CN111944682A - Nucleic acid detection chip, preparation method and nucleic acid detection method - Google Patents

Abstract

The invention relates to the technical field of biomolecule detection, in particular to a nucleic acid detection chip, a preparation method and a nucleic acid detection method. The nucleic acid detection chip includes: a reaction substrate and a temperature control substrate, the reaction substrate is provided with a reaction chamber; the reaction substrate is further provided with an injection port, and the injection port is communicated with the reaction chamber; the reaction substrate It includes a first reaction substrate and a second reaction substrate, the reaction chamber is at least partially arranged on the first reaction substrate, and the first reaction substrate is covered with the second reaction substrate so that the reaction chamber forms a closed A cavity structure; a substrate mounting structure is provided on the first reaction substrate and/or the second reaction substrate, and the temperature control substrate is arranged in the substrate mounting structure. The nucleic acid detection chip has a simple structure, integrates a reaction substrate and a temperature control substrate, and can complete the detection process without additional supporting equipment.

Description

Nucleic acid detection chip, preparation method and nucleic acid detection method

technical field

The present invention relates to the technical field of biomolecule detection, in particular to a nucleic acid detection chip, a preparation method and a nucleic acid detection method.

Background technique

A microfluidic chip is a technology that precisely controls and manipulates micro-scale fluids. It can integrate basic operation units such as sample preparation, reaction, separation, and detection in the analysis process into a centimeter-square chip to automatically complete the entire analysis process. It has the advantages of less sample and reagent consumption, fast detection speed, high sensitivity and low cost, so the current application research in pathogen detection is wide and progressing rapidly. The introduction of microfluidic chip technology into nucleic acid detection for biomedical analysis, environmental detection and forensic identification can simplify and integrate the tedious steps of sample pretreatment and amplification products in traditional nucleic acid detection, which not only shortens detection time, but also reduces reagents. and sample consumption, and make up for the cumbersome operation and high cost of traditional methods, and can be used for instant detection in biological laboratories and field portable applications.

Among various nucleic acid amplification detection methods, the nucleic acid detection system combining loop-mediated isothermal amplification (LAMP) and microfluidic technology has the most potential to be applied to the rapid diagnosis of biomolecules on site. LAMP is a new isothermal amplification technology developed by Notomi et al. This technology designs four specific primers based on six gene fragments. Under the action of Bst polymerase, heating at 60-65 ℃, and the amplification can be completed in 20-60 minutes. It is easy to operate, fast and time-saving, specific Sexual characteristics. Compared to traditional polymerase chain reaction (PCR), isothermal nucleic acid amplification does not require cycling between different temperatures. Each thermal cycling step of the PCR reaction requires precise temperature control, and only a single reaction temperature is required in the entire process of nucleic acid isothermal amplification, which makes nucleic acid isothermal amplification get rid of the dependence on complex instruments, and the reaction process is also easier. Simple and effective. In recent years, many studies have combined loop-mediated isothermal amplification with microfluidic chips to detect pathogenic microorganisms, cancer biomarkers, and other target genes.

For most microfluidic systems, the channel network is fixed at the time of microfabrication, often requiring pumping, valve control, or external forces using electric and magnetic fields. Therefore, the fluid drive and control technology in the microchannel is still a challenge, and how to reduce the peripherals and increase the intelligent integration in the microfluidic system is an urgent problem to be solved.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is the complex structure of the nucleic acid detection chip in the prior art and the problems of many peripheral devices.

In order to solve the above technical problems, in the first aspect, the embodiments of the present application disclose a nucleic acid detection chip, which includes: a reaction substrate and a temperature control substrate,

a reaction chamber is provided on the reaction substrate;

The reaction substrate is further provided with an injection port, and the injection port is communicated with the reaction chamber;

The reaction substrate includes a first reaction substrate and a second reaction substrate, the reaction chamber is at least partially disposed on the first reaction substrate, and the first reaction substrate is covered with the second reaction substrate to make the reaction The cavity forms a closed cavity structure;

A substrate mounting structure is provided on the first reaction substrate and/or the second reaction substrate, and the temperature control substrate is arranged in the substrate mounting structure.

Further, the reaction chamber is provided with a plurality of reaction functional zones, and different reaction functional zones are pre-embedded with reaction reagents with different functions;

The reaction functional area includes a cleavage area, a pre-amplification area and an amplification detection area, the cleavage area and the pre-amplification area are connected through a first microfluidic channel, and the pre-amplification area is connected with the amplification detection area. The regions are communicated through a second microfluidic channel, and a control valve is provided in the first microfluidic channel and the second microfluidic channel.

