A method for preparing a gene sequencing substrate

Abstract

The present invention provides a method for preparing a gene sequencing substrate, which can eliminate the fluorescence of nano-imprint glue on glass, so that a micro-pit structure prepared by ultraviolet nano-imprint glue can be applied to a gene sequencing chip. The fluorescence of the thin layer of nano-imprint glue on the glass can be eliminated by irradiating the thin layer of nano-imprint glue on the glass with an ultraviolet light source for a long time. The method is simple and does not cause damage to the nano-imprint glue.

Description

Technical Field

[0001] The invention relates to a method for preparing a gene sequencing chip, and belongs to the field of gene sequencing. Background Art

[0002] High-throughput sequencers are a technology that has developed rapidly in recent years. Compared with traditional Sanger sequencing, the biggest advantage of high-throughput sequencing is that it can read a large amount of sequence information at the same time. Although the accuracy is not as good as traditional sequencing methods, due to the analysis of massive data, information beyond the sequence itself, such as gene expression and copy number variation, can be obtained. Today's mainstream sequencers all use the SBS (sequencing by synthesis) method, such as Solexa / Illumina, 454, iontorrent, etc. The structures of these sequencers are similar, including fluid systems, optical systems, and chip systems. The sequencing reaction occurs in the chip. The sequencing process is also very similar, including: passing the reaction solution into the chip, SBS reaction, signal collection, washing, and the next round. General sequencing chips include micro-reaction pools, mainly quartz and other materials as substrates. Nanoimprint technology is a technology that transfers the micro-nano structure on the template to the material to be processed with the assistance of photoresist. This technology is expected to replace the existing photolithography technology. Nanoimprint technology has low cost, high precision and good repeatability. The present invention provides a method for preparing organic micro-pits on glass by nano-imprinting. When the micro-pits are used for gene sequencing, the influence of the original fluorescence on the sequencing reaction can be eliminated through certain treatment. Summary of the invention

[0003] The present invention discloses a method for preparing a gene sequencing substrate, comprising the following steps:

[0004] Applying glue on the substrate, and then preparing the microstructure substrate by using a soft embossing method;

[0005] Place the prepared microstructured substrate under the irradiation device.

[0006] The irradiation equipment includes an LED light source and a Fresnel lens;

[0007] irradiating the substrate with an irradiation device;

[0008] The wavelength of the LED light source is 300-600nm.

[0009] According to a preferred embodiment, the microstructure refers to micro-pits arranged in an array.

[0010] According to a preferred embodiment, the period of the microstructure is 1-10 microns, preferably 1.2-5 microns.

[0011] According to a preferred embodiment, the coating of glue on the substrate refers to spin coating of ultraviolet nanoimprint glue.

[0012] According to a preferred embodiment, the substrate is one of glass, quartz, sapphire glass, silicon wafer, and metal plate.

[0013] According to a preferred embodiment, the substrate is placed under the irradiation device, and the light from the LED light source is irradiated onto the substrate through the Fresnel lens; the irradiation time of the substrate is 1-8 hours, preferably 2-6 hours.

[0014] According to a preferred embodiment, the substrate is packaged to form a gene sequencing chip.

[0015] According to a preferred embodiment, after the substrate is irradiated, the emission light of the coating material at 450-650nm is significantly reduced, preferably the emission light at 480-600nm is significantly reduced, and the significant reduction refers to a reduction of the emission light intensity by more than 50%, preferably more than 60%, more preferably more than 70%, more preferably more than 80%, and more preferably more than 90%.

[0016] The present invention discloses a gene sequencing method, characterized in that:

[0017] Sequencing is performed using a gene sequencing chip; the sequencing chip has a fluid outlet, a fluid inlet, and a fluid chamber;

[0018] At least one surface of the fluid chamber has micro-pits prepared in advance by a nano-imprinting method;

[0019] The gene sequencing chip is irradiated with light having a wavelength of 300-600 nm, preferably 400-550 nm.

[0020] The power of the LED is 10-500W, preferably 50-100W.

[0021] A device for removing fluorescence from a gene sequencing substrate, characterized in that it comprises 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 embossing method; the surface of the substrate has an ultraviolet nano-imprinting glue; the light source is an LED light source; the light from the LED light source is irradiated onto the substrate through the Fresnel lens; and the substrate is placed on the platform.

