CN210030701U

CN210030701U - Gene sequencing chip

Info

Publication number: CN210030701U
Application number: CN201920262700.6U
Authority: CN
Inventors: 石磊; 陈子天
Original assignee: Saina Biological Technology (beijing) Co Ltd
Current assignee: Saina Biological Technology (beijing) Co Ltd
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2020-02-07
Anticipated expiration: 2029-03-01

Abstract

A gene sequencing chip, the gene sequencing chip comprising: the device comprises a first solid substrate, a second solid substrate, a glue coating area and a reaction chamber, wherein the glue coating area and the reaction chamber are positioned between the first solid substrate and the second solid substrate; the glue coating area directly surrounds the reaction chamber; and a supporting layer is arranged between the first solid substrate and the second solid substrate. Performing surface chemical modification on areas corresponding to the first solid substrate and the second solid substrate to form a glue coating area and a reaction chamber, so that the glue coating area directly surrounds the reaction chamber; and a supporting layer is arranged between the first solid substrate and the second solid substrate to limit the distance between the two substrates. In the gene sequencing chip, an isolation strip is not needed between the glue coating area and the reaction chamber, so that the packaging strength is improved on the premise of the same reaction chamber area compared with a method using the isolation strip; compared with the method of etching the substrate, the method reduces the production cost.

Description

Gene sequencing chip

Technical Field

The utility model relates to a biochemical detection chip belongs to biology, chemistry detection area. More specifically, the utility model relates to a gene sequencing chip.

Background

In recent years, research on biochips or microfluidic chips has attracted more and more attention. The typical microfluidic chip generally refers to a chip with micron-sized detection units for integrating biological and chemical reaction, analysis, detection, etc. In the production process of the microfluidic chip, chip packaging is an important part. Microfluidic chips or biochemical detection chips are increasingly required for chip packaging processes because many fluids are involved and high temperature or high pressure conditions are applied as required. Common microfluidic chips are PDMS type or glass type chips. The general PDMS chip utilizes the active group on the surface of the polymer to be covalently bonded with other surfaces so as to achieve the purpose of encapsulation. A typical glass chip can be encapsulated by thermal bonding. At present, with the increase of various chip preparation materials, the common chip packaging method is not suitable for special materials.

Gene sequencing is a novel gene detection technology, and can analyze and determine gene sequences from blood or human appendages to predict the possibility of suffering from various diseases, such as cancers or leukemia. Products and techniques related to gene sequencing have evolved from laboratory studies to clinical use. Gene chips or sequencing chips are chips for gene sequencing. A variety of gene sequencing chips are currently available. The prototype of the gene chip was proposed in the middle of the 80 s. The sequencing principle of gene chips is the hybridization sequencing method, i.e., the method of determining the sequence of nucleic acid by hybridization with a group of nucleic acid probes of known sequence, wherein the probes of target nucleotides of known sequence are immobilized on the surface of a substrate. When the nucleic acid sequences with fluorescent labels in the solution are complementarily matched with the nucleic acid probes at corresponding positions on the gene chip, a group of probe sequences with completely complementary sequences is obtained by determining the fluorescence intensity. The sequence of the nucleic acid to be detected can be recombined according to the sequence. In practice, the sequencing chip has many indexes, such as pressure resistance, fluorescence, etc. The existing sequencing chip has the production modes of double-sided adhesive tape packaging, liquid adhesive tape packaging and the like, wherein the double-sided adhesive tape packaged chip has poor temperature and pressure resistance; the conventional sequencing chip packaged by liquid glue mainly adopts two modes, namely defining a glue coating area and a reaction chamber area by using a separation strip (such as a double-sided adhesive tape), and etching a groove or forming a lug boss on a solid substrate to limit the glue coating area and the reaction chamber area. In the first method, the isolation belt and the reaction chamber have the same plane in the gluing area, so that the glue area and the gluing area can compete with each other, and the available area of the reaction chamber is smaller under the condition of achieving a certain pressure resistance. The second method requires etching a large area and a specific shape on a solid substrate, and is expensive.

SUMMERY OF THE UTILITY MODEL

The utility model aims at providing a gene sequencing chip of liquid glue encapsulation, through the surface chemistry modification who divides the region on solid substrate, use the lower liquid glue of viscosity, after the rubber coating, automatic diffusion forms appointed shape, need not the median, need not the sculpture base plate, can improve available reaction chamber area, reduce cost simultaneously.

