Chip, application and method of manufacturing a chip

Abstract

The application discloses a chip, an application and a method for preparing the chip. The chip comprises a first substrate and a second substrate, the second substrate is arranged in a stacked manner with the first substrate, the second substrate comprises 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 further comprises a first coating arranged on the second surface of the second substrate, the first coating has a spontaneous fluorescence intensity less than a preset intensity; and a second coating is formed in the fluid channel. The second surface of the second substrate of the chip is provided with a specific first coating, and the first coating has weak spontaneous fluorescence characteristics. The chip is particularly suitable for a platform for imaging the chip based on an optical system to detect biomolecules in the chip. In addition, the second coating in the chip can reduce the adsorption of impurities in the air in the fluid channel, and reduce the sequencing error rate of the chip.

Description

Technical Field

[0001] This application relates to the field of nucleic acid detection, and in particular to a chip, its application, and a method for preparing the chip. Background Technology

[0002] The topics discussed in this section should not be considered prior art simply because they are mentioned here. Similarly, the technical problems mentioned in this section or related to the topics provided as background art should not be considered as having been previously recognized in the prior art. The topics in this section merely represent different methods, which themselves may correspond to specific embodiments of the technical solutions in the claims.

[0003] A chip adapted to a sequencing platform is a reaction device that can hold the nucleic acid to be tested and contain a solution to provide a reaction environment or detection environment for the nucleic acid to be tested. It is also called a flow cell.

[0004] Two pieces of glass (at least one of the two opposing surfaces of the two pieces of glass is etched) and an opaque substrate can be bonded together using an adhesive to create a chip with internal space / cavity / channel.

[0005] On a sequencing platform (sometimes simply called a sequencer) that uses an optical imaging system to detect chips, the base sequence of the nucleic acid molecule is identified and determined by imaging a designated location on the chip (the surface connected to the nucleic acid molecule to be tested, sometimes also called the field of view / reaction region). For example, specifically in a platform that uses fluorescently labeled nucleotides and performs sequencing by synthesis, a high-energy laser emitted by a laser in the sequencer illuminates the reaction region of the chip through a lens. The nucleic acid molecule to be tested is placed in a reagent solution, and the laser irradiates the fluorescent molecules in the reagent solution, exciting them to emit fluorescent signals. These fluorescent signals are then collected, for example, by taking a picture to obtain an image. The base sequence is then identified and determined based on the information in these images to achieve the sequencing purpose.

[0006] The aforementioned sequencing platform uses an optical system to image the chip and performs detection based on the image. Understandably, the higher the signal-to-noise ratio of the image, the more accurate and reliable the sequencing results. The imaging target, the nucleic acid molecule to be tested (e.g., a single molecule or cluster), is located on a designated surface of the chip. Understandably, during the chip's fabrication, storage, transportation, and / or use, any behavior that may affect or alter the surface properties or the behavior of the analyte molecules on it will influence the signal of the acquired image, thus affecting image-based molecular detection. For example, during chip fabrication and storage, substances in the air or unexpected environments may non-specifically adsorb onto the chip channels or designated surfaces. In chip-based imaging sequencing platforms, such non-specific adsorption introduces unexpected substances that affect sequencing biochemistry and / or imaging, for example, leading to a decrease in the signal-to-noise ratio of the captured image, thereby increasing the error rate of base identification based on this image information or reducing high-quality sequencing data.

[0007] Furthermore, the designated surfaces of a chip are typically chemically modified surfaces with reactive sites, sometimes also referred to as (active) functional groups. During chip fabrication and storage, unintended substances adsorbed onto these functional groups effectively occupy a portion of the functional groups / reactive sites, thus reducing the available functional groups on the surface, lowering its loading capacity, and decreasing detection throughput. Simultaneously, the functional groups or their polymers on the designated chip surfaces are often susceptible to oxidation or hydrolysis reactions with oxygen and moisture in the air, leading to polymer degradation. This can potentially introduce further noise and increase the error rate of sequencing data. Summary of the Invention

[0008] This application provides a chip, an application, and a method for fabricating the chip.

[0009] The chip in this embodiment includes a first substrate; a second substrate stacked on the first substrate, the second substrate including a first surface and a second surface facing away from each other, the first surface of the second substrate facing the first substrate, one or more fluid channels being provided between the first surface of the second substrate and the first substrate; a first coating being provided on the second surface of the second substrate, the autofluorescence intensity of the first coating being less than a preset intensity; and a second coating being formed in the fluid channels.

[0010] The chip according to this application includes a basic structure with fluid channels formed by stacking a first substrate and a second substrate. It includes a first coating on a second surface of the second substrate and a second coating within the fluid channels to cover surfaces corresponding to the fluid channels, such as the lower surface of the first substrate (the surface of the first substrate facing the first surface of the second substrate) and / or the upper surface of the second substrate (the first surface of the second substrate). This makes the chip particularly suitable for a platform for biomolecular detection based on chip imaging. Specifically, target molecules can be attached to designated surfaces of the chip, such as the lower surface of the first substrate and / or the upper surface of the second substrate. Multiple imaging of the target molecules on the designated surface at different time points can yield stable images with high signal-to-noise ratios. In applications involving the detection of signals from test samples from the chip using an optical imaging system, such as sequencing platforms for nucleic acid sequencing based on the detection of fluorescence signals of nucleic acid molecules in the chip using optical imaging, this chip facilitates obtaining high signal-to-noise ratio (SNR) images and the identification of target signals based on these images, thereby facilitating accurate base identification and, consequently, obtaining high-quality sequencing results. Moreover, chips with the above characteristics have stable and controllable surface properties, making them particularly suitable for mass production. They also have low requirements for storage and transportation. In other words, even after a period of storage and / or conventional biochemical detection reagent logistics transportation, the performance of the chip remains relatively stable, making it highly practical for industrial use.

[0011] In addition, according to embodiments of this application, the chip may also have at least one of the following additional technical features.

[0012] In some embodiments, the components of the material forming the second coating include a temperature-sensitive substance / material.

[0013] In some embodiments, the temperature-sensitive substance / material is selected from at least one of gelatin, pectin, ethylene glycol, glycerin, and trehalose. Specifically, in some examples, the temperature-sensitive substance / material comprises gelatin. Gelatin can be dissolved in water to prepare a solution at a relatively high temperature, which will then exhibit a gel-like or semi-solid state at a relatively low temperature.

[0014] In some embodiments, the temperature-sensitive substance / material is selected from temperature-sensitive hydrogels.

[0015] In some embodiments, the temperature-sensitive substance / material is provided in the form of a solution. In some examples, the temperature-sensitive substance / material is provided in the form of an aqueous solution.

[0016] In some examples, the second coating is a hydrogel at the operating temperature; the hydrogel is, for example, a solution of gelatin and trehalose dissolved in water, which may be solid at a relatively low temperature, such as 4°C, and gel or liquid at a relatively high temperature.

[0017] In some embodiments, the mass concentration of the temperature-sensitive substance / material in the solution is 0.5% to 5%.

[0018] In some embodiments, the material forming the second coating also includes an anti-corrosion material.

[0019] In some embodiments, the corrosion-resistant material is selected from at least one of ethylenediaminetetraacetic acid (EDTA), ethylene glycol phenyl ether, and sodium thimerosal.

[0020] In some embodiments, the first coating is applied to 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 beneath the second substrate when excited by the transmitted excitation light. The coating process includes smearing, brushing, printing, etc.

[0021] In some embodiments, the thickness of the first coating ranges from 5 μm to 20 μm. A first coating of this thickness provides good shielding against excitation light, ensuring that images of specific areas of the chip acquired after excitation light irradiation meet sequencing requirements.

[0022] Preferably, the thickness of the first coating ranges from 8 μm to 15 μm. A first coating with this thickness provides good blocking effect against excitation light transmitted through the substrate.

[0023] In some embodiments, the first coating has a light-shielding rate of not less than 80% under operating conditions. This light-shielding rate facilitates the acquisition of images of specific areas of the chip with a high signal-to-noise ratio, thus improving the quality of sequencing results. For sequencing platforms that determine nucleic acid sequences based on the fluorescence signal of nucleic acid molecules in the chip using optical imaging, the operating environment here includes lasers of specific wavelengths and intensities; in one example, the operating environment refers to an 800–1000 mW red or green laser (e.g., a laser with an emission wavelength of 532 nm or 635 nm).

[0024] In some embodiments, the flatness deviation of the side of the first coating facing away from the second substrate is allowed to be no more than 0.1 μm. In this way, after being stacked and connected to other structures such as substrates for packaging, it can be ensured that the mechanically accumulated surface flatness deviation / tolerance is within a preset range, so that the flatness of the chip surface meets the predetermined requirements. It also facilitates the stable and firm connection between the first coating and the corresponding surface of the third substrate, which is beneficial to the chip structure being firm and stable.

[0025] In some embodiments, the material of the first coating includes ink. This results in a chip with the first coating exhibiting high light-shielding properties and weak luminescence characteristics under operating conditions, and is also easy to fabricate. Specifically, in one example, the first coating is black ink, which allows for the acquisition of high-quality images of specific areas of the chip. Furthermore, by printing on the corresponding surface of a second substrate to obtain a second substrate with the black first coating, it is possible to quickly and controllably fabricate a first coating that meets the requirements, or a chip containing a second substrate with the first coating.

[0026] In some embodiments, the first substrate includes a first surface and a second surface facing away from each other, a 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 chip image is less than or equal to a preset value, and the chip image is an image of the second surface of the first substrate and / or an image of the first surface of the second substrate under operating conditions. Preferably, the background intensity of the chip image is less than or equal to 400.

[0027] The chip image refers to an image of one or more regions / fields of view (FOV) of the chip containing the analyte molecule. The preset value is set by the inventors through comparison and calculation of images corresponding to a large number of better and worse sequencing results obtained under the same working environment. The acquired chip image is evaluated and judged by the preset value, and the image is quickly evaluated and fed back to help decide whether to further process the image, whether to continue acquiring images, whether to adjust the optical imaging system, whether to refocus, and to evaluate the chip quality, etc.

[0028] In some embodiments, the chip includes a third substrate disposed on the first coating, the third substrate being made of metal. This ensures the planarity stability and temperature conduction stability of the first and second substrates.

[0029] In some embodiments, the chip includes an interposer layer disposed between a first substrate and a second substrate, the interposer layer connecting the first substrate and the second substrate, and one or more fluid channels disposed in the interposer layer.

[0030] In some embodiments, an interposer is bonded to the first substrate and the second substrate. The interposer has a hollow structure and includes:

[0031] The base layer has a first surface and a second surface that are opposite to each other;

[0032] 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;

[0033] A second adhesive layer is disposed on the second surface of the substrate and is bonded to the second substrate; and a fluid channel is a perforated structure that penetrates the substrate, the first adhesive layer, and the second adhesive layer. This eliminates the need to etch the fluid channel onto the first and second substrates, simplifying the manufacturing process of the fluid channel and also simplifying the chip fabrication process.

[0034] In some embodiments, the first substrate and / or the second substrate are provided with through holes communicating with a fluid channel. This allows fluids such as reaction reagents to enter the fluid channel through the first substrate and / or the second substrate, and allows chemical reactions to occur within the fluid channel before flowing out through the first substrate and / or the second substrate. Furthermore, it facilitates the connection of pipes or manifolds to connect valve bodies and reaction reagent containers.

[0035] In some embodiments, the peel force of the first adhesive layer to the first substrate and / or the peel force of the second adhesive layer to the second substrate is not less than 560g. This ensures that the adhesive strength of the first adhesive layer to the first substrate and / or the second adhesive layer to the second substrate meets operational requirements; for example, if the adhesive peel force between the adhesive layer and the substrate is not less than this specified value, it ensures that the chip structures are firmly connected, the chip structure is stable, and the sequencing requirements are met.

[0036] Preferably, the peel force of the first adhesive layer to the first substrate and / or the peel force of the second adhesive layer to the second substrate is not less than 800g. This ensures that the bonding strength of the first adhesive layer to the first substrate and / or the second adhesive layer to the second substrate meets the operational requirements, enabling the chip to be used well for sequencing.

[0037] In some embodiments, the fluid channel has a larger dimension in a first direction than in a second direction, with the first and second directions perpendicular to each other, both perpendicular to the thickness direction of the interposer. This standardizes the approximate shape of the fluid channel formed in the interposer, facilitating fluid control within the channel and enabling the positioning and imaging of these areas of the chip.

[0038] In some embodiments, there are multiple fluid channels extending along a first direction within the interposer; and / or, the fluid channels are arrayed along a second direction on the interposer. Thus, multiple fluid channels can make the sequence determination process more efficient, facilitate control of the fluid within the fluid channels, and also facilitate the positioning and imaging of these regions of the chip.

[0039] In some embodiments, the fluid channel includes an intermediate section, a first end, and a second end, which are located at opposite ends of the fluid channel. The dimension of the first end in the second direction and / or the dimension of the second end in the second direction are smaller than the dimension of the intermediate section in the second direction. This further standardizes the shape of the fluid channel, facilitating the control of the fluid within the channel and the positioning and imaging of these areas of the chip.

[0040] In some implementations, the dimension of the intermediate segment in the second direction is constant. That is, the length of the intermediate segment is equal everywhere in the second direction.

[0041] In some embodiments, the intermediate section has a dimension ranging from 4.4 mm to 8.4 mm in the second direction. This reasonable dimension range allows the fluid channel to have a certain width to accommodate the reaction reagents, which is beneficial for fluid control and for efficient biochemical reactions within it.

