CN115537307A

CN115537307A - Chip, application and method for preparing chip

Info

Publication number: CN115537307A
Application number: CN202110737992.6A
Authority: CN
Inventors: 李广
Original assignee: Genemind Biosciences Co Ltd
Current assignee: Genemind Biosciences Co Ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2022-12-30

Abstract

The application discloses a chip, application and a method for preparing the chip. The chip comprises a first substrate and a second substrate, wherein the second substrate and the first substrate are arranged in a stacked mode, the second substrate comprises a first surface and a second surface which are opposite to each other, the first surface of the second substrate faces the first substrate, and one or more fluid channels are arranged between the first surface of the second substrate and the first substrate; the chip also includes a coating disposed on the second surface of the second substrate, the coating having an autofluorescence intensity less than a predetermined intensity. The chip in the embodiment of the application comprises a basic structure which is formed by laminating a first substrate and a second substrate and contains a fluid channel, and the second surface of the second substrate is provided with a specific coating which has weaker autofluorescence property. The chip is particularly suitable for use in a platform for imaging the chip based on an optical system for detecting biomolecules within the chip.

Description

Chip, application and method for preparing chip

Technical Field

The application relates to the field of nucleic acid detection, in particular to a chip, application and a method for preparing the chip.

Background

The chip adapted to the sequencing platform is a reaction device, also called a flow cell or flow cell, capable of supporting a nucleic acid to be detected, and capable of accommodating a solution to provide a reaction environment or a detection environment for the nucleic acid to be detected.

Two pieces of glass (at least one of two opposite surfaces of the two pieces of glass is subjected to etching treatment) and one opaque substrate are adhesively packaged with an adhesive to produce a chip having a space/chamber inside.

On a platform (sometimes referred to as a sequencer) for sequencing based on an optical imaging system detection chip, imaging is performed on a specific position (a position to which a nucleic acid molecule to be detected is connected, and sometimes referred to as a reaction region or a fluid channel) of the chip, and then the base sequence of the nucleic acid molecule to be detected is identified and determined based on information of the images. For example, in a platform for sequencing by using fluorescence labeled nucleotides based on the principle of sequencing by synthesis, in the sequencing, high-energy laser emitted by a laser in a sequencer is irradiated onto a reaction region of a chip through a lens, nucleic acid molecules to be detected in the reaction region are placed in a reagent solution, the laser irradiates the fluorescent molecules in the reagent solution and excites the fluorescent molecules to emit fluorescent signals, and then the fluorescent signals are collected, for example, photographed to obtain images, and the base arrangement order is identified and determined based on information on the images to achieve the purpose of sequencing.

The sequencing platform images the chip based on the optical system and realizes detection based on the image, and understandably, the higher the signal-to-noise ratio of the image is, the more accurate and reliable the sequencing result is. In the chip, the laser irradiates fluorescent molecules, and at the same time, the laser also irradiates on the opaque substrate and/or the adhesive of the substrate and the glass through the glass, and the molecules in the substrate and/or the adhesive are excited to emit light to generate noise interference, which affects the final sequencing result.

Therefore, the structure and/or the manufacturing method of the chip need to be improved.

Disclosure of Invention

The embodiment of the application provides a chip, application and a chip preparation method.

The chip of the embodiment of the present application includes a first substrate, a second substrate, and a coating layer. The second substrate and the first substrate are arranged in a stacked mode, the second substrate comprises a first surface and a second surface which are opposite to each other, 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 coating is disposed on the second surface of the second substrate, and the autofluorescence intensity of the coating is less than a preset intensity.

The chip of the embodiment of the application comprises a basic structure which is formed by laminating a first substrate and a second substrate and is provided with a fluid channel, and a specific coating is arranged on the second surface of the second substrate. In applications involving detecting signals of a sample to be detected from the chip by using an optical imaging system, such as a sequencing platform for detecting fluorescent signals of nucleic acid molecules in the chip based on optical imaging to realize nucleic acid sequencing, the chip itself is irradiated, i.e. the fluorescent signals (background signals) emitted by excitation light are weak, which is beneficial to obtain images with high signal-to-noise ratio (SNR), is beneficial to identification of target fluorescent signals, and is beneficial to obtain high-quality sequencing results.

Furthermore, according to the embodiments of the present application, the chip may further have at least one of the following additional technical features.

In some embodiments, the coating is coated on the second surface of the second substrate. Therefore, the exciting light passing through the second substrate is reduced, and the fluorescence emitted by the structure below the second substrate under the excitation of the passing exciting light is weakened. The coating includes painting, brushing, printing, and the like.

In certain embodiments, the coating has a thickness in the range of 5 μm to 20 μm. The coating with the thickness has a good shielding effect on the exciting light, so that an image of a specific area of the chip acquired by irradiation of the exciting light can meet the sequencing requirement.

Preferably, the thickness of the coating is in the range of 8 μm to 15 μm. The coating with the thickness has a good blocking effect on exciting light penetrating through the substrate.

In certain embodiments, the coating has a light obscuration of not less than 80% under a working environment. The coating has the shading rate under the working environment, is beneficial to acquiring the image of a specific area of the chip with higher signal-to-noise ratio, and is beneficial to obtaining a high-quality sequencing result. For a sequencing platform based on optical imaging to detect fluorescence signals of nucleic acid molecules in a chip to realize nucleic acid sequence determination, the working environment comprises laser with specific wavelength and intensity; in one example, the operating environment refers to 800-1000mW of red or green laser light (e.g., laser light emitting at 532nm or 635 nm).

In some embodiments, the flatness of the side of the coating facing away from the second substrate is allowed to deviate by no more than 0.1 μm. Thus, after the packaging is connected with other structures such as a substrate in a stacking mode, the deviation/tolerance of the mechanical accumulated surface flatness can be ensured to be within a preset range, so that the flatness of the chip surface meets the preset requirement, the stable and firm connection of the coating and the corresponding surface of the third substrate is facilitated, and the firm and stable structure of the chip is facilitated.

In certain embodiments, the material of the coating comprises ink. Therefore, the chip with the coating has high light shading performance and weak light emitting characteristic under the working environment, and is easy to prepare. In particular, in one example, the coating is a black ink, which can capture images of specific areas of the chip of higher quality. Moreover, by printing on the corresponding surface of the second substrate to obtain the second substrate with the black coating, a coating satisfying the requirements or a chip including the second substrate with the coating can be rapidly and easily obtained.

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

An image of a chip is an image of one or more areas/fields of view (FOV) of the chip containing molecules to be measured. The preset value is set by comparing and calculating a large number of images corresponding to better and worse sequencing results obtained under the same working environment, and the acquired images of the chip are evaluated and judged through the preset value, so that the images are quickly evaluated and fed back, and the decision of whether to further process the images, whether to continuously acquire the images, whether to adjust an optical imaging system, whether to refocus, the quality of the chip and the like is facilitated.

In some embodiments, the chip includes a third substrate attached to the coating, and the third substrate is made of metal. Therefore, the flatness stability and the temperature conduction stability of the first substrate and the second substrate can be ensured.

In some embodiments, the chip includes an interposer disposed between the first substrate and the second substrate, the interposer connecting the first substrate and the second substrate, the one or more fluid channels being disposed in the interposer.

In some embodiments, an interposer is bonded to a first substrate and a second substrate, the interposer having a hollow structure, the interposer comprising:

a base layer having opposing first and second surfaces;

the first adhesive layer is arranged on the first surface of the base layer and is bonded with the first substrate;

the second adhesive layer is arranged on the second surface of the base layer and is bonded with the second substrate; and the fluid channel is a hollow structure formed by penetrating through the base layer, the first adhesive layer and the second adhesive layer. Therefore, the fluid channel does not need to be etched on the first substrate and the second substrate, the manufacturing procedure of the fluid channel is simplified, and the preparation process of the chip is also simplified.

In some embodiments, the first substrate and/or the second substrate is provided with a through hole communicating with the fluid channel. Therefore, fluids such as reaction reagents can enter the fluid channel through the first substrate and/or the second substrate, can also flow out through the first substrate and/or the second substrate after chemical reaction occurs in the fluid channel, and is convenient for connecting a pipeline or a manifold to connect the valve body and the reaction reagent container.

In certain 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. Therefore, the bonding strength of the first bonding agent layer to the first substrate and/or the second bonding agent layer to the second substrate can be ensured to meet the operation requirement; for example, the adhesive peel force between the adhesive layer and the substrate is not less than the predetermined value, and the chip structure can be firmly connected and stabilized, thereby satisfying the sequencing requirement.

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. Therefore, the bonding strength of the first adhesive layer to the first substrate and/or the bonding strength of the second adhesive layer to the second substrate can be ensured to meet the operation requirement, so that the chip can be well used for sequencing.

In some embodiments, the fluid channels have a dimension in a first direction that is greater than their dimension in a second direction, the first direction being perpendicular to the second direction, and both the first direction and the second direction being perpendicular to the thickness direction of the interposer. In this manner, the general shape of the fluid channels formed in the interposer is normalized, which facilitates control of the fluid in the fluid channels, as well as positioning and imaging of the regions of the die.

In some embodiments, the number of the fluid channels is multiple, and the fluid channels extend along the first direction and are arranged in the interposer; and/or the fluid channels are arranged on the interposer in the second direction array. Therefore, the multiple fluid channels can enable the sequence determination process to be more efficient, control over fluid in the fluid channels is facilitated, and positioning and imaging of the areas of the chip are facilitated.

In some embodiments, the fluid channel comprises an intermediate section, a first end and a second end, the first end and the second end being located at both ends of the fluid channel, respectively, the dimension of the first end in the second direction and/or the dimension of the second end in the second direction being smaller than the dimension of the intermediate section in the second direction. Therefore, the shape of the fluid channel is further normalized, the fluid in the fluid channel is favorably controlled, and the areas of the chip are favorably positioned and imaged.

