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 detecting chip includes: the device comprises a reaction substrate and a temperature control substrate, wherein a reaction cavity is arranged on the reaction substrate; the reaction substrate is also provided with a sample inlet which is communicated with the reaction cavity; the reaction substrate comprises a first reaction substrate and a second reaction substrate, at least part of the reaction cavity is arranged on the first reaction substrate, and the first reaction substrate and the second reaction substrate are covered to form a closed cavity structure in the reaction cavity; and a substrate mounting structure is arranged 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 is simple in structure, integrates the reaction substrate and the temperature control substrate together, and can complete the detection process without additionally arranging matched equipment.

Description

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

Technical Field

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.

Background

The micro-fluidic chip is a technology for accurately controlling and controlling micro-scale fluid, and can integrate basic operation units of sample preparation, reaction, separation, detection and the like in the analysis process on a chip with a square centimeter, so as to automatically complete the whole analysis process. The method has the advantages of less sample and reagent consumption, high detection speed, high sensitivity and low cost, so that the method is wide in application and research range and rapid in progress in pathogen detection at present. The micro-fluidic chip technology is introduced into nucleic acid detection for biomedical analysis, environmental detection and forensic identification, the complicated sample pretreatment and amplification product steps in the traditional nucleic acid detection can be simplified and integrated, the detection time can be shortened, the reagent and sample consumption can be reduced, the defects of complicated operation, high cost and the like in the traditional method are overcome, and the method can be used for instant detection in a biological laboratory and field portable application.

Among various nucleic acid amplification detection methods, a nucleic acid detection system combining a loop-mediated isothermal amplification (LAMP) technology and a microfluidic technology has the most potential to be applied to the field rapid diagnosis of biomolecules. LAMP is a novel isothermal amplification technique developed by Notomi et al, Japan. The technology designs four specific primers according to six gene segments, and the primers are heated at 60-65 ℃ under the action of Bst polymerase, and amplification can be completed within 20-60 min. Isothermal amplification of nucleic acids does not require cycling between different temperatures, as compared to conventional Polymerase Chain Reaction (PCR). Each thermal cycle step of the PCR reaction needs accurate temperature control, and only a single reaction temperature is needed in the whole process of the isothermal amplification of the nucleic acid, so that the isothermal amplification of the nucleic acid is free from the dependence on complex instruments, and the reaction process is simpler and more effective. In recent years, the combination of loop-mediated isothermal amplification with microfluidic chips has been studied for the detection of pathogenic microorganisms, cancer biomarkers and other target genes.

For most microfluidic systems, the channel network is fixed at the time of micromachining, often requiring pumping, valve control, or control using external forces of electric and magnetic fields. Therefore, the fluid driving and controlling technology in the micro-channel is still a challenge, and how to reduce the peripheral devices and increase the intelligent integration in the micro-fluidic system is an urgent problem to be solved.

Disclosure of Invention

The invention aims to solve the technical problems of complex structure and more peripheral devices of a nucleic acid detection chip in the prior art.

In order to solve the above technical problem, in a first aspect, an embodiment of the present application discloses a nucleic acid detecting chip, including: a reaction substrate and a temperature control substrate,

a reaction cavity is arranged on the reaction substrate;

the reaction substrate is also provided with a sample inlet which is communicated with the reaction cavity;

the reaction substrate comprises a first reaction substrate and a second reaction substrate, at least part of the reaction cavity is arranged on the first reaction substrate, and the first reaction substrate and the second reaction substrate are covered to form a closed cavity structure in the reaction cavity;

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

Furthermore, a plurality of reaction functional areas are arranged in the reaction cavity, and reaction reagents with different functions are pre-embedded in different reaction functional areas;

the reaction functional zone comprises a cracking zone, a pre-amplification zone and an amplification detection zone, wherein the cracking zone is communicated with the pre-amplification zone through a first micro-channel, the pre-amplification zone is communicated with the amplification detection zone through a second micro-channel, and a control valve is arranged in the first micro-channel and the second micro-channel.