[0042] In some implementations, the spacing between two adjacent fluid channels in the second direction ranges from 0.8 mm to 1.5 mm. This facilitates the fabrication of multiple fluid channels on the interlayer while ensuring a sufficient number of fluid channels to achieve the most efficient sequencing possible.

[0043] In some embodiments, the thickness of the substrate ranges from 30 μm to 90 μm; and / or,

[0044] The thickness of the first adhesive layer ranges from 75 μm to 85 μm; and / or,

[0045] The thickness of the second adhesive layer ranges from 75 μm to 85 μm. Thus, the first and second adhesive layers have a certain thickness, ensuring the peel force of the first / second adhesive layer on the first / second substrate, thereby guaranteeing the normal progress of the biochemical reaction in the interlayer.

[0046] In some embodiments, the base layer, the first adhesive layer, and / or the second adhesive layer are resistant to temperatures not less than 80°C. This ensures that the base layer, the first adhesive layer, and / or the second adhesive layer do not undergo significant deformation during biochemical reactions, guaranteeing the normal progress of biochemical reactions within the intermediate layer.

[0047] In some embodiments, the first adhesive layer and / or the second adhesive layer withstand temperatures of not less than 110°C. Thus, the first adhesive layer and / or the second adhesive layer do not exhibit significant deformation during biochemical reactions, ensuring the normal progress of the biochemical reactions within the intermediate layer.

[0048] 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 experience problems such as peeling or failure of the adhesive or film layers in the specified solvent, thereby ensuring the normal progress of the biochemical reactions in the intermediate layer.

[0049] In some implementations, the base material comprises polyimide or PET. This allows the base material to withstand certain high temperatures and also meets the requirement of withstanding specified solvents.

[0050] In some embodiments, the first adhesive layer and the second adhesive layer are made of the same material. This simplifies the manufacturing process of both the first and second adhesive layers, allowing them to be manufactured together.

[0051] 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). Thus, the first adhesive layer and / or the second adhesive layer can withstand certain high temperatures and also meet the requirement of resistance to specified solvents.

[0052] This application also provides a method for fabricating a chip, which can be used to fabricate 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 facing away from each other; stacking the second substrate on the first substrate, the second substrate including a first surface and a second surface facing away from each other, such that the first surface of the second substrate faces the first substrate; forming one or more fluid channels between the first surface of the second substrate and the first substrate; forming a first coating on the second surface of the second substrate, the autofluorescence intensity of the first coating being less than a preset intensity; and forming a second coating within the fluid channels.

[0053] This method can be used to prepare chips in any of the above embodiments. It is simple to operate, suitable for automated control, and facilitates the batch preparation or production of chips with stable and consistent surface properties, thus promoting the industrialization of chip-based detection applications. The chip prepared by this method includes a basic structure with fluid channels formed by stacking a first substrate and a second substrate. A first coating is disposed on the second surface of the second substrate, and a second coating is disposed within the fluid channels to cover the surfaces corresponding to the fluid channels. This makes the chip particularly suitable as a platform for biomolecular detection based on chip imaging. Specifically, target molecules can be attached to designated surfaces of the chip, such as the lower surface of the first substrate and / or the upper surface of the second substrate. Multiple imaging of the target molecules on the designated surface at different time points can yield stable images with high signal-to-noise ratio (SNR). In applications involving the detection of signals from test samples from the chip using an optical imaging system, such as sequencing platforms that detect the fluorescence signals of nucleic acid molecules in the chip using optical imaging to achieve nucleic acid sequencing, this method facilitates obtaining high SNR images and identifying target signals based on these images, thereby enabling accurate base identification and high-quality sequencing results.

[0054] In some embodiments, the second coating is formed after the first and second substrates have been stacked and a channel has been formed between them. That is, the second coating is applied to a designated surface of the chip after the components are assembled. In one example, a solution containing a temperature-sensitive substance / material is introduced into the fluid channel at a flow rate of 1500 μL / min to 2000 μL / min and a pressure of 0.01 MPa to 0.1 MPa to form the second coating. This process can be automated, which improves preparation efficiency and reduces contamination or individualization factors that may result from manual operation, leading to a batch of chips with stable and consistent performance. Chips prepared using this method can achieve a high signal-to-noise ratio for nucleic acid detection, facilitating accurate and stable detection.

[0055] Understandably, the process of connecting or assembling components to form a chip often involves the use of adhesives. For example, adhesives are used to bond designated surfaces of a first substrate and a second substrate to create a stacked arrangement. Structures formed in this way are prone to deformation under stress. When chips prepared in this way are used for detection, such as imaging a designated surface or target molecules on a designated surface through biochemical reactions in channels, deformation can cause leakage or seepage between channels, potentially affecting the biochemical reactions and the imaging process or the quality of the acquired image, such as making it difficult to focus or track focus to obtain a clear image. In this embodiment, by controlling the flow rate and pressure within a suitable range, it is beneficial to form a uniform second coating covering the designated surface within the fluid channel. It is also beneficial to control the force applied to the assembled structure during the process, thereby controlling the degree of deformation of the chip surface or related structures. This results in a higher yield of the prepared chip, and the chip's structure, surface performance parameters, etc., are more consistent and stable.

[0056] In some embodiments, the method further includes: providing a third substrate; and disposing the third substrate under the first coating. Thus, the first substrate, the second substrate, and the third substrate are bonded together to form the body of the chip, and the third substrate helps to ensure the planarity stability and temperature conductivity stability of the first substrate and the second substrate.

[0057] In some embodiments, the method further includes: providing an interposer; disposing the interposer between a first substrate and a second substrate, and disposing one or more fluid channels in the interposer. This eliminates the need to etch fluid channels onto the first and second substrates, simplifying the manufacturing process of the fluid channels.

[0058] In some embodiments, an interposer is provided, comprising: providing a base layer having opposing first and second surfaces; 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 comprising: bonding the first adhesive layer to a first substrate, and bonding the second adhesive layer to a second substrate. This allows the interposer to bond the first and second substrates, and the hollowed-out structure of the bonded interposer forms the corresponding fluid channel, eliminating the need to etch the fluid channel onto the first and second substrates, thus simplifying the manufacturing process of the chip containing the fluid channel.

[0059] The application of the chip in any of the above embodiments, or the chip prepared by the method in any of the above embodiments, in sequence determination.

[0060] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0061] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, wherein:

[0062] Figure 1 This is a schematic diagram of the chip structure in the embodiments of this application;

[0063] Figure 2 This is a cross-sectional schematic diagram of the chip in the embodiments of this application;

[0064] Figure 3 This is an embodiment of the present application. Figure 2 Enlarged view of point P in the middle;

[0065] Figure 4 This is a schematic diagram of the structure of the intermediary layer in the embodiments of this application;

[0066] Figure 5 This is a cross-sectional schematic diagram of the intermediary layer in the embodiments of this application;

[0067] Figure 6 This is a schematic diagram of the background intensity of the image acquired in the embodiments of this application;

[0068] Figure 7 This is a schematic flowchart of the chip fabrication method in the embodiments of this application;

[0069] Figure 8 This is another schematic flowchart of the chip fabrication method in the embodiments of this application;

[0070] Figure 9 This is another schematic flowchart of the chip fabrication method in the embodiments of this application;

[0071] Figure 10 This is another schematic flowchart of the chip fabrication method in the embodiments of this application;

[0072] Figure 11A This is a comparison chart of the error rates of the chip in the embodiments of this application and the comparative chip;

[0073] Figure 11B This is a comparison chart of the error rates of a chip in another embodiment of this application and a comparative chip;

[0074] Figure 12 This is a comparison diagram of the 640 adsorption results between the chip in the embodiment of this application and the comparative chip;

[0075] Figure 13 This is a box plot showing the test results of density, throughput, read length, and error rate of the chip in the embodiments of this application after being stored for 180 days. Detailed Implementation

[0076] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0077] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the stated features.

[0078] In this application, "multiple" means two or more, unless otherwise expressly defined.

[0079] Most of the specific data / values ​​mentioned in the description of this application are statistically significant. Therefore, unless otherwise specified, any value expressed in a precise manner represents a range, that is, an interval including plus or minus 10% of the value. This will not be repeated below.

[0080] In this application, the term "chip" refers to a reaction chamber containing a solid substrate, having a space to contain liquid, and capable of immobilizing the sample to be tested; it is also called a flow cell or flow-cell. The "solid substrate," such as the substrate described here, can be any solid support that can be used to immobilize nucleic acid sequences, such as nylon membranes, glass slides, plastics, silicon wafers, magnetic beads, etc. In some examples, the first and second substrates are transparent, for example, both being glass slides / glass layers.

[0081] In this application, "thermosensitive substance / material" refers to substances or materials that are soluble in water or other solvents, responsive to temperature stimuli, and whose state or physicochemical properties change when the external or internal ambient temperature changes. For example, substances / materials that dissolve in water at relatively high temperatures to form an aqueous solution, but gradually solidify in the aqueous solution to form a gel or semi-solid state at relatively low temperatures; or substances / materials that dissolve in water at relatively low temperatures to form a solution, but gradually solidify in the aqueous solution to form a gel or semi-solid state at relatively high temperatures. Exemplarily, thermosensitive substances / materials include, but are not limited to, gelatin, pectin, ethylene glycol, glycerin, trehalose, and thermosensitive hydrogels (e.g., polymers of acrylamide derivatives, acetalized polyvinyl alcohol), etc.

[0082] In this application, "solution containing thermosensitive substance / material" refers to a homogeneous and stable solution formed by dissolving the thermosensitive substance / material in a solvent (e.g., water).

[0083] In this application, "coating" refers to a coating layer formed by applying a coating material to a surface through methods such as deposition, dip coating, spraying, spin coating, printing, or flow coating.

[0084] In this application, "sequencing" refers to sequence determination, the same as "nucleic acid sequencing" or "gene sequencing," which refers to the determination of the base order in a nucleic acid sequence; including sequencing by synthesis (sequencing-by-synthesis, SBS) and / or sequencing by ligation (sequencing-by-ligation, SBL); including DNA sequencing and / or RNA sequencing; including long fragment sequencing and / or short fragment sequencing, the terms "long fragment" and "short fragment" 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; including paired-end sequencing, single-end sequencing, and / or paired-end sequencing, etc., the term "paired-end sequencing" or "paired-end sequencing" can refer to the readout of any two segments or parts of the same nucleic acid molecule that do not completely overlap.

[0085] Sequencing, as it is called, involves the process of binding nucleotides (including nucleotide analogs) to a template and acquiring the corresponding reaction signals. In some sequencing platforms where the binding of nucleotides to the template and the acquisition of the corresponding reaction signals are asynchronous, multiple rounds of sequencing are generally used to determine the order of multiple nucleotides / bases on the template. One sequencing cycle, also called a sequencing round, can be defined as one base extension of four nucleotides / bases; in other words, it can be defined as the process of determining the base type at any specified position on the template. For sequencing platforms that achieve sequencing based on controlled polymerization or ligation reactions, one round of sequencing includes the process of binding four nucleotides to the template once and acquiring the corresponding reaction signals. For platforms that achieve sequencing based on polymerization reactions, the reaction system includes reaction substrate nucleotides, polymerase, and template, so that a predetermined sequence (sequencing primer) is bound to the template, based on the base pairing principle. Following the principle of polymerization, the added reaction substrate (nucleotide) is controllably linked to the 3' end of the sequencing primer under the catalysis of polymerase, achieving base pairing with the corresponding bases in the template. Typically, a sequencing run may include one or more base extensions (repeat). For example, four nucleotides are added to the reaction system sequentially, and base extension and corresponding reaction signal acquisition are performed separately, resulting in four base extensions in one sequencing run. Alternatively, four nucleotides can be added to the reaction system in any combination, such as in pairs or in a one-to-three combination, with each combination performing base extension and corresponding reaction signal acquisition separately, resulting in two base extensions in one sequencing run. Yet another example is that four nucleotides are added to the reaction system simultaneously for base extension and reaction signal acquisition, resulting in one base extension in one sequencing run.

[0086] Sequencing can be performed using a sequencing platform, which can be selected from, but is not limited to, Illumina's HiSeq / MiSeq / NextSeq / Novaseq sequencing platforms, Thermo Fisher / Life Technologies' IonTorrent platform, BGI Genomics' BGISEQ and MGISEQ / DNBSEQ platforms, as well as single-molecule sequencing platforms; the sequencing method can be either single-end sequencing or paired-end sequencing.

[0087] Typically, sequencing chips adapted to SBS platforms contain one or more parallel channels / fluidic channels. These channels are used for the entry, exit, and carrying of reagents to create the environment required for sequencing reactions. The chip body can be encapsulated by bonding two pieces of glass and a metal substrate. The sequencing process involves an imaging system, such as a camera, taking multiple pictures of one or more areas of the chip. Each pictured area is called the field of view (FOV). Between two cycles, reagents are reintroduced for biochemical reactions.

[0088] Please see Figures 1-4 This application provides a chip 100, which includes a first substrate 20 and a second substrate 30, which are stacked on top of the first substrate 20. The second substrate 30 includes a first surface 31 and a second surface 32 facing away from each other. The first surface 31 of the second substrate 30 faces the first substrate 20. One or more fluid channels 70 are provided between the first surface 31 of the second substrate 30 and the first substrate 20. A first coating 50 is provided on the second surface 32 of the second substrate 30. The autofluorescence intensity of the first coating 50 is less than a preset intensity.