In certain embodiments, the dimension of the middle segment in the second direction is constant. That is, the lengths of the intermediate sections in the second direction are equal everywhere.

In certain embodiments, the intermediate section has a dimension in the second direction in the range of 4.4mm to 8.4mm. Therefore, the reasonable size range enables the fluid channel to have a certain width to contain the reaction reagent, thereby being beneficial to controlling the fluid in the fluid channel and carrying out efficient biochemical reaction in the fluid channel.

In certain embodiments, the spacing between two adjacent fluid channels in the second direction is in the range of 0.8mm to 1.5mm. Therefore, a plurality of fluid channels can be conveniently processed on the interposer, and the number of the fluid channels is ensured to realize efficient sequencing as much as possible.

In certain embodiments, the base layer has a thickness in the range of 30 μm to 50 μm; and/or the presence of a gas in the atmosphere,

the first adhesive layer has a thickness in the range of 75 μm to 85 μm; and/or the presence of a gas in the gas,

the thickness of the second adhesive layer ranges from 75 μm to 85 μm. In this way, the first adhesive layer and the second adhesive layer have certain thicknesses, so that the peeling force of the first adhesive layer/the second adhesive layer to the first substrate/the second substrate is ensured, and the normal operation of biochemical reaction in the intermediate layer is ensured.

In certain embodiments, the base layer, the first adhesive layer, and/or the second adhesive layer withstand a temperature of not less than 80 ℃. Therefore, the base layer, the first adhesive layer and/or the second adhesive layer are not obviously deformed during biochemical reaction, and the normal running of the biochemical reaction in the intermediate layer can be ensured.

In certain embodiments, the first adhesive layer and/or the second adhesive layer is resistant to temperatures of not less than 110 ℃. Therefore, the first adhesive layer and/or the second adhesive layer have no obvious deformation during biochemical reaction, and normal biochemical reaction in the intermediate layer can be ensured.

In certain embodiments, the base layer, the first adhesive layer, and/or the second adhesive layer are resistant to a specified solvent. Therefore, the problems of glue layer and film layer shedding, failure and the like do not occur in the specified solvent of the base layer, the first adhesive layer and/or the second adhesive layer, so that the normal running of biochemical reaction in the intermediate layer is ensured.

In certain embodiments, the material of the base layer comprises polyimide. In this way, the base layer can withstand certain high temperatures and can also meet the requirements for resistance to a given solvent.

In certain embodiments, the first adhesive layer and the second adhesive layer are made of the same material. Thus, the first adhesive layer and the second adhesive layer can be manufactured in a single process.

In certain embodiments, the material of the first adhesive layer and/or the second adhesive layer comprises a silicone gel, such as a pressure sensitive silicone gel (PSA silicone gel). As such, the first adhesive layer and/or the second adhesive layer are able to withstand certain high temperatures and may also meet the need to withstand specified solvents.

Use of a chip according to any of the embodiments above in a sequence assay.

Embodiments of the present application also provide a method for preparing a chip, which can be used to prepare a chip according to any of the above embodiments. The method comprises the following steps: providing a first substrate; providing a second substrate, wherein the second substrate comprises a first surface and a second surface which are opposite; stacking a second substrate on the first substrate, wherein the second substrate comprises a first surface and a second surface which are opposite to each other, and the first surface of the second substrate faces the first substrate; disposing one or more fluid channels between the first surface of the second substrate and the first substrate; and arranging a coating on the second surface of the second substrate, wherein the autofluorescence intensity of the coating is less than the preset intensity.

The preparation method is simple in process, and the coating is arranged on the second surface of the second substrate, so that the preparation method is beneficial to obtaining images with high signal-to-noise ratio, identifying target fluorescent signals and obtaining high-quality sequencing results.

In certain embodiments, the method further comprises: providing a third substrate; a third substrate is disposed under the coating. Therefore, the first substrate, the second substrate and the third substrate are bonded to form a main body of the chip, and the third substrate is favorable for ensuring the flatness stability and the temperature conductivity stability of the first substrate and the second substrate.

In certain embodiments, the method further comprises: providing an interposer; an interposer is disposed between the first substrate and the second substrate, and one or more fluid channels are disposed in the interposer. Therefore, the fluid channel does not need to be etched on the first substrate and the second substrate, and the manufacturing process of the fluid channel is simplified.

In some embodiments, an interposer is provided, comprising: providing a base layer, wherein the base layer is provided with a first surface and a second surface which are opposite; disposing a first adhesive layer on a first surface of a base layer; disposing a second adhesive layer on a second surface of the base layer; forming a fluid channel through the base layer, the first adhesive layer, and the second adhesive layer; the method further comprises the following steps: the first adhesive layer is bonded to the first substrate, and the second adhesive layer is bonded to the second substrate. Therefore, the interposer can be bonded with the first substrate and the second substrate, and the hollow structure of the bonded interposer forms the corresponding fluid channel, so that the fluid channel does not need to be etched on the first substrate and the second substrate, and the manufacturing process of the chip comprising the fluid channel is simplified.

Additional aspects and advantages of the present 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 the present application.

Drawings

The above and/or additional aspects and advantages of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic diagram of a chip according to an embodiment of the present application;

FIG. 2 is a schematic cross-sectional view of a chip in an embodiment of the present application;

FIG. 3 is an enlarged schematic view at P of FIG. 2 in an embodiment of the present application;

FIG. 4 is a schematic structural diagram of an interposer in an embodiment of the present application;

FIG. 5 is a cross-sectional schematic view of an interposer in an embodiment of the present application;

FIG. 6 is a schematic diagram of background intensity of an acquired image according to an embodiment of the present disclosure;

FIG. 7 is a schematic flow chart of a method for manufacturing a chip according to an embodiment of the present application;

FIG. 8 is another schematic flow chart of a method of manufacturing a chip in an embodiment of the present application;

FIG. 9 is a schematic flow chart of a method for manufacturing a chip according to an embodiment of the present application;

fig. 10 is a schematic flow chart of a method for manufacturing a chip in an embodiment of the present application.

Description of the main element symbols:

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

Detailed Description

Reference will now be made in detail to embodiments of the present application, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative and are only for the purpose of explaining the present application and are not to be construed as limiting the present application.

In the present application, the terms "first", "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defined as "first", "second", may explicitly or implicitly include one or more of the described features.

In this application, "plurality" means two or more unless explicitly defined otherwise.

The specific data/values referred to in the description of the present application are in most cases statistically significant, and thus, unless otherwise indicated, any numerical value expressed in a precise manner is intended to mean a range including values within plus or minus 10%, and will not be repeated below.

In the present application, the term "chip" is a reaction cell comprising a solid substrate with a space for holding a liquid, which can be used to immobilize a sample to be measured, also called a flow cell or flow cell (Flowcell). The "solid substrate" such as the substrate herein can be any solid support that can be used to immobilize nucleic acid sequences, such as nylon membrane, glass slide, plastic, silicon wafer, magnetic beads, etc. In some examples, the first substrate and the second substrate are light transmissive, e.g., both glass sheets/layers.

In this application, the term "sequencing" is used to refer to sequence determination, as in "nucleic acid sequencing" or "gene sequencing", and refers to the determination of the order of bases in a nucleic acid sequence; including sequencing by synthesis (sequencing by synthesis, SBS) and/or sequencing by ligation (sequencing by ligation, SBL); comprises DNA sequencing and/or RNA sequencing; including long and/or short fragment sequencing, the long and short fragments being referred to in opposition, e.g., nucleic acid molecules longer than 1Kb, 2Kb, 5Kb or 10Kb may be referred to as long fragments and nucleic acid molecules shorter than 1Kb or 800bp may be referred to as short fragments; including double-ended sequencing, single-ended sequencing, and/or paired-end sequencing, etc., the term double-ended sequencing or paired-end sequencing can refer to the reading of any two segments or portions of the same nucleic acid molecule that do not completely overlap.

So-called sequencing includes the process of binding nucleotides (including nucleotide analogs) to the template and collecting the corresponding reaction signals. In some sequencing platforms in which nucleotides are bound to a template and corresponding reaction signals are collected asynchronously, determination of the order of multiple nucleotides/bases on the template is typically accomplished by multiple rounds of sequencing, a round of sequencing (cycle), also known as a sequencing round, which may be defined as one base extension of four nucleotides/bases, or, put another way, as a process to complete determination of the base type at any given position on the template; for a sequencing platform for realizing sequencing based on control polymerization or connection reaction, one round of sequencing comprises the processes of realizing the combination of four nucleotides to a named template once and collecting corresponding reaction signals; for a platform for realizing sequencing based on polymerization reaction, a reaction system comprises reaction substrate nucleotide, polymerase and a template, a section of preset sequence (sequencing primer) is combined on the template, and the added reaction substrate (nucleotide) is controllably connected to the 3' end of the sequencing primer under the catalysis of the polymerase on the basis of a base pairing principle and a polymerization reaction principle to realize the base pairing with the corresponding position of the template; generally, one round of sequencing may comprise one or more base extensions (repeat), for example, four nucleotides are sequentially added to the reaction system to perform base extension and corresponding acquisition of reaction signals, respectively, and one round of sequencing comprises four base extensions; for another example, four kinds of nucleotides are added to the reaction system in any combination, such as two-two combination or one-three combination, the two combinations are respectively used for base extension and corresponding reaction signal collection, and one round of sequencing comprises two times of base extension; as another example, four nucleotides are added simultaneously to the reaction system for base extension and reaction signal acquisition, and one round of sequencing involves one base extension.

