CN112195227A

CN112195227A - Preparation method of gene sequencing substrate

Info

Publication number: CN112195227A
Application number: CN202010984826.1A
Authority: CN
Inventors: 李文涛; 陈子天; 段海峰
Original assignee: Sina Biotechnology Beijing Co ltd
Current assignee: Sina Biotechnology Beijing Co ltd
Priority date: 2020-09-18
Filing date: 2020-09-18
Publication date: 2021-01-08

Abstract

The invention provides a preparation method of a gene sequencing substrate, which can eliminate the fluorescence of nano-imprinting glue on glass, so that a micro-pit structure prepared by ultraviolet nano-imprinting glue can be applied to a gene sequencing chip. The ultraviolet light source is utilized to irradiate the thin layer of the nano imprinting glue on the glass for a long time, so that the fluorescence of the thin layer of the nano imprinting glue can be eliminated. The method is simple, and the nano-imprint glue cannot be damaged.

Description

Preparation method of gene sequencing substrate

Technical Field

The invention relates to a preparation method of a gene sequencing chip, belonging to the field of gene sequencing.

Background

High throughput sequencers are a technology that has developed at a rapid pace in recent years. Compared with the traditional Sanger sequencing, the high-throughput sequencing has the greatest advantage that massive sequence information can be read simultaneously. Although the accuracy is not as good as that of the traditional sequencing method, information beyond the sequence itself, such as gene expression amount and copy number variation, can be obtained due to massive data analysis. Currently, the mainstream sequencers use the SBS (sequencing by synthesis sequencing) method, such as solexa/illumina, 454, iontorent, etc. These sequencers are structurally similar and include fluidic systems, optical systems, and chip systems. The sequencing reaction takes place on-chip. The sequencing process is also similar, and comprises the following steps: and introducing the reaction liquid into the chip to perform SBS reaction, collecting signals, washing and performing the next round. The general sequencing chip includes a micro-reaction cell, mainly including quartz and the like as a substrate. The nanoimprint technology is a technology for transferring a micro-nano structure on a template to a material to be processed by assistance of photoresist. This technique is expected to replace existing lithography techniques. The nano-imprinting technology has low cost, high precision and good repeatability. The invention provides a method for preparing organic matter micro-pits on glass by utilizing nano-imprinting, and when the micro-pits are used for gene sequencing, the influence of original fluorescence on sequencing reaction can be eliminated through certain treatment.

Disclosure of Invention

The invention discloses a preparation method of a gene sequencing substrate, which comprises the following steps:

gluing a substrate, and preparing a microstructure substrate by using a soft stamping method;

the prepared microstructured substrate is placed under an illumination device,

the irradiation equipment comprises an LED light source and a Fresnel lens;

irradiating the substrate with an irradiation device;

wherein the wavelength of the LED light source is 300-600 nm.

According to a preferred embodiment, the microstructures refer to micro-pits arranged in an array.

According to a preferred embodiment, the period of the microstructure is between 1 and 10 microns, preferably between 1.2 and 5 microns.

According to a preferred embodiment, said gluing on the substrate refers to spin-coating of an ultraviolet nanoimprint glue.

According to a preferred embodiment, the substrate is one of glass, quartz, sapphire glass, a silicon wafer, a metal plate.

According to a preferred embodiment, the substrate is placed under an illumination device, and the light of the LED light source is illuminated through the fresnel lens onto the substrate; the irradiation time of the substrate is 1 to 8 hours, preferably 2 to 6 hours.

According to a preferred embodiment, the substrate is packaged to form a gene sequencing chip.

According to a preferred embodiment, after the substrate is irradiated, the emission of the coating material at 450-650nm is significantly reduced, preferably 480-600nm, wherein the significant reduction means that the emission light intensity is reduced by more than 50%, preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%.

The invention discloses a gene sequencing method, which is characterized in that,

sequencing by using a gene sequencing chip; the sequencing chip has a fluid outlet, a fluid inlet, and a fluid chamber;

at least one surface of the fluid chamber is provided with a micro pit prepared by a nano-imprinting method in advance;

the gene sequencing chip is irradiated by light with the wavelength of 300-600nm, preferably 400-550 nm.

The power of the LED is 10-500W, preferably 50-100W.