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

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-controlled substrate comprises a supporting layer and a temperature control layer, and the temperature control layer is arranged on the supporting 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 configured to heat the first micro flow channel and the pre-amplification region, and the second heating electrode is configured to heat the second micro flow channel and the amplification detection region.

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

obtaining a reaction substrate, wherein the reaction substrate comprises a first reaction substrate and a second reaction substrate;

manufacturing reaction cavities on the first reaction substrate and the second reaction substrate; 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 and the second reaction substrate;

the method comprises the steps of obtaining a temperature control substrate, wherein the temperature control substrate comprises a supporting layer and a temperature control layer, and the temperature control layer is provided with a temperature sensor and a heating electrode;

manufacturing a substrate mounting groove on the reaction substrate;

and installing the temperature control substrate 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, and includes:

introducing a sample to be detected into a cracking area through an injection port for cracking to obtain a cracked sample;

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

heating the second micro-channel to enable the control valve to melt and conduct the pre-amplification area and the amplification detection area, and enabling the pre-amplification sample to enter a reaction tank in the amplification detection area for reaction;

and detecting whether the specific target exists in each reaction pool in the amplification detection area.

Further, before the pre-amplified sample enters the reaction cell in the amplification detection zone for reaction, the method further comprises controlling the liquid overflowing the reaction cell to enter a waste liquid zone.

Further, the pre-amplification sample is subjected to isothermal amplification reaction with a primer for isothermal amplification reaction, DNTP and enzyme required by the reaction, which are pre-embedded in the reaction tank, in a preset temperature environment.

Further, the detecting whether the specific target exists in each reaction cell in the amplification detection area comprises:

calibrating each reaction tank in the amplification detection area by adopting 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.

By adopting the technical scheme, the nucleic acid detection chip, the preparation method and the nucleic acid detection method have the following beneficial effects:

according to the nucleic acid detection chip, the reaction cavity is formed in the reaction substrate, a nucleic acid detection process can be integrally completed in the reaction cavity, the temperature control substrate is arranged in the substrate mounting structure on the reaction substrate, and the temperature control substrate can directly regulate and control the reaction substrate according to the requirement of the detection process; the nucleic acid detection chip is simple in structure, integrates the reaction substrate and the temperature control substrate together, and can complete the detection process without additionally arranging matched equipment.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a schematic diagram of a nucleic acid detecting chip according to an embodiment of the present application;

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

FIG. 3 is a schematic diagram of a reaction cell configuration according to one embodiment of the present application;

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

fig. 5 is a schematic flow chart of a temperature control method according to an embodiment of the present disclosure;

FIG. 6 is a schematic flow chart of a method for preparing a nucleic acid detecting chip according to an embodiment of the present disclosure;

FIG. 7 is a schematic flow chart of a method for detecting nucleic acid according to an embodiment of the present disclosure.

The following is a supplementary description of the drawings:

101-a first reaction substrate; 102-a second reaction substrate; 103-substrate mounting groove; 104-a sample inlet; 111-a cleavage zone; 112-a pre-amplification region; 113-amplifying the detection zone; 114-a waste liquid zone; 115-a first microchannel; 116-a second microchannel; 120-a reaction plate; 121-a first film; 122-a second film; 2-temperature control substrate; 201-heating electrodes; 202-temperature sensor.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It is to be understood that the embodiments described are only a few embodiments of the present application and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic may be included in at least one implementation of the present application. In the description of the present application, it is to be understood that the terms "upper", "lower", "top", "bottom", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are only for convenience in describing the present application and simplifying the description, and do not indicate or imply that the referred devices or elements must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present application. Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Moreover, the terms "first," "second," and the like are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the application described herein are capable of operation in sequences other than those illustrated or described herein.