Sequencing can be carried out by a sequencing platform, and the sequencing platform can be selected from but not limited to Hiseq/Miseq/Nextseq/Novaseq sequencing platform of Illumina, ion Torrent platform of Thermo Fisher/Life Technologies, BGISEQ and MGISEQ/DNBSEQ platforms of Huada genes and single-molecule sequencing platform; the sequencing mode can be single-ended sequencing or double-ended sequencing.

Typically, SBS-adapted sequencing platforms chips may contain one or more parallel channels/fluidic channels (channels) for access and support of reagents to form the environment required for sequencing reactions. The chip main body can be formed by bonding and packaging two pieces of glass and a bottom plate made of metal materials, the sequencing process comprises the step of taking pictures for multiple times in one or more areas of the chip by an imaging system such as a camera, the area taken each time can be called as Fov (field of view), and the two cycles comprise the step of introducing reagents again for carrying out biochemical reaction.

Referring to fig. 1-4, in an embodiment of the present invention, a chip 100 is provided, where the chip 100 includes a first substrate 20 and a second substrate 30, and the second substrate 30 is stacked on the first substrate 20. The second substrate 30 includes a first surface 31 and a second surface 32 opposite to each other, the first surface 31 of the second substrate 30 faces the first substrate 20, one or more fluid channels 70 are disposed between the first surface 31 of the second substrate 30 and the first substrate 20, the second surface 32 of the second substrate 30 is provided with a coating 50, and the autofluorescence intensity of the coating 50 is less than a predetermined intensity.

In the chip 100 of the embodiment of the present application, the first substrate 20 and the second substrate 30 are stacked to form a basic structure of the chip 100, and one or more fluid channels 70 are disposed between the first surface 31 of the first substrate 20 and the second surface 31 of the second substrate 30, so that the chip 100 can carry a sample to be tested, including a holding solution or a reagent, to provide a solution environment for biochemical reaction or specific detection, further, the excitation light transmitted through the second substrate 30 is reduced by providing the coating 50 on the second surface 32 of the second substrate 30, the excitation light transmitted through the second substrate 30 is reduced to facilitate reducing fluorescence emitted by a structure under the second substrate 30 excited by the transmitted excitation light, and since the autofluorescence intensity of the coating 50 is less than a preset intensity, the fluorescence intensity generated by the coating 50 irradiated by the excitation light is weak, therefore, under a working environment, the fluorescence signal generated by the chip 100 itself is weak, the chip 100 is suitable for carrying a biological sample to realize biological macromolecule detection, and is suitable for an optical imaging platform for realizing detection of the sample to be tested based on a detection chip.

Referring to fig. 1 and 4, in one example, the chip 100 is placed under an optical system such as a microscope for detection, the nucleic acid molecules to be detected are labeled with fluorescence, and the nucleic acid molecules to be detected are located in the fluid channel 70 and have one end connected to the second surface 22 of the first substrate 20 and/or one 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, the camera includes a lens 200, the laser with a specific wavelength emitted by the laser irradiates the fluid channel 70 of the chip 100 to excite the fluorescent mark therein to emit fluorescence, and the lens 200 included in the camera is used to photograph the areas emitting fluorescence to obtain images, thereby realizing signal acquisition of the sample to be measured. Detection of the sample to be tested can then be achieved based on processing and analyzing the image.

Specifically, on a sequencing platform for implementing nucleic acid sequencing based on chip detection, a chip adapted to the sequencing platform may be generally stacked and packaged with two layers of glass and a metal bottom plate with good thermal conductivity, such as an aluminum plate, and the three-piece structure is bonded by using an adhesive, such as a water gel or a double-sided adhesive. Wherein, the lower layer glass can be bonded with the aluminum plate through an adhesive, so that the current material science and process, such as surface processing, etching and bonding packaging process, can be developed, and the requirements of a sequencing platform on the plane flatness of the upper glass layer and the lower glass layer of the chip and the stability of the temperature conductivity of the chip can be generally ensured.

The requirements of the sequencing platform referred to herein, it is understood, for automated sequencing platforms comprising optical systems, generally comprise taking successive photographs of multiple regions on a chip; in particular, for a sequencing platform comprising an optical system with a high magnification lens, such as 20 × or more, the range capable of clearly imaging the sample to be tested is typically micro-scale or nano-scale (focal plane/clear plane), and thus the requirement for the flatness of the surface of the adapted chip is high, and the sequencing reaction generally involves various biochemical reactions, involving the use of corrosive solutions and short-time temperature increases and decreases, etc., and thus has more and higher requirements for the physicochemical properties of the structure containing/contacting the solutions, the connection strength, etc.; in addition, the chip generally needs to apply a large force at a high temperature during the preparation process to make the structures tightly attached, so that there are many requirements and limitations on the pressure tolerance degree, the temperature tolerance degree, the adhesive strength, and the like of the chip adapted to the sequencing platform and the various constituent structures thereof. If it is further desired that the chip consumes less reagents, for example, rapid detection of nanoliter or smaller volume samples, the requirements for structure, connection, material and processing thereof, etc. are much more important in the field of microfluidic technology.

However, the inventor finds that, in the structural design, preparation and testing of the chip 100 adapted to the sequencing platform, when the laser emitted from the laser of the sequencing platform is irradiated into the fluid channel 70 of the chip 100 through the lens 200, the laser is also irradiated onto the glue layer connecting the lower glass and the aluminum plate through the lower glass, and molecules of the glue layer are excited to emit fluorescence, thereby generating a large interference on the identification and detection of a target signal, i.e., a signal from a nucleic acid molecule to be detected in the fluid channel 70.

The inventors have set expectations/requirements for the surface of a metal base plate such as an aluminum plate and expectations/requirements for a glue used to adhere the aluminum plate based on a large amount of prior test data including image data of better and poorer quality chips obtained under a specific working environment, such as thickness, surface flatness, adhesive bonding force, glue chemical property, optical property, etc., of each coating of the chip, and have conducted processing and testing by various suppliers in the field.

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

Specifically, the first substrate 20 and the second substrate 30 may include any suitable material, such as glass, silicon dioxide, crystal, quartz glass, plastic, ceramic, PET (poly terephthalic acid), PMMA (poly methyl methacrylate), or any other suitable material.

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

The shapes of the first substrate 20 and the second substrate 30 may be various regular shapes such as a square, a rectangle, a circle, a triangle, etc., of course, the first substrate 20 and the second substrate 30 may also be irregular shapes, in this embodiment, the first substrate 20 and the second substrate 30 are long rectangular shapes.

The first substrate 20 and the second substrate 30 may have the same size or different sizes, and in one embodiment, the thickness of the first substrate 20 is smaller than that of the second substrate 30.

The second substrate 30 comprises a first surface 31 of the second substrate 30 and a second surface 32 of the second substrate 30, which are opposite to each other, wherein the first surface 31 of the second substrate 30 faces the first substrate 20, and the second surface 32 of the second substrate 30 is disposed away from the first surface 31 of the second substrate 30 as a bottom surface of the second substrate 30. A fluid channel 70 is disposed between the first surface 31 of the second substrate 30 and the first substrate 20, and the fluid channel 70 can be used as a fluid reagent chemical reaction site, i.e., a region where a target signal is located.

In order to reduce the laser light from being irradiated through the second substrate 30 onto other structures below the second substrate 30 of the chip 100, such as a glue layer connecting the second substrate 30 and the structures below the second substrate 30, the coating layer 50 may be disposed on the second surface 32 of the second substrate 30, and it is desirable that the autofluorescence intensity of the coating layer 50 is less than a preset intensity.

It is understood that many substances in nature have autofluorescence property, which means that the substances are excited to emit fluorescence under or after irradiation with light (absorption energy). If the autofluorescence of the coating 50 is stronger, it means that the molecules in the coating 50 are excited by the laser to generate stronger fluorescence, that is, it means that the stronger the noise is, which may cause the signal-to-noise ratio of the acquired image to be reduced, and affect the implementation of the sequencing. When the coating layer 50 is selected, a material having an autofluorescence intensity smaller than a predetermined intensity is preferably selected as the coating layer 50. The preset intensity can be calculated and determined according to actual operation requirements. Specifically, the autofluorescence intensity of the coating 50 is related to the irradiation conditions, i.e., the intensity of laser light emitted from a laser, objective lens barrel parameters, etc., and also to the autofluorescence properties of the material of the coating 50. The inventors have found through extensive comparative tests that under certain working conditions, such as 532nm or 635nm laser irradiation of 800-1000mW, the background intensity of the image (16-bit map) of the chip 100 is less than or equal to 400, and high-quality sequencing results can be obtained based on the images. Based on this specific preset value and the specific working environment, the preset intensity here can be determined.

Referring to fig. 2 and 3, in some embodiments, a coating 50 may be applied to the second surface.

Thus, by coating the coating 50 on the second surface, when the chip 100 is irradiated by the laser, since the coating 50 is coated on the second surface 32 of the second substrate 30 and the autofluorescence intensity of the coating 50 is smaller than the preset intensity, the excitation light passing through the second substrate 30 is reduced, i.e., the laser is shielded on the second surface 32 of the second substrate 30, which is beneficial to reducing the fluorescence emitted by the structure below the second substrate 30 excited by the transmitted excitation light, so as to meet the requirement of the gene sequencer on the fluorescent background characteristic of the chip 100.

Specifically, as described above, the second substrate 30 may be made of glass, and when the coating layer 50 is disposed on the second surface 32 of the second substrate 30, the coating layer 50 may be selectively disposed on the second surface 32 of the second substrate 30 in a spraying manner, wherein the spraying may be performed a plurality of times to form a coating layer 50 with a certain thickness; of course, the coating 50 may also be printed on the second surface 32 of the second substrate 30 by silk-screen printing, and the coating 50 may be disposed on the second surface 32 of the second substrate 30 by spraying if the second surface 32 of the second substrate 30 is not a plane and the coating 50 cannot be disposed on the second surface 32 of the second substrate 30 by silk-screen printing.

