CN117949262A

CN117949262A - Membrane-mediated imprinting chip and preparation method and application thereof

Info

Publication number: CN117949262A
Application number: CN202211335477.6A
Authority: CN
Inventors: 邬建敏; 刘星月; 陈晓明
Original assignee: Hangzhou Well Healthcare Technologies Co ltd; Zhejiang University ZJU
Current assignee: Hangzhou Well Healthcare Technologies Co ltd; Zhejiang University ZJU
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2024-04-30

Abstract

The invention discloses a membrane-mediated imprinting chip and a preparation method and application thereof, and belongs to the technical field of mass spectrum detection. The chip comprises a substrate, wherein a porous film is attached to the substrate, the porous film is provided with micropores penetrating through the upper surface and the lower surface, and the aperture of the micropores is 150-300 nm. The chip of the invention is easy to prepare and low in cost, not only can effectively isolate a tissue layer from an imaging substrate, but also can ensure that target molecules on the tissue are efficiently imprinted on the imaging substrate in situ, and the spatial distribution of the target molecules is reserved to the greatest extent. The chip and the method for detecting have good detection sensitivity and coverage rate, strong operability and universality, and are expected to become a universal technology for preparing mass spectrum samples.

Description

Membrane-mediated imprinting chip and preparation method and application thereof

Technical Field

The invention belongs to the technical field of mass spectrum detection, and particularly relates to a membrane-mediated imprinting chip and a preparation method and application thereof.

Background

The mass spectrum imaging technology (MSI) can provide the spatial distribution and content information of molecules on the surface of a sample, and has the characteristics of no marking, high sensitivity, wide dynamic range, wide molecular species coverage, high analysis speed and the like. In the life science field, MSI can realize spatial distribution imaging of proteins, lipids and small molecule organic metabolites on the surface of a tissue slice. Matrix-assisted laser desorption/ionization (MALDI) is one of the most commonly used mass spectrometry imaging techniques that typically use an organic Matrix (e.g., organic acid, etc.) to assist in laser desorption and ionization of analytes. Under laser irradiation, the analyte and matrix are simultaneously desorbed and ionized. The matrix molecules are further fragmented to generate fragments, which are informative of background peaks in the low mass-to-charge ratio (m/z) range, interfering with the detection of small molecule metabolites (< 1000 da). And the spray coating of the matrix before imaging is easy to cause the analyte to be delocalized or uneven crystals to cause the deviation of molecular imaging spatial distribution information. Therefore, the development of mass spectrum imaging technology and material of the spraying-free matrix has important significance for practical application of MSI.

The Surface-assisted laser desorption ionization mass spectrometry imaging (Surface-ASSISTED LASER Desorption and Ionization Mass Spectrometry Imaging, SALDI-MSI) utilizes the absorption and charge transfer capability of inorganic nano materials to laser, so that the rapid mass spectrometry detection of small molecules is better realized, and the existing SALDI materials comprise: graphene, carbon quantum dots, noble metal nanoparticles, porous silicon, silicon nanowires, and the like. Inorganic nanomaterials are ideal candidates for supplementing or replacing organic matrices. The interaction of the laser with the nanostructure can result in a higher heating rate, an extended interaction time, and most importantly, improved ionization efficiency. In addition, the ideal "non-matrix" ionization promoter effectively uses the laser pulse energy to generate ions from the adsorbate, thereby generating minimal to no debris. SALDI-MSI creates new prospects for imaging of low molecular weight compounds, especially in the metabonomics and lipidomic era.