[0089] In the chip 100 of this application embodiment, the basic structure of the chip 100 is formed by stacking a first substrate 20 and a second substrate 30. One or more fluid channels 70 are provided between the first surfaces 31 of the first substrate 20 and the second substrate 30, so that the chip 100 can carry the sample to be tested, including containing solutions or reagents, to provide a solution environment for biochemical reactions or specific detections. Furthermore, by providing a first coating 50 on the second surface 32 of the second substrate 30, the excitation light transmitted through the second substrate 30 is reduced. The reduction of the excitation light transmitted through the second substrate 30 helps to weaken the fluorescence emitted by the structure below the second substrate 30 when excited by the transmitted excitation light. Since the autofluorescence intensity of the first coating 50 is less than a preset intensity, the fluorescence intensity generated by the first coating 50 when irradiated by the excitation light is weak. Therefore, in the working environment, the fluorescence signal generated by the chip 100 itself is very weak. The chip 100 is suitable for carrying biological samples to realize the detection of biological macromolecules and is adapted to an optical imaging platform for realizing the detection of the sample to be tested based on the detection chip.

[0090] Please see Figures 1-4 In one example, chip 100 is placed under an optical system such as a microscope for detection. The nucleic acid molecule to be tested is labeled with fluorescence and is located in a fluid channel 70, with one end connected to the second surface 22 of the first substrate 20 and / or the other end 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, which includes a lens 200. A laser of a specific wavelength emitted by the laser irradiates the fluid channel 70 of chip 100, exciting the fluorescent label therein to emit fluorescence. The lens 200 of the camera is used to photograph these fluorescent areas to obtain an image, thereby realizing signal acquisition of the sample to be tested. Then, based on image processing and analysis, the sample to be tested can be detected.

[0091] Specifically, in sequencing platforms that enable nucleic acid sequencing based on chip detection, the chip adapted to the sequencing platform is typically constructed by stacking two layers of glass and a thermally conductive metal substrate, such as an aluminum plate, and bonding these three structures together using adhesives such as water-based glue or double-sided tape. The lower glass layer can be bonded to the aluminum plate using adhesives. Current materials science and processes, such as surface processing, etching, and bonding encapsulation techniques, generally ensure that the planar flatness of the upper and lower glass layers and the stability of the chip's temperature conductivity meet the requirements of the sequencing platform.

[0092] The requirements for sequencing platforms mentioned here can be understood as follows: For automated sequencing platforms that include optical systems, this generally involves continuously photographing multiple regions on the chip. In particular, for sequencing platforms that include high-magnification lenses, such as 20× or higher optical systems, the area capable of clearly imaging the sample is typically at the micrometer or nanometer scale (focal plane / clear plane), thus placing high demands on the compatible chip. For example, the flatness of the chip surface is crucial. Furthermore, sequencing reactions often involve multiple biochemical reactions, the use of corrosive solutions, and short-term temperature increases and decreases, thus placing numerous and high demands on the physicochemical properties and bonding strength of the structures containing / contacting the solution. Additionally, chip fabrication typically requires applying significant forces at high temperatures to ensure tight adhesion between structures, thus imposing numerous requirements and limitations on the pressure tolerance, temperature tolerance, and bonding strength of the chip and its constituent structures compatible with the sequencing platform. If a further goal is to achieve low reagent consumption, such as rapid detection of samples in nanoliters or smaller volumes, microfluidics technology is involved, placing even more demands on the structure, connectivity, materials, and fabrication processes.

[0093] However, during the structural design, fabrication, and testing of the chip 100 adapted to the sequencing platform, the inventors discovered that when the laser emitted by the sequencing platform's laser irradiates the fluid channel 70 of the chip 100 through the lens 200, it also irradiates the adhesive layer connecting the lower glass and the aluminum plate through the lower glass. As a result, the molecules in the adhesive layer are excited to emit fluorescence, which greatly interferes with the recognition and detection of the target signal, namely the signal of the nucleic acid molecules to be tested from the fluid channel 70.

[0094] Based on extensive prior test data, including image data of chips of varying quality obtained under specific working conditions, the inventors measured and compared the characteristics and parameters of the corresponding chips, such as the thickness of each chip coating, surface flatness, adhesive strength, adhesive chemical properties, and optical properties. They set expectations / requirements for the surface of the metal substrate, such as an aluminum plate, and for the adhesive used to bond the aluminum plate. However, investigations and commissioning of multiple suppliers in the field for processing and testing revealed that virtually no suppliers on the market can provide the processing technology and / or adhesive to meet the set expectations / requirements. In other words, the yield of chips meeting the expectations / requirements prepared using commercially available technologies / processes is very low, resulting in high chip manufacturing costs. This high cost and difficulty in controlling structural parameters are highly detrimental to the industrial production of chips adapted to sequencing platforms and further performance improvements.

[0095] The chip 100 with the above-mentioned features in this embodiment can solve this problem well. The first coating 50 on the chip 100 can ensure that the chip 100 has good fluorescence characteristics to meet the requirements of gene sequencers for the fluorescence background characteristics of the chip 100. Moreover, the processing technology for preparing the first coating 50 on the second surface 32 of the second substrate 30 is mature and simple, and the performance of the prepared first coating 50 is controllable, which is very beneficial for the large-scale preparation of the chip 100 adapted to the sequencing platform.

[0096] Specifically, the first substrate 20 and the second substrate 30 may comprise any suitable material, such as glass, silicon dioxide, crystal, quartz glass, plastic, ceramic, PET (polyterephthalic acid), PMMA (polymethyl methacrylate), or any other suitable material.

[0097] The first substrate 20 and the second substrate 30 can be optically transparent.

[0098] The first substrate 20 and the second substrate 30 can be in 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.

[0099] The first substrate 20 and the second substrate 30 may be the same size or different. In one embodiment, the thickness of the first substrate 20 is less than the thickness of the second substrate 30.

[0100] The second substrate 30 includes a first surface 31 and a second surface 32 of opposite surfaces, 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 and serves as the bottom surface of the second substrate 30. A fluid channel 70 is provided between the first surface 31 of the second substrate 30 and the first substrate 20, and the fluid channel 70 can serve as the site for chemical reaction of fluid reagents, i.e., the area where the target signal is located.

[0101] In order to reduce the amount of laser light that passes through the second substrate 30 and irradiates other structures below the second substrate 30 of the chip 100, such as the adhesive layer connecting the second substrate 30 and the structure below the second substrate 30, a first coating 50 can be provided on the second surface 32 of the second substrate 30, and it is desirable that the autofluorescence intensity of the first coating 50 is less than a preset intensity.

[0102] Understandably, many substances in nature possess autofluorescence properties, which refers to the fluorescence emitted by a substance when excited by light or after absorbing energy. A stronger autofluorescence property of the first coating 50 indicates stronger fluorescence generated by the molecules in the first coating 50 when excited by the laser, which in turn indicates stronger noise, leading to a lower signal-to-noise ratio in the acquired image and affecting sequencing. Therefore, when selecting the first coating 50, materials with autofluorescence intensities lower than a preset intensity are preferred. The preset intensity can be calculated and determined based on actual operational requirements. Specifically, the autofluorescence intensity of the first coating 50 is related to the irradiation conditions, such as the laser intensity emitted by the laser and the objective lens parameters, as well as the autofluorescence properties of the material in the first coating 50. Through extensive comparative testing, the inventors discovered that under specific working conditions, such as irradiation with an 800–1000 mW 532 nm or 635 nm laser, 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. Based on this specific preset value and the specific working environment, the preset intensity here can be determined.

[0103] Please see Figure 2 and Figure 3 In some embodiments, the first coating 50 may be applied to the second surface.

[0104] Thus, by coating the first coating 50 onto the second surface, when the laser irradiates the chip 100, since the first coating 50 is coated on the second surface 32 of the second substrate 30 and the autofluorescence intensity of the first coating 50 is less than the preset intensity, the excitation light transmitted through the second substrate 30 is reduced, that is, the laser is blocked on the second surface 32 of the second substrate 30. This helps to reduce the fluorescence emitted by the structure below the second substrate 30 when 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.

[0105] Specifically, as mentioned above, the second substrate 30 can be made of glass. When the first coating 50 is disposed on the second surface 32 of the second substrate 30, the first coating 50 can be disposed on the second surface 32 of the second substrate 30 by spraying, wherein the spraying can be repeated multiple times to form a first coating 50 of a certain thickness. Of course, the first coating 50 can also be printed on the second surface 32 of the second substrate 30 by screen printing. Spraying is more applicable than screen printing. When the second surface 32 of the second substrate 30 is not flat, the first coating 50 cannot be disposed on the second surface 32 of the second substrate 30 by screen printing. In this case, the first coating 50 can be disposed on the second surface 32 of the second substrate 30 by spraying.

[0106] It is understandable that, since the autofluorescence intensity of the first coating 50 is less than the preset intensity, coating the second surface 32 of the second substrate 30 with the first coating 50 can block the laser from the side of the second substrate 30, so as to meet the requirements of the gene sequencer for the fluorescence background characteristics of the chip 100.

[0107] Please see Figure 3 In some embodiments, the thickness A of the first coating 50 can be in the range of 5 μm to 20 μm. Preferably, the thickness A of the first coating 50 can be in the range of 8 μm to 15 μm. Thus, by applying a first coating 50 of a certain thickness, the first coating 50 provides better shielding against laser light, ensuring that the image of a specific area of ​​the chip 100 acquired after excitation light irradiation meets the sequencing requirements.

[0108] In this application, it should be understood that the terms "length", "width", "thickness", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation.

[0109] Specifically, since the first coating 50 is repeatedly applied to the second surface, and the purpose of applying the first coating 50 is to shield the laser from the second substrate 30, the first coating 50 needs to have a certain thickness. If the thickness is too small, the first coating 50 cannot play a good shielding role. If the thickness is too large, it is understandable that too many times of spraying are required. If the first coating 50 is too thick, it is easy to make it difficult to ensure the uniformity of the thickness of the first coating 50, and it is easy to have uneven surface, wrinkles, or even cracks.

[0110] In other embodiments, the thickness A of the first coating 50 can be set to 10 μm to 40 μm, so that the first coating 50 can better shield the laser, and the image of a specific area of ​​the chip 100 acquired by the excitation light can meet the sequencing requirements.

[0111] In some embodiments, the first coating 50 has a light-shielding rate of not less than 80% in the working environment. Thus, by setting the light-shielding rate of the first coating 50 to not less than 80% in the working environment, it is sufficient to ensure that the first coating 50 can provide good light-shielding for the laser in the working environment.

[0112] Specifically, the working environment includes lasers of specific wavelengths and intensities; in one example, the working environment refers to 800–1000 mW of red or green laser (e.g., lasers emitting wavelengths of 532 nm or 635 nm). To prevent the laser from penetrating the second substrate 30 and illuminating the rest of the chip 100, the first coating 50 needs to have a high light-shielding rate. For example, in this embodiment, when the first coating 50 is applied to the second surface 32 of the second substrate 30 and during the sequencing process, the light-shielding rate of the first coating 50 should be no less than 80%, thus ensuring that the first coating 50 can effectively shield the laser.

[0113] Preferably, the first coating 50 has a light-shielding rate of not less than 95% in the working environment, thereby ensuring that the first coating 50 can play a better light-shielding role for the laser in the working environment.

[0114] In some embodiments, the flatness deviation of the side of the first coating 50 facing away from the second substrate 30 is allowed to be no more than 0.1 μm. In this way, after being stacked and packaged with the remaining structures, it can be ensured that the mechanically accumulated surface flatness deviation / tolerance is within a preset range, so that the flatness of the chip 100 surface meets the predetermined requirements. It also facilitates the stable and firm connection between the first coating 50 and the corresponding surface of the third substrate 60, and contributes to the robustness and stability of the chip 100 structure.

[0115] Specifically, flatness refers to the fact that during the processing or production of certain workpieces, the surface of the workpiece is not absolutely flat, and the difference between its unevenness and its absolute flatness is the flatness. Based on the certain relationship between flatness and scattering rate, since uneven surfaces have a scattering effect on incident light, it can be understood that in order to ensure the quality of the fluorescence signal image, the flatness value of the side of the first coating 50 facing away from the second substrate 30 should be as small as possible. Therefore, the allowable deviation of the flatness of the side of the first coating 50 facing away from the second substrate 30 is set to no more than 0.1 μm.

[0116] Furthermore, it should be noted that setting the allowable flatness deviation of the side of the first coating 50 facing away from the second substrate 30 to no more than 0.1 μm ensures a low scattering rate of the first coating 50. Scattering, as a physical concept, describes the phenomenon where radiant energy (various electromagnetic waves, including light waves and sound waves) encounters localized non-uniform regions along its propagation path, causing its propagation direction to deviate from its original direction. Moreover, as the roughness of the non-uniform region gradually increases, the mirror scattering component gradually decreases, while the diffuse scattering component increases accordingly.

[0117] In this embodiment, the local non-uniformity refers to the fact that the surface of the first coating 50 is not completely smooth and has certain slight height undulations. During gene sequencing, the laser emitted by the laser irradiates the reaction reagents within the fluid channel 70, causing the irradiated fluorescent molecules to emit corresponding fluorescent signals. When the lens 200 captures a photograph of the fluorescent signal, to ensure higher quality and easier analysis, the allowable flatness deviation of the side of the first coating 50 facing away from the second substrate 30 is set to no more than 0.1 μm. This reduces scattering interference that occurs when the incident laser irradiates the first coating 50, thus reducing the difficulty in capturing the fluorescent signal image.