It can be understood that, since the autofluorescence intensity of the coating 50 is less than the preset intensity, the coating 50 is coated on the second surface 32 of the second substrate 30, and the laser can be shielded on the second substrate 30 side, so as to meet the requirement of the gene sequencer on the fluorescence background characteristic of the chip 100.

Referring to FIG. 3, in some embodiments, the thickness A of the coating 50 may range from 5 μm to 20 μm. Preferably, the thickness A of the coating 50 may range from 8 μm to 15 μm. Thus, the coating 50 with a certain thickness is coated, so that the coating 50 has a better shielding effect on laser, and an image of a specific region of the chip 100 acquired by irradiation of exciting light can meet sequencing requirements.

In this application, it is to be understood that the terms "length," "width," "thickness," and the like, indicate an orientation or positional relationship based on that shown in the drawings, merely for convenience in describing the application and for simplicity in description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation.

Specifically, since the coating layer 50 is repeatedly coated on the second surface for multiple times, and the purpose of coating the coating layer 50 is to shield the laser from the second substrate 30 side, the coating layer 50 needs to have a certain thickness, when the thickness is too small, the coating layer 50 cannot play a good shielding role, and when the thickness is too large, it can be understood that the number of times of repeatedly coating is too large, and the coating layer 50 is too thick, which easily causes difficulty in ensuring the uniform thickness of the coating layer 50, and is prone to surface unevenness, wrinkles, even bursting, and the like.

In other embodiments, the thickness range A of the coating 50 can be set between 10 μm and 40 μm, so that the coating 50 can better shield the laser light, and the image of a specific region of the chip 100 collected by the excitation light irradiation can satisfy the sequencing requirement.

In certain embodiments, the coating 50 has a shading rate of not less than 80% under a working environment. Thus, the light shielding rate of the coating 50 in the working environment is set to be not less than 80%, which is enough to ensure that the coating 50 in the working environment can play a good light shielding role on the laser.

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

Preferably, the light shielding rate of the coating 50 in the working environment may be not less than 95%, so as to ensure that the coating 50 in the working environment can play a better light shielding role on the laser.

In some embodiments, the flatness tolerance of the side of the coating 50 facing away from the second substrate 30 is not more than 0.1 μm. Thus, after the package is connected to the rest of the structure in a stacked manner, the deviation/tolerance of the mechanically accumulated surface flatness can be ensured within a preset range, so that the flatness of the surface of the chip 100 meets the preset requirement, and the stable and firm connection of the coating layer 50 and the corresponding surface of the third substrate 60 is also facilitated, thereby facilitating the firm and stable structure of the chip 100.

Specifically, flatness means that when some workpieces are machined or produced, the surface of the workpiece is not absolutely flat, and the difference data between the uneven surface and the absolute level is the flatness. Since the uneven surface has a scattering effect on incident light according to a certain relationship between the flatness and the scattering rate, it can be understood that the smaller the value of the flatness of the side of the coating layer 50 facing away from the second substrate 30 is, the better in order to ensure the quality of the fluorescent signal image, and thus the allowable deviation of the flatness of the side of the coating layer 50 facing away from the second substrate 30 is set to be not more than 0.1 μm.

In addition, it should be noted that setting the allowable deviation of the flatness of the side of the coating layer 50 away from the second substrate 30 to be not more than 0.1 μm can ensure that the scattering rate of the coating layer 50 is low. Scattering is a physical concept describing a phenomenon in which radiation energy (various electromagnetic waves, including light waves, sound waves, and the like, for example) encounters a locally uneven area on a propagation path so that the propagation direction deviates from its original direction. When the roughness of the uneven area gradually increases, the specular scattering component gradually attenuates when scattering occurs, and the diffuse scattering component increases accordingly.

The local unevenness in this embodiment is: the surface of the coating 50 is not a completely smooth surface, with some minor height undulations. Because the laser emitted by the laser in the gene sequencing process irradiates the reaction reagent in the fluid channel 70, the irradiated fluorescent molecules are excited to generate corresponding fluorescent signals, and at the moment, the lens 200 is used for photographing and taking a fluorescent signal photo, so that the quality of the obtained fluorescent signal photo is higher, the analysis and calculation are convenient, the flatness allowable deviation of the surface of the coating 50, which is far away from the second substrate 30, is set to be not more than 0.1 μm, and the scattering interference generated when the incident laser irradiates on the coating 50 can be reduced to take a fluorescent signal image.

In certain embodiments, the material of the coating 50 may include ink. Thus, the chip 100 having the coating layer 50 has high light-shielding property and weak light-emitting property under a working environment, and is easily manufactured.

Specifically, the ink is a homogeneous mixture mainly comprising pigment, binder, filler, additive, etc. and may be printed and dried on the printed matter.

Wherein, the colorant can comprise pigment and dye, the pigment comprises organic pigment and inorganic pigment, the former has bright color tone, strong tinting strength and short drying time, so the colorant is widely applied to the printing ink; the latter has better light resistance, heat resistance, solvent resistance and hiding power. Pigments are colored in a particulate state and are not dissolved, and are the most commonly used colorants in inks. The dye is prepared into a solution when in use, is in a molecular state for coloring, and has no effect like a pigment.

The binder plays a role of dispersing the colorant, and may be prepared by dissolving a small amount of natural resin, synthetic resin, cellulose, rubber derivative, etc. in a drying oil or solvent. The vehicle can make the ink form a uniform thin layer after being sprayed on an object, and a film layer with certain strength is formed after being dried so as to protect the pigment.

The filling material and the additive are used as auxiliary components of the ink, the former can be used as an auxiliary agent for adjusting the concentration of the ink and can also increase the thickness of an ink film layer, the auxiliary components mainly comprise materials such as barium sulfate, talcum powder, calcium carbonate and the like, and the latter can be an additional part of a pigment or an additional part of a bonding material according to the requirements of products.

In certain embodiments, it is preferred that the coating 50 be a black ink. In this way, a higher quality image of a specific area of the chip 100 can be acquired. Moreover, by printing on the corresponding surface of the second substrate 30 to obtain the second substrate 30 with the black coating 50, it is possible to quickly and easily control the coating 50 or the chip 100 including the second substrate 30 with the coating 50 to meet the requirements.

In particular, there is a high demand for the light-shielding property of the coating layer 50 in the present application. While the pigment is used as a solid component in the ink, and is a color developing substance of the ink, and is generally a pigment insoluble in water, it is preferable that a black ink is used to achieve a high light-shielding effect in consideration of the light-shielding requirement of the coating layer 50, and the ink used as one of the materials for forming the coating layer 50 is required to have a high temperature resistance in consideration of the working environment and stability.

Referring to fig. 3 and 4, in some embodiments, the first substrate 20 may include a first surface and a second surface, and the first surface 21 of the first substrate 20 is disposed opposite to the second surface 22 of the first substrate 20. The fluid channel 70 may be formed on the second surface 22 of the first substrate 20 and the first surface 31 of the second substrate 30, 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 the image of the second surface 22 of the first substrate 20 and/or the image of the first surface 31 of the second substrate 30 in the working environment.

Specifically, in one example, a sample to be tested, such as a solution containing a fluorescence-labeled nucleic acid molecule, 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, the lens module is moved to find a medium interface where the nucleic acid molecule is located, that is, to find the second surface 21 of the first substrate 20, and a focal plane/clear plane is determined to acquire a clear image of the nucleic acid molecule. In the imaging process, when excited by the irradiation of the excitation light, the first surface 31, the second surface 32 and/or the coating 50 of the second substrate 30 of the chip 100 emit fluorescence, and the fluorescence appears as a background signal or an interference signal that is difficult to distinguish from a target signal in the acquired image of the chip 100, except for the fluorescence emitted by the nucleic acid molecules containing the fluorescent label.

In another example, a sample to be tested, such as a solution containing nucleic acid molecules, is located in the fluid channel 70, one end of the nucleic acid molecules is connected to the second surface 21 of the first substrate 20 and the first surface 31 of the second substrate 30, an image of the nucleic acid molecules is captured by an optical imaging system including a lens module, and during a focusing stage, the lens module is moved to find a medium interface where the nucleic acid molecules are located, that is, to find the second surface 22 of the first substrate 20 or the first surface 31 of the second substrate 30, so as to determine a corresponding focal plane/sharp plane to capture a sharp image of the nucleic acid molecules. In the imaging process, when excited by the irradiation of the excitation light, the first surface 31, the second surface 32 and/or the coating 50 of the second substrate 30 of the chip 100 emit fluorescence, and the fluorescence appears as a background signal or an interference signal that is difficult to distinguish from a target signal in the acquired image of the chip 100, except for the fluorescence emitted by the nucleic acid molecules containing the fluorescent label.

In a certain example, under a specific working environment, such as 532nm or 635nm laser irradiation of 800-1000mW, the acquired image is a 16-bit map, and the preset value is set to 400 (int), that is, the background intensity of the image of the chip 100 is smaller than or equal to 400. Therefore, an image with a higher signal-to-noise ratio can be obtained, and a high-quality sequencing result is favorably obtained. Fig. 6 illustrates the background intensities of the acquired images of four chips 100, the second surface 32 of the second substrate 30 of the chips 100 is printed with black ink, and the fluorescence background intensities of the images are all less than 400.

It will be appreciated that those skilled in the art can determine appropriate preset values in other working environments, such as lasers of different intensities and wavelengths, by way of determining preset values as exemplified herein.