In order to achieve the above object, the present invention provides a gene sequencing chip, comprising: the device comprises a first solid substrate, a second solid substrate, a glue coating area and a reaction chamber, wherein the glue coating area and the reaction chamber are positioned between the first solid substrate and the second solid substrate;

a fluid inlet and a fluid outlet are arranged on the first solid substrate;

the reaction chamber is positioned in the middle, the glue coating area is positioned at the periphery of the reaction chamber, and the glue coating area directly surrounds the reaction chamber;

and a supporting layer is arranged between the first solid substrate and the second solid substrate.

Further, the glue coating area is filled with solidified liquid glue.

Further, the viscosity of the liquid glue is less than 1000 cps.

Further, the fluid inlet and the fluid outlet correspond to a region where the reaction chamber is located.

Further, the support layer comprises a plurality of support posts for supporting and defining a distance between the first and second solid substrates.

Further, the first solid substrate and the second solid substrate are transparent solid flat sheets, and are preferably selected from any one of glass, quartz, and organic high polymer materials.

Further, the support layer is selected from any one of double-sided adhesive tape, PI material and PET material.

In summary, the present invention provides a gene sequencing chip, which performs surface chemical modification on the corresponding regions of a first solid substrate and a second solid substrate to form a glue coating region and a reaction chamber, such that the glue coating region directly surrounds the reaction chamber; and a supporting layer is arranged between the first solid substrate and the second solid substrate to limit the distance between the two substrates. In the gene sequencing chip, an isolation belt is not needed between the gluing area and the reaction chamber, and the packaging strength is improved on the premise of the same reaction chamber area compared with a method using the isolation belt; compared with the method of etching the substrate, the method reduces the production cost.

Drawings

FIG. 1(a) is a schematic structural view of a first solid substrate; FIG. 1(b) is a schematic structural view of a second solid substrate; FIG. 1(c) is a schematic view of a glue application zone and a reaction chamber between a first solid substrate and a second solid substrate; FIG. 1(d) is a schematic structural diagram of the gene sequencing chip of the present invention in which the first solid substrate, the second solid substrate, the glue coating region and the reaction chamber are superposed together;

FIG. 2 is a schematic diagram showing the cross-sectional structure of the gene sequencing chip of the present invention;

FIG. 3 is a schematic flow chart of the method for preparing the gene sequencing chip of the present invention.

Reference numerals:

101: a first solid substrate; 102: a second solid substrate; 103: a support pillar; 104: a glue spreading area; 105: a reaction chamber; 106: the finished chip; 107: a fluid inlet, a fluid outlet.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the description is intended to be illustrative only and is not intended to limit the scope of the present invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.

The first aspect of the present invention provides a gene sequencing chip, as shown in fig. 1-2, comprising a first solid substrate 101 (as shown in fig. 1 (a)), a second solid substrate 102 (as shown in fig. 1 (b)), and a glue coating region 104 and a reaction chamber 105 (as shown in fig. 1 (c)) located between the first solid substrate 101 and the second solid substrate 102. Fig. 1(d) shows an entire chip formed by stacking the first solid substrate 101, the second solid substrate 102, the glue coating region 104, and the reaction chamber 105. A fluid inlet and a fluid outlet 107 are provided on the first solid substrate 101.

The reaction chamber 105 is located in the middle, the glue coating area 104 is located around the reaction chamber 105, the glue coating area 104 directly surrounds the reaction chamber 105, an isolation belt is not arranged in the middle, and solidified liquid glue is arranged in the glue coating area 104. Compared with the prior art, the utility model, need not set up the median between rubber coating district and reaction chamber, improved encapsulation intensity under the prerequisite of the same reaction chamber area for the method that uses the median. For example, the long side width of the reaction chamber is 1.5 mm, if the reaction chamber is packaged by a 0.5 mm isolation strip and 1mm liquid glue, the utility model discloses with the liquid glue encapsulation of 1.5 mm, the packaging strength improves about 50%. And adopt the liquid glue packaging method of median, when liquid glue solidification, probably because the liquid glue shrink leads to the plane degree variation of base plate, the utility model discloses do not have this kind of hidden danger.

