阅读说明：本技术 一种核酸扩增检测微流控芯片 (Micro-fluidic chip for nucleic acid amplification detection ) 是由 董思聪 刘毅 于 2021-09-23 设计创作，主要内容包括：本发明涉及体外医学领域,尤其为一种核酸扩增检测微流控芯片,包括：微流控芯片；膜,分别覆盖在微流控芯片的上下表面；引物腔室,设于所述微流控芯片上,用于预置引物；反应扩增腔室,设于所述微流控芯片上,与所述引物腔室连接,用于预置扩增反应混合物；检测腔室,设于所述微流控芯片上,与所述反应扩增腔室联通,用于预置荧光检测试剂。本发明,核酸扩增检测微流控芯片采用膜结构封闭,样品不易外泄,不易造成气溶胶污染,影响检测结果,加样方便、样品用量少,且无需使用PCR管,操作方便,适合快速检测,可以精准控制前端与后端反应腔室之间的联通状态,整体结构更轻量化。(The invention relates to the field of in vitro medicine, in particular to a nucleic acid amplification detection microfluidic chip, which comprises: a microfluidic chip; the membranes are respectively covered on the upper surface and the lower surface of the microfluidic chip; the primer chamber is arranged on the microfluidic chip and used for presetting a primer; the reaction amplification chamber is arranged on the microfluidic chip, is connected with the primer chamber and is used for presetting an amplification reaction mixture; and the detection chamber is arranged on the microfluidic chip, is communicated with the reaction amplification chamber and is used for presetting a fluorescence detection reagent. According to the invention, the nucleic acid amplification detection micro-fluidic chip is sealed by adopting a membrane structure, a sample is not easy to leak, aerosol pollution is not easy to cause, the detection result is not influenced, the sample adding is convenient, the sample using amount is less, a PCR tube is not required, the operation is convenient, the micro-fluidic chip is suitable for rapid detection, the communication state between the front-end reaction chamber and the rear-end reaction chamber can be accurately controlled, and the whole structure is lighter.)

1. A nucleic acid amplification detection microfluidic chip is characterized by comprising:

a microfluidic chip (24);

membranes (15) respectively covering the upper and lower surfaces of the microfluidic chip (24);

the primer chamber (16) is arranged on the microfluidic chip (24) and is used for presetting a primer;

the reaction amplification chamber (17) is arranged on the microfluidic chip (24), is connected with the primer chamber (16) and is used for presetting an amplification reaction mixture;

and the detection chamber (18) is arranged on the microfluidic chip (24), is communicated with the reaction amplification chamber (17), and is used for presetting a fluorescence detection reagent.

2. The nucleic acid amplification detection microfluidic chip according to claim 1, further comprising:

the valve hole is arranged on the microfluidic chip (24);

the drain valve (22) and the waterproof breathable film (23) are arranged on the microfluidic chip (24), the drain valve (22) is used for controlling the communication state of the reaction amplification chamber (17) and the detection chamber (18), and the waterproof breathable film (23) is used for controlling the communication state of the primer chamber (16), the reaction amplification chamber (17) and the detection chamber (18) with the external atmosphere.

3. The microfluidic chip for nucleic acid amplification detection according to claim 2, wherein the valve hole comprises:

the sample inlet (20) is connected with the primer chamber (16) and is used for accessing positive pressure sample introduction;

a membrane valve hole (19) connected to the trap (22) and closed at positive pressure;

the air hole (21) is connected with the waterproof breathable film (23) and is used for communicating the primer chamber (16), the reaction amplification chamber (17) and the detection chamber (18) with the outside atmosphere by controlling the waterproof breathable film (23).

4. The microfluidic chip for nucleic acid amplification detection according to claim 3, wherein the microfluidic chip (24) has a valve groove (32) and a membrane groove (31) for correspondingly accommodating the drain valve (22) and the waterproof breathable membrane (23), respectively.

5. The microfluidic chip for nucleic acid amplification detection according to claim 4, wherein the membrane groove (31) further contains a pressure-sensitive membrane (28), and the hydrophobic valve (22) comprises a valve portion pressure-sensitive membrane (29) and a valve portion hydrophobic membrane (30) in the valve groove (32).

Technical Field

The invention relates to the technical field of in-vitro medical detection, in particular to a micro-fluidic chip for nucleic acid amplification detection.

