Method, device and medium for analyzing distribution of biomolecule spatial signals

Abstract

The application discloses a biomolecule spatial signal distribution analysis method, device, equipment and medium, relates to the pathological diagnosis technical field, and will be analyzed tissue section adhere to the tissue sample adhering end, and carry out tissue lysis and dye incubation; the target three-dimensional high-throughput amplification micropore array meeting the preset high-throughput condition is placed in the unhardened concentrated glue, until the concentrated glue is hardened, the biomolecule in the tissue section after lysis and incubation completes aggregation in the concentrated glue, is input from the input end of amplification array to the solidified separation glue, continues three-dimensional electrophoresis to realize the separation of biomolecule, when three-dimensional electrophoresis ends, the solidified separation glue section is cut, and multiple layer gel sections are obtained, the multiple layer gel sections are analyzed layer by layer, the spatial signal distribution of biomolecule is obtained, the problem that the segmentation accuracy of tissue section cannot meet the accurate pathological diagnosis of small tissue and small structure in tissue is solved, and the resolution and accuracy of biomolecule spatial signal distribution analysis are improved.

Description

Technical Field

[0001] This invention relates to the field of pathological diagnostic technology, and in particular to a method, apparatus, equipment and medium for analyzing the spatial signal distribution of biomolecules. Background Technology

[0002] Three-dimensional gel electrophoresis (3D GEE) is a novel detection method in the field of pathological diagnosis. Using 3D GEE, proteins or nucleic acids are separated in a gel according to their molecular weight, achieving thorough separation of biological samples with molecular weights ranging from 10 to 180 kilodaltons within a separation distance of 1 cm. Because the electric field exists only in the z-axis direction, the spatial distribution of biomolecular signals in the x and y axes is maintained. Multilayer section analysis of the gel after 3D GEE allows for the acquisition of the spatial signal distribution of biomolecules in each molecular weight layer. Currently available 3D GEE devices are equipped with microwell arrays with variable throughput, containing 10 to 100 microwells per square centimeter. Higher throughput microwell arrays can achieve more precise regional segmentation of the tissue sample, resulting in higher accuracy of the xy-plane spatial distribution information of biomolecules in the final tissue section. By using the spatial distribution information of biomolecules to replace visual diagnosis of tissue morphology and structure, it can be effectively applied to the diagnosis of benign and malignant resections of surgically removed tissues, as well as the diagnosis of malignant margins in frozen sections of surgically removed tissues. However, current three-dimensional gel electrophoresis devices have the following drawbacks: The current microwell arrays used in 3D gel electrophoresis have low throughput and limited spatial segmentation accuracy for tissue sections, resulting in low signal resolution for pathological diagnosis and making it difficult to meet the needs for accurate pathological diagnosis of micro-tissues and structures within micro-tissues; the large pore size of the microwell arrays makes tissue sections prone to breakage under excessive local tension, leading to signal distortion within the microwells. Signal diffusion within individual microwells causes signal overlap and spatial signal distortion, making it difficult for the imaging signal distribution at a single signal point to correspond to the actual tissue distribution, thus hindering more accurate pathological diagnosis of micro-tissues and structures within micro-tissues; the small spacing between the microwells in the molecular signal input separating gel of the microwell array causes signal overlap between microwells under the influence of signal diffusion, disrupting the spatial signal distribution and making it difficult to meet the needs for accurate pathological diagnosis of micro-tissues and structures within micro-tissues.

[0003] As can be seen from the above, how to improve the analytical capabilities of three-dimensional gel electrophoresis systems for tissue sections, solve the problem that the current segmentation accuracy of tissue sections cannot meet the requirements for accurate pathological diagnosis of micro-tissues and micro-structures within tissues, and improve the resolution and accuracy of analyzing the spatial signal distribution of biomolecules are problems that need to be solved in this field. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method, apparatus, device, and medium for analyzing the spatial distribution of biomolecules, which can improve the analytical capabilities of three-dimensional gel electrophoresis systems for tissue sections, solve the problem that the segmentation accuracy of existing tissue sections cannot meet the requirements for accurate pathological diagnosis of micro-tissues and micro-structures within tissues, and improve the resolution and accuracy of analyzing the spatial distribution of biomolecules. The specific solution is as follows:

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

[0006] Obtain tissue sections to be analyzed, attach the tissue sections to the local tissue sample attachment end, and perform tissue lysis and dye incubation to obtain lysed and incubated tissue sections.

[0007] A target three-dimensional high-throughput microwell array meeting preset high-throughput conditions is placed in a local uncured stacking gel until the stacking gel is completely cured. Based on the target three-dimensional high-throughput microwell array, lysed and incubated tissue sections are subjected to three-dimensional electrophoresis. Biomolecules in the lysed and incubated tissue sections aggregate in the stacking gel and are input from the input end of the amplification array to a local cured separation gel for further three-dimensional electrophoresis to achieve separation of biomolecules. The target three-dimensional high-throughput microwell array contains 100 to 400 microwells per square centimeter, and the area magnification of each microwell array unit is 1 to 2.25 times. The biomolecules are proteins or nucleic acids.

