A dPCR chip and its preparation method

Abstract

This application discloses a dPCR chip and its preparation method, relating to the field of biotechnology, which simplifies the preparation process and reduces preparation costs. A dPCR chip includes: a substrate; and a base adhesive disposed on one side of the substrate. The base adhesive includes a plurality of pores arranged in a non-array configuration on the base adhesive, the pores being obtained by dissolving exposed particles mixed in the base adhesive.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a dPCR chip and its preparation method. Background Technology

[0002] PCR (Polymerase Chain Reaction) is a molecular biology technique that utilizes the principle of DNA (deoxyribonucleic acid) double-strand replication to amplify a large number of specific DNA fragments in vitro. Its key feature is the ability to rapidly amplify a large number of copies using minute amounts of DNA as templates. dPCR (Digital PCR) is an absolute quantification technique for nucleic acid molecules. Compared to qPCR (quantitative real-time PCR), dPCR can directly count the number of DNA molecules, providing absolute quantification of the starting sample. The working principle of dPCR involves dividing a DNA or cDNA (complementary DNA) sample into many individual, parallel PCR reactions. Some of these reactions contain the target molecule (positive), while others do not (negative). The dPCR process generally includes three main steps: liquid distribution, amplification, and fluorescence data reading and analysis. Currently, the mainstream liquid distribution methods are divided into two categories: microwell-based and droplet-based. Existing microwell chips are all array microwell chips, which are mainly prepared directly or indirectly through photolithography. After preparation, the sample to be reacted is injected into the well and PCR reaction is performed. After the reaction is completed, the chip area is imaged or scanned to record the fluorescence intensity in each well, thereby determining the initial concentration of the sample.

[0003] However, microporous dPCR chips are disposable and must be used once. Existing microporous dPCR chips are mainly prepared by photolithography, either directly or indirectly, depending on the materials used. Photolithography is a complicated process with high costs. Summary of the Invention

[0004] This application provides a method for preparing a dPCR chip, which simplifies the preparation process and reduces preparation costs.

[0005] A first aspect of this application provides a dPCR chip, comprising:

[0006] Substrate;

[0007] A base adhesive is disposed on one side of the substrate. The base adhesive includes a plurality of pores arranged in a non-array manner on the base adhesive. The pores are obtained by dissolving exposed particles mixed in the base adhesive.

[0008] In some embodiments, on the surface of the base adhesive on the side away from the substrate, the ratio of the sum of the opening areas of the plurality of holes to the total area of ​​the base adhesive surface ranges from 1% to 7.2%.

[0009] In some embodiments, the opening diameter of the hole on the surface of the substrate adhesive away from the substrate is smaller than the maximum diameter of the hole.

[0010] The maximum aperture of the hole ranges from 20 μm to 200 μm.

[0011] A second aspect of this application provides a method for preparing a dPCR chip, comprising:

[0012] A base adhesive containing multiple particles is deposited on one side of a substrate, wherein the material of the particles is different from the material of the base adhesive.

[0013] The base adhesive is cured.

[0014] The exposed particles in the cured base adhesive are dissolved to form pores.

[0015] In some embodiments, the particle size ranges from 20 μm to 200 μm; and / or,

[0016] The number of particles ranges from 8,000 to 100,000.

[0017] In some embodiments, the particles are hollow spheres.

[0018] In some embodiments, the dissolution treatment of the exposed particles in the cured substrate to form pores includes:

[0019] The exposed portion of the hollow sphere in the cured base adhesive is dissolved to form the pores; or,

[0020] The hollow spheres in the cured base adhesive are completely dissolved to form the pores.

[0021] In some embodiments, the particles are solid spheres;

[0022] The process of dissolving the exposed particles in the cured base adhesive to form pores includes:

[0023] The solid spheres in the cured base adhesive are completely dissolved to form the pores.