A device for removing fluorescence of a gene sequencing substrate is characterized by comprising a substrate, a platform, a light source and a Fresnel lens; wherein the substrate is a component of a gene sequencing chip; the substrate is a microstructure substrate prepared by a soft imprinting method; the surface of the substrate is provided with ultraviolet nano-imprinting glue; the light source is an LED light source; the light of the LED light source irradiates the substrate through the Fresnel lens; the substrate is placed on a stage.

According to a preferred embodiment, the platform is rotating.

According to a preferred embodiment, the wavelength of the light source is 300-600 nm.

According to a preferred embodiment, the nanoimprint paste has a thickness of 0.5 to 5 microns.

The principle of the method is that a high-power LED light source is used for uniformly irradiating the surface of a chip to obtain a low-fluorescence substrate. In a general nanoimprint method, UV glue or hot-press glue is the main nanoimprint glue. UV glue or hot-pressed glue generally have a very strong fluorescence in a specific wavelength band. This is unacceptable for sequencing reactions. It is therefore necessary to remove the fluorescence from the surface of the substrate. The method is simple, does not damage the nanoimprint glue, can remove over 90% of fluorescence of the nanoimprint glue, and can meet the requirement of gene sequencing.

All words referred to in this application are commonly understood in the art and for the sake of clarity, the words referred to in this application are explained in further detail below.

The substrates of the present invention are commonly used in the art. The method refers to a transparent or opaque flat substrate such as a glass sheet, a silicon wafer, a quartz sheet and the like which is directly used for experiments in biochemical experiments, or a flat substrate for forming a sealed chip after packaging. On the substrate, a selective partial area can be formed as a functional area by micromachining or other means.

The low fluorescence or no fluorescence described in the present invention is a relative concept. However, it is obvious to those skilled in the art that the conventional ultraviolet glue and the like have obvious fluorescence, such as US8 and the like, and when irradiated by an ultraviolet light source, the fluorescent glue emits obvious light, and partial biochemical experiments cannot be used. Low fluorescence is also a relative term. Even quartz with very high purity (above 99.99%) is observed by sensitive fluorescence microscopy, but quartz slides do not affect the observation of certain biochemical experiments due to fluorescence. Except where specifically stated, all terms used in the present application are to be interpreted as commonly understood in the art.

Drawings

FIG. 1 is a schematic view of the apparatus.

Detailed Description

To further illustrate the core of the present invention, the following examples are given as illustrations of the invention. The examples are intended to further illustrate the summary of the invention and are not intended to limit the invention.

The nano-imprinting technology, as a next-generation lithography technology, has the advantages of high resolution, high yield, low cost and the like. Different from the traditional photoetching technology, the nanometer stamping technology directly adopts a mechanical stamping method to transfer the micro-nano graph instead of adopting light to form the photoresist. Common nanoimprint techniques can be classified into thermal imprint (HEL) and ultraviolet imprint (UV-NIL) depending on materials and processes. Thermoplastic nanoimprinting (i.e., thermal imprinting) specifically uses a thermoplastic material whose pattern is transferred to a substrate by pressure when the thermoplastic material is heated above its glass transition temperature. Ultraviolet imprinting uses ultraviolet light to irradiate a template, so that photoresist filled in gaps of the template is solidified, and thus patterns on the template are transferred onto a substrate. The research of the ultraviolet nano-imprinting glue focuses on the aspects of molding, separation, etching and the like, and few people research the fluorescence property of the imprinting glue. Firstly, this fluorescence property is not of interest to many people, since it is weak, typically only below 50% of photoresists, such as SU8, but in special application areas this level of fluorescence property is still strongly affected. On the other hand, nanoimprint resist is often used as an intermediate, for example, for etching, and direct application is limited because the material is specific. Ultraviolet printing paste has many different properties from hot printing paste, for example, it can be made into smaller patterns and filled better. In summary, there is relatively little research in the field of direct use of patterns made of uv nanoimprint resist.

The invention relates to a biochemical chip, in particular to a gene sequencing chip. The applicant previously filed several patents on sequencing methods or techniques, such as CN201510155218.9, CN201510212788.7, CN201510212789.1, CN201510822361.9, cn201510815685.x, CN201510944878.5, cn201610899880.x, etc., which are multi-base sequencing methods belonging to fluorescence switching. The contents of the above patents may be incorporated by reference into this patent, where necessary. The applicant previously filed a series of patents on sequencing chips, such as CN2017105741742, CN2017105741441, 201910156547.3, the contents of which may be incorporated herein by reference, if necessary.