[0008] After three-dimensional electrophoresis, the solidified separation gel is sliced ​​to obtain multilayer gel slices. The multilayer gel slices are then analyzed layer by layer to obtain the spatial signal distribution of biomolecules of various molecular weights in the tissue slices.

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

[0010] Optionally, the step of obtaining the tissue section to be analyzed, attaching the tissue section to the local tissue sample adhesion end, and performing tissue lysis and dye incubation includes:

[0011] Obtain the initial tissue to be analyzed, and perform freezing and sectioning on the initial tissue to be analyzed to obtain tissue sections.

[0012] The tissue slice to be analyzed was attached to the local tissue sample attachment end, and the tissue was lysed and incubated with dye at room temperature.

[0013] Optionally, before placing the target three-dimensional high-throughput magnified micropore array that meets the preset high-throughput conditions into the local uncured stacked adhesive, the method further includes:

[0014] The separation gel curing process was carried out in a local electric swimming pool;

[0015] Add uncured binder to the top of the cured release gel.

[0016] Optionally, placing the target three-dimensional high-throughput magnified micropore array that meets the preset high-throughput conditions in a local uncured stacked adhesive includes:

[0017] The target three-dimensional high-throughput amplified micropore array that meets the preset high-throughput conditions is placed in the local uncured thickener, and the current thickener is monitored to see if it has filled the target three-dimensional high-throughput amplified micropore array.

[0018] Optionally, the three-dimensional electrophoresis of the lysed and incubated tissue sections includes:

[0019] Electrophoresis solution was added between the local cathode plate and the cured concentrated gel, and between the anode plate and the bottom of the electrophoresis pool. A constant voltage power supply was then connected to perform three-dimensional electrophoresis on the lysed and incubated tissue sections.

[0020] Optionally, the solidified separating gel input from the amplification array input terminal is further processed by three-dimensional electrophoresis to achieve the separation of biomolecules; after the three-dimensional electrophoresis is completed, the solidified separating gel is sliced ​​to obtain multilayer gel slices, including:

[0021] The biomolecules aggregated in the stacking gel are input into the local curing and separating gel using the local amplified signal input terminal, and the curing and separating gel is subjected to three-dimensional electrophoresis.

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

[0023] Secondly, this application discloses a device for analyzing the spatial signal distribution of biomolecules, applied to a target three-dimensional high-throughput amplified micropore array sample loading device based on a three-dimensional gel electrophoresis apparatus, comprising:

[0024] The slicing lysis and incubation module is used to obtain tissue slices to be analyzed, attach the tissue slices to be analyzed to the local tissue sample attachment end, and perform tissue lysis and dye incubation to obtain lysed and incubated tissue slices.

[0025] A three-dimensional electrophoresis module is used to place a target three-dimensional high-throughput magnified microwell array that meets preset high-throughput conditions into a local uncured stacking gel until the stacking gel is completely cured. Based on the target three-dimensional high-throughput magnified microwell array, three-dimensional electrophoresis is performed on lysed and incubated tissue sections. Biomolecules in the lysed and incubated tissue sections aggregate in the stacking gel and are input from the magnification array input end to a local cured separation gel for continued three-dimensional electrophoresis to achieve separation of biomolecules. The target three-dimensional high-throughput magnified microwell array contains 100 to 400 microwells per square centimeter, and the area magnification factor 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 three-dimensional electrophoresis to obtain multilayer gel slices, and to perform layer-by-layer analysis on the multilayer gel slices to obtain the spatial signal distribution of each molecular weight biomolecule in the tissue slices.

[0027] Thirdly, this application discloses an electronic device, including:

[0028] Memory, used to store computer programs;

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

[0030] Fourthly, this application discloses a computer storage medium for storing a computer program; wherein, when the computer program is executed by a processor, it implements the steps of the aforementioned disclosed method for analyzing the spatial distribution of biomolecular signals.

