Surface-modified full-process nucleic acid detection chip

Abstract

The invention relates to the technical field of nucleic acid detection, and discloses a surface-modified full-process nucleic acid detection chip, which comprises a detection chip body, the detection chip body comprises a silicon substrate and a cover plate, the cover plate is adhered to the top of the silicon substrate, and a cracking cavity, a first washing cavity, a second washing cavity and an elution cavity are respectively arranged above the silicon substrate; the cracking cavity is used for cracking pathogens; the first washing cavity is used for washing nucleic acid molecules; the second washing cavity is used for secondary washing of nucleic acid molecules; the elution cavity is used for elution and PCR amplification of the adsorbed nucleic acid molecules; the problem of reagent transfer in a traditional method can be avoided, the nucleic acid extraction efficiency can be improved, and aerosol pollution is avoided; by integrating nucleic acid extraction, washing, elution and detection into one chip, automatic control is realized, the detection efficiency is improved, and the dependence on professional laboratories and operators is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and specifically to a surface-modified full-process nucleic acid detection chip. Background Technique

[0002] Nucleic acid detection technology is currently the main means for direct detection of pathogens, and the detection limit can reach dozens or even a few copies. Compared with immunology-based detection methods, it can fundamentally detect whether the target sequence of the detected pathogen exists, effectively reducing the existence of false negatives in the detection results.

[0003] Nucleic acid detection is of great significance in aspects such as food safety, environmental monitoring, and clinical diagnosis. Traditional nucleic acid extraction and detection have high requirements for equipment and operators, take a long time, and cannot meet the requirements of POCT. In addition, the aerosol generated during the open pipetting process may pollute the environment, especially highly infectious viruses, increasing the risk of infection for technicians. Therefore, a surface-modified full-process nucleic acid detection chip is proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a surface-modified full-process nucleic acid detection chip to solve the problems raised in the above background technique that traditional nucleic acid extraction and detection have high requirements for equipment and operators, take a long time, cannot meet the requirements of POCT, and in addition, the aerosol generated during the open pipetting process may pollute the environment, especially highly infectious viruses, increasing the risk of infection for technicians.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A surface-modified full-process nucleic acid detection chip, including

[0006] A detection chip body;

[0007] The detection chip body includes a silicon substrate and a cover plate, the cover plate is bonded to the top of the silicon substrate, and a lysis chamber, a first washing chamber, a second washing chamber, and an elution chamber are respectively arranged above the silicon substrate;

[0008] The lysis chamber is used for lysis of pathogens;

[0009] The first washing chamber is used for washing of nucleic acid molecules;

[0010] The second washing chamber is used for secondary washing of nucleic acid molecules;

[0011] The elution chamber is used for eluting and PCR amplifying the adsorbed nucleic acid molecules.

[0012] A detection method for a surface-modified full-process nucleic acid detection chip includes the following steps:

[0013] S1. First, fill the internal space of the chip with oil, and then inject the nucleic acid lysis solution, the first washing solution, the second washing solution, the elution solution, and the PCR amplification reagent mixture into the corresponding injection ports in sequence, and separate them with the oil to form a lysis chamber, a first washing chamber, a second washing chamber, and an elution chamber;

[0014] S2. Inject the sample containing the pathogen into the lysis chamber. Under the action of the nucleic acid lysis solution, the pathogen is lysed. Put magnetic beads inside the lysis chamber, and the released nucleic acid molecules are adsorbed by the magnetic beads;

[0015] S3. Place the handheld permanent magnet under the silicon substrate, and then move the permanent magnet to drag the magnetic beads into the first washing chamber and the second washing chamber in sequence to complete the washing of the nucleic acid molecules;

[0016] S4. Then, move the permanent magnet to drag the magnetic beads into the elution chamber. After the elution solution elutes the nucleic acid molecules adsorbed by the magnetic beads, use the permanent magnet to drag the magnetic beads to the previous chamber, place the chip on the heating module, and under the action of the heating module, the nucleic acid molecules complete the PCR reaction in the microchamber reaction chamber. By detecting the fluorescence intensity emitted by the fluorescent group with an instrument, accurate quantitative detection of the pathogen can be achieved.

[0017] Preferably, in S1 above, the oil is silicone oil.

[0018] Preferably, in S1 above, the nucleic acid lysis solution, the first washing solution, and the second washing solution are respectively injected into the lysis chamber, the first washing chamber, and the second washing chamber.

[0019] Preferably, in S1 above, the elution solution and the PCR amplification reagent mixture are injected into the elution chamber.

[0020] Preferably, in S3 above, the first washing solution and the second washing solution are used to remove impurities and proteins during nucleic acid extraction.

[0021] Preferably, in S4 above, when eluting the nucleic acid molecules, it is necessary to hold the permanent magnet and move it continuously within the chamber range to keep the magnetic beads in a moving and stirring state in the chamber.

