Preparation method and application of copper-based conductive MOF mass spectrum chip

Abstract

The invention discloses a preparation method and application of a copper-based conductive MOF mass spectrum chip, and relates to the technical field of tissue space multi-omics detection.The preparation method comprises the steps that a mixed ethanol solution of copper acetate and ferrous acetate, a 2, 3, 6, 7, 10, 11-hexahydroxy triphenyl solution and an 80% ethanol solution are prepared; dipping and pulling the ITO slide in a mixed ethanol solution of copper acetate and ferrous acetate, a 2, 3, 6, 7, 10, 11-hexahydroxy triphenyl solution and a 80% ethanol solution in sequence; repeating the dipping and pulling operations, and circularly coating the surface of the ITO slide with a film; and washing with 80% ethanol solution, and blow-drying with nitrogen at room temperature for 10 minutes to obtain the copper-based conductive MOF mass spectrum chip. By designing the copper-based conductive MOF mass spectrum chip, the copper-based conductive MOF mass spectrum chip has the advantages of high electron transmission efficiency and photoelectric conversion capability and fusion of the components, and shows more excellent mass spectrum detection performance.

Description

Technical Field

[0001] This invention relates to the field of tissue spatial multi-omics detection technology, and in particular to a method for preparing a copper-based conductive MOF mass spectrometry chip and its application. Background Technology

[0002] Colorectal cancer is a common malignant tumor of the digestive tract, with over 1.9 million new cases worldwide each year. The five-year survival rate is less than 65%, and advanced-stage patients often fail treatment due to tumor metastasis and drug resistance. Studies have shown that the synergistic effect of metabolic microenvironment dysregulation and immune escape mechanisms within the tumor microenvironment is a core factor driving the malignant progression of colorectal cancer: on the one hand, cancer cells produce large amounts of metabolites such as lactic acid and ketone bodies (at concentrations 3-5 times higher than normal tissues) through abnormal metabolism, inducing suppression of immune cell function; on the other hand, for every 10% decrease in the spatial distribution density of key immune cells (such as CD8+ T cells), the risk of postoperative recurrence increases by more than 37%. Therefore, simultaneously acquiring spatial co-localization information from metabolomics and immunomics is of great value in elucidating tumor evolution mechanisms and developing precision treatment plans.

[0003] Current clinical testing systems face significant bottlenecks: 1) Traditional mass spectrometry imaging requires the spraying of organic matrices such as α-cyanocinic acid, whose crystallization process can damage the integrity of tissue surface antigens (loss rate > 60%), leading to the failure of immunofluorescence detection; while the fixation and blocking process necessary for immunoassay introduces salt contamination, increasing mass spectrometry background noise by 4-8 times. 2) Existing technologies require separate metabolic and immunological analyses of adjacent slices, and due to tissue heterogeneity, spatial positioning deviations of the same pathological region exceed 200 μm, making it impossible to achieve single-cell-scale metabolic-immune correlation analysis. Existing solutions also have shortcomings in biomarker detection: proteomics-based (such as PD-L1, Ki67) detection systems have limited accuracy (AUC 0.65-0.75) and cannot simultaneously reflect metabolic status; conventional metabolomics detection relies on liquid chromatography-mass spectrometry, which suffers from complex pretreatment (time > 3 hours) and low throughput (< 10 samples / batch). While laser desorption / ionization mass spectrometry (LDI-MS) enables rapid detection, traditional metal nanoparticle matrices (such as Au and Pt) face limitations such as severe interference from biomolecules and low ionization efficiency, resulting in insufficient sensitivity for metabolite detection in clinical samples (LOQ > 1). mol).

[0004] Therefore, those skilled in the art are dedicated to developing a method for preparing a copper-based conductive MOF mass spectrometry chip and its application for LDI-MS metabolic imaging and multiplex immunofluorescence imaging of tumor tissues from colorectal cancer patients, while simultaneously acquiring the spatial distribution of metabolites and protein markers in biological tissues. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to achieve LDI-MS metabolic imaging and multiplex immunofluorescence imaging of the same tissue slice.

[0006] To achieve the above objectives, the present invention provides a method for preparing a copper-based conductive MOF mass spectrometry chip, the method comprising the following steps: Step 1: Prepare a mixed ethanol solution of copper acetate and ferrous acetate, a 2,3,6,7,10,11-hexahydroxytribenzene solution, and an 80% ethanol solution; Step 2: ITO glass slides are immersed and lifted sequentially in a mixed ethanol solution of copper acetate and ferrous acetate, a 2,3,6,7,10,11-hexahydroxytribenzene solution, and an 80% ethanol solution. Step 3: Repeat the immersion and lifting operation of Step 2 to continuously coat the ITO glass slide surface. Step 4: Wash with 80% ethanol solution and dry with nitrogen gas at room temperature for 10 minutes to obtain a copper-based conductive MOF mass spectrometer chip.