[0031] As can be seen, this application provides a method for analyzing the spatial signal distribution of biomolecules, including obtaining tissue slices to be analyzed, attaching the tissue slices to a local tissue sample attachment end, and performing tissue lysis and dye incubation to obtain lysed and incubated tissue slices; placing a target three-dimensional high-throughput microwell array meeting preset high-throughput conditions in a local uncured stacking gel until the stacking gel is completely cured; and performing three-dimensional electrophoresis on the lysed and incubated tissue slices based on the target three-dimensional high-throughput microwell array, thereby analyzing the biomolecules within the lysed and incubated tissue slices. The biomolecules are aggregated in a stacking gel and fed into a locally cured separating gel from the input of the amplification array for further three-dimensional electrophoresis to achieve separation. The target three-dimensional high-throughput amplification micropore array contains 100 to 400 micropores per square centimeter, and the area magnification of each micropore array unit is 1 to 2.25 times. The biomolecules are proteins or nucleic acids. After three-dimensional electrophoresis, the cured separating gel is sliced ​​to obtain multilayer gel slices. The multilayer gel slices are analyzed layer by layer to obtain the spatial signal distribution of biomolecules of various molecular weights in the tissue slices. This application applies to a target three-dimensional high-throughput microwell array sample loading device based on a three-dimensional gel electrophoresis apparatus. Tissue slices to be analyzed are adhered to the local tissue sample attachment end, and tissue lysis and dye incubation are performed to obtain lysed and incubated tissue slices. The target three-dimensional high-throughput microwell array is placed in a local uncured stacking gel until it solidifies. Based on the lysed and incubated tissue slices and the target three-dimensional high-throughput microwell array, three-dimensional electrophoresis is performed on the solidified stacking gel. The resulting electrophoretically separated biomolecules are input into a local solidified separating gel for slicing. This slicing enables high-precision in-situ segmentation, providing high-precision signal information for tissue analysis. It solves the problem that the segmentation accuracy of existing tissue slices cannot meet the requirements for accurate pathological diagnosis of micro-tissues and micro-structures within tissues, and improves the accuracy of analysis and diagnosis of micro-tissues and micro-structures within tissues. This expands the practicality of three-dimensional gel electrophoresis devices in the field of pathological diagnosis, improves the resolution of the microwell array, and enhances the resolution and accuracy of analyzing the spatial signal distribution of biomolecules. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

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

[0034] Figure 2 This is a structural diagram of a three-dimensional electrophoresis apparatus and a target three-dimensional high-throughput amplification micropore array sample loading device disclosed in this application;

[0035] Figure 3 This is a flowchart illustrating the spatial distribution analysis of biomolecular signals disclosed in this application.

[0036] Figure 4 This is a schematic diagram of a tissue section subjected to zonal analysis at different micropore array flux levels as disclosed in this application;

[0037] Figure 5 This is a schematic diagram showing the lateral diffusion of tissue components in a tissue section disclosed in this application under both magnified and non-magnified microporous array conditions.

[0038] Figure 6 This application discloses a method for 4T1 multi-sample detection with different micropore array throughputs and their effects on judging the contours of small tumors under magnification and non-magnification.

[0039] Figure 7 This is an image illustrating the effect of identifying tumor heterogeneity in breast cancer adenocarcinoma tissue as disclosed in this application.

[0040] Figure 8 This is an image illustrating the effect of identifying the distribution of cancer cells in a micro-tissue biopsy lymph node as disclosed in this application;

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

[0042] Figure 10 This application provides a structural diagram of an electronic device. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Three-dimensional gel electrophoresis (3D GEE) is a novel detection method in the field of pathological diagnosis. Using 3D GEE, proteins or nucleic acids are separated in a gel according to their molecular weight, achieving thorough separation of biological samples with molecular weights ranging from 10 to 180 kilodaltons within a separation distance of 1 cm. Because the electric field exists only in the z-axis direction, the spatial distribution of biomolecular signals in the x and y axes is maintained. Multilayer section analysis of the gel after 3D GEE allows for the acquisition of the spatial signal distribution of biomolecules in each molecular weight layer. Currently available 3D GEE devices are equipped with microwell arrays with variable throughput, containing 10 to 100 microwells per square centimeter. Higher throughput microwell arrays can achieve more precise regional segmentation of the tissue sample, resulting in higher accuracy of the xy-plane spatial distribution information of biomolecules in the final tissue section. By using the spatial distribution information of biomolecules to replace visual diagnosis of tissue morphology and structure, it can be effectively applied to the diagnosis of benign and malignant resections of surgically removed tissues, as well as the diagnosis of malignant margins in frozen sections of surgically removed tissues. However, current three-dimensional gel electrophoresis devices have the following drawbacks: The current microwell arrays used in 3D gel electrophoresis have low throughput and limited spatial segmentation accuracy for tissue sections, resulting in low signal resolution for pathological diagnosis and making it difficult to meet the needs for accurate pathological diagnosis of micro-tissues and structures within micro-tissues; the large pore size of the microwell arrays makes tissue sections prone to breakage under excessive local tension, leading to signal distortion within the microwells. Signal diffusion within individual microwells causes signal overlap and spatial signal distortion, making it difficult for the imaging signal distribution at a single signal point to correspond to the actual tissue distribution, thus hindering more accurate pathological diagnosis of micro-tissues and structures within micro-tissues; the small spacing between the microwells in the molecular signal input separating gel of the microwell array causes signal overlap between microwells under the influence of signal diffusion, disrupting the spatial signal distribution and making it difficult to meet the needs for accurate pathological diagnosis of micro-tissues and structures within micro-tissues. As can be seen from the above, how to improve the analytical capabilities of three-dimensional gel electrophoresis systems for tissue sections, solve the problem that the current segmentation accuracy of tissue sections cannot meet the requirements for accurate pathological diagnosis of micro-tissues and micro-structures within tissues, and improve the resolution and accuracy of analyzing the spatial signal distribution of biomolecules are problems that need to be solved in this field.