At present, four main ways of preparing samples in SALDI-MSI are respectively a deposition method, an imprinting method, a spraying method and a metal sputtering method. Both the spraying method and the metal sputtering method are continuation and improvement of the traditional MALDI matrix spraying, the spraying or sputtering process is tedious and time-consuming, the problem of sample molecule delocalization cannot be essentially improved, and further innovation and popularization are difficult. Deposition and imprinting are the two sample preparation methods that are currently most likely to drive the development and application of SALDI-MSI. The deposition method is to simply deposit a tissue sample on the surface of the nano substrate and then directly image the tissue sample, and the method has the defect that laser is difficult to penetrate a tissue layer to act with a nano structure to generate high desorption ionization efficiency. Ionization with higher laser intensities can result in lower detection sensitivity and can cause partial tissue ablation, possibly with observed imaging artifacts, and is difficult to popularize into biological tissue imaging applications. The blotting principle is to transfer target molecules on a sample to the surface of the nano-substrate, the sample is removed before analysis, and a molecular blot is left on the surface. Although it well eliminates the effect of tissue layers on imaging in deposition methods, the process of directly imprinting tissue with a substrate tends to foul spatial details, limiting imaging spatial resolution. And the characteristics of the nano-substrate significantly determine which analytes are imprinted on the surface, creating a large barrier to the improvement of detection sensitivity and coverage. Therefore, in the existing sample preparation processes, there is no universal and universal method, and there is a great development space for SALDI-MSI.

Currently, DIUTHAME chips are developed by the japanese koku pine company, using aluminum oxide thin films as imaging substrates. The film is composed of closely packed sub-micron channels and is coated with a platinum layer on the side exposed to the laser (promoting efficient laser desorption ionization). By capillary action, tissue metabolites permeate in situ from the surface opposite to the exposure surface, and imaging experiments can be performed immediately. Although the method provides a convenient mode for mass spectrum imaging, the aluminum oxide film substrate is fragile and is easy to crack in the operation process. In addition, the noble metal platinum coating has weak surface controllability, has certain bias to the desorption ionization efficiency of different types of metabolic molecules, and has low mass spectrum peak coverage rate of the metabolic molecules. And DIUTAHME chips are expensive, which limits their use in mass spectrometry imaging applications.

Disclosure of Invention

In order to solve at least one of the technical problems, the invention adopts the following technical scheme:

The first aspect of the present invention provides an application of a porous film in preparing a chip for biological tissue imprinting, the chip comprising a substrate, the porous film having micropores penetrating through the upper and lower surfaces, the pore diameter of the micropores being 150-300 nm, and the porous film being attached to the substrate when the mass spectrometry chip is prepared.

In the invention, the porous film is used as a mediated imprinting layer, and analyte molecules on the surface of the biological tissue slice can permeate to the substrate through micropores in use so as to complete imprinting process. Therefore, the invention has higher requirement on the micropore aperture of the porous film. On the one hand, the pore diameter of the micropore cannot be less than 100nm, otherwise, the permeability of the micropore is poor, the analyte cannot be effectively transferred to the surface of the substrate, so that a mass spectrum detection signal is weak, and the detection sensitivity is reduced; on the other hand, the pore diameter of the micropores cannot be larger than 400nm, otherwise, the molecules of the analyte are severely displaced, the in-situ property of the analyte cannot be reserved, and the spatial resolution is low. The present invention has unexpectedly found that the detection effect is better when the pore diameter of the microwells is 150 to 300nm, and further unexpectedly, the detection effect is better when the pore diameter of the microwells is 200 nm.

In some embodiments of the invention, the porous membrane is a nuclear pore membrane. The nuclear pore membrane is a membrane which is obtained by irradiating a homogeneous membrane of polycarbonate with high energy rays, such as alpha particles of 4-5 MeV, to break chemical bonds in the polymer and then corroding the polymer, and has a cylindrical pore with a pore diameter controllable in the range of 100-1000 nm.

In the present invention, the pore density of the micropores is also required, and cannot be too dense or too sparse. In some embodiments of the invention, the microwells have a cell density of 2 x 10 ⁸～4×10⁸ cells/cm ². In some preferred embodiments of the invention, the microwells have a cell density of 2.9X10 ⁸/cm ².