Further, the control valve is a solid phase change material encapsulated in the first microchannel and the second microchannel, and the melting point of the solid phase change material is lower than the tolerance of the nucleic acid detection chip. temperature.

Further, the solid phase change material is at least one of paraffin, butter, rosin, and asphalt.

Further, the substrate mounting structure is a substrate mounting groove, and the temperature control substrate is arranged in the substrate mounting groove.

Further, the temperature control substrate includes a support layer and a temperature control layer, and the temperature control layer is disposed on the support layer;

The temperature control layer is provided with a temperature sensor and a heating electrode.

Further, the heating electrode includes a first heating electrode and a second heating electrode, the first heating electrode is used for heating the first microfluidic channel and the pre-amplification region, and the second heating electrode is used for heating The second microfluidic channel and the amplification detection zone are heated.

In a second aspect, the embodiments of the present application disclose a method for preparing a nucleic acid detection chip, the preparation method comprising:

obtaining a reaction substrate, the reaction substrate includes a first reaction substrate and a second reaction substrate;

A reaction chamber is fabricated on the first reaction substrate and the second reaction substrate; wherein, the reaction chamber includes a plurality of reaction functional areas, and the plurality of reaction functional areas are communicated with each other through a microfluidic channel;

loading reaction reagents in the reaction chamber;

A control valve is arranged in the microfluidic channel, and the control valve is a solid phase change material potted in the microfluidic channel;

sealingly combining the first reaction substrate and the second reaction substrate;

obtaining a temperature control substrate, the temperature control substrate includes a support layer and a temperature control layer, and the temperature control layer is provided with a temperature sensor and a heating electrode;

making a substrate mounting groove on the reaction substrate;

The temperature control substrate is installed in the substrate installation groove.

In a third aspect, the embodiments of the present application disclose a nucleic acid detection method, which is applied to a nucleic acid detection chip, including:

The sample to be tested is introduced into the lysis zone through the injection port for lysis to obtain a lysis sample;

Heating the first microchannel to melt the control valve to conduct the lysis zone and the pre-amplification zone, and the lysed sample enters the pre-amplification zone for pre-amplification to obtain a pre-amplification sample;

Heating the second microchannel to melt the control valve to conduct the pre-amplification area and the amplification detection area, and the pre-amplification sample enters the reaction pool in the amplification detection area for reaction;

Detect whether a specific target exists in each reaction pool in the amplification detection area.

Further, before the pre-amplified sample enters the reaction tank in the amplification detection area for reaction, it also includes controlling the liquid overflowing the reaction tank to enter the waste liquid area.

Further, the pre-amplified sample is subjected to an isothermal amplification reaction with primers, DNTPs and enzymes required for the reaction that are pre-buried in the reaction pool for the isothermal amplification reaction under a preset temperature environment.

Further, whether there is a specific target in each reaction pool in the detection amplification detection zone, including:

using optical method to calibrate each reaction pool in the amplification detection zone;

Detecting the calibration reaction intensity in each of the reaction pools;

The presence of a specific target is determined based on the calibrated reaction intensity in each of the reaction cells.

Using the above technical solution, the nucleic acid detection chip, the preparation method and the nucleic acid detection method described in the embodiments of the present application have the following beneficial effects:

In the nucleic acid detection chip described in the embodiments of the present application, a reaction chamber is provided in the reaction substrate, and the nucleic acid detection process can be integrated in the reaction chamber. The temperature control substrate is arranged in the substrate mounting structure on the reaction substrate, and the temperature control substrate can The requirements of the detection process directly regulate the reaction substrate; the nucleic acid detection chip has a simple structure, integrates the reaction substrate and the temperature control substrate, and can complete the detection process without additional supporting equipment.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present application more clearly, the following briefly introduces the drawings that are used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative effort.

1 is a schematic structural diagram of a nucleic acid detection chip according to an embodiment of the application;

FIG. 2 is a schematic structural diagram of a reaction chamber according to an embodiment of the present application;

3 is a schematic structural diagram of a reaction tank according to an embodiment of the application;

4 is a schematic structural diagram of a temperature control system according to an embodiment of the application;

5 is a schematic flowchart of a temperature control method provided by an embodiment of the present application;

6 is a schematic flowchart of a method for preparing a nucleic acid detection chip provided in an embodiment of the present application;

FIG. 7 is a schematic flowchart of a nucleic acid detection method provided in an embodiment of the present application.

