Array type space microsphere chip as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of space omics chips, and particularly relates to an array type space microsphere chip as well as a preparation method and application thereof. According to the spatial microsphere chip provided by the invention, the circular through holes in array distribution of the through hole film are used as a microsphere limiting area, the diameter and depth of the circular through holes are matched with those of the microspheres, and the microspheres are limited at unique positions after entering the circular through holes under the action of gravity or fluid spreading in cooperation with a regular array distribution structure and a double-sided adhesive film; a round through hole is formed in the substrate, and the round through hole is adhered to the substrate through a double-sided adhesive film, so that a chip bearing system with a stable structure can be formed, the structural stability in the subsequent blowing and beating, biological reaction and slide glass operation processes is ensured, and the risk of microsphere stacking, position deviation or falling off is avoided, so that the spreading and hole falling effects of the microspheres are improved, and the hole falling defect is not easy to occur; the technical problem that an existing space microsphere chip is prone to hole falling is solved.

Description

Technical Field

[0001] This application belongs to the field of space omics chip technology, and in particular relates to an array-type space microsphere chip, its preparation method and application. Background Technology

[0002] Spatial omics technology provides a new research dimension for understanding complex tissue structures and functions by analyzing the spatial distribution of gene expression in tissues and the interactions of cells in the microenvironment, and has become an important development direction in the field of life science and medical research.

[0003] Space omics technology relies on space omics chips to provide tissue localization information. There are various methods to achieve this localization. Microsphere-based space omics chips, namely space microsphere chips, are one of the methods for achieving tissue localization using microspheres. After randomly spreading microspheres with spatial barcodes within a confined area, oligonucleotide probes, capture molecules, or other functional chemical groups modified with microspheres to identify target molecules are used. Through covalent bonds, affinity pairing, or adsorption, the probes or functional molecules are stably immobilized, capturing or labeling nucleic acids or proteins in tissue sections. Combined with high-throughput sequencing and computational reconstruction, the spatial distribution of biomolecules in tissues can be accurately analyzed, thereby obtaining high-resolution spatial expression maps.

[0004] However, during the random spreading process of spatial microsphere chips, the random distribution of microspheres makes it difficult to control the gaps between microspheres and the microsphere positioning accuracy is insufficient, resulting in unsatisfactory microsphere spreading and well placement. While spreading adjustments through methods such as blowing can be effective, the stability of microsphere spreading may lead to microsphere stacking or positional shifts, creating gaps or the risk of detachment. This results in defects such as voids, multiple beads, and abnormal well placement in spatial microsphere chips. These defects can also affect the adhesion stability and incubation conditions of tissue sections, leading to uneven contact between the probe layer and the tissue region, local detachment, or uneven molecular diffusion. Ultimately, this can cause spatial information loss or a decrease in sequencing signal quality, affecting the continuity of spatial signals and data accuracy. Therefore, it is necessary to develop a spatial microsphere chip that allows microspheres to automatically fall into wells under physical constraints within a defined area during random spreading, improving microsphere positioning accuracy and ensuring stable, non-detachment during spreading adjustments such as blowing, thus minimizing well placement defects. Summary of the Invention

[0005] In view of this, this application provides an array-type spatial microsphere chip, its fabrication method, and its application, to solve the technical problem that existing spatial microsphere chips are prone to hole defects.

[0006] The first aspect of this application provides an array-type spatial microsphere chip, including a substrate, a double-sided adhesive film, a through-hole film, and microspheres;

[0007] The substrate and the perforated membrane are bonded together by adhesion on both sides of a double-sided adhesive membrane;

[0008] The microsphere-limiting region of the perforated membrane is an array of circular perforations that accommodate the microspheres.

[0009] Preferably, the substrate is selected from at least one of silicon wafer substrate, glass substrate, polymethyl methacrylate substrate, and quartz substrate.

[0010] Preferably, the double-sided adhesive film is selected from at least one of silicone-based double-sided adhesive tape film and acrylic double-sided adhesive tape film.

[0011] Preferably, the material of the through-hole film is selected from at least one of photoresist material, polymer film material, and inorganic film material.

[0012] Preferably, the circular through holes distributed in the through-hole membrane are at least one of the following: circular through holes distributed in a rectangular array, circular through holes distributed in a circular array, and circular through holes distributed in a ring array.

[0013] Preferably, the circular through holes in the array are evenly spaced, with a spacing of 5~30μm; for example, 12μm or 18μm.

