Chip, application and method for preparing chip

Abstract

The present application discloses a chip, application and method for preparing the chip. The chip includes a first substrate and a second substrate, the second substrate is stacked with the first substrate, the second substrate includes a first surface and a second surface opposite to each other, wherein the first surface of the second substrate faces the first substrate, and one or more fluid channels are arranged between the first surface of the second substrate and the first substrate; the chip also includes a coating arranged on the second surface of the second substrate, and the coating has a spontaneous fluorescence intensity less than a preset intensity. The chip in the embodiment of the present application includes a basic structure including a fluid channel formed by stacking the first substrate and the second substrate, and a specific coating is arranged on the second surface of the second substrate, and the coating has a weak spontaneous fluorescence characteristic. The chip is particularly suitable for a platform for imaging the chip based on an optical system to detect biological molecules in the chip.

Description

Technical Field

[0001] The present application relates to the field of nucleic acid detection, and in particular to a chip, an application and a method for preparing the chip. Background Art

[0002] The chip adapted to the sequencing platform is a reaction device that can carry the nucleic acid to be tested and can contain a solution to provide a reaction environment or a detection environment for the nucleic acid to be tested, also known as a flow chamber or a flow cell.

[0003] Two pieces of glass (at least one of the two opposite surfaces of the two pieces of glass is etched) and an opaque substrate may be bonded and packaged using an adhesive to form a chip with a space / cavity inside.

[0004] On a platform that realizes sequencing based on an optical imaging system detection chip (sometimes referred to as a sequencer), by imaging a specific position of the chip (a position connected to the nucleic acid molecule to be tested, sometimes also referred to as a reaction area or a fluid channel), the base arrangement order of the nucleic acid molecule to be tested is identified and determined based on the information of these images. For example, specifically, in a platform that uses fluorescently labeled nucleotides and performs sequencing based on the principle of sequencing by synthesis, during sequencing, a high-energy laser emitted by a laser in the sequencer is irradiated to the reaction area of ​​the chip through a lens, and the nucleic acid molecule to be tested in the reaction area is placed in a reagent solution. The laser irradiates the fluorescent molecules in the reagent solution and excites them to emit fluorescent signals, and then these fluorescent signals are collected, such as taking pictures to obtain images, and the base arrangement order is identified and determined based on the information on these images to achieve the purpose of sequencing.

[0005] The above sequencing platform uses an optical system to image the chip and detect based on the image. It is understandable that the higher the signal-to-noise ratio of the image, the more accurate and reliable the sequencing result. For the above chip, while irradiating the fluorescent molecules, the laser will also shine through the glass onto the opaque substrate and / or the adhesive between the substrate and the glass, and excite the molecules in the substrate and / or the adhesive to emit light, generating noise interference, which will affect the final sequencing results.

[0006] Therefore, the structure and / or preparation method of the chip needs to be improved. Summary of the invention

[0007] Embodiments of the present application provide a chip, an application, and a method for preparing the chip.

[0008] The chip of the embodiment of the present application includes a first substrate, a second substrate and a coating. The second substrate is stacked with the first substrate, the second substrate includes a first surface and a second surface opposite to each other, the first surface of the second substrate faces the first substrate, and one or more fluid channels are provided between the first surface of the second substrate and the first substrate; the coating is provided on the second surface of the second substrate, and the autofluorescence intensity of the coating is less than a preset intensity.

[0009] The chip of the embodiment of the present application includes a basic structure with a fluid channel formed by stacking a first substrate and a second substrate, and a specific coating is provided on the second surface of the second substrate. In applications involving the use of an optical imaging system to detect signals from a sample to be tested on the chip, such as a sequencing platform based on optical imaging to detect the fluorescent signals of nucleic acid molecules in the chip to achieve nucleic acid sequence determination, the fluorescent signal (background signal) emitted by the chip itself when irradiated, i.e., excited by the excitation light, is very weak, which is conducive to obtaining images with a high signal-to-noise ratio (SNR), facilitating the identification of target fluorescent signals, and facilitating the acquisition of high-quality sequencing results.

[0010] In addition, according to the implementation of the present application, the chip may also have at least one of the following additional technical features.

[0011] In some embodiments, the coating is applied on the second surface of the second substrate. This reduces the amount of excitation light transmitted through the second substrate, which helps to reduce the fluorescence emitted by the structure below the second substrate when excited by the transmitted excitation light. The so-called coating includes smearing, brushing, printing, etc.

[0012] In some embodiments, the thickness of the coating ranges from 5 μm to 20 μm. The coating with this thickness has a good shielding effect on the excitation light, so that the image of the specific area of ​​the chip collected by irradiation with the excitation light can meet the sequencing requirements.

[0013] Preferably, the thickness of the coating layer is in the range of 8 μm to 15 μm. The coating layer with this thickness has a good blocking effect on the excitation light passing through the substrate.

[0014] In some embodiments, the light shielding rate of the coating under the working environment is not less than 80%. The coating has such a light shielding rate under the working environment, which is conducive to collecting images of specific areas of the chip with a high signal-to-noise ratio, and is conducive to obtaining high-quality sequencing results. For a sequencing platform based on optical imaging to detect the fluorescent signals of nucleic acid molecules in the chip to achieve nucleic acid sequence determination, the working environment here includes a laser of a specific wavelength and intensity; in one example, the working environment refers to a red or green laser of 800-1000mW (for example, a laser with a light emission wavelength of 532nm or 635nm).

[0015] In some embodiments, the flatness of the coating layer facing away from the second substrate is allowed to deviate by no more than 0.1 μm. In this way, after being stacked, connected and packaged with other structures such as substrates, the deviation / tolerance of the mechanically accumulated surface flatness can be ensured to be within a preset range, so that the flatness of the chip surface meets the predetermined requirements, and it is also beneficial for the coating layer to be stably and firmly connected to the corresponding surface of the third substrate, and for the chip structure to be firm and stable.

[0016] In some embodiments, the material of the coating includes ink. In this way, the chip with the coating has high light-shielding properties and weak luminescence properties in a working environment, and is easy to prepare. In particular, in one example, the coating is black ink, and a high-quality image of a specific area of ​​the chip can be collected. Moreover, by printing on the corresponding surface of the second substrate to obtain a second substrate with a black coating, a coating that meets the requirements or a chip including a second substrate with a coating can be quickly and easily obtained.

[0017] In some embodiments, the first substrate includes a first surface and a second surface opposite to each other, the fluid channel is formed between the second surface of the first substrate and the first surface of the second substrate, the background intensity of the image of the chip is less than or equal to a preset value, and the image of the chip is an image of the second surface of the first substrate and / or an image of the first surface of the second substrate in the working environment. Preferably, the background intensity of the image of the chip is less than or equal to 400.

[0018] The so-called chip image is an image of one or more regions / field of view (FOV) of the chip containing the molecules to be detected. The so-called preset value is set by the inventors by comparing and calculating a large number of images corresponding to better and worse sequencing results obtained under the same working environment. The preset value is used to evaluate and determine the image of the collected chip, and the image is quickly evaluated and fed back, which is conducive to deciding whether to further process the image, whether to continue to acquire the image, whether to adjust the optical imaging system, whether to refocus, and evaluate the chip quality, etc.

[0019] In some embodiments, the chip includes a third substrate attached to the coating, and the third substrate is made of metal, so as to ensure the stability of the planarity and the temperature conduction between the first substrate and the second substrate.

[0020] In some embodiments, the chip includes an interposer disposed between a first substrate and a second substrate, the interposer connects the first substrate and the second substrate, and one or more fluid channels are disposed in the interposer.

[0021] In some embodiments, the intermediary layer is bonded to the first substrate and the second substrate, the intermediary layer has a hollow structure, and the intermediary layer includes:

[0022] A base layer having a first surface and a second surface opposite to each other;

[0023] A first adhesive layer is disposed on the first surface of the base layer, and the first adhesive layer is bonded to the first substrate;

[0024] The second adhesive layer is disposed on the second surface of the base layer, and the second adhesive layer is bonded to the second substrate; and the fluid channel is a hollow structure formed by penetrating the base layer, the first adhesive layer, and the second adhesive layer. In this way, it is not necessary to etch the fluid channel on the first substrate and the second substrate, which simplifies the manufacturing process of the fluid channel and the preparation process of the chip.

[0025] In some embodiments, the first substrate and / or the second substrate is provided with a through hole connected to the fluid channel, so that the reaction reagent and other fluids can enter the fluid channel through the first substrate and / or the second substrate, and can also flow out through the first substrate and / or the second substrate after a chemical reaction occurs in the fluid channel, and it is also convenient to connect a pipeline or a manifold to connect the valve body and the reaction reagent container.

[0026] In some embodiments, the peeling force of the first adhesive layer on the first substrate and / or the peeling force of the second adhesive layer on the second substrate is not less than 560 g. In this way, it can be ensured that the bonding strength of the first adhesive layer on the first substrate and / or the second adhesive layer on the second substrate meets the operation requirements; for example, the bonding peeling force between the adhesive layer and the substrate is not less than the specified value, which can ensure that the various structures of the chip are firmly connected and the chip structure is stable, meeting the sequencing requirements.

[0027] Preferably, the peeling force of the first adhesive layer on the first substrate and / or the peeling force of the second adhesive layer on the second substrate is not less than 800 g. In this way, it can be ensured that the bonding strength of the first adhesive layer on the first substrate and / or the second adhesive layer on the second substrate meets the operation requirements, so that the chip can be well used for sequencing.

[0028] In some embodiments, the size of the fluid channel in the first direction is greater than its size in the second direction, the first direction is perpendicular to the second direction, and the first direction and the second direction are both perpendicular to the thickness direction of the interposer. In this way, the general shape of the fluid channel formed in the interposer is standardized, which is conducive to controlling the fluid in the fluid channel and facilitating the positioning and imaging of these areas of the chip.

[0029] In some embodiments, there are multiple fluid channels, and the fluid channels are arranged in the interposer along a first direction; and / or the fluid channels are arranged in an array on the interposer along a second direction. In this way, multiple fluid channels can make the sequence determination process more efficient, facilitate the control of the fluid in the fluid channels, and facilitate the positioning and imaging of those areas of the chip.

[0030] In some embodiments, the fluid channel includes a middle section, a first end, and a second end, the first end and the second end are located at two ends of the fluid channel, respectively, and the size of the first end in the second direction and / or the size of the second end in the second direction is smaller than the size of the middle section in the second direction. In this way, the shape of the fluid channel is further standardized, which is conducive to controlling the fluid in the fluid channel and facilitating the positioning and imaging of these areas of the chip.

[0031] In some embodiments, the size of the middle segment in the second direction is constant, that is, the length of the middle segment in the second direction is the same everywhere.