The following supplementary descriptions are provided for the accompanying drawings:

101-first reaction substrate; 102-second reaction substrate; 103-substrate installation groove; 104-injection port; 111-lysis area; 112-pre-amplification area; 113-amplification detection area; 115-the first microchannel; 116-the second microchannel; 120-reaction plate; 121-the first film; 122-the second film; 2-temperature control substrate; 201-heating electrode; 202-temperature sensor.

Detailed ways

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of this application.

Reference herein to "one embodiment" or "an embodiment" refers to a particular feature, structure, or characteristic that may be included in at least one implementation of the present application. In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "top", "bottom", etc. is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the purpose of It is convenient to describe the application and to simplify the description, rather than indicating or implying that the device or element referred to must have a particular orientation, be constructed and operate in a particular orientation, and therefore should not be construed as limiting the application. In addition, the terms "first" and "second" are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature defined as "first" or "second" may expressly or implicitly include one or more of that feature. Also, the terms "first," "second," etc. are used to distinguish between similar objects, and are not necessarily used to describe a particular order or precedence. It is to be understood that data so used may be interchanged under appropriate circumstances so that the embodiments of the application described herein can be practiced in sequences other than those illustrated or described herein.

Nucleic acid-based diagnostic technology is one of the most promising directions in molecular diagnostic technology, and has a wide range of applications in disease detection, food safety and other fields. Among the nucleic acid detection methods, the polymerase chain reaction (PCR) technology achieves the purpose of detecting specific target nucleic acid fragments by amplifying specific nucleic acid fragments. Usually, nucleic acid detection needs to go through three steps: nucleic acid extraction, nucleic acid amplification and nucleic acid detection. The three steps of traditional nucleic acid detection methods are discrete, and there are problems such as long preparation and analysis time, susceptibility to contamination, sensitivity limitations, and complicated procedures and operations. The integration of nucleic acid extraction, amplification and detection through microfluidic technology Helps to address the technical and analytical limitations associated with nucleic acid detection in practical applications.

As shown in FIG. 1 , an embodiment of the present application discloses a nucleic acid detection chip, including: a reaction substrate and a temperature control substrate 2 , a reaction chamber is provided on the reaction substrate; a sample inlet 104 is further provided on the reaction substrate, and the sample inlet 104 communicates with the reaction chamber; the reaction substrate includes a first reaction substrate 101 and a second reaction substrate 102, the reaction chamber is at least partially arranged on the first reaction substrate 101, and the first reaction substrate 101 and the second reaction substrate 102 are covered to make the reaction chamber A closed cavity structure is formed; the first reaction substrate 101 and/or the second reaction substrate 102 are provided with a substrate mounting structure, and the temperature control substrate 2 is provided in the substrate mounting structure.

In the nucleic acid detection chip described in the embodiments of the present application, a reaction chamber is provided in the reaction substrate, and the nucleic acid detection process can be integrated in the reaction chamber. The temperature control substrate 2 is arranged in the substrate mounting structure on the reaction substrate, and the temperature control substrate 2 The reaction substrate can be directly regulated according to the needs of the detection process; the nucleic acid detection chip has a simple structure, the reaction substrate and the temperature control substrate 2 are integrated together, and the detection process can be completed without additionally setting up ancillary equipment.

In the embodiment of the present application, as shown in FIG. 1 , the reaction substrate is a thin plate made of a transparent material. The transparency mainly facilitates the technician to visually track the reaction state of the sample during the operation. The material of the reaction substrate can be an inorganic material, such as glass. ; Can also be organic polymer materials, such as polyethylene PE, polypropylene PP, polycarbonate PC, polyethylene terephthalate (PET), polystyrene (PS), polymethyl dimethacrylate (PMMA), acrylonitrile-butadiene-polyethylene copolymer (ABS), etc. In order to reduce the difficulty of the manufacturing process and the use cost, the material of the reaction substrate is preferably a transparent polymer material. The reaction substrate can be an integral structure, which is formed by an injection molding process, and the interior of the reaction substrate is a cavity structure, and the cavity structure is a reaction cavity in the nucleic acid detection process. The reaction substrate can also be formed by buckling and sealing two thin plates, and the reaction chamber structure can be partially or completely arranged on the first reaction substrate 101. Correspondingly, the reaction chamber structure can be entirely or partially arranged on the second reaction substrate 102. The two reaction substrates are buckled together to form a reaction chamber structure, and then sealed to form a whole. The above-mentioned method of sealing the two reaction substrates includes one of laser welding, thermal compression sealing, high-strength chemical adhesive bonding or ultrasonic welding. One or more methods, and other sealing methods can also be used, as long as the two reaction substrates can be sealed and connected as a whole. A sample inlet 104 is provided on the reaction substrate to communicate with the reaction chamber. Optionally, a part of the sample inlet 104 is arranged on the first reaction substrate 101, and the other part is arranged on the second reaction substrate 102; 104 are all arranged on any one of the reaction substrates. The sample to be detected enters the reaction chamber through the sample inlet 104, and steps such as sample cracking, mixing, and reaction can be integrated in the reaction chamber. The substrate mounting structure is a cavity or groove structure arranged on the reaction substrate, and the temperature control substrate 2 is arranged in the substrate mounting structure by clamping, bonding, screwing or embedding.