The diagnosis technology based on nucleic acid is one of the most potential methods in the current molecular diagnosis technology, and has wide application in the fields of disease detection, food safety and the like. In the detection method of nucleic acid, Polymerase Chain Reaction (PCR) technology is used to amplify a specific nucleic acid fragment so as to achieve the purpose of detecting a specific target nucleic acid fragment. Typically, the detection of nucleic acids requires three steps: nucleic acid extraction, nucleic acid amplification and nucleic acid detection. The three steps of the traditional nucleic acid detection method are separated, the problems of long preparation and analysis time, easy pollution, limited sensitivity, complex procedure and operation and the like exist, and the integration of nucleic acid extraction, amplification and detection by a microfluid technology is helpful for solving the technical and analysis limitations related to nucleic acid detection in practical application.

As shown in fig. 1, the embodiment of the present application discloses a nucleic acid detection chip, including: the reaction substrate is provided with a reaction cavity; 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, at least part of the reaction cavity is arranged on the first reaction substrate 101, and the first reaction substrate 101 and the second reaction substrate 102 are covered to form a closed cavity structure; 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.

According to the nucleic acid detection chip disclosed by the embodiment of the application, the reaction cavity is arranged in the reaction substrate, the nucleic acid detection process can be integrally completed in the reaction cavity, the temperature control substrate 2 is arranged in the substrate mounting structure on the reaction substrate, and the temperature control substrate 2 can directly regulate and control the reaction substrate according to the requirement of the detection process; the nucleic acid detection chip is simple in structure, integrates the reaction substrate and the temperature control substrate 2 together, and can complete the detection process without additionally arranging matched 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, which is mainly transparent and facilitates technicians to visually track the reaction state of the sample during the operation process, and the reaction substrate may be made of an inorganic material, such as glass; it may also be an organic polymer material such as Polyethylene (PE), polypropylene (PP), Polycarbonate (PC), polyethylene terephthalate (PET), Polystyrene (PS), polymethyl methacrylate (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 of an integral structure and is formed through an injection molding process, the interior of the reaction substrate is of 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, the reaction cavity structure can be partially or completely arranged on the first reaction substrate 101, correspondingly, the reaction cavity structure can be completely or partially arranged on the second reaction substrate 102, the two reaction substrates are buckled together to form the reaction cavity structure inside, and then a whole is formed by sealing, the mode for sealing the two reaction substrates comprises one or more modes of laser welding, hot-press sealing, high-strength chemical adhesive bonding or ultrasonic welding, and other sealing modes can be adopted as long as the two reaction substrates can be sealed and connected into a whole. The reaction substrate is provided with a sample inlet 104 communicated with the reaction cavity, optionally, one 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; optionally, the sample inlet 104 is disposed on any one of the reaction substrates. The sample to be detected enters the reaction cavity through the sample inlet 104, and the steps of sample cracking, mixing, reaction and the like can be integrally completed in the reaction cavity. 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 in a clamping, bonding, screwing or embedding mode.

In the embodiment of the present application, the nucleic acid detecting chip is usually a disposable product, i.e., the nucleic acid detecting chip is discarded after a nucleic acid detection, and therefore the material of the reaction substrate should comprehensively consider factors such as use cost, processing difficulty and degradation difficulty. Temperature control base plate 2 can recycle as the temperature control core part, therefore, temperature control base plate 2 can adopt detachable mode and reaction substrate to be connected, and when nucleic acid detection accomplished the back, can pull down cyclic utilization with temperature control base plate 2.

As shown in fig. 2, a plurality of reaction functional regions are arranged in the reaction chamber, and different reaction reagents with different functions are pre-embedded in different reaction functional regions; the reaction functional region comprises a lysis region 111, a pre-amplification region 112 and an amplification detection region 113, the lysis region 111 is communicated with the pre-amplification region 112 through a first microchannel 115, the pre-amplification region 112 is communicated with the amplification detection region 113 through a second microchannel 116, and control valves are arranged in the first microchannel 115 and the second microchannel 116.

