Biomolecule space signal distribution condition analysis method, device, equipment and medium

Abstract

The invention discloses a biological molecule space signal distribution condition analysis method, device, equipment and medium, and relates to the technical field of pathological diagnos.The method comprises the steps that a to-be-analyzed tissue slice adheres to the adhesion end of a tissue sample, and tissue lysis and dye incubation are conducted; placing a target three-dimensional high-throughput amplification micropore array meeting a preset high-throughput condition in uncured concentrated gel until the concentrated gel is cured, biological molecules in the cracked and incubated tissue slices are gathered in the concentrated gel, inputting the biological molecules into the cured separation gel from the input end of the amplification array, and continuing three-dimensional electrophoresis to realize separation of the biological molecules, and after the three-dimensional electrophoresis is finished, slicing the solidified separation gel to obtain a multi-layer gel slice, and analyzing the multi-layer gel slice layer by layer to obtain the spatial signal distribution condition of the biomolecules, thereby solving the problem that the segmentation precision of the tissue slice cannot meet the precise pathological diagnosis of the micro tissue and the micro structure in the tissue. And the analysis resolution and accuracy of the spatial signal distribution condition of the biomolecules are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of pathological diagnosis, and in particular to a method, device, equipment and medium for analyzing the spatial signal distribution of biological molecules. Background Art

[0002] The three-dimensional gel electrophoresis device is a new detection method in the field of pathological diagnosis. Using the three-dimensional gel electrophoresis method, proteins or nucleic acids are separated in the gel according to their molecular weight, and biological samples with a molecular weight range of 10 to 180 kilodaltons can be fully separated within a separation distance of 1 cm. Since the electric field exists only in the z-axis direction, the spatial distribution of the biomolecule signal in the horizontal and vertical directions can be maintained. Multi-layer slice analysis of the gel after three-dimensional gel electrophoresis can obtain the spatial signal distribution of biomolecules in each molecular weight layer. The micropore array loading device currently equipped with the three-dimensional gel electrophoresis device has a variable array flux, containing 10 to 100 micropores per square centimeter. The higher the throughput of the micropore array loading device, the more accurate the regional segmentation of the slice sample, and the higher the accuracy of the spatial distribution information of the biomolecules in the xy plane of the tissue slice presented in the end. Replacing the visual diagnosis of the morphology and structure of the tissue with the spatial distribution information of biomolecules can be effectively applied to the diagnosis of benign and malignant surgically resected tissues and the diagnosis of malignant resection margins of frozen sections of surgically resected tissues. However, the current three-dimensional gel electrophoresis device has the following disadvantages: the flux of the micropore array equipped with existing three-dimensional gel electrophoresis is low, and the spatial segmentation accuracy of tissue slices is limited, resulting in low signal resolution of tissues for pathological diagnosis, which makes it difficult to meet the needs of accurate pathological diagnosis of tiny tissues and structures within tiny tissues; the aperture of the equipped micropore array is large, and tissue slices are prone to rupture when local tension is excessive, resulting in distortion of signals inside the micropores, and signal diffusion within a single micropore leads to signal overlap and spatial signal distortion within the micropores. It is difficult to correspond the imaging signal distribution in a single signal point with the actual tissue distribution, which makes it difficult to meet the needs of more accurate pathological diagnosis of tiny tissues and structures within tiny tissues; the micropore spacing of the molecular signal input separation gel in the micropore array is small, resulting in overlapping signals between micropores under the influence of signal diffusion, and the spatial distribution of signals is destroyed, which makes it difficult to meet the needs of accurate pathological diagnosis of tiny tissues and structures within tiny tissues.

[0003] As can be seen from the above, how to improve the analysis ability of the three-dimensional gel electrophoresis system for tissue sections, solve the problem that the existing segmentation accuracy of tissue sections cannot meet the accurate pathological diagnosis of tiny tissues and tiny structures within tissues, and improve the resolution and accuracy of the analysis of the spatial signal distribution of biological molecules are problems to be solved in this field. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method, device, equipment and medium for analyzing the spatial signal distribution of biomolecules, which can improve the analysis ability of the three-dimensional gel electrophoresis system for tissue sections, solve the problem that the existing segmentation accuracy of tissue sections cannot meet the accurate pathological diagnosis of micro-tissues and micro-structures in tissues, and improve the resolution and accuracy of the analysis of the spatial signal distribution of biomolecules. The specific scheme is as follows:

[0005] In a first aspect, the present application discloses a method for analyzing the spatial signal distribution of biomolecules, which is applied to a target three-dimensional high-throughput amplified microwell array sample loading device based on a three-dimensional gel electrophoresis device, comprising:

[0006] Obtaining a tissue section to be analyzed, adhering the tissue section to be analyzed to a local tissue sample adhering end, and performing tissue lysis and dye incubation to obtain a lysed and incubated tissue section;

[0007] A target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions is placed in a local uncured concentrated gel until the concentrated gel is completely cured, and three-dimensional electrophoresis is performed on the lysed and incubated tissue slices on the basis of the target three-dimensional high-throughput amplification microwell array, and the biomolecules in the lysed and incubated tissue slices are aggregated in the concentrated gel and input from the input end of the amplification array to the local cured separation gel, and three-dimensional electrophoresis is continued to achieve separation of biomolecules; the number of microwells contained in each square centimeter of the target three-dimensional high-throughput amplification microwell array is 100 to 400, and the area magnification of each microwell array unit is 1 to 2.25 times; the biomolecules are proteins or nucleic acids;

[0008] After the three-dimensional electrophoresis is completed, the solidified separation gel is sliced ​​to obtain multi-layer gel slices, and the multi-layer gel slices are analyzed layer by layer to obtain the spatial signal distribution of each molecular weight biomolecule in the tissue slice.