[0022] According to a preferred embodiment, the platform is rotating.

[0023] According to a preferred embodiment, the wavelength of the light source is 300-600 nm.

[0024] According to a preferred embodiment, the thickness of the nanoimprint glue is 0.5-5 microns.

[0025] The principle of this method is to use a high-power LED light source to evenly illuminate the surface of the sheet to obtain a low-fluorescence substrate. In general nanoimprint methods, UV glue or hot-pressed glue is the main nanoimprint glue. UV glue or hot-pressed glue generally has very strong fluorescence in a specific band. This is unacceptable for sequencing reactions. Therefore, it is necessary to remove the fluorescence on the surface of the substrate. The method of the present invention is simple, will not cause damage to the nanoimprint glue, and can remove more than 90% of the fluorescence of the nanoimprint glue, which can meet the needs of gene sequencing.

[0026] The terms involved in the present invention are all commonly used in the art. For further clarity, the terms used in the present invention are further explained below.

[0027] The substrate described in the present invention is a common usage in the art. It refers to a transparent or opaque flat substrate such as a glass sheet, silicon wafer, quartz sheet, etc. directly used in biochemical experiments, or a flat substrate that forms a sealed chip after packaging. Part of the substrate can be selectively patterned as a functional area through micromachining or other means.

[0028] 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 common UV glue and other materials have obvious fluorescence, such as US8, etc., which will emit obvious light when irradiated with ultraviolet light, and will cause some biochemical experiments to be unusable. Low fluorescence is also a relative concept. Even quartz with very high purity (more than 99.99%) can be observed by a sensitive fluorescence microscope, but quartz sheets will not affect the observation of certain biochemical experiments due to fluorescence. Unless otherwise specified, the words involved in the present invention are general terms in the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 .Diagram of the device. DETAILED DESCRIPTION

[0030] In order to further illustrate the core content of the present invention, the present invention is now illustrated by the following examples. The examples are for further explaining the invention content and do not limit the present invention.

[0031] As a next-generation lithography technology, nanoimprint technology has the advantages of high resolution, high yield, and low cost. Unlike traditional lithography, nanoimprint technology directly uses mechanical imprinting to transfer micro-nano patterns instead of using light to shape photoresist. According to different materials and processes, commonly used nanoimprint technologies can be divided into hot embossing (HEL) and ultraviolet embossing (UV-NIL). Thermoplastic nanoimprinting (i.e. hot embossing) specifically uses thermoplastic materials. When the thermoplastic material is heated to above the glass transition temperature, the pattern of the template is transferred to the substrate through pressure. UV imprinting uses ultraviolet light to irradiate the template to solidify the photoresist filling the template gaps, thereby transferring the pattern on the template to the substrate. The research focus of ultraviolet nanoimprint glue is on molding, detachment, etching, etc., and few people study the fluorescence properties of the imprint glue itself. First of all, this fluorescence property is not paid attention to by many people because it is relatively weak. Generally speaking, it is only less than 50% of photoresist such as SU8, but in special application fields, this level of fluorescence performance still has a great influence. On the other hand, nanoimprint adhesive is often used as an intermediate medium, such as for etching, and its direct application is limited because its material is relatively special. Many properties of UV imprint adhesive are different from those of hot stamp adhesive, such as being able to make smaller graphics and fill better. Overall, there is relatively little research in the field of direct use of patterns made with UV nanoimprint adhesive.

[0032] The present invention relates to a biochemical chip, more specifically a gene sequencing chip. The applicant has previously applied for some patents on sequencing methods or technologies, such as CN201510155218.9, CN201510212788.7, CN201510212789.1, CN201510822361.9, CN201510815685.X, CN201510944878.5, CN201610899880.X, etc. These patents belong to fluorescent switching multi-base sequencing methods. When necessary, the contents of the above patents can be incorporated into this patent by reference. The applicant has previously applied for a series of patents on sequencing chips, such as CN2017105741742, CN2017105741441, 201910156547.3. When necessary, the contents of the above patents can be incorporated into this patent by reference.