[0045] See Figure 1 As shown, this invention discloses a method for analyzing the spatial signal distribution of biomolecules, applied to a target three-dimensional high-throughput amplified micropore array sample loading device based on a three-dimensional gel electrophoresis apparatus, specifically including:

[0046] Step S11: Obtain the tissue section to be analyzed, attach the tissue section to the local tissue sample attachment end, and perform tissue lysis and dye incubation to obtain the lysed and incubated tissue section.

[0047] In this embodiment, an initial tissue to be analyzed is obtained, and the initial tissue to be analyzed is subjected to freezing and sectioning to obtain tissue sections to be analyzed. The tissue sections to be analyzed are attached to the local tissue sample attachment end, and tissue lysis and dye incubation are performed at room temperature to obtain lysed and incubated tissue sections.

[0048] This application applies to a target three-dimensional high-throughput amplified microwell array sample loading device based on a three-dimensional gel electrophoresis apparatus. The three-dimensional gel electrophoresis process and the structure of the target three-dimensional high-throughput amplified microwell array sample loading device are as follows: Figure 2 As shown, the device includes a tissue sample adhesion end, a target three-dimensional high-throughput amplified microwell array meeting preset high-throughput conditions, and an amplified signal input end. The three-dimensional gel electrophoresis apparatus includes a target three-dimensional high-throughput amplified microwell array sample loading device. The tissue sample adhesion end is a microwell array plane for solution drop loading or tissue sample attachment and lysis. The number of microwells per square centimeter of the microwell array plane is 100 to 400. The height of each microwell in the target three-dimensional high-throughput amplified microwell array is 0.7 cm. The diameter of the microwells along the x and y axes remains unchanged in any cross-section of the z-axis of each microwell column, and the area is the same, only the microwell spacing changes; the area amplification factor of each microwell array unit is 1 to 2.25 times. The amplified signal input end is a microwell array plane perpendicular to the electric field and already amplified.

[0049] Step S12: Place the target three-dimensional high-throughput microwell array that meets the preset high-throughput conditions into the local uncured stacking gel until the stacking gel is completely cured. Based on the target three-dimensional high-throughput microwell array, perform three-dimensional electrophoresis on the lysed and incubated tissue sections. The biomolecules in the lysed and incubated tissue sections aggregate in the stacking gel and are input from the input end of the amplification array to the local cured separation gel for continued three-dimensional electrophoresis to achieve separation of biomolecules. The target three-dimensional high-throughput microwell array contains 100 to 400 micropores per square centimeter, and the area magnification factor of each microwell array unit is 1 to 2.25 times. The biomolecules are proteins or nucleic acids.

[0050] In this embodiment, the separating gel is cured in a local electrophoresis pool. Uncured stacking gel is added to the top of the cured separating gel. The target three-dimensional high-throughput amplified micropore array that meets the preset high-throughput conditions is placed in the local uncured stacking gel. The current stacking gel is monitored to see if it is filled with the target three-dimensional high-throughput amplified micropore array until the stacking gel is cured. Based on the target three-dimensional high-throughput amplified micropore array, electrophoresis solution is added between the local cathode plate and the cured stacking gel, and between the anode plate and the bottom of the electrophoresis pool. A constant voltage power supply is connected to perform three-dimensional electrophoresis on the lysed and incubated tissue sections. This can ensure the xy-plane spatial distribution of the tissue sections while realizing the in-situ separation of biomolecules in the tissue sections. The biomolecules in the lysed and incubated tissue sections are aggregated in the stacking gel. The biomolecules aggregated in the stacking gel are input to the local cured separating gel using the local amplified signal input terminal, and three-dimensional electrophoresis is performed on the cured separating gel.

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

[0052] In this embodiment, after three-dimensional electrophoresis, the solidified separating gel is frozen, and the frozen separating 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 multilayer gel slices. The multilayer gel slices are analyzed layer by layer to obtain the spatial signal distribution of the biomolecules.

[0053] The specific process of this application for analyzing the spatial distribution of biomolecular signals using a target three-dimensional high-throughput amplification micropore array sample loading device based on a three-dimensional gel electrophoresis apparatus 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. Frozen sections of tissue: The tissue is frozen and embedded and then frozen sectioned.

[0055] 3. Tissue adhesion and lysis: Frozen tissue sections were adhered to the tissue sample adhesion end of the high-throughput microwell array sample loading device, and tissue lysis and dye incubation were performed at room temperature.