In the process of preparing the gene sequencing chip, the substrate belongs to an important component of the sequencing chip. The substrate in combination with other packaging materials may form a complete chip. For example, in 2017105741441, the

serial number

102 or 101 in picture 2 may correspond to the location of the substrate. The specific packaging method of the chip is described in the above patent, and is not described herein. It is contemplated that a two-layer chip may be formed using the substrate of the present invention with another glass sheet or an opaque metal or non-metal sheet. It should be noted that, the gene sequencing chip requires optical signal transmission due to the conditions of its use, and therefore, at least one of its packaging surfaces is transparent.

In gene detection, high-throughput sequencing is generally referred to as second-generation sequencing. In most of the second generation sequencing, a substrate with a micro-pit structure is used. The applicant has specifically described in previous patents. Typical micro-pits refer to an array of highly dense structures. Typically, the diameter of the micropits is from 0.2 to 5 microns, preferably from 0.3 to 2 microns. The period of the micro-pits is 0.5-10 microns, preferably 0.6-4 microns. The diameter to depth ratio of the micro-pits is 0.3 to 3, preferably 0.5 to 2.

The nanoimprint technology belongs to the conventional technology, and generally comprises the steps of providing a substrate, and forming a polymer layer on the surface of the substrate; and (3) enabling the polymer layer to be in a softened state, embossing the side of the polymer layer away from the substrate, heating and/or ultraviolet exposing and hardening, and demolding and molding. The nano-imprint glue is various, and mainly comprises a UV type and a heat-sensitive type. However, the conventional nanoimprint glue has certain fluorescence under extreme test conditions. In general applications, the fluorescence is not affected, but in the fields of gene sequencing and the like, the weak fluorescence directly causes higher background fluorescence, and affects the sequencing result.

The invention relates to a gene sequencing chip prepared from ultraviolet nano imprinting glue. In particular to a second generation gene sequencing chip. Second generation gene sequencing is high throughput gene sequencing, requiring dense data points within a small chip area. The data points may be generally spaced apart using micro-pits. Therefore, nanoimprint preparation of gene sequencing chips is an ideal field. In the hot stamping, there are many problems. Gene sequencing chips typically have a micropit size in the range of 0.1-5 microns, preferably 0.2-3 microns. That is, there are dense very small-sized micro-pits on the chip. At this size level, uv glue nanoimprint technology performs better, while thermal nanoimprint is more difficult. Such small dimensions and high pattern density present significant challenges in both mold release and pattern integrity during hot embossing.

It has been described above that the UV nanoimprint resist, due to its intrinsic characteristics, performs strongly in the absorption/emission range of 300-650 nm. That is, on the substrate, the micro-pit pattern directly prepared by the nanoimprint paste is not easy to be directly used, and has a great influence on the signal of gene sequencing. Therefore, the method of removing such signals is a significant matter.

In general, fluorescence of quartz substrates (up to 99.9% purity) is not considered to affect the sequencing process. The nanoimprint resist is the largest additional influence in the overall system.

The invention provides a method for removing fluorescence of nanoimprint glue by using a high-power UV lamp. Collimated light is generated using a uniform ultraviolet lamp and directed at the surface of the substrate. Under the condition of reaching a certain irradiation time, the fluorescent background of the nano imprinting glue on the surface of the substrate can be removed. Typically, large area, high power UV lamps are used as the light source. Collimated light can be generated using a fresnel lens and the light intensity distribution adjusted. The background fluorescence of the nanoimprint glue can be removed by using the combination of the UV lamp and the Fresnel lens and irradiating for a long time.

Typically, the nanoimprint resist is spin coated on the substrate and then imprinted. The substrate may be transparent or opaque. Such as glass, quartz, silicon wafers, BF33 glass, and the like. The thickness of the substrate is preferably 0.1 to 1 mm.

Fresnel lenses (Fresnel lenses), also known as screw lenses, are mostly sheets of polyolefin material that are injection-molded, and are also made of glass, one surface of the lens is smooth, and the other surface is inscribed with concentric circles from small to large, and the texture of the lens is designed according to the requirements of light interference and interference, relative sensitivity and receiving angle. With a fresnel lens a relatively uniform light field can be formed on the other side of the light source.