[0024] In some embodiments, prior to curing the base adhesive, the process further includes:

[0025] The base adhesive laid on one side of the substrate is subjected to thickness homogenization treatment so that the thickness of the base adhesive falls within a set size range, wherein the set size range is the sum of the outer diameter of the particle and the thickness homogenization process error.

[0026] In some embodiments, the curing process of the base adhesive includes:

[0027] The base adhesive is cured to cause it to shrink, and the ends of the particles that are away from the substrate extend beyond the interface of the cured base adhesive.

[0028] In some embodiments, the volume shrinkage rate of the base adhesive ranges from 3% to 20%.

[0029] In some embodiments, the step of uniformizing the thickness of the base adhesive deposited on one side of the substrate so that the thickness of the base adhesive falls within a predetermined thickness range includes:

[0030] Press the cover plate against the side of the base adhesive away from the substrate, so that the distance between the cover plate and the substrate falls within the set size range;

[0031] After the base adhesive has been cured, the cover plate is removed.

[0032] In some embodiments, pressing the cover plate against the side of the adhesive away from the substrate includes:

[0033] Press the side of the cover plate with the release agent onto the side of the base adhesive away from the substrate.

[0034] The dPCR chip and its fabrication method provided in this application involve laying a substrate containing multiple particles of a base adhesive on one side of the substrate and then curing the base adhesive. After curing, the base adhesive shrinks to a certain extent, exposing some of the particles within the shrunken adhesive; that is, the particles extend beyond the substrate interface. The curing process transforms a flowable colloid into a non-flowable solid, fixing it to the substrate. The exposed particles in the shrunken, cured base adhesive are then dissolved. Since the particle material differs from the base adhesive material, the base adhesive remains unchanged during dissolution, leaving pores at the locations of the dissolved particles. The dPCR chip fabrication steps provided in this application are simple, requiring no photomasks or photolithography equipment, resulting in low production costs. The randomly arranged pores in the chip allow for imaging during subsequent fluorescence signal reading, and this random pore distribution also satisfies the Poisson statistical principle. Attached Figure Description

[0035] Figure 1 A schematic partial structural diagram of a dPCR chip provided in an embodiment of this application;

[0036] Figure 2 A schematic flowchart illustrating the fabrication of a hole, provided for an embodiment of this application;

[0037] Figure 3 This is a schematic diagram of the structure of a hollow sphere after partial dissolution, provided in an embodiment of this application.

[0038] Figure 4 A schematic diagram of the structure of another hollow sphere after partial dissolution, provided in an embodiment of this application;

[0039] Figure 5 A schematic flowchart illustrating a method for preparing a dPCR chip according to an embodiment of this application;

[0040] Figure 6 This is a schematic diagram illustrating the structural changes in a dPCR chip preparation method provided in an embodiment of this application.

[0041] Figure 7 A schematic flowchart illustrating another method for preparing a dPCR chip provided in this application embodiment;

[0042] Figure 8 This is a schematic flowchart illustrating another method for preparing a dPCR chip provided in this application embodiment. Detailed Implementation

[0043] To better understand the technical solutions provided in the embodiments of this specification, the technical solutions of the embodiments of this specification will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this specification and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this specification, rather than limitations on the technical solutions of this specification. In the absence of conflict, the embodiments of this specification and the technical features in the embodiments can be combined with each other.

[0044] In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying 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 limitation, 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. The term "two or more" includes two or more cases.