[0009] Optionally, the target three-dimensional high-throughput amplification microwell array loading device includes a tissue sample adhesion end, a target three-dimensional high-throughput amplification microwell array that meets preset high-throughput conditions, and an amplification signal input end; the three-dimensional gel electrophoresis device includes a target three-dimensional high-throughput amplification microwell array loading device.

[0010] Optionally, obtaining a tissue section to be analyzed, adhering the tissue section to be analyzed to a local tissue sample adhering end, and performing tissue lysis and dye incubation includes:

[0011] Acquiring an initial tissue to be analyzed, and freezing and slicing the initial tissue to be analyzed to obtain tissue slices to be analyzed;

[0012] The tissue slice to be analyzed is adhered to the local tissue sample adhesion end, and tissue lysis and dye incubation are performed at room temperature.

[0013] Optionally, before placing the target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions in the local unsolidified concentrated gel, the method further includes:

[0014] The separation gel solidification operation is carried out in a local electrophoresis pool;

[0015] Add unsolidified stacking gel on top of the solidified separation gel.

[0016] Optionally, placing the target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions in a local unsolidified concentrated gel comprises:

[0017] The target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions is placed in the local unsolidified concentrated gel, and the three-dimensional high-throughput amplification microwell array in the target three-dimensional high-throughput amplification microwell array is monitored to see whether the current concentrated gel is full of the three-dimensional high-throughput amplification microwell array.

[0018] Optionally, performing three-dimensional electrophoresis on the lysed and incubated tissue sections comprises:

[0019] Electrophoresis fluid is added between the local cathode plate and the solidified concentrated gel and between the anode plate and the bottom of the electrophoresis pool, and a constant voltage power supply is connected to perform three-dimensional electrophoresis on the lysed and incubated tissue sections.

[0020] Optionally, the solidified separation gel input from the input end of the amplification array is input to the local solidified separation gel, and three-dimensional electrophoresis is continued to achieve separation of biomolecules; after the three-dimensional electrophoresis is completed, the solidified separation gel is sliced ​​to obtain multi-layer gel slices, including:

[0021] The biomolecules aggregated in the concentrated gel are input into the local solidified separation gel by using the local amplification signal input end, and the solidified separation gel is subjected to three-dimensional electrophoresis;

[0022] After the three-dimensional electrophoresis is completed, the solidified separation gel is frozen, and the frozen separation gel is sliced ​​and separated along a direction perpendicular to the vertical axis according to the molecular weight to obtain multi-layer gel slices.

[0023] In a second aspect, the present application discloses a biomolecule spatial signal distribution analysis device, which is applied to a target three-dimensional high-throughput amplified microwell array sample loading device based on a three-dimensional gel electrophoresis device, comprising:

[0024] A slice lysis and incubation module, used to obtain a tissue slice to be analyzed, adhere the tissue slice to be analyzed to a local tissue sample adhesion end, and perform tissue lysis and dye incubation to obtain a lysed and incubated tissue slice;

[0025] A three-dimensional electrophoresis module is used to place a target three-dimensional high-throughput amplified microwell array that meets the preset high-throughput conditions in a local uncured concentrated gel until the concentrated gel is completely cured, and on the basis of the target three-dimensional high-throughput amplified microwell array, three-dimensional electrophoresis is performed on the lysed and incubated tissue slices, and the biomolecules in the lysed and incubated tissue slices are aggregated in the concentrated gel and input from the input end of the amplified array to the local cured separation gel, and three-dimensional electrophoresis is continued to achieve separation of biomolecules; the number of microwells contained in each square centimeter of the target three-dimensional high-throughput amplified microwell array is 100 to 400, and the area magnification of each microwell array unit is 1 to 2.25 times; the biomolecules are proteins or nucleic acids;

[0026] The slicing and analysis module is used to slice the solidified separation gel after the three-dimensional electrophoresis is completed to obtain multi-layer gel slices, and analyze the multi-layer gel slices layer by layer to obtain the spatial signal distribution of each molecular weight biological molecule in the tissue slice.

[0027] In a third aspect, the present application discloses an electronic device, comprising:

[0028] Memory, used to store computer programs;

[0029] The processor is used to execute the computer program to implement the aforementioned method for analyzing the spatial signal distribution of biomolecules.

[0030] In a fourth aspect, the present application discloses a computer storage medium for storing a computer program; wherein, when the computer program is executed by a processor, the steps of the aforementioned method for analyzing the spatial signal distribution of biological molecules disclosed above are implemented.