A support layer is arranged between the first solid substrate 101 and the second solid substrate 102, and specifically, the support layer is provided with double-sided adhesive support columns 103 located at four corners of the adhesive coating area 104. The height of the supporting column 103 determines the height of the glue application area 104 and the fluid reaction chamber 105 (which are the same height).

Further, the glue coating area 104 is filled with solidified liquid glue, and preferably, the viscosity of the liquid glue is less than 1000 cps.

Further, the fluid inlet and the fluid outlet correspond to the region where the reaction chamber 105 is located, so as to facilitate the injection and outflow of the analyte liquid during the gene sequencing test.

Further, the support layer comprises a plurality of support columns for supporting and defining the distance between the first solid substrate 101 and the second solid substrate 102.

Further, the first solid substrate 101 and the second solid substrate 102 are transparent solid flat sheets, and are preferably selected from any one of glass, quartz, and organic high molecular polymer materials.

Further, the supporting layer is selected from any one of double-sided adhesive tape, PI material and PET material.

Another aspect of the present invention provides a method for preparing a gene sequencing chip, which comprises performing surface chemical modification on two solid substrates in different areas to diffuse liquid glue into a predetermined shape, thereby forming a glue spreading area and a reaction chamber having a predetermined shape; the analyte is detected by a biochemical reaction in the reaction chamber. The utility model discloses the principle of control liquid glue diffusion does: the surface of the clean glass/quartz (solid substrate) is hydrophilic and the liquid glue diffuses freely on the surface, the surface of the glass/quartz modified with fluoro-silanes [ such as trichloro (1H,1H,2H, 2H-perfluorooctyl) silane, chloro (1H,1H,2H, 2H-perfluorooctyl) dimethylsilane or (1H,1H,2H, 2H-perfluorooctyl) trimethoxysilane ] exhibits hydrophobicity, the liquid glue diffuses on the hydrophilic surface, and the diffusion stops when encountering the hydrophobic surface or diffuses in the hydrophilic area along the boundary line of the hydrophilic and hydrophobic surfaces. Specifically, the flow 300 of the method for preparing a gene sequencing chip of the present invention is shown in fig. 3, and comprises the following steps:

step 310, preparing a first solid substrate 101, a second solid substrate 102 and a support layer. Specifically, the method comprises the steps of cleaning and drying a first solid substrate 101 and a second solid substrate 102; the support layer is a plurality of double-sided adhesive support columns with preset size, which are cut and can be square or rectangular or other shapes, such as 1X1mm small pieces, and the thickness can be adjusted according to the requirements of the reaction chamber, such as 50 microns, 100 microns, 150 microns and the like.

The first and second solid substrates may be transparent solid flat sheets, such as glass and quartz; or an organic high molecular polymer material such as PMMA; the substrate may or may not have microstructures such as micro pits. The supporting layer can be made of double-sided adhesive tape to facilitate later assembly, and can also be made of other film materials with fixed thickness, such as PI, PET and the like.

Step 320, performing surface chemical modification on corresponding positions of the first solid substrate 101 and the second solid substrate 102 to divide the glue coating area 104 and the reaction chamber 105 area; the areas that are not chemically modified at the surface are glue coated areas 104.

The glue coating area is a hydrophilic area which is not subjected to surface chemical modification, and the reaction chamber area is a hydrophobic area which is subjected to surface chemical modification. The chamber region is designed to include fluid inlets and outlets on the substrate.

Specifically, two methods can be employed:

(1) a masking glue is arranged in a glue coating area 104 of the first solid substrate 101 and the second solid substrate 102: attaching masking glue (e.g., acrylic nitto HJ3160W) to areas (glue areas) where surface chemical modification is not desired;

performing Chemical Vapor Deposition (CVD) treatment on the first solid substrate 101 and the second solid substrate 102 with fluorosilane; the fluoro silane can be selected from trichloro (1H,1H,2H, 2H-perfluorooctyl) silane, monochloro (1H,1H,2H, 2H-perfluorooctyl) dimethylsilane or (1H,1H,2H, 2H-perfluorooctyl) trimethoxysilane;

the masking tape is then removed.

(2) Performing Chemical Vapor Deposition (CVD) treatment on the first solid substrate 101 and the second solid substrate 102 with fluorosilane; the fluoro silane is selected from trichloro (1H,1H,2H, 2H-perfluorooctyl) silane, monochloro (1H,1H,2H, 2H-perfluorooctyl) dimethylsilane or (1H,1H,2H, 2H-perfluorooctyl) trimethoxysilane;

shields are provided in the reaction chamber 105 regions of the first solid substrate 101 and the second solid substrate 102: attaching a mask (such as a PDMS flat sheet) to the region where the surface modification is desired to be preserved (the reaction chamber region), and performing plasma treatment;

the mask is then removed.