[0014] Preferably, the diameter of the circular through holes arrayed in the through-hole membrane is 1.02 to 1.1 times the diameter of the microspheres; for example, 1.05 times the diameter, while the depth of the circular through holes is greater than the radius of the microspheres, for example, the depth is not less than the radius of the microspheres + 1.5 μm.

[0015] Preferably, the microspheres are selected from at least one of polystyrene microspheres, polyacrylate microspheres, agarose microspheres, silica microspheres, magnetic bead microspheres, and fluorescent microspheres.

[0016] The second aspect of this application provides a method for fabricating an array-type spatial microsphere chip, which can fabricate the array-type spatial microsphere chip described in the first aspect, comprising the following steps:

[0017] A perforated film with an array of circular vias is prepared by attaching a peelable release film layer to the surface of a substrate and covering it with one of photoresist, polymer thin film material or inorganic thin film material as raw material, using micro-nano manufacturing technology.

[0018] A perforated membrane with an array of circular through-holes is peeled off from the release film layer and adhered to the substrate surface using a double-sided adhesive film.

[0019] By employing techniques such as fabric spreading and blowing, electrostatic spraying, or magnetic field adsorption, microspheres in a microsphere suspension are spread into the circular pores of an array distributed in a porous membrane, and then dried to obtain an array-type spatial microsphere chip based on a porous membrane and an adhesive substrate.

[0020] A third aspect of this application provides another method for fabricating an array-type spatial microsphere chip, which can fabricate the array-type spatial microsphere chip described in the first aspect, comprising the following steps:

[0021] A perforated membrane with an array of circular vias is prepared by adhering a double-sided adhesive film to the substrate surface and covering it with one of photoresist, polymer thin film material or inorganic thin film material as raw material, using micro-nano manufacturing technology.

[0022] By employing techniques such as fabric spreading and blowing, electrostatic spraying, or magnetic field adsorption, microspheres in a microsphere suspension are spread into the circular pores of an array distributed in a porous membrane, and then dried to obtain an array-type spatial microsphere chip based on a porous membrane and an adhesive substrate.

[0023] Preferably, in the preparation method described in the second aspect, the peelable release film is selected from NFEP release film, PET release film, PMMA release film, or fluorinated polymer release film.

[0024] Preferably, in the preparation method described in the second or third aspect, the micro / nano manufacturing technology is selected from at least one of two-photon direct writing, nanoimprinting, photolithography, and physical or chemical film deposition combined with etching.

[0025] Preferably, in the preparation method described in the second or third aspect, the resuspending solvent used in the microsphere suspension is selected from at least one of water, ethanol, isopropanol, and DMSO.

[0026] Preferably, the number of microspheres in the microsphere suspension is 2 to 4 times, for example, 3 times, the number of circular pores in the porous membrane.

[0027] The fourth aspect of this application provides the application of an array-type spatial microsphere chip as described in the first aspect in life science research, preparation of medical diagnostic equipment, or preparation of biochips.

[0028] Among them, life science research is used to analyze the spatial distribution of gene expression in tissues and study the cellular microenvironment; while medical diagnostic equipment is used to conduct tumor heterogeneity analysis or discover disease biomarkers; and biochips provide an efficient and accurate chip platform for high-throughput sequencing and spatial omics experiments.

[0029] Compared with the prior art, the array-type spatial microsphere chip provided in this application has at least the following beneficial effects:

[0030] 1. This application provides an array-type spatial microsphere chip, which uses an array of circular vias in a perforated membrane as the microsphere confinement area. The diameter and depth of the circular vias match the microspheres. Combined with a regular array distribution structure and a double-sided adhesive membrane, the microspheres are confined to a single position after entering the circular vias under gravity or fluid spreading. The circular vias are then adhered to the substrate by the double-sided adhesive membrane, forming a structurally stable chip carrier system. This ensures structural stability during subsequent blowing, bioreaction, and wafer loading operations, avoiding the risks of microsphere stacking, positional displacement, or detachment. As a result, the microsphere spreading and placement within the vias is improved, and the defects of placement within the vias are less likely to occur. This allows it to provide explicit spatial coordinates as a regular microsphere array, making downstream space omics experimental data traceable, reducing systematic data loss, and improving data processing efficiency and accuracy.

[0031] 2. The array-type spatial microsphere chip provided in this application can also improve the microsphere material, using magnetic material microspheres in conjunction with magnetic field adsorption technology to improve the microsphere spreading accuracy and efficiency.