[0033] In the process of preparing gene sequencing chips, the substrate is an important component of the sequencing chip. The substrate can be combined with other packaging materials to form a complete chip. For example, in 2017105741441, serial number 102 or 101 in picture 2 can correspond to the position of the substrate. The specific packaging method of the chip has been described in the previous patents and will not be repeated here. It is conceivable that the substrate described in the present invention is used to form a two-layer chip with another glass sheet or an opaque metal or non-metal sheet. It should be noted that the gene sequencing chip requires the conduction of optical signals due to its use conditions, so at least one of its packaging surfaces is transparent.

[0034] When performing genetic testing, high-throughput sequencing generally refers to second-generation sequencing. In most second-generation sequencing, a substrate with a micro-pit structure is required. This has been described in detail in the applicant's previous patents. Generally, micro-pits refer to highly dense structures arranged in an array. Generally, the diameter of the micro-pits is 0.2-5 microns, preferably 0.3-2 microns. The period of the micro-pits is 0.5-10 microns, preferably 0.6-4 microns. The ratio of the diameter to the depth of the micro-pits is 0.3-3, preferably 0.5-2.

[0035] Nanoimprint technology is a conventional technology, and its general steps include providing a substrate, forming a polymer layer on the surface of the substrate; making the polymer layer in a softened state, and imprinting the side of the polymer layer away from the substrate, heating and / or UV exposure hardening, and demolding. There are many kinds of nanoimprint glues, mainly UV type and heat-sensitive type. However, conventional nanoimprint glues will have a certain degree of fluorescence under extreme test conditions. In general applications, this fluorescence has no effect, but in fields such as gene sequencing, this weak fluorescence will directly lead to high background fluorescence, affecting the sequencing results.

[0036] The present invention relates to a gene sequencing chip prepared by ultraviolet nanoimprint glue. It particularly refers to a second-generation gene sequencing chip. Second-generation gene sequencing is high-throughput gene sequencing, which requires dense data points in a small-sized chip area. The data points can generally be separated by micropits. Therefore, nanoimprinting is an ideal field for preparing gene sequencing chips. There will be many problems during hot embossing. The general micropit size range of gene sequencing chips is 0.1-5 microns, preferably 0.2-3 microns. In other words, there are dense and very small micropits on the chip. At this size level, ultraviolet glue nanoimprinting technology has better performance, while hot embossing is more difficult. With such a small size and a very high graphic density, there are great challenges in demoulding and graphic integrity during hot embossing.

[0037] As described above, due to its own characteristics, UV nanoimprint glue has a strong performance in the absorption / emission range of 300-650nm. In other words, on the substrate, the micro-pit pattern prepared directly with nanoimprint glue is not easy to use directly, and will have a great impact on the signal of gene sequencing. Therefore, a method to remove this signal is very meaningful.

[0038] Generally speaking, the fluorescence of the quartz substrate (purity of more than 99.9%) can be considered to have no effect on the sequencing process. In the entire system, the nanoimprint glue has the greatest additional impact.

[0039] The present invention provides a method for removing the fluorescence of nano-imprint glue by using a high-power UV lamp. A uniform ultraviolet lamp is used to generate collimated light, which is directly irradiated on the surface of a substrate. When a certain irradiation time is reached, the fluorescent background of the nano-imprint glue on the surface of the substrate can be removed. Generally, a large-area, high-power UV lamp is used as a light source. A Fresnel lens can be used to generate collimated light and adjust the light intensity distribution. By using a combination of a UV lamp and a Fresnel lens, the background fluorescence of the nano-imprint glue can be removed by irradiating for a long time.

[0040] Generally, the nanoimprint glue is spin-coated on a substrate and then imprinted. The substrate can be transparent or opaque, such as glass, quartz, silicon wafer, BF33 glass, etc. The thickness of the substrate is preferably 0.1-1 mm.

[0041] Fresnel lens, also known as spiral lens, is mostly made of thin sheets of polyolefin material injection molding, and some are made of glass. One side of the lens surface is smooth, and the other side is engraved with concentric circles from small to large. Its texture is designed according to the interference and disturbance of light, as well as the relative sensitivity and receiving angle requirements. Using Fresnel lens, a relatively uniform light field can be formed on the other side of the light source.