Background

Nucleic acid detection is widely applied to the fields of disease diagnosis, food safety and environmental detection at present, and the general nucleic acid detection process is as follows: weighing a sample, extracting nucleic acid by using the kit, mixing the extracted nucleic acid with an amplification reagent, putting the mixture into a PCR (polymerase chain reaction) instrument for amplification reaction, and then carrying out data analysis. The operation process is complex, human errors are easily caused when a plurality of samples and a plurality of indexes are detected, and the consumed time and the long acting rate are low.

Microfluidic chips are an emerging technology for manipulating and detecting chemical or biological samples on a chip. The microfluidic chip can integrate operation units of sample preparation, reaction, separation, detection and the like on a chip with a few square centimeters, and automatically complete the analysis process by controlling and controlling the fluid in the microchannel network. The micro-fluidic chip technology has the characteristics of small sample volume, high integration level and easy realization of automatic control and high-throughput analysis, so that the biochemical reaction operation on the micro-fluidic chip is more convenient and rapid than the conventional analysis sample pretreatment, and the cost is low. But the UNICOM state between current micro-fluidic chip still can't accurate control front end and the rear end reaction chamber when detecting, is mostly the mutual lock of stereoplasm casing in the aspect of the structure moreover, is unfavorable for the lightweight, also remains further to be improved.

Disclosure of Invention

The present invention aims at providing a micro-fluidic chip for nucleic acid amplification detection to solve the problems in the background art.

In order to achieve the purpose, the invention provides the following technical scheme:

an integrated nucleic acid amplification detection apparatus comprising:

a base frame;

a nucleic acid extraction device mounted on the base frame for lysing and extracting nucleic acid;

the nucleic acid amplification detection micro-fluidic chip is arranged on the base frame and used for detecting the negative and positive of the extracted nucleic acid;

the electromagnetic valve I is arranged on the base frame and is used for controlling the communication state between the nucleic acid extraction device and the nucleic acid amplification detection microfluidic chip;

the electromagnetic valve II is arranged on the base frame and is used for controlling the communication state of the nucleic acid amplification detection micro-fluidic chip and the outside atmosphere;

the pressure supply device is arranged on the base frame and is used for providing positive pressure for the drain valve on the nucleic acid amplification detection micro-fluidic chip to be used as a valve control;

and the control panel is used for driving and controlling the electromagnetic valve I, the electromagnetic valve II and the pressure supply device.

Further, the nucleic acid extraction apparatus includes:

a lysis cassette;

the temperature sensor clamp is sleeved at the bottom of the cracking box and detachably installs the cracking box on the base;

the heating plate is arranged in the cracking box and used for heating the cracking liquid injected into the cracking box;

and the temperature sensing module is arranged on the temperature sensor clamp and used for detecting the temperature of the heating piece for heating the cracking box.

Further, the method also comprises the following steps:

the heat conducting pad is positioned on the heating sheet;

the glass sheet is positioned at the bottom of the cracking box and positioned on the heat conducting pad;

a cushion pad positioned under the heating sheet;

the cracking box cover is arranged at the top of the cracking box;

and the spring clamp is sleeved on the temperature sensor clamp and then connected to the base through a screw.

Furthermore, the bottom of the cracking box is provided with a cracking box outlet.

Further, the nucleic acid amplification detection microfluidic chip comprises:

a microfluidic chip;

the membranes are respectively covered on the upper surface and the lower surface of the microfluidic chip;

the primer chamber is arranged on the microfluidic chip and used for presetting a primer;

the reaction amplification chamber is arranged on the microfluidic chip, is connected with the primer chamber and is used for presetting an amplification reaction mixture;

and the detection chamber is arranged on the microfluidic chip, is communicated with the reaction amplification chamber and is used for presetting a fluorescence detection reagent.

Further, the method also comprises the following steps:

the valve hole is arranged on the microfluidic chip;

the drain valve is used for controlling the communication state of the reaction amplification chamber and the detection chamber, and the waterproof breathable film is used for controlling the communication state of the primer chamber, the reaction amplification chamber and the detection chamber with the external atmosphere.

Further, the valve hole includes:

the sample inlet is connected with the primer chamber and used for accessing positive pressure sample introduction;

the film valve hole is connected with the drain valve and is closed under positive pressure;

and the air hole is connected with the waterproof breathable film and is used for realizing the communication of the primer cavity, the reaction amplification cavity and the detection cavity with the external atmosphere by controlling the waterproof breathable film.

Further, the microfluidic chip is provided with a valve groove and a membrane groove which are respectively used for correspondingly accommodating the drain valve and the waterproof and breathable membrane.