In the embodiments of the present application, the nucleic acid detection chip is usually a disposable product, that is, the nucleic acid detection chip is discarded after one nucleic acid detection. Therefore, the material of the reaction substrate should comprehensively consider factors such as use cost, processing difficulty, and degradation difficulty. As the core component of temperature control, the temperature control substrate 2 can be recycled. Therefore, the temperature control substrate 2 can be detachably connected to the reaction substrate. After the nucleic acid detection is completed, the temperature control substrate 2 can be removed and recycled.

As shown in FIG. 2 , there are multiple reaction functional areas in the reaction chamber, and different reaction functional areas are pre-embedded with reaction reagents with different functions; the reaction functional areas include a lysis area 111 , a pre-amplification area 112 and an amplification detection area Zone 113, the cracking zone 111 and the pre-amplification zone 112 are connected through the first micro-channel 115, the pre-amplification zone 112 and the amplification detection zone 113 are communicated through the second micro-channel 116, the first micro-channel 115 and the second A control valve is provided in the microfluidic channel 116 .

In the embodiment of the present application, a lysis zone 111, a pre-amplification zone 112 and an amplification detection zone 113 are arranged in the reaction chamber, and reaction reagents for realizing related functions are pre-embedded in each reaction functional zone, wherein the types of the reaction reagents include cleavage solution, primers, enzymes, etc. for isothermal amplification reactions. The above-mentioned reaction reagents are usually made into lyophilized powder by freeze-drying technology, which is pre-sealed in the chip, so that the reaction reagents can be stored at room temperature for a long time and maintain high enzymatic activity. When the nucleic acid detection chip is used, after the sample enters the reaction chamber through the sample inlet 104, a closed space is formed inside the chip, and lysis, pre-amplification, and amplification detection are performed in sequence. Each reaction functional area is a closed cavity structure, and each reaction functional area is connected through a micro-channel, and a control valve is arranged in the micro-channel. When the sample completes the reaction in the previous reaction functional area, the valve, so that the sample enters the next reaction function area to react.

As shown in FIG. 2 , the control valve is a solid phase change material potted in the first microchannel 115 and the second microchannel 116 , and the melting point of the solid phase change material is lower than the tolerance temperature of the nucleic acid detection chip.

In the examples of this application, the solid phase change material is a solid compound or mixture with a relatively low melting point under normal conditions, and its melting point should be lower than the limit test temperature of the nucleic acid detection chip. Preferably, the melting point of the solid phase change material is not higher than that of the nucleic acid detection chip. The reaction temperature at the time of detection. The solid phase change material with low melting point under normal conditions is used as the control valve to block the microchannel, and the control valve is integrated into the detection chip, eliminating the need for external microfluidic pumps, control valves, etc., reducing the number of peripheral devices and simplifying the nucleic acid detection process. , so that the nucleic acid detection chip can be applied to more scenarios and expand the scope of use of the chip. In addition, compared with various physical valve devices in the prior art, the control valve of the present application is easier to embed in the chip, which greatly reduces the difficulty of the manufacturing process. Meanwhile, phase change materials with low melting point are easy to obtain, which greatly reduces the The cost of making the chip.

The solid phase change material is at least one of paraffin, butter, rosin, and asphalt.

In the examples of this application, there are many types of low-melting phase change materials that are solid under normal conditions. Since the control valve is built into the reaction chamber, the solid phase change material should be selected with relatively stable properties, and it is not easy for the reaction reagents and reaction samples to produce chemical reactions. The material to be reacted, such as paraffin, butter, rosin, asphalt, etc., can also be a mixture of a plurality of the above-mentioned materials.

As shown in FIG. 1 , the substrate mounting structure is a substrate mounting groove 103 , and the temperature control substrate 2 is arranged in the substrate mounting groove 103 .

In the embodiment of the present application, the reaction substrate is provided with a substrate mounting groove 103, and the substrate mounting groove 103 may be provided on the first reaction substrate 101, or may be provided on the second reaction substrate 102, or both reaction substrates may be provided . The substrate installation groove 103 is a groove structure with at least one side open, and the temperature control substrate 2 is installed in the substrate installation groove 103 by clamping, bonding, screwing or embedding. For example, the temperature control substrate 2 is in close contact with the substrate mounting structure by a plug-in structure, which can be directly inserted and pulled out for easy replacement.