[0045] PCR is a molecular biology technique that utilizes the principle of DNA double-strand replication to amplify a large number of specific DNA fragments in vitro. Its key feature is the ability to rapidly amplify a large number of copies using minute amounts of DNA as templates. dPCR is an absolute quantification technique for nucleic acid molecules. Compared to qPCR, dPCR can directly count the number of DNA molecules, providing absolute quantification of the starting sample. The working principle of dPCR involves dividing the DNA or cDNA sample into many individual, parallel PCR reactions. Some of these reactions contain the target molecule (positive), while others do not (negative). The dPCR process generally includes three main steps: liquid distribution, amplification, and fluorescence data reading and analysis. Currently, the mainstream liquid distribution methods are divided into two categories: microwell-based and droplet-based. Existing microwell-based chips are all array microwell chips, mainly prepared directly or indirectly through photolithography. After preparation, the sample to be reacted is injected into the wells for PCR. After the reaction is complete, the chip area is imaged or scanned to record the fluorescence intensity in each well, thereby determining the initial concentration of the sample. However, microporous dPCR chips are disposable and must be used once. Existing microporous dPCR chips are mainly prepared by photolithography, either directly or indirectly, depending on the materials used. Photolithography is a complicated process with high costs.

[0046] In view of this, the present application provides a dPCR chip and its preparation method, which can simplify the preparation process and reduce the preparation cost.

[0047] A first aspect of this application provides a dPCR chip. Figure 1 A schematic partial structural diagram of a dPCR chip provided in an embodiment of this application; Figure 2 This is a schematic flowchart illustrating the fabrication of a hole, provided as an embodiment of this application. (In conjunction with...) Figure 1 and Figure 2The dPCR chip provided in this application includes: a substrate 600 and a base adhesive 500. The base adhesive 500 is disposed on one side of the substrate 600 and includes a plurality of holes 700. The plurality of holes 700 are arranged in a non-array on the base adhesive 500. The holes 700 are obtained by dissolving the exposed particles 400 mixed in the base adhesive 500.

[0048] like Figure 2 As shown, the particle 400 is spherical, but its shape can also be ellipsoidal, cubic, cuboid, or other polygonal shapes, etc., which are not specifically limited in this embodiment. Multiple particles 400 can be mixed into the base adhesive 500, uniformly mixed, and then coated onto one side of the substrate 600. Since the material of the particles 400 is different from that of the base adhesive 500, the base adhesive 500 remains unchanged while the particles 400 are dissolved. The dissolved particles 400 leave pores 700 in the base adhesive 500, thus obtaining a dPCR chip. Because the distribution of the particles 400 in the base adhesive 500 is not absolutely uniform, the pores on the dPCR chip are also non-arrayed. During the use of the dPCR chip, samples can be injected into the pores 700.

[0049] The dPCR chip fabrication method provided in this application involves laying a base adhesive containing multiple particles on one side of a substrate and curing the base adhesive. After curing, the base adhesive 500 shrinks to a certain extent, and the particles 400 are partially exposed in the shrunken base adhesive 500, meaning that a portion of the particles 400 extends beyond the interface of the base adhesive 500. The curing process of the base adhesive 500 transforms a flowable colloid into a non-flowable solid, fixing it onto the substrate 600. The exposed particles 400 in the cured and shrunken base adhesive 500 are dissolved. Since the material of the particles 400 is different from that of the base adhesive 500, the base adhesive 500 remains unchanged while the particles 400 are dissolved. The dissolved particles 400 leave pores 700 in the base adhesive 500. The dPCR chip provided in this application has a simple preparation process, does not require the use of a mask and photolithography equipment, has low production cost, and the holes of the chip are randomly arranged, which can be used in imaging mode when reading the fluorescence signal in the later stage. At the same time, this random hole distribution can also satisfy the Poisson statistical principle.

[0050] In some embodiments, on the surface of the base adhesive away from the substrate, the ratio of the sum of the opening areas of the plurality of pores to the total surface area of ​​the base adhesive ranges from 1% to 7.2%. The base adhesive 500 is typically cured before the dissolved particles 400, and the cured base adhesive 500 undergoes volume shrinkage.