In the embodiment of the present application, a lysis zone 111, a pre-amplification zone 112, and an amplification detection zone 113 are disposed in the reaction chamber, and a reaction reagent for implementing a related function is pre-embedded in each reaction functional zone, wherein the types of the reaction reagent include a lysate, a primer for isothermal amplification reaction, an enzyme, and the like. The above-mentioned reaction reagent is usually made into freeze-dried powder by means of freeze-drying technology, and is pre-sealed in the chip so as to make the reaction reagent be stored at normal temp. for a long time and retain high enzyme activity. When the nucleic acid detection chip is used, a sample enters the reaction cavity from the sample inlet 104, so that a closed space is formed inside the chip, and cracking, pre-amplification and amplification detection are sequentially performed. Each reaction functional area is of a closed cavity structure, the reaction functional areas are communicated through a micro-channel, a control valve is arranged in the micro-channel, and after a sample finishes reaction in the previous reaction functional area, the control valve is opened through control, so that the sample enters the next reaction functional area for reaction.

As shown in fig. 2, the control valve is a solid phase-change material encapsulated in the first micro flow channel 115 and the second micro flow channel 116, and the melting point of the solid phase-change material is lower than the temperature tolerance of the nucleic acid detection chip.

In the embodiment of the present application, the solid phase-change material is a solid compound or a mixture with a lower melting point in a normal state, and the melting point of the solid phase-change material should be lower than the limit test temperature of the nucleic acid detection chip, and preferably, the melting point of the solid phase-change material is not higher than the reaction temperature of the nucleic acid detection chip during detection. The micro-flow channel is plugged by using a low-melting-point solid phase-change material as a control valve in a normal state, the control valve is integrated in the detection chip, a micro-flow pump, a control valve and the like do not need to be arranged outside, the number of devices outside is reduced, the nucleic acid detection process is simplified, the nucleic acid detection chip can be applied to more scenes, and the application range of the chip is expanded. In addition, compare various entity valving among the prior art, the control valve of this application is changeed in realizing embedding in the chip, and the greatly reduced preparation technology degree of difficulty, the phase change material of low melting point easily obtains simultaneously, has greatly reduced the cost of manufacture of chip.

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

In the embodiment of the present application, the low melting point phase change material which is solid in a normal state has more types, and the control valve is disposed in the reaction chamber, so that the solid phase change material has more stable properties and is not easy to react with the reaction sample, such as paraffin, butter, rosin, and asphalt, or a mixture of a plurality of materials.

As shown in fig. 1, the substrate mounting structure is a substrate mounting groove 103, and the temperature-controlled substrate 2 is disposed in the substrate mounting groove 103.

In the embodiment of the present application, a substrate mounting groove 103 is formed in the reaction substrate, and the substrate mounting groove 103 may be disposed on the first reaction substrate 101, the second reaction substrate 102, or both the first and second reaction substrates. The substrate mounting groove 103 is a groove structure with at least one side opened, and the temperature control substrate 2 is mounted in the substrate mounting groove 103 in a clamping, bonding, screwing or embedding manner. For example, the temperature control substrate 2 and the substrate mounting structure adopt a card insertion type structure to be in close contact, and can be directly inserted and pulled out, thereby being convenient for replacement.

The temperature control substrate 2 comprises a support layer and a temperature control layer, and the temperature control layer is arranged 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 detachment and recovery of the temperature control substrate 2, the temperature control layer for temperature control may be disposed on a supporting layer for supporting. The supporting layer can be made of transparent ITO glass or high polymer materials. One or more temperature sensors 202 are arranged on the temperature control layer, and the temperature sensors 202 are used for feeding back the temperature of each reaction functional area in real time; the temperature control layer is also provided with a plurality of heating electrodes 201 for heating the reaction functional area and the control valve. The temperature control layer is attached to the supporting layer through a sputtering process, and the material of the temperature control layer comprises Au/Ti, Pt/Ti and the like.