The temperature control substrate 2 includes a support layer and a temperature control layer, the temperature control layer is provided on the support layer; the temperature control layer is provided with a temperature sensor 202 and a heating electrode 201 .

In the embodiment of the present application, in order to facilitate the disassembly and recovery of the temperature control substrate 2, the temperature control layer used for temperature control may be arranged on a support layer that plays a supporting role. The support layer can be made of transparent ITO glass or polymer material. One or more temperature sensors 202 are arranged on the temperature control layer, and the temperature sensors 202 are used for real-time feedback of the temperature of each reaction function area; a plurality of heating electrodes 201 are also arranged on the temperature control layer, which are used for the reaction function area and the control valve. heating. The temperature control layer is attached on the support layer through a sputtering process, and the material of the temperature control layer includes Au/Ti, Pt/Ti and the like.

As shown in FIG. 1 , the heating electrode 201 includes a first heating electrode and a second heating electrode, the first heating electrode is used for heating the first microfluidic channel 115 and the pre-amplification region 112 , and the second heating electrode is used for heating the second microchannel Flow channel 116 and amplification detection zone 113 .

In the embodiment of the present application, two heating electrodes 201 are provided on the temperature control layer. When the detection chip is used for detection, after the sample to be tested is cracked in the cracking area 111, the first microfluidic electrode is controlled by the first heating electrode. The channel 115 and the pre-amplification zone 112 are heated, so that the control valve in the first micro-channel 115 is heated and melted, and the sample to be tested enters the pre-amplification zone 112 from the lysis zone 111 through the first micro-channel 115 for reaction; After the pre-amplification of the test sample in the pre-amplification area 112 is completed, the second micro-channel 116 and the amplification detection area 113 are heated by controlling the second heating electrode, so that the control valve in the second micro-channel 116 is heated and melted, The sample to be tested enters the amplification detection area 113 from the pre-amplification area 112 through the second microfluidic channel 116 for reaction.

In the embodiment of the present application, the temperature control system is arranged on the temperature control layer. FIG. 4 is a schematic structural diagram of the temperature control system according to an embodiment of the present application. As shown in FIG. 4 , the micro control unit MCU and the temperature control unit interactively control and each reaction The heating electrodes 201 corresponding to the functional areas are switched and heated in sequence, so that the detection chip can complete lysis, mixing, pre-amplification, mixing and amplification detection. FIG. 5 is a schematic flowchart of a temperature control method provided by an embodiment of the present application. Referring to FIG. 5, the temperature control method includes:

S501: Initialize the temperature control system;

S503: The temperature sensor 202 feeds back the temperature of the corresponding reaction functional area;

S505: Calculate the deviation value between the feedback temperature and the preset temperature;

S507: Whether the deviation value is smaller than the initial setting value; if yes, go to step S509; if not, go to step S511;

S509: Enter the timer interrupt program;

S511: call the heating program and output the heating control signal;

S513: Control the heating electrode 201 according to the control signal.

In the embodiment of the present application, the micro-control unit MCU outputs a PWM signal to control the opening and closing of the triode, thereby controlling each temperature control unit. When the triode is turned on, the temperature control functional area is energized and starts to heat; when the triode is disconnected, the temperature control functional area is powered off and stops heating.

As shown in FIG. 3 , the amplification detection area 113 is provided with a reaction plate 120, and the reaction plate 120 is provided with a plurality of through holes; one side of the reaction plate 120 is provided with a first film 121, and the other side of the reaction plate 120 is provided with a first film 121 There is a second film 122; the first film 121 is provided with a plurality of small holes, the number of small holes and through holes are equal, and the positions of the small holes correspond to the positions of the through holes one-to-one; the first film 121 and the second film 122 will pass through. The pores are closed to form a set of reaction cells, which includes a plurality of reaction cells.

In the embodiment of the present application, the film seals the two sides of the through hole, so that each through hole forms a reaction cell, and the small holes on the first film 121 are used to allow the liquid with the nucleic acid of the test sample to enter the reaction cell, and To a certain extent, the outflow of the liquid entering the reaction tank can be restricted, and at the same time, the interaction of the liquid between different through holes can be prevented, so as to further ensure the smooth progress of the amplification reaction.

The reaction pool set includes at least two reaction pool groups, and any one reaction pool group is provided with at least three reaction pools.