[0032] 3. The array-type spatial microsphere chip provided in this application also improves the fabrication process by directly fabricating a through-hole film on a double-sided adhesive film, which simplifies the fabrication process and meets the needs of large-scale industrial fabrication. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram of the microsphere spreading and hole-setting effect in the array-type spatial microsphere chip based on a porous membrane and an adhesive substrate prepared in Example 1 of this application;

[0035] Figure 2 This is a schematic diagram of the microsphere spreading and hole-setting effect in the array-type spatial microsphere chip based on a porous membrane and an adhesive substrate prepared in Example 2 of this application;

[0036] Figure 3 This is a schematic diagram of the microsphere spreading and hole-forming effect in the spatial microsphere chip prepared in Comparative Example 1 of this application. Detailed Implementation

[0037] This application provides an array-type spatial microsphere chip, its fabrication method, and its application, which addresses the technical problem of existing spatial microsphere chips being prone to hole defects.

[0038] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] Example 1

[0040] This embodiment provides a method for fabricating an array of spatial microsphere chips, including a porous membrane fabrication step, a porous membrane transfer and fixation step, and a microsphere spreading and drying step.

[0041] The preparation steps of the through-pore membrane include:

[0042] Select silicon wafer substrate;

[0043] A bubble-free NFEP release film layer was attached to a silicon wafer substrate and SU-8 2000 series photoresist was spin-coated. A through-hole film with an array of circular through holes was prepared by photolithography using a photolithography machine. The through-hole film is arranged in a rectangular, equally spaced array. The diameter of the circular through holes on the through-hole film is 10.5 μm, the depth (film thickness) is 6.5 μm, and the spacing is 12 μm.

[0044] The through-hole membrane transfer and fixation steps include:

[0045] Select a glass slide substrate;

[0046] A perforated membrane with an array of circular through holes is adhered to one side of a high-temperature resistant silicone double-sided adhesive tape, and then peeled off from the NFEP release film layer, transferring the perforated membrane with an array of circular through holes onto the silicone double-sided adhesive tape.

[0047] The other side of the silicone-based double-sided adhesive tape film, that is, the side facing away from the perforated film with an array of circular through holes, is adhered and fixed to the glass slide substrate to complete the transfer and fixation of the perforated film.

[0048] The microsphere spreading and drying steps include:

[0049] Based on 3 times the number of circular holes in the perforated membrane transferred and fixed to the glass slide substrate.

[0050] Polystyrene microspheres with DNA probes were prepared and had a diameter of 10 μm. Anhydrous ethanol was used as the resuspension solvent to prepare a microsphere suspension.

[0051] First, the microsphere suspension is spread onto the porous membrane and dried in an oven at 50°C for 30 minutes. Then, anhydrous ethanol is used to remove the stacked microspheres by blowing, and the microspheres are dried in an oven at 50°C for 10 minutes to obtain an array of spatial microsphere chips based on the porous membrane and the adhesive substrate.

[0052] Example 2

[0053] This embodiment provides a method for fabricating an array of spatial microsphere chips, including a porous membrane fabrication step, a porous membrane transfer and fixation step, and a microsphere spreading and drying step.

[0054] The preparation steps of the through-pore membrane include:

[0055] Select the glass substrate;

[0056] A bubble-free PMMA release film layer was attached to a glass substrate and spin-coated with IP series photoresist. A through-hole film with an array of circular through holes was prepared by a two-photon printer using a two-photon printing process. The through-hole film has a rectangular, equally spaced array distribution. The diameter of the circular through holes on the through-hole film is 15.75 μm, the depth (film thickness) is 9 μm, and the spacing is 18 μm.

[0057] The through-hole membrane transfer and fixation steps include:

[0058] Select a glass slide substrate;

[0059] A perforated membrane with an array of circular through-holes is adhered to one side of a high-temperature resistant acrylic double-sided tape film and peeled off from the PMMA release film layer, transferring the perforated membrane with an array of circular through-holes onto the acrylic double-sided tape film.

[0060] The other side of the acrylic double-sided adhesive tape film, that is, the side facing away from the perforated film with an array of circular through holes, is adhered and fixed to the glass slide substrate to complete the transfer and fixation of the perforated film.

[0061] The microsphere spreading and drying steps include:

[0062] Based on 3 times the number of circular holes in the perforated membrane transferred and fixed to the glass slide substrate.

[0063] Polystyrene microspheres with RNA probes were prepared and had a diameter of 15 μm. The prepared polystyrene microspheres were then suspended in a microsphere suspension using a 10% DMSO aqueous solution as the resuspension solvent.