[0032] In some embodiments, the size of the middle section in the second direction ranges from 4.4 mm to 8.4 mm. Thus, a reasonable size range enables the fluid channel to have a certain width to accommodate the reaction reagents, facilitates the fluid control therein, and facilitates efficient biochemical reactions therein.

[0033] In some embodiments, the spacing between two adjacent fluid channels in the second direction ranges from 0.8 mm to 1.5 mm. In this way, it is convenient to process multiple fluid channels on the interposer, while ensuring the number of fluid channels to achieve efficient sequencing as much as possible.

[0034] In certain embodiments, the thickness of the base layer ranges from 30 μm to 50 μm; and / or,

[0035] The thickness of the first adhesive layer is in the range of 75 μm to 85 μm; and / or,

[0036] The thickness of the second adhesive layer ranges from 75 μm to 85 μm. Thus, the first adhesive layer and the second adhesive layer have a certain thickness, thereby ensuring the peeling force of the first adhesive layer / the second adhesive layer on the first substrate / the second substrate, thereby ensuring the normal progress of the biochemical reaction in the intermediate layer.

[0037] In some embodiments, the base layer, the first adhesive layer and / or the second adhesive layer can withstand a temperature of not less than 80° C. Thus, the base layer, the first adhesive layer and / or the second adhesive layer will not deform significantly during the biochemical reaction, thus ensuring the normal progress of the biochemical reaction in the intermediary layer.

[0038] In some embodiments, the first adhesive layer and / or the second adhesive layer can withstand a temperature of not less than 110° C. Thus, the first adhesive layer and / or the second adhesive layer will not be significantly deformed during the biochemical reaction, thus ensuring the normal progress of the biochemical reaction in the intermediate layer.

[0039] In some embodiments, the base layer, the first adhesive layer and / or the second adhesive layer are resistant to a specified solvent. Thus, the base layer, the first adhesive layer and / or the second adhesive layer do not have problems such as adhesive layer and film layer shedding and failure in the specified solvent, thereby ensuring the normal progress of the biochemical reaction in the intermediary layer.

[0040] In some embodiments, the material of the base layer includes polyimide. Thus, the base layer can withstand a certain high temperature and can also meet the requirement of withstanding a specified solvent.

[0041] In some embodiments, the first adhesive layer and the second adhesive layer are made of the same material, so that the manufacturing process of the first adhesive layer and the second adhesive layer is simple and can be manufactured together.

[0042] In some embodiments, the material of the first adhesive layer and / or the second adhesive layer includes silicone, such as pressure sensitive silicone (PSA silicone). In this way, the first adhesive layer and / or the second adhesive layer can withstand a certain high temperature and can also meet the requirement of withstanding a specified solvent.

[0043] Use of the chip in any of the above embodiments in sequence determination.

[0044] The present application also provides a method for preparing a chip, which can be used to prepare the chip in any of the above embodiments. The method includes: providing a first substrate; providing a second substrate, the second substrate including a first surface and a second surface opposite to each other; stacking the second substrate on the first substrate, the second substrate including a first surface and a second surface opposite to each other, so that the first surface of the second substrate faces the first substrate; setting one or more fluid channels between the first surface of the second substrate and the first substrate; and setting a coating on the second surface of the second substrate, wherein the autofluorescence intensity of the coating is less than a preset intensity.

[0045] The preparation method has a simple process, and because a coating is provided on the second surface of the second substrate, it is advantageous to obtain an image with a high signal-to-noise ratio, to identify a target fluorescent signal, and to obtain high-quality sequencing results.

[0046] In some embodiments, the method further includes: providing a third substrate; and placing the third substrate under the coating. In this way, the first substrate, the second substrate and the third substrate are bonded to form the main body of the chip, and the third substrate is conducive to ensuring the stability of the planarity and temperature conductivity of the first substrate and the second substrate.

[0047] In some embodiments, the method further includes: providing an interposer; arranging the interposer between the first substrate and the second substrate, and arranging one or more fluid channels in the interposer. In this way, it is not necessary to etch the fluid channels on the first substrate and the second substrate, thereby simplifying the manufacturing process of the fluid channels.

[0048] In some embodiments, an interposer is provided, including: providing a base layer, the base layer having a first surface and a second surface opposite to each other; disposing a first adhesive layer on the first surface of the base layer; disposing a second adhesive layer on the second surface of the base layer; forming a fluid channel penetrating the base layer, the first adhesive layer, and the second adhesive layer; the method further includes: bonding the first adhesive layer to the first substrate, and bonding the second adhesive layer to the second substrate. In this way, the interposer can bond the first substrate and the second substrate, and the hollow structure of the interposer after bonding forms a corresponding fluid channel, so that there is no need to etch the fluid channel on the first substrate and the second substrate, simplifying the manufacturing process of the chip containing the fluid channel.

[0049] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0051] Figure 1 is a schematic diagram of the structure of a chip in an embodiment of the present application;

[0052] Figure 2 is a cross-sectional schematic diagram of a chip in an embodiment of the present application;

[0053] Figure 3 In the embodiment of this application Figure 2 The enlarged schematic diagram of P in the middle;

[0054] Figure 4 is a schematic structural diagram of an intermediary layer in an embodiment of the present application;

[0055] Figure 5 is a cross-sectional schematic diagram of an interposer in an embodiment of the present application;

[0056] Figure 6 is a schematic diagram of the background intensity of the image collected in the implementation manner of the present application;

[0057] Figure 7 is a schematic flow chart of a method for preparing a chip in an embodiment of the present application;

[0058] Figure 8 is another schematic flow chart of the method for preparing a chip in an embodiment of the present application;

[0059] Fig. 9 is another schematic flow chart of the method for preparing a chip in an embodiment of the present application;

[0060] Fig.10It is another schematic flow chart of the method for preparing a chip in an embodiment of the present application.

[0061] Description of main component symbols:

[0062] Chip 100, lens 200, housing 10, first substrate 20, second substrate 30, first surface 21 of first substrate 20, second surface 22 of first substrate 20, first surface 31 of second substrate 30, second surface 32 of second substrate 30, through hole 33, intermediate layer 40, base layer 41, first surface 411 of base layer 41, second surface 412 of base layer 41, first adhesive layer 42, second adhesive layer 43, coating 50, third substrate 60, adhesive 61, fluid channel 70, middle section 71, first end 72, second end 73. DETAILED DESCRIPTION

[0063] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0064] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0065] In the present application, "plurality" means two or more than two, unless clearly defined otherwise.

[0066] Most of the specific data / values ​​involved in the description of this application are statistically significant. Therefore, unless otherwise specified, any numerical value expressed in a precise manner represents a range, that is, an interval including plus or minus 10% of the numerical value, which will not be repeated below.

[0067] In the present application, the so-called "chip" is a reaction chamber containing a solid phase substrate, having a space for accommodating liquid, and can be used to fix the sample to be tested, also known as a flow cell or flow cell. "Solid substrate" such as the substrate here can be any solid support that can be used to fix nucleic acid sequences, such as nylon membrane, glass sheet, plastic, silicon wafer, magnetic beads, etc. In some examples, the first substrate and the second substrate are light-transmissive, for example, both are glass sheets / glass layers.

[0068] In the present application, the term "sequencing" refers to sequence determination, which is the same as "nucleic acid sequencing" or "gene sequencing", and refers to the determination of the base order in a nucleic acid sequence; it includes synthesis sequencing (sequencing by synthesis, SBS) and / or ligation sequencing (sequencing by ligation, SBL); it includes DNA sequencing and / or RNA sequencing; it includes long fragment sequencing and / or short fragment sequencing, and the so-called long fragments and short fragments are relative, such as nucleic acid molecules longer than 1Kb, 2Kb, 5Kb or 10Kb can be called long fragments, and those shorter than 1Kb or 800bp can be called short fragments; it includes double-end sequencing, single-end sequencing and / or paired-end sequencing, etc., and the so-called double-end sequencing or paired-end sequencing can refer to the reading of any two segments or two parts of the same nucleic acid molecule that do not completely overlap.

[0069] The so-called sequencing includes the process of binding nucleotides (including nucleotide analogs) to the template and collecting the corresponding reaction signals. In some sequencing platforms that asynchronously bind nucleotides to the template and collect the corresponding reaction signals, the order of multiple nucleotides / bases on the template is generally determined through multiple rounds of sequencing. A round of sequencing (cycle) is also called a sequencing round, which can be defined as a base extension of four nucleotides / bases. In other words, it can be defined as the process of completing the determination of the base type at any specified position on the template; for sequencing platforms that realize sequencing based on controlled polymerization or ligation reactions, a round of sequencing includes the process of realizing the binding of four nucleotides to the so-called template once and collecting the corresponding reaction signals; for platforms that realize sequencing based on polymerization reactions, the reaction system includes reaction substrate nucleotides, polymerase and template, so that a preset sequence (sequencing primer) is bound to the template, based on the base pairing principle. According to the polymerization reaction principle, the added reaction substrate (nucleotide) is controllably connected to the 3' end of the sequencing primer under the catalysis of the polymerase to achieve pairing with the base at the corresponding position of the template; usually, one round of sequencing may include one or more base extensions (repeat), for example, four nucleotides are added to the reaction system in sequence, and base extensions and corresponding reaction signals are collected respectively, and one round of sequencing includes four base extensions; for another example, the four nucleotides are added to the reaction system in any combination, such as two-by-two combinations or one-by-three combinations, and base extensions and corresponding reaction signals are collected respectively in two combinations, and one round of sequencing includes two base extensions; for another example, the four nucleotides are added to the reaction system at the same time for base extension and reaction signal collection, and one round of sequencing includes one base extension.

[0070] Sequencing can be performed through a sequencing platform, which can be selected from but not limited to Illumina's HiSeq / Miseq / Nextseq / Novaseq sequencing platforms, Thermo Fisher / Life Technologies' IonTorrent platforms, BGISEQ and MGISEQ / DNBSEQ platforms of BGI, and single-molecule sequencing platforms; the sequencing method can be single-end sequencing or double-end sequencing.

[0071] Commonly, a chip adapted to the SBS sequencing platform may include one or more parallel channels / fluid channels (channels), which are used for the entry and exit and for carrying reagents to form the environment required for the sequence determination reaction. The chip body can be made of two pieces of glass and a metal base plate bonded and packaged. The sequencing process includes an imaging system such as a camera taking multiple photos of one or more areas of the chip. Each area photographed can be called Fov (field of view). Between two cycles, reagents are reintroduced for biochemical reactions.

[0072] See also Figure 1-Figure 4 The embodiment of the present application provides a chip 100, which includes a first substrate 20 and a second substrate 30, and the second substrate 30 is stacked with the first substrate 20. The second substrate 30 includes a first surface 31 and a second surface 32 opposite to each other, the first surface 31 of the second substrate 30 faces the first substrate 20, one or more fluid channels 70 are arranged between the first surface 31 of the second substrate 30 and the first substrate 20, and a coating 50 is arranged on the second surface 32 of the second substrate 30, and the autofluorescence intensity of the coating 50 is less than a preset intensity.