In the embodiment of the present application, at least two groups are set in the reaction pool, including a negative control group and one or more test groups, each group is not less than three duplicate wells, to realize single-target or multi-target detection of a sample, reduce external Factors that affect the reaction process, which result in inaccurate subsequent test results, occur.

The reaction functional area further includes a waste liquid area 114 , and the waste liquid area 114 communicates with the amplification detection area 113 .

In the embodiment of the present application, the waste liquid area 114 is mainly used to collect excess liquid in the amplification chamber, so as to reduce a large amount of liquid remaining in the amplification detection area 113 and affect the nucleic acid amplification reaction process. The number of waste liquid areas 114 can be at most one One, preferably, two waste liquid areas 114 are provided on both sides of the amplification detection area 113, so that the excess mixed liquid entering the amplification detection area 113 can be collected simultaneously on both sides in the subsequent operation process, which is further beneficial to Shorten the overall time of the reaction process.

In the nucleic acid detection chip described in the embodiments of the present application, the reaction functional areas are arranged in order from top to bottom, and are respectively a lysis area 111 , a pre-amplification area 112 , an amplification area detection area and a waste liquid area 114 . The sample inlet 104, the lysis zone 111, the pre-amplification zone 112, the detection zone of the amplification zone and the waste liquid zone 114 are connected through a microfluidic channel; The built-in control valve between the detection zones 113 is closed and connected. Combined with the pre-embedded reagent of LAMP reaction, the chip can complete the steps of sample lysis, mixing, reaction, etc. in an integrated manner, and realize sample input and result output, which solves the problems of complex structure of detection chips and many peripherals in the prior art, and reduces the cost of The probability of contamination during the detection process saves the detection time for the experimenter and improves the detection efficiency.

The embodiment of the present application also discloses a method for preparing a nucleic acid detection chip. FIG. 6 is a schematic flowchart of a method for preparing a nucleic acid detection chip provided by the embodiment of the present application. Please refer to FIG. 6 , and the preparation method includes:

S601 : Obtain a reaction substrate, where the reaction substrate includes a first reaction substrate 101 and a second reaction substrate 102 .

In the embodiments of the present application, a transparent polymer material is used as the preparation material of the chip reaction substrate, and the material includes polyethylene PE, polypropylene PP, polycarbonate PC, polyethylene terephthalate (PET), polystyrene Ethylene (PS), polymethyl dimethacrylate (PMMA), acrylonitrile-butadiene-polyethylene copolymer (ABS), etc.

S603 : forming a reaction chamber on the first reaction substrate 101 and the second reaction substrate 102 .

In the embodiment of the present application, a hot pressing process is used to fabricate a reaction chamber in the first reaction substrate 101 and the second reaction substrate 102 . The reaction chamber includes a plurality of reaction functional regions, and the plurality of reaction functional regions are communicated with each other through a microfluidic channel. Specifically, draw the desired pattern according to the structure of the reaction cavity, then make a mold according to the pattern, then cut, wash and dry the transparent polymer material, and transfer the pattern on the mold onto it to obtain a cavity containing The first reaction substrate 101 and the second reaction substrate 102 of the structure.

S605: Load the reaction reagent in the reaction chamber.

In the examples of the present application, the LAMP reaction detection reagents are loaded into each reaction functional zone in the reaction chamber of any reaction substrate, and then the reaction substrate is placed in a freeze dryer for freeze drying at different temperatures to obtain a prepackaged The substrate for burying the reagents.

S607: A control valve is arranged in the microchannel, and the control valve is a solid phase change material encapsulated in the microchannel.

In the embodiment of the present application, a low-melting-point solid-state phase change material is used as a physical separation between the reaction functional areas on the detection chip, and serves as a control valve inside the chip. The solid phase change material should be made of materials that are relatively stable in nature and are not easy to react with the reaction reagents and reaction samples, such as paraffin, butter, rosin, tar and other materials, and can also be a mixture of a variety of the above materials. Specifically, the melted solid phase change material, such as paraffin, is spot-coated in the microfluidic channel connected to the reaction chamber.

S609: Seal the first reaction substrate 101 and the second reaction substrate 102 together.

In the embodiment of the present application, the first reaction substrate 101 and the second reaction substrate 102 are combined by thermocompression, that is, the preparation of the reaction functional area in the detection chip is completed, and a sealing cover is added to the sample inlet 104 to complete the packaging.

S611: Obtain a temperature control substrate 2, where the temperature control substrate 2 includes a support layer and a temperature control layer.