[0022] Preferably, the stirring time is 30 s.

[0023] Compared with the prior art, the present invention adopts the above technical solutions and has the following technical effects: The present invention can avoid the reagent transfer problem of the traditional method, is conducive to improving the nucleic acid extraction efficiency, and avoids aerosol pollution; by integrating nucleic acid extraction, washing, elution, and detection into one chip, automatic control is realized, the detection efficiency is improved, and the dependence on professional laboratories and operators is reduced. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 It is a schematic top view structure diagram of the chip of the present invention;

[0026] Figure 2 It is a schematic front view structure diagram of the chip of the present invention;

[0027] Figure 3 It is a schematic overall structure diagram of the chip of the present invention;

[0028] Figure 4 It is a process flow diagram of the Lift-off process of the present invention;

[0029] Figure 5 It is a schematic diagram of a fluorescence microscope for negative samples of the present invention;

[0030] Figure 6 It is a schematic diagram of a fluorescence microscope for positive samples of the present invention.

[0031] Explanation of reference numerals: 1, silicon substrate; 2, lysis chamber; 3, first washing chamber; 4, second washing chamber; 5, elution chamber. Detailed implementation manners

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0033] It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those who are familiar with this technology to understand and read, and are not used to limit the limiting conditions that the present application can be implemented. Therefore, they do not have a substantial technical meaning. Any modification of the structure, change of the proportional relationship, or adjustment of the size, without affecting the effects that the present application can produce and the purposes that can be achieved, should still fall within the scope that can be covered by the technical content disclosed in the present application.

[0034] Embodiment

[0035] Please refer to Figures 1-6, the present invention provides a technical solution: a surface-modified full-process nucleic acid detection chip, including a detection chip body;

[0036] The detection chip body includes a silicon substrate 1 and a cover plate, the cover plate is bonded to the top of the silicon substrate 1, and a lysis chamber 2, a first washing chamber 3, a second washing chamber 4 and an elution chamber 5 are respectively arranged above the silicon substrate 1;

[0037] The lysis chamber 2 is used for lysis of pathogens;

[0038] The first washing chamber 3 is used for washing of nucleic acid molecules;

[0039] The second washing chamber 4 is used for secondary washing of nucleic acid molecules;

[0040] The elution chamber 5 is used for eluting the adsorbed nucleic acid molecules and PCR amplification.

[0041] A detection method for a surface-modified full-process nucleic acid detection chip includes the following steps:

[0042] S1. First, fill the internal space of the chip with oil, and then inject a nucleic acid lysis solution, a first washing solution, a second washing solution, an elution solution and a PCR amplification reagent mixture from the corresponding injection ports in sequence, and separate them by the oil to form a lysis chamber 2, a first washing chamber 3, a second washing chamber 4 and an elution chamber 5;

[0043] S2. Inject a sample containing pathogens into the lysis chamber 2. Under the action of the nucleic acid lysis solution, the pathogens are lysed. Put magnetic beads inside the lysis chamber 2, and the released nucleic acid molecules are adsorbed by the magnetic beads;

[0044] S3. Place a handheld permanent magnet under the silicon substrate 1, and then move the permanent magnet to drag the magnetic beads into the first washing chamber 3 and the second washing chamber 4 in sequence to complete the washing of nucleic acid molecules;

[0045] S4. Then move the permanent magnet to drag the magnetic beads into the elution chamber 5. After the elution solution elutes the nucleic acid molecules adsorbed by the magnetic beads, then use the permanent magnet to drag the magnetic beads to the previous chamber, place the chip on the heating module, and under the action of the heating module, the nucleic acid molecules complete the PCR reaction in the microchamber reaction chamber, and the fluorescence intensity emitted by the fluorescent group is detected by the instrument to achieve precise quantitative detection of pathogens.

[0046] In S1, the oil is silicone oil, the nucleic acid lysis solution, the first washing solution, and the second washing solution are respectively injected into the interior of the lysis chamber 2, the first washing chamber 3, and the second washing chamber 4, and the elution solution and the PCR amplification reagent mixture are injected into the interior of the elution chamber 5; the entire chamber is filled with silicone oil, and then the corresponding reagents are added to the corresponding chambers. The density of the reagents is greater than that of the silicone oil, so they will sink to the bottom. The bottom is just hydrophilized, and the liquid can be fixed in the corresponding position without flowing; the lysis solution can effectively lyse cells and release nucleic acids;

[0047] In S3, the first washing solution and the second washing solution are used to remove impurities and proteins during nucleic acid extraction.

[0048] In S4, when eluting nucleic acid molecules, a permanent magnet needs to be held and continuously moved within the chamber range to keep the magnetic beads in a moving and stirring state within the chamber. The stirring time is 30 s; the elution solution ensures the maximization of the elution efficiency and recovery rate of nucleic acids, while protecting nucleic acids from nuclease degradation and oxidative damage and improving the purity of nucleic acids.