[0056] 4. Three-dimensional gel electrophoresis: The separating gel is cured in an electrophoresis pool. Then, an uncured stacking gel is added to the top of the cured separating gel, and the target three-dimensional high-throughput microarray is placed in the uncured stacking gel. Curing is completed once the stacking gel fills the target three-dimensional high-throughput microarray. Electrophoresis buffer is then added between the cathode plate and the stacking gel, and between the anode plate and the bottom of the electrophoresis pool. Finally, a 40V constant voltage power supply is connected to the three-dimensional gel electrophoresis apparatus for three-dimensional electrophoresis. Under the influence of an electric field, proteins or nucleic acids in the tissue are fully separated along the z-axis of the separating gel according to their molecular weight. Larger molecular weight proteins or nucleic acids have shorter electromigration distances and move more closely along the z-axis in the separating gel, thus bringing them closer to the amplified signal input while preserving two-dimensional spatial information in the xy-plane.

[0057] 5. Layer-by-layer gel analysis: The separating gel is frozen, and the frozen gel is sliced ​​along the direction perpendicular to the z-axis. The number of layers of the frozen gel, from smallest to largest, corresponds to the molecular weight range of proteins or nucleic acids from high to low. The distribution of protein or nucleic acid signal points in the slices of each layer of gel can reflect the two-dimensional spatial information of biomolecules of the corresponding molecular weight range in the xy plane.

[0058] Taking the diagnosis of positive margins in multiple small-volume samples of 4T1 xenograft tumors in mice (or rats) under different parameters as an example, specifically, based on a three-dimensional gel electrophoresis apparatus, proteins in 4T1 tumor samples are separated and detected using different numbers of microwells and magnifications of the sample loading device, and the tumor contour is predicted. A flowchart of the three-dimensional gel electrophoresis and detection process for tissue samples is attached. Figure 3 As shown.

[0059] Specifically, the sample loading devices were selected as 20×20, 30×30, and 40×40, corresponding to microwell array densities of 100 wells / cm², 225 wells / cm², and 400 wells / cm², respectively. The tissue sample partitioning is shown in the attached figure. Figure 4 As shown. The magnification of the sampling device is selected to be 1 to 1.5 times the period side length magnification and 1 to 2.25 times the area magnification.

[0060] 4T1 tumor sample acquisition is approximately Balb / c mice were injected intradermally with 4T1 cells. All tumor-bearing mice were sacrificed on day 35, and 4T1 tumor tissue was surgically collected. The obtained tumor tissue was then surgically dissected into sections, processed, and then embedded and frozen using OCT (occlusive tissue embedding medium) to establish a model for simultaneous detection of multiple spatial locations of surgically removed tumors. To demonstrate the performance differences between different microwell array sampling devices, the sample size was controlled to be approximately 2 mm. The tumor samples were distributed in a 2×2 pattern within the OCT.

[0061] Subsequently, the frozen tumor samples were sliced ​​using a cryostat and adhered to the tissue attachment ends of different types of microwell array sampling devices, followed by tissue lysis. The tumor samples were serially sliced ​​on the various types of microwell array sampling devices, with each slice being 60 micrometers thick. RIPA Lysis Buffer was used as the lysis buffer, but not limited to this. The tissue lysis process was performed simultaneously with protein or nucleic acid staining incubation.

[0062] After tissue lysis, three-dimensional gel electrophoresis was performed on different microwell array sample loading devices. Under a constant voltage plate electric field, proteins were separated in the separating gel medium according to their molecular weight. A schematic diagram of the lateral diffusion of proteins during three-dimensional gel electrophoresis is shown below. Figure 5 As shown.

[0063] After electrophoresis, the separating gel medium was frozen and then sectioned. Proteins were distributed in their respective gel layers according to molecular weight, and the protein distribution within the gel layers was then detected and analyzed. (See attached image) Figure 6 As shown, Figure 7 Three-dimensional gel electrophoresis was performed using three different microwell loading devices: 20×20 (1×), 30×30 (1×), and 40×40 (2×). Within the same gel layer, increasing the throughput of the loading device allowed for more precise partitioning of tissue samples, enabling effective reconstruction of the internal tissue distribution within small tissues. Enlarging the spacing between the microwell signal output ends effectively suppressed signal crosstalk caused by signal diffusion between microwells, thus preventing distortion and ensuring the accuracy of signal points. Through the combination of high throughput and amplification of the microwell array, the three-dimensional gel electrophoresis device's ability to determine tumor contours in small samples was optimized.

[0064] This application proposes a target three-dimensional high-throughput magnified microwell array sample loading device based on a three-dimensional gel electrophoresis apparatus to identify tumor heterogeneity in human breast cancer adenocarcinoma tissue. For example, a patient's breast cancer tumor underwent three-dimensional gel electrophoresis with a microwell array throughput of 40×40 and a magnification of 2.25x. First, the tumor tissue was embedded and frozen sectioned (thickness: 60 micrometers), then gel electrophoresis was performed, followed by freezing and sectioning analysis of the separated gel medium after electrophoresis. Next, the inventors combined the molecular signal point distribution of the tenth layer in the gel section with the H&E (hematoxylin-eosin staining) of the frozen section of the tumor tissue for analysis, such as... Figure 7 As shown. Frozen sections of tumor tissue obtained by H&E were 10 micrometers thick and were connected to sections obtained by three-dimensional gel electrophoresis. Figure 7In the H&E magnification of the tumor tissue, region 1 represents the stromal tissue. In this region, after proteins bind to dye molecules, there is a significant difference in signal intensity compared to the breast ductal carcinoma tissue in regions 2, 3, and 4. The fluorescence signal intensity in the breast tumor tissue is higher. However, in regions 5, 6, and 7, H&E analysis shows that the tumor tissue in these areas exhibits differentiation into apocrine glands. Correspondingly, the molecular signal intensity in these regions is significantly higher than that of the surrounding cancerous tissue.