[0118] In some embodiments, the material of the first coating 50 may include ink. This results in a chip 100 having high light-shielding properties and weak luminescence characteristics under operating conditions, and is also easy to manufacture.

[0119] Specifically, ink is a homogeneous mixture that mainly consists of pigments, binders, fillers, additives, and other substances. Ink can be printed and dried on the printed surface.

[0120] Pigments can include both pigments and dyes. Pigments are divided into organic and inorganic pigments. Organic pigments have bright colors, strong tinting strength, and short drying time, so they are widely used in inks. Inorganic pigments have better lightfastness, heat resistance, solvent resistance, and hiding power. Pigments color in a particulate state and do not dissolve, making them the most commonly used pigments in inks. Dyes, on the other hand, are formulated into solutions and color in a molecular state, resulting in a less effective color than pigments.

[0121] The binder acts as a dispersant for the pigments and can be made by dissolving small amounts of natural resins, synthetic resins, cellulose, rubber derivatives, etc., in drying oils or solvents. The binder allows the ink to form a uniform thin layer after being sprayed onto an object, and after drying, it forms a film layer with a certain strength to protect the pigments.

[0122] 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, talc, and calcium carbonate. The latter may be an additional part of the pigment or an additional part of the binder, depending on the product requirements.

[0123] In some embodiments, the first coating 50 is preferably black ink. This allows for the acquisition of higher quality images of specific areas of the chip 100. Furthermore, by printing on the corresponding surface of the second substrate 30 to obtain a second substrate 30 with the black first coating 50, it is possible to quickly and controllably produce a first coating 50 that meets the requirements, or a chip 100 including a second substrate 30 with the first coating 50.

[0124] Specifically, this application requires a high degree of light-blocking properties for the first coating 50. Pigments, as solid components of ink and the colorants in ink, are generally water-insoluble pigments. Therefore, considering the light-blocking requirements of the first coating 50, preferably, in one embodiment, black ink can be used to achieve a high level of light-blocking effect. Furthermore, considering the working environment and stability, the ink used as one of the materials for the first coating 50 also needs to possess high-temperature resistance.

[0125] Please see Figure 3 and Figure 4 In some embodiments, the first substrate 20 may include a first surface and a second surface, with the first surface 21 of the first substrate 20 and the second surface 22 of the first substrate 20 disposed opposite to each other. Fluid channels 70 may be formed on the second surface 22 of the first substrate 20 and the first surface 31 of the second substrate 30. 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 operating conditions.

[0126] Specifically, in one 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. An optical imaging system including a lens module is used to acquire an image of the nucleic acid molecule. During 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 21 of the first substrate 20, and then to determine the focal plane / sharp plane to acquire a clear image of the nucleic acid molecule. During this imaging process, when excited by excitation light, in addition to the fluorescently labeled nucleic acid molecules emitting fluorescence, the first surface 31, the second surface 32, and / or the first coating 50 of the second substrate 30 of the chip 100 also emit fluorescence, which appears as background signals or interference signals that are difficult to distinguish from the target signal in the acquired image of the chip 100.

[0127] In another example, the sample to be tested, such as a solution containing nucleic acid molecules, is located in 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. An optical imaging system including a lens module is used to acquire an image of the nucleic acid molecule. During 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, thereby determining the corresponding focal plane / sharp plane to acquire a clear image of the nucleic acid molecule. During this imaging process, when excited by excitation light, in addition to the fluorescence emitted by the nucleic acid molecules containing fluorescent labels, the first surface 31, the second surface 32, and / or the first coating 50 of the second substrate 30 of the chip 100 will also emit fluorescence, which appears as background signals or interference signals that are difficult to distinguish from the target signal in the acquired image of the chip 100.

[0128] In one example, under specific working conditions, such as irradiation with a 532nm or 635nm laser at 800–1000mW, the acquired image is a 16-bit image with a preset value of 400 (int), meaning the background intensity of the image from chip 100 is less than or equal to 400. This allows for the acquisition of images with a high signal-to-noise ratio, which is beneficial for obtaining high-quality sequencing results. Figure 6 The background intensity of the images of four chips 100 is shown in the diagram. The second surface 32 of the second substrate 30 of these chips 100 is printed with black ink, and the fluorescence background intensity of these images is less than 400.

[0129] It is understood that those skilled in the art, through the method of determining the preset values ​​in this example, can determine suitable preset values ​​in other working environments, such as lasers of different intensities and wavelengths.

[0130] Specifically, image intensity represents the intensity of image pixels. In a grayscale image, image intensity is the gray level of the image. In the RGB color space, it can be understood as the pixel gray level value of the R channel, G channel, or B channel. Other color spaces are similar.

[0131] In a grayscale image, the gray level represents the brightness of the image. The higher the gray level, the brighter the pixel. Thus, in this embodiment, the background intensity of the image of chip 100 is less than or equal to a preset value, which means that the background brightness of the obtained image of chip 100 is low, that is, the signal-to-noise ratio of the image is high, which is conducive to the identification of the target signal and the identification of bases based on the target signal. It can be understood that the preset value can be set according to the actual needs during sequencing.

[0132] Specifically, in one example, on a sequencing platform containing an optical imaging system, the optical system includes a laser and a camera, the camera including a lens 200, and the image of the chip 100 can be acquired by: turning on the laser to emit a laser, so that the laser shines through the lens 200 onto the test reagent in the fluid channel 70, the test reagent including a nucleic acid molecule with a fluorescent label, the fluorescent label being excited to emit fluorescence, and then using the camera to collect and photograph the fluorescence passing through the lens 200 to obtain an image containing the target signal.

[0133] In another example, on a sequencing platform incorporating 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, such as a total internal reflection objective. Images of the chip 100 can be acquired by: activating the laser to emit a laser beam, which is then directed through the lens 200 at an angle greater than the critical angle to the medium interface in the chip 100, such as a solid-liquid interface, for example, the second surface 22 of the first substrate 20. An evanescent wave / evanescent field is generated at this interface. The test reagent includes a nucleic acid molecule with a fluorescent label, one end of which is attached to the second surface 22 of the first substrate 20. The fluorescent label emits fluorescence in the evanescent field. The camera then captures and photographs the fluorescence passing through the lens 200 to obtain an image containing the target signal.

[0134] In addition, the background intensity of the image of chip 100 is less than or equal to the preset value because the first coating 50 coated on the second surface 32 of the second substrate 30 blocks the laser from the side of the second substrate 30. Since the autofluorescence intensity of the first coating 50 itself is low, the fluorescence signal generated by the molecules in the first coating 50 when irradiated by the laser is weak, that is, the noise is low. As a result, the signal-to-noise ratio of the acquired image is improved, so that the background intensity of the finally obtained fluorescence signal image can be less than or equal to the preset value.

[0135] In some implementations, the background intensity of the image of chip 100 can be less than or equal to 400. Thus, by controlling the background intensity of the image of chip 100 to less than or equal to 400, the brightness of the background portion of the image is lower, facilitating the comparison and display of effective fluorescence signals, thereby improving sequencing quality.

[0136] 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. This means that when irradiated with an 800mW red-green laser, the background intensity of the image of the chip 100 acquired by the lens 200 is required to be less than or equal to 400 in the 16-bit image, thereby improving the sequencing quality.

[0137] Furthermore, the second coating in this chip isolates the air from the fluid channels during chip storage, reducing the adsorption of airborne impurities within the fluid channels, thereby reducing the chip's background signal and sequencing error rate. Moreover, this second coating solution is washed away before chip use, leaving no residue and not affecting subsequent reactions within the chip.

[0138] In some embodiments, the components forming the second coating include a temperature-sensitive substance / material. In some embodiments, the temperature-sensitive substance / material is provided in the form of an aqueous solution. Thus, an aqueous solution of the temperature-sensitive substance / material (e.g., gelatin, pectin, ethylene glycol, trehalose described below) can be introduced into the fluid channels of the chip at a higher temperature, and then the temperature can be lowered to solidify the aqueous solution, thereby forming a wet film-like second coating. Before chip use, the second coating is re-dissolved by raising the temperature, thereby removing the temperature-sensitive substance / material without affecting subsequent reactions in the chip. Alternatively, in some embodiments, an aqueous solution of the temperature-sensitive substance / material (e.g., temperature-sensitive hydrogel described below) can be introduced into the fluid channels of the chip at a lower temperature, and then the temperature can be raised to solidify the aqueous solution, thereby forming a wet film-like second coating. Before chip use, the second coating is re-dissolved by lowering the temperature, thereby removing the temperature-sensitive substance / material without affecting subsequent reactions in the chip.

[0139] Specifically, in some embodiments, the temperature-sensitive material / substance can be removed by cleaning as follows: the temperature of the cryogenically stored chip is equilibrated to room temperature to allow the temperature-sensitive material / substance to re-dissolve, and then a cleaning solution is introduced into the fluid channel to remove the material / substance. In some embodiments, different cleaning solutions can be used to clean the fluid channel twice, with heating applied during the cleaning process to further improve the cleaning effect of the temperature-sensitive material / substance. For example, 1 mL of sodium dodecyl sulfate solution (SDS) can be first introduced into the fluid channel and reacted at 50–60°C for 20–40 min; then, 3 × 1 mL of sodium citrate buffer (SSC) can be rinsed and reacted at 50–60°C for 20–40 min to complete the removal of the second coating. By performing a two-step cleaning, the physical adsorption on the chip substrate surface can be removed while restoring the three-dimensional structure of the functional groups on the chip surface, thereby facilitating the connection of hybridization chains and improving hybridization efficiency.

[0140] In some embodiments, the temperature-sensitive substance / material can be selected from at least one of gelatin, pectin, ethylene glycol, glycerin, and trehalose, preferably two of them, such as a combination of trehalose and gelatin, or a combination of glycerin and gelatin. In some embodiments, the temperature-sensitive substance / material can also be selected from temperature-sensitive hydrogels. Taking the combination of trehalose and gelatin as an example, the mass ratio of the two can be (10-20):(80-90), more preferably 15:85; gelatin can provide excellent water barrier properties, reduce surface water vapor flow, and prevent changes in the surface microenvironment, while trehalose increases the surface's antioxidant properties, making surface molecules less susceptible to oxygen influence and increasing the stability of surface genes and groups. Taking the combination of glycerin and gelatin as an example, the mass ratio of the two can be (40-60):(40-60), more preferably 50:50; gelatin can provide water barrier properties, while glycerin provides certain antibacterial properties and improves the low-temperature stability of the surface. In addition, it should be noted that in the multi-component thermosensitive substance / material, the proportion of gelatin component is preferably 10% to 100%, which plays a role in stabilizing the surface moisture and humidity microenvironment and ensures that the surface will not be excessively dried, which would cause significant changes in the structure of surface macromolecules and genetic components (structural collapse). The proportions of trehalose, glycerol, ethylene glycol and pectin are preferably 0% to 90%, so that the protective ability of the surface protective layer is not too singular and has a more comprehensive protective ability.

[0141] Preferably, the mass concentration of the thermosensitive substance / material in the aqueous solution is 0.5% to 5%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc. By setting the mass concentration of the thermosensitive substance / material in the aqueous solution within the above range, the overall solidification and melting temperature of the thermosensitive substance / material aqueous solution can be kept within an easily operable range, facilitating the formation and removal of the second coating. If the concentration of the thermosensitive substance / material in the aqueous solution is too high, the formed second coating may have a strong adhesion to the designated surface, making it difficult to remove or rinse off during use, thereby affecting the performance of the designated surface, the biochemical reactions on the designated surface, and detection; in addition, it may also have other adverse effects on the performance of the second coating and / or the chip. For example, when glycerol is used as the thermosensitive substance / material, an excessively high glycerol concentration will result in an excessively strong water absorption capacity of the formed second coating, causing a decrease in the concentration of the second coating solution, and excessive water residue will accelerate the hydrolysis reaction on the chip substrate surface, leading to the hydrolysis of specific components on the substrate surface and affecting the sequencing biochemical reaction. If the concentration of the temperature-sensitive substance / material in the aqueous solution is too low, it will be difficult to form a second coating, or the formed second coating will not provide adequate protection for the designated surface within the fluid channel. Therefore, controlling the content of the temperature-sensitive substance / material within the above-mentioned range is beneficial for obtaining chips with long-term stable surface properties and easy storage and transportation.

[0142] In the typical coating industry, coatings are at the micron level and engineering grade. The adhesion of coatings, such as paint films, to surfaces can be measured using industry standards, for example, by scratching the paint film thousands of times with a pencil. Similarly, the adhesion of adhesive films, similar to tape, to surfaces can be measured using standard testing methods, such as a peel force of 1-2 N / cm. However, it is understood that the second coating in this application is a hydrogel, which is at the molecular or nanoscale level. The interaction between this coating and the surface falls under the category of molecular-to-surface interactions. The intermolecular distance in this hydrogel is likely approximately 0.3-0.5 nm. Currently, there are no testing standards for this interaction; it is a matter of scientific research scale. Based on the specific substance content used to form the hydrogel coating in this example, including the description of the influencing factors and their degree of influence discovered by the inventors, it is believed that those skilled in the art can, based on this and in conjunction with the state of the specified chip surface in a specific application scenario, formulate a suitable solution to form an effective protective coating on that specified surface.

[0143] In some embodiments, the material forming the second coating also includes a corrosion-resistant material. By using a corrosion-resistant material, not only can certain temperature-sensitive substances / materials (such as gelatin) be prevented from being decomposed and deteriorated by bacteria, but bacteria can also be prevented from acting on the PO sites of the functional group phosphodiester bonds on the inner surface of the chip, thereby avoiding problems such as primer detachment and loss of hybridization sites.