Step 330, the first solid substrate 101 and the second solid substrate 102 are assembled in an aligned manner, a support layer is adhered between the first solid substrate 101 and the second solid substrate 102, and the reaction chambers 105 of the first solid substrate 101 and the second solid substrate 102 are arranged corresponding to the glue spreading area 104. Specifically, double faced adhesive tape support columns 103 are attached to four corners of the second solid substrate, release paper on the support columns 103 is removed, and then the two substrates are assembled on the first solid substrate in an aligning manner (aligning according to specific chip design), so that it is required to ensure that the surfaces of the two substrates which are chemically modified in different areas are opposite, and the hydrophobic areas are aligned.

Step 340, glue filling is performed on the glue spreading area 104, and liquid glue is injected. Both manual and automatic methods may be employed.

(1) Manual glue pouring: the manual glue filling method comprises the steps of gluing by using a syringe, filling liquid glue (the liquid glue with lower viscosity needs to be selected, for example, the viscosity is not more than 1000cps) into the syringe, pasting a syringe needle in an included angle between the side face of the second solid substrate and the first solid substrate, enabling the tip of the syringe needle to be located between the second solid substrate and the first solid substrate, slowly pushing out the liquid glue and moving along the long edge, and paying attention to avoid the excessive overflow of the liquid glue to the upper surface of the second solid substrate. Glue was applied to the two long sides in sequence, about 20 microliters per side (this value was calculated from the specific support post height and glue area). And waiting for the liquid glue to spread to the whole glue spreading area.

(2) And (5) automatically pouring glue by using a glue dispenser.

Step 350, curing the liquid glue. If the ultraviolet glue is used, the ultraviolet lamp is used for curing.

Obtaining a gene sequencing chip through the above steps, as shown in fig. 1(d) and 2, 106 is a finished chip, 101 is a first solid substrate, 107 is a sample inlet or a sample outlet, 102 is a second solid substrate, 103 is a support column, 104 is a glue coating area, i.e., a hydrophilic area, on the second solid substrate, and in the complete chip, the glue coating area is a thin layer (e.g., 0.1mm thick) after curing; 105 is the reaction chamber area on the second solid substrate, i.e. the hydrophobic area, which is a cavity with a certain thickness (e.g. 0.1 mm) in the complete chip.

Compared with the method for etching the substrate, the method of the utility model reduces the production cost. The large area etching of the substrate, for example, the wet etching method, has higher processing cost. And the two solid substrates of the method can use flat sheets, which are the cheapest of all shapes, thereby further reducing the production cost.

In order to further explain the present invention, the following detailed description is made in conjunction with specific examples to describe the preparation method of the gene sequencing chip provided by the present invention, but it should be understood that the embodiment is implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, only for further explaining the features and advantages of the present invention, but not for limiting the claims of the present invention, and the scope of the present invention is not limited to the following embodiments.

In the case of the example 1, the following examples are given,

the chip structure is divided into 3 layers, from bottom to top, a first solid substrate 101, a glue coating region 104+ a reaction chamber 105+ a support column 103, and a second solid substrate 102, as shown in fig. 2.

The first solid substrate 101 is glass of size 25X75X1mm with two 1mm diameter wells 107; the second solid substrate 102 is a quartz plate with a micro-pit structure, and the size is 20 X45X0.7mm; the supporting column 103 is 4 double-sided adhesive tapes of 1X1mm, and the thickness is 0.1 mm; the glue is ultraviolet glue with certain low viscosity; the surface of the reaction chamber area is chemically modified by trichloro (1H,1H,2H, 2H-perfluorooctyl) silane. The production is carried out according to the steps of the chip production method in the content of the utility model.

1) A first solid substrate 101, a second solid substrate 102, and a support layer are prepared. The method comprises the steps of cleaning and drying a first solid substrate and a second solid substrate; and cutting the double-sided adhesive tape support column.