In some embodiments of the invention, the substrate is a substrate having a nanowire structure, and the porous film is for attachment to a side of the substrate having a nanowire structure. At this time, the micropores of the nanowire structure on the surface of the substrate are communicated with the outside of the other surface of the porous film. When a tissue slice is placed on the porous membrane, lipid and/or metabolite molecules can permeate through the micropores onto the nanowire structures.

In some embodiments of the invention, the nanowire structure is a one-dimensional structure having a dimension that is limited to below 100nm in the lateral direction (without limitation in the longitudinal direction).

Further, the nanowires are vertical nanowires, and the substrate having the vertical nanowire structure is also referred to as a vertical nanowire substrate. Preferably, the vertical nanowire substrate is selected from at least one of the group consisting of a silicon vertical nanowire substrate, a titanium oxide nanowire, and a silicon carbide vertical nanowire substrate, in which the materials of the substrates are different.

In some preferred embodiments of the invention, the vertical nanowire substrate is a silicon vertical nanowire substrate. In some more preferred embodiments of the present invention, the silicon vertical nanowire substrate is prepared and obtained using the following steps:

s11, cutting p-type monocrystalline silicon, and etching in a solution containing hydrofluoric acid and a silver catalyst;

And S12, washing the etched p-type monocrystalline silicon with deionized water, and immersing in dilute nitric acid to remove the silver catalyst, thereby obtaining the silicon vertical nanowire substrate with the vertical nanowire array.

The second aspect of the invention provides a chip for biological tissue imprinting, comprising a substrate, wherein a porous film is attached to the substrate, the porous film is provided with micropores penetrating through the upper surface and the lower surface, and the aperture of the micropores is 150-300 nm.

In some embodiments of the invention, the substrate includes a blotting region and a molecular weight calibration region.

A third aspect of the present invention provides a method for manufacturing a chip according to the second aspect of the present invention, comprising the step of attaching the porous film to the surface of the substrate.

The explanation and further definition of the micro-holes and the substrate of the first aspect of the present invention are equally applicable to the second and third aspects of the present invention, and are not repeated here.

A fourth aspect of the invention provides a method of biological tissue blotting based on the chip according to the second aspect of the invention, comprising the steps of:

S1, precooling the chip;

S2, fully expanding the biological tissue slice onto the surface of the porous film of the mass spectrum chip, heating the back of the chip to melt the slice, and drying the mass spectrum chip at room temperature;

And S3, taking down the tissue slice and the porous film on the mass spectrum chip to obtain the substrate containing the tissue print.

In some embodiments of the invention, the tissue imprinted substrate may be subjected to mass spectrometry imaging, in particular by feeding the imprinted substrate into a mass spectrometer for mass spectrometry imaging scanning. The chip provided by the invention is used for carrying out biological tissue imprinting mass spectrometry imaging, so that the transverse diffusion of metabolic molecules in the process of contacting a tissue slice with a nano solid substrate can be avoided, and the high-fidelity metabolic molecule mass spectrometry imaging result can be obtained.

From the above description, the person skilled in the art may also image the tissue print using other imaging methods, such as raman spectroscopy.

In some embodiments of the invention, the tissue section has a thickness of 20 to 50 μm.

In the present invention, the method is for non-diagnostic non-therapeutic purposes.

A fifth aspect of the invention provides the use of a chip according to the second aspect of the invention in surface-assisted laser desorption ionization mass spectrometry imaging

In a sixth aspect, the invention provides the use of a chip according to the second aspect of the invention for the preparation of a kit for detecting lipids and/or metabolites in a tissue.

In a seventh aspect, the invention provides a kit for detecting lipids and/or metabolites in a tissue, comprising a chip according to the second aspect of the invention.

In the present invention, the lipid includes a polar lipid and a neutral lipid. In some embodiments of the invention, the polar lipids include phospholipids and sphingolipids and the neutral lipids include glycerides and sterol lipids.