[0144] For example, the corrosion inhibitor may be selected from at least one of ethylenediaminetetraacetic acid (EDTA), ethylene glycol phenyl ether (EDP), and sodium thimerosal. When disodium EDTA is used as the corrosion inhibitor, its preferred concentration is 0.05–0.15 mmol / L; when ethylene glycol phenyl ether is used as the corrosion inhibitor, its preferred concentration is 0.5%–1.5% (mass-volume ratio); and when sodium thimerosal is used as the corrosion inhibitor, its preferred concentration is 0.005%–0.015% (mass-volume ratio). It should be noted that the above concentrations refer to the final concentration of the corrosion inhibitor in the mixed solution after being added to the aqueous solution containing the temperature-sensitive substance / material.

[0145] In some embodiments, the primers / probes involved in sequencing in the chip of this application are covalently linked to specific components (such as epoxy silane) on the surface of the chip fluid channels. For this connection method, the protective effect of the second coating can be further improved by employing the second coating material as described above.

[0146] In some embodiments, the solution used to form the second coating is pre-sterilized before use. For example, sterilization may be performed at 115–125°C for 10–20 minutes.

[0147] Please see Figure 1 and Figure 2 In some embodiments, the chip 100 may include a third substrate 60 disposed on the first coating 50, wherein the third substrate 60 is made of metal.

[0148] Thus, by setting a third substrate 60 attached to the first coating 50, the flatness stability and temperature conductivity stability of the first substrate 20 and the second substrate 30 can be guaranteed.

[0149] Specifically, the chip 100 also includes a housing 10 and a third substrate 60. The housing 10 can provide a certain degree of protection for the chip 100. The housing 10 can be made of resin, that is, the housing 10 is formed by injection molding, which makes the manufacturing cost low and the manufacturing process simple.

[0150] The third substrate 60 can be attached to the first coating 50 and disposed on opposite sides of the second substrate 30. The third substrate 60 is made of a metal with good thermal conductivity, such as an aluminum plate, and the third substrate 60 and the first coating 50 can be bonded together using adhesive 61.

[0151] It is understandable that, in the packaging of chip 100, the superimposed packaging method of using the first substrate 20, the second substrate 30, and the third substrate 60 and bonding them together with adhesive 61 can give chip 100 a packaging structure with good sealing and heat dissipation. Specifically, using an aluminum plate as the third substrate 60 is lightweight, provides good heat dissipation, and ensures 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 to the first coating 50, thereby fixing the third substrate 60 to the first substrate 20 and the second substrate 30, resulting in good sealing of chip 100.

[0152] Specifically, since a first coating 50 is provided on the second surface 32 of the second substrate 30, the first coating 50 can prevent the laser from irradiating the adhesive 61 between the aluminum plate and the first coating 50, thereby preventing the molecules in the aluminum plate or adhesive 61 from being excited to form fluorescence signal-to-noise ratio. In this way, the chip 100's dependence on the surface quality of the aluminum plate is reduced, thereby reducing the aluminum plate processing cost, and also expanding the selection range of adhesive 61, no longer limited by fluorescence performance indicators.

[0153] Please see 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. One or more fluid channels 70 may be disposed in the interposer 40. Thus, by disposing of 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, simplifying the manufacturing process of the fluid channels 70 and also simplifying the fabrication process of the chip 100.

[0154] In this application, unless otherwise expressly specified or limited, the terms "connected" or "linked" should be interpreted broadly. For example, it can refer to a fixed connection, a detachable connection, or an integral connection; it can refer to a mechanical connection, an electrical connection, or a connection that allows communication between the components; it can refer to a direct connection or an indirect connection through an intermediate medium; it can refer to the internal communication between two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0155] Specifically, an interposer 40 is formed between the first substrate 20 and the second substrate 30. The interposer 40 can serve as a channel adhesive 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 encapsulated together through the interposer 40. The interposer 40 is provided with fluid channels 70, so that the interposer 40 can serve as a site for chemical reactions of fluid reagents, that is, the region where beneficial fluorescence signals are generated by laser irradiation.

[0156] Therefore, 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 the outlet or inlet of fluid pumped out from the fluid channel 70 of the intermediate layer 40.

[0157] Specifically, 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 formation process of the fluid channel 70. In this application, the fluid channel 70 may be formed by stamping the interposer 40, laser cutting, or other methods.

[0158] Please see Figure 4 and Figure 5 In some embodiments, the intermediary layer 40 can bond the first substrate 20 and the second substrate 30. The intermediary layer 40 can have a hollow structure. The intermediary layer 40 can include a base layer 41, a first adhesive layer 42, a second adhesive layer 43, and the hollow structure penetrates the base layer 41, the first adhesive layer 42, and the second adhesive layer 43 to form one or more fluid channels 70.

[0159] The base layer 41 has a first surface and a second surface facing away from each other. A first adhesive layer 42 is disposed on the first surface of the base layer 41, and a second adhesive layer 43 is disposed on the second surface 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.

[0160] Thus, by providing the first adhesive layer 42 and the second adhesive layer 43, the intermediate layer 40 can be disposed between the first substrate 20 and the second substrate 30. By forming a perforated structure on the intermediate layer 40, the perforated 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. This allows reactive reagents to enter the fluid channels 70 through the first substrate 20 and / or the second substrate 30, undergo a chemical reaction within the fluid channels 70, and then flow out through the first substrate 20 and / or the second substrate 30.

[0161] Specifically, the interposer 40 includes a base layer 41, and the base layer 41 can be a major component of the interposer 40. The material of the base layer 41 can include black PET, clear PET, or any other plastic or polymer, providing high contrast for the fluorescent image ultimately captured by the lens 200.

[0162] The interposer layer 40 further includes a first adhesive layer 42 and a second adhesive layer 43. The first adhesive layer 42 is disposed on a first surface of the substrate 41, and the second adhesive layer 43 is disposed on a second surface of the substrate 41, wherein the first surface and the second surface of the substrate 41 are opposite to each other. The first adhesive layer 42 and the second adhesive layer 43 are adhesive, thereby connecting the first substrate 20 and the second substrate 30 to the interposer layer 40. It is easy to understand that the first adhesive layer 42 can be bonded to the first substrate 20 or to the second substrate 30, and correspondingly, the second adhesive layer 43 can be bonded to the second substrate 30 or to the first substrate 20.

[0163] Furthermore, the first adhesive layer 42 can be bonded to the second surface 22 of the first substrate 20, and the second adhesive layer 43 can 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 an integral whole.

[0164] The first adhesive layer 42 and the second adhesive layer 43 should also have low autofluorescence, so that the influence of the intermediate layer 40 formed by the combination of the base layer 41, the first adhesive layer 42 and the second adhesive layer 43 on the fluorescence signal generated in the region of the intermediate layer 40 can be ignored, thereby improving the signal-to-noise ratio and improving the sequencing quality.

[0165] There can be one or more fluid channels 70. Multiple fluid channels 70 can make the biochemical reaction of the reaction reagents more uniform and the sequencing more efficient. The fluid channels 70 can be formed on the intermediate layer 40 by means of stamping or laser cutting. In particular, the fluid channels 70 penetrate through the first adhesive layer 42, the base layer 41 and the second adhesive layer 43, that is, the fluid channels 70 extend through each of the first adhesive layer 42, the base layer 41 and the second adhesive layer 43.

[0166] Please see Figure 4 and Figure 5 In some embodiments, the first substrate 20 and / or the second substrate 30 are provided with through holes 33 communicating with the fluid channel 70.

[0167] Thus, by providing through holes 33 connected to the fluid channel 70 on the first substrate 20 and / or the second substrate 30, fluids such as reaction reagents can enter the fluid channel 70 through the first substrate 20 and / or the second substrate 30, and can also undergo a chemical reaction in the fluid channel 70 before flowing out through the first substrate 20 and / or the second substrate 30. Moreover, it is also convenient to connect pipes or manifolds to connect valve bodies and reaction reagent containers.

[0168] Specifically, the through hole 33 can be formed on the first substrate 20, or on the second substrate 30, or on both the first substrate 20 and the second substrate 30. In this embodiment, the through hole 33 is formed on the second substrate 30 so that the fluid such as the reaction reagent flows into the fluid channel 70 through the through hole 33 and flows out through the through hole 33 after the reaction.

[0169] Furthermore, it can be understood that there can be multiple vias 33, some of which serve as fluid inlets and the rest as fluid outlets. The vias 33 can be formed in an array on the first substrate 20 and the second substrate 30. Moreover, the array of vias can be etched on either the first substrate 20 or the second substrate 30 by wet etching or dry etching.

[0170] In some embodiments, the peel force of the first adhesive layer 42 to the first substrate 20 and / or the peel force of the second adhesive layer 43 to the second substrate 30 may be not less than 560g. Preferably, the peel force of the first adhesive layer 42 to the first substrate 20 and / or the peel force of the second adhesive layer 43 to the second substrate 30 may be not less than 800g.

[0171] Thus, by minimizing the peel force of the first adhesive layer 42 on the first substrate 20 and / or the peel force of the second adhesive layer 43 on the second substrate 30, 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 to meet the operational requirements. For example, if the adhesive peel force of the first adhesive layer 42 / second adhesive layer 43 on the first substrate 20 / second substrate 30 is not less than the specified value, the various structures of the chip 100 can be firmly connected and the structure of the chip 100 can be stable, thus meeting the sequencing requirements.

[0172] Specifically, during gene sequencing, the chip 100 undergoes multiple pressure cycles due to pressurized fluid flow through the fluid channel 70, meaning the interposer 40 with the fluid channel 70 is exposed to high pressure. Therefore, it is necessary to ensure that the interposer 40 can withstand the pressure. Thus, at least one of the peel forces of the first adhesive layer 42 to the first substrate 20 and the second adhesive layer 43 to the second substrate 30 needs to be set to not less than 560g. In other embodiments, this can be set to not less than 800g, ensuring a sufficiently strong bond between the first adhesive layer 42 and the first substrate 20, and between the second adhesive layer 43 and the second substrate 30.

[0173] It is understandable that the key indicator for bonding quality is adhesive strength. Therefore, ensuring that the peel force of the first adhesive layer 42 to the first substrate 20 and the peel force of the second adhesive layer 43 to the second substrate 30 are minimized can guarantee the adhesive strength of the first adhesive layer 42 to the first substrate 20 and / or the second adhesive layer 43 to the second substrate 30.

[0174] Please see Figure 4 In some embodiments, the fluid channel 70 has a larger dimension in the first direction X than in the second direction Y, where 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. This standardizes the approximate shape of the fluid channel 70 formed in the interposer 40, facilitating fluid control within the fluid channel 70 and enabling the positioning and imaging of these areas of the chip 100.

[0175] Specifically, such as Figure 4 As shown, Figure 4 A schematic diagram of the planar structure of the intermediary layer 40 is shown. In this diagram, the first direction X can be the length direction of the intermediary layer 40, and the second direction Y can be the width direction of the intermediary layer 40. Figure 4 It can be easily seen that the fluid channel 70 has an irregular shape, and the size of the fluid channel 70 in the first direction X is larger than the size in the second direction Y. Furthermore, the first direction X is perpendicular to the second direction Y. In addition, both the first direction X and the second direction Y are perpendicular to the thickness direction of the interposer layer 40.

[0176] Of course, the fluid channel 70 can also have a dimension in the first direction X that is smaller than the dimension in the second direction Y. In this case, the first direction X is still perpendicular to the second direction Y. Furthermore, both the first direction X and the second direction Y are still perpendicular to the thickness direction of the interposer 40.

[0177] Please see Figure 4 In some embodiments, the number of fluid channels 70 is multiple, and the fluid channels 70 are disposed in the intermediate layer 40 extending along a first direction X; and / or, the fluid channels 70 are arrayed on the intermediate layer 40 along a second direction Y. In this way, by forming multiple fluid channels 70, the gene sequencing process becomes more efficient, it is easier to control the fluid in the fluid channels 70, and it is also easier to locate and image these regions of the chip 100.

[0178] Specifically, such as Figure 4As shown, there are four fluid channels 70. The fluid channels 70 are arranged in a first direction X, i.e., the length direction of the intermediate layer 40, and simultaneously arrayed on the intermediate layer 40 along the second direction Y, i.e., the width direction of the intermediate layer 40. In particular, setting multiple fluid channels 70 can make the sequencing process more efficient. At the same time, the array arrangement ensures that the spacing of the fluid channels 70 is consistent, thereby ensuring that the reaction reagents and other fluids pumped into the fluid channels 70 are uniform.

[0179] Please see Figure 4 In some embodiments, the fluid channel 70 includes an intermediate section 71, a first end 72, and a second end 73, with the first end 72 and the second end 73 located at opposite ends of the fluid channel 70. The dimension of the first end 72 in the second direction Y and / or the dimension of the second end 73 in the second direction Y are smaller than the dimension of the intermediate section 71 in the second direction Y.

[0180] This further standardizes the shape of the fluid channel 70, which is beneficial for controlling the fluid in the fluid channel 70 and for locating and imaging these areas of the chip 100.

[0181] Specifically, the fluid channel 70 can be irregular in 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 both ends of the fluid channel 70 and are both triangular in shape. The middle section 71 is a long, narrow rectangle. This 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.

[0182] Of course, the first end 72 and the second end 73 can also be different shapes, as long as the first end 72 and the second end 73 are located at the two ends of the middle section 71 respectively, and one of them has a dimension in the second direction Y that is smaller than the dimension of the middle section 71 in the second direction Y.