[0064] First, the microsphere suspension is spread onto the porous membrane and dried in an oven at 50°C for 40 minutes. Then, the stacked microspheres are removed by pipetting with a 10% DMSO aqueous solution and dried in an oven at 50°C for 15 minutes to obtain an array of spatial microsphere chips based on the porous membrane and the adhesive substrate.

[0065] Example 3

[0066] This embodiment provides a method for fabricating an array of spatial microsphere chips, including a porous membrane fabrication step, microsphere spreading, and drying steps.

[0067] The preparation steps of the through-pore membrane include:

[0068] Select the silicon substrate;

[0069] A layer of bubble-free, highly solvent-resistant, and low-emission silicone-based double-sided adhesive tape (preferably made of polyimide and with the silicone adhesive layer highly cross-linked to resist subsequent photoresist solvents and developer) is attached to the surface of a silicon wafer substrate. SU-8 2000 series photoresist is then spin-coated, and a through-hole film with an array of circular vias is prepared using a photolithography machine. The array is rectangular and evenly spaced. The diameter of the circular vias on the through-hole film is 10.5 μm, the depth (film thickness) is 6.5 μm, and the spacing is 12 μm.

[0070] The microsphere spreading and drying steps include:

[0071] Polystyrene microspheres were prepared based on three times the number of circular vias in the via-film on a silicon wafer substrate.

[0072] The polystyrene microspheres, each 10 μm in diameter, were equipped with DNA probes and were suspended in a microsphere suspension using anhydrous ethanol as the resuspension solvent.

[0073] First, the microsphere suspension is spread onto the porous membrane and dried in an oven at 50°C for 30 minutes. Then, anhydrous ethanol is used to remove the stacked microspheres by blowing, and the microspheres are dried in an oven at 50°C for 10 minutes to obtain an array of spatial microsphere chips based on the porous membrane and the adhesive substrate.

[0074] Comparative Example 1

[0075] This comparative example provides a method for preparing a spatial microsphere chip, including a through-hole film preparation step, a through-hole film transfer step, a microsphere spreading and drying step.

[0076] The preparation steps of the through-pore membrane include:

[0077] Select silicon wafer substrate;

[0078] A bubble-free NFEP release film layer was attached to a silicon wafer substrate and SU-8 2000 series photoresist was spin-coated. A through-hole film with an array of circular through holes was prepared by photolithography using a photolithography machine. The through-hole film is arranged in a rectangular, equally spaced array. The diameter of the circular through holes on the through-hole film is 10.5 μm, the depth (film thickness) is 6.5 μm, and the spacing is 12 μm.

[0079] The through-hole membrane transfer step includes:

[0080] Select a glass slide substrate;

[0081] The NFEP release film is peeled off from the silicon wafer substrate, and the side of the NFEP release film facing away from the perforated film with an array of circular vias is attached to the glass slide substrate to complete the perforated film transfer.

[0082] The microsphere spreading and drying steps include:

[0083] Polystyrene was prepared based on three times the number of circular pores in the porous membrane transferred to a glass slide substrate.

[0084] Ethylene microspheres, each containing a DNA probe, are 10 μm in diameter. Anhydrous ethanol is used as the resuspending solvent to prepare a microsphere suspension from the polystyrene microspheres.

[0085] First, the microsphere suspension is spread onto the perforated membrane and dried in an oven at 50°C for 30 minutes. Then, anhydrous ethanol is used to remove the stacked microspheres by blowing, and the membrane is dried in an oven at 50°C for 10 minutes to obtain the spatial microsphere chip.

[0086] Experimental Example 1

[0087] This experiment used a microscope to observe the microsphere spreading and pore-forming effect in the array-type spatial microsphere chips based on porous membranes and adhesive substrates prepared in Examples 1-2 and the spatial microsphere chip prepared in Comparative Example 1. The results are as follows: Figures 1-3 As shown.

