Gene sequencing chip, gene sequencing device and gene sequencing method

Abstract

The present invention provides a gene sequencing chip, a gene sequencing device and a gene sequencing method. The gene sequencing chip comprises: a transparent first substrate (1); a second substrate (2) arranged opposite to the first substrate (1); a first electrode (3) and / or a second electrode (4), wherein the first electrode (3) is arranged on the first substrate (1) and is a transparent electrode, and the second electrode (4) is arranged on the second substrate (2); an electronic ink layer (5) arranged between the first substrate (1) and the second substrate (2); a microporous layer (6) arranged on a side of the second substrate (2) away from the first substrate (1), and micropores (7) are arranged on the microporous layer (6) at positions corresponding to the first electrode (3) or the second electrode (4). The gene sequencing chip in the present invention does not require any field effect transistor, has a simple manufacturing process, and can greatly reduce the manufacturing difficulty and cost.

Description

Technical Field

[0001] The present invention relates to the field of gene sequencing, and in particular to a gene sequencing chip, a gene sequencing device and a gene sequencing method. Background Art

[0002] Gene sequencing technology is the most commonly used technology in modern molecular biology research. Since the development of the first generation of gene sequencing in 1977, gene sequencing technology has made considerable progress, mainly including the first generation Sanger sequencing technology, the second generation high-throughput sequencing technology, the third generation single-molecule sequencing technology and the fourth generation nanopore sequencing technology. However, the mainstream sequencing technology in the market is still dominated by the second generation high-throughput sequencing.

[0003] The second generation of high-throughput sequencing technologies mainly include Illumina's sequencing by synthesis technology, ThermoFisher's ion semiconductor sequencing technology, ligation sequencing technology and Roche's pyrophosphate sequencing technology. Among them, Illumina's sequencing by synthesis and Thermo Fisher's ligation sequencing both require fluorescent labeling, laser light sources and optical systems. Although Roche's pyrophosphate sequencing does not have a laser light source and optical system, it also requires fluorescent labeling. The ion semiconductor sequencing method requires the use of CMOS technology to produce an ion sensor and two field effect transistors.

[0004] Since both Illumina's sequencing by synthesis and Thermo Fisher's ligation sequencing require fluorescent labeling, laser light sources and optical systems, sequencing becomes complicated, increasing sequencing time and cost. Ion semiconductor sequencing uses CMOS technology to make an ion sensor and two field-effect transistors, so the process is complicated and difficult to manufacture. Summary of the invention

[0005] Technical problem to be solved by the invention

[0006] In order to solve the above problems in the prior art, the present invention provides a gene sequencing chip, which does not require a laser light source and an optical system, nor does it require any field effect transistors, has a simple manufacturing process, and can greatly reduce the manufacturing difficulty and cost. The present invention also relates to a gene sequencing device including the gene sequencing chip.

[0007] In addition, the present invention also provides a gene sequencing method, which uses the gene sequencing chip of the present invention, does not require fluorescent labeling of deoxyribonucleotides, and can perform gene sequencing simply and conveniently.

[0008] Technical solution of the invention

[0009] The present invention provides a gene sequencing chip, comprising: a transparent first substrate; a second substrate, the second substrate is arranged opposite to the first substrate; a first electrode and / or a second electrode, wherein the first electrode is arranged on the first substrate and is a transparent electrode, and the second electrode is arranged on the second substrate; an electronic ink layer, the electronic ink layer is arranged between the first substrate and the second substrate; a microporous layer, the microporous layer is arranged on a side of the second substrate away from the first substrate, and micropores are arranged at positions corresponding to the first electrode or the second electrode on the microporous layer. The electronic ink layer includes a plurality of microcapsules, each of which includes positively charged white particles and negatively charged black particles.

[0010] Preferably, an ion-sensitive membrane is disposed on a side of the micropores close to the second substrate, and the ion-sensitive membrane can make the color change of the electronic ink layer more obvious.

[0011] Preferably, the ion sensitive membrane is made of silicon nitride, and the ion sensitive membrane made of silicon nitride is more sensitive to hydrogen ions.

