitivity in the analysis of small metabolites in complex biological fluids.

[0064] ③CBZ and CBZ-E were selected as standard samples to investigate the repeatability of Mn@Ti3C2 assisted LDI-MS. Two concentrations of CBZ and CBZ-E were prepared, 10 μg / mL and 50 μg / mL. First, the repeatability of CBZ and CBZ-E was detected within the points at two concentrations. The samples were divided into ten areas on the target plate, and the laser intensity was set to 55%. The mass spectrometry data was obtained by adjusting the laser beam, and the coefficient of variation (CV) of [M+Na] + intensity of the ten area samples was calculated. The repeatability of CBZ and CBZ-E was investigated between the points at two concentrations by different ten samples, and the CV value of [M+Na] + intensity of the ten samples was calculated. Figure 9 a-d, the CV of CBZ and CBZ-E within and between points at 10 μg / mL and 50 μg / mL was within 8%, respectively, showing that the Mn@Ti3C2 matrix has good mass spectrometry stability, solving the problems of uneven crystallization and poor reproducibility of traditional organic matrix and analyte co-crystallization, and realizing the quantitative analysis of small molecule metabolites by LDI-MS.

[0065] ④The detection limit (LODs) and the limit of quantification (LOQs) were detected with a signal-to-noise ratio (S / N) greater than or equal to 3 and 10 as the standard, respectively. The detection limit of CBZ and CBZ-E was 0.02 ng / μL and 0.01 ng / μL, respectively, and the limit of quantification of CBZ and CBZ-E was 0.6 ng / μL and 0.3 ng / μL, respectively, with Mn@Ti3C2 as the matrix.

[0066] ⑤An internal standard quantitative method was developed to solve the problem of unstable quantification of analytes in MALDI-TOF-MS. Isopropylazine was selected as the internal standard, and the concentration range of CBZ and CBZ-E was set to 1-45 μg / mL. CBZ and CBZ-E were mixed with 30 μg / mL isopropylazine and healthy rat serum at the same proportion, respectively, and the CBZ [M+Na] + peak (m / z 259.27), CBZ-E [M+Na] + peak (m / z 275.27), isopropylazine [M+Na] + peak (m / z 307.42) were obtained from the mass spectrum. As Figure 10As shown in Figure a, with the concentration of CBZ on the x-axis and the peak area ratio of the sodium adduct of CBZ to promethazine on the y-axis, the regression equation for the CBZ standard curve is obtained as Y = 0.5287X + 0.1181(R²). 2 =0.9997), and using the same method, the regression equation for the CBZ-E standard curve is Y = 0.3017X + 0.0584(R = 0.9997). 2 =0.9999)( Figure 10 b), R 2 All values ​​are greater than 0.99, indicating that there is a good linear relationship between the concentration of the analyte and the peak area.

[0067] Example 3

[0068] (1) Distribution characteristics of procaine and other metabolites on fingerprint surface

[0069] Figure 11 a shows the MS spectrum of procaine solution with 1 mg / mL Mn@Ti3C2 solution as the substrate and the MSI image of procaine with Mn@Ti3C2 self-supporting film as the substrate, [M+H]. + Peak (m / z 237.3), [M+Na] + Peak (m / z 259.3) and [M+K] + All peaks (m / z 275.3) were detectable. Figure 11 b is the 3D PCA score map of fingerprint metabolites, where each point represents a compound in the obtained dataset. It is evident that there are significant differences in metabolites between the two regions. Furthermore, from... Figure 11 In c, we also found the endogenous metabolite glutamine (m / z 147.1 [M+H]). + ) and lysoPG(16:0 / 0:0)(m / z 523.0[M+H] + ).

[0070] (2) Distribution characteristics of cheeks and chin

[0071] The cheeks have a higher water content and thicker skin than the chin, a specific difference that leads to significant differences in metabolism between the cheeks and chin. Furthermore, the skin on the cheeks and chin is richer in sweat, meets the morphological requirements of curved skin, and is easy to sample. This invention selected the cheeks and chins of volunteers for research; three-dimensional PCA score maps showed the differences in compounds between the cheeks and chin. Figure 12 a) This indicates the existence of diverse and heterogeneous chemical environments on the surface of human facial skin. Glycolic acid, glycerol, and inositol from sebum, and lactic acid from sweat were detected in the cheeks and chins of volunteers. These molecules are all involved in skin metabolism and maintaining the balance of the skin's microecology. Figure 12b-i). glyceric acid (m / z 107.1 [M+H] + ), phenylpropionamide (m / z 165.2 [M+H] + ), myo-inositol (m / z 181.2 [M+H] + ), galactosamine (m / z 202.2 [M+Na] + ), and lysoPE (18:3(6Z,9Z,12Z) / 0:0) (m / z 514.0 [M+Na] + ) were more abundant on the cheeks, which might be related to the high metabolic activity of the volunteers' cheeks. Lactic acid (m / z 113.1 [M+Na] + ) and glycerol (m / z 115.1 [M+Na] + ) were more secreted on the chin. In addition, glycine-L-threonine (m / z 1992 [M+Na] + ) was detected on both the cheeks and chin.