In the present invention, the metabolites include, but are not limited to, sugars, nucleosides, organic acids, ketones, polypeptides, amino acids, organic amines, aldehydes, terpenes, steroids, alkaloids. In some embodiments of the invention, the metabolites refer to metabolic intermediate and metabolic end product molecules having a molecular weight of less than 1000Da produced by the metabolic process of an organism. In some preferred embodiments of the invention, the metabolites refer to metabolic intermediates and metabolic end product molecules having a molecular weight of less than 300Da, such as uric acid, taurine, glucose.

In the present invention, the biological tissue sample is a tissue sample for various scientific researches or clinical diagnosis and treatment, not for organ transplantation.

In some embodiments of the invention, the biological tissue sample is a clinical tissue sample including, but not limited to, heart, liver, spleen, lung, intestine, stomach, kidney, and brain tissue.

In some embodiments of the invention, the biological tissue sample is a tumor tissue sample derived from at least one of the group comprising lung cancer, liver cancer, stomach cancer, colorectal cancer, breast cancer, cervical cancer, prostate cancer.

In some embodiments of the invention, the biological tissue sample is used to prepare tissue sections using methods that are common to the present invention and well known to those skilled in the art.

The beneficial effects of the invention are that

Compared with the prior art, the invention has the following beneficial effects:

(1) The chip of the invention can utilize any porous film meeting the limiting conditions, such as a nuclear pore film, has wide sources of raw materials, is easy to prepare and has low cost.

(2) The chip and the method can effectively isolate the tissue layer from the imaging substrate, cannot lose any metabolite molecules, and overcomes the defects of a deposition method: the laser can reach the surface of the substrate after penetrating the tissue layer, and the desorption is very low; the defect that the imprinting method is easy to cause molecular displacement and loss of partial metabolites in the process of removing the tissue slices by using solvent cleaning is also avoided.

(3) Compared with a blotting method, the chip and the method of the invention have the advantages that the tissue surface metabolite is reserved in situ without being washed, and the loss of small metabolic molecules is avoided. And the high-density independent pore channels of the porous film can ensure that target molecules on tissues are efficiently imprinted on an imaging substrate in situ, so that the spatial distribution of the target molecules is reserved to the greatest extent.

(4) Compared with a Binsong DIUTHAME chip, the chip provided by the invention is easier to operate (the substrate is not fragile), can be compatible with multiple SALDI material substrates, and has a larger space on the promotion of desorption ionization. In addition, by utilizing DIUTHAME chips, the process of attaching tissues is easy to damage, and the chips can be effectively avoided.

(5) The chip and the method for mass spectrum imaging detection have good detection sensitivity and coverage rate, and have strong operability and universality, and are expected to become a universal technology for mass spectrum imaging sample preparation.

Drawings

Fig. 1 shows a structure of a film-attached chip in an embodiment of the present invention. a: the film-sticking chip forms a schematic diagram; b: nuclear pore membrane (aperture 200 nm) scanning electron microscopy; c: siNWs chip cross section scanning electron microscope.

FIG. 2 shows a schematic diagram of a membrane-mediated imprinting mass spectrometry imaging technique in an embodiment of the invention. a: freezing the tissue, and precooling the film-sticking chip; b: the temperature difference causes the slice to melt, the metabolite generates transmembrane movement, macromolecules such as protein and the like are intercepted, and a membrane-mediated imprinting process occurs; c: tearing off the film and the tissue layer attached to the film, and leaving marks on the surface of the chip; d: scanning mass spectrum imaging; e: and outputting a mass spectrometry imaging result.

Figure 3 shows the effect of nuclear pore membranes of different pore sizes on membrane-mediated blotting imaging. a: kidney tissue mass spectrum imaging images obtained using nuclear pore membranes of different pore sizes; b: scanning electron microscope pictures of nuclear pore membranes with different apertures; c: the nuclear pore membranes with different pore diameters were used to obtain a kidney tissue average mass spectrum.