[0012] Preferably, the first electrode is arranged on a side of the first substrate facing the second substrate, and / or the second electrode is arranged on a side of the second substrate facing the first substrate.

[0013] Preferably, both the first electrode and the second electrode are block electrodes, and projections of the first electrode and the second electrode on the first substrate or the second substrate overlap with each other.

[0014] Preferably, projections of the first electrode, the second electrode, and the microhole on the first substrate or the second substrate overlap with each other.

[0015] Preferably, the first electrode is a planar electrode covering the entire first substrate, and the second electrode is a block electrode;

[0016] Alternatively, the second electrode is a planar electrode covering the entire second substrate, and the first electrode is a block electrode, which helps to further reduce manufacturing difficulty and cost.

[0017] Preferably, a first signal lead for applying a voltage to the first electrode is disposed on the first substrate, and / or a second signal lead for applying a voltage to the second electrode is disposed on the second substrate.

[0018] Preferably, the first electrode is made of indium tin oxide (ITO), the second electrode, the first signal lead and the second signal lead are made of ITO, molybdenum, aluminum, copper, etc., and the microporous layer is made of silicon nitride or silicon oxide.

[0019] The present invention also provides a gene sequencing device, including a gene sequencing chip, the gene sequencing chip including: a first substrate; a second substrate, the second substrate is arranged opposite to the first substrate; an electronic ink layer, the electronic ink layer is arranged between the first substrate and the second substrate; a first electrode, the first electrode is arranged on the first substrate, or the first electrode is arranged on the second substrate; an insulating layer, the insulating layer is arranged on the side of the second substrate away from the first substrate, and a micropore is arranged on the insulating layer at a position corresponding to the first electrode. Wherein, the electronic ink layer includes a plurality of microcapsules, each of which includes positively charged white particles and negatively charged black particles.

[0020] The present invention also provides a gene sequencing method, which comprises the following steps:

[0021] Adding DNA microbeads containing DNA chains into the microwells for PCR amplification;

[0022] Applying a voltage to the first electrode and / or applying a voltage to the second electrode so that an electric field is formed between the first substrate and the second substrate, with the electric field direction pointing from the first substrate to the second substrate;

[0023] adding four deoxyribonucleoside triphosphates into the microwells in sequence, and detecting whether the color of the electronic ink layer on one side of the first substrate changes;

[0024] The base type on the DNA chain is determined according to the deoxyribonucleoside triphosphate added when the color of the electronic ink layer on one side of the first substrate changes.

[0025] Preferably, the deoxyribonucleoside triphosphate is a reversibly terminated deoxyribonucleoside triphosphate, and the gene sequencing method further comprises: washing away the reversibly terminated deoxyribonucleoside triphosphate added to the microwell, and adding a thiol reagent.

[0026] Advantageous Effects of the Invention

[0027] The gene sequencing chip of the present invention does not require a laser light source and an optical system, nor does it require any field effect transistors, and has a simple manufacturing process, which can greatly reduce the manufacturing difficulty and cost. The present invention also relates to a gene sequencing device including the gene sequencing chip.

[0028] The gene sequencing method of the present invention uses the gene sequencing chip of the present invention, does not require fluorescent labeling of deoxyribonucleotides, and can perform gene sequencing simply and conveniently. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A cross-sectional view of a gene sequencing chip of the present invention is shown, with the section line being Figure 2 and Figure 3 middle A-A' line;

[0030] Figure 2 Show Figure 1 A plan view of the first substrate of the gene sequencing chip;

[0031] Figure 3 Show Figure 1 A plan view of the second substrate of the gene sequencing chip;

[0032] Figure 4 Show Figure 1 A cross-sectional view of a gene sequencing chip during base pairing reactions;

[0033] Figure 5-1 Show Figure 1 The color of the electronic ink layer on one side of the first substrate when no base pairing reaction occurs in the gene sequencing chip;

[0034] Figure 5-2 Show Figure 1 The color of the electronic ink layer on one side of the first substrate when a base pairing reaction occurs in the gene sequencing chip.