[0073] In the chip 100 of the embodiment of the present application, the basic structure of the chip 100 is formed by stacking the first substrate 20 and the second substrate 30, and one or more fluid channels 70 are provided between the first substrate 20 and the first surface 31 of the second substrate 30, so that the chip 100 can carry the sample to be tested, including the solution or reagent, to provide a solution environment for biochemical reactions or specific detection. Furthermore, by providing a coating 50 on the second surface 32 of the second substrate 30, the excitation light passing through the second substrate 30 is reduced. The reduction of the excitation light passing through the second substrate 30 is conducive to weakening the fluorescence emitted by the structure below the second substrate 30 due to the excitation light passing through. Since the autofluorescence intensity of the coating 50 is less than the preset intensity, the fluorescence intensity generated by the coating 50 irradiated with the excitation light is relatively weak. Therefore, under the working environment, the fluorescence signal generated by the chip 100 itself is very weak. The chip 100 is suitable for carrying biological samples for realizing biomacromolecule detection, and is suitable for an optical imaging platform for realizing detection of samples to be tested based on a detection chip.

[0074] See also Figure 1 and Figure 4 In one example, the chip 100 is placed under an optical system such as a microscope for detection. The nucleic acid molecule to be detected is fluorescently labeled. The nucleic acid molecule to be detected is located in the fluid channel 70 and one end is connected to the second surface 22 of the first substrate 20 and / or one end is connected to the first surface 31 of the second substrate 30. The optical system includes a light source such as a laser and a camera. The camera includes a lens 200. The laser emits a laser of a specific wavelength to irradiate the fluid channel 70 of the chip 100, stimulating the fluorescent marker therein to emit fluorescence. The lens 200 included in the camera is used to take pictures of the fluorescent areas to obtain images, thereby realizing signal acquisition of the sample to be tested. Then, based on processing and analyzing the image, the detection of the sample to be tested can be realized.

[0075] Specifically, on a sequencing platform that realizes nucleic acid sequencing based on chip detection, a chip adapted to the sequencing platform can usually be made by stacking and packaging two layers of glass and a metal base plate with good thermal conductivity, such as an aluminum plate, and bonding the three-piece structure using an adhesive such as a water-based adhesive or a double-sided adhesive. Among them, the lower layer of glass can be bonded to the aluminum plate by an adhesive, and the development of current material science and technology, such as surface processing, etching and bonding packaging technology, generally ensures that the plane flatness of the upper and lower glass layers of the chip and the stability of the temperature conductivity of the chip meet the requirements of the sequencing platform.

[0076] The requirements of the sequencing platform referred to here are understandable. For an automated sequencing platform including an optical system, it generally includes continuous photography of multiple areas on the chip; in particular, for a sequencing platform including a high-magnification lens such as an optical system of 20× or more, the range in which the sample to be tested can be clearly imaged is usually micrometer-level or nanometer-level (focal plane / clear plane), so there are high requirements for the adapted chip, for example, the surface flatness of the chip is very high. Moreover, the sequencing reaction usually involves a variety of biochemical reactions, the use of corrosive solutions and short-term temperature rise and fall, etc., so there are more and higher requirements for the physical and chemical properties of the structure containing / contacting the solution, the connection strength, etc.; and, during the preparation process of the chip, it is usually necessary to apply a large force at a high temperature to make the various structures fit closely, so there are many requirements and restrictions on the pressure tolerance, temperature tolerance, bonding strength, etc. of the chip adapted to the sequencing platform and its various component structures. If it is further desired that the chip consumes low reagents, for example, to achieve rapid detection of samples of nanoliters or smaller volumes, microfluidic technology is involved, and there are even more requirements for the structure, connection, material and its processing technology.

[0077] However, the inventors discovered during the structural design, preparation and testing of the chip 100 adapted to the sequencing platform that when the laser emitted by the laser of the sequencing platform is irradiated into the fluid channel 70 of the chip 100 through the lens 200, it will also be irradiated through the lower layer of glass onto the glue layer connecting the lower layer of glass and the aluminum plate. The molecules of the glue layer are therefore excited to emit fluorescence, which greatly interferes with the recognition and detection of the target signal, i.e., the signal from the nucleic acid molecule to be tested in the fluid channel 70.

[0078] Based on a large amount of prior test data, including image data of chips with better and worse quality obtained under specific working environments, the inventors measured and compared the characteristics and parameters of the corresponding chips, such as the thickness of each coating on the chip, surface flatness, adhesive adhesion, adhesive chemical properties and optical properties, and set expectations / requirements for the surface of a metal base plate such as an aluminum plate and expectations / requirements for the glue used to bond the aluminum plate. However, after investigating and entrusting a number of suppliers in this field to perform processing and testing, it was found that there are basically no suppliers on the market that can provide processing technology and / or glue to achieve the set expectations / requirements. In other words, the yield rate of chips that meet the expectations / requirements prepared by the technology / process provided on the market is very low, which means that the cost of preparing the chip is very high. As a result, the high cost and difficulty in controlling the structural parameters are very unfavorable for the industrial production of chips adapted to sequencing platforms and the further improvement of their performance.

[0079] However, the chip 100 with the above-mentioned features of this embodiment can solve this problem well. The setting of the coating 50 on the chip 100 can ensure that the chip 100 has good fluorescence characteristics to meet the requirements of the gene sequencer for the fluorescence background characteristics of the chip 100. Moreover, the processing technology for preparing the coating 50 on the second surface 32 of the second substrate 30 is relatively mature and simple and can make the performance of the prepared coating 50 controllable, which is very beneficial to the large-scale preparation of the chip 100 adapted to the sequencing platform.

[0080] Specifically, the first substrate 20 and the second substrate 30 may include any suitable material, such as glass, silicon dioxide, crystal, quartz glass, plastic, ceramic, PET (polyethylene terephthalate), PMMA (polymethyl methacrylate) or any other suitable material.

[0081] The first substrate 20 and the second substrate 30 may be optically transparent.

[0082] The shapes of the first substrate 20 and the second substrate 30 can be various regular shapes such as square, rectangle, circle, triangle, etc. Of course, the first substrate 20 and the second substrate 30 can also be irregular shapes. In this embodiment, the first substrate 20 and the second substrate 30 are long rectangles.

[0083] The sizes of the first substrate 20 and the second substrate 30 may be the same or different. In one embodiment, the thickness of the first substrate 20 is smaller than the thickness of the second substrate 30 .

[0084] The second substrate 30 includes a first surface 31 of the second substrate 30 and a second surface 32 of the second substrate 30 facing each other, wherein the first surface 31 of the second substrate 30 faces the first substrate 20, and the second surface 32 of the second substrate 30 faces away from the first surface 31 of the second substrate 30 and is arranged as the bottom surface of the second substrate 30. A fluid channel 70 is arranged between the first surface 31 of the second substrate 30 and the first substrate 20, and the fluid channel 70 can be used as a fluid reagent chemical reaction site, that is, the area where the target signal is located.

[0085] In order to reduce the laser from irradiating through the second substrate 30 to other structures below the second substrate 30 of the chip 100, such as irradiating the glue layer connecting the second substrate 30 and the structure below the second substrate 30, a coating 50 can be set on the second surface 32 of the second substrate 30, and it is desired that the spontaneous fluorescence intensity of the coating 50 is less than a preset intensity.

[0086] It is understandable that many substances in nature have autofluorescence properties, and autofluorescence refers to the fact that a substance is excited to emit fluorescence under or after the irradiation of light (absorbing energy). If the autofluorescence property of the coating 50 is strong, it means that the fluorescence generated by the molecules in the coating 50 when excited by the laser is stronger, that is, the noise is stronger, which will cause the signal-to-noise ratio of the collected image to decrease, affecting the realization of sequencing. Then when selecting the coating 50, it is preferred to use a material with an autofluorescence intensity less than a preset intensity as the coating 50. The so-called preset intensity can be calculated and determined according to actual operation requirements. Specifically, the autofluorescence intensity of the coating 50 is related to the irradiation conditions, that is, the laser intensity emitted by the laser, the objective lens parameters, etc., and is also related to the autofluorescence property of the material of the coating 50. After a large number of comparative tests, the inventor found that under a specific working environment, such as after irradiation with a 532nm or 635nm laser of 800-1000mW, the background intensity of the image (16-bit image) of the chip 100 is less than or equal to 400, and high-quality sequencing results can be obtained based on these images. The preset strength here can be determined based on the specific preset value and the specific working environment.

[0087] See also Figure 2 and Figure 3 In some embodiments, the coating 50 may be applied on the second surface.

[0088] In this way, by applying the coating 50 on the second surface, when the laser irradiates the chip 100, since the coating 50 is applied on the second surface 32 of the second substrate 30 and the spontaneous fluorescence intensity of the coating 50 is less than the preset intensity, the excitation light passing through the second substrate 30 is reduced, that is, the laser is blocked on the second surface 32 of the second substrate 30, which is beneficial to weaken the fluorescence emitted by the structure under the second substrate 30 excited by the transmitted excitation light, so as to meet the requirements of the gene sequencer for the fluorescence background characteristics of the chip 100.

[0089] Specifically, as described above, the second substrate 30 can be made of glass. Then, when the coating 50 is set on the second surface 32 of the second substrate 30, the coating 50 can be set on the second surface 32 of the second substrate 30 by spraying, wherein the number of spraying can be multiple times to form a coating 50 of a certain thickness; of course, the coating 50 can also be printed on the second surface 32 of the second substrate 30 by silk screen printing. The spraying method has a wider range of applications than the silk screen printing method. When the second surface 32 of the second substrate 30 is not a plane, the coating 50 cannot be set on the second surface 32 of the second substrate 30 by silk screen printing. Then, the coating 50 can be set on the second surface 32 of the second substrate 30 by spraying.

[0090] It can be understood that since the spontaneous fluorescence intensity of the coating 50 is less than the preset intensity, coating the coating 50 on the second surface 32 of the second substrate 30 can shield the laser on the second substrate 30 side to meet the requirements of the gene sequencer for the fluorescence background characteristics of the chip 100.

[0091] See also Figure 3 In some embodiments, the thickness range A of the coating 50 may be 5 μm to 20 μm. Preferably, the thickness range A of the coating 50 may be 8 μm to 15 μm. In this way, a coating 50 of a certain thickness is applied, so that the coating 50 has a better shielding effect on the laser, so that the image of a specific area of ​​the chip 100 collected by irradiation with the excitation light can meet the sequencing requirements.

[0092] In the present application, it is necessary to understand that the orientation or positional relationship indicated by terms such as "length", "width", and "thickness" are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation.