[0042] Other techniques for removing fluorescence may damage the structure of the nanoimprint glue. Experimentally, treating the nanoimprint glue with 84 will cause serious deformation and damage to the microstructure, or even fall off. Other organic substances, such as acetone, cannot achieve the purpose of removing fluorescence. In addition, chemical solvents such as acetone can also affect the nanoimprint glue. When the pattern is larger than 100 microns, the impact may not be too serious, but smaller patterns may cause obvious effects. Other possible experiments have also been attempted, including irradiation with sunlight. Simple results show that long-term exposure to sunlight can reduce the fluorescence of the nanoimprint glue, but there will be many effects. First, the time is extremely long, requiring 1-6 months. Second, the surface structure of the substrate after treatment is prone to cracks, etc., and the reduction in fluorescence cannot reach 80% or even 90%.

[0043] Nanoimprint adhesives are divided into many types, including general hot-stamp adhesives, ultraviolet-stamp adhesives, composite nanoimprint adhesives, and the like. In general, ultraviolet nanoimprint adhesives are more widely used, with mild conditions, and can produce smaller graphics. The imprinting described in the present invention refers to the use of ultraviolet nanoimprint adhesives. Ultraviolet nanoimprint adhesives can be divided into acrylic-type imprint adhesives, epoxy-type imprint adhesives, vinyl ether-type imprint adhesives, and the like. However, it is certain that ultraviolet nanoimprint adhesives have the problem of residual fluorescent substances after imprinting. The present invention adopts a method of removing the fluorescence in the nano-prepared pattern by light irradiation, which is simple, low-cost, and easy to operate.

[0044] In general biochemical experiments, such as gene sequencing, the requirements for the fluorescence background of the chip are relatively high. For example, when observing fluorescence staining, the background fluorescence may seriously affect the accuracy of the observation. For example, in gene sequencing, since the general signal intensity is very weak, the intensity of the background signal is a very important indicator, which will lead to inaccurate fluorescence sequencing. After being treated by the method described in the present invention, the sequencing reaction will not be affected.

[0045] It can be understood that in Example 1 described in the present invention, the fluorescence background intensity of quartz is 500-1000. The background intensity of quartz is also a result of comparison. Different detection conditions correspond to different background intensities. In the present invention, the values ​​of the fluorescence background intensity are basically measured according to unified experimental conditions. Even if a general chip is made of low-fluorescence glass or quartz, the presence of multiple structures or micro-processing patterns will also lead to an increase in the fluorescence background. It can be understood that the general background intensity is 2-3 times lower than the quartz intensity to meet most of the biochemical detection conditions. General quartz glass meets the above description, and its quartz purity should be above 99.99%. In addition to high-purity quartz, the fluorescence background values ​​of other glasses are not uniform. The results of laboratory tests show that the possible background value of ordinary float glass is about 1.5-5 times higher than that of quartz; this value range is very large, meeting the needs of general sequencing, but it has potential interference, lower fluorescence background, and comprehensive consideration of cost is the preferred solution for industrialization.

[0046] The power of the LED light source can be selected. It is selected from 10-500W, preferably 50-100W. Of course, this condition can be changed. It is understandable that when the power is relatively low, the irradiation time is longer, and when the power is relatively high, the irradiation time is shorter. The irradiation amount and the treatment time are not in a simple linear proportional relationship, but the deviation is not obvious.

[0047] The LED light source is composed of multiple lamp beads. The power of a single lamp bead is selected from 1-500W, preferably 1-10W.

[0048] The irradiation time of a single substrate is 1-8 hours, preferably 2-6 hours.

[0049] When irradiating a single substrate, considering the uneven light field caused by the uneven light source, it is preferred to change the relative position of the substrate and the light source. The substrate is fixed at a constant distance from the light source, and the influence of the uneven light field can be eliminated by rotation or other methods.

[0050] The side of the substrate coated with glue faces the direction of the LED light source. Overall, if quartz is used as the substrate, the obstruction of light in many bands is relatively small, and the nanoimprint glue can also be processed through the substrate material.