[0183] Please see Figure 4 In some embodiments, the dimension of the intermediate segment 71 in the second direction Y is constant. Thus, since the dimension of the intermediate segment 71 in the second direction Y is constant, that is, the length of the intermediate segment 71 in the second direction Y is equal everywhere, for example, the length of the intermediate segment 71 in the second direction Y is always 5 mm. Specifically, as described above, in Figure 4 In the embodiment shown, the middle segment 71 is a long, narrow rectangle, meaning that the length of the middle segment 71 in the second direction Y is constant. Therefore, it can be concluded that the size of the middle segment 71 in the second direction Y is constant.

[0184] Please see Figure 4In some embodiments, the size range L1 of the intermediate segment 71 in the second direction Y is 4.4 mm to 8.4 mm. Thus, by controlling the size range L1 of the intermediate segment 71 in the second direction Y to be 4.4 mm to 8.4 mm, the fluid channel 70 has a certain width to reasonably accommodate the reaction reagents, which is beneficial for fluid control within the fluid channel 70 and for carrying out efficient biochemical reactions within the fluid channel 70.

[0185] Specifically, the middle section 71 can be understood as the main region where the reactants undergo biochemical reactions. Therefore, the middle section 71 should have an appropriate width, since the second direction Y is... Figure 4 As shown in the diagram, when the size of the intermediate segment 71 in the second direction Y is less than 4.4 mm, it may be difficult to process and form the fluid channel 70. When the size of the intermediate segment 71 in the second direction Y is greater than 8.4 mm, the excessive width makes it impossible to form more fluid channels 70 on the intermediate layer 40, the sequencing reaction cannot be carried out efficiently, and the amount of reaction reagent carried by a single fluid channel 70 is also large, which may lead to uneven reaction of the reaction reagent.

[0186] Please see Figure 4 In some embodiments, the spacing L2 between two adjacent fluid channels 70 in the second direction Y can be 0.8 mm to 1.5 mm. This facilitates the fabrication of multiple fluid channels 70 on the interposer layer 40 and ensures a sufficient number of fluid channels 70, thereby enabling efficient sequencing as much as possible.

[0187] Specifically, when the distance between two adjacent fluid channels 70 is less than 0.8 mm, it is difficult to process and form multiple fluid channels 70. When the distance between two adjacent fluid channels 70 is greater than 1.5 mm, the number of fluid channels 70 with the hollow structure formed on the intermediate layer 40 will decrease. Therefore, by controlling the distance L2 between two adjacent fluid channels 70 in the second direction Y to be between 0.8 mm and 1.5 mm, the number of fluid channels 70 is guaranteed while facilitating processing.

[0188] In some embodiments, the thickness B of the base layer 41 can be 30 μm to 90 μm; and / or, the thickness C of the first adhesive layer 42 can be 75 μm to 85 μm; and / or, the thickness D of the second adhesive layer 43 can be 75 μm to 85 μm. Thus, by reasonably setting the thickness B of the base layer 41 and the thickness D of 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.

[0189] Specifically, the thickness range B of the base layer 41 can be 30μm to 90μm, and the thickness range C of the first adhesive layer 42 and the thickness range D of the second adhesive layer 43 are 75μm to 85μm; or, the thickness range B of the base layer 41 can be 30μm to 90μm, and the thickness range C or D of either the second adhesive layer 43 or the first adhesive layer 42 is 75μm to 85μm; or, only the thickness range requirements are set for the first adhesive layer 42 and the second adhesive layer 43, and the thickness range C and the thickness range D of the first adhesive layer 42 and the second adhesive layer 43 are set to 75μm to 85μm; or, only the thickness range C or the thickness range D of the first adhesive layer 42 or the second adhesive layer 43 are required, and the thickness range of one of the adhesive layers is set to 75μm to 85μm.

[0190] It should be noted that, as mentioned above, at least one of the peel force of the first adhesive layer 42 to the first substrate 20 and the peel force of the second adhesive layer 43 to 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 formulation and its thickness relative to the adhesive layer.

[0191] When the adhesive layer is too thin, the first adhesive layer 42 and the second adhesive layer 43 may not provide sufficient peel and shear stress; while when the adhesive layer is too thick, voids may form in the first adhesive layer 42 and the second adhesive layer 43, leading to bubble formation, which weakens the bonding strength. Furthermore, most of the stress and shear stress may act on the first adhesive layer 42 and the second adhesive layer 43 and not be transferred to the substrate 41, making the first adhesive layer 42 and the second adhesive layer 43 prone to cracking and causing chip 100 failure. In summary, the thickness range B of the substrate 41 should be set to 30μm to 90μm, and / or the thickness range C of the first adhesive layer 42 should be set to 75μm to 85μm, and / or the thickness range D of the second adhesive layer 43 should be set to 75μm to 85μm.

[0192] In some embodiments, the base layer 41, the first adhesive layer 42, and / or the second adhesive layer 43 are resistant to temperatures not less than 80°C. Thus, by setting certain temperature tolerances for the base layer 41, the first adhesive layer 42, and the second adhesive layer 43, the normal progress of biochemical reactions in the intermediate layer 40 can be ensured.

[0193] Specifically, during gene sequencing, the chip 100 may also be exposed to a thermal cycling environment. Therefore, 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 the tolerance to a certain temperature.

[0194] It is 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 do not exhibit significant deformation at a specified temperature. Therefore, the base layer 41 needs to withstand a temperature of not less than 80°C, and the first adhesive layer 42 and / or the second adhesive layer 43 need to withstand a temperature of not less than 80°C.

[0195] In some embodiments, the first adhesive layer 42 and / or the second adhesive layer 43 are resistant to temperatures not less than 110°C. Thus, by setting a certain temperature resistance for the first adhesive layer 42 and the second adhesive layer 43, the normal progress of biochemical reactions in the intermediate layer 40 can be ensured.

[0196] Similarly, as mentioned above, in order to ensure that the first adhesive layer 42 and the second adhesive layer 43 do not deform significantly when the chip 100 is exposed to a thermal cycling environment during gene sequencing, the temperature tolerance 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.

[0197] 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 requirement that the base layer 41, the first adhesive layer 42, and / or the second adhesive layer 43 be resistant to a specified solvent, the normal progress of biochemical reactions in the intermediate layer 40 can be ensured.

[0198] During gene sequencing, the chip 100 may also be exposed to corrosive reagents (such as formamide). Therefore, at least one of the base layer 41, the first adhesive layer 42, and the second adhesive layer 43 that support the biochemical reaction needs to meet the requirements of resistance to a specified solvent. Resistance means that no problems such as peeling or failure of the adhesive or film layers occur in the specified solvent, which can be water, DMSO, formamide solution, etc.

[0199] In some embodiments, the material of the base layer 41 may include polyimide. In this way, using polyimide material for the base layer 41 can meet the requirements of the base layer 41 being able to withstand certain high temperatures and also meet the requirements of being able to withstand specified solvents, so as to avoid problems such as film peeling and failure in high-temperature environments and certain solvents.

[0200] Specifically, polyimide materials are characterized by high temperature resistance, low coefficient of thermal expansion, and good solvent resistance. Therefore, using polyimide materials in the base layer 41 can meet the requirement that the base layer 41 can withstand a temperature of not less than 80°C, and can also withstand specified solvents, so as to avoid problems such as film peeling and failure in the above solvents.

[0201] 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, etc.

[0202] 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 bond instantly with slight pressure. Currently, commonly used pressure-sensitive adhesives mainly include rubber-based pressure-sensitive adhesives, acrylic-based pressure-sensitive adhesives, and silicone-based pressure-sensitive adhesives. Rubber-based pressure-sensitive adhesives are currently the most widely used type, and rubber includes natural rubber, synthetic rubber, and reclaimed rubber. Silicone-based pressure-sensitive adhesives are made by combining silicone rubber and silicone resin, with silicone rubber as the basic component and silicone resin as a tackifier. The properties of the pressure-sensitive adhesive change with the ratio of the two components. Preferably, in this application, a pressure-sensitive adhesive made of silicone rubber may be used.

[0203] In some embodiments, at least one of the first adhesive layer 42 and the second adhesive layer 43 may be made of silicone. Specifically, at least one of the first adhesive layer 42 and the second adhesive layer 43 may be made of silicone. Silicone can withstand temperatures above 110°C, and will not deform under conditions of 80°C for three consecutive hours, thus preventing deformation or cross-channeling of the fluid channel 70. Furthermore, the silicone material has an adhesive-peel force greater than 800g on the glass surfaces, i.e., the first substrate 20 and the second substrate 30, which meets the requirement that the peel force of the first adhesive layer 42 on the first substrate 20 and / or the peel force of the second adhesive layer 43 on the second substrate 30 is not less than 800g. Simultaneously, the silicone material also possesses solvent resistance, ensuring that the fluid channel 70 will not deform or cross-channel after 350 cycles of sequencing in the modification and sequencing reagents.

[0204] Chip 100 can be used to perform sequence determination.

[0205] Please see Figure 7 This application provides a method for fabricating a chip 100, wherein the method may include the following steps:

[0206] Step S10: Provide the first substrate 20;

[0207] Step S20: Provide a second substrate 30, the second substrate 30 including a first surface 31 and a second surface 32 facing away from each other;

[0208] Step S30: The second substrate 30 is stacked on the first substrate 20, such that the first surface 31 of the second substrate 30 faces the first substrate 20;

[0209] Step S40: Set one or more fluid channels 70 between the first surface 31 of the second substrate 30 and the first substrate 20;

[0210] Step S50: A first coating 50 is applied to the second surface 32 of the second substrate 30, and the autofluorescence intensity of the first coating 50 is less than a preset intensity.

[0211] Step S51: Form a second coating within the fluid channel 70.

[0212] Thus, by performing this method, a basic structure with fluid channels 70 is formed by stacking the first substrate 20 and the second substrate 30, and a specific first coating 50 is provided on the second surface 32 of the second substrate 30. In applications involving the detection of signals from the sample to be tested from the chip 100 using an optical imaging system, such as a sequencing platform that uses optical imaging to detect the fluorescence signal of nucleic acid molecules in the chip to achieve nucleic acid sequencing, the fluorescence signal (background signal) emitted by the chip 100 itself when irradiated (i.e., excited by excitation light) is very weak. This is beneficial for obtaining images with a high signal-to-noise ratio, for identifying target fluorescence signals, and for obtaining high-quality sequencing results. In addition, the second coating in the chip 100 can isolate the contact between air and the fluid channels during the storage of the chip 100, thereby reducing the background signal of the chip and reducing the sequencing error rate of the chip 100.

[0213] Specifically, the chip 100 can be a reactor with a liquid space containing nucleic acid molecules bearing optical detection labels, capable of immobilizing the sample; it is also called a flow cell or flow chamber. In steps S10 to S40, the first substrate 20 and the second substrate 30 are any solid support suitable for immobilizing nucleic acid sequences, such as nylon membranes, glass sheets, plastics, silicon wafers, magnetic beads, etc. Therefore, the provided first substrate 20 and second substrate 30 can include any suitable material, such as glass, silica, crystal, quartz glass, plastics, ceramics, PET (polyterephthalic acid), PMMA (polymethyl methacrylate), or any other suitable material. In one embodiment, the provided first substrate 20 and second substrate 30 are optically transparent, for example, glass sheets / glass layers.

[0214] 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 faces 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 for entering, exiting and carrying reagents to form the environment required for sequencing reactions. 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.

[0215] In this application, unless otherwise expressly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature being directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature.

[0216] In step S50, the inventors discovered during the structural design, fabrication, and testing of the chip adapted to the sequencing platform that when the laser emitted by the sequencing platform's laser shines through the lens into the chip's fluid channel, it also shines through the lower glass onto the adhesive layer connecting the lower glass and the aluminum plate. As a result, the molecules in the adhesive layer are excited to emit fluorescence, which greatly interferes with the identification and detection of the target signal, i.e., the signal from the nucleic acid molecules to be tested in the fluid channel.

[0217] Therefore, a first coating 50 with an autofluorescence intensity lower than a preset intensity needs to be provided on the second surface 32 of the second substrate 30. This reduces the amount of excitation light transmitted through the second substrate 30, which helps to weaken the fluorescence emitted by the structure below the second substrate 30 when excited by the transmitted excitation light. Since the autofluorescence intensity of the first coating 50 is lower than the preset intensity, the fluorescence intensity generated by the first coating 50 when irradiated by the excitation light is weak. Under working conditions, the fluorescence signal generated by the chip itself is very weak. Thus, the chip 100 prepared by this method is suitable for carrying biological samples to realize the detection of biological macromolecules and is compatible with optical imaging platforms that realize the detection of samples based on detection chips.

[0218] The first coating 50 can be applied to the second surface 32 of the second substrate 30, or it can be printed on the second surface 32 of the second substrate 30. Specific requirements for the first coating 50 are as described above. For example, the material of the first coating 50 may include black ink to provide high light-blocking properties; the thickness A of the first coating 50 can be 5μm to 20μm or 8μm to 15μm; the light-blocking rate of the first coating 50 in the working environment is not less than 80%; and the flatness deviation of the side of the first coating 50 facing away from the second substrate 30 is allowed to be no more than 0.1μm.

[0219] In step S51, a second coating can be formed by introducing an aqueous solution containing a temperature-sensitive substance / material into the fluid channel 70 at a higher temperature and then lowering the temperature to allow the aqueous solution of the temperature-sensitive substance / material to solidify.