[0093] Specifically, since the coating 50 is repeatedly applied on the second surface, and the purpose of applying the coating 50 is to shield the laser from the second substrate 30 side, the coating 50 needs to have a certain thickness. When the thickness is too small, the coating 50 cannot play a good shielding role. When the thickness is too large, it can be understood that too many repeated spraying times are required. If the coating 50 is too thick, it is easy to make it difficult to ensure the uniform thickness of the coating 50, and it is easy to have an uneven surface, wrinkles, or even cracks.

[0094] In other embodiments, the thickness range A of the coating 50 can be set to 10 μm to 40 μm, so that the coating 50 can better shield the laser, so that the image of a specific area of ​​the chip 100 collected by irradiation with excitation light can meet the sequencing requirements.

[0095] In some embodiments, the light shielding rate of the coating 50 in the working environment is not less than 80%. Thus, by setting the light shielding rate of the coating 50 in the working environment to not less than 80%, it is sufficient to ensure that the coating 50 in the working environment can play a good light shielding role for the laser.

[0096] Specifically, the working environment includes lasers of specific wavelengths and intensities; in one example, the working environment refers to 800-1000mW red or green lasers (e.g., lasers with a light emission wavelength of 532nm or 635nm). In order to prevent the laser from irradiating the remaining structures of the chip 100 through the second substrate 30, the coating 50 needs to have a high light shielding rate. For example, in this embodiment, the coating 50 is in the working environment, that is, when the coating 50 is coated on the second surface 32 of the second substrate 30 and is in the sequencing process, the light shielding rate of the coating 50 should be not less than 80%, so that it can be ensured that the coating 50 can have a good shielding effect on the laser.

[0097] Preferably, the light shielding rate of the coating 50 in the working environment may be not less than 95%, thereby ensuring that the coating 50 in the working environment can play a better light shielding role for the laser.

[0098] In some embodiments, the flatness of the coating 50 facing away from the second substrate 30 is allowed to deviate by no more than 0.1 μm. In this way, after being stacked, connected and packaged with the rest of the structure, the deviation / tolerance of the mechanically accumulated surface flatness can be ensured to be within a preset range, so that the flatness of the surface of the chip 100 meets the predetermined requirements, and it is also beneficial for the coating 50 to be stably and firmly connected to the corresponding surface of the third substrate 60, which is beneficial for the chip 100 to be structurally firm and stable.

[0099] Specifically, flatness refers to the fact that when processing or producing certain workpieces, the surface of the workpiece is not absolutely flat, and the difference between its unevenness and the absolute level is the flatness. According to a certain relationship between flatness and scattering rate, since the uneven surface has a scattering effect on the incident light, it can be understood that in order to ensure the quality of the fluorescent signal image, the flatness value of the side of the coating 50 away from the second substrate 30 is as small as possible, so the flatness allowable deviation of the side of the coating 50 away from the second substrate 30 is set to no more than 0.1μm.

[0100] In addition, it should be noted that setting the flatness allowable deviation of the side of the coating 50 facing away from the second substrate 30 to no more than 0.1 μm can ensure that the scattering rate of the coating 50 is low. Scattering, as a physical concept, describes the phenomenon that the propagation direction deviates from its original direction due to the local uneven areas encountered by radiation energy (various electromagnetic waves, such as light waves, sound waves, etc.) on the propagation path. In addition, when the roughness of the uneven area gradually increases, the mirror scattering component gradually decays when scattering occurs, and the diffuse scattering component increases accordingly.

[0101] Among them, the situation of local unevenness in this embodiment is that the surface of the coating 50 is not a completely smooth surface, and has certain small height fluctuations. Since the laser emitted by the laser during the gene sequencing process will irradiate the reaction reagent in the fluid channel 70, the irradiated fluorescent molecules are excited to emit corresponding fluorescent signals. At this time, the lens 200 is used to take a photo of the fluorescent signal. In order to make the obtained fluorescent signal photo higher in quality and convenient for analysis and calculation, the flatness allowable deviation of the side of the coating 50 away from the second substrate 30 is set to no more than 0.1μm, which can reduce the scattering interference of the incident laser on the coating 50 when taking the fluorescent signal image.

[0102] In some embodiments, the material of the coating layer 50 may include ink. Thus, the chip 100 having the coating layer 50 has a higher light shielding property and a weaker luminous property under a working environment, and is easy to manufacture.

[0103] Specifically, ink is a uniform mixture, which mainly includes colorants, binders, fillers, additives and other substances. The ink can be printed and dried on the printed object.

[0104] Among them, colorants can include pigments and dyes. Pigments are divided into organic pigments and inorganic pigments. The former has bright colors, strong coloring power, and short drying time, so it is widely used in inks; the latter has good light resistance, heat resistance, solvent resistance, and hiding power. Pigments are colored in particulate form and do not dissolve. They are the most commonly used colorants in inks. Dyes are prepared into solutions when used, and color in molecular form, and the effect is not as good as pigments.

[0105] The binder plays the role of dispersing the pigment and can be made by dissolving a small amount of natural resin, synthetic resin, cellulose, rubber derivatives, etc. in drying oil or solvent. The binder can make the ink form a uniform thin layer after spraying on the object, and form a film layer with a certain strength after drying to protect the pigment.

[0106] Fillers and additives are auxiliary components of inks. The former can be used as an auxiliary agent to adjust the ink concentration and increase the thickness of the ink film. They mainly include materials such as barium sulfate, talcum powder, and calcium carbonate. The latter may be an additional part of the colorant or an additional part of the binder, depending on product requirements.

[0107] In some embodiments, preferably, the coating 50 is black ink. In this way, a high-quality image of a specific area of ​​the chip 100 can be collected. Moreover, by printing on the corresponding surface of the second substrate 30 to obtain the second substrate 30 with the black coating 50, the coating 50 that meets the requirements or the chip 100 including the second substrate 30 with the coating 50 can be quickly and easily obtained.

[0108] Specifically, the present application has a high requirement for the light-shielding property of the coating 50. The pigment is a solid component in the ink, which is a color-developing substance of the ink and is generally a water-insoluble pigment. Considering the light-shielding requirement of the coating 50, preferably, in one embodiment, black ink can be used to achieve a high light-shielding effect. In addition, considering the working environment and stability, the ink as one of the materials for making the coating 50 also needs to have high temperature resistance.

[0109] See also Figure 3 and Figure 4 In some embodiments, the first substrate 20 may include a first surface and a second surface, and the first surface 21 of the first substrate 20 is disposed opposite to the second surface 22 of the first substrate 20. The fluid channel 70 may be formed on the second surface 22 of the first substrate 20 and the first surface 31 of the second substrate 30, and the background intensity of the image of the chip 100 may be less than or equal to a preset value, and the image of the chip 100 is an image of the second surface 22 of the first substrate 20 and / or an image of the first surface 31 of the second substrate 30 under the working environment.

[0110] Specifically, in a certain example, the sample to be tested, such as a solution containing fluorescently labeled nucleic acid molecules, is located in the fluid channel 70, one end of the nucleic acid molecule is connected to the second surface 21 of the first substrate 20, and an optical imaging system including a lens module is used to collect images of the nucleic acid molecules. In the focusing stage, the lens module is moved to find the medium interface where the nucleic acid molecules are located, that is, to find the second surface 21 of the first substrate 20, and then determine the focal plane / clear plane to collect a clear image of the nucleic acid molecules. In this imaging process, in addition to the fluorescently labeled nucleic acid molecules emitting fluorescence, the first surface 31, the second surface 32 and / or the coating 50 of the second substrate 30 of the chip 100 will emit fluorescence under the irradiation of the excitation light, which will appear as a background signal or an interference signal that is difficult to distinguish from the target signal in the collected image of the chip 100.

[0111] In another example, the sample to be tested, such as a solution containing nucleic acid molecules, is located in the fluid channel 70, one end of the nucleic acid molecule is connected to the second surface 21 of the first substrate 20 and the first surface 31 of the second substrate 30, and the image of the nucleic acid molecule is collected using an optical imaging system including a lens module. In the focusing stage, the lens module is moved to find the medium interface where the nucleic acid molecule is located, that is, to find the second surface 22 of the first substrate 20 or the first surface 31 of the second substrate 30, and then determine the corresponding focal plane / clear plane to collect a clear image of the nucleic acid molecule. In this imaging process, in addition to the fluorescently labeled nucleic acid molecules emitting fluorescence, the first surface 31, the second surface 32 and / or the coating 50 of the second substrate 30 of the chip 100 will emit fluorescence under the irradiation of the excitation light, which appears as a background signal or an interference signal that is difficult to distinguish from the target signal in the collected image of the chip 100.

[0112] In a certain example, under a specific working environment, such as after irradiation with a 532nm or 635nm laser of 800-1000mW, the collected image is a 16-bit image, and the preset value is set to 400 (int), that is, the background intensity of the image of the chip 100 is less than or equal to 400. In this way, an image with a higher signal-to-noise ratio can be obtained, which is conducive to obtaining high-quality sequencing results. Figure 6 The background intensity of the collected images of four chips 100 is shown. The second surface 32 of the second substrate 30 of the chips 100 is printed with black ink. The fluorescence background intensity of the images is less than 400.

[0113] It is understandable that those skilled in the art can determine appropriate preset values ​​in other working environments, such as lasers of different intensities and wavelengths, by using the method of determining the preset values ​​exemplified herein.

[0114] Specifically, image intensity represents the intensity of image pixels. In a grayscale image, image intensity is the grayscale of the image. In the RGB color space, it can be understood as the pixel grayscale value of the R channel, G channel or B channel. Other color spaces are similar.

[0115] In a grayscale image, the grayscale of an image is the brightness of the image. The larger the grayscale value, the brighter the pixel. Thus, the background intensity of the image of the chip 100 in this embodiment is less than or equal to the preset value, which means that the background brightness of the image of the chip 100 obtained is low, that is, the signal-to-noise ratio of the image is high, which is conducive to the recognition of the target signal and the realization of base recognition based on the target signal. It can be understood that the preset value can be set according to the actual needs during sequencing.

[0116] Specifically, in a certain example, on a sequencing platform including an optical imaging system, the optical system includes a laser and a camera, the camera includes a lens 200, and the image of the chip 100 can be collected in the following manner: turn on the laser to emit laser light, so that the laser light passes through the lens 200 to irradiate the test agent in the fluid channel 70, the test agent includes a nucleic acid molecule with a fluorescent marker, the fluorescent marker is excited to emit fluorescence, and then the camera is used to collect and photograph the fluorescence passing through the lens 200 to obtain an image containing the target signal.

[0117] In another example, on a sequencing platform including a total internal reflection optical imaging system, the total internal reflection fluorescence imaging system includes a laser and a camera, the camera includes a lens 200, for example, a total internal reflection objective lens, and the image of the chip 100 can be collected in the following manner: turn on the laser to emit laser light, so that the laser light is irradiated through the lens 200 at an angle greater than the critical angle to the medium interface in the chip 100, such as the solid-liquid interface, for example, the second surface 22 of the first substrate 20, and an evanescent wave / evanescent field is generated on the interface. The test agent includes a nucleic acid molecule with a fluorescent marker, and one end of the nucleic acid molecule is connected to the second surface 22 of the first substrate 20. The fluorescent marker emits fluorescence in the evanescent field, and then the camera is used to collect and photograph the fluorescence passing through the lens 200 to obtain an image containing the target signal.