[0031] It can be seen that the present application provides a method for analyzing the spatial signal distribution of biological molecules, including obtaining a tissue slice to be analyzed, adhering the tissue slice to be analyzed to a local tissue sample adhesion end, and performing tissue lysis and dye incubation to obtain a tissue slice after lysis and incubation; placing a target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions in a local uncured concentrated gel until the concentrated gel is completely cured, and performing three-dimensional electrophoresis on the tissue slice after lysis and incubation on the basis of the target three-dimensional high-throughput amplification microwell array, and the biological molecules in the tissue slice after lysis and incubation are The aggregation is completed in the concentrated gel and input into the local solidified separation gel from the input end of the amplification array, and the three-dimensional electrophoresis is continued to realize the separation of biological molecules; the number of micropores contained in each square centimeter in the target three-dimensional high-throughput amplification micropore array is 100 to 400, and the area magnification of each micropore array unit is 1 to 2.25 times; the biological molecules are proteins or nucleic acids; after the three-dimensional electrophoresis is completed, the solidified separation gel is sliced ​​to obtain multi-layer gel slices, and the multi-layer gel slices are analyzed layer by layer to obtain the spatial signal distribution of each molecular weight biological molecule in the tissue slice. The present application is applied to a target three-dimensional high-throughput amplified microwell array loading device based on a three-dimensional gel electrophoresis device, wherein a tissue slice to be analyzed is adhered to a local tissue sample adhesion end, and tissue lysis and dye incubation are performed to obtain a tissue slice after lysis and incubation, and the target three-dimensional high-throughput amplified microwell array is placed in a local unsolidified concentrated gel until solidified, and three-dimensional electrophoresis is performed on the solidified concentrated gel based on the tissue slice after lysis and incubation and the target three-dimensional high-throughput amplified microwell array, and the obtained biomolecules separated by electrophoresis are input into the local solidified separation gel for slicing, and high-precision in-situ segmentation can be achieved through slicing, providing high-precision signal information for tissue analysis, solving the problem that the existing segmentation accuracy of tissue slices cannot meet the accurate pathological diagnosis of microtissues and microstructures in tissues, and can improve the analysis and diagnosis accuracy of microtissues and microstructures in tissues, expand the practicality of the three-dimensional gel electrophoresis device in the field of pathological diagnosis, improve the resolution of the microwell array, and improve the resolution and accuracy of the analysis of the spatial signal distribution of biomolecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 This is a flow chart of a method for analyzing the spatial signal distribution of biomolecules disclosed in this application;

[0034] Figure 2 A structural diagram of a three-dimensional electrophoresis device and a target three-dimensional high-throughput amplification microwell array loading device disclosed in the present application;

[0035] Figure 3 A specific flow chart for analyzing the spatial distribution of a biomolecule signal disclosed in this application;

[0036] Figure 4 A schematic diagram of partition analysis of a tissue slice disclosed in the present application under different microwell array fluxes;

[0037] Figure 5 A schematic diagram of lateral diffusion of tissue components in a tissue slice disclosed in the present application without magnification and with magnification of a microwell array;

[0038] Figure 6 This is a diagram showing different microwell array fluxes in a 4T1 multi-sample test disclosed in the present application and the effect of determining the contour of a small-sized tumor under magnification and without magnification;

[0039] Figure 7 This is a rendering of identifying tumor heterogeneity in breast cancer adenocarcinoma tissue disclosed in the present application;

[0040] Figure 8 This is a rendering of the effect of identifying the distribution of cancer cells in tiny tissue puncture lymph nodes disclosed in the present application;

[0041] Fig. 9 This is a schematic diagram of the structure of a biomolecule spatial signal distribution analysis device disclosed in this application;

[0042] Fig.10 A structural diagram of an electronic device provided for this application. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The three-dimensional gel electrophoresis device is a new detection method in the field of pathological diagnosis. Using the three-dimensional gel electrophoresis method, proteins or nucleic acids are separated in the gel according to their molecular weight, and biological samples with a molecular weight range of 10 to 180 kilodaltons can be fully separated within a separation distance of 1 cm. Since the electric field exists only in the z-axis direction, the spatial distribution of the biomolecule signal in the horizontal and vertical directions can be maintained. Multi-layer slice analysis of the gel after three-dimensional gel electrophoresis can obtain the spatial signal distribution of biomolecules in each molecular weight layer. The micropore array loading device currently equipped with the three-dimensional gel electrophoresis device has a variable array flux, containing 10 to 100 micropores per square centimeter. The higher the throughput of the micropore array loading device, the more accurate the regional segmentation of the slice sample, and the higher the accuracy of the spatial distribution information of the biomolecules in the xy plane of the tissue slice presented in the end. Replacing the visual diagnosis of the morphology and structure of the tissue with the spatial distribution information of biomolecules can be effectively applied to the diagnosis of benign and malignant surgically resected tissues and the diagnosis of malignant resection margins of frozen sections of surgically resected tissues. However, the current three-dimensional gel electrophoresis device has the following disadvantages: the flux of the micropore array equipped with existing three-dimensional gel electrophoresis is low, and the spatial segmentation accuracy of tissue slices is limited, resulting in low signal resolution of tissues for pathological diagnosis, which makes it difficult to meet the needs of accurate pathological diagnosis of tiny tissues and structures within tiny tissues; the aperture of the equipped micropore array is large, and tissue slices are prone to rupture when local tension is excessive, resulting in distortion of signals inside the micropores, and signal diffusion within a single micropore leads to signal overlap and spatial signal distortion within the micropores. It is difficult to correspond the imaging signal distribution in a single signal point with the actual tissue distribution, which makes it difficult to meet the needs of more accurate pathological diagnosis of tiny tissues and structures within tiny tissues; the micropore spacing of the molecular signal input separation gel in the micropore array is small, resulting in overlapping signals between micropores under the influence of signal diffusion, and the spatial distribution of signals is destroyed, which makes it difficult to meet the needs of accurate pathological diagnosis of tiny tissues and structures within tiny tissues. As can be seen from the above, how to improve the analysis ability of the three-dimensional gel electrophoresis system for tissue sections, solve the problem that the existing segmentation accuracy of tissue sections cannot meet the accurate pathological diagnosis of tiny tissues and tiny structures within tissues, and improve the resolution and accuracy of the analysis of the spatial signal distribution of biological molecules are problems to be solved in this field.

[0045] See also Figure 1 As shown, the embodiment of the present invention discloses a method for analyzing the spatial signal distribution of biomolecules, which is applied to a target three-dimensional high-throughput amplified microwell array sample loading device based on a three-dimensional gel electrophoresis device, and specifically may include:

[0046] Step S11: obtaining a tissue section to be analyzed, adhering the tissue section to be analyzed to a local tissue sample adhering end, and performing tissue lysis and dye incubation to obtain a tissue section after lysis and incubation.