Specifically, the image intensity represents the intensity of an image pixel, and in a grayscale image, the image intensity is the grayscale of the image, and in an RGB color space, it can be understood as the pixel grayscale value of an R channel, a G channel, or a B channel, and other color spaces are similar.

In the grayscale image, the grayscale of the image is the brightness of the image, and the larger the grayscale value is, the brighter the pixel point is, so that the background intensity of the image of the chip 100 in this embodiment is smaller than or equal to the preset value, that is, the obtained background brightness of the image of the chip 100 is lower, that is, the signal-to-noise ratio of the image is high, which is beneficial to the identification of the target signal, and the base identification is realized based on the target signal, it can be understood that the preset value can be set according to the actual requirement in the sequencing.

Specifically, in a certain example, on a sequencing platform comprising an optical imaging system, the optical system comprises a laser and a camera, the camera comprises a lens 200, and an image of the chip 100 can be acquired by: the laser is started to emit laser, so that the laser irradiates the to-be-tested reagent in the fluid channel 70 through the lens 200, the to-be-tested reagent comprises nucleic acid molecules with fluorescent marks, the fluorescent marks are excited to emit fluorescence, and then the camera is used for collecting and photographing the fluorescence passing through the lens 200 to obtain an image containing a target signal.

In another example, on a sequencing platform containing 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 lens, and an image of the chip 100 can be acquired by: the laser is started to emit laser, the laser irradiates a medium interface such as a solid-liquid interface in the chip 100, for example, the second surface 22 of the first substrate 20, at an angle larger than the critical angle through the lens 200, an evanescent wave/evanescent field is generated at the interface, the test agent includes nucleic acid molecules with fluorescent labels, one end of the nucleic acid molecules is connected to the second surface 22 of the first substrate 20, the fluorescent labels emit fluorescence in the evanescent field, and the fluorescence passing through the lens 200 is collected and photographed by a camera to obtain an image containing a target signal.

In addition, the background intensity of the image of the chip 100 is less than or equal to the predetermined value, which is caused by the coating 50 coated on the second surface 32 of the second substrate 30 shielding the laser from the second substrate 30 side, and since the autofluorescence intensity of the coating 50 itself is low, when the coating 50 is irradiated by the laser, the fluorescence signal generated by the molecules in the coating 50 excited by the laser is weak, i.e., the noise is low, so that the signal-to-noise ratio of the acquired image is improved, and the background intensity of the finally obtained fluorescence signal image can be less than or equal to the predetermined value.

In some embodiments, the background intensity of the image of the chip 100 may be less than or equal to 400. Thus, by controlling the background intensity of the image of the chip 100 to be less than or equal to 400, the brightness of the background portion of the image is low, so that an effective fluorescence signal can be conveniently displayed in a contrast manner, and the purpose of improving the sequencing quality can be achieved.

Specifically, the sequencer has a certain requirement on the fluorescent background characteristic of the chip 100, for example, in an embodiment, the preset value may be 400, which means that when a red-green laser of 800mW is used for illumination, the background intensity of the image of the chip 100 captured by the lens 200 is required to be less than or equal to 400 in a 16-bit map, so as to improve the sequencing quality.

Referring to fig. 1 and 2, in some embodiments, the chip 100 may include a third substrate 60 attached on the coating layer 50, wherein the third substrate 60 is made of a metal material.

In this way, by providing the third substrate 60 attached to the coating layer 50, the flatness stability and the temperature conductivity stability of the first and

second substrates

20 and 30 can be ensured.

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

The third substrate 60 can be attached to the coating layer 50 and disposed on two opposite sides of the coating layer 50 from the second substrate 30. The third substrate 60 is made of metal with good thermal conductivity, for example, the third substrate 60 may be an aluminum plate, and the third substrate 60 and the coating layer 50 may be bonded together by using an adhesive 61.

It can be understood that, when the chip 100 is packaged, the chip 100 may have a package structure with good sealing performance and good heat dissipation by using a stacked package manner in which the first substrate 20, the second substrate 30, and the third substrate 60 are bonded and fixed by the adhesive 61. The aluminum plate is used as the third substrate 60, so that the weight is light, the heat dissipation is good, and the flatness stability and the temperature conductivity stability of the first substrate 20 and the second substrate 30 can be ensured; the third substrate 60 is bonded to the coating 50 by the adhesive 61, so that the third substrate 60 is fixedly connected to the first substrate 20 and the second substrate 30, and the chip 100 has good sealing performance.

In particular, since the coating layer 50 is disposed on the second surface 32 of the second substrate 30, the coating layer 50 can prevent the laser from irradiating the adhesive glue 61 between the aluminum plate and the coating layer 50, thereby preventing molecules in the aluminum plate or the adhesive glue 61 from being excited to form fluorescence signal noise. Thus, the dependence of the chip 100 on the surface quality of the aluminum plate is reduced, so that the processing cost of the aluminum plate can be reduced, the material selection range of the adhesive 61 is expanded, and the limitation of the fluorescent characteristic index is avoided.

Referring to fig. 4, in some embodiments, the chip 100 may further include an interposer 40, the interposer 40 may be disposed between the first substrate 20 and the second substrate 30, the interposer 40 connects the first substrate 20 and the second substrate 30, and one or more fluid channels 70 may be disposed in the interposer 40. In this way, by disposing one or more fluid channels 70 on the interposer 40, the fluid channels 70 do not need to be etched on the first substrate 20 and the second substrate 30, which simplifies the manufacturing process of the fluid channels 70 and the manufacturing process of the chip 100.

In this application, the terms "connected" and "connected," unless expressly stated or limited otherwise, are to be construed broadly, e.g., as meaning a fixed connection, a removable connection, or an integral connection; may be mechanically, electrically or may be in communication with each other; they may be directly connected or indirectly connected through intervening media, or may be connected through the use of two elements or the interaction of two elements. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art as appropriate.

Specifically, the interposer 40 is formed between the first substrate 20 and the second substrate 30, and the interposer 40 may be a channel glue between the first substrate 20 and the second substrate 30, or the first substrate 20 and the second substrate 30 are adhesively packaged together through the interposer 40. The interposer 40 is provided with a fluid channel 70, such that the interposer 40 can be used as a chemical reaction site for the fluid reagent, i.e. a region where the laser irradiation generates a beneficial fluorescence signal.

It is understood that the first substrate 20 and/or the second substrate 30 may be used to pump fluids, such as reactive agents, into the fluid channels 70 provided in the interposer 40 and/or as an outlet or inlet for fluids pumped from the fluid channels 70 of the interposer 40.

In particular, the fluid passages 70 are formed in the interposer 40, which eliminates the need to physically or chemically etch the first substrate 20 and/or the second substrate 30, thereby simplifying the formation process of the fluid passages 70. In the present application, the fluid channels 70 may be formed by stamping the interposer 40, laser cutting, or the like.

Referring to fig. 4 and 5, in some embodiments, the interposer 40 may bond the first substrate 20 and the second substrate 30, the interposer 40 may have a hollow structure, and the interposer 40 may include a base layer 41, a first adhesive layer 42, a second adhesive layer 43, and one or more fluid channels 70 formed through the base layer 41, the first adhesive layer 42, and the second adhesive layer 43 by the hollow structure.

Wherein the base layer 41 has a first surface 411 and a second surface 412 opposite to each other, the first adhesive layer 42 is disposed on the first surface 411 of the base layer 41, the second adhesive layer 43 is disposed on the second surface 412 of the base layer 41, the first adhesive layer 42 can be adhered to the first substrate 20, and the second adhesive layer 43 can be adhered to the second substrate 30.

As such, by providing the first adhesive layer 42 and the second adhesive layer 43, it is possible to dispose the interposer 40 between the first substrate 20 and the second substrate 30. Through the hollow structure formed on the interposer 40, one or more fluid channels 70 may be formed through the base layer 41, the first adhesive layer 42 and the second adhesive layer 43, so that the reaction reagent may enter the fluid channels 70 through the first substrate 20 and/or the second substrate 30, and then exit through the first substrate 20 and/or the second substrate 30 after the chemical reaction occurs in the fluid channels 70.

Specifically, the interposer 40 includes a base layer 41, and the base layer 41 may be a main component of the interposer 40. The material of the substrate 41 may comprise black PET, clear PET, or any other plastic or polymer that provides high contrast to the fluorescence image ultimately captured by the lens 200.

The interposer 40 also includes a first adhesive layer 42 and a second adhesive layer 43, the first adhesive layer 42 being disposed on a first surface 411 of the base layer 41, the second adhesive layer 43 being disposed on a second surface 412 of the base layer 41, wherein the first surface 411 of the base layer 41 is in an opposing relationship to the second surface 412 of the base layer 41. The first adhesive layer 42 and the second adhesive layer 43 have adhesive properties so as to connect the first substrate 20 and the second substrate 30 with the interposer 40, and it can be easily understood that the first adhesive layer 42 can be bonded to the first substrate 20 or the second substrate 30, and correspondingly, the second adhesive layer 43 can be bonded to the second substrate 30 or the first substrate 20.

Further, the first adhesive layer 42 may be adhered to the second surface 22 of the first substrate 20, and the second adhesive layer 43 may be adhered to the first surface 31 of the second substrate 30, thereby integrally adhering the first substrate 20, the interposer 40, and the second substrate 30.

The first adhesive layer 42 and the second adhesive layer 43 should also have low autofluorescence, so that the effect of the interposer 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 area of the interposer 40 is negligible, thereby improving the signal-to-noise ratio and improving the sequencing quality.

The number of the fluid channels 70 may be one or more, and the plurality of fluid channels 70 may make the biochemical reaction of the reaction reagent more uniform and the sequencing more efficient. The fluid channels 70 may be formed on the interposer 40 by stamping or laser cutting, and the fluid channels 70 may extend 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.