In the embodiment of the present application, the temperature control layer is provided on the support layer, and the temperature control layer is provided with a temperature sensor 202 and a heating electrode 201 . According to the position of each reaction functional area on the reaction substrate, design the structure of the heating electrode 201 and the temperature sensor 202 in the corresponding temperature control layer, draw the required pattern, and then make a chrome mask. The heating electrode 201 preferably has a complementary symmetrical structure. . A commercialized ITO glass sheet is used as the temperature control substrate 2, and the ITO glass sheet includes a glass substrate and an ITO thin film indium tin oxide semiconductor transparent conductive film layer, which is prepared by a photolithography method. Specifically, first, a layer of positive photoresist is coated on the surface of the ITO glass, and the required temperature control layer structure is obtained through process steps such as exposure, development, etching, and degumming. In the above process, the etching solution is preferably prepared by mixing dilute hydrochloric acid and ferric chloride in a mass ratio of 10:1.

S613: Form the substrate mounting groove 103 on the reaction substrate.

In the embodiment of the present application, a laser engraving machine is used to engrave grooves on any reaction substrate for placing the temperature control substrate 2 .

S615 : Install the temperature control substrate 2 in the substrate mounting groove 103 .

In the embodiment of the present application, the temperature control substrate 2 with the electrode pattern is in close contact with the reaction functional area by a plug-in structure, which can be directly inserted and pulled out, which is convenient for replacement. It is connected to the heating electrode 201 to realize heating control.

The examples of this application also disclose a nucleic acid detection method, which is applied to a nucleic acid detection chip. It should be noted that the sample in the following embodiments refers to cells, cell lysates or cell extracts, cell materials or Solutions of one or more molecules of viral material such as polypeptides or nucleic acids; or containing non-naturally occurring nucleic acids such as cDNA, but also any external solution that may or may not contain pathogen cells, cellular components or nucleic acids. FIG. 7 is a schematic flowchart of a nucleic acid detection method provided in an embodiment of the present application. Please refer to FIG. 7 . The method includes:

S701: The sample to be tested is introduced into the lysis zone 111 through the injection port 104 for lysis to obtain a lysis sample.

In the embodiment of the present application, the sample to be tested is dissolved in the diluent, introduced into the lysis zone 111 through the injection port 104, heated in the lysis zone 111, and fully reacted for a period of time to obtain the lysed sample.

S703: Heating the first microchannel 115 to melt the control valve to conduct the lysis zone 111 and the pre-amplification zone 112, and the lysed sample enters the pre-amplification zone 112 for pre-amplification to obtain a pre-amplified sample.

In the embodiment of the present application, after the sample to be tested is completely cracked, the first microchannel 115 is heated, so that the solid phase change material serving as the control valve in the first microchannel 115 is melted, and the cracking area 111 and the pre-amplification area are connected. 112, allowing the lysed sample to enter the pre-amplification area 112 for reaction.

S705: Heating the second microchannel 116 to melt the control valve to conduct the pre-amplification region 112 and the amplification detection region 113, and the pre-amplification sample enters the amplification detection region 113 for reaction.

In the embodiment of the present application, the second microfluidic channel 116 is heated to melt the solid phase change material serving as the control valve in the second microfluidic channel 116, and the pre-amplification region 112 and the amplification detection region 113 are connected to make the pre-amplification region 113 connected. The latter test sample enters the amplification detection area 113 for reaction.

S707: Detect whether a specific target exists in each reaction pool in the amplification detection area 113.

In the embodiment of the present application, the pre-amplified sample further includes controlling the liquid overflowing the reaction tank to enter the waste liquid area 114 before the reaction in the reaction tank, and then the pre-amplified sample is subjected to isothermal amplification pre-buried in the reaction tank under a preset temperature environment. The primers, DNTPs and enzymes required for the reaction are used for isothermal amplification reaction. Each reaction cell in the amplification detection area 113 is calibrated by an optical method. Optionally, a fluorescent light source is used to excite each reaction cell in the amplification detection area 113; optionally, a dye is used for each reaction cell in the amplification detection area 113. Calibration is performed. Then, the calibration reaction intensity in each reaction cell is detected, that is, the fluorescence intensity or the dye absorption degree in each reaction cell is detected, and whether there is a specific target is determined based on the calibration reaction intensity in each reaction cell.

The above are only preferred embodiments of the present application, and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection of the present application. within the range.