[0051] For example, Figure 3 This is a schematic diagram of the structure of a hollow sphere after partial dissolution, provided in an embodiment of this application. Figure 4 This is a schematic diagram of the structure of another hollow sphere partially dissolved according to an embodiment of this application; particle 400 can be a hollow sphere, and the particle size of particle 400 is the outer diameter of the hollow sphere. The volume shrinkage rate of the base adhesive 500 is 3%-20%, and the upper surface of the hollow sphere can be exposed. The substrate carrying the cured UV adhesive and hollow sphere is immersed in HF for a certain period of time to etch away the upper surface of the hollow silica sphere, thus obtaining pores, which can form a non-array dPCR chip. For example, as shown... Figure 3 As shown, the volume shrinkage rate of the corresponding base adhesive 500 is 3%, so the volume of the corresponding base adhesive 500 after shrinkage is 97%, the original thickness is 100%, and the corresponding thickness after shrinkage is 99%, so the thickness shrinkage rate is 1%. Figure 3 The dashed line shown represents the interface of the base adhesive 500 before shrinkage. For example, as... Figure 4 As shown, the volume shrinkage rate of the corresponding base adhesive 500 is 20%, so the volume of the corresponding base adhesive 500 after shrinkage is 80%, the original thickness is 100%, and the corresponding thickness after shrinkage is 92.8%, so the thickness shrinkage rate is 7.2%. Figure 4 The dashed line shown represents the interface of the base adhesive 500 before shrinkage. Figure 3 and Figure 4 It can be seen that the area ratio of the holes obtained after dissolving the hollow spheres on the substrate is affected by the shrinkage rate of the base adhesive 500. That is, the ratio of the sum of the opening areas of multiple holes to the total area of ​​the base adhesive surface ranges from 1% to 7.2%, which is the same as the thickness shrinkage rate of the base adhesive.

[0052] In some embodiments, the opening diameter of the hole 700 on the surface of the base adhesive 500 away from the substrate 600 is smaller than the maximum pore diameter of the hole 700; the maximum pore diameter of the hole 700 ranges from 20 μm to 200 μm. For example, Figure 3 and Figure 4 The opening of hole 700 shown is smaller than the diameter of the hole.

[0053] A second aspect of this application provides a method for preparing a dPCR chip. Figure 5 This is a schematic flowchart illustrating a method for preparing a dPCR chip, as provided in an embodiment of this application. Figure 5 As shown in the embodiments of this application, the method for preparing a dPCR chip includes:

[0054] S100: A base adhesive containing multiple particles is laid on one side of a substrate, wherein the material of the particles is different from the material of the base adhesive.

[0055] For example, Figure 6This is a schematic diagram illustrating the structural changes in a dPCR chip preparation method provided in an embodiment of this application. Figure 6 As shown, in step S100, the particle 400 is spherical in shape. The particle 400 can also be an ellipsoid, cube, cuboid, or other polygonal shape, etc., and this embodiment does not impose specific limitations. Multiple particles 400 can be mixed into the base adhesive 500, uniformly mixed, and then coated onto one side of the substrate 600.

[0056] S200: Curing treatment for the base adhesive. (Reference) Figure 6 After the base adhesive 500 cures, it will shrink to a certain extent, and the particles 400 will be partially exposed in the shrunken base adhesive 500, that is, part of the particles 400 will extend beyond the interface of the base adhesive 500. The curing process of the base adhesive 500 can transform the flowable colloid into a non-flowable solid, so as to fix it on the substrate 600.

[0057] S300: Dissolves exposed particles in the cured substrate to form pores. The exposed particles 400 in the cured and shrunken substrate 500 are dissolved. Since the material of particles 400 is different from that of substrate 500, the substrate 500 remains unchanged while the particles 400 are dissolved. The dissolved particles 400 leave pores 700 in the substrate 500, thus forming a dPCR chip. Because the distribution of particles 400 within the substrate 500 is not perfectly uniform, the pores on the dPCR chip are also non-arrayed. During the use of the dPCR chip, samples can be injected into the pores 700.

[0058] For example, such as Figure 1 As shown, in the local structure of the dPCR chip prepared by the dPCR chip preparation method provided in the embodiments of this application, the holes 700 are not arranged in an array.