[0118] In addition, the background intensity of the image of the chip 100 is less than or equal to the preset value because the coating 50 coated on the second surface 32 of the second substrate 30 blocks the laser on the side of the second substrate 30, and because the coating 50 itself has a low autofluorescence intensity, when the coating 50 is irradiated with the laser, the fluorescence signal generated by the molecules in the coating 50 excited by the laser is weak, that is, the noise is low, thereby improving the signal-to-noise ratio of the collected image, so that the background intensity of the fluorescent signal image finally obtained can be less than or equal to the preset value.

[0119] In some embodiments, the background intensity of the image of the chip 100 may be less than or equal to 400. Thus, by controlling the background intensity of the image of the chip 100 to be less than or equal to 400, the brightness of the background part of the image is lowered, which facilitates comparison and presentation of effective fluorescent signals, thereby achieving the purpose of improving sequencing quality.

[0120] Specifically, the sequencer has certain requirements for the fluorescence background characteristics of the chip 100. For example, in one embodiment, the preset value can be 400, which means that when irradiated with 800mW red and green laser, the background intensity of the image of the chip 100 captured by the lens 200 is required to be less than or equal to 400 in a 16-bit image, thereby improving the sequencing quality.

[0121] See also Figure 1 and Figure 2 In some embodiments, the chip 100 may include a third substrate 60 attached to the coating layer 50 , wherein the third substrate 60 is made of metal.

[0122] In this way, by providing the third substrate 60 attached to the coating layer 50 , the planarity stability and the temperature conductivity stability of the first substrate 20 and the second substrate 30 can be ensured.

[0123] Specifically, the chip 100 further includes a housing 10 and a third substrate 60. The housing 10 can protect the chip 100 to a certain extent, and the housing 10 can be made of resin, that is, the housing 10 is formed by injection molding, so that the manufacturing cost is low and the manufacturing process is simple.

[0124] The third substrate 60 can be attached to the coating 50, and is disposed on two sides opposite to the coating 50 together with the second substrate 30. The third substrate 60 is made of a metal with good thermal conductivity, for example, the third substrate 60 can be an aluminum plate, and the third substrate 60 and the coating 50 can be connected together by bonding with adhesive 61.

[0125] It can be understood that when the chip 100 is packaged, the stacking packaging method of the first substrate 20, the second substrate 30 and the third substrate 60 bonded and fixed by the adhesive 61 can make the chip 100 have a packaging structure with good sealing and heat dissipation. Among them, the aluminum plate is used as the third substrate 60, which is light and has good heat dissipation and can ensure the flatness stability and temperature conductivity stability of the first substrate 20 and the second substrate 30; the adhesive 61 is used to bond the third substrate 60 and the coating 50 so that the third substrate 60 is fixedly connected with the first substrate 20 and the second substrate 30, so that the sealing of the chip 100 is good.

[0126] In particular, since the coating 50 is disposed on the second surface 32 of the second substrate 30, the coating 50 can prevent the laser from irradiating the adhesive 61 between the aluminum plate and the coating 50, thereby preventing the molecules in the aluminum plate or the adhesive 61 from being excited to form fluorescent signal noise. In this way, the chip 100 is less dependent on the surface quality of the aluminum plate, thereby reducing the processing cost of the aluminum plate, and at the same time, the material selection range of the adhesive 61 is expanded, and is no longer limited by the fluorescence characteristic index.

[0127] See also Figure 4 In some embodiments, the chip 100 may further include an interposer 40, which may be disposed between the first substrate 20 and the second substrate 30, and the interposer 40 connects the first substrate 20 and the second substrate 30, and one or more fluid channels 70 may be disposed in the interposer 40. Thus, by disposing one or more fluid channels 70 on the interposer 40, it is not necessary to etch the fluid channels 70 on the first substrate 20 and the second substrate 30, thereby simplifying the manufacturing process of the fluid channels 70 and also simplifying the preparation process of the chip 100.

[0128] In this application, unless otherwise clearly specified or limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or mutual communication; it can be a direct connection, or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0129] Specifically, the intermediary layer 40 is formed between the first substrate 20 and the second substrate 30. The intermediary layer 40 can be a channel glue between the first substrate 20 and the second substrate 30, or in other words, the first substrate 20 and the second substrate 30 are bonded and packaged together through the intermediary layer 40. The intermediary layer 40 is provided with a fluid channel 70, so that the intermediary layer 40 can be used as a fluid reagent chemical reaction site, that is, a region where a beneficial fluorescent signal is generated under laser irradiation.

[0130] It can be understood that the first substrate 20 and / or the second substrate 30 can be used to pump fluids such as reaction reagents into the fluid channel 70 provided in the intermediate layer 40, and / or serve as an outlet or inlet for fluids pumped out of the fluid channel 70 of the intermediate layer 40.

[0131] In particular, the fluid channel 70 is formed in the interposer 40, which eliminates the need for physical or chemical etching on the first substrate 20 and / or the second substrate 30, thereby simplifying the process of forming the fluid channel 70. In the present application, the fluid channel 70 can be formed by stamping the interposer 40, laser cutting, etc.

[0132] See also Figure 4 and Figure 5 In some embodiments, the intermediary layer 40 can be bonded to the first substrate 20 and the second substrate 30, the intermediary layer 40 can have a hollow structure, and the intermediary layer 40 can include a base layer 41, a first adhesive layer 42, a second adhesive layer 43, and a hollow structure that penetrates the base layer 41, the first adhesive layer 42, and the second adhesive layer 43 to form one or more fluid channels 70.

[0133] Among them, the base layer 41 has a first surface 411 and a second surface 412 opposite to each other, the first adhesive layer 42 is arranged on the first surface 411 of the base layer 41, and the second adhesive layer 43 is arranged on the second surface 412 of the base layer 41. The first adhesive layer 42 can be bonded to the first substrate 20, and the second adhesive layer 43 can be bonded to the second substrate 30.

[0134] Thus, by providing the first adhesive layer 42 and the second adhesive layer 43, the intermediary layer 40 can be provided between the first substrate 20 and the second substrate 30. By forming a hollow structure on the intermediary layer 40, the hollow structure can penetrate the base layer 41, the first adhesive layer 42 and the second adhesive layer 43 to form one or more fluid channels 70, so that the reaction reagent can enter the fluid channel 70 through the first substrate 20 and / or the second substrate 30, and then flow out through the first substrate 20 and / or the second substrate 30 after a chemical reaction occurs in the fluid channel 70.

[0135] Specifically, the intermediate layer 40 includes a base layer 41, and the base layer 41 can be a main component of the intermediate layer 40. The material of the base layer 41 can include black PET, transparent PET or any other plastic or polymer, which provides high contrast for the fluorescent image finally captured by the lens 200.

[0136] The intermediary layer 40 further includes a first adhesive layer 42 and a second adhesive layer 43, wherein the first adhesive layer 42 is disposed on the first surface 411 of the base layer 41, and the second adhesive layer 43 is disposed on the second surface 412 of the base layer 41, wherein the first surface 411 of the base layer 41 is opposite to the second surface 412 of the base layer 41. The first adhesive layer 42 and the second adhesive layer 43 are adhesive, so that the first substrate 20 and the second substrate 30 can be connected and arranged together with the intermediary layer 40. It can be easily understood that the first adhesive layer 42 can be bonded to the first substrate 20 or the second substrate 30, and correspondingly, the second adhesive layer 43 is bonded to the second substrate 30 or the first substrate 20.

[0137] Furthermore, the first adhesive layer 42 may be bonded to the second surface 22 of the first substrate 20 , and the second adhesive layer 43 may be bonded to the first surface 31 of the second substrate 30 , thereby bonding the first substrate 20 , the intermediate layer 40 and the second substrate 30 to form a whole.

[0138] The first adhesive layer 42 and the second adhesive layer 43 should also have low spontaneous fluorescence, so that the intermediate layer 40 formed by the combination of the base layer 41, the first adhesive layer 42 and the second adhesive layer 43 has a negligible effect on the fluorescence signal generated in the intermediate layer 40 area, thereby improving the signal-to-noise ratio and improving the sequencing quality.

[0139] There may be one or more fluid channels 70. Multiple fluid channels 70 can make the biochemical reaction of the reaction reagent more uniform and the sequencing more efficient. The fluid channel 70 can be formed on the interposer 40 by stamping or laser cutting. In particular, the fluid channel 70 runs through the first adhesive layer 42, the base layer 41 and the second adhesive layer 43, that is, the fluid channel 70 extends through each of the first adhesive layer 42, the base layer 41 and the second adhesive layer 43.

[0140] See also Figure 4 and Figure 5 In some embodiments, the first substrate 20 and / or the second substrate 30 is provided with a through hole 33 communicating with the fluid channel 70 .

[0141] In this way, by providing a through hole 33 connected to the fluid channel 70 on the first substrate 20 and / or the second substrate 30, the reaction reagent and other fluids can enter the fluid channel 70 through the first substrate 20 and / or the second substrate 30, and can also flow out through the first substrate 20 and / or the second substrate 30 after a chemical reaction occurs in the fluid channel 70. Moreover, it is also convenient to connect a pipeline or a manifold to connect the valve body and the reaction reagent container.

[0142] Specifically, the through hole 33 can be formed on the first substrate 20, or on the second substrate 30, or on the first substrate 20 and the second substrate 30. In this embodiment, the through hole 33 is formed on the second substrate 30 to allow fluids such as reaction reagents to flow into the fluid channel 70 from the through hole 33 and flow out of the through hole 33 after reaction.

[0143] In addition, it can be understood that the number of through holes 33 can be multiple, some of which serve as fluid inlets, and the remaining through holes 33 serve as fluid outlets. The through holes 33 can be formed in an array on the first substrate 20 and the second substrate 30, and further, the hole array can be etched on any one of the first substrate 20 and the second substrate 30 by wet etching or dry etching.

[0144] In some embodiments, the peeling force of the first adhesive layer 42 on the first substrate 20 and / or the peeling force of the second adhesive layer 43 on the second substrate 30 may be no less than 560g. Preferably, the peeling force of the first adhesive layer 42 on the first substrate 20 and / or the peeling force of the second adhesive layer 43 on the second substrate 30 may be no less than 800g.