[0088] from Figures 1-3 The performance test results shown indicate that the microsphere spreading and hole-forming effects differ in different spatial microsphere chips. Figure 3 The microsphere chip provided in Comparative Example 1 exhibits obvious defects such as voids and poor microsphere spreading and placement. This can easily lead to spatial information loss or decreased sequencing signal quality, which is detrimental to spatial signal continuity and data accuracy. Figure 1-2In the array-type spatial microsphere chips based on perforated membranes and adhesive substrates prepared in Examples 1-2, the microspheres exhibit good spreading and hole-setting effects, without defects such as voids, multiple beads, or abnormal hole-setting. This indicates that in the array-type spatial microsphere chips based on perforated membranes and adhesive substrates prepared in Examples 1-2 of this application, the circular perforations distributed in the perforated membrane serve as microsphere-limiting areas. The diameter and depth of the circular perforations match the microspheres. Combined with the regular array distribution structure and the double-sided adhesive membrane, the microspheres are effectively dispersed and set within the circular perforations under gravity or fluid spreading. Limited to a single location, a flat interface with uniform height, single-layer, regular, and fixed-point structure is formed. At the same time, the through-hole membrane is adhered to the substrate through a double-sided adhesive film, forming a structurally stable chip carrier system. This ensures structural stability during subsequent blowing, bioreaction, and wafer loading operations, avoiding the risks of microsphere stacking, positional displacement, or detachment. As a result, the microsphere spreading and hole-laying effect is improved. It can provide explicit spatial coordinates for regular microsphere arrays, making downstream space omics experimental data traceable, reducing systematic data loss, and improving data processing efficiency and accuracy.

[0089] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. An array-type spatial microsphere chip, characterized in that, This includes substrates, double-sided adhesive membranes, porous membranes, and microspheres; The substrate and the perforated membrane are bonded together by adhesion on both sides of a double-sided adhesive membrane; The microsphere-limiting region of the perforated membrane is an array of circular perforations that accommodate the microspheres.

2. The array-type spatial microsphere chip according to claim 1, characterized in that, The substrate is selected from at least one of silicon wafer substrate, glass substrate, polymethyl methacrylate substrate, and quartz substrate.

3. The array-type spatial microsphere chip according to claim 1, characterized in that, The double-sided adhesive film is selected from at least one of silicone-based double-sided adhesive tape film and acrylic double-sided adhesive tape film.

4. The array-type spatial microsphere chip according to claim 1, characterized in that, The circular through-holes in the through-hole membrane are distributed in an array, and are at least one of the following: a rectangular array of circular through-holes, a circular array of circular through-holes, and a ring array of circular through-holes.

5. An array-type spatial microsphere chip according to claim 1, characterized in that, The diameter of the circular through-holes distributed in the array in the porous membrane is 1.02 to 1.1 times the diameter of the microspheres, and the depth is greater than the radius of the microspheres.

6. The array-type spatial microsphere chip according to claim 1, characterized in that, The microspheres are selected from at least one of the following: polystyrene microspheres, polyacrylate microspheres, agarose microspheres, silica microspheres, magnetic beads microspheres, and fluorescent microspheres.

7. A method for fabricating an array of spatial microsphere chips, characterized in that, An array-type spatial microsphere chip according to any one of claims 1-6 can be prepared by the following steps: A perforated film with an array of circular vias is prepared by attaching a peelable release film layer to the surface of a substrate and covering it with one of photoresist, polymer thin film material or inorganic thin film material as raw material, using micro-nano manufacturing technology. A perforated membrane with an array of circular through-holes is peeled off from the release film layer and adhered to the substrate surface using a double-sided adhesive film. By employing techniques such as fabric spreading and blowing, electrostatic spraying, or magnetic field adsorption, microspheres in a microsphere suspension are spread into the circular pores of an array distributed in a porous membrane, and then dried to obtain an array-type spatial microsphere chip based on a porous membrane and an adhesive substrate.

8. A method for fabricating an array of spatial microsphere chips, characterized in that, An array-type spatial microsphere chip according to any one of claims 1-6 can be prepared by the following steps: A perforated membrane with an array of circular vias is prepared by adhering a double-sided adhesive film to the substrate surface and covering it with one of photoresist, polymer thin film material or inorganic thin film material as raw material, using micro-nano manufacturing technology. By employing techniques such as fabric spreading and blowing, electrostatic spraying, or magnetic field adsorption, microspheres in a microsphere suspension are spread into the circular pores of an array distributed in a porous membrane, and then dried to obtain an array-type spatial microsphere chip based on a porous membrane and an adhesive substrate.

9. A method for fabricating an array-type spatial microsphere chip according to claim 7 or 8, characterized in that, The micro / nano manufacturing technology is selected from at least one of two-photon direct writing, nanoimprinting, photolithography, and physical or chemical film deposition combined with etching.

10. The application of an array-type spatial microsphere chip according to any one of claims 1-9 in life science research, preparation of medical diagnostic equipment, or preparation of biochips.

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