As shown in fig. 1, 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.

In the embodiment of the application, two heating electrodes 201 are arranged on the temperature control layer, when the detection chip is used for detection, after the sample to be detected is cracked in the cracking region 111, the first heating electrode is controlled to heat the first micro-channel 115 and the pre-amplification region 112, so that the control valve in the first micro-channel 115 is heated and melted, and the sample to be detected enters the pre-amplification region 112 from the cracking region 111 through the first micro-channel 115 for reaction; after the pre-amplification of the sample to be detected in the pre-amplification region 112 is completed, the second heating electrode is controlled to heat the second microchannel 116 and the amplification detection region 113, so that the control valve in the second microchannel 116 is heated and melted, and the sample to be detected enters the amplification detection region 113 from the pre-amplification region 112 through the second microchannel 116 to react.

In the embodiment of the present application, the temperature control system is disposed on the temperature control layer, fig. 4 is a schematic structural diagram of the temperature control system in one embodiment of the present application, and as shown in fig. 4, the micro control unit MCU and the temperature control unit interactively control the heating electrodes 201 corresponding to each reaction functional region to sequentially switch and heat, so that the detection chip completes lysis, mixing, pre-amplification, mixing, and amplification detection. Fig. 5 is a schematic flow chart of a temperature control method according to an embodiment of the present application, please refer to fig. 5, in which the temperature control method includes:

s501: initializing a temperature control system;

s503: the temperature sensor 202 feeds back the temperature of the corresponding reaction functional region;

s505: calculating a deviation value between the feedback temperature and a preset temperature;

s507: whether the deviation value is smaller than an initial set value or not; if yes, go to step S509; if not, go to step S511;

s509: entering a timer interrupt program;

s511: calling a heating program and outputting a heating control signal;

s513: the heating electrode 201 is controlled according to the control signal.

In this application embodiment, through the switching of miniature control unit MCU output PWM signal control triode, and then control each temperature control unit. When the triode is conducted, the temperature control functional area is electrified and starts to be heated; when the triode is disconnected, the temperature control functional area is powered off to stop heating.

As shown in FIG. 3, a reaction plate 120 is disposed in the amplification detection zone 113, and a plurality of through holes are formed in the reaction plate 120; a first film 121 is arranged on one side of the reaction plate 120, and a second film 122 is arranged on the other side of the reaction plate 120; the first film 121 is provided with a plurality of small holes, the number of the small holes is equal to that of the through holes, and the positions of the small holes correspond to the positions of the through holes one by one; the first film 121 and the second film 122 close the through holes to form a reaction cell set, and the reaction cell set comprises a plurality of reaction cells.

In the embodiment of the present application, the two sides of the through holes are sealed by the film, so that each through hole forms a reaction chamber, the small hole on the first film 121 is used for allowing the liquid with the nucleic acid of the test sample to enter the reaction chamber, and limits the outflow of the liquid entering the reaction chamber to a certain extent, and meanwhile, the interaction of the liquid between different through holes can be prevented, thereby further ensuring the smooth progress of the amplification reaction.

The reaction pool set comprises at least two reaction pool groups, and at least three reaction pools are arranged in any one reaction pool group.

In the embodiment of the application, at least two groups of reaction pools are arranged, each group of reaction pools comprises a negative control group and one or more test groups, each group is not lower than three multiple holes, single target or multiple target detection of one sample is realized, and the condition that the subsequent test result is inaccurate due to the influence of external factors on the reaction process is reduced.

The reaction functional region further includes a waste liquid region 114, and the waste liquid region 114 is in communication with the amplification detection region 113.