[0145] In this way, by placing a minimum limit on the peeling force of the first adhesive layer 42 on the first substrate 20 and / or the peeling force of the second adhesive layer 43 and the second substrate 30, it is possible to ensure that the bonding strength of the first adhesive layer 42 on the first substrate 20 and / or the second adhesive layer 43 on the second substrate 30 meets the operational requirements; for example, the bonding peeling force between the first adhesive layer 42 / the second adhesive layer 43 and the first substrate 20 / the second substrate 30 is not less than the specified value, which can ensure that the various structures of the chip 100 are firmly connected and the structure of the chip 100 is stable, thereby meeting the sequencing requirements.

[0146] Specifically, during the gene sequencing process, the chip 100 undergoes multiple pressure cycles due to the flow of pressurized fluid through the fluid channel 70, that is, the intermediary layer 40 provided with the fluid channel 70 is exposed to high pressure, so it is necessary to ensure that the intermediary layer 40 can withstand the pressure. Therefore, it is necessary to set at least one of the peeling force of the first adhesive layer 42 on the first substrate 20 and the peeling force of the second adhesive layer 43 on the second substrate 30 to be no less than 560g. Of course, in other embodiments, it can be set to no less than 800g, so that the first adhesive layer 42 and the first substrate 20, and the second adhesive layer 43 and the second substrate 30 form a sufficiently strong bond.

[0147] It can be understood that an important indicator for bonding quality is bonding strength, so it is necessary to ensure that the peeling force of the first adhesive layer 42 on the first substrate 20 and the peeling force of the second adhesive layer 43 on the second substrate 30 have minimum restrictions, that is, the bonding strength of the first adhesive layer 42 on the first substrate 20 and / or the second adhesive layer 43 on the second substrate 30 can be guaranteed.

[0148] See also Figure 4 In some embodiments, the size of the fluid channel 70 in the first direction X is greater than its size in the second direction Y, the first direction X is perpendicular to the second direction Y, and both the first direction X and the second direction Y are perpendicular to the thickness direction of the interposer 40. In this way, the general shape of the fluid channel 70 formed in the interposer 40 is standardized, which is conducive to controlling the fluid in the fluid channel 70 and facilitating the positioning and imaging of these areas of the chip 100.

[0149] Specifically, Figure 4 As shown, Figure 4FIG. 4 shows a schematic diagram of a planar structure of the intermediary layer 40. In this case, the first direction X may be the length direction of the intermediary layer 40, and the second direction Y may be the width direction of the intermediary layer 40. Figure 4 It can be easily seen that the shape of the fluid channel 70 is an irregular figure, and the size of the fluid channel 70 in the first direction X is greater than the size in the second direction Y, and the first direction X is perpendicular to the second direction Y. Further, the first direction X and the second direction Y are both perpendicular to the thickness direction of the intermediate layer 40.

[0150] Of course, the size of the fluid channel 70 in the first direction X can also be smaller than the size in the second direction Y. In this case, the first direction X is still perpendicular to the second direction Y. Furthermore, the first direction X and the second direction Y are still perpendicular to the thickness direction of the intermediate layer 40.

[0151] See also Figure 4 In some embodiments, there are multiple fluid channels 70, and the fluid channels 70 are arranged in the interposer 40 along the first direction X; and / or, the fluid channels 70 are arranged in an array on the interposer 40 along the second direction Y. In this way, by forming multiple fluid channels 70, the gene sequencing process is more efficient, which is conducive to controlling the fluid in the fluid channels 70 and locating and imaging those areas of the chip 100.

[0152] Specifically, Figure 4 As shown, the number of fluid channels 70 is 4, wherein the arrangement of the fluid channels 70 is to extend in the intermediary layer 40 along a first direction X, i.e., the length direction of the intermediary layer 40, and to be arrayed on the intermediary layer 40 along a second direction Y, i.e., the width direction of the intermediary layer 40. In particular, the provision of multiple fluid channels 70 can make the sequencing process more efficient, and at the same time, by means of array arrangement, the intervals of the fluid channels 70 are made consistent, so that the fluids such as reaction reagents pumped into the fluid channels 70 are uniform.

[0153] See also Figure 4 In some embodiments, the fluid channel 70 includes a middle section 71, a first end 72 and a second end 73, and the first end 72 and the second end 73 are respectively located at two ends of the fluid channel 70, and the size of the first end 72 in the second direction Y and / or the size of the second end 73 in the second direction Y is smaller than the size of the middle section 71 in the second direction Y.

[0154] In this way, the shape of the fluid channel 70 is further standardized, which is beneficial to control the fluid in the fluid channel 70 and also beneficial to positioning and imaging those areas of the chip 100.

[0155] Specifically, the shape of the fluid channel 70 may be an irregular shape, for example, the fluid channel 70 may include a middle section 71, a first end 72, and a second end 73. The first end 72 and the second end 73 are symmetrically arranged at the two ends of the fluid channel 70, and are both triangular in shape, and the middle section 71 is in a long narrow rectangular shape, which means that the dimensions of the first end 72 and the second end 73 in the second direction Y are smaller than the dimensions of the middle section 71 in the second direction Y.

[0156] Of course, the first end 72 and the second end 73 may also be of different shapes, as long as the first end 72 and the second end 73 are respectively located at the two ends of the middle section 71 and the dimension of one of them in the second direction Y is smaller than the dimension of the middle section 71 in the second direction Y.

[0157] See also Figure 4 In some embodiments, the size of the middle section 71 in the second direction Y is constant. Thus, since the size of the middle section 71 in the second direction Y is constant, it means that the length of the middle section 71 in the second direction Y is the same everywhere. For example, the length of the middle section 71 in the second direction Y is 5 mm. Specifically, as described above, Figure 4 In the illustrated embodiment, the middle section 71 is a long narrow rectangle, that is, the length of the middle section 71 in the second direction Y is the same everywhere, so it can be concluded that the size of the middle section 71 in the second direction Y is constant.

[0158] See also Figure 4 In some embodiments, the size range L1 of the middle section 71 in the second direction Y is 4.4 mm-8.4 mm. Thus, the size range L1 of the middle section 71 along the second direction Y is controlled within 4.4 mm-8.4 mm, so that the fluid channel 70 has a certain width to reasonably accommodate the reaction reagents, which is beneficial to the fluid control in the fluid channel 70 and to the efficient biochemical reaction in the fluid channel 70.

[0159] Specifically, it can be understood that the middle section 71 is the main area where the biochemical reaction of the reaction reagent occurs, so the middle section 71 should have a suitable width. Figure 4 If the size of the middle section 71 in the second direction Y is smaller than 4.4 mm, the fluid channel 70 may be difficult to form; if the size of the middle section 71 in the second direction Y is larger than 8.4 mm, the width is too large to form more fluid channels 70 on the intermediary layer 40, and the sequencing reaction cannot be carried out efficiently. In addition, the amount of reaction reagents carried by a single fluid channel 70 is large, which may cause uneven reaction of the reaction reagents.

[0160] See also Figure 4In some embodiments, the spacing range L2 between two adjacent fluid channels 70 in the second direction Y may be 0.8 mm to 1.5 mm. In this way, it is convenient to process multiple fluid channels 70 on the interposer 40 and the number of fluid channels 70 is guaranteed, so that efficient sequencing can be achieved as much as possible.

[0161] Specifically, when the distance between two adjacent fluid channels 70 is less than 0.8 mm, it is difficult to form a plurality of fluid channels 70, and when the distance between two adjacent fluid channels 70 is greater than 1.5 mm, the number of fluid channels 70 of the hollow structure formed on the intermediary layer 40 will be reduced. Therefore, the distance range L2 between two adjacent fluid channels 70 in the second direction Y is controlled to be 0.8 mm-1.5 mm, which facilitates processing while ensuring the number of fluid channels 70.

[0162] In some embodiments, the thickness range B of the base layer 41 may be 30 μm-50 μm; and / or the thickness range C of the first adhesive layer 42 may be 75 μm-85 μm; and / or the thickness range D of the second adhesive layer 43 may be 75 μm-85 μm. Thus, by reasonably setting the thickness range B of the base layer 41, the thickness range D of the first adhesive layer 42 and the second adhesive layer 43, the normal progress of the biochemical reaction in the intermediary layer 40 can be ensured.

[0163] Specifically, the thickness range B of the base layer 41 can be 30μm-50μm, and the thickness range C of the first adhesive layer 42 and the thickness range D of the second adhesive layer 43 are 75μm-85μm; or, the thickness range B of the base layer 41 can be 30μm-50μm, and one of the thickness ranges C or thickness range D of the second adhesive layer 43 or the first adhesive layer 42 is 75μm-85μm; or, only the thickness range requirements are made for the first adhesive layer 42 and the second adhesive layer 43, and the thickness range C and thickness range D of the first adhesive layer 42 and the second adhesive layer 43 are set to 75μm-85μm; or, only the thickness range C or thickness range D of the first adhesive layer 42 or the second adhesive layer 43 is required, and the thickness range of one of the adhesive layers is set to 75μm-85μm.

[0164] It should be noted that, as described above, at least one of the peeling force of the first adhesive layer 42 on the first substrate 20 and the peeling force of the second adhesive layer 43 on the second substrate 30 is not less than 800 grams, wherein the shear strength and peel strength of the adhesive layer depend on its chemical formula and its thickness relative to the agent layer.

[0165] When the adhesive layer is too thin, the first adhesive layer 42 and the second adhesive layer 43 may not provide sufficient peeling and shearing pressure; when the adhesive layer is too thick, gaps may be formed in the first adhesive layer 42 and the second adhesive layer 43, resulting in bubble formation, which may weaken the bonding strength, and most of the stress and shear stress may act on the first adhesive layer 42 and the second adhesive layer 43, and will not be transferred to the base layer 41, so the first adhesive layer 42 and the second adhesive layer 43 are prone to rupture and cause the chip 100 to fail. In summary, the thickness range B of the base layer 41 should be set to 30μm-50μm, and / or the thickness range C of the first adhesive layer 42 should be set to 75μm-85μm, and / or the thickness range D of the second adhesive layer 43 should be set to 75μm-85μm.

[0166] In some embodiments, the base layer 41, the first adhesive layer 42 and / or the second adhesive layer 43 can withstand a temperature of not less than 80° C. Thus, by setting a certain tolerance temperature for the base layer 41, the first adhesive layer 42 and the second adhesive layer 43, the normal biochemical reaction in the intermediate layer 40 can be ensured.

[0167] Specifically, during the gene sequencing process, the chip 100 may also be exposed to a thermal cycle environment, so the base layer 41 of the intermediate layer 40 that supports the biochemical reaction and the first adhesive layer 42 and the second adhesive layer 43 need to meet a certain temperature tolerance.

[0168] It can be understood that the high temperature resistance mentioned above means that the base layer 41, the first adhesive layer 42 and the second adhesive layer 43 have no obvious deformation at a specified temperature. Therefore, it is necessary to set the base layer 41 to have a temperature resistance of not less than 80°C, and the first adhesive layer 42 and / or the second adhesive layer 43 to have a temperature resistance of not less than 80°C.