Referring to fig. 4 and 5, in some embodiments, the first substrate 20 and/or the second substrate 30 are provided with a through hole 33 in communication with the fluid channel 70.

In this way, the through holes 33 connected to the fluid channel 70 are formed in the first substrate 20 and/or the second substrate 30, so that a fluid such as a reaction reagent can enter the fluid channel 70 through the first substrate 20 and/or the second substrate 30, and can flow out through the first substrate 20 and/or the second substrate 30 after a chemical reaction occurs in the fluid channel 70, and it is also convenient to connect a pipe or a manifold to connect the valve body and the reaction reagent container.

Specifically, the through hole 33 may be formed in the first substrate 20, the second substrate 30, or both the first substrate 20 and the second substrate 30, and in this embodiment, the through hole 33 is formed in the second substrate 30, so that a fluid such as a reaction reagent flows into the fluid channel 70 from the through hole 33 and flows out from the through hole 33 after the reaction.

In addition, it is understood that the number of the through holes 33 may be plural, wherein some of the through holes 33 are used as the fluid inlets, and the rest of the through holes 33 are used as the fluid outlets. The through holes 33 may be formed in an array on the first substrate 20 and the second substrate 30, and further, the array of holes may be etched in any one of the first substrate 20 and the second substrate 30 by wet etching or dry etching.

In certain 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 no 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.

In this way, by minimally limiting the magnitude of the peeling force of the first adhesive layer 42 to the first substrate 20 and/or the peeling force of the second adhesive layer 43 and the second substrate 30, it is possible to ensure that the 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 meets the operational requirements; for example, the adhesion peel force between the first adhesive layer 42/second adhesive layer 43 and the first substrate 20/second substrate 30 is not less than the predetermined value, and the respective structures of the chip 100 are firmly connected, the structure of the chip 100 is stabilized, and the sequencing requirements are satisfied.

In particular, since the inside of the chip 100 undergoes a plurality of pressure cycles due to the flow of the pressurized fluid through the fluid channel 70 during the gene sequencing process, i.e., the interposer 40 provided with the fluid channel 70 is exposed to a high pressure, it is necessary to ensure that the interposer 40 can withstand the pressure. It is therefore necessary to set at least one of the peeling force of the first adhesive layer 42 to the first substrate 20 and the peeling force of the second adhesive layer 43 and the second substrate 30 to not less than 560g, and of course, in other embodiments, to not less than 800g, so that the first adhesive layer 42 forms a sufficiently strong bond with the first substrate 20 and the second adhesive layer 43 with the second substrate 30.

It is understood that an important index for the bonding quality is the adhesive strength, and then there is a minimum limit to ensure the peeling force of the first adhesive layer 42 to the first substrate 20 and the peeling force of the second adhesive layer 43 and the second substrate 30, that is, 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 can be ensured.

Referring to fig. 4, in some embodiments, the fluid passages 70 have a larger dimension in a first direction X than in a second direction Y, the first direction X is perpendicular to the second direction Y, and both the first direction X and the second direction Y are perpendicular to the thickness direction of the interposer 40. As such, the general shape of the fluid channels 70 formed in the interposer 40 is dictated, which facilitates control of the fluid in the fluid channels 70, as well as positioning and imaging of the areas of the chip 100.

Specifically, as shown in fig. 4, fig. 4 shows a schematic plan view of the interposer 40, in which case the first direction X may be a length direction of the interposer 40, and the second direction Y may be a width direction of the interposer 40. It can be easily seen from fig. 4 that the shape of the fluid channels 70 is irregular, and the dimension of the fluid channels 70 in the first direction X is larger than the dimension in the second direction Y, and the first direction X is perpendicular to the second direction Y, and further, the first direction X and the second direction Y are perpendicular to the thickness direction of the interposer 40.

Of course, the fluid passages 70 may also have a dimension in the first direction X smaller than a dimension in the second direction Y, and the first direction X is still perpendicular to the second direction Y, and further, both the first direction X and the second direction Y are still perpendicular to the thickness direction of the interposer 40

Referring to fig. 4, in some embodiments, the number of the fluid passages 70 is multiple, and the fluid passages 70 extend along the first direction X and are disposed in the interposer 40; and/or, the fluid channels 70 are arranged on the interposer 40 in an array along the second direction Y. Thus, by forming a plurality of fluid channels 70, the gene sequencing process is more efficient, facilitating control of the fluid in the fluid channels 70 and also facilitating the positioning and imaging of the regions of the chip 100.

Specifically, as shown in fig. 4, the number of the fluid channels 70 is 4, wherein the fluid channels 70 are arranged in the interposer 40 in a first direction X, i.e., the length direction of the interposer 40, and are arranged on the interposer 40 in a second direction Y, i.e., the width direction of the interposer 40. In particular, the arrangement of the plurality of fluid channels 70 can make the sequencing process more efficient, and at the same time, the intervals of the fluid channels 70 are consistent by the arrangement of the array, so that the fluids such as the reaction reagents pumped into the fluid channels 70 are uniform.

Referring to fig. 4, in some embodiments, the fluid channel 70 includes a middle section 71, a first end 72 and a second end 73, the first end 72 and the second end 73 are respectively located at two ends of the fluid channel 70, and a dimension of the first end 72 in the second direction Y and/or a dimension of the second end 73 in the second direction Y are smaller than a dimension of the middle section 71 in the second direction Y.

As such, the shape of fluid channel 70 is further normalized to facilitate control of the fluid in fluid channel 70 and to facilitate locating and imaging of the regions of chip 100.

In particular, the shape of the fluid passage 70 may be irregular, for example the fluid passage 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 disposed at two ends of the fluid channel 70, and are both in a triangular shape, and the middle section 71 is in a long narrow rectangular shape, which also means that the size of the first end 72 and the second end 73 in the second direction Y is smaller than that of the middle section 71 in the second direction Y.

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

Referring to fig. 4, in some embodiments, the dimension of the middle section 71 in the second direction Y is constant. Thus, since the dimension of the middle section 71 in the second direction Y is constant, that is, the length of the middle section 71 in the second direction Y is equal everywhere, for example, the length of the middle section 71 in the second direction Y is 5mm. In particular, as described above, in the embodiment shown in fig. 4, the intermediate section 71 is a long narrow rectangle, that is to say the lengths of the intermediate sections 71 in the second direction Y are all equal, and then it can be obtained that the dimension of the intermediate section 71 in the second direction Y is constant.

Referring to fig. 4, in some embodiments, the dimension L1 of the middle section 71 in the second direction Y is in a range of 4.4mm to 8.4mm. In this way, the dimension L1 of the middle section 71 along the second direction Y is controlled to be 4.4mm-8.4mm, so that the fluid channel 70 has a certain width to reasonably contain the reagent, thereby facilitating the fluid control in the fluid channel 70 and the efficient biochemical reaction in the fluid channel 70.

In particular, it can be understood that the middle section 71 is the main area where the biochemical reaction of the reaction reagent occurs, and then the middle section 71 should have a suitable width, and since the second direction Y is the width direction of the interposer 40 shown in fig. 4, when the dimension of the middle section 71 in the second direction Y is less than 4.4mm, the fluid channel 70 may not be easily formed; when the size of the middle section 71 in the second direction Y is greater than 8.4mm, the width is too large to form more fluid channels 70 on the interposer 40, the sequencing reaction cannot be performed efficiently, and the amount of the reaction reagent carried by a single fluid channel 70 is also large, so that the reaction of the reaction reagent may not be uniform.

Referring to fig. 4, in some embodiments, the distance L2 between two adjacent fluid channels 70 in the second direction Y may be in a range of 0.8mm to 1.5mm. Thus, a plurality of fluid channels 70 can be easily processed on the interposer 40, and the number of the fluid channels 70 can be ensured, so that efficient sequencing can be realized as much as possible.

Specifically, when the pitch of two adjacent fluid channels 70 is smaller than 0.8mm, it is difficult to machine a plurality of fluid channels 70, and when the pitch of two adjacent fluid channels 70 is larger than 1.5mm, the number of fluid channels 70 with the hollow structure formed on the interposer 40 will be reduced. Therefore, the distance L2 between the adjacent two fluid passages 70 in the second direction Y is controlled to be 0.8mm to 1.5mm, and the number of the fluid passages 70 is ensured while facilitating the processing.

In some embodiments, the thickness B of the base layer 41 may range from 30 μm to 50 μm; and/or, the first adhesive layer 42 may have a thickness C in the range of 75 μm to 85 μm; and/or the thickness D of the second adhesive layer 43 may be in the range of 75 μm to 85 μm. Thus, by properly setting the thickness range B of the base layer 41 and the thickness ranges D of the first adhesive layer 42 and the second adhesive layer 43, the normal progress of the biochemical reaction in the interposer 40 can be ensured.

Specifically, the thickness range B of the base layer 41 may be 30 μm to 50 μm, and the thickness range C of the first adhesive layer 42 and the thickness range D of the second adhesive layer 43 are 75 μm to 85 μm; alternatively, the thickness range B of the base layer 41 may be 30 μm to 50 μm, and one of the thickness range C or the thickness range D of the second adhesive layer 43 or the first adhesive layer 42 is 75 μm to 85 μm; or, only the thickness range of the first adhesive layer 42 and the second adhesive layer 43 is required, 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 75 μm to 85 μm; further alternatively, only the thickness range C or the thickness range D of the first adhesive layer 42 or the second adhesive layer 43 is required, and the thickness range of one of the adhesive layers is set to 75 μm to 85 μm.

It is noted that, as described 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 and 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 agent layer.