[0059] It should be noted that microporous dPCR chips are typically fabricated, depending on the materials used, through direct or indirect photolithography. This involves transferring the desired aperture pattern onto a photoresist coated on a substrate using a photomask, followed by exposure and development using a photolithography machine to etch the apertures. The photolithography process is complex and requires both a photomask and a photolithography machine, resulting in high costs. Since dPCR chips are disposable, reducing their production costs is of paramount importance.

[0060] The dPCR chip fabrication method provided in this application involves laying a base adhesive containing multiple particles on one side of a substrate and curing the base adhesive. After curing, the base adhesive 500 shrinks to a certain extent, and the particles 400 are partially exposed in the shrunken base adhesive 500, meaning that a portion of the particles 400 extends beyond the interface of the base adhesive 500. The curing process of the base adhesive 500 transforms a flowable colloid into a non-flowable solid, fixing it onto the substrate 600. The exposed particles 400 in the cured and shrunken base adhesive 500 are dissolved. Since the material of the particles 400 is different from that of the base adhesive 500, the base adhesive 500 remains unchanged while the particles 400 are dissolved. The dissolved particles 400 leave pores 700 in the base adhesive 500. The dPCR chip provided in this application has a simple preparation process, does not require the use of a mask and photolithography equipment, has low production cost, and the holes of the chip are randomly arranged, which can be used in imaging mode when reading the fluorescence signal in the later stage. At the same time, this random hole distribution can also satisfy the Poisson statistical principle.

[0061] In some implementations, the particle size of particles 400 ranges from 20 μm to 200 μm; the number of particles 400 ranges from 8,000 to 100,000. Typically, the detection requirement for dPCR chips ranges from 8,000 to 100,000 pores, so the number of particles can be roughly within this range. The particle size of particles 400 generally determines the pore size. For example, the particle size of particles 400 can be 100 μm. Particle sizes larger than 200 μm would result in excessively large pores, increasing the amount of sample needed and wasting sample. Particle sizes smaller than 20 μm are difficult to control in terms of pore formation precision, increasing the difficulty of dPCR chip fabrication.

[0062] In some embodiments, particle 400 can be a hollow sphere, and the particle size of particle 400 is the outer diameter of the hollow sphere.

[0063] In some implementations, step S300 includes:

[0064] The exposed portion of the hollow sphere in the cured base adhesive is dissolved to form pores.

[0065] refer to Figure 1 The holes 700 obtained by dissolving the hollow spheres can be connected without creating a connection between the two holes, thus ensuring the independence of each hole 700 and the quantity of holes 700.

[0066] In some implementations, step S300 includes:

[0067] The hollow spheres in the cured base adhesive are completely dissolved to form pores.

[0068] For example, Figure 7 This is a schematic flowchart illustrating another method for preparing a dPCR chip provided in this application embodiment. Figure 7 As shown, step S300 can be to dissolve part of the exposed hollow sphere 401 in the cured base adhesive 500. After the part of the hollow sphere 401 is dissolved, the hollow is exposed. The undissolved part of the hollow sphere 401 can be used as a hole 700. The diameter of the hole is the hollow inner diameter of the hollow sphere 401.

[0069] For example, the hollow sphere 401 can be completely dissolved. After the hollow sphere 401 is dissolved, the original space will be left with a hole 700, and the diameter of the hole 700 is the outer diameter of the hollow sphere 401.

[0070] The embodiments of this application use hollow spheres to prepare dPCR chips. Even if the hollow spheres are connected, it does not affect the spatial independence of the holes. More holes 700 can be prepared in a smaller area, thereby improving the detection accuracy and available data of the chip.

[0071] In some implementations, particle 400 may be a solid sphere.

[0072] Step S300 may include:

[0073] The solid spheres in the cured base adhesive are completely dissolved to form holes.