The first solid substrate and the second solid substrate were first ultrasonically cleaned with acetone for 5 minutes, then with isopropyl alcohol for 5 minutes, and then with ultrapure water for 5 minutes. Then sequentially using 10% NaOH/45% water/45% ethanol solution, 4% HCl/12% H2O 2/84% water solution and ultra-pure water for cleaning; then blowing the mixture by high-purity nitrogen.

The double-sided adhesive tape is cut into 1X1mm small pieces by using Nitto HJ 3160W.

2) And performing surface chemical modification on the first and second solid substrates in different regions. The second solid substrate glue coating area 104 is a hydrophilic area without surface chemical modification, and the reaction chamber area 105 is a hydrophobic area with surface chemical modification; the glue spreading area and the reaction chamber area of the first solid substrate should correspond to the same position as the second solid substrate. The chamber area is designed to include fluid ports 107 on the substrate.

Two methods are available:

in the region where surface chemical modification is not required (the gumming region 104), masking paste (for example, acrylic nitto HJ3160W) is applied, CVD (chemical vapor deposition) is performed on the masking paste, and then trichlorosilane (for example, trichloro (1H, 2H-perfluorooctyl) silane is applied, treated, and then the masking paste is removed.

Alternatively, the whole wafer is chemically modified chemically, treated with CVD (chemical vapor deposition) fluorosilane (e.g., trichloro (1H, 2H-perfluorooctyl) silane), then masked (e.g., PDMS slab) in the area where surface modification is desired to be retained (reaction chamber area), subjected to air plasma treatment, e.g., for 10 to 400 seconds, and then the mask is removed.

3) The first solid substrate 101 and the second solid substrate 102 are assembled in alignment. The double-sided adhesive support posts 103 are attached to four corners of the second solid substrate 102, the release paper on the support posts 103 is removed, and then the two substrates are assembled on the first solid substrate 101 in an aligned manner (aligned according to specific chip design), so that it is required to ensure that the surfaces of the two substrates modified in different areas are opposite, and the hydrophilic area is aligned with the hydrophobic area.

4) And (5) pouring glue in the glue spreading area. Both manual and automatic methods may be employed.

The manual method is to glue with a syringe, pour liquid glue (with a lower viscosity, for example, not more than 1000cps) into the syringe, attach the syringe needle to the included angle between the side surface of the second solid substrate and the first solid substrate, and move along the long edge while slowly pushing out the glue, so as to avoid the glue from overflowing to the upper surface of the second solid substrate. Glue was applied to the two long sides in sequence, approximately 30 microliters per side (this value was calculated from the specific support post height and glue area). And waiting for the glue to spread to the whole gluing area.

Or automatic glue pouring is adopted, and a glue dispenser can be adopted for automatic glue pouring.

5) And (6) curing the glue. If the ultraviolet glue is used, the ultraviolet lamp is used for curing.

The method does not need to arrange an isolation strip and etch the substrate, so that the obtained gene sequencing chip can increase the available reaction chamber area and reduce the cost.

It is to be understood that the above-described embodiments of the present invention are merely illustrative of or explaining the principles of the invention and are not to be construed as limiting the invention. Therefore, any modification, equivalent replacement, improvement and the like made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. Further, it is intended that the appended claims cover all such variations and modifications as fall within the scope and boundaries of the appended claims or the equivalents of such scope and boundaries.

Claims

1. A gene sequencing chip, comprising: the device comprises a first solid substrate, a second solid substrate, a glue coating area and a reaction chamber, wherein the glue coating area and the reaction chamber are positioned between the first solid substrate and the second solid substrate;

a fluid inlet and a fluid outlet are arranged on the first solid substrate;

2. The gene sequencing chip of claim 1, wherein the glue coating area is filled with solidified liquid glue.

3. The gene sequencing chip of claim 2, wherein the viscosity of the liquid gel is less than 1000 cps.

4. The gene sequencing chip of claim 1, wherein the fluid inlet and the fluid outlet correspond to a region where the reaction chamber is located.

5. The gene sequencing chip of claim 1, wherein the support layer comprises a plurality of support posts for supporting and defining a distance between the first solid substrate and the second solid substrate.

6. The gene sequencing chip of any one of claims 1 to 5, wherein the first solid substrate and the second solid substrate are transparent solid flat sheets, preferably selected from any one of glass, quartz or organic high molecular polymer materials.

7. The gene sequencing chip of any one of claims 1 to 5, wherein the support layer is selected from any one of double-sided tape, PI material and PET material.