[0047] In this embodiment, an initial tissue to be analyzed is obtained, and the initial tissue to be analyzed is frozen and sliced ​​to obtain tissue slices to be analyzed. The tissue slices to be analyzed are adhered to the local tissue sample adhesion end, and tissue lysis and dye incubation are performed at room temperature to obtain tissue slices after lysis and incubation.

[0048] The present application is applied to a target three-dimensional high-throughput amplified microporous array sample loading device based on a three-dimensional gel electrophoresis device. The three-dimensional gel electrophoresis process and the structure of the target three-dimensional high-throughput amplified microporous array sample loading device are as follows: Figure 2 As shown, it includes a tissue sample adhesion end, a target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions, and an amplification signal input end; the three-dimensional gel electrophoresis device includes a target three-dimensional high-throughput amplification microwell array loading device. The tissue sample adhesion end is a microwell array plane for solution dripping or tissue sample attachment and lysis. The number of microwells contained in each square centimeter of the microwell array plane is 100 to 400. The height of each microwell in the target three-dimensional high-throughput amplification microwell array is 0.7 cm, and the diameter of the xy-axis microwell of each microwell column in any section of the z-axis remains unchanged, the area is the same, and only the microwell spacing changes; the area magnification of each microwell array unit is 1 to 2.25 times. The amplification signal input end is a microwell array plane that is perpendicular to the electric field and has completed amplification.

[0049] Step S12: placing a target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions in a local uncured concentrated gel until the concentrated gel is completely cured, and performing three-dimensional electrophoresis on the lysed and incubated tissue slices on the basis of the target three-dimensional high-throughput amplification microwell array, the biological molecules in the lysed and incubated tissue slices are aggregated in the concentrated gel, and are input from the input end of the amplification array to the local cured separation gel, and the three-dimensional electrophoresis is continued to achieve the separation of the biological molecules; the number of microwells contained in each square centimeter of the target three-dimensional high-throughput amplification microwell array is 100 to 400, and the area magnification of each microwell array unit is 1 to 2.25 times; the biological molecules are proteins or nucleic acids.

[0050] In this embodiment, a separation gel solidification operation is performed in a local electrophoresis pool, unsolidified concentrated gel is added to the upper end of the solidified separation gel, a target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions is placed in the local unsolidified concentrated gel, and it is monitored whether the current concentrated gel is full of the three-dimensional high-throughput amplification microwell array in the target three-dimensional high-throughput amplification microwell array until the concentrated gel is completely solidified. On the basis of the target three-dimensional high-throughput amplification microwell array, electrophoresis fluid is added between the local cathode plate and the solidified concentrated gel and between the anode plate and the bottom of the electrophoresis pool, and a constant voltage power supply is connected to perform three-dimensional electrophoresis on the lysed and incubated tissue slices, which can ensure the xy plane spatial distribution of the tissue slices while realizing the in situ separation of biological molecules in the tissue slices, the biological molecules in the lysed and incubated tissue slices are aggregated in the concentrated gel, and the local amplification signal input end is used to input the concentrated gel aggregated biological molecules into the local solidified separation gel, and the solidified separation gel is subjected to three-dimensional electrophoresis.

[0051] Step S13: After the three-dimensional electrophoresis is completed, the solidified separation gel is sliced ​​to obtain multi-layer gel slices, and the multi-layer gel slices are analyzed layer by layer to obtain the spatial signal distribution of each molecular weight biomolecule in the tissue slice.

[0052] In this embodiment, after the three-dimensional electrophoresis is completed, the solidified separation gel is frozen, and the frozen separation gel is sliced ​​and separated according to the molecular weight along the direction perpendicular to the vertical axis (i.e., the horizontal-vertical plane direction) to obtain multi-layer gel slices, and the multi-layer gel slices are analyzed layer by layer to obtain the spatial signal distribution of the biological molecules.

[0053] The specific process of analyzing the spatial distribution of biomolecule signals by using a target three-dimensional high-throughput amplification microwell array loading device based on a three-dimensional gel electrophoresis device is as follows: Figure 3 As shown, the steps are as follows: 1. Surgical removal of tissue: obtain the tissue to be tested.

[0054] 2. Tissue frozen sections: Cryo-embed and freeze-section the tissue.

[0055] 3. Tissue adhesion and lysis: The frozen sectioned tissue is adhered to the tissue sample adhesion end of the high-throughput amplification microwell array loading device, and the tissue is lysed and incubated with dye at room temperature.

[0056] 4. Three-dimensional gel electrophoresis: solidify the separation gel in the electrophoresis pool, then add the unsolidified concentrated gel to the top of the solidified separation gel, and place the target three-dimensional high-throughput amplification microwell array in the unsolidified concentrated gel. After the concentrated gel fills the three-dimensional high-throughput amplification microwell array in the target three-dimensional high-throughput amplification microwell array, solidify. Then add electrophoresis liquid between the cathode plate and the concentrated gel and between the anode plate and the bottom of the electrophoresis pool. Finally, connect a 40V constant voltage power supply to the three-dimensional gel electrophoresis device for three-dimensional electrophoresis. Under the action of the electric field, the proteins or nucleic acids in the tissue are fully separated along the z-axis direction in the separation gel according to the size of the molecular weight. The larger the molecular weight of the protein or nucleic acid, the smaller the electrical migration distance, the shorter the moving distance along the z-axis in the separation gel, and the closer to the input end of the amplification signal while retaining the two-dimensional spatial information of the xy plane.