[0074] For example, refer to Figure 2 , Figure 2 The particle 400 shown can be regarded as a solid sphere. During the dissolution process, the entire solid sphere can be dissolved. Therefore, the diameter of the hole 700 is the outer diameter of the solid sphere.

[0075] In some embodiments, the material of particle 400 includes silicon oxide. The substrate 500 may include photoresist; the substrate 500 may be made of a low-fluorescence background material, such as black adhesive, to avoid interfering with the fluorescence signal during fluorescence signal readout.

[0076] In some implementations, before step S200, the following may also be included:

[0077] The base adhesive laid on one side of the substrate undergoes a thickness homogenization process to ensure its thickness falls within a predetermined range. This predetermined range is the sum of the particle outer diameter and the thickness homogenization process error. The purpose of the thickness homogenization process is to ensure that particles 400 are distributed in a single layer within the base adhesive 500, preventing particle overlap. This prepares the substrate for subsequent dissolution of particles 400, allowing some of the single-layer particles 400 to be exposed on the surface of the base adhesive 500. Dissolving the exposed particles 400 or dissolving all of them leaves pores 700 in the base adhesive 500, ensuring that the pores 700 are distributed in a single layer. It should be noted that fluctuations within the predetermined range primarily depend on the particle size variation of the particles 400 and the process error in the thickness homogenization process of the base adhesive 500.

[0078] In some implementations... Figure 8 This is a schematic flowchart illustrating another method for preparing a dPCR chip, as provided in this application embodiment. Figure 8 As shown, the base adhesive laid on one side of the substrate undergoes a thickness homogenization treatment to ensure that the thickness of the base adhesive falls within a predetermined thickness range, including:

[0079] S001: Press the cover plate 800 onto the side of the base adhesive 500 away from the substrate 600, so that the distance between the cover plate 800 and the substrate 600 falls within a set size range. Then the outer surface of the hollow sphere 401 rests between the cover plate 800 and the substrate 600.

[0080] After step S200, the cover plate 800 is removed.

[0081] Pressing the cover plate against the side of the base adhesive away from the substrate can include:

[0082] Press the side of the cover plate with the release agent onto the side of the base adhesive away from the substrate.

[0083] The cover plate 800 is coated with a release agent, which helps to remove the cover plate 800 from the base adhesive 500 and prevents the cover plate 800 from sticking to the base adhesive 500 during the removal process.

[0084] In some implementations, step 200 may include:

[0085] The base adhesive 500 is cured so that the end of the particle 400 away from the substrate 600 extends beyond the interface of the cured base adhesive 500.

[0086] In some embodiments, the curing process of the base adhesive 500 includes UV curing or thermosetting, and the corresponding base adhesive 500 can be a UV-curable adhesive or a thermosetting adhesive. The thickness of the cured base adhesive 500 is reduced by 3% to 20% compared to the volume of the base adhesive 500 before curing, which allows the particles 400 to be exposed on the surface of the base adhesive 400, facilitating subsequent dissolution steps.

[0087] For example, the steps in preparing a dPCR chip can be:

[0088] Uniformly sized hollow silica spheres are dissolved evenly in uncured UV-cured photoresist. The hollow sphere material should be different from the UV-cured photoresist material to facilitate subsequent dissolution and sacrificial treatment of the hollow spheres. The etching solution required for dissolution must not react with the UV-cured photoresist. The size of the hollow silica spheres is generally related to the size of the holes to be prepared. The number of hollow silica spheres is approximately 20,000, but can be more than 20,000. The UV-cured photoresist should be chosen with minimal background fluorescence; for example, a black photoresist can be selected. The UV-cured photoresist should also have low viscosity to facilitate easier dispersion of the hollow silica spheres. The hollow silica spheres are selected so that the cavities formed after the outer surface dissolves become micropores.

[0089] After the hollow silica spheres are mixed with the UV adhesive, the UV adhesive can be spread evenly on a glass slide of a certain area, such as a 2cm×2cm substrate. The size of the substrate can be determined according to the imaging area later.