Other techniques for removing fluorescence may disrupt the structure of the nanoimprint paste. Experimentally, treatment of the nanoimprint resist with 84 results in severe deformation damage to the microstructure and even peeling. Other organic substances, such as acetone, do not achieve the goal of removing fluorescence. Furthermore, chemical solvents such as acetone may also have an effect on the nanoimprint resist, which may be less severe when the pattern is larger than, for example, 100 microns, and may have a significant effect on smaller patterns. Other possible experiments have been tried, including sunlight exposure, and simple results show that the long-term exposure to sunlight can reduce the fluorescence of nanoimprint resist, but there are many effects, firstly, the time is extremely long, which requires 1-6 months, the surface structure of the substrate after the second treatment is easy to crack, and the fluorescence can not be reduced to 80% or even 90%.

The nano-imprinting glue is divided into various types, such as general hot-imprinting glue, ultraviolet-imprinting glue, composite nano-imprinting glue and the like. Generally, the ultraviolet nano-imprint glue has wider application and mild condition, and can be used for making smaller patterns. The imprinting of the invention refers to the adoption of ultraviolet nano-imprinting glue. The ultraviolet nanoimprint resist may be classified into an acrylic type imprint resist, an epoxy type imprint resist, a vinyl ether type imprint resist, and the like. However, it was confirmed that the uv nanoimprint paste had a problem that fluorescent substance remained after imprinting. The method for removing the fluorescence in the prepared pattern by light irradiation is simple, low in cost and easy to operate.

In general biochemical experiments, such as gene sequencing, the requirements for the fluorescence background of the chip are high. For example, in the case of observation of fluorescent stains, the background fluorescence may seriously affect the accuracy of observation. For example, in gene sequencing, since the signal intensity is usually very weak, the intensity of the background signal is a very important index, which may lead to inaccurate fluorescent sequencing. After the treatment by the method, the sequencing reaction is not influenced.

It can be understood that in the embodiment 1 of the present invention, the fluorescence background intensity of the quartz is 500-1000. The background intensity of quartz is also a result of comparison, and different detection conditions correspond to different background intensities, and in the present invention, the value related to the fluorescence background intensity is determined substantially according to the unified experimental conditions. Even if a general chip is made of low-fluorescence glass or quartz, the fluorescence background is increased due to the existence of multiple structures or micro-machined patterns. It will be appreciated that background intensities typically 2-3 times lower than quartz intensities are satisfactory for most biochemical detection conditions. The general silica glass satisfies the foregoing description, and the silica purity should be 99.99% or more. Except for high-purity quartz, the fluorescence background values of other glasses are not uniform, and laboratory detection results show that the possible background value of the common float glass is about 1.5 to 5 times higher than that of the quartz; in the case of a large number range, the requirement of general sequencing is met, but the potential interference is certain, the fluorescence background is lower, and the comprehensive consideration of the cost is the preferable scheme for industrialization.

The power of the LED light source is selectable. Selected from 10-500W, preferably 50-100W. Of course this condition can be varied, it being understood that at lower power the illumination is longer and at higher power the illumination is shorter. The exposure dose is not simply in a straight-line proportional relationship with the treatment time, but the deviation is not significant.

The LED light source consists of a plurality of lamp beads. The power of the single lamp bead is selected from 1-500W, preferably 1-10W.

The irradiation time of the individual substrates is from 1 to 8 hours, preferably from 2 to 6 hours.

When a single substrate is irradiated, it is preferable to change the relative positions of the substrate and the light source in consideration of the unevenness of the light field caused by the unevenness of the light source. The substrate is held at a constant distance from the light source and the effect of the optical field non-uniformity can be rotated or otherwise eliminated.

The glue coated side of the substrate faces the direction of the LED light source. Overall, if quartz is used as the substrate, the blocking of light for many bands is small and the nanoimprint resist can also be processed through the substrate material.

Example 1

An LED group with TAYLAN actual power of 50W is selected as a light source. Select Shenzhen city meiying science and technology screw thread Fresnel lens for use, diameter 255mm, focus 1000 mm. And respectively fixing the Fresnel lens and the light source on a pre-built platform. And (3) placing the nanoimprinted substrate on a platform, starting a light source, irradiating for 120min, recording the fluorescence value, then irradiating for 120min, and recording the fluorescence value. See fig. 1. 101 is an LED light source, 102 is a nanoimprinted substrate, 103 is a platform, and 104 is a fresnel lens.