In the embodiment of this application, waste liquid district 114 mainly used collects and augments unnecessary liquid in the chamber, reduce a large amount of liquid and be detained and influence nucleic acid amplification reaction process in augmenting the detection zone 113, waste liquid district 114's quantity can be one to a plurality of, and is preferred, waste liquid district 114 sets up two and corresponds the both sides that set up at augmenting the detection zone 113, so that follow-up operation in-process both sides are collected simultaneously and are got into unnecessary mixed liquid in augmenting the detection zone 113, further be favorable to shortening reaction process's total time.

In the nucleic acid detecting chip of the embodiment of the present application, the reaction functional regions are sequentially arranged from top to bottom and are a lysis region 111, a pre-amplification region 112, an amplification region detection region and a waste liquid region 114. The sample inlet 104, the lysis zone 111, the pre-amplification zone 112, the amplification zone detection zone and the waste liquid zone 114 are communicated through a micro-channel; control valves are arranged between the lysis zone 111 and the pre-amplification zone 112, and between the pre-amplification zone 112 and the amplification detection zone 113 to realize closing and communication. The chip is combined with the pre-embedded reagent of LAMP reaction, so that the steps of sample cracking, mixing, reaction and the like can be integrally completed, the sample can be fed and discharged, the problems of complex structure and more peripheral devices of the detection chip in the prior art are solved, the probability of pollution in the detection process is reduced, the detection time is saved for experimenters, and the detection efficiency is improved.

The embodiment of the present application further discloses a method for preparing a nucleic acid detection chip, fig. 6 is a schematic flow chart of the method for preparing a nucleic acid detection chip provided in the embodiment of the present application, please refer to fig. 6, and the method includes:

s601: reaction substrates are obtained, which include a first reaction substrate 101 and a second reaction substrate 102.

In the embodiment of the present application, a transparent polymer material is used as a material for preparing a chip reaction substrate, and the material includes polyethylene PE, polypropylene PP, polycarbonate PC, polyethylene terephthalate (PET), Polystyrene (PS), polymethyl methacrylate (PMMA), acrylonitrile-butadiene-polyethylene copolymer (ABS), and the like.

S603: reaction chambers are fabricated on the first reaction substrate 101 and the second reaction substrate 102.

In the embodiment of the present application, a reaction cavity is formed in the first reaction substrate 101 and the second reaction substrate 102 by using a hot pressing process, the reaction cavity includes a plurality of reaction functional regions, and the plurality of reaction functional regions are communicated with each other by micro channels. Specifically, drawing a required graph according to the structure of the reaction cavity, then manufacturing a mold according to the graph, cutting, washing and drying the transparent high polymer material, and transferring the graph on the mold to obtain a first reaction substrate 101 and a second reaction substrate 102 which comprise cavity structures.

S605: a reaction chamber is loaded with a reaction reagent.

In the embodiment of the application, the LAMP reaction detection reagent is loaded in each reaction functional region in the reaction cavity of any reaction substrate, and then the reaction substrate is placed in a freeze dryer to be freeze-dried at different temperatures, so as to obtain the substrate in which the reaction reagent is pre-embedded.

S607: and a control valve is arranged in the micro-channel and is made of a solid phase-change material encapsulated in the micro-channel.

In the embodiment of the application, the reaction functional areas on the detection chip are physically separated by low-melting-point solid phase change materials, and the reaction functional areas play a role of a control valve inside the chip. The solid phase-change material should be a material which has stable properties and is not easy to react with the reaction reagent and the reaction sample, such as paraffin, butter, rosin, asphalt and the like, or a mixture of a plurality of materials. Specifically, a molten solid phase-change material, such as paraffin, is spotted in a microchannel connecting the reaction chambers.

S609: the first reaction substrate 101 and the second reaction substrate 102 are hermetically bonded.

In the embodiment of the present application, the first reaction substrate 101 and the second reaction substrate 102 are thermally pressed to complete the preparation of the reaction functional region in the detection chip, and a sealing cap is added to the sample inlet 104 to complete the packaging.