[0169] In some embodiments, the first adhesive layer 42 and / or the second adhesive layer 43 can withstand a temperature of not less than 110° C. Thus, by setting a certain tolerance temperature for the first adhesive layer 42 and the second adhesive layer 43 , the normal progress of the biochemical reaction in the intermediate layer 40 can be ensured.

[0170] Similarly, as described above, during the gene sequencing process, in order to ensure that the first adhesive layer 42 and the second adhesive layer 43 have no obvious deformation when the chip 100 is exposed to a thermal cycle environment, the tolerance temperature of at least one of the first adhesive layer 42 and the second adhesive layer 43 can be set to be not less than 110°C.

[0171] In some embodiments, the base layer 41, the first adhesive layer 42 and / or the second adhesive layer 43 need to be resistant to a specified solvent. Thus, by setting the base layer 41, the first adhesive layer 42 and / or the second adhesive layer 43 to be resistant to a specified solvent, the normal progress of the biochemical reaction in the intermediary layer 40 can be ensured.

[0172] During the gene sequencing process, the chip 100 may also be exposed to corrosive reagents (such as formamide) and other environments, so at least one of the base layer 41 of the intermediate layer 40 that supports the biochemical reaction and the first adhesive layer 42 and the second adhesive layer 43 needs to meet the tolerance to the specified solvent. The tolerance means that in the specified solvent, there will be no problems such as the glue layer and the film layer falling off and failure. The specified solvent can be water, DMSO, formamide solution, etc.

[0173] In some embodiments, the material of the base layer 41 may include polyimide. Thus, the base layer 41 using polyimide material can meet the requirement that the base layer 41 can withstand a certain high temperature and can also meet the requirement of withstanding a specified solvent, so as to avoid problems such as film shedding and failure in a high temperature environment and certain solvents.

[0174] Specifically, the characteristics of polyimide materials are high temperature resistance, small thermal expansion coefficient, and good solvent resistance. Therefore, the use of polyimide material for the base layer 41 can meet the condition that the base layer 41 can withstand a temperature of not less than 80°C, and can also meet the tolerance to specified solvents, so as to avoid problems such as film shedding and failure in the above-mentioned solvents.

[0175] In some embodiments, the first adhesive layer 42 and the second adhesive layer 43 may be made of the same material. Specifically, the first adhesive layer 42 and the second adhesive layer 43 may be made of materials including acrylic adhesive, butyl rubber, silicone, and the like.

[0176] In some embodiments, at least one of the first adhesive layer 42 and the second adhesive layer 43 may be made of a pressure-sensitive adhesive. For example, it may be pressure-sensitive silicone (PSA silicone). Specifically, pressure-sensitive adhesive refers to an adhesive that can be instantly bonded by applying a little pressure. Currently, commonly used pressure-sensitive adhesives mainly include rubber-type pressure-sensitive adhesives, acrylate-type pressure-sensitive adhesives, and silicone pressure-sensitive adhesives. Rubber-type pressure-sensitive adhesives are currently the most widely used type of pressure-sensitive adhesives. Rubber includes natural rubber, synthetic rubber, and recycled rubber. Silicone pressure-sensitive adhesives are made of silicone rubber and silicone resin. Silicone rubber is the basic component of the pressure-sensitive adhesive, and silicone resin is the tackifier. The performance of the pressure-sensitive adhesive changes with the change in the ratio of the two. Preferably, a pressure-sensitive adhesive made of silicone rubber can be used in this application.

[0177] In some embodiments, the material of at least one of the first adhesive layer 42 and the second adhesive layer 43 may include silicone. Specifically, at least one of the first adhesive layer 42 and the second adhesive layer 43 may be made of silicone. The silicone material can withstand high temperatures greater than 110°C. When kept at 80°C for three consecutive hours, the silicone will not deform and cause deformation and crosstalk of the fluid channel 70; and the silicone material has an adhesive peeling force greater than 800g on the glass surface, that is, the first substrate 20 and the second substrate 30, which can meet the requirements that the peeling force of the first adhesive layer 42 on the first substrate 20 and / or the peeling force of the second adhesive layer 43 on the second substrate 30 is not less than 800g; at the same time, the silicone material is also solvent-resistant. In the modification and sequencing reagents, after completing 350 cycles of sequencing, the fluid channel 70 will not deform or crosstalk.

[0178] The chip 100 can be used to implement sequence determination.

[0179] See also Figure 7 The present application provides a method for preparing a chip 100, wherein the method may include the following steps:

[0180] Step S10: providing a first substrate 20;

[0181] Step S20: providing a second substrate 30, wherein the second substrate 30 comprises a first surface 31 and a second surface 32 opposite to each other;

[0182] Step S30 : stacking the second substrate 30 on the first substrate 20 , with the first surface 31 of the second substrate 30 facing the first substrate 20 ;

[0183] Step S40: disposing one or more fluid channels 70 between the first surface 31 of the second substrate 30 and the first substrate 20;

[0184] Step S50: disposing a coating 50 on the second surface 32 of the second substrate 30, wherein the autofluorescence intensity of the coating 50 is less than a preset intensity;

[0185] Thus, by executing the present method, the first substrate 20 and the second substrate 30 are stacked to form a basic structure having a fluid channel 70, and a specific coating 50 is provided on the second surface 32 of the second substrate 30. In applications involving the use of an optical imaging system to detect signals from a sample to be tested on the chip 100, such as a sequencing platform based on optical imaging to detect the fluorescent signals of nucleic acid molecules in the chip to achieve nucleic acid sequence determination, the fluorescent signal (background signal) emitted by the chip 100 itself when irradiated, i.e., excited by the excitation light, is very weak, which is conducive to obtaining an image with a high signal-to-noise ratio, facilitating the identification of target fluorescent signals, and facilitating the acquisition of high-quality sequencing results.

[0186] Specifically, the chip 100 can be a reactor fixed with a nucleic acid molecule to be tested with an optical detection mark, with a space for accommodating liquid, which can be used to fix the sample to be tested, also known as a flow cell or a flow cell. In step S10-step S40, the first substrate 20 and the second substrate 30 are any solid supports that can be used to fix nucleic acid sequences, such as nylon membranes, glass sheets, plastics, silicon chips, magnetic beads, etc. Then the first substrate 20 and the second substrate 30 provided can include any suitable material, such as glass, silicon dioxide, crystal, quartz glass, plastics, ceramics, PET (polyterephthalic acid), PMMA (polymethyl methacrylate) or any other suitable material. In one embodiment, the first substrate 20 and the second substrate 30 provided are optically transparent, such as glass sheets / glass layers.

[0187] The second substrate 30 is stacked on the first substrate 20, and the first substrate 20 is disposed on the second substrate 30, that is, the first surface 31 of the second substrate 30 is disposed toward the first substrate 20. In addition, as mentioned above, the chip 100 adapted to the SBS sequencing platform may include one or more parallel channels, which are used for entering and exiting and carrying reagents to form an environment required for the sequence determination reaction, that is, one or more fluid channels 70 need to be provided between the first surface 31 of the second substrate 30 and the first substrate 20.

[0188] In the present application, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include that the first and second features are in direct contact, or may include that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, a first feature being "above", "above" or "above" a second feature includes that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature.

[0189] In step S50, the inventors discovered during the structural design, preparation and testing of the chip adapted to the sequencing platform that when the laser emitted by the laser of the sequencing platform is irradiated into the chip fluid channel through the lens, it will also pass through the lower glass and irradiate onto the glue layer connecting the lower glass and the aluminum plate. The molecules of the glue layer are therefore excited to emit fluorescence, which greatly interferes with the recognition and detection of the target signal, i.e., the signal from the nucleic acid molecule to be tested in the fluid channel.

[0190] Therefore, it is necessary to set a coating 50 with a spontaneous fluorescence intensity less than a preset intensity on the second surface 32 of the second substrate 30, so that the excitation light passing through the second substrate 30 is reduced, which is beneficial to weakening the fluorescence emitted by the structure below the second substrate 30 when excited by the transmitted excitation light. Moreover, since the spontaneous fluorescence intensity of the coating 50 is less than the preset intensity, the fluorescence intensity generated by the coating 50 irradiated with the excitation light is relatively weak. Under the working environment, the fluorescence signal generated by the chip itself is very weak. Therefore, the chip 100 prepared by the present preparation method is suitable for carrying biological samples for realizing the detection of biological macromolecules, and is suitable for an optical imaging platform for realizing the detection of samples to be tested based on the detection chip.

[0191] The coating 50 may be coated on the second surface 32 of the second substrate 30, or may be printed on the second surface 32 of the second substrate 30. The specific requirements of the coating 50 are as described above for the coating 50, for example, the material of the coating 50 may include black ink to provide a higher light shielding property; the thickness range A of the coating 50 may be 5 μm to 20 μm or 8 μm to 15 μm; the light shielding rate of the coating 50 in the working environment is not less than 80%; the flatness of the side of the coating 50 away from the second substrate 30 is allowed to have a deviation of no more than 0.1 μm.

[0192] See also Figure 8 In some embodiments, the method for preparing a chip further comprises:

[0193] Step S60: providing a third substrate 60;

[0194] Step S70 : disposing the third substrate 60 under the coating layer 50 .

[0195] In this way, the first substrate 20 , the second substrate 30 and the third substrate 60 disposed under the coating layer 50 are bonded together to form the main body of the chip 100 .

[0196] Specifically, in step S50 and step S60, the third substrate 60 may be an aluminum plate with good thermal conductivity, and the third substrate 60 is disposed under the coating 50. In this way, the main body of the chip 100 may be formed by bonding two glass-made first substrates 20 and second substrates 30, and a metal-made bottom plate, namely the third substrate 60, so as to ensure the stability of the planarity and the temperature conductivity of the first substrate 20 and the second substrate 30.

[0197] See also Fig. 9 In some embodiments, the method for preparing a chip further comprises the following steps:

[0198] Step S80: providing an interposer 40; Step S90: disposing the interposer 40 between the first substrate 20 and the second substrate 30, and disposing one or more fluid channels 70 in the interposer 40. In this way, the method disposes one or more fluid channels 70 in the interposer 40, so that the fluid channels 70 do not need to be etched on the first substrate 20 and the second substrate 30, thereby simplifying the manufacturing process of the fluid channels 70.

[0199] Specifically, in step S80 and step S90, an intermediary layer 40 is provided, and one or more fluid channels 70 are set in the intermediary layer 40. The fluid channels 70 can be formed by stamping the intermediary layer 40, laser cutting, etc. The size specifications of the fluid channels 70, the spacing between multiple fluid channels 70, and other specific data have been described in detail above and will not be repeated here. In this way, the intermediary layer 40 can be used as a fluid reagent chemical reaction site, that is, the interval where the laser irradiation generates a beneficial fluorescent signal, and there is no need to etch the fluid channels 70 on the first substrate 20 and the second substrate 30, which simplifies the manufacturing process of the fluid channels 70.