Fig. 4 shows the results of contrast between nuclear Kong Mojie guide blotting and deposition, blotting imaging. a: the kidney tissue section carries out mass spectrum imaging through a membrane-mediated imprinting method, an imprinting method and a deposition method to obtain an average positive ion spectrogram; b: comparing the number of effective peaks (signal to noise ratio > 5) of the spectrograms obtained by the three methods; c: the spectra obtained by the three methods have mean intensity contrast of effective peaks (signal to noise ratio > 5).

Fig. 5 shows a contrast of nuclear Kong Mojie guide blotting with renal tissue mass spectrometry imaging of blotting.

Fig. 6 shows a contrast of kidney tissue mass spectrometry imaging of the nuclear Kong Mojie guide-blotting method with the conventional MALDI method.

Fig. 7 shows a sagittal plane mass spectrometry imaging of mouse brain tissue obtained by nuclear Kong Mojie guide blotting method.

Figure 8 shows a horizontal mass spectrometry imaging of mouse brain tissue obtained by nuclear Kong Mojie guide blotting method.

Fig. 9 shows HE staining images and mass spectrometry imaging images of different subtypes of liver cancer tissue obtained by a nuclear Kong Mojie guide blotting method.

FIG. 10 shows a graph of kidney tissue metabolites obtained by the alumina membrane mediated blotting method under different detection modes.

Detailed Description

Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this disclosure is incorporated herein by reference in its entirety, and the equivalent patents are incorporated herein by reference, especially with respect to the definitions of synthetic techniques, product and process designs, polymers, comonomers, initiators or catalysts, etc. in the art, as disclosed in these documents. If the definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present application, the definition of the term provided in the present application controls.

The numerical ranges in the present application are approximations, so that it may include the numerical values outside the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For example, if a component, physical or other property (e.g., molecular weight, melt index, etc.) is recited as being 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or process, and are not related to whether or not such other component, step or process is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising", "including" or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant or compound. Rather, the term "consisting essentially of … …" excludes any other component, step, or process from the scope of any of the terms recited below, as those out of necessity for performance of the operation. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

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 invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1 preparation of film-attached imaging chip

The silicon nanowire substrate is used as a base material of the film-sticking chip and is prepared by a metal-assisted chemical etching method. Briefly, p-type single crystal silicon (resistivity 5.about.10Ω. Cm) was subjected to oxygen plasma cleaning for 10min, and then reactive etched in an AgNO ₃ solution containing 4.8M HF and 0.02M in a fume hood at room temperature (23 ℃) for 10min. Repeated washing with water after etching, followed by immersing in dilute HNO ₃(HNO₃：H₂ o=1:1) for 1h to dissolve Ag catalyst. And (5) thoroughly cleaning with ultrapure water after etching is finished, and drying at room temperature to obtain the silicon nanowire substrate.

Subsequently, a nuclear pore membrane (purchased from Whatman company, model 10417006) is tightly attached to the silicon nanowire substrate, and no gap is ensured between the nuclear pore membrane and the silicon nanowire substrate as much as possible, so that a film-attached imaging chip is manufactured.

As shown in fig. 1, the film-attached chip is mainly composed of two parts (a): the first nuclear pore membrane with real pore channels and uniform pore distribution is shown in a scanning electron microscope image as a b diagram in fig. 1, the pore diameter is 200nm, and the pore density is 2.9X10 ⁸/cm^2;. In this example, a silicon nanowire substrate was used, the cross-sectional scanning electron microscope of which is shown in fig. 1c, and the nanowire length was 1 μm.

Example 2 Membrane mediated imprinting Mass Spectrometry imaging

Film-mediated blotting mass spectrometry imaging was performed using the film-attached chip prepared in example 1, and the technical flow chart is shown in fig. 2, specifically as follows:

(1) And (3) placing the film-adhered chip into a box body of a frozen microtome for precooling, and simultaneously performing frozen tissue section, wherein the thickness of the frozen tissue section is controlled to be 20-50 mu m.