A quartz substrate was used as a control. A488 nm light source is used as exciting light, and 532 nm is used as central value to absorb light. Under the same conditions, the fluorescence background value of quartz was recorded as 500. The fluorescence background of the untreated substrate with nanoimprint resist ranged from about 23000 to about 30000. There will be some difference in the fluorescence background of the different substrates. The test treated 120min substrates and found a reduction in fluorescence, approximately 3600-6000. After 120min treatment, the fluorescence value is approximately in the range of 1200 and 2500. The analysis shows that the result is an average result of 50 slices, and although the interval value of the result is still larger, the descending trend or descending proportion is more obvious. The better results show that the fluorescence background is reduced to about 95%, and the average value is more than 90%.

Compared with a quartz plate, the fluorescence value is slightly higher, however, it can be known that the surface of the nanoimprint plate is provided with a microstructure, so that certain fluorescence rise is caused during measurement, and the nanoimprint plate can be applied to basically all known biochemical experiments. In addition, the intensity of this fluorescence value substantially reaches the detection limit of most devices. In this embodiment, a plurality of batches of nano-imprinting sheets are tested, all of which show the same range, and the difference between the nano-imprinting sheets is relatively large. Depending on the results of the sequencing experiment, this fluorescent background can be used under the sequencing conditions. It can be known that it is meaningless to simply provide the fluorescence background value, and the detection conditions of all fluorescence background values in the invention are consistent, so that the accuracy of the relative intensity measurement is ensured.

Example 2

The treatment method described in example 1. After the two treatments, the nanoimprint sheet was placed on the platen again for 240 min. As a result, it was found that the fluorescence background of the nanoimprint sheet was not significantly reduced. The invention adopts uniform conditions for all fluorescence value measurement.

Example 3

The nano-imprinting sheet prepared by SU8 had a micro-structure of micro-pits with a size of 5 microns in diameter and 5 microns in height. Under the conditions of light irradiation described in example 1, the fluorescence value after the treatment was 1500. Meets the requirements of common biochemical experiments.

Example 4

The nanoimprint sheet described in example 3, treated with acetone, was not effective. Treatment with 84 sterilizing liquid is substantially ineffective.

The specific examples given in this detailed description of the invention are intended to be illustrative of the invention and are not intended to be limiting.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A method for preparing a gene sequencing substrate comprises the following steps: gluing a substrate, and preparing a microstructure substrate by using a soft stamping method; placing the prepared substrate with the microstructure below irradiation equipment; irradiating the substrate with an irradiation device; the irradiation equipment comprises an LED light source and a Fresnel lens; the wavelength of the LED light source is 300-600 nm.

2. The method of claim 1, wherein the microstructures are micro-pits arranged in an array.

3. Method according to any of claims 1-2, wherein the period of the microstructure is 1-10 microns, preferably 1.2-5 microns.

4. The method according to any of claims 1-2, wherein the coating of the paste on the substrate is a spin-on uv nanoimprint paste.

5. The method according to any one of claims 1 to 4, wherein the substrate is one of glass, quartz, sapphire glass, a silicon wafer, and a metal plate.

6. The method of claim 5, wherein the substrate is placed under an illumination device, and light from the LED light source is illuminated onto the substrate through a fresnel lens; the irradiation time of the substrate is 1 to 8 hours, preferably 2 to 6 hours.

7. The method according to any one of claims 1 to 6, wherein after the substrate is irradiated, the coating material exhibits a significant reduction in the emitted light at 450-650nm, preferably at 480-600nm, said significant reduction being a reduction in the emitted light intensity of more than 50%, preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%.

8. A gene sequencing chip is characterized in that a reaction chamber is manufactured by utilizing a substrate; at least one surface of the fluid chamber is provided with a micro pit prepared by a nano-imprinting method in advance; the gene sequencing chip is irradiated by light with the wavelength of 300-600nm, preferably 400-550 nm.

9. A device for removing fluorescence of a gene sequencing substrate is characterized by comprising a substrate, a platform, a light source and a Fresnel lens; wherein the substrate is a component of a gene sequencing chip; the substrate is a microstructure substrate prepared by a soft imprinting method; the surface of the substrate is provided with ultraviolet nano-imprinting glue; the light source is an LED light source; the light of the LED light source irradiates the substrate through the Fresnel lens; the substrate is placed on a stage. .

10. The apparatus as claimed in claim 9, wherein the light source has a wavelength of 300-600 nm.