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 pressure; when the adhesive layer is too thick, voids may be formed in the first adhesive layer 42 and the second adhesive layer 43 to cause air bubbles to form, which may weaken the bonding strength, and most of the stress and shear stress may act on the first adhesive layer 42 and the second adhesive layer 43 and may not be transferred to the base layer 41, so that the first adhesive layer 42 and the second adhesive layer 43 may be easily broken to cause the chip 100 to malfunction. In summary, the thickness range B of the base layer 41 should be set to 30 μm to 50 μm, and/or the thickness range C of the first adhesive layer 42 should be set to 75 μm to 85 μm, and/or the thickness range D of the second adhesive layer 43 should be set to 75 μm to 85 μm.

In certain embodiments, the base layer 41, the first adhesive layer 42, and/or the second adhesive layer 43 withstand a temperature of no less than 80 ℃. In this way, by setting a certain withstand temperature for the base layer 41, the first adhesive layer 42, and the second adhesive layer 43, the normal progress of the biochemical reaction in the interposer 40 can be ensured.

In particular, in the gene sequencing process, the chip 100 may be exposed to thermal cycling, and the substrate 41 carrying the mediator layer 40 for biochemical reaction and the first adhesive layer 42 and the second adhesive layer 43 are required to satisfy the tolerance to a certain temperature.

It will be appreciated that the above-mentioned high temperature resistance means that there is no significant deformation of the base layer 41, the first adhesive layer 42 and the second adhesive layer 43 at a given temperature. It is necessary to set the temperature resistance of the base layer 41 to not less than 80 c and the temperature resistance of the first adhesive layer 42 and/or the second adhesive layer 43 to not less than 80 c.

In certain embodiments, first adhesive layer 42 and/or second adhesive layer 43 withstand a temperature of not less than 110 ℃. In this way, by setting a certain withstand temperature for the first adhesive layer 42 and the second adhesive layer 43, the normal progress of the biochemical reaction in the interposer 40 can be ensured.

Also, as described above, in order that there is no significant deformation of the first adhesive layer 42 and the second adhesive layer 43 when the chip 100 is exposed to the environment of thermal cycling during gene sequencing, the resistant temperature of at least one of the first adhesive layer 42 and the second adhesive layer 43 may be set to be not less than 110 ℃.

In certain 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. In this manner, by providing the base layer 41, the first adhesive layer 42, and/or the second adhesive layer 43 with resistance to a prescribed solvent, it is possible to ensure normal progress of biochemical reactions in the interposer 40.

During gene sequencing, the chip 100 may be exposed to corrosive reagents (such as formamide), and the like, and the substrate 41 carrying the mediator layer 40 for biochemical reaction and at least one of the first adhesive layer 42 and the second adhesive layer 43 are required to be resistant to a given solvent. Wherein, the tolerance represents that the problems of glue layer and film layer shedding, invalidation and the like do not occur in a specified solvent, and the specified solvent can be water, DMSO, formamide solution and the like.

In some embodiments, the material of the base layer 41 may comprise polyimide. Thus, the base layer 41 made of the polyimide material can meet the requirement that the base layer 41 can resist certain high temperature and can also meet the requirement of resisting a specified solvent, so that the problems of film falling, failure and the like in a high-temperature environment and certain solvents are avoided.

Specifically, the polyimide material is characterized by high temperature resistance, a small thermal expansion coefficient and good solvent resistance, so that the use of the polyimide material for the base layer 41 can satisfy the condition that the base layer 41 can withstand a temperature of not less than 80 ℃, and can also satisfy the requirement of withstanding a specified solvent, so that the problems of film falling, failure and the like in the above solvent are avoided.

In certain 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 a material including acrylic adhesive, butyl rubber, silicone rubber, and the like.

In some embodiments, the material of at least one of the first adhesive layer 42 and the second adhesive layer 43 may be a pressure sensitive adhesive. For example, the pressure-sensitive adhesive is a pressure-sensitive silica (PSA silica), specifically, the pressure-sensitive adhesive refers to an adhesive that can be instantly bonded with a slight pressure, and currently, the commonly used pressure-sensitive adhesives mainly include rubber-type pressure-sensitive adhesives, acrylate-type pressure-sensitive adhesives, and silicone pressure-sensitive adhesives, and the rubber-type pressure-sensitive adhesives are currently widely used pressure-sensitive adhesives, and the rubbers include natural rubbers, synthetic rubbers, and reclaimed rubbers; the organic silicon pressure-sensitive adhesive is prepared by matching silicon rubber and silicon resin, wherein the silicon rubber is used as a basic component of the pressure-sensitive adhesive, and the silicon resin is used as a tackifier. The properties of the pressure-sensitive adhesive vary with the ratio of the two, and preferably, a pressure-sensitive adhesive made of silicone rubber may be used herein.

In some embodiments, the material of at least one of the first adhesive layer 42 and the second adhesive layer 43 may include silicone. Specifically, at least one of the first adhesive layer 42 and the second adhesive layer 43 may be made of silicone. The silica gel material can resist the high temperature of more than 110 ℃, and the silica gel can not deform to cause the deformation and channel crossing of the fluid channel 70 under the condition of continuously keeping at 80 ℃ for three hours; moreover, the silicone material has an adhesion peel force greater than 800g to the glass material surface, i.e., the first substrate 20 and the second substrate 30, and can meet the requirement that 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 is not less than 800g; meanwhile, the silica gel material also has solvent resistance, and in the modifying and sequencing reagent, after the 350-cycle sequencing process is completed, the fluid channel 70 cannot be deformed and cross-linked.

The chip 100 may be used to perform sequencing.

Referring to fig. 7, the present application provides a method for manufacturing a chip 100, wherein the method may include the following steps:

step S10: providing a first substrate 20;

step S20: providing a second substrate 30, the second substrate 30 comprising a first surface 31 and a second surface 32 opposite to each other;

step S30: laminating the second substrate 30 on the first substrate 20 such that the first surface 31 of the second substrate 30 faces the first substrate 20;

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

step S50: disposing a coating 50 on the second surface 32 of the second substrate 30, the coating 50 having an autofluorescence intensity less than a predetermined intensity;

thus, by performing the method, the first substrate 20 and the second substrate 30 are stacked to form a basic structure having the fluid channel 70, and the second surface 32 of the second substrate 30 is provided with the specific coating layer 50. In applications involving the detection of signals from a sample to be tested of the chip 100 by an optical imaging system, such as a sequencing platform for detecting fluorescence signals of nucleic acid molecules in the chip based on optical imaging to realize nucleic acid sequencing, the chip 100 itself is irradiated, i.e., the fluorescence signals (background signals) emitted by excitation light are weak, which is beneficial to obtain images with high signal-to-noise ratio, is beneficial to the identification of target fluorescence signals, and is beneficial to obtain high-quality sequencing results.

Specifically, the chip 100 may be a reactor in which a nucleic acid molecule to be detected having an optical detection label is fixed, has a space for containing a liquid, and can be used to fix a sample to be detected, and is also called a flow cell or a flow cell (Flowcell). In steps S10-S40, the first substrate 20 and the second substrate 30 are any solid support for fixing nucleic acid sequences, such as nylon membrane, glass sheet, plastic, silicon wafer, magnetic beads, etc. The first substrate 20 and the second substrate 30 may be provided to comprise any suitable material, such as glass, silica, crystal, quartz glass, plastic, ceramic, PET (poly terephthalic acid), PMMA (poly methyl methacrylate), or any other suitable material. In one embodiment, the first substrate 20 and the second substrate 30 are provided to be optically transparent, such as a glass sheet/layer.

The second substrate 30 is stacked on the first substrate 20, and the first substrate 20 is disposed on the second substrate 30, that is, the first surface 31 of the second substrate 30 is disposed toward the first substrate 20. In addition, as mentioned above, the SBS compliant sequencing platform chip 100 may comprise one or more parallel channels for ingress and egress of reagents to form the environment required for the sequencing reaction, i.e., one or more fluidic channels 70 need to be provided between the first surface 31 of the second substrate 30 and the first substrate 20.

In this application, unless expressly stated or limited otherwise, the first feature "on" or "under" the second feature may comprise direct contact of the first and second features, or may comprise contact of the first and second features not directly but through another feature in between. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature.

In step S50, as the inventor finds in the structural design, preparation and test of the chip adapted to the sequencing platform that when the laser emitted from the laser of the sequencing platform is irradiated into the fluid channel of the chip through the lens, the laser also passes through the lower glass and is irradiated onto the glue layer connecting the lower glass and the aluminum plate, and molecules of the glue 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 molecule to be detected in the fluid channel.

Therefore, the coating 50 with autofluorescence intensity smaller than the preset intensity needs to be disposed on the second surface 32 of the second substrate 30, so that the excitation light passing through the second substrate 30 is reduced, which is beneficial to weakening the fluorescence emitted by the structure below the second substrate 30 excited by the transmitted excitation light, and because the autofluorescence intensity of the coating 50 is smaller than the preset intensity, the fluorescence intensity generated by the coating 50 irradiated by the excitation light is weaker, and the fluorescence signal generated by the chip itself is weaker in the working environment, so that the chip 100 prepared by the preparation method is suitable for bearing a biological sample to realize biological macromolecule detection, and is suitable for an optical imaging platform for realizing detection of a sample to be detected based on a detection chip.

The coating 50 may be applied to the second surface 32 of the second substrate 30 or may be printed on the second surface 32 of the second substrate 30. The specific requirements of the coating 50 are as described above for the coating 50, for example, the material of the coating 50 may include black ink for providing high opacity; the thickness range A of the coating 50 may be 5 μm to 20 μm or 8 μm to 15 μm; the light shading rate of the coating 50 in the working environment is not less than 80%; the flatness of the side of the coating 50 facing away from the second substrate 30 is allowed to deviate by no more than 0.1 μm.