[0051] Example 1

[0052] A TAYLAN LED group with an actual power of 50W was selected as the light source. A Shenzhen Meiying Technology threaded Fresnel lens with a diameter of 255mm and a focal length of 1000mm was selected. The Fresnel lens and the light source were fixed on the pre-built platform. The nanoimprinted substrate was placed on the platform, the light source was turned on, and the fluorescence value was recorded after irradiation for 120 minutes, and then irradiation for another 120 minutes, and the fluorescence value was recorded. Figure 1 101 is an LED light source, 102 is a nano-imprint substrate, 103 is a platform, and 104 is a Fresnel lens.

[0053] A quartz substrate was used as a control. A 488nm light source was used as the excitation light, and 532nm was used as the center value as the absorption light. Under the same conditions, the fluorescence background value of quartz was recorded as 500. The fluorescence background value of the substrate with nanoimprint glue that was not treated was about 23,000 to 30,000. There will be certain differences in the fluorescence background of different substrates. After testing the substrate treated for 120 minutes, it was found that its fluorescence value decreased to about 3,600-6,000. After another 120 minutes of treatment, its fluorescence value ranged from 1,200 to 2,500. Analysis shows that this result is the average result of 50 pieces. Although the interval value of the result is still relatively large, its downward trend or the proportion of decline is relatively obvious. The better results show that the reduction of the fluorescence background has reached about 95%, and the average value is also above 90%.

[0054] Compared with quartz sheets, this fluorescence value is slightly higher. However, it can be known that due to the microstructure on the surface of the nanoimprint sheet, a certain increase in fluorescence will occur during measurement, which can be applied to basically all known biochemical experiments. In addition, the intensity of this fluorescence value basically reaches the detection limit of most equipment. In this embodiment, multiple batches and multiple nanoimprint sheets were tested, all showing the same range, and the differences between the nanoimprint sheets are relatively large. According to the results of the sequencing experiment, this fluorescence background can be used under sequencing conditions. It can be known that it is meaningless to simply propose a fluorescence background value. The detection conditions of all fluorescence background values ​​in the present invention are consistent, which ensures the accuracy of the relative intensity measurement.

[0055] Example 2

[0056] According to the treatment method described in Example 1. After two treatments, the nanoimprint sheet was placed on the platform and treated for 240 minutes. It was found that the fluorescence background of the nanoimprint sheet was not significantly reduced. All fluorescence values ​​in the present invention were measured under uniform conditions.

[0057] Example 3

[0058] The nanoimprint sheet prepared by SU8 has a microstructure of micropits with a diameter of 5 microns and a height of 5 microns. Using the light irradiation conditions described in Example 1, it is found that the fluorescence value after treatment is 1500, which meets the requirements of common biochemical experiments.

[0059] Example 4

[0060] The nanoimprint sheet described in Example 3 was not effectively treated with acetone, and was basically ineffective with 84 disinfectant.

[0061] The specific embodiments in the specific implementation manner of the present invention are merely further explanations of the present invention and are not sufficient to constitute limiting factors of the present invention.

[0062] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing a gene sequencing substrate, comprising the following steps: applying glue on a substrate, and then preparing a microstructure substrate by a soft embossing method; placing the substrate with the prepared microstructure under an irradiation device; irradiating the substrate with the irradiation device; wherein the irradiation device comprises an LED light source and a Fresnel lens; and the wavelength of the LED light source is 300-600nm; in, The coating of the substrate with glue refers to spin coating of ultraviolet nanoimprint glue; the irradiation time of the substrate is 1-8 hours.

2. The method according to claim 1, It is characterized in that The microstructure refers to micropits arranged in an array.

3. The method according to any one of claims 1 to 2, It is characterized in that The period of the microstructure is 1-10 microns.

4. The method according to any one of claims 1 to 2, It is characterized in that The substrate is one of glass, quartz, sapphire glass, silicon wafer and metal plate.

5. The method according to claim 4, It is characterized in that The substrate is placed under the irradiation device, and the light from the LED light source is irradiated onto the substrate through the Fresnel lens; the irradiation time of the substrate is 2-6 hours.

6. The method according to any one of claims 1 to 2, It is characterized in that After the substrate is irradiated, the emission light of the coating material in the range of 450-650nm is significantly reduced; the significant reduction refers to a reduction of more than 50% in the intensity of the emission light.

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