[0035] Description of Reference Numerals

[0036] 1 first substrate, 2 second substrate, 3 first electrode, 4 second electrode, 5 electronic ink layer, 6 microporous layer, 7 micropores, 8 ion sensitive membrane, 90 microcapsules, 91 positively charged white particles, 92 negatively charged black particles, 10 first signal lead, 11 second signal lead DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0038] Unless otherwise defined, the technical or scientific terms used herein shall have the usual meanings understood by persons of ordinary skill in the field to which the invention belongs. The words "first", "second" and similar terms used in the patent application specification and claims of the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. Similarly, words such as "one" or "one" do not indicate a quantity limitation, but indicate the existence of at least one. Words such as "connect" or "connected" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the object being described changes, the relative positional relationship also changes accordingly.

[0039] Figure 1 A cross-sectional view of a gene sequencing chip of the present invention is shown, with the section line being Figure 2 and Figure 3 A-A' line. Figure 1 As shown, a gene sequencing chip of the present invention includes a transparent first substrate 1, a second substrate 2 is arranged opposite to the first substrate 1, a first electrode 3 is arranged on the first substrate 1, and the first electrode 3 is a transparent electrode, and the second electrode 4 is arranged on the second substrate 2. An electronic ink layer 5 is arranged between the first substrate 1 and the second substrate 2, and the electronic ink layer 5 includes a plurality of microcapsules 90, each microcapsule 90 includes positively charged white particles 91 and negatively charged black particles 92. A microporous layer 6 is arranged on the side of the second substrate 2 away from the first substrate 1, and micropores 7 are arranged at positions corresponding to the first electrode 3 or the second electrode 4 in the microporous layer 6. The projections of the first electrode 3, the second electrode 4 and the micropore 7 on the first substrate 1 or the second substrate 2 overlap with each other.

[0040] An ion-sensitive membrane 8 is provided on one side of the micropore 7 close to the second substrate 2. Preferably, the ion-sensitive membrane 8 is made of silicon nitride. When base complementary pairing occurs in the micropore 7, hydrogen ions are released, which induces a Nernst potential on the surface of the ion-sensitive membrane 8, thereby affecting the electric field between the first substrate 1 and the second substrate 2, and changing the distribution of charged particles.

[0041] Figure 2 Show Figure 1 A plan view of the first substrate of the gene sequencing chip. Figure 3 Show Figure 1 A plan view of the second substrate of the gene sequencing chip. Figure 2 As shown, the first electrode 3 is a block electrode, which is disposed on the first substrate 1 and is connected by a first signal lead 10 on the first substrate 1 to which a voltage is applied. Figure 3As shown, the second electrode 4 is also a block electrode, which is disposed on the second substrate 2 and connected by a second signal lead 11 on the second substrate 2 to apply a voltage thereto.

[0042] like Figures 1 to 3 As shown, the first electrode 3 and the second electrode 4 are block electrodes, and the first electrode 3 is arranged on the side of the first substrate 1 facing the second substrate 2, and the second electrode 4 is arranged on the side of the second substrate 2 facing the first substrate 1. It should be noted that the above is a preferred embodiment, and the shapes and positions of the first electrode 3 and the second electrode 4 are not limited in the present invention. Specifically, the first electrode 3 can be a block electrode or a planar electrode. The second electrode 4 can be a block electrode or a planar electrode. The first electrode 3 can be arranged on the side of the first substrate 1 close to the second substrate 2, or on the side of the first substrate 1 away from the second substrate 2. The second electrode 4 can be arranged on the side of the second substrate 2 close to the first substrate 1, or on the side of the second substrate 2 away from the first substrate 1. Among them, the first electrode 3 is made of indium tin oxide (Indium Tin Oxide, ITO), the second electrode 4, the first signal lead 10 and the second signal lead 11 are made of ITO, molybdenum, aluminum, copper, etc., and the microporous layer is made of silicon nitride or silicon oxide.

[0043] Likewise, the first electrode 3 and the second electrode 4 in the present invention may exist at the same time, or only the first electrode 3 or only the second electrode 4 may exist.