[0090] A cover plate with a release agent, such as PTFE or glass, is used to press the UV adhesive flat. The distance between the cover plate and the substrate is the diameter of the hollow sphere. This step is to ensure that the hollow spheres are all on the same plane and do not form a stacked state.

[0091] After curing the UV adhesive, remove the cover plate. The volume of the cured UV adhesive will shrink to a certain extent, while the volume shrinkage rate of the base adhesive is 3%-20%, and the upper surface of the hollow sphere can be exposed.

[0092] The substrate, carrying the cured UV adhesive and hollow spheres, is immersed in HF for a certain period of time. The upper surface of the hollow silica spheres is then etched away to obtain pores, which can form a non-array dPCR chip.

[0093] For example, such as Figure 3 As shown, the volume shrinkage rate of the corresponding base adhesive 500 is 3%, so the volume of the corresponding base adhesive 500 after shrinkage is 97%, the original thickness is 100%, and the corresponding thickness after shrinkage is 99%, so the thickness shrinkage rate is 1%. Figure 3 The dashed line shown represents the interface of the base adhesive 500 before shrinkage.

[0094] For example, such as Figure 4 As shown, the volume shrinkage rate of the corresponding base adhesive 500 is 20%, so the volume of the corresponding base adhesive 500 after shrinkage is 80%, the original thickness is 100%, and the corresponding thickness after shrinkage is 92.8%, so the thickness shrinkage rate is 7.2%. Figure 4 The dashed line shown represents the interface of the base adhesive 500 before shrinkage.

[0095] It should be noted that the software control program corresponding to the dPCR chip preparation method can be burned into the controller, and the controller can be integrated into the dPCR chip preparation device. This application does not make specific limitations.

[0096] It should be noted that the descriptions of each embodiment in the above embodiments have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0097] Those skilled in the art will understand that embodiments of this application can be provided as methods, systems, or computer program products. Therefore, this application can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, this application can take the form of a computer program product embodied on one or more computer-readable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) containing computer-readable program code.

[0098] This application is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this application. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create a machine for implementing the flowchart illustrations. Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0099] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0100] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0101] This application also provides a computer program product, which includes computer software instructions that, when executed on a processing device, cause the processing device to execute the process of a dPCR chip preparation method.

[0102] A computer program product includes one or more computer instructions. When the computer program instructions are loaded and executed on a computer, all or part of the flow or function according to the embodiments of this application is generated. The computer may be a general-purpose computer, a special-purpose computer, a computer network, or other programmable device. The computer instructions may be stored in a computer-readable storage medium or transmitted from one computer-readable storage medium to another. For example, computer instructions may be transmitted from one website, computer, server, or data center to another website, computer, server, or data center via wired (e.g., coaxial cable, fiber optic, digital subscriber line (DSL)) or wireless (e.g., infrared, wireless, microwave, etc.) means. The computer-readable storage medium may be any available medium that a computer can store or a data storage device such as a server or data center that integrates one or more available media. The available medium may be a magnetic medium (e.g., floppy disk, hard disk, magnetic tape), an optical medium (e.g., DVD), or a semiconductor medium (e.g., solid-state disk (SSD)).

[0103] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, devices, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0104] In the several embodiments provided in this application, it should be understood that the disclosed devices, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces, or indirect coupling or communication connection between devices or units, and may be electrical, mechanical, or other forms.

[0105] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0106] Furthermore, the functional units in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0107] If the integrated unit is implemented as a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, or all or part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods of the various embodiments of this application. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0108] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit it. 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 of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

[0109] Although preferred embodiments have been described in this specification, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this specification.

[0110] Obviously, those skilled in the art can make various modifications and variations to this specification without departing from its spirit and scope. Therefore, if such modifications and variations fall within the scope of the claims and their equivalents, this specification is also intended to include such modifications and variations.