[0057] 5. Gel layer-by-layer detection: Freeze the separation gel and slice the frozen gel in the direction perpendicular to the z-axis. The number of layers of frozen gel from small to large corresponds to the molecular weight range of proteins or nucleic acids from high to low. The distribution of signal points of proteins or nucleic acids in the slices of each layer of gel can reflect the two-dimensional spatial information of biological molecules in the molecular weight range corresponding to the tissue slice in the xy plane.

[0058] Taking the positive margin diagnosis of multiple small-volume samples of mouse (or rat) 4T1 transplanted tumor samples under different parameters as an example, the protein of 4T1 tumor samples is separated and detected based on a three-dimensional gel electrophoresis device, using different numbers of micropores and magnifications of the sample loading device, and the tumor contour is predicted. The flow diagram of three-dimensional gel electrophoresis and detection of tissue samples is shown in the attached figure. Figure 3 shown.

[0059] Specifically, the sample loading devices are selected to be 20×20, 30×30 and 40×40, and the corresponding microwell array densities are 100 / cm2, 225 / cm2 and 400 / cm2 respectively. Figure 4 The magnification of the sample adding device is selected to be 1 to 1.5 times the periodic side length and 1 to 2.25 times the area magnification.

[0060] 4T1 tumor samples were obtained by 4T1 cells were injected intradermally into Balb / c mice. All tumor-bearing mice were killed on the 35th day, and 4T1 tumor tissues were collected by surgery. Next, the obtained tumor tissue was divided into blocks by surgical cutting and then embedded and frozen in OCT (frozen tissue embedding medium) to establish a model for simultaneous detection of multiple spatial locations of surgically removed tumors. In order to reflect the performance differences of different microwell array loading devices, the size of small volume samples was controlled to be about 2 mm. The distribution of tumor samples in the frozen tissue embedding medium (OCT) was 2×2.

[0061] Subsequently, the frozen tumor samples were sliced ​​by a cryostat and adhered to the tissue sample adhesion end of different types of microwell array loading devices, and tissue lysis was performed. Tumor samples were sliced ​​continuously on different types of microwell array loading devices, and the thickness of each slice was 60 microns. The tissue lysis buffer used was RIPA Lysis Buffer and was not limited to this lysis buffer. The tissue lysis process was carried out simultaneously with the protein or nucleic acid staining incubation process.

[0062] After tissue lysis, three-dimensional gel electrophoresis is performed on different microwell array loading devices. Under the constant voltage flat plate electric field, proteins are separated according to molecular weight in the separation gel medium. The schematic diagram of the lateral diffusion of proteins during three-dimensional gel electrophoresis is shown in Figure 2. Figure 5 shown.

[0063] After electrophoresis, the separation gel medium is frozen and then sliced. The proteins are distributed in the corresponding gel layer according to their molecular weight, and the distribution of proteins in the gel layer is then detected and analyzed. Figure 6 As shown, Figure 7 Three-dimensional gel electrophoresis of three types of microporous sample loading devices, 20×20 (1×), 30×30 (1×), and 40×40 (2×), was performed. In the same gel layer, by increasing the throughput of the sample loading device, the tissue sample can be partitioned with higher precision, which can effectively restore the tissue distribution inside the micro-tissue; by enlarging the spacing of the microporous signal output end, the signal crosstalk caused by signal diffusion between the micropores is effectively suppressed, thereby ensuring the accuracy of the signal point. Through the combination of high throughput and amplification of the microporous array, the three-dimensional gel electrophoresis device has optimized its ability to judge the tumor contour of small samples.

[0064] The target three-dimensional high-throughput amplified microwell array loading device based on the three-dimensional gel electrophoresis device proposed in this application has the ability to identify tumor heterogeneity in human breast cancer adenocarcinoma tissue. For example, a patient's breast cancer tumor was subjected to three-dimensional gel electrophoresis with a microwell array throughput of 40×40 and a magnification of 2.25 times. First, the tumor tissue was embedded and frozen (thickness: 60 microns), and then gel electrophoresis was performed. After that, the separation gel medium after electrophoresis was frozen and sliced ​​for analysis. Next, the inventor combined the distribution of molecular signal points in the tenth layer of the gel slice with the H&E (hematoxylin-eosin staining) of the frozen section of the tumor tissue for analysis, such as Figure 7 As shown in Figure 1, the frozen sections of tumor tissue taken by H&E are 10 microns thick and are connected to the sections taken by 3D gel electrophoresis. Figure 7In the figure, the H&E local magnification area 1 of the tumor tissue is the matrix tissue. After the protein in this area binds to the dye molecules, there is a significant difference in signal intensity compared with the breast ductal carcinoma tissue in the local magnification areas 2, 3, and 4. The corresponding fluorescence signal intensity in the breast tumor tissue is high. In the local magnification areas 5, 6, and 7, the H&E analysis results show that the tumor tissue in this area is accompanied by apocrine differentiation. Corresponding to the H&E analysis results, the molecular signal intensity in this area is significantly greater than that of the surrounding cancer tissue.

[0065] Therefore, it was demonstrated that after adopting the targeted three-dimensional high-throughput amplified microwell array loading device, the three-dimensional gel electrophoresis device could distinguish the benign and malignant nature of the excised tissue and determine the tumor contour. The three-dimensional electrophoresis method also demonstrated its ability to identify the heterogeneity of tumor tissue and the strong connection between tissue type and molecular signal points.