[0044] It should also be noted that the ion-sensitive membrane 8 is not necessary. When the ion-sensitive membrane 8 is not present, the hydrogen ions generated by base complementary pairing in the micropores 7 will also affect the electric field between the first electrode 1 and the second electrode 2, thereby changing the distribution of charged particles.

[0045] The following describes a gene sequencing method using the gene sequencing chip of the present invention. The gene sequencing method comprises the following steps:

[0046] Adding DNA microbeads containing DNA chains into the microwells 7 for PCR amplification;

[0047] Applying a voltage to the first electrode 3, and / or applying a voltage to the second electrode 4, so that an electric field is formed between the first substrate 1 and the second substrate 2, with the electric field direction pointing from the first substrate 1 to the second substrate 2;

[0048] Add four kinds of deoxyribonucleoside triphosphates into the micropores 7 in sequence, and detect whether the color of the electronic ink layer 5 on one side of the first substrate 1 changes;

[0049] The base type on the DNA chain is determined according to the deoxyribonucleoside triphosphate added when the color of the electronic ink layer 5 on one side of the first substrate 1 changes.

[0050] Preferably, the above-mentioned deoxyribonucleoside triphosphates are reversibly terminated deoxyribonucleoside triphosphates, specifically including reversibly terminated adenine deoxyribonucleotide triphosphate, reversibly terminated thymine deoxyribonucleotide triphosphate, reversibly terminated cytosine deoxyribonucleotide triphosphate and reversibly terminated guanine deoxyribonucleotide triphosphate.

[0051] After the DNA microbeads containing the DNA chain are added to the microwells 7 for PCR amplification, a voltage signal is applied to the first electrode 3 through the first signal lead 10, and a voltage signal is applied to the second electrode 4 through the second signal lead 11, so that there is an electric field between the first substrate 1 and the second substrate 2, which points from the first substrate 1 to the second substrate 2. At this time, the positively charged white particles 91 in the microcapsules 90 will gather on the side of the second substrate 2, and the negatively charged black particles 92 will gather on the side of the first substrate 1. Figure 1 and Figure 5-1 As shown, when observed from the first substrate 1 side, the color of the electronic ink layer 5 is black.

[0052] When the deoxyribonucleoside triphosphate in the micropore 7 is synthesized into the DNA molecule, hydrogen ions are released, thereby generating an electric field from the second substrate 2 to the first substrate 1. This electric field causes the positively charged white particles 91 in the microcapsule 90 to move toward the first substrate 1 and the negatively charged black particles 92 to move toward the second substrate 2. At this time, Figure 4 and Figure 5-2 As shown, when observed from the first substrate 1 side, the color of the electronic ink layer 5 is white.

[0053] If an ion-sensitive membrane 8 is provided in the micropore 7, hydrogen ions will induce a Nernst potential on the surface of the ion-sensitive membrane 8, which will also generate an electric field directed from the second substrate 2 to the first substrate 1, thereby causing the positively charged white particles 91 in the microcapsule 90 to move toward the first substrate 1 and the negatively charged black particles 92 to move toward the second substrate 2. The base type on the DNA chain is determined by the deoxyribonucleoside triphosphate added when the color of the electronic ink layer 5 on one side of the first substrate 1 changes.

[0054] Specifically, when the color of the electronic ink layer 5 on one side of the first substrate 1 changes, if the deoxyribonucleoside triphosphate added to the micropore 7 is adenine triphosphate deoxyribonucleotide, then the base on the DNA chain to be tested is thymine; if the deoxyribonucleoside triphosphate added to the micropore 7 is thymine triphosphate deoxyribonucleotide, then the base on the DNA chain to be tested is adenine; if the deoxyribonucleoside triphosphate added to the micropore 7 is cytosine triphosphate deoxyribonucleotide, then the base on the DNA chain to be tested is guanine; if the deoxyribonucleoside triphosphate added to the micropore 7 is guanine triphosphate deoxyribonucleotide, then the base on the DNA chain to be tested is cytosine.