[0007] Furthermore, the copper / iron ratio in the mixed ethanol solution of copper acetate and ferrous acetate was 2:1, and the concentration was 0.3 mg / mL; the concentration of the 2,3,6,7,10,11-hexahydroxytribenzene solution was 0.05 mg / mL.

[0008] Furthermore, step 2 also includes: an impregnation speed of 300 mm / min.

[0009] Furthermore, step 2 also includes: a lifting speed of 500 mm / min.

[0010] Furthermore, step 2 also includes: an immersion time of 20 seconds.

[0011] Furthermore, step 3 also includes: cyclically coating the ITO glass slide surface 50 times.

[0012] An application of a copper-based conductive MOF mass spectrometry chip for the simultaneous extraction and co-localization of tissue spatial metabolic information and multiplex immunofluorescence information includes: Biological tissue samples are frozen and then sectioned directly to obtain tissue sections. Tissue slices are covered onto the substrate of a copper-based conductive MOF mass spectrometry chip prepared by any one of claims 1 to 6; Spatial metabolic imaging data of tissue sections were acquired using laser desorption / ionization mass spectrometry. Multiplex immunofluorescence imaging data were collected by treating the same tissue sections with fluorescently modified antibodies. The collected spatial metabolic imaging data and multiplex immunofluorescence imaging data are overlaid to construct a spatial data model and achieve spatial colocalization analysis.

[0013] Furthermore, the slice thickness is 10 micrometers.

[0014] Furthermore, the spatial metabolic imaging data includes: m / z signals covering the range of 80-1000 Da and a spatial resolution range of 50 μm × 50 μm.

[0015] Furthermore, the fluorescently modified antibody is either CK20 antibody or EpCAM antibody.

[0016] Compared with the prior art, the present invention has at least the following beneficial technical effects: 1. This invention utilizes an improved dip-coating method to grow conductive MOFs layer by layer at room temperature. The resulting copper-based conductive MOF mass spectrometry chip possesses high electron transport efficiency and photoelectric conversion capability, integrates the advantages of each component, and exhibits superior mass spectrometry detection performance. The chip surface is flat and uniform, with small differences in detection performance in different regions, which is beneficial for imaging and detection of large-area tissues. 2. This invention utilizes a copper-based conductive MOF mass spectrometry chip for LDI-MS metabolic imaging and multiplex immunofluorescence imaging, which can continuously perform LDI-MS metabolic imaging and multiplex immunofluorescence imaging on the same tissue sample. It has the advantages of simple operation and high throughput, meets the clinical needs of imaging the same tissue sample, and can significantly improve detection efficiency.

[0017] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of a copper-based conductive MOF chip structure according to a preferred embodiment of the present invention; Figure 2 This is a comparison diagram of mass spectrometry signals according to a preferred embodiment of the present invention; Figure 3 This is a schematic diagram of spatial metabolic imaging data according to a preferred embodiment of the present invention; Figure 4 This is a schematic diagram of multiplex immunofluorescence imaging data according to a preferred embodiment of the present invention. Detailed Implementation

[0019] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0020] In the accompanying drawings, components with the same structure are indicated by the same numerical designation, and components with similar structures or functions are indicated by similar numerical designations. The dimensions and thicknesses of each component shown in the drawings are arbitrary, and the present invention does not limit the dimensions and thicknesses of each component. To make the illustrations clearer, the thickness of some components has been appropriately exaggerated in the drawings.

[0021] This embodiment provides a method for fabricating a copper-based conductive MOF mass spectrometry chip, which uses an improved Czochralski deposition method to synthesize copper-based conductive MOF thin films layer by layer, including the following steps: Step 1: Prepare a mixed ethanol solution of copper acetate and ferrous acetate, a 2,3,6,7,10,11-hexahydroxytribenzene solution, and an 80% ethanol solution; wherein, the copper / iron ratio in the mixed ethanol solution of copper acetate and ferrous acetate is 2:1 and the concentration is 0.3 mg / mL; the concentration of the 2,3,6,7,10,11-hexahydroxytribenzene solution is 0.05 mg / mL.

[0022] Step 2: Immerse and lift the ITO glass slide in the three solutions mentioned above in sequence. The immersion speed is 300 mm / min, the lifting speed is 500 mm / min, and the immersion time is 20 s.

[0023] Step 3: Repeat the immersion and lifting operation of Step 2, and circulate the coating on the ITO glass slide 50 times. Step 4: Wash with 80% ethanol solution, and dry with nitrogen gas at room temperature for 10 minutes to obtain a copper-based conductive MOF mass spectrometry chip, as shown below. Figure 1 As shown.

[0024] The resulting conductive MOF mass spectrometry chip possesses a large, flat surface area, exhibits high reproducibility of detection signals in each region, and demonstrates excellent electron transfer efficiency and photoelectric properties, making it suitable as an LDI-MS matrix for small molecule metabolic imaging. Furthermore, since tissue sections are directly applied to the chip substrate, immunofluorescence imaging can be performed without the need for washing after metabolic imaging.