Referring to fig. 8, in some embodiments, the method for manufacturing a chip further includes:

step S60: providing a third substrate 60;

step S70: a third substrate 60 is disposed under the coating 50.

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

Specifically, in step S50 and step S60, the third substrate 60 may be an aluminum plate with good thermal conductivity, and the third substrate 60 is disposed under the coating layer 50. In this way, the chip 100 body may be formed by bonding two glass first and

second substrates

20 and 30 and a metal bottom plate, i.e., the third substrate 60, so as to ensure the stability of flatness and temperature conductivity of the first and

second substrates

20 and 30.

Referring to fig. 9, in some embodiments, the method for manufacturing a chip further includes the following steps:

step S80: providing an interposer 40; 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. In this way, the method provides one or more fluid channels 70 in the interposer 40, so that the fluid channels 70 do not need to be etched on the first substrate 20 and the second substrate 30, and the manufacturing process of the fluid channels 70 is simplified.

Specifically, in step S80 and step S90, an interposer 40 is provided, and one or more fluid channels 70 are disposed in the interposer 40, the fluid channels 70 may be formed by stamping the interposer 40, laser cutting, and the like, and specific data such as the size of the fluid channels 70 and the distance between the fluid channels 70 are described in detail above, which is not described herein again. The intermediate layer 40 can be used as a chemical reaction site of the fluid reagent, i.e., a region where the laser irradiation generates a beneficial fluorescent signal, and the fluid channel 70 does not need to be etched on the first substrate 20 and the second substrate 30, thereby simplifying the manufacturing process of the fluid channel 70.

Since one or more fluid channels 70 are formed between the first substrate 20 and the second substrate 30, and thus the interposer 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 may be used to pump a fluid such as a reactive agent into the fluid channels 70 disposed in the interposer 40, and/or to serve as an outlet or an inlet of the fluid pumped from the fluid channels 70 of the interposer 40.

Referring to fig. 10, in some embodiments, providing the interposer 40 (step S80) includes:

step S81: providing a base layer 41 having opposing first and second surfaces 411, 412;

step S82: providing a first adhesive layer 42 on a first surface 411 of the base layer 41;

step S83: disposing a second adhesive layer 43 on the second surface 412 of the base layer 41;

step S84: forming fluid channels 70 through the base layer 41, the first adhesive layer 42, and the second adhesive layer 43;

the method further comprises the following steps: step S100: the first adhesive layer 42 is bonded to the first substrate 20, and the second adhesive layer 43 is bonded to the second substrate 30.

Specifically, in step S81, the provided base layer 41 may be a main component of the interposer 40. The material of the base layer 41 may comprise black PET, clear PET or any other plastic or polymer. The black PET has low autofluorescence so that a high signal-to-noise ratio can be obtained during sequencing and provides high contrast to the fluorescence image finally taken by the lens 200.

In steps S82 and S83, the first adhesive layer 42 and the second adhesive layer 43 should also be provided with low autofluorescence. The first adhesive layer 42 is disposed on the first surface 411 of the base layer 41, the second adhesive layer 43 is provided on the second surface 412 of the base layer 41, and the first adhesive layer 42 and the second adhesive layer 43 have adhesiveness such that the first substrate 20 and the second substrate 30 can be attached and disposed together with the interposer 40.

In step S84, since the hollow structure is formed on the interposer 40, the interposer 40 includes the base layer 41, the first adhesive layer 42, and the second adhesive layer 43, so that the hollow structure can penetrate through 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 channels 70 through the first substrate 20 and/or the second substrate 30, and flow out through the first substrate 20 and/or the second substrate 30 after the chemical reaction occurs in the fluid channels 70.

In step S100, the first adhesive layer 42 is bonded to the second surface 22 of the first substrate 20, and the second adhesive layer 43 is bonded to the first surface 31 of the second substrate 30, so as to integrally bond the first substrate 20, the interposer 40 and the second substrate 30.

In the description of the present specification, reference to the description of "one embodiment", "certain embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that: numerous changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the application, the scope of which is defined by the claims and their equivalents.

Claims

1. A chip, comprising:

a first substrate;

the second substrate is stacked with the first substrate and comprises a first surface and a second surface which are opposite, 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;

a coating disposed on the second surface of the second substrate, the coating having an autofluorescence intensity less than a preset intensity.

2. The die of claim 1, wherein the coating is disposed on the second surface of the second substrate;

optionally, the coating has a thickness in the range of 5 μm to 20 μm;

optionally, the coating has a thickness in the range of 8 μm to 15 μm;

optionally, the coating has a light shading rate of not less than 80% in a working environment;

optionally, the flatness tolerance of the side of the coating facing away from the second substrate is not more than 0.1 μm;

optionally, the material of the coating comprises an ink;

optionally, the material of the coating is a black ink.

3. The chip according to any one of claims 1-2, wherein the first substrate comprises a first surface and a second surface opposite to each other, the fluid channel is formed between the second surface of the first substrate and the first surface of the second substrate, the background intensity of the image of the chip is less than or equal to a preset value, and the image of the chip is the image of the second surface of the first substrate and/or the image of the first surface of the second substrate in a working environment;

optionally, the background intensity of the image of the chip is less than or equal to 400.

4. The chip of any one of claims 1-3, further comprising a third substrate attached to the coating, wherein the third substrate is made of metal.

5. The die of any of claims 1-4, further comprising an interposer disposed between the first substrate and the second substrate, the interposer connecting the first substrate and the second substrate, the one or more fluid channels being disposed in the interposer.

6. The die of claim 5, wherein the interposer adheres the first substrate and the second substrate, the interposer having a hollowed-out structure, the interposer comprising:

a base layer having opposing first and second surfaces;

a first adhesive layer disposed on a first surface of the base layer, the first adhesive layer being bonded to the first substrate;

a second adhesive layer disposed on a second surface of the base layer, the second adhesive layer being bonded to the second substrate;

and the number of the first and second groups,

the fluid channel is the hollow structure formed by penetrating through the base layer, the first adhesive layer and the second adhesive layer;

optionally, the first substrate and/or the second substrate is provided with a through hole communicating with the fluid channel;

optionally, 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;

optionally, 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;

optionally, the fluid channel has a dimension in a first direction that is greater than its dimension in a second direction, the first direction being perpendicular to the second direction, the first direction and the second direction both being perpendicular to the thickness direction of the interposer;

optionally, the number of the fluid channels is multiple, and the fluid channels extend along the first direction and are arranged in the interposer; and/or the fluid channels are arranged in the interposer along the second direction array;

optionally, the fluid channel comprises a middle section, a first end and a second end, the first end and the second end being located at both ends of the fluid channel, respectively, the dimension of the first end in the second direction and/or the dimension of the second end in the second direction being smaller than the dimension of the middle section in the second direction;

optionally, the dimension of the middle section in the second direction is constant;

optionally, the intermediate section has a dimension in the second direction in the range of 4.4mm to 8.4mm;

optionally, the distance between two adjacent fluid channels in the second direction is in the range of 0.8mm to 1.5mm;

optionally, the base layer has a thickness in the range of 30 μm to 50 μm; and/or the presence of a gas in the gas,

the first adhesive layer has a thickness in the range of 75 μm to 85 μm; and/or the presence of a gas in the atmosphere,

the thickness of the second adhesive layer ranges from 75 μm to 85 μm;

optionally, the base layer, the first adhesive layer, and/or the second adhesive layer withstand a temperature of not less than 80 ℃;

optionally, the first adhesive layer and/or the second adhesive layer withstand a temperature of not less than 110 ℃;

optionally, the base layer, the first adhesive layer, and/or the second adhesive layer are resistant to a specified solvent;

optionally, the material of the base layer comprises polyimide;

optionally, the first adhesive layer and the second adhesive layer are made of the same material;

optionally, the material of the first adhesive layer and/or the second adhesive layer comprises silicone.

7. Use of a chip according to any one of claims 1 to 6 in a sequence assay.

8. A method of fabricating a chip, comprising:

providing a first substrate;

providing a second substrate comprising a first surface and a second surface opposite to each other;

laminating the second substrate on the first substrate so that the first surface of the second substrate faces the first substrate;

providing one or more fluid channels between the first surface of the second substrate and the first substrate;

disposing a coating on a second surface of the second substrate, the coating having an autofluorescence intensity less than a predetermined intensity;

the method further comprises the following steps:

providing a third substrate;

disposing the third substrate under the coating;

the method further comprises the following steps:

providing an interposer;

disposing the interposer between the first substrate and the second substrate, disposing the one or more fluid channels in the interposer.

9. The method of claim 8, wherein the providing an interposer comprises:

providing a base layer having first and second opposing surfaces;

disposing a first adhesive layer on a first surface of the base layer;

disposing a second adhesive layer on a second surface of the base layer;

forming the fluid channel through the base layer, the first adhesive layer, and the second adhesive layer;

the method further comprises the following steps: bonding the first adhesive layer to the first substrate and the second adhesive layer to the second substrate.

10. The method of claim 9, wherein the base layer has a thickness in the range of 30 μ ι η to 50 μ ι η; and/or the presence of a gas in the gas,

the thickness of the second adhesive layer ranges from 75 μm to 85 μm;

optionally, the base layer is a polyimide;

optionally, the first adhesive layer and the second adhesive layer are the same material;

optionally, the first adhesive layer and/or the second adhesive layer is a silicone gel;

optionally, the material of the first adhesive layer and/or the second adhesive layer is a pressure sensitive adhesive;

optionally, the first substrate and/or the second substrate is provided with a through hole communicating with the fluid channel.