[0066] Taking the axillary lymph node puncture samples of three breast cancer patients as an example, the tumor tissue was first embedded and continuously frozen (thickness: 60 microns), then gel electrophoresis was performed, and then the separation gel medium after electrophoresis was frozen and sliced ​​for analysis. The specific steps are consistent with those described in the above embodiment. Next, the inventor combined the molecular signal point distribution in the tenth layer of the gel slice with the frozen section H&E (hematoxylin-eosin staining) of the tumor tissue for analysis. Figure 8 As shown. Tumor tissue serial sections were alternately sliced ​​with frozen sections taken for H&E, so that each tissue section used for 3D gel electrophoresis corresponded to adjacent tissues stained with hematoxylin-eosin (H&E). The thickness of the frozen sections taken for H&E was 10 μm. Figure 8 In the figure, after three-dimensional gel electrophoresis using a microwell array high-throughput amplification loading device, malignant samples 1 and 2 with a width of about 1 mm can be distinguished from benign sample 3. It is worth noting that for the non-tumor area in sample 2, the corresponding molecular signal intensity is also lower than that of the tumor area.

[0067] The innovation of this application is that: the flux of the micropore array is increased, specifically, the number of micropore arrays at the tissue sample adhesion end is increased to 100 to 400 per square centimeter, and the ratio of micropore spacing to micropore diameter is 4:3. The micropore array with increased flux has a higher pattern analysis capability. As the flux of the micropore array increases, the cutting of the same pattern is finer and the degree of restoration is higher; the micropore array spacing is enlarged, specifically, the micropore spacing at the micropore array signal input and output end is enlarged without changing the micropore spacing at the tissue sample adhesion end and the micropore diameter of the micropore array, so that the area of ​​each micropore array unit in the amplified signal input and output end is enlarged by 1 to 2.25 times. The amplified micropore array ensures the accuracy of the signal point by effectively suppressing the signal crosstalk between the micropores. After the amplified micropore array, the distance between the signal points is increased, which effectively alleviates the signal overlap phenomenon between the micropores; a high-throughput amplified micropore array loading device is used to realize three-dimensional gel electrophoresis fluorescence pathology analysis.

[0068] In this embodiment, a tissue slice to be analyzed is obtained, and the tissue slice to be analyzed is adhered to a local tissue sample adhesion end, and tissue lysis and dye incubation are performed to obtain a tissue slice after lysis and incubation; a target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions is placed in a local uncured concentrated gel until the concentrated gel is completely cured, and three-dimensional electrophoresis is performed on the tissue slice after lysis and incubation on the basis of the target three-dimensional high-throughput amplification microwell array, and the biomolecules in the tissue slice after lysis and incubation are aggregated in the concentrated gel and input from the input end of the amplification array to the local cured separation gel, and three-dimensional electrophoresis is continued to achieve separation of biomolecules; the number of micropores contained in each square centimeter in the target three-dimensional high-throughput amplification microwell array is 100 to 400, and the area magnification of each micropore array unit is 1 to 2.25 times; the biomolecule is a protein or a nucleic acid; after the three-dimensional electrophoresis is completed, the cured separation gel is sliced ​​to obtain a multi-layer gel slice, and the multi-layer gel slice is analyzed layer by layer to obtain the spatial signal distribution of each molecular weight biomolecule in the tissue slice. The present application is applied to a target three-dimensional high-throughput amplified microwell array loading device based on a three-dimensional gel electrophoresis device, wherein a tissue slice to be analyzed is adhered to a local tissue sample adhesion end, and tissue lysis and dye incubation are performed to obtain a tissue slice after lysis and incubation, and the target three-dimensional high-throughput amplified microwell array is placed in a local unsolidified concentrated gel until solidified, and three-dimensional electrophoresis is performed on the solidified concentrated gel based on the tissue slice after lysis and incubation and the target three-dimensional high-throughput amplified microwell array, and the obtained biomolecules separated by electrophoresis are input into the local solidified separation gel for slicing, and high-precision in-situ segmentation can be achieved through slicing, providing high-precision signal information for tissue analysis, solving the problem that the existing segmentation accuracy of tissue slices cannot meet the accurate pathological diagnosis of microtissues and microstructures in tissues, and can improve the analysis and diagnosis accuracy of microtissues and microstructures in tissues, expand the practicality of the three-dimensional gel electrophoresis device in the field of pathological diagnosis, improve the resolution of the microwell array, and improve the resolution and accuracy of the analysis of the spatial signal distribution of biomolecules.

[0069] See also Fig. 9 As shown, the embodiment of the present invention discloses a biomolecule spatial signal distribution analysis device, which is applied to a target three-dimensional high-throughput amplification microwell array sample loading device based on a three-dimensional gel electrophoresis device, and specifically may include:

[0070] The slice lysis and incubation module 11 is used to obtain the tissue slice to be analyzed, adhere the tissue slice to be analyzed to the local tissue sample adhesion end, and perform tissue lysis and dye incubation to obtain the tissue slice after lysis and incubation;

[0071] The three-dimensional electrophoresis module 12 is used to place the target three-dimensional high-throughput amplified microwell array that meets the preset high-throughput conditions in a local uncured concentrated gel until the concentrated gel is completely cured, and on the basis of the target three-dimensional high-throughput amplified microwell array, perform three-dimensional electrophoresis on the lysed and incubated tissue slices, and the biomolecules in the lysed and incubated tissue slices are aggregated in the concentrated gel and input from the input end of the amplified array to the local cured separation gel, and continue the three-dimensional electrophoresis to achieve the separation of biomolecules; the number of microwells contained in each square centimeter of the target three-dimensional high-throughput amplified microwell array is 100 to 400, and the area magnification of each microwell array unit is 1 to 2.25 times; the biomolecules are proteins or nucleic acids;

[0072] The slicing and analyzing module 13 is used to slice the solidified separation gel after the three-dimensional electrophoresis is completed to obtain multi-layer gel slices, and analyze the multi-layer gel slices layer by layer to obtain the spatial signal distribution of each molecular weight biological molecule in the tissue slice.