S611: and acquiring a temperature control substrate 2, wherein the temperature control substrate 2 comprises a supporting layer and a temperature control layer.

In the embodiment of the present application, the temperature control layer is disposed 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, the structures of the heating electrode 201 and the temperature sensor 202 in the corresponding temperature control layer are designed, the required graph is drawn, then a chromium plate mask is manufactured, and the heating electrode 201 is preferably of a complementary symmetrical structure. A commercialized ITO glass sheet is used as the temperature control substrate 2, the ITO glass sheet comprises a glass substrate and an ITO thin film indium tin oxide semiconductor transparent conductive film layer, and the ITO glass sheet is prepared by a photoetching method. Specifically, a layer of positive photoresist is coated on the surface of the ITO glass, and the required temperature control layer structure is obtained through the process steps of exposure, development, etching, photoresist removal and the like. In the above process, the etching solution is preferably diluted hydrochloric acid and ferric chloride according to a mass ratio of 10: 1, mixing and preparing.

S613: a substrate mounting groove 103 is formed in the reaction substrate.

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

S615: the temperature controlled substrate 2 is mounted in the substrate mounting groove 103.

In the embodiment of the application, the temperature control substrate 2 with the electrode pattern and the reaction functional area adopt the card insertion type structure to be in close contact with each other, so that the electrode pattern can be directly inserted and pulled out, the replacement is convenient, and the external circuit is connected with the temperature sensor 202 and the heating electrode 201 on the temperature control substrate 2 through the metal shrapnel to realize the heating control.

The embodiment of the application also discloses a nucleic acid detection method, which is applied to a nucleic acid detection chip, and it should be noted that in the following embodiments, the sample means a solution of one or more molecules of cells, cell lysate or cell extract, cell material or virus material such as polypeptide or nucleic acid; or contain non-naturally occurring nucleic acids such as cDNA, and may be any external solution that may or may not contain pathogen cells, cellular components, or nucleic acids. Fig. 7 is a schematic flow chart of a method for detecting nucleic acid according to an embodiment of the present application, please refer to fig. 7, which includes:

s701: and introducing the sample to be detected into a cracking area 111 through the sample inlet 104 for cracking to obtain a cracked sample.

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

S703: and heating the first micro-channel 115 to enable the 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.

In the embodiment of the present application, after the sample to be tested is completely cracked, the first micro flow channel 115 is heated to melt the solid phase-change material serving as the control valve in the first micro flow channel 115, and the cracking region 111 and the pre-amplification region 112 are conducted to allow the cracked sample to enter the pre-amplification region 112 for reaction.

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

In the embodiment of the present application, the second micro flow channel 116 is heated to melt the solid phase change material serving as the control valve in the second micro flow channel 116, and the pre-amplification region 112 and the amplification detection region 113 are conducted, so that the pre-amplified test sample enters the amplification detection region 113 for reaction.

S707: detecting the presence of the specific target in each reaction cell within the amplification detection zone 113.

In the embodiment of the present application, before the pre-amplification sample reacts in the reaction tank, the method further includes controlling the liquid overflowing from the reaction tank to enter the waste liquid region 114, and then performing isothermal amplification reaction on the pre-amplification sample, the primer for isothermal amplification reaction, the DNTP, and the enzyme required for reaction, which are pre-embedded in the reaction tank, in a preset temperature environment. Calibrating each reaction cell in the amplification detection zone 113 by an optical method, and optionally exciting each reaction cell in the amplification detection zone 113 by a fluorescent light source; optionally, a dye is used to calibrate each reaction cell in the amplification detection zone 113. Then, the calibration reaction intensity in each reaction tank is detected, namely the fluorescence intensity or the absorption degree of the dye in each reaction tank is detected, and whether the specific target exists is judged based on the calibration reaction intensity in each reaction tank.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

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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