[0065] Therefore, it is demonstrated that after using the target three-dimensional high-throughput amplified micropore array sample loading device, the three-dimensional gel electrophoresis device can distinguish between benign and malignant excised tissues and determine the tumor outline. In addition, the three-dimensional electrophoresis method also demonstrates the ability to identify the heterogeneity of tumor tissues and the strong correlation between tissue type and molecular signal points.

[0066] Taking axillary lymph node biopsy samples from three breast cancer patients as an example, the tumor tissue was first embedded and serially frozen sectioned (thickness: 60 micrometers), followed by gel electrophoresis. The separated gel media after electrophoresis was then frozen and sectioned for analysis, with the specific steps consistent with the above-described embodiments. Next, the inventors combined the distribution of molecular signal points in the tenth layer of the gel section with the H&E (hematoxylin-eosin staining) of the frozen tumor tissue sections for analysis, such as... Figure 8 As shown. Serial sections of tumor tissue were alternated with frozen sections taken for H&E, ensuring that each tissue section used for three-dimensional gel electrophoresis corresponded to hematoxylin-eosin staining (H&E) of adjacent tissues. The tissue thickness of the frozen section H&E sections was 10 micrometers. (See attached...) Figure 8 In the three-dimensional gel electrophoresis using a micropore array high-throughput amplification sample loading device, malignant samples 1 and 2, with a width of approximately 1 mm, were distinguishable from benign sample 3. Notably, the molecular signal intensity of the non-tumor regions in sample 2 was also lower than that of the tumor regions.

[0067] The innovations of this application are as follows: Increased throughput of the microwell array, specifically increasing the number of microwells at the tissue sample adhesion end to 100-400 per square centimeter, with a microwell spacing to microwell diameter ratio of 4:3. The increased throughput of the microwell array results in higher pattern analysis capabilities. With increased throughput, the cutting of the same pattern becomes more precise and the reproduction accuracy is higher. The microwell array spacing is also enlarged; specifically, without changing the microwell spacing at the tissue sample adhesion end or the microwell diameter, the microwell spacing at the signal input and output ends of the microwell array is enlarged, resulting in an area of ​​1 to 2.25 times larger for each microwell array unit in the amplified signal input and output ends. The amplified microwell array effectively suppresses signal crosstalk between microwells to ensure the accuracy of signal points. The increased distance between signal points after amplification effectively alleviates signal overlap between microwells. Three-dimensional gel electrophoresis fluorescence pathological analysis is achieved using a high-throughput amplified microwell array sample loading device.

[0068] In this embodiment, tissue slices to be analyzed are obtained, and the tissue slices are attached to the local tissue sample attachment end. Tissue lysis and dye incubation are then performed to obtain lysed and incubated tissue slices. A target three-dimensional high-throughput microwell array meeting preset high-throughput conditions is placed in a local uncured stacking gel until the stacking gel is completely cured. Based on the target three-dimensional high-throughput microwell array, three-dimensional electrophoresis is performed on the lysed and incubated tissue slices. Biomolecules within the lysed and incubated tissue slices aggregate in the stacking gel and are input from the amplification array input end to a local cured separation gel for further three-dimensional electrophoresis to achieve biomolecule separation. The target three-dimensional high-throughput microwell array contains 100 to 400 micropores per square centimeter, and the area magnification of each microwell array unit is 1 to 2.25 times. The biomolecules are proteins or nucleic acids. After three-dimensional electrophoresis, the cured separation gel is sliced ​​to obtain multilayer gel slices. Layer-by-layer analysis is performed on the multilayer gel slices to obtain the spatial signal distribution of biomolecules of various molecular weights in the tissue slices. This application applies to a target three-dimensional high-throughput microwell array sample loading device based on a three-dimensional gel electrophoresis apparatus. Tissue slices to be analyzed are adhered to the local tissue sample attachment end, and tissue lysis and dye incubation are performed to obtain lysed and incubated tissue slices. The target three-dimensional high-throughput microwell array is placed in a local uncured stacking gel until it solidifies. Based on the lysed and incubated tissue slices and the target three-dimensional high-throughput microwell array, three-dimensional electrophoresis is performed on the solidified stacking gel. The resulting electrophoretically separated biomolecules are input into a local solidified separating gel for slicing. This slicing enables high-precision in-situ segmentation, providing high-precision signal information for tissue analysis. It solves the problem that the segmentation accuracy of existing tissue slices cannot meet the requirements for accurate pathological diagnosis of micro-tissues and micro-structures within tissues, and improves the accuracy of analysis and diagnosis of micro-tissues and micro-structures within tissues. This expands the practicality of three-dimensional gel electrophoresis devices in the field of pathological diagnosis, improves the resolution of the microwell array, and enhances the resolution and accuracy of analyzing the spatial signal distribution of biomolecules.