Claims (12)

1. A nucleic acid detecting chip characterized by comprising: a reaction substrate and a temperature control substrate (2),

a reaction cavity is arranged on the reaction substrate;

the reaction substrate is also provided with a sample inlet (104), and the sample inlet (104) is communicated with the reaction cavity;

the reaction substrate comprises a first reaction substrate (101) and a second reaction substrate (102), the reaction cavity is at least partially arranged on the first reaction substrate (101), and the first reaction substrate (101) and the second reaction substrate (102) are covered to enable the reaction cavity to form a closed cavity structure;

and a substrate mounting structure is arranged on the first reaction substrate (101) and/or the second reaction substrate (102), and the temperature control substrate (2) is arranged in the substrate mounting structure.

2. The nucleic acid detecting chip according to claim 1, wherein a plurality of reaction functional regions are provided in the reaction chamber, and different reaction reagents having different functions are pre-embedded in different reaction functional regions;

the reaction functional zone comprises a lysis zone (111), a pre-amplification zone (112) and an amplification detection zone (113), wherein the lysis zone (111) is communicated with the pre-amplification zone (112) through a first micro-channel (115), the pre-amplification zone (112) is communicated with the amplification detection zone (113) through a second micro-channel (116), and a control valve is arranged in the first micro-channel (115) and the second micro-channel (116).

3. The nucleic acid detecting chip according to claim 2, wherein the control valve is a solid phase change material potted in the first micro flow channel (115) and the second micro flow channel (116), and a melting point of the solid phase change material is lower than a temperature which the nucleic acid detecting chip is resistant to.

4. The nucleic acid detecting chip of claim 3, wherein the solid phase change material is at least one of paraffin, butter, rosin, and asphalt.

5. The nucleic acid detecting chip according to claim 1, wherein the substrate mounting structure is a substrate mounting groove (103), and the temperature-controlled substrate (2) is disposed in the substrate mounting groove (103).

6. The nucleic acid detecting chip according to claim 5, wherein the temperature-controlled substrate (2) includes a support layer and a temperature control layer, the temperature control layer being disposed on the support layer;

the temperature control layer is provided with a temperature sensor (202) and a heating electrode (201).

7. The nucleic acid detecting chip according to claim 6, wherein: the heating electrode (201) includes a first heating electrode for heating the first microchannel (115) and the pre-amplification zone (112), and a second heating electrode for heating the second microchannel (116) and the amplification detection zone (113).

8. A method for preparing a nucleic acid detection chip, the method comprising:

obtaining a reaction substrate, wherein the reaction substrate comprises a first reaction substrate (101) and a second reaction substrate (102);

manufacturing reaction cavities on the first reaction substrate (101) and the second reaction substrate (102); the reaction cavity comprises a plurality of reaction functional areas which are communicated through a micro-channel;

loading a reaction reagent in the reaction chamber;

arranging a control valve in the micro flow channel, wherein the control valve is made of a solid phase-change material encapsulated in the micro flow channel;

hermetically bonding the first reaction substrate (101) and the second reaction substrate (102);

obtaining a temperature control substrate (2), wherein the temperature control substrate (2) comprises a supporting layer and a temperature control layer, and the temperature control layer is provided with a temperature sensor (202) and a heating electrode (201);

manufacturing a substrate mounting groove (103) on the reaction substrate;

and installing the temperature control substrate (2) in the substrate installation groove (103).

9. A method for detecting a nucleic acid, which is applied to the nucleic acid detecting chip according to any one of claims 1 to 8, comprising:

a sample to be detected is led into a cracking area (111) through a sample inlet (104) to be cracked to obtain a cracked sample;

heating the first micro-channel (115) to enable a control valve to melt and conduct the cracking region (111) and the pre-amplification region (112), and enabling the cracking sample to enter the pre-amplification region (112) for pre-amplification to obtain a pre-amplification sample;

heating the second micro-channel (116), so that the control valve is melted and conducted with the pre-amplification area (112) and the amplification detection area (113), and the pre-amplification sample enters a reaction pool in the amplification detection area (113) for reaction;

detecting the presence of a specific target in each reaction cell within the amplification detection zone (113).

10. The method of claim 9, further comprising controlling liquid overflowing the reaction cell to enter a waste liquid zone (114) before the pre-amplified sample enters the reaction cell in the amplification detection zone for reaction.

11. The method for detecting nucleic acid according to claim 10, wherein the pre-amplified sample is subjected to isothermal amplification reaction with the pre-embedded primer for isothermal amplification reaction, DNTP and enzyme required for reaction in the reaction cell under a preset temperature environment.

12. The method for detecting nucleic acid according to claim 9, wherein the detecting whether a specific target exists in each reaction cell in the amplification detection zone (113) comprises:

calibrating each reaction cell in the amplification detection zone (113) by an optical method;

detecting the calibration reaction intensity in each reaction tank;

and judging whether a specific target exists or not based on the calibrated reaction intensity in each reaction pool.

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