[0220] Specifically, in some embodiments, an aqueous solution containing a temperature-sensitive substance / material can be prepared first. This aqueous solution is then introduced into the fluid channel 70, allowed to stand, and then air is introduced. Subsequently, any remaining residue can be removed by nitrogen blowing. The nitrogen blowing pressure can be 0.02 MPa to 0.08 MPa, and the nitrogen blowing time can be 1 s to 3 s. The standing process allows the aqueous solution to be effectively adsorbed on the surface of the fluid channel 70; the introduction of air removes most of the liquid within the fluid channel 70, facilitating subsequent nitrogen blowing. Alternatively, in some embodiments, for chips with excessively large fluid channel 70 volumes, the aqueous solution containing the temperature-sensitive substance / material can be introduced directly with air and nitrogen blowing without standing, to avoid excessive residue within the fluid channel 70.

[0221] It should be noted that the volume of the above-mentioned aqueous solution and air introduced is not particularly limited and can be determined based on the space of the fluid channel 70.

[0222] In some embodiments, an aqueous solution containing a temperature-sensitive substance / material can be introduced into the fluid channel 70 at a flow rate of 1500 μL / min to 2000 μL / min and a pressure of 0.01 MPa to 0.1 MPa.

[0223] In some embodiments, the second coating is formed after the other structures of the chip 100 have been fabricated, by introducing an aqueous solution containing a temperature-sensitive substance / material into the fluid channel 70. This avoids contamination of the first coating 50 by airborne impurities during the second coating formation step, reducing the sequencing error rate of the chip 100. Simultaneously, it allows for adjustment of the flow rate and pressure of the aqueous solution during the second coating formation step according to the specific structure of the chip 100, further enhancing the protective effect of the second coating.

[0224] Furthermore, in some embodiments, the chip after the second coating is formed can be stored or transported in an environment of 2°C to 8°C after vacuuming.

[0225] Please see Figure 8 In some embodiments, the method for fabricating the chip further includes:

[0226] Step S60: Provide a third substrate 60;

[0227] Step S70: Place the third substrate 60 under the first coating 50;

[0228] Step S71: Form a second coating within the fluid channel 70.

[0229] Thus, the first substrate 20, the second substrate 30 and the third substrate 60 disposed under the first coating 50 are bonded together to form the main body of the chip 100.

[0230] Specifically, in steps S50 and S60, the third substrate 60 can be an aluminum plate with good thermal conductivity, and the third substrate 60 is disposed under the first coating 50. In this way, the main body of the chip 100 can be formed by bonding two glass substrates, the first substrate 20 and the second substrate 30, and a metal base plate, namely the third substrate 60, thereby ensuring the flatness stability and temperature conductivity stability of the first substrate 20 and the second substrate 30.

[0231] Additionally, it should be noted that step S71 can be performed with reference to step S51, and will not be repeated here.

[0232] Please see Figure 9 In some embodiments, the method for fabricating the chip further includes the following steps:

[0233] Step S80: Provide an intermediary layer 40;

[0234] Step S90: An interposer 40 is disposed between the first substrate 20 and the second substrate 30, and one or more fluid channels 70 are disposed in the interposer 40.

[0235] Step S91: Form a second coating within the fluid channel 70.

[0236] Thus, this method provides one or more fluid channels 70 in the interposer layer 40, eliminating the need 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.

[0237] Specifically, in steps S80 and S90, an interposer layer 40 is provided, and one or more fluid channels 70 are formed in the interposer layer 40. The fluid channels 70 can be formed by stamping the interposer layer 40, laser cutting, etc. The specific data such as the size specifications of the fluid channels 70 and the spacing between multiple fluid channels 70 have been described in detail above and will not be repeated here. This interposer layer 40 can serve as the site for the chemical reaction of the fluid reagent, that is, the region where beneficial fluorescence signals are generated by laser irradiation, 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.

[0238] 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 disposed between the first substrate 20 and the second substrate 30. 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 disposed in the intermediate layer 40, and / or serve as outlets or inlets for fluids pumped out from the fluid channels 70 of the intermediate layer 40.

[0239] Additionally, it should be noted that step S91 can be performed with reference to step S51, and will not be repeated here.

[0240] Please see Figure 10 In some implementations, providing the intermediary layer 40 (step S80) includes the following steps:

[0241] Step S81: Provide a base layer 41, the base layer having a first surface and a second surface facing away from each other;

[0242] Step S82: A first adhesive layer 42 is formed on the first surface of the base layer 41;

[0243] Step S83: A second adhesive layer 43 is formed on the second surface of the base layer 41;

[0244] Step S84: Form a fluid channel 70 that penetrates the base layer 41, the first adhesive layer 42, and the second adhesive layer 43;

[0245] 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.

[0246] Specifically, in step S81, the provided base layer 41 can serve as a major component of the interposer 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, which can provide a high signal-to-noise ratio during sequencing and provide high contrast for the fluorescence image finally captured by the lens 200.

[0247] In steps S82 and S83, the provided first adhesive layer 42 and second adhesive layer 43 should also have low autofluorescence. The first adhesive layer 42 is disposed on the first surface of the substrate 41, and the provided second adhesive layer 43 is disposed on the second surface of the substrate 41. The first adhesive layer 42 and the second adhesive layer 43 are adhesive, thereby connecting the first substrate 20 and the second substrate 30 to the interposer layer 40 together.

[0248] 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. Thus, 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, undergo a chemical reaction in the fluid channel 70, and then flow out through the first substrate 20 and / or the second substrate 30.

[0249] 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, thereby bonding the first substrate 20, the intermediate layer 40 and the second substrate 30 to form a whole.

[0250] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0251] Example 1:

[0252] (1) Preparation of the second coating solution

[0253] Prepare 1 L of a solution with an EDTA concentration of 0.1 mmol / L and a gelatin mass fraction of 0.5% using ultrapure water.

[0254] High-temperature sterilization. After sterilization, observe whether it is a clear and transparent liquid (if not clear, shake to enhance dissolution), cool to room temperature, let stand, and then dispense.

[0255] (2) Forming a second coating

[0256] Take 350 μL of the solution prepared in step (1) above and introduce it into the fluid channel of the chip (the chip has a probe fixed on a specified surface) at a flow rate of 1800 μL / min. After standing, introduce air into the fluid channel at a flow rate of 2000 μL / min under the condition of ambient temperature ≤26℃ to form the coating.

[0257] (3) Remove the second coating

[0258] The second coating is removed by manually or using a syringe or automated device to purify the fluid channel with a buffer solution, such as SDS, at 50°C for 40 minutes. Alternatively, purging with, for example, 3×SSC at 50°C for 40 minutes may be necessary to remove the second coating as completely as possible.

[0259] This embodiment does not impose any particular limitation on the solution used to remove the second coating. Understandably, a solution with a relatively stable pH value that does not affect the surface covered by the second coating or the characteristics of the functional groups / reaction sites on the surface can generally be used to remove or rinse away the second coating.

[0260] Example 2:

[0261] (1) Preparation of the second coating solution

[0262] Prepare 1 L of a solution containing 0.1 mmol / L EDTA, 0.5% glycerol, and 0.5% gelatin using ultrapure water.

[0263] High-temperature sterilization. After sterilization, observe whether it is a clear and transparent liquid (if not clear, shake to enhance dissolution), cool to room temperature, let stand, and then dispense.

[0264] (2) Forming a second coating

[0265] Take 350 μL of the solution prepared in step (1) above and pass it into the fluid channel of the chip (the chip substrate surface is fixed with probes (e.g., DNA nucleic acid fragments)) at a flow rate of 1500 μL / min. After standing, introduce air into the fluid channel at a flow rate of 2000 μL / min under the condition of ambient temperature ≤26℃ to form the coating.

[0266] (3) Remove the second coating

[0267] The second coating is removed by manually or using a syringe or automated device to circulate a buffer solution, such as SDS solution, into the fluid channel at 60°C for 20 minutes. Optionally, 3×SSC is then circulated in for 20 minutes at 60°C to remove the second coating as completely as possible. This embodiment does not impose any particular limitation on the solution used to remove the second coating. Understandably, a solution with a relatively stable pH value that does not affect the surface covered by the second coating or the characteristics of the functional groups / reaction sites on the surface can generally be used to remove or rinse away the second coating.

[0268] Example 3

[0269] Error rate test

[0270] 1. Take, for example, the chip with the second coating formed in Example 1 (2) (with 0.5% gelatin) and the chip without the second coating (without 0.5% gelatin) for testing. Probes (oligonucleotide fragments) are immobilized on the substrate surface of both chips. The substrate surface referred to here is, for example, the lower surface of the first substrate and / or the upper surface of the second substrate (for the first substrate, second substrate, lower surface, upper surface, etc., please refer to the following). Figure 1 The relative positions are shown in the diagram; the surface closest to the objective lens is the upper surface of the first substrate. The test method is as follows:

[0271] (1) At room temperature, chips with a second coating (with 0.5% gelatin) and chips without a second coating (without 0.5% gelatin) were stored for one month to simulate aging (the chips / surfaces were exposed to a specific environment for a certain period of time, so that various characterization parameters of the surface, such as non-specific adsorption and free group activity, gradually tended to remain relatively unchanged or within a specific range).

[0272] (2) Remove the second coating from the chip with the second coating (with 0.5% gelatin) using the operation method in Example 1 (3);

[0273] (3) Sequencing of the same nucleic acid samples was performed using both the chip with the second coating (with 0.5% gelatin) and the chip without the second coating (without 0.5% gelatin). For example, following the requirements and operating instructions of the sequencing platform used, the nucleic acid to be tested was hybridized with the probe to ligate to the designated surface. Optionally, the nucleic acid molecules to be tested were amplified. Then, 50 cycles of two-color or four-color sequencing were performed. After sequencing, the error rate was calculated. Here, "error rate" refers to the percentage of bases read incorrectly or unread (identified incorrectly or unidentified) in the sequencing data out of the total number of bases.

[0274] Surface treatment, probe immobilization, hybridization, etc., can be performed according to the methods disclosed in CN109610006A, CN111100786A, CN111100785A, etc., which are incorporated herein by reference. Test results are shown in Table 1A and... Figure 11A .

[0275] Table 1A

[0276] 1A 2.74％ 4.67％ 2A 2.81％ 4.22％ 3A 3.23％ 4.43％ 4A 3.70％ 5.65％ 5A 2.84％ 5.03％ 6A 2.56％ 5.41％

[0277] 2. An error rate test was conducted on the chip with the second coating (with 0.5% gelatin and 0.5% glycerin) and the chip without the second coating (without 0.5% gelatin and 0.5% glycerin) from Example 2 (2). The error rate test method was the same as above, and the test results are shown in Table 1B and 1C. Figure 11B .

[0278] Table 1B

[0279] 1B 2.72％ 4.66％ 2B 2.80％ 4.23％ 3B 3.25％ 4.41％ 4B 3.68％ 5.64％ 5B 2.86％ 4.87％ 6B 2.55％ 4.61％

[0280] Conclusion: The test results show that the error rate of the chip with the second coating is reduced by about 41.32% compared with the chip without the second coating, indicating that passing gelatin or a combination of gelatin and glycerin through the chip has a significant effect on chip preservation.

[0281] The inventors anticipate that after the chip is filled with gelatin or a combination of gelatin and glycerin, the coating covering the inner surface of the channel will isolate the adsorption of impurities in the air, thus reducing the error rate. The error rate is caused by the physical adsorption of the glass background and the adsorption of surface functional groups within the chip channel. The protection of the coating can reduce the adsorption of air impurities by functional groups, thereby reducing the error rate and improving test quality.

[0282] Example 4

[0283] 640 Adsorption Test

[0284] Without introducing the target nucleic acid (i.e., without hybridizing the target nucleic acid molecule with the probe to attach to the designated chip surface), a single-molecule SBS sequencing process was simulated. Reagent inflow or outflow for 72 rounds of sequencing was used to simulate SBS sequencing. This included influencing nucleotides with fluorescent groups in each round and allowing them to stand for a period to simulate a single-base color reaction; influencing detection reagents to excite and acquire signals from the fluorescent groups (e.g., for imaging); influencing surface washing / buffer; and passing reagents that can cleave the fluorescent groups on the nucleotides. After completion, the number of bright spots adsorbed on the designated chip surface was counted. This process was repeated multiple times. Some of the statistical test results are shown below. Figure 12 As shown, the vertical axis represents the number of bright spots, and the horizontal axis "room temperature", "0.5% gelatin", and "alcohol" represent the processing conditions used for the fluid channels. "room temperature" means that the fluid channels of the chip are not processed; "0.5% gelatin" means that the fluid channels of the chip are treated with 0.5% gelatin, that is, a second coating is formed in the fluid channels; "alcohol" means that the fluid channels of the chip are treated with alcohol.

[0285] The fluorescent group attached to the nucleotide is, for example, atto647N; the "number of dots" or "number of bright spots" here refers to the number of locations with relatively strong signals in the image obtained by exciting and image acquisition of the FOV (field of view) / reaction area on a specified surface of the chip at a specific wavelength, such as 640nm laser wavelength. It is sometimes also called the "number of fluorescent dots". The number of bright spots can reflect the adsorption capacity of the specified surface to a certain extent; the inventors found that, within the same batch of chips, the longer the chip has been stored, the more bright spots are detected after imaging. The bright spots may come from fluorescently labeled nucleotides, or they may come from substances in other unexpected environments such as air impurities. These substances adsorb onto the channel or the specified surface in a non-specific adsorption manner.