Further, the membrane groove is also provided with a pressure-sensitive membrane, and the drain valve comprises a valve part pressure-sensitive membrane and a valve part drain membrane which are positioned in the valve groove.

Further, the nucleic acid amplification detection microfluidic chip is mounted on the base frame through a chip clamp, a ceramic heating sheet is arranged at a position, corresponding to the lower portion of the reaction amplification chamber, on the chip clamp and used for heating the reaction amplification chamber, exciting light is emitted from the side face of the nucleic acid amplification detection microfluidic chip through an LED lamp, and fluorescence in the detection chamber is conducted to a photodiode through an optical fiber on the chip clamp to obtain an optical signal.

Compared with the prior art, the invention has the beneficial effects that:

the device can complete three steps of nucleic acid extraction, nucleic acid amplification and nucleic acid detection at one time, has the characteristics of smooth whole nucleic acid detection process, convenient operation, high efficiency and the like, is small in size and convenient to carry, and is disposable in disposable design.

According to the nucleic acid extraction device, the lysis solution is cracked in the completely closed chamber formed by the cracking box, and the nucleic acid in the sample is released.

The nucleic acid extraction device disclosed by the invention is small in size after being assembled, relatively simple in structure, convenient to carry and transport, simple and convenient in nucleic acid extraction operation steps, and capable of improving the extraction efficiency.

According to the nucleic acid extraction device, part of parts in the device need to be replaced after being used, the rest parts can be reused, and the device can also be used as an integrated disposable consumable, and the pressure can be used as a subsequent liquid driving source due to the fact that a large positive pressure exists in the cracking box after the sample is cracked.

The micro-fluidic chip for nucleic acid amplification detection is sealed by adopting a membrane structure, a sample is not easy to leak, aerosol pollution is not easy to cause, the detection result is not influenced, the sample adding is convenient, the sample using amount is small, a PCR tube is not needed, the operation is convenient, the micro-fluidic chip is suitable for rapid detection, the communication state between the front end reaction chamber and the rear end reaction chamber can be accurately controlled, and the whole structure is lighter.

Drawings

FIG. 1 is a schematic diagram of the (disposable) kit with nucleic acid extraction amplification qualitative detection of the present invention.

Fig. 2 is a schematic view of the overall structure of the present invention.

FIG. 3 is a schematic view showing a structure of a chip holder detecting part according to the present invention.

FIG. 4 is a schematic view of the operation control of the apparatus of the present invention.

FIG. 5 is a schematic view of the nucleic acid isolation apparatus according to the present invention.

FIG. 6 is a schematic view of the present invention shown in FIG. 5 with the heat patch, base, etc. removed.

FIG. 7 is a schematic exploded view of FIG. 6 according to the present invention.

FIG. 8 is a schematic exploded view of FIG. 5 according to the present invention.

FIG. 9 is a schematic diagram of the front side structure of the nucleic acid amplification detection microfluidic chip according to the present invention.

FIG. 10 is a schematic diagram of the back structure of a microfluidic chip for nucleic acid amplification detection according to the present invention.

FIG. 11 is a schematic diagram of a structure of a nucleic acid amplification detection microfluidic chip with a membrane removed.

Fig. 12 is an exploded view in a top view of the present invention.

Fig. 13 is an exploded view from the bottom of the present invention.

In the figure: 1-cracking box, 2-temperature sensor clamp, 3-spring clamp, 4-base, 5-buffer pad, 6-heat conducting pad, 7-glass sheet, 8-heating sheet, 9-temperature sensing module, 10-cracking box cover, 11-screw, 12-cracking box outlet, 13-notch groove, 14-antenna, 15-membrane, 16-primer chamber, 17-reaction amplification chamber, 18-detection chamber, 19-membrane valve hole, 20-sample inlet, 21-air hole, 22-drain valve, 23-waterproof air-permeable membrane, 24-microfluidic chip, 25-joint, 26-upper membrane, 27-bottom membrane, 28-pressure-sensitive membrane, 29-pressure-sensitive membrane, 30-valve part drain membrane, 31-membrane groove, 31-valve part, 32-valve groove, 33-nucleic acid extraction device, 34-nucleic acid amplification detection micro-fluidic chip, 35-base frame, 36-control board, 37-pressure supply device, 38-electromagnetic valve I, 39-electromagnetic valve II, 40-chip clamp, 41-optical fiber, 42-photodiode and 43-LED lamp.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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 invention.