[0049] Lysis points, first washing points, second washing points, and elution and PCR amplification points are sequentially arranged on the silicon substrate 1. A straight line is formed between each point, and each point is a layer of SiO deposited by lift-off and PECVD processes 2 , with a thickness of about 200 - 500 nm. Plasma-enhanced chemical vapor deposition (PECVD) is a thin-film deposition technology widely used in the semiconductor industry. It combines the principle of chemical vapor deposition (CVD) with plasma technology, and can generate high-quality thin films and precisely control their properties. SiO 2 is hydrophilic, which can make the reagents for each step of the reaction better retained in place. Each step site is separated by an oil phase, and the reaction reagents do not interfere with each other.

[0050] Above the silicon substrate 1 is a cavity cover plate made of plastic material, which is bonded to the silicon base through glue to form a space for accommodating and separating the oil phase. There are four round holes on the top layer of the plastic, which are vertically corresponding to the lysis point, the first washing point, the second washing point, and the elution and PCR amplification points respectively, and serve as the reagent inlets for the four points.

[0051] Those skilled in the art can understand that the features recited in the various embodiments and / or claims of the present invention can be combined or combined in various ways, even if such combinations or combinations are not explicitly recited in the present invention. In particular, without departing from the spirit and teachings of the present invention, the features recited in the various embodiments and / or claims of the present invention can be combined and combined in various ways. All such combinations and / or combinations fall within the scope of the present invention.

Claims

1. A surface-modified full-process nucleic acid detection chip, characterized in that: include Detection chip body; The detection chip body comprises a silicon substrate (1) and a cover plate, wherein the cover plate is bonded to the top of the silicon substrate (1), and a lysis chamber (2), a first washing chamber (3), a second washing chamber (4) and an elution chamber (5) are respectively arranged above the silicon substrate (1); The lysis chamber (2) is used for lysis of pathogens; The first washing chamber (3) is used for washing nucleic acid molecules; The second washing chamber (4) is used for secondary washing of nucleic acid molecules; The elution chamber (5) is used for eluting the adsorbed nucleic acid molecules and performing PCR amplification.

2. A detection method for a surface-modified full-process nucleic acid detection chip, characterized in that: The following steps are involved: S1. First, the internal space of the chip is filled with oil, and then the nucleic acid lysis solution, the first washing solution, the second washing solution, the elution solution and the PCR amplification reagent mixture are injected from the corresponding injection ports in sequence, and separated by the oil to form a lysis chamber (2), a first washing chamber (3), a second washing chamber (4) and an elution chamber (5); S2, injecting a sample containing the pathogen into the lysis chamber (2), the pathogen is lysed under the action of a nucleic acid lysis solution, placing magnetic beads inside the lysis chamber (2), and the released nucleic acid molecules are adsorbed by the magnetic beads; S3, holding a permanent magnet under the silicon substrate (1), then moving the permanent magnet to drag the magnetic beads into the first washing chamber (3) and the second washing chamber (4) in sequence, to complete the washing of the nucleic acid molecules; S4, then use the moving permanent magnet to drag the magnetic beads to the inside of the elution chamber (5), after the elution liquid elutes the nucleic acid molecules adsorbed by the magnetic beads, use the permanent magnet to drag the magnetic beads to the front chamber, place the chip on the heating module, under the action of the heating module, the nucleic acid molecules complete the PCR reaction in the micro-cavity reaction chamber, and use the instrument to detect the fluorescence intensity emitted by the fluorescent group, so as to achieve accurate quantitative detection of pathogens.

3. The detection method of a surface-modified full-process nucleic acid detection chip according to claim 2, characterized in that: In S1, the oil is silicone oil.

4. The detection method of a surface-modified full-process nucleic acid detection chip according to claim 2, characterized in that: In S1, the nucleic acid lysing solution, the first washing solution and the second washing solution are respectively injected into the interior of the lysing chamber (2), the first washing chamber (3) and the second washing chamber (4).

5. The detection method of a surface-modified full-process nucleic acid detection chip according to claim 2, characterized in that: In S1, the elution liquid and the PCR amplification reagent mixture are injected into the elution chamber (5).

6. The detection method of a surface-modified full-process nucleic acid detection chip according to claim 2, characterized in that: In S3, the first washing solution and the second washing solution are used to remove impurities and proteins during the nucleic acid extraction process.

7. The detection method of a surface-modified full-process nucleic acid detection chip according to claim 2, characterized in that: In S4, when eluting nucleic acid molecules, a handheld permanent magnet needs to be moved continuously within the chamber to keep the magnetic beads in a moving and stirring state within the chamber.

8. The detection method of a surface-modified full-process nucleic acid detection chip according to claim 7, characterized in that: The stirring time is 30s.

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