[0286] Conclusion: The freshly made chip had 230 adsorption points. After being left at room temperature for one month, the number of adsorption points in the untreated fluid channels increased by 15.8 times, the number in the gelatin-treated fluid channels increased by 1.93 times, and the number in the alcohol-treated fluid channels increased by 61 times. Therefore, the gelatin treatment was effective and had the best results, significantly reducing adsorption on the chip.

[0287] Example 5

[0288] Throughput, read length, density, and error rate were tested across different fluid channels (lanes) on the same chip.

[0289] The testing method is as follows:

[0290] (1) Select two fluid channels from the four fluid channels of chips numbered 1, 2 and 3 respectively and process them according to the operation method in Example 1 (2) to form a second coating in the fluid channels; the remaining fluid channels are not processed.

[0291] (2) Store numbers 1, 2 and 3 at 4°C for 180 days;

[0292] (3) Remove the second coating from the fluid channel with the second coating formed using the operation method in Example 1 (3);

[0293] (4) These chips / fluidic channels were used for routine single-molecule SBS sequencing. For example, sequencing was performed using Illumina's sequencing platform. Following the platform's requirements and operating instructions, 100 rounds of two-color or four-color sequencing were performed. After sequencing, throughput, read length, density, and error rate were statistically analyzed. Sequencing quality was judged based on throughput, read length, density, and error rate, with particular emphasis on error rate and throughput. Low error rate and high throughput were the desired sequencing results. Specific results are shown in Table 2 and... Figure 13 As shown.

[0294] Table 2

[0295]

[0296] It can be seen that the fluid channels with the second coating can still function normally after being stored at 4°C for 6 months (180 days). This is mainly manifested in the fact that as the chip's storage time increases, the error rate hardly increases or increases very slowly, the read length is unaffected, and the throughput does not decrease significantly. Specifically, Figure 13 middle:

[0297] Density refers to the density (the density of a fresh chip is 5.45). Figure A shows that, from the perspective of chip density, the chip density will slowly decrease with the extension of storage time. Chips that have been stored for too long will have their surface functional groups folded or detached, which will lead to a decrease in subsequent hybridization efficiency. As can be seen from Figure A, with the increase of storage time, the gelatin-treated chips can maintain the slow decrease of the chip substrate without affecting the performance.

[0298] Unique mapped reads represent throughput (5.4M throughput for a fresh chip). Figure B shows that throughput is an indicator of the success of the test results. The fact that a throughput of ≥3.5M can still be obtained after six months of storage indicates that the gelatin coating preservation method is effective.

[0299] Average length represents the read length (39.88 reads for fresh chips). Figure C shows the change in read length of sequencing results over time; compared to fresh chips, chips protected by gelatin coating show little difference.

[0300] Error rate (the error rate of fresh chips is 0.0675317): The chip error rate in Figure D does not change significantly, indicating that gelatin has a good effect on reducing background adsorption caused by preservation.

Claims

1. A chip, characterized in that, include: First substrate; A second substrate is stacked on the first substrate, the second substrate includes a first surface and a second surface facing away from 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; A first coating is disposed on the second surface of the second substrate, wherein the autofluorescence intensity of the first coating is less than a preset intensity; A second coating is formed within the fluid channel; The first coating has a light-shielding rate of not less than 80% under working conditions; The components that form the second coating include a temperature-sensitive material and a corrosion-resistant material.

2. The chip according to claim 1, characterized in that, The temperature-sensitive material is selected from at least one of gelatin, pectin, ethylene glycol, glycerin, and trehalose.

3. The chip according to claim 1, characterized in that, The temperature-sensitive material is selected from temperature-sensitive hydrogels.

4. The chip according to claim 1, characterized in that, The temperature-sensitive material is provided in the form of a solution.

5. The chip according to claim 4, characterized in that, The mass concentration of the temperature-sensitive material in the solution is 0.5% to 5%.

6. The chip according to claim 1, characterized in that, The corrosion-resistant material is selected from at least one of ethylenediaminetetraacetic acid, ethylene glycol phenyl ether, and sodium thimerosal.

7. The chip according to any one of claims 1-6, characterized in that, The thickness of the first coating ranges from 5 μm to 20 μm.

8. The chip according to claim 7, characterized in that, The thickness of the first coating ranges from 8 μm to 15 μm.

9. The chip according to any one of claims 1-6, characterized in that, The allowable deviation of the flatness of the side of the first coating away from the second substrate is no more than 0.1 μm.

10. The chip according to claim 7, characterized in that, The allowable deviation of the flatness of the side of the first coating away from the second substrate is no more than 0.1 μm.

11. The chip according to any one of claims 1-6, characterized in that, The material of the first coating includes ink.

12. The chip according to claim 7, characterized in that, The material of the first coating includes ink.

13. The chip according to claim 11, characterized in that, The material of the first coating is black ink.

14. The chip according to claim 12, characterized in that, The material of the first coating is black ink.

15. The chip according to any one of claims 1-6, characterized in that, The first substrate includes a first surface and a second surface facing away from 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 chip image is less than or equal to a preset value. The chip image is an image of the second surface of the first substrate and / or an image of the first surface of the second substrate under the working environment.

16. The chip according to claim 7, characterized in that, The first substrate includes a first surface and a second surface facing away from 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 chip image is less than or equal to a preset value. The chip image is an image of the second surface of the first substrate and / or an image of the first surface of the second substrate under the working environment.

17. The chip according to claim 15, characterized in that, The background intensity of the image from the chip is less than or equal to 400.

18. The chip according to any one of claims 1-6, characterized in that, The chip also includes a third substrate attached to the first coating, the third substrate being made of metal.

19. The chip according to claim 7, characterized in that, The chip also includes a third substrate attached to the first coating, the third substrate being made of metal.

20. The chip according to any one of claims 1-6, characterized in that, The chip further includes an intermediary layer disposed between the first substrate and the second substrate, the intermediary layer connecting the first substrate and the second substrate, and the one or more fluid channels disposed in the intermediary layer.

21. The chip according to claim 7, characterized in that, The chip further includes an intermediary layer disposed between the first substrate and the second substrate, the intermediary layer connecting the first substrate and the second substrate, and the one or more fluid channels disposed in the intermediary layer.

22. The chip according to claim 20, characterized in that, The interposer layer bonds the first substrate and the second substrate, and the interposer layer has a hollow structure. The interposer layer includes: The base layer has a first surface and a second surface that are opposite to each other; 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. A second adhesive layer is disposed on the second surface of the base layer, and the second adhesive layer is bonded to the second substrate; as well as, The fluid channel is the hollow structure formed through the base layer, the first adhesive layer and the second adhesive layer.

23. The chip according to claim 21, characterized in that, The interposer layer bonds the first substrate and the second substrate, and the interposer layer has a hollow structure. The interposer layer includes: The base layer has a first surface and a second surface that are opposite to each other; 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. A second adhesive layer is disposed on the second surface of the base layer, and the second adhesive layer is bonded to the second substrate; as well as, The fluid channel is the hollow structure formed through the base layer, the first adhesive layer and the second adhesive layer.

24. The chip according to claim 22 or 23, characterized in that, The first substrate and / or the second substrate are provided with through holes communicating with the fluid channel.

25. The chip according to claim 22 or 23, characterized in that, The peel force of the first adhesive layer on the first substrate and / or the peel force of the second adhesive layer on the second substrate is not less than 560g.

26. The chip according to claim 25, characterized in that, The peel force of the first adhesive layer on the first substrate and / or the peel force of the second adhesive layer on the second substrate is not less than 800g.

27. The chip according to claim 22 or 23, characterized in that, The fluid channel has a larger dimension in a first direction than in a second direction, the first direction being perpendicular to the second direction, and both the first and second directions being perpendicular to the thickness direction of the interlayer.

28. The chip according to claim 27, characterized in that, The fluid channels are multiple, and the fluid channels extend along the first direction and are disposed in the intermediate layer; And / or, the fluid channels are arrayed in the intermediate layer along the second direction.

29. The chip according to claim 28, characterized in that, The fluid channel includes an intermediate section, a first end, and a second end, with the first end and the second end located at opposite ends of the fluid channel. The dimension of the first end in the second direction and / or the dimension of the second end in the second direction is smaller than the dimension of the intermediate section in the second direction.

30. The chip according to claim 29, characterized in that, The dimension of the intermediate segment in the second direction is constant.

31. The chip according to claim 29, characterized in that, The dimensions of the intermediate segment in the second direction range from 4.4 mm to 8.4 mm.

32. The chip according to claim 28, characterized in that, The spacing between two adjacent fluid channels in the second direction ranges from 0.8 mm to 1.5 mm.

33. The chip according to claim 22 or 23, characterized in that, The thickness of the base layer ranges from 30 μm to 90 μm; and / or, The thickness of the first adhesive layer ranges from 75 μm to 85 μm; and / or, The thickness of the second adhesive layer ranges from 75 μm to 85 μm.

34. The chip according to claim 22 or 23, characterized in that, The base layer, the first adhesive layer and / or the second adhesive layer are resistant to temperatures of not less than 80 °C.

35. The chip according to claim 22 or 23, characterized in that, The first adhesive layer and / or the second adhesive layer can withstand a temperature of not less than 110 °C.

36. The chip according to claim 22 or 23, characterized in that, The base layer, the first adhesive layer, and / or the second adhesive layer are resistant to the specified solvent.

37. The chip according to claim 22 or 23, characterized in that, The base layer material contains polyimide or PET.

38. The chip according to claim 22 or 23, characterized in that, The first adhesive layer and the second adhesive layer are made of the same material.

39. The chip according to claim 22 or 23, characterized in that, The material of the first adhesive layer and / or the second adhesive layer includes silicone.

40. A method for fabricating a chip, characterized in that, include: Provide a first substrate; A second substrate is provided, the second substrate including a first surface and a second surface opposite to each other; The second substrate is stacked on the first substrate, such that the first surface of the second substrate faces the first substrate; One or more fluid channels are provided between the first surface of the second substrate and the first substrate; A first coating is applied to the second surface of the second substrate, wherein the autofluorescence intensity of the first coating is less than a preset intensity. A second coating is formed within the fluid channel; The first coating has a light-shielding rate of not less than 80% under working conditions; The components that form the second coating include a temperature-sensitive material and a corrosion-resistant material.

41. The method according to claim 40, characterized in that, The second coating is formed by introducing a solution containing a temperature-sensitive material into the fluid channel at a flow rate of 1500 μL / min to 2000 μL / min and a pressure of 0.01 MPa to 0.1 MPa.

42. The method according to claim 40, characterized in that, The method further includes: Provide a third substrate; The third substrate is disposed under the first coating.

43. The method according to claim 40, characterized in that, The method further includes: Provide an intermediary layer; The interlayer is disposed between the first substrate and the second substrate, and the one or more fluid channels are disposed in the interlayer.

44. The method according to any one of claims 40-43, characterized in that, The intermediary layer includes: A base layer is provided, the base layer having a first surface and a second surface facing away from each other; A first adhesive layer is disposed on the first surface of the base layer; A second adhesive layer is provided on the second surface of the base layer; Forming fluid channels that penetrate 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.

45. The method according to claim 44, characterized in that, The thickness of the base layer ranges from 30 μm to 90 μm; and / or, The thickness of the first adhesive layer ranges from 75 μm to 85 μm; and / or, The thickness of the second adhesive layer ranges from 75 μm to 85 μm.

46. ​​The method according to claim 44, characterized in that, The base layer, the first adhesive layer and / or the second adhesive layer are resistant to temperatures of not less than 80 °C.

47. The method according to claim 46, characterized in that, The first adhesive layer and / or the second adhesive layer can withstand a temperature of not less than 110 °C.

48. The method according to claim 44, characterized in that, The base layer, the first adhesive layer, and / or the second adhesive layer are resistant to the specified solvent.

49. The method according to claim 44, characterized in that, The base layer is polyimide.

50. The method according to any one of claims 45-49, characterized in that, The first adhesive layer and the second adhesive layer are made of the same material.

51. The method according to claim 44, characterized in that, The first adhesive layer and the second adhesive layer are made of the same material.

52. The method according to any one of claims 45-49, characterized in that, The first adhesive layer and / or the second adhesive layer are silicone.

53. The method according to claim 44, characterized in that, The first adhesive layer and / or the second adhesive layer are silicone.

54. The method according to any one of claims 45-49, characterized in that, The material of the first adhesive layer and / or the second adhesive layer is pressure-sensitive adhesive.

55. The method according to claim 44, characterized in that, The material of the first adhesive layer and / or the second adhesive layer is pressure-sensitive adhesive.

56. The method according to any one of claims 45-49, characterized in that, The peel force of the first adhesive layer on the first substrate and / or the peel force of the second adhesive layer on the second substrate is not less than 560g.

57. The method according to claim 44, characterized in that, The peel force of the first adhesive layer on the first substrate and / or the peel force of the second adhesive layer on the second substrate is not less than 560g.

58. The method according to claim 56, characterized in that, The peel force of the first adhesive layer on the first substrate and / or the peel force of the second adhesive layer on the second substrate is not less than 800g.

59. The method according to claim 57, characterized in that, The peel force of the first adhesive layer on the first substrate and / or the peel force of the second adhesive layer on the second substrate is not less than 800g.

60. The method according to any one of claims 45-49, characterized in that, The first substrate and / or the second substrate are provided with through holes communicating with the fluid channel.

61. The method according to claim 44, characterized in that, The first substrate and / or the second substrate are provided with through holes communicating with the fluid channel.

62. The application of a chip as described in any one of claims 1-39 or a chip prepared by the method described in any one of claims 40-61 in sequence determination.

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