(2) The tissue slice is completely unfolded to the surface of the film-sticking chip by using a hairbrush, the back of the chip is heated by using the temperature of fingers, the temperature difference causes the slice to melt, metabolites to perform transmembrane motion, macromolecules such as protein and the like are intercepted, the film-mediated imprinting process occurs, the chip is taken out from the box body of the frozen slice machine, and the chip is dried at room temperature.

(3) After the chip is completely dried, the film and the tissue layer attached to the film are torn off. At this point the imaging substrate surface has left a tissue metabolite footprint.

(4) And (5) feeding the imprinted imaging substrate into a mass spectrometer for mass spectrometry imaging scanning.

(5) And outputting a mass spectrometry imaging result.

In order to verify the effect of nuclear pore membrane pore diameters on tissue imaging, the inventors selected three pore diameter nuclear pore membranes for experiments, 100nm, 200nm, 400nm respectively, using mouse kidney tissue imaging for membrane mediated blotting. From the mass spectrometric imaging images of kidney tissue obtained from nuclear pore membranes of different pore sizes (panel a in fig. 3), it can be seen that: the 200nm aperture nuclear pore membrane has the best spatial resolution effect, and can clearly distinguish the structures of the outer cortex area, the inner cortex area, the medulla area and the renal pelvis area of the kidney tissue. However, no clear demarcation of the fine structure of kidney tissue was seen with both 100nm and 400nm pore size nuclear pore membranes. Analysis shows that the method is mainly due to the fact that the 100nm nuclear pore membrane has smaller pore diameter (shown as a diagram B in fig. 3), the permeability is poor, metabolites cannot be completely and effectively transferred to the surface of the silicon nanowire chip, the mass spectrum detection signal sensitivity is low, and the average mass spectrum diagram of the diagram C in fig. 3 can also prove the problem. And for the 400nm aperture nuclear pore membrane, although the aperture is larger (B diagram in figure 3), the permeability is good, the metabolite on the tissue slice can be effectively transferred, and the mass spectrum signal sensitivity is stronger (C diagram in figure 3). But the molecular shift is severe due to the too large pore size, resulting in lower spatial resolution.

In conclusion, the 200nm aperture nuclear pore membrane shows the clearest spatial resolution and stronger detection sensitivity, and is the best choice of the nuclear pore membrane in the membrane-mediated imprinting mass spectrometry imaging technology. Further research by the inventor proves that nuclear pore membranes with the aperture of 150-300 nm can better complete imprinting.

Example 3 comparison of Membrane-mediated blotting and deposition

The inventors performed comparative experiments using mouse kidney tissue imaging membrane-mediated blotting and deposition. Wherein, the steps of the imprinting method and the deposition method are respectively as follows:

(1) Deposition method SALDI mass spectrum imaging flow

Frozen sections of tissue 5 μm thick were directly attached to the pre-chilled SALDI chip surface, thawed at finger temperature and dried. After the chip is completely dried, the SALDI chip with the ultrathin tissue slice attached to the surface is directly sent into a mass spectrometer for mass spectrum imaging scanning.

(2) Imprinting SALDI mass spectrum imaging process

Tissue frozen sections 50 μm thick were directly attached to the pre-chilled SALDI chip surface, thawed at finger temperature and blotted for 1 minute. The tissue sections on the chip surface were then washed with deionized water, leaving only the metabolite blots. After drying, the SALDI chip is sent into a mass spectrometer for mass spectrum imaging scanning.

As shown in fig. 4, it can be seen from the positive ion spectrum obtained from the kidney tissue of the mice (fig. 4, a diagram) that the number and intensity of peaks detected by the membrane-mediated blotting method are far higher than those of the other two methods, and the unique advantages are achieved in terms of detection coverage rate and sensitivity.