[0073] In some specific embodiments, the target three-dimensional high-throughput amplification microwell array loading device includes a tissue sample adhesion end, a target three-dimensional high-throughput amplification microwell array that meets preset high-throughput conditions, and an amplification signal input end; the three-dimensional gel electrophoresis device includes a target three-dimensional high-throughput amplification microwell array loading device.

[0074] In some specific embodiments, the slice lysis and incubation module 11 may specifically include:

[0075] A tissue processing module, used for obtaining an initial tissue to be analyzed, freezing and slicing the initial tissue to be analyzed to obtain a tissue slice to be analyzed;

[0076] The adhesion module is used to adhere the tissue slice to be analyzed to the local tissue sample adhesion end, and perform tissue lysis and dye incubation at room temperature.

[0077] In some specific embodiments, the three-dimensional electrophoresis module 12 may specifically include:

[0078] Separation gel solidification module, used to perform separation gel solidification operation in the local electrophoresis pool;

[0079] The liquid concentrated gel adding module is used to add unsolidified concentrated gel on the upper end of the solidified separation gel.

[0080] In some specific embodiments, the 3D electrophoresis module 12 may specifically include:

[0081] The monitoring module is used to place the target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions in the local unsolidified concentrated gel, and monitor whether the current concentrated gel is full of the three-dimensional high-throughput amplification microwell array in the target three-dimensional high-throughput amplification microwell array.

[0082] In some specific embodiments, the three-dimensional electrophoresis module 12 may specifically include:

[0083] The electrophoresis fluid adding module is used to add electrophoresis fluid between the local cathode plate and the solidified concentrated gel and between the anode plate and the bottom of the electrophoresis pool, and connect to a constant voltage power supply to perform three-dimensional electrophoresis on the tissue sections after lysis and incubation.

[0084] In some specific embodiments, the slicing and analyzing module 13 may specifically include:

[0085] The slicing and separation module is used to input the biomolecules aggregated in the concentrated gel into the local solidified separation gel by using the local amplification signal input end, and perform three-dimensional electrophoresis on the solidified separation gel;

[0086] After the three-dimensional electrophoresis is completed, the solidified separation gel is frozen, and the frozen separation gel is sliced ​​and separated along a direction perpendicular to the vertical axis according to the molecular weight to obtain multi-layer gel slices.

[0087] Fig.10 A schematic diagram of the structure of an electronic device provided in an embodiment of the present application. The electronic device 20 may specifically include: at least one processor 21, at least one memory 22, a power supply 23, a communication interface 24, an input / output interface 25, and a communication bus 26. The memory 22 is used to store a computer program, which is loaded and executed by the processor 21 to implement the relevant steps in the method for analyzing the spatial signal distribution of biomolecules performed by the electronic device disclosed in any of the aforementioned embodiments.

[0088] In this embodiment, the power supply 23 is used to provide working voltage for each hardware device on the electronic device 20; the communication interface 24 can create a data transmission channel between the electronic device 20 and the external device, and the communication protocol it follows is any communication protocol that can be applied to the technical solution of the present application, and is not specifically limited here; the input and output interface 25 is used to obtain external input data or output data to the outside world, and its specific interface type can be selected according to specific application needs and is not specifically limited here.

[0089] In addition, the memory 22, as a carrier for storing resources, can be a read-only memory, a random access memory, a disk or an optical disk, etc. The resources stored thereon include an operating system 221, a computer program 222 and data 223, etc. The storage method can be temporary storage or permanent storage.

[0090] The operating system 221 is used to manage and control the hardware devices and computer programs 222 on the electronic device 20, so as to realize the operation and processing of the data 223 in the memory 22 by the processor 21, which can be Windows, Unix, Linux, etc. In addition to including a computer program that can be used to complete the biomolecule spatial signal distribution analysis method performed by the electronic device 20 disclosed in any of the aforementioned embodiments, the computer program 222 can also further include a computer program that can be used to complete other specific tasks. In addition to data transmitted from an external device received by the biomolecule spatial signal distribution analysis device, the data 223 can also include data collected by its own input and output interface 25.

[0091] The steps of the method or algorithm described in conjunction with the embodiments disclosed herein may be implemented directly using hardware, a software module executed by a processor, or a combination of the two. The software module may be placed in a random access memory (RAM), a memory, a read-only memory (ROM), an electrically programmable ROM, an electrically erasable programmable ROM, a register, a hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art.

[0092] Furthermore, an embodiment of the present application also discloses a computer-readable storage medium, in which a computer program is stored. When the computer program is loaded and executed by a processor, the steps of the method for analyzing the spatial signal distribution of biological molecules disclosed in any of the aforementioned embodiments are implemented.

[0093] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0094] The above is a detailed introduction to a method, device, equipment and storage medium for analyzing the spatial signal distribution of biological molecules provided by the present invention. Specific examples are used in this article to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for a person skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A method for analyzing the spatial signal distribution of biomolecules, characterized in that: The target three-dimensional high-throughput amplification microwell array loading device applied to the three-dimensional gel electrophoresis device includes: Obtaining a tissue section to be analyzed, adhering the tissue section to be analyzed to a local tissue sample adhering end, and performing tissue lysis and dye incubation to obtain a lysed and incubated tissue section; A target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions is placed in a local uncured concentrated gel until the concentrated gel is completely cured, and three-dimensional electrophoresis is performed on the lysed and incubated tissue slices on the basis of the target three-dimensional high-throughput amplification microwell array, and the biomolecules in the lysed and incubated tissue slices are aggregated in the concentrated gel and input from the input end of the amplification array to the local cured separation gel, and three-dimensional electrophoresis is continued to achieve separation of biomolecules; the number of microwells contained in each square centimeter of the target three-dimensional high-throughput amplification microwell array is 100 to 400, and the area magnification of each microwell array unit is 1 to 2.25 times; the biomolecules are proteins or nucleic acids; After the three-dimensional electrophoresis is completed, the solidified separation gel is sliced ​​to obtain multi-layer gel slices, and the multi-layer gel slices are analyzed layer by layer to obtain the spatial signal distribution of each molecular weight biomolecule in the tissue slice.