[0072] (3) Comparison of the metabolic profiles of the forehead regions sprayed and unsprayed with dihydromyricetin

[0073] Dihydromyricetin is an extract of Ampelopsis grossedentata, and as one of the flavonoids, it has anti-inflammatory, antioxidant, and anti-tumor effects. The three-dimensional PCA score plot showed the differences between the compounds in the two regions of the forehead skin after spraying dihydromyricetin, which indicated that spraying dihydromyricetin could change the small molecule metabolic changes of the skin Figure 13 b) MSI results Figure 13 c-d) showed that the [M+Na] + peak (m / z 343.3) and [M+K] + peak (m / z 359.3) of dihydromyricetin were detected on the skin. In addition, some small molecule metabolites were detected on the skin without spraying dihydromyricetin, such as acetic acid (m / z 99.1 [M+K] + ), lactic acid (m / z 113.1 [M+Na] + ), purine (m / z 121.1 [M+H] + ), myo-inositol (m / z 181.2 [M+H] + ), and lactyl-CoA (m / z 858.1 [M+K] + ).

[0074] The application applies a new type of Mn@Ti3C2 self-supporting film which is bendable and has high adsorption, improves the molecular coverage and resolution of the MALDI mass spectrum imaging technology, establishes a new method for in vivo visual analysis based on the in situ visualization advantage of the MALDI mass spectrum imaging technology, can detect the distribution of various endogenous metabolites such as amino acids, lipids and exogenous drug small molecules such as procaine and dihydro myricetin, realizes in situ, visual and non-invasive research on the complex in vivo curved surface metabolism mechanism from the microscale, and reveals the spatiotemporal dynamic change mechanism of human skin metabolism.

[0075] The above-described embodiments are only used to describe the preferred modes of the application, and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements of the technical solutions of the application made by those skilled in the art shall fall within the protection scope determined by the claims of the application.

Claims

1. A freestanding film of monatomic manganese-doped Ti3C2 nanomaterial, characterized in that: In-situ doping Mn in the synthesis of Ti3AlC2; the prepared self-supporting film can be used for in-situ visual analysis of skin metabolites; The application relates to a preparation method of a monatomic manganese-doped Ti3C2 nanomaterial self-supporting film, which comprises the following steps: (1) Ti, Al, C and Mn elements are used as raw materials, mixed uniformly through ball milling, heated under the action of Ar protection, and Mn@Ti3AlC2 powder is obtained; (2) HF is used to etch the Mn@Ti3AlC2, Al atoms are removed, and DMSO is used to strip the multilayer Mn@Ti3C2 solution to make it into a monolayer Mn@Ti3C2; (3) The monolayer Mn@Ti3C2 solution is added into a suction filter bottle, a vacuum pump is opened for suction filtration, a filter paper is taken out after suction filtration, and the Mn@Ti3C2 self-supporting film is obtained after drying.

2. The monatomic manganese-doped Ti3C2 nanomaterial free-standing film according to claim 1, characterized in that, In the step (1), the molar ratio of Ti, Al, C and Mn is 3:1.2:2:0.1, the temperature rising rate is 10 DEG C / min, the temperature is raised to 1500 DEG C, and the temperature is kept for 5 h.

3. The monatomic manganese-doped Ti3C2 nanomaterial free-standing film of claim 1, wherein, In the step (2), the etching step is as follows: the Mn@Ti3AlC2 powder is slowly added into a 50% HF solution, continuously stirred at room temperature for 24 h, the obtained suspension is cleaned with ultrapure water, placed in a centrifuge and centrifuged for 8 times, each time for 5 min, the rotating speed is set to 4000 rpm, and the pH of the final solution is greater than or equal to 6; the precipitate after centrifugation is dissolved in DMSO, continuously stirred for 24 h under the protection of argon, and then the solution is centrifuged for 1 h, the rotating speed is set to 5000 rpm, and the supernatant is collected.

4. The monatomic manganese-doped Ti3C2 nanomaterial free-standing film of claim 1, wherein, In the step (3), the volume of the Mn@Ti3C2 solution for suction filtration is 5 mL, the concentration is 10 mg / mL, and the suction filtration time is 20-30 min.

5. The application of the monatomic manganese-doped Ti3C2 nanomaterial self-supporting film according to any one of claims 1-4 in small molecule compound detection.

6. The application according to claim 5, mass spectrometry imaging spatial metabolomics analysis of fingerprints and facial skin, comprising the following steps: (1) Preparation of fingerprint samples: the right index finger is coated with procaine standard powder, the left index finger is blank, then the two groups of fingerprint molecules are simultaneously pressed on the Mn@Ti3C2 film by using a direct pressing method, and the Mn@Ti3C2 film is directly detected by MSI technology; (2) Preparation of chin and cheek skin samples: two rectangles are randomly selected on the cheeks and chin of a volunteer, and two pieces of Mn@Ti3C2 film are tightly pasted on the two selected areas; (3) Preparation of forehead skin samples: a rectangle is selected on the forehead of a volunteer, a 2% dihydroquercetin solution is uniformly sprayed on 1 / 2 area of the rectangle by using a spray bottle, and the other 1 / 2 area is blank, and a Mn@Ti3C2 film with the same area as the rectangle is pasted on the selected area; (4) MALDI mass spectrometry imaging analysis and detection, and the mass-to-charge ratio detected is 100-1000.

7. Use according to claim 6, characterized in that: The imprinting time in the step (1) is 10 min.

8. Use according to claim 6, characterized in that: The sampling time of chin and cheek skin in the step (2) is 10 min.

9. Use according to claim 6, characterized in that: Dihydromyricetin was sprayed every 2 min in step (3), 5 times, 3 mL in total, and the interval time between spraying dihydromyricetin and resampling was 2 h. Dihydromyricetin was sprayed every 2 min in step (3), 5 times, 3 mL in total, and the interval time between spraying dihydromyricetin and resampling was 2 h.

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