Statistical data shows (panel b in fig. 4) that the number of effective peaks (signal to noise ratio greater than 5) obtained by the membrane-mediated blotting method and the average intensity are 4-5 times that of those obtained by the blotting method and the deposition method. Particularly in the deposition method, the obtained effective signal is very small, because the tissue slice remains on the chip surface, the laser is difficult to penetrate the tissue layer to reach the surface of the silicon nanowire, and desorption ionization is very low.

Subsequently, the inventors compared the effects of renal tissue mass spectrometry imaging with effective peak signaling and membrane-mediated blotting (fig. 5). Membrane-mediated imprinting mass spectrometry imaging is found to clearly distinguish the structures of the outer cortex region, the inner endothelio region, the medulla region and the renal pelvis region of renal tissues, while mass spectrometry imaging images obtained by the imprinting method are difficult to distinguish the fine structures of the renal tissues. This is because blotting tends to result in molecular displacement and loss of part of the metabolite during removal of tissue sections by solvent washing. The membrane-mediated imprinting method can effectively separate a tissue slice layer from a substrate chip and can realize high-fidelity in-situ imprinting of metabolite molecules. Thus, the membrane-mediated blotting method exhibits excellent detection sensitivity, coverage, relative to the deposition method and blotting method.

Example 4 comparison of Membrane-mediated imprinting Mass Spectrometry imaging with traditional MALDI Mass Spectrometry imaging

To verify the advantages of the application of the membrane-mediated imprinting mass spectrometry imaging technique in the MALDI imaging platform, the inventors performed a contrast experiment using mouse kidney tissue imaging for membrane-mediated imprinting mass spectrometry imaging with conventional MALDI mass spectrometry imaging.

The steps of conventional MALDI mass spectrometry imaging are as follows:

Frozen sections of tissue 12 μm thick were attached to the ITO glass surface, thawed at finger temperature and dried. DHB was dissolved to 10mg/mL in 50% methanol and sprayed onto tissue sections using IMAGEPREP (bruk's instrument). After drying, the ITO glass is sent into a mass spectrometer for mass spectrum imaging scanning.

As shown in fig. 6, it can be seen that the resolution of the fine structure of kidney tissue obtained by the conventional MALDI mass spectrometry imaging method using the matrix spray method is far lower than that of the membrane-mediated blotting method. The membrane-mediated imprinting method can improve the metabolite imaging resolution in a MALDI imaging platform relative to conventional MALDI mass spectrometry imaging because of unavoidable molecular displacement and crystallization non-uniformity caused by the matrix spraying process. In addition, the matrix spraying time in the traditional MALDI mass spectrum imaging is longer (0.5-1 hour is required), and the overall pretreatment time of the membrane-mediated imprinting method can be controlled within 2 minutes, so that the membrane-mediated imprinting mass spectrum imaging has the advantages of high resolution, ultrasensitive, convenience, rapidness in pretreatment and the like in the MALDI imaging platform.

Example 5 mouse brain tissue sagittal plane Mass Spectrometry imaging based on Membrane mediated blotting

In order to verify the applicability of the membrane-mediated blotting method to mass spectrometry imaging of different tissues, the inventor adopts a mouse brain tissue sagittal plane slice with a relatively complex fine structure to perform experiments.

As a result, as shown in FIG. 7, it was found that the structures of mouse brain tissue thalamus, inferior colliculus, superior colliculus, striatum, hippocampus, cerebral cortex, cerebellum were clearly observed at a resolution of 200. Mu.m, and the specific distribution of characteristic lipids in these regions was seen in both positive and negative ion modes. The membrane-mediated imprinting mass spectrum imaging is fully proved to control the molecular displacement in the imprinting process, and high-resolution and ultrasensitive metabolite mass spectrum imaging is realized.

Example 6 Membrane-mediated blotting-based mouse brain tissue level Mass Spectrometry imaging

Similarly, the inventors performed validation experiments using horizontal slices of mouse brain tissue. As a result, as shown in FIG. 8, the structures of the cerebral cortex, striatum, hippocampus, superior colliculus and cerebellum of the brain tissue of the mouse were clearly observed at a resolution of 200. Mu.m, and the left-right structure was symmetrical. The result further fully proves that the membrane-mediated imprinting mass spectrometry imaging can control the molecular displacement in the imprinting process, and high-resolution and ultrasensitive metabolite mass spectrometry imaging is realized.