[0055] After completing the base type detection at one position of DNA, it is necessary to wash off the reversibly terminated deoxyribonucleoside triphosphate added to the micropore 7 and add a thiol reagent. Unlike ordinary deoxyribonucleoside triphosphates, the 3′ end of the reversibly terminated deoxyribonucleoside triphosphate is connected to an azide group, which cannot form a phosphodiester bond during DNA synthesis, thereby interrupting DNA synthesis. If a thiol reagent is added, the azide group will break and form a hydroxyl group at the original position. After adding the thiol reagent, the base type detection at the subsequent position can be continued. The detection method is the same as the above method and will not be repeated here.

[0056] Preferably, an image acquisition device may be provided on one side of the first substrate 1 , and the image acquisition device may be used to capture the color change of the electronic ink layer 5 on one side of the first substrate 1 .

[0057] Preferably, the image acquisition device is a CCD camera.

[0058] The above is a preferred embodiment of the present invention. It should be noted that a person skilled in the art can make several improvements and modifications without departing from the principles of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A gene sequencing chip, characterized in that: include: A transparent first substrate (1); a second substrate (2), the second substrate (2) being arranged opposite to the first substrate (1); a first electrode (3) and a second electrode (4), wherein the first electrode (3) is arranged on a side of the first substrate (1) facing the second substrate (2) and is a transparent electrode, and the second electrode (4) is arranged on a side of the second substrate (2) facing the first substrate (1); An electronic ink layer (5), the electronic ink layer (5) being arranged between the first substrate (1) and the second substrate (2); a microporous layer (6), the microporous layer (6) being arranged on a side of the second substrate (2) away from the first substrate (1), and micropores (7) being arranged on the microporous layer (6) at positions corresponding to the first electrode (3) and the second electrode (4); An ion-sensitive membrane (8) is arranged in the micropore (7) on a side close to the second substrate (2), and the ion-sensitive membrane (8) is made of silicon nitride; The first electrode (3) and the second electrode (4) are both block electrodes, and the projections of the first electrode (3) and the second electrode (4) on the first substrate (1) and the second substrate (2) overlap with each other; The projections of the first electrode (3), the second electrode (4) and the micropore (7) on the first substrate (1) and the second substrate (2) overlap with each other.

2. The gene sequencing chip according to claim 1, characterized in that: The first electrode (3) is a planar electrode covering the entire first substrate (1), and the second electrode (4) is a block electrode; Alternatively, the second electrode (4) is a planar electrode that covers the entire second substrate (2), and the first electrode (3) is a block electrode.

3. The gene sequencing chip according to claim 1, characterized in that: A first signal lead (10) for applying a voltage to the first electrode (3) is provided on the first substrate (1), and a second signal lead (11) for applying a voltage to the second electrode (4) is provided on the second substrate (2).

4. A gene sequencing device, characterized in that: A gene sequencing chip comprising any one of claims 1-3.

5. A gene sequencing method using the gene sequencing chip according to any one of claims 1 to 3, characterized in that: The gene sequencing method comprises the following steps: Adding DNA microbeads containing DNA chains into the microwells (7) for PCR amplification; Applying a voltage to the first electrode (3) and applying a voltage to the second electrode (4) so ​​that an electric field is formed between the first substrate (1) and the second substrate (2), with the electric field direction pointing from the first substrate (1) to the second substrate (2); Adding four deoxyribonucleoside triphosphates into the micropores (7) in sequence, and detecting whether the color of the electronic ink layer (5) on one side of the first substrate (1) changes; The base type on the DNA chain is determined according to the deoxyribonucleoside triphosphate added when the color of the electronic ink layer (5) on one side of the first substrate (1) changes.

6. The gene sequencing method according to claim 5, characterized in that: The deoxyribonucleoside triphosphate is a reversibly terminated deoxyribonucleoside triphosphate, and the gene sequencing method further comprises: The reversibly terminated deoxyribonucleoside triphosphate added to the microwell (7) is washed away, and a thiol reagent is added.

Images