In the description of the present invention, it should be noted that the terms "upper/lower end", "inner", "outer", "front end", "rear end", "both ends", "one end", "the other end", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be configured in a specific orientation, and operate, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed/sleeved," "connected," and the like are to be construed broadly, e.g., "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; either directly or indirectly through intervening media, or may be communicated between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

Referring to fig. 1-13, the present invention provides a technical solution:

an integrated nucleic acid amplification detection device comprises a base frame 35, a nucleic acid extraction device 33, an electromagnetic valve I38, an electromagnetic valve II 39, a pressure supply device 37, a control panel 36 and the like, wherein the nucleic acid extraction device 33, the electromagnetic valve I38, the electromagnetic valve II 39, the pressure supply device 37, the control panel 36 and the like are arranged on the base frame 35, and the pressure supply device 37 adopts an air pump.

The following is a detailed description:

a nucleic acid extraction device 33, which is used for nucleic acid cracking, wherein the parts in the device need to be replaced after use, and the rest parts can be reused or can be all used as disposable consumables. The top of schizolysis box 1 has schizolysis lid 10, and the bottom has base 4, and schizolysis box 1 will be placed in proper order through base 4 glass piece 7, heat conduction pad 6, heating plate 8, temperature sensing module 9 and blotter 5 and the nucleic acid extraction element of temperature sensor anchor clamps 2 suit one-piece. Wherein, temperature sensing module 9 adopts infrared temperature sensor, blotter 5 adopts the foam pad, schizolysis box 1 and base 4 are the dismantlement formula installation, after placing the base with temperature sensor anchor clamps 2 cover on schizolysis box 1, again sheathe spring clamp 3 on temperature sensor anchor clamps 2, the feeler 14 of spring clamp 3 blocks temperature sensor anchor clamps 2, rethread screw 11 is fixed spring clamp 3 and base 4, thereby with device assembly shaping, and leave the breach groove 13 that is used for holding 8 connecting wires of heating plate and schizolysis box export 12 on base 4.

When the device is used, 3ml of lysis solution can be preset in the lysis box 1, the downstream of a pipeline connected with an outlet 13 of the lysis box is closed, a throat sampling swab head is arranged in the lysis box 1, and a lysis box cover 10 is covered to enable the lysis box 1 to form a completely closed chamber. The heating plate 8 is electrified to heat so that the temperature of the lysis solution rises and is maintained at 93 ℃, the lysis solution starts to work at the temperature, the sample is cracked to release the nucleic acid in the sample, the process lasts for 3 minutes, and the air pressure in the cracking box 1 also gradually rises. After the cracking is finished, the power supply of the heating plate 8 is closed, the downstream valve of the outlet 12 of the cracking box is opened, and the cracking liquid in the cracking box 1 is released.

The device's advantage has, one be that in the device after the part uses need change all the other parts can used repeatedly, also can regard as the disposable consumptive material that the integral type can be thrown, another be because there is great malleation in the schizolysis box 1 behind the sample schizolysis, and this pressure can be regarded as follow-up liquid drive source.

The micro-fluidic chip 34 for nucleic acid amplification detection comprises a micro-fluidic chip 24, wherein the upper surface and the lower surface of the micro-fluidic chip 24 are respectively covered with an upper membrane 26 and a bottom membrane 27, and the micro-fluidic chip 24 is provided with a valve hole, a primer chamber 16, a reaction amplification chamber 17, a detection chamber 18, a drain valve 22, a waterproof breathable membrane 23 and the like. Wherein, the valve hole comprises a sample inlet 20, a film valve hole 19 and an air hole 21 which are respectively corresponding to the installation joint 25. The air hole 20 is communicated with the atmosphere, the sample inlet 20 is used for positive pressure driving, and the membrane valve 19 is closed under positive pressure.

When the chip is in operation, the chip is placed in a manner that the direction A in FIG. 11 is vertically upward, and air bubbles are prevented from entering the reaction amplification chamber 17 by gravity.

The whole working flow of the chip is as follows:

in the initial state, the membrane valve cavity (air hole 21) is connected with an air source, and the air source provides positive pressure to close the drain valve 22, so that liquid cannot pass through. The primer chamber 16 is preset with a primer, the reaction amplification chamber 17 is preset with an amplification reaction mixture, and the detection chamber 18 is preset with a fluorescence detection reagent.

The sample liquid containing nucleic acid enters the sample inlet 20 through positive pressure, and respectively enters the 5 primer chambers 16 to be mixed with the primers in the chambers.

Because the chip is vertically positioned, as the sample enters, the liquid level in the primer chamber 16 gradually rises to the height of the inlet of the reaction amplification chamber 17, and the sample flows into the reaction amplification chamber 17 and mixes with the amplification reactants.