[0025] like Figure 2 The image shows a comparison of LDI-MS mass spectrometry signals acquired from the copper-based conductive MOF mass spectrometry chip (a), iron oxide matrix (b), and commercial organic matrix (c) prepared in this embodiment for the same colorectal cancer tissue sample.

[0026] This embodiment also provides an application of a copper-based conductive MOF mass spectrometry chip for the simultaneous extraction and co-localization of tissue spatial metabolic information and multiplex immunofluorescence information, to study the molecular mechanisms of colorectal cancer, including: Step 1: After freezing, the biological tissue sample is directly sectioned to obtain tissue sections; the biological tissue sample is a fresh tumor tissue sample of colorectal cancer, and the section thickness is 10 micrometers. Step 2: Cover the substrate of the copper-based conductive MOF mass spectrometry chip with tissue sections; Step 3: Acquire spatial metabolic imaging data of tissue sections using LDI-MS, specifically including m / z signals covering the range of 80-1000 Da, with a spatial resolution of 50 μm × 50 μm. The acquired spatial metabolic images are as follows: Figure 3 As shown; Step 4: The tumor tissue sections that have been imaged by LDI-MS and attached to the surface of the copper-based conductive MOF chip are treated with fluorescent group-modified antibodies. The antibody combination used is fluorescent group-modified CK20 antibody and EpCAM antibody. Step 5: Acquire multiplex immunofluorescence imaging data, such as... Figure 4 As shown; Step 6: Overlay the collected spatial metabolic imaging data and multiplex immunofluorescence imaging data to construct a spatial data model and achieve spatial co-localization analysis.

[0027] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for fabricating a copper-based conductive MOF mass spectrometry chip, characterized in that, The method includes the following steps: Step 1: Prepare a mixed ethanol solution of copper acetate and ferrous acetate, a 2,3,6,7,10,11-hexahydroxytribenzene solution, and an 80% ethanol solution; Step 2: ITO glass slides are immersed and lifted sequentially in a mixed ethanol solution of copper acetate and ferrous acetate, a 2,3,6,7,10,11-hexahydroxytribenzene solution, and an 80% ethanol solution. Step 3: Repeat the immersion and lifting operation of Step 2 to continuously coat the ITO glass slide surface. Step 4: Wash with 80% ethanol solution and dry with nitrogen gas at room temperature for 10 minutes to obtain a copper-based conductive MOF mass spectrometer chip.

2. The method for preparing a copper-based conductive MOF mass spectrometry chip as described in claim 1, characterized in that, In step 1, the copper / iron ratio in the mixed ethanol solution of copper acetate and ferrous acetate is 2:1, and the concentration is 0.3 mg / mL; the concentration of the 2,3,6,7,10,11-hexahydroxytribenzene solution is 0.05 mg / mL.

3. The method for preparing a copper-based conductive MOF mass spectrometry chip as described in claim 1, characterized in that, Step 2 further includes: an impregnation speed of 300 mm / min.

4. The method for preparing a copper-based conductive MOF mass spectrometry chip as described in claim 1, characterized in that, Step 2 also includes: a lifting speed of 500 mm / min.

5. The method for preparing a copper-based conductive MOF mass spectrometry chip as described in claim 1, characterized in that, Step 2 also includes: soaking time of 20 seconds.

6. The method for preparing a copper-based conductive MOF mass spectrometry chip as described in claim 1, characterized in that, Step 3 also includes: cyclically coating the ITO glass slide surface 50 times.

7. An application of a copper-based conductive MOF mass spectrometry chip, characterized in that, Simultaneous extraction and co-localization of tissue spatial metabolic information and multiplex immunofluorescence information, including: Biological tissue samples are frozen and then sectioned directly to obtain tissue sections. Tissue slices are covered onto the substrate of a copper-based conductive MOF mass spectrometry chip prepared by any one of claims 1 to 6; Spatial metabolic imaging data of tissue sections were acquired using laser desorption / ionization mass spectrometry. Multiplex immunofluorescence imaging data were collected by treating the same tissue sections with fluorescently modified antibodies. The collected spatial metabolic imaging data and multiplex immunofluorescence imaging data are overlaid to construct a spatial data model and achieve spatial colocalization analysis.

8. The application of the copper-based conductive MOF mass spectrometry chip as described in claim 7, characterized in that, The slice is 10 micrometers thick.

9. The application of the copper-based conductive MOF mass spectrometry chip as described in claim 7, characterized in that, The spatial metabolic imaging data includes: m / z signals covering the range of 80-1000 Da and a spatial resolution range of 50 μm × 50 μm.

10. The application of the copper-based conductive MOF mass spectrometry chip as described in claim 7, characterized in that, The fluorescently modified antibodies are CK20 antibody and EpCAM antibody.

Images