[0069] See Figure 9 As shown, this invention discloses a device for analyzing the spatial signal distribution of biomolecules, applied to a target three-dimensional high-throughput amplified micropore array sample loading device based on a three-dimensional gel electrophoresis apparatus, specifically including:

[0070] The slicing lysis and incubation module 11 is used to obtain tissue slices to be analyzed, attach the tissue slices to be analyzed to the local tissue sample attachment end, and perform tissue lysis and dye incubation to obtain lysed and incubated tissue slices.

[0071] The three-dimensional electrophoresis module 12 is used to place a target three-dimensional high-throughput magnified microwell array that meets preset high-throughput conditions into a local uncured stacking gel until the stacking gel is completely cured. Based on the target three-dimensional high-throughput magnified microwell array, three-dimensional electrophoresis is performed on lysed and incubated tissue sections. Biomolecules in the lysed and incubated tissue sections aggregate in the stacking gel and are input from the magnification array input end to the local cured separation gel for continued three-dimensional electrophoresis to achieve separation of biomolecules. The target three-dimensional high-throughput magnified microwell array contains 100 to 400 microwells per square centimeter, and the area magnification factor of each microwell array unit is 1 to 2.25 times. The biomolecules are proteins or nucleic acids.

[0072] The slicing and analysis module 13 is used to slice the solidified separation gel after three-dimensional electrophoresis to obtain multilayer gel slices, and to perform layer-by-layer analysis on the multilayer gel slices to obtain the spatial signal distribution of each molecular weight biomolecule in the tissue slices.

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

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

[0075] The tissue processing module is used to obtain the initial tissue to be analyzed, and to perform freezing and sectioning on the initial tissue to be analyzed to obtain tissue sections.

[0076] The adhesion module is used to adhere the tissue slices 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] Separating adhesive curing module for performing separating adhesive curing operations in local electric swimming pools;

[0079] The liquid concentrate addition module is used to add uncured concentrate to the top of the cured release adhesive.

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

[0081] The monitoring module is used to place a target three-dimensional high-throughput amplified micropore array that meets the preset high-throughput conditions in a local uncured thickener, and monitor whether the thickener has filled the target three-dimensional high-throughput amplified micropore array.

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

[0083] The electrophoresis solution addition module is used to add electrophoresis solution between the local cathode plate and the cured thickened gel, and between the anode plate and the bottom of the electrophoresis pool, and to connect a constant voltage power supply to perform three-dimensional electrophoresis on the lysed and incubated tissue sections.

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

[0085] The slicing and separation module is used to input the biomolecules aggregated in the stacking gel into the local curing separation gel using the local amplified signal input terminal, and to perform three-dimensional electrophoresis on the cured separation gel.

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

[0087] Figure 10 This is a schematic diagram of an electronic device provided in an embodiment of this 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 stores a computer program, which is loaded and executed by the processor 21 to implement the relevant steps in the biomolecular spatial signal distribution analysis method performed by the electronic device disclosed in any of the foregoing embodiments.

[0088] In this embodiment, the power supply 23 is used to provide operating 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 external devices, and the communication protocol it follows can be any communication protocol applicable to the technical solution of this application, and is not specifically limited here; the input / output interface 25 is used to acquire 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 resource storage, can be a read-only memory, random access memory, disk or optical disk, etc. The resources stored on it include operating system 221, computer program 222 and data 223, etc., and the storage method can be temporary storage or permanent storage.

[0090] The operating system 221 manages and controls the various hardware devices on the electronic device 20 and the computer program 222 to enable the processor 21 to perform calculations and processing on the data 223 in the memory 22. It can be Windows, Unix, Linux, etc. The computer program 222, in addition to including a computer program capable of performing the biomolecular spatial signal distribution analysis method executed by the electronic device 20 as disclosed in any of the foregoing embodiments, may further include computer programs capable of performing other specific tasks. The data 223 may include data received by the biomolecular spatial signal distribution analysis device from external devices, as well as data collected by its own input / output interface 25.

[0091] The steps of the methods or algorithms described in conjunction with the embodiments disclosed herein can be implemented directly by hardware, a software module executed by a processor, or a combination of both. The software module can be located in random access memory (RAM), main memory, read-only memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, removable disk, CD-ROM, or any other form of storage medium known in the art.

[0092] Furthermore, embodiments of this application also disclose a computer-readable storage medium storing a computer program. When the computer program is loaded and executed by a processor, it implements the steps of the method for analyzing the spatial distribution of biomolecules disclosed in any of the foregoing embodiments.