Claims

1. A dPCR chip, characterized in that, include: Substrate; A base adhesive is disposed on one side of the substrate. The base adhesive includes a plurality of pores, which are arranged in a non-array manner on the base adhesive. The pores are obtained by dissolving exposed particles mixed in the base adhesive. Before dissolving the particles, the base adhesive needs to be cured. After curing, the base adhesive will shrink in volume, and the particles will be partially exposed in the shrunken base adhesive. The base adhesive is fixed on the substrate, and the base adhesive is made of a low fluorescence background material; the thickness of the base adhesive falls within a set size range, wherein the set size range is the sum of the outer diameter of the particle and the thickness homogenization process error, and the thickness homogenization process is used to make the particle monolayer distributed within the base adhesive. The opening diameter of the hole on the surface of the substrate away from the base adhesive is smaller than the maximum diameter of the hole; the particle includes a hollow sphere or a solid sphere, and the inner diameter of the hole is the inner diameter or outer diameter of the hollow sphere, or the inner diameter of the hole is the outer diameter of the solid sphere.

2. The dPCR chip according to claim 1, characterized in that, On the surface of the base adhesive away from the substrate, the ratio of the sum of the opening areas of the plurality of holes to the total surface area of ​​the base adhesive ranges from 1% to 7.2%.

3. The dPCR chip according to claim 1, characterized in that, The maximum aperture of the hole ranges from 20 μm to 200 μm.

4. A method for preparing a dPCR chip, characterized in that, The method for preparing a dPCR chip as described in any one of claims 1 to 3 comprises: A base adhesive containing multiple particles is deposited on one side of a substrate, wherein the material of the particles is different from the material of the base adhesive. The base adhesive is cured. The exposed particles in the cured base adhesive are dissolved to form pores.

5. The method for preparing a dPCR chip according to claim 4, characterized in that, The particle size ranges from 20 μm to 200 μm; and / or, The number of particles ranges from 8,000 to 100,000.

6. The method for preparing a dPCR chip according to claim 4, characterized in that, The particles are hollow spheres.

7. The method for preparing a dPCR chip according to claim 6, characterized in that, The process of dissolving the exposed particles in the cured base adhesive to form pores includes: The exposed portion of the hollow sphere in the cured base adhesive is dissolved to form the pores; or, The hollow spheres in the cured base adhesive are completely dissolved to form the pores.

8. The method for preparing a dPCR chip according to claim 4, characterized in that, The particles are solid spheres; The process of dissolving the exposed particles in the cured base adhesive to form pores includes: The solid spheres in the cured base adhesive are completely dissolved to form the pores.

9. The method for preparing a dPCR chip according to claim 4, characterized in that, Before curing the base adhesive, the process further includes: The base adhesive laid on one side of the substrate is subjected to thickness homogenization treatment so that the thickness of the base adhesive falls within a set size range, wherein the set size range is the sum of the outer diameter of the particles and the thickness homogenization process error.

10. The method for preparing a dPCR chip according to claim 9, characterized in that, The curing process of the base adhesive includes: The base adhesive is cured so that the end of the particle away from the substrate extends beyond the interface of the cured base adhesive.

11. The method for preparing a dPCR chip according to claim 10, characterized in that, The volume shrinkage rate of the base adhesive ranges from 3% to 20%.

12. The method for preparing a dPCR chip according to claim 10, characterized in that, The step of uniformizing the thickness of the base adhesive laid on one side of the substrate to ensure that the thickness of the base adhesive falls within a predetermined thickness range includes: Press the cover plate against the side of the base adhesive away from the substrate, so that the distance between the cover plate and the substrate falls within the set size range; After the base adhesive has been cured, the cover plate is removed.

13. The method for preparing a dPCR chip according to claim 12, characterized in that, The step of pressing the cover plate onto the side of the base adhesive away from the substrate includes: Press the side of the cover plate with the release agent onto the side of the base adhesive away from the substrate.

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