2. The method for analyzing the spatial signal distribution of biomolecules according to claim 1, characterized in that: The target three-dimensional high-throughput amplification microporous array loading device includes a tissue sample adhesion end, a target three-dimensional high-throughput amplification microporous array that meets preset high-throughput conditions, and an amplification signal input end; the three-dimensional gel electrophoresis device includes a target three-dimensional high-throughput amplification microporous array loading device.

3. The method for analyzing the spatial signal distribution of biomolecules according to claim 1, characterized in that: The step of obtaining a tissue section to be analyzed, adhering the tissue section to be analyzed to a local tissue sample adhering end, and performing tissue lysis and dye incubation includes: Acquiring an initial tissue to be analyzed, and freezing and slicing the initial tissue to be analyzed to obtain tissue slices to be analyzed; The tissue slice to be analyzed is adhered to the local tissue sample adhesion end, and tissue lysis and dye incubation are performed at room temperature.

4. The method for analyzing the spatial signal distribution of biomolecules according to claim 1, characterized in that: Before placing the target three-dimensional high-throughput amplified microwell array that meets the preset high-throughput conditions in the local unsolidified concentrated gel, the method further includes: The separation gel solidification operation is carried out in a local electrophoresis pool; Add unsolidified stacking gel on top of the solidified separation gel.

5. The method for analyzing the spatial signal distribution of biomolecules according to claim 1, characterized in that: The method of placing the target three-dimensional high-throughput amplified microwell array that meets the preset high-throughput conditions in a local unsolidified concentrated gel comprises: The target three-dimensional high-throughput amplification microwell array that meets the preset high-throughput conditions is placed in the local unsolidified concentrated gel, and the three-dimensional high-throughput amplification microwell array in the target three-dimensional high-throughput amplification microwell array is monitored to see whether the current concentrated gel is full of the three-dimensional high-throughput amplification microwell array.

6. The method for analyzing the spatial signal distribution of biomolecules according to claim 1, characterized in that: The three-dimensional electrophoresis of the lysed and incubated tissue sections comprises: Electrophoresis fluid is added between the local cathode plate and the solidified concentrated gel and between the anode plate and the bottom of the electrophoresis pool, and a constant voltage power supply is connected to perform three-dimensional electrophoresis on the lysed and incubated tissue sections.

7. The method for analyzing the spatial signal distribution of biomolecules according to any one of claims 1 to 6, characterized in that: The solidified separation gel is input from the input end of the amplification array to the local area, and three-dimensional electrophoresis is continued to achieve separation of biomolecules; After the three-dimensional electrophoresis is completed, the solidified separation gel is sliced ​​to obtain multi-layer gel slices, including: The biomolecules aggregated in the concentrated gel are input into the local solidified separation gel by using the local amplification signal input end, and the solidified separation gel is subjected to three-dimensional electrophoresis; After the three-dimensional electrophoresis is completed, the solidified separation gel is frozen, and the frozen separation gel is sliced ​​and separated along a direction perpendicular to the vertical axis according to the molecular weight to obtain multi-layer gel slices.

8. A biomolecule spatial signal distribution analysis device, characterized in that: The target three-dimensional high-throughput amplification microwell array loading device applied to the three-dimensional gel electrophoresis device includes: A slice lysis and incubation module, used to obtain a tissue slice to be analyzed, adhere the tissue slice to be analyzed to a local tissue sample adhesion end, and perform tissue lysis and dye incubation to obtain a lysed and incubated tissue slice; A three-dimensional electrophoresis module is used to place a target three-dimensional high-throughput amplified microwell array that meets the preset high-throughput conditions in a local uncured concentrated gel until the concentrated gel is completely cured, and on the basis of the target three-dimensional high-throughput amplified microwell array, three-dimensional electrophoresis is performed on the lysed and incubated tissue slices, and the biomolecules in the lysed and incubated tissue slices are aggregated in the concentrated gel and input from the input end of the amplified array to the local cured separation gel, and three-dimensional electrophoresis is continued to achieve separation of biomolecules; the number of microwells contained in each square centimeter of the target three-dimensional high-throughput amplified microwell array is 100 to 400, and the area magnification of each microwell array unit is 1 to 2.25 times; the biomolecules are proteins or nucleic acids; The slicing and analysis module is used to slice the solidified separation gel after the three-dimensional electrophoresis is completed to obtain multi-layer gel slices, and analyze the multi-layer gel slices layer by layer to obtain the spatial signal distribution of each molecular weight biological molecule in the tissue slice.

9. An electronic device, characterized in that: include: Memory, used to store computer programs; A processor, configured to execute the computer program to implement the method for analyzing the spatial signal distribution of biomolecules according to any one of claims 1 to 7.

10. A computer-readable storage medium, characterized in that: Used to store a computer program; wherein, when the computer program is executed by a processor, the method for analyzing the spatial signal distribution of biomolecules according to any one of claims 1 to 7 is implemented.

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