[0093] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0094] The present invention provides a detailed description of a method, apparatus, device, and storage medium for analyzing the spatial distribution of biomolecules. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, those skilled in the art will recognize that there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for analyzing the spatial signal distribution of biomolecules, characterized in that, Applications include target three-dimensional high-throughput amplification micropore array sample loading devices based on three-dimensional gel electrophoresis apparatus, including: Obtain tissue sections to be analyzed, attach the tissue sections to the local tissue sample attachment end, and perform tissue lysis and dye incubation to obtain lysed and incubated tissue sections. A target three-dimensional high-throughput microwell array meeting preset high-throughput conditions is placed in a local uncured stacking gel until the stacking gel is completely cured. Based on the target three-dimensional high-throughput microwell array, lysed and incubated tissue sections are subjected to three-dimensional electrophoresis. Biomolecules in the lysed and incubated tissue sections aggregate in the stacking gel and are input from the input end of the amplification array to a local cured separation gel for further three-dimensional electrophoresis to achieve separation of biomolecules. The target three-dimensional high-throughput microwell array contains 100 to 400 microwells per square centimeter, and the area magnification of each microwell array unit is 1 to 2.25 times. The biomolecules are proteins or nucleic acids. After three-dimensional electrophoresis, the solidified separation gel is sliced ​​to obtain multilayer gel slices. The multilayer gel slices are then analyzed layer by layer to obtain the spatial signal distribution of biomolecules of various molecular weights in the tissue slices.

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

3. The method for analyzing the spatial distribution of biomolecules according to claim 1, characterized in that, The process of obtaining the tissue section to be analyzed, attaching the tissue section to the local tissue sample attachment end, and performing tissue lysis and dye incubation includes: Obtain the initial tissue to be analyzed, and perform freezing and sectioning on the initial tissue to be analyzed to obtain tissue sections. The tissue slice to be analyzed was attached to the local tissue sample attachment end, and the tissue was lysed and incubated with dye 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 magnified micropore array that meets the preset high-throughput conditions into the local uncured stacked adhesive, the method further includes: The separation gel curing process was carried out in a local electric swimming pool; Add uncured binder to the top of the cured release gel.

5. The method for analyzing the spatial signal distribution of biomolecules according to claim 1, characterized in that, The step of placing the target three-dimensional high-throughput magnified micropore array that meets the preset high-throughput conditions in a local uncured concentrate includes: The target three-dimensional high-throughput amplified micropore array that meets the preset high-throughput conditions is placed in the local uncured thickener, and the current thickener is monitored to see if it has filled the target three-dimensional high-throughput amplified micropore 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 includes: Electrophoresis solution was added between the local cathode plate and the cured concentrated gel, and between the anode plate and the bottom of the electrophoresis pool. A constant voltage power supply was then 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 cured separation gel, input from the amplification array input terminal, is then used for three-dimensional electrophoresis to achieve the separation of biomolecules. After three-dimensional electrophoresis, the solidified separating gel is sliced ​​to obtain multilayer gel slices, including: The biomolecules aggregated in the stacking gel are input into the local curing and separating gel using the local amplified signal input terminal, and the curing and separating gel is subjected to three-dimensional electrophoresis. After three-dimensional electrophoresis, the solidified separating gel is frozen, and the frozen separating gel is sliced ​​and separated according to molecular weight along a direction perpendicular to the vertical axis to obtain multilayer gel slices.

8. A device for analyzing the spatial distribution of biomolecules, characterized in that, Applications include target three-dimensional high-throughput amplification micropore array sample loading devices based on three-dimensional gel electrophoresis apparatus, including: The slicing lysis and incubation module is used to obtain tissue slices to be analyzed, attach the tissue slices to be analyzed to the local tissue sample attachment end, and perform tissue lysis and dye incubation to obtain lysed and incubated tissue slices. A three-dimensional electrophoresis module is used to place a target three-dimensional high-throughput magnified microwell array that meets preset high-throughput conditions into a local uncured stacking gel until the stacking gel is completely cured. Based on the target three-dimensional high-throughput magnified microwell array, three-dimensional electrophoresis is performed on lysed and incubated tissue sections. Biomolecules in the lysed and incubated tissue sections aggregate in the stacking gel and are input from the magnification array input end to a local cured separation gel for continued three-dimensional electrophoresis to achieve separation of biomolecules. The target three-dimensional high-throughput magnified microwell array contains 100 to 400 microwells per square centimeter, and the area magnification factor 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 three-dimensional electrophoresis to obtain multilayer gel slices, and to perform layer-by-layer analysis on the multilayer gel slices to obtain the spatial signal distribution of each molecular weight biomolecule in the tissue slices.

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

10. A computer-readable storage medium, characterized in that, Used to store computer programs; wherein, when the computer programs are executed by a processor, they implement the method for analyzing the spatial distribution of biomolecules as described in any one of claims 1 to 7.

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