Example 7 liver cancer tissue subtype discrimination based on Membrane-mediated blotting

The inventor further performs space metabonomics analysis on liver cell carcinoma (HCC), cholangiocellular carcinoma (ICC) and intestinal cancer liver Metastasis Liver Cancer (MLC) through a membrane-mediated imprinting mass spectrometry imaging technology, and discovers that the lipid fingerprint spectrograms of the liver cancer liver Metastasis Liver Cancer (MLC) have significant differences (shown in figure 9). Obvious difference distribution of the biomarkers can be observed on the two-dimensional mass spectrum image, the capability of the method in discriminating the subtype of liver cancer tissues is shown, and meanwhile, the membrane-mediated imprinting mass spectrum technology is proved to be hopefully applied to the pathological diagnosis of cancer tissues based on metabolites.

Example 8 alumina Membrane mediated imprinting Mass Spectrometry

To verify the advantages of nuclear pore membranes in mediating blots, the inventors tried to use an aluminum oxide membrane (purchased from whatman company, cat No. 6809-6022) for mediating blots. The procedure of mediating blotting was identical to that described above, except that the nuclear pore membrane was replaced with an alumina membrane, and kidney tissue metabolites were mediating blotted and mass spectrometric detection was performed.

As a result, as shown in FIG. 10, the alumina film was good in wettability, but was stronger in adsorption property, and the metabolite was hard to permeate onto the chip substrate. Therefore, under the positive ion and negative ion modes, only small-molecule metabolites before 600Da have a small amount of signals, lipid metabolites after 600Da are almost detected without signals, the detection sensitivity and the coverage rate are far lower than those of nuclear Kong Mojie guide marks, and the subsequent imaging effect is more unsatisfactory.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. The application of the porous film in preparing a chip for biological tissue imprinting, wherein the chip comprises a substrate, and the chip is characterized in that the porous film is provided with micropores penetrating through the upper surface and the lower surface, the aperture of the micropores is 150-300 nm, and the porous film is attached to the substrate when the chip is prepared.

2. The use according to claim 1, wherein the porous membrane is a nuclear pore membrane.

3. The use according to claim 1 or 2, wherein the microwell has a cell density of 2 x 10 ⁸～4×10⁸ cells/cm ².

4. The use according to claim 1 or 2, wherein the substrate is a substrate with a nanowire structure and the porous film is adapted to be attached to the substrate on the side with the nanowire structure.

5. The chip for biological tissue imprinting comprises a substrate, and is characterized in that a porous film is attached to the substrate, wherein the porous film is provided with micropores penetrating through the upper surface and the lower surface, and the aperture of the micropores is 150-300 nm.

6. The chip of claim 5, wherein the substrate comprises a footprint area and a molecular weight calibration area.

7. The method of manufacturing a chip according to claim 5, comprising the step of attaching the porous film to the surface of the substrate.

8. A method of performing biological tissue blotting based on the chip of claim 5, comprising the steps of:

s1, precooling the chip of claim 5;

s2, fully expanding the biological tissue slice onto the surface of the porous film of the chip, heating the back of the chip to melt the slice, and drying the chip at room temperature;

And S3, taking down the tissue slice and the porous film on the chip to obtain the substrate containing the tissue print.

9. The method of claim 8, wherein the tissue slice has a thickness of 20-50 μm.

10. Use of the chip of claim 5 in surface-assisted laser desorption ionization mass spectrometry imaging.

11. Use of the chip of claim 5 for the preparation of a kit for detecting tissue lipids and/or metabolites.

12. A kit for detecting lipids and/or metabolites in a tissue, comprising the chip of claim 5.