[0200] Since one or more fluid channels 70 are formed between the first substrate 20 and the second substrate 30, the intermediate layer 40 needs to be arranged between the first substrate 20 and the second substrate 30. Then, the first substrate 20 and / or the second substrate 30 can be used to pump fluids such as reaction reagents into the fluid channels 70 arranged in the intermediate layer 40, and / or, as an outlet or inlet for fluids pumped out of the fluid channels 70 of the intermediate layer 40.

[0201] See also Fig.10 In some embodiments, providing the interposer 40 (step S80) includes the following steps:

[0202] Step S81: providing a base layer 41, wherein the base layer has a first surface 411 and a second surface 412 opposite to each other;

[0203] Step S82: disposing a first adhesive layer 42 on the first surface 411 of the base layer 41;

[0204] Step S83: disposing a second adhesive layer 43 on the second surface 412 of the base layer 41;

[0205] Step S84: forming a fluid channel 70 penetrating the base layer 41, the first adhesive layer 42 and the second adhesive layer 43;

[0206] The method further includes: step S100 : bonding the first adhesive layer 42 to the first substrate 20 , and bonding the second adhesive layer 43 to the second substrate 30 .

[0207] Specifically, in step S81, the provided base layer 41 can be used as a main component of the intermediate layer 40. The material of the base layer 41 can include black PET, transparent PET or any other plastic or polymer. Black PET has low autofluorescence, so that a high signal-to-noise ratio can be obtained during the sequencing process, and a high contrast can be provided for the fluorescent image finally captured by the lens 200.

[0208] In step S82 and step S83, the first adhesive layer 42 and the second adhesive layer 43 provided should also have low autofluorescence. The first adhesive layer 42 is disposed on the first surface 411 of the base layer 41, and the second adhesive layer 43 provided is disposed on the second surface 412 of the base layer 41. The first adhesive layer 42 and the second adhesive layer 43 are adhesive, so that the first substrate 20 and the second substrate 30 can be connected and disposed together with the intermediate layer 40.

[0209] In step S84, since a hollow structure is formed on the intermediary layer 40, the intermediary layer 40 includes a base layer 41, a first adhesive layer 42, and a second adhesive layer 43, so that the hollow structure can penetrate the base layer 41, the first adhesive layer 42, and the second adhesive layer 43 to form one or more fluid channels 70, so that the reaction reagent can enter the fluid channel 70 through the first substrate 20 and / or the second substrate 30, and then flow out through the first substrate 20 and / or the second substrate 30 after a chemical reaction occurs in the fluid channel 70.

[0210] In step S100 , the first adhesive layer 42 is bonded to the second surface 22 of the first substrate 20 , and the second adhesive layer 43 is bonded to the first surface 31 of the second substrate 30 , so that the first substrate 20 , the intermediate layer 40 and the second substrate 30 are bonded together to form a whole.

[0211] In the description of this specification, the description with reference to the terms "one embodiment", "certain embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0212] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.

Claims

1. A chip, characterized in that: include: a first substrate; a second substrate stacked with the first substrate, the second substrate comprising a first surface and a second surface opposite to each other, the first surface of the second substrate facing the first substrate, and one or more fluid channels being provided between the first surface of the second substrate and the first substrate; A coating disposed on the second surface of the second substrate, wherein the autofluorescence intensity of the coating is less than a preset intensity; The shading rate of the coating in a working environment is not less than 80%.

2. The chip according to claim 1, characterized in that: The coating is coated on the second surface of the second substrate.

3. The chip according to claim 1, characterized in that: The thickness of the coating ranges from 5 μm to 20 μm.

4. The chip according to claim 3, characterized in that: The thickness of the coating ranges from 8 μm to 15 μm.

5. The chip according to any one of claims 1 to 4, characterized in that: The flatness of the coating layer facing away from the second substrate is allowed to have a deviation of no more than 0.1 μm.

6. The chip according to any one of claims 1 to 4, characterized in that: The material of the coating layer includes ink.

7. The chip according to claim 6, characterized in that ,The material of the coating is black ink.

8. The chip according to any one of claims 1 to 4, characterized in that: The first substrate includes a first surface and a second surface opposite to each other, the fluid channel is formed between the second surface of the first substrate and the first surface of the second substrate, the background intensity of the image of the chip is less than or equal to a preset value, and the image of the chip is an image of the second surface of the first substrate and / or an image of the first surface of the second substrate under a working environment.

9. The chip according to claim 8, characterized in that: The background intensity of the image of the chip is less than or equal to 400.

10. The chip according to any one of claims 1 to 4, characterized in that: The chip further comprises a third substrate attached to the coating layer, and the third substrate is made of metal.

11. The chip according to any one of claims 1 to 4, characterized in that: The chip further includes an interposer disposed between the first substrate and the second substrate, the interposer connecting the first substrate and the second substrate, and the one or more fluid channels are disposed in the interposer.

12. The chip according to claim 11, characterized in that: The intermediary layer is bonded to the first substrate and the second substrate, the intermediary layer has a hollow structure, and the intermediary layer includes: A base layer having a first surface and a second surface opposite to each other; A first adhesive layer disposed on a first surface of the base layer, the first adhesive layer being bonded to the first substrate; a second adhesive layer disposed on the second surface of the base layer, the second adhesive layer being bonded to the second substrate; as well as, The fluid channel is the hollow structure formed by penetrating the base layer, the first adhesive layer and the second adhesive layer.

13. The chip according to claim 12, characterized in that: The first substrate and / or the second substrate is provided with a through hole communicating with the fluid channel.

14. The chip according to claim 12, characterized in that: The peeling force of the first adhesive layer on the first substrate and / or the peeling force of the second adhesive layer on the second substrate is not less than 560 g.

15. The chip according to claim 12, characterized in that: The peeling force of the first adhesive layer on the first substrate and / or the peeling force of the second adhesive layer on the second substrate is not less than 800 g.

16. The chip according to claim 11, characterized in that: The size of the fluid channel in a first direction is greater than that in a second direction, the first direction is perpendicular to the second direction, and both the first direction and the second direction are perpendicular to the thickness direction of the intermediate layer.

17. The chip according to claim 16, characterized in that: There are multiple fluid channels, and the fluid channels are arranged in the intermediate layer extending along the first direction; And / or, the fluid channels are arranged in an array in the intermediate layer along the second direction.

18. The chip according to claim 16, characterized in that: The fluid channel includes a middle section, a first end, and a second end, wherein the first end and the second end are respectively located at two ends of the fluid channel, and a size of the first end in the second direction and / or a size of the second end in the second direction is smaller than a size of the middle section in the second direction.

19. The chip according to claim 18, characterized in that: The size of the middle segment in the second direction is constant.

20. The chip according to claim 19, characterized in that: The size of the middle section in the second direction ranges from 4.4 mm to 8.4 mm.

21. The chip according to claim 17, characterized in that: The distance between two adjacent fluid channels in the second direction ranges from 0.8 mm to 1.5 mm.

22. The chip according to claim 12, characterized in that: The thickness of the base layer is in the range of 30 μm to 50 μm; and / or, The thickness of the first adhesive layer is in the range of 75 μm to 85 μm; and / or, The thickness of the second adhesive layer is in the range of 75 μm to 85 μm.

23. The chip according to claim 12, characterized in that: The base layer, the first adhesive layer and / or the second adhesive layer can withstand a temperature of not less than 80°C.

24. The chip according to claim 12, characterized in that: The first adhesive layer and / or the second adhesive layer can withstand a temperature of not less than 110°C.

25. The chip according to claim 12, characterized in that: The base layer, the first adhesive layer, and / or the second adhesive layer are resistant to a specified solvent.

26. The chip according to claim 12, characterized in that: The material of the base layer includes polyimide.

27. The chip according to claim 12, characterized in that: The first adhesive layer and the second adhesive layer are made of the same material.

28. The chip according to claim 12, characterized in that: The material of the first adhesive layer and / or the second adhesive layer includes silicone.

29. Use of the chip according to any one of claims 1 to 28 in sequence determination.

30. A method for preparing a chip, characterized in that: include: providing a first substrate; Providing a second substrate, the second substrate comprising a first surface and a second surface opposite to each other; The second substrate is stacked on the first substrate so that the first surface of the second substrate faces the first substrate. Disposing one or more fluid channels between the first surface of the second substrate and the first substrate; Disposing a coating on the second surface of the second substrate, wherein the autofluorescence intensity of the coating is less than a preset intensity; The shading rate of the coating in a working environment is not less than 80%.

31. The method according to claim 30, characterized in that Also includes: providing a third substrate; The third substrate is disposed under the coating layer.

32. The method according to claim 30, characterized in that Also includes: Provide an intermediary layer; The intermediary layer is disposed between the first substrate and the second substrate, and the one or more fluid channels are disposed in the intermediary layer.

33. The method according to claim 32, characterized in that Providing the intermediary layer includes: Providing a base layer having a first surface and a second surface opposite to each other; Disposing a first adhesive layer on the first surface of the base layer; Disposing a second adhesive layer on the second surface of the base layer; forming the fluid channel through the base layer, the first adhesive layer and the second adhesive layer; The method further includes bonding the first adhesive layer to the first substrate and bonding the second adhesive layer to the second substrate.

34. The method according to claim 33, characterized in that The thickness of the base layer is in the range of 30 μm to 50 μm; and / or, The thickness of the first adhesive layer is in the range of 75 μm to 85 μm; and / or, The thickness of the second adhesive layer is in the range of 75 μm to 85 μm.

35. The method according to claim 33, characterized in that The base layer, the first adhesive layer and / or the second adhesive layer can withstand a temperature of not less than 80°C.

36. The method according to claim 33, characterized in that The first adhesive layer and / or the second adhesive layer can withstand a temperature of not less than 110°C.

37. The method according to claim 33, characterized in that The base layer, the first adhesive layer, and / or the second adhesive layer are resistant to a specified solvent.

38. The method according to claim 33, characterized in that The base layer is polyimide.

39. The method according to claim 33, characterized in that The first adhesive layer and the second adhesive layer are made of the same material.

40. The method according to any one of claims 33 to 39, characterized in that The first adhesive layer and / or the second adhesive layer is silicone.

41. The method according to claim 40, characterized in that The material of the first adhesive layer and / or the second adhesive layer is pressure-sensitive adhesive.

42. The method according to claim 33, characterized in that The peeling force of the first adhesive layer on the first substrate and / or the peeling force of the second adhesive layer on the second substrate is not less than 560 g.

43. The method according to claim 42, characterized in that The peeling force of the first adhesive layer on the first substrate and / or the peeling force of the second adhesive layer on the second substrate is not less than 800 g.

44. The method according to claim 30, characterized in that The first substrate and / or the second substrate is provided with a through hole communicating with the fluid channel.

Images