The positive pressure at the injection port 20 was closed, and the reaction amplification region was heated to a temperature of 65 ℃ to amplify the nucleic acid at this temperature for 30 minutes.

After 30 minutes, the gas source connected to the diaphragm valve port 19 is closed, and the trap 22 is opened. Providing positive pressure to the sample inlet 20 causes the liquid in the reaction amplification zone to enter the detection chamber 18 through the hydrophobic valve and mix with the detection reagent. The negative and positive of the sample can be obtained by irradiating the detection hole with light of a specified wavelength to excite fluorescence and receiving the fluorescence intensity of the detection hole.

Specifically, as shown in FIGS. 9-13, in the initial state, the gas source provides positive pressure to close trap 22 through membrane valve opening 19: the valve portion water-repellent film 30 positively presses the valve portion pressure-sensitive film in the valve groove 32 to block the flow path formed by the valve portion pressure-sensitive film, thereby closing the space between the reaction amplification chamber 17 (reaction amplification region) and the detection chamber 18. The air source connected with the membrane valve hole 19 is closed, the positive pressure is relieved, the membrane valve hole 19 and the drain valve 22 are opened, and the reaction amplification area and the detection chamber 18 are in a communication state.

The heating temperature control of the nucleic acid extracting device is realized by the temperature fed back by the infrared temperature control on the nucleic acid extracting device 33, the outlet 12 of the cracking box is connected with the sample inlet 20 of the nucleic acid amplification detection micro-fluidic chip 34 through a hose, the hose is controlled to be in a communicated state through an electromagnetic valve I38, and the hose is kept normally closed in a standby state. The valve hole of the micro-fluidic chip 24 is connected with a three-way connector through a hose, the other two connectors are respectively connected with an inflator pump and external air, the external air pipe controls the communication state through an electromagnetic valve II 39, and the standby state is normally closed.

The chip holder 40 is responsible for holding the microfluidic chip 24 in an upright position, and a ceramic heating plate (not shown) is located at a position corresponding to the position below the reaction amplification chamber 17 on the holder and is responsible for heating the amplification chamber 18. As shown in FIG. 3, the exciting light is emitted from the side of the chip through the LED, and the fluorescence in the detection chamber 18 is conducted to the photodiode 42 through the optical fiber 41 on the clamp, so as to obtain an optical signal.

All heating, valve switches, air pump switches, LED lamps 43, photodiode signals and the like on the equipment are controlled and processed by the control panel 36 on the back of the equipment, and automatic control is achieved.

The whole equipment works according to the following procedures:

1. solenoid valve I38 is closed, and the pharyngeal swab sample is inserted into the chamber of the lysis cassette 1 and mixed with the lysate preloaded in the chamber.

2. The lid 10 of the lysis cassette is closed, the apparatus is started and the chamber is heated to 93 ℃ for 3 minutes to allow the sample to lyse and release nucleic acid.

After 3.3 minutes, the pressure supply device 37 (air pump) is opened, the electromagnetic valve II 39 is closed, the chip membrane valve 19 is closed, and the electromagnetic valve I38 is opened.

4. Due to the high temperature and the high air pressure in the chamber of the lysis box 1, the lysate sample enters the primer chamber 16 and the reaction amplification chamber 17 of the chip under the action of the pressure difference and is mixed with the reagents in the chambers, and the liquid finally stays in the reaction amplification chamber 17 due to the existence of the membrane valve 19.

5. Closing the electromagnetic valve I38, closing the heating of the lysis chamber, opening the reaction amplification heating sheet (i.e. ceramic heating sheet) on the chip clamp 40, stabilizing the temperature of the reaction amplification chamber 17 on the chip at 65 ℃ for 30 minutes, and entering the nucleic acid amplification stage.

After 6.30 minutes, the reaction amplification heating sheet is closed, the air pump is closed, the electromagnetic valve II 39 is opened, the chip membrane valve 19 is opened, the electromagnetic valve I38 is opened, the heating sheet of the cracking chamber is opened, the air pressure in the cracking chamber is increased, the liquid is pushed to flow forwards, enters the detection chamber and is mixed with the reagent in the detection chamber.

7. The electromagnetic valve 1 and the heating sheet 8 of the lysis box 1 are closed, so that the liquid stays in the detection chamber 18, the LED lamp 42 is turned on, and the photodiode 42 receives the fluorescence excited by the sample in the detection chamber 18.

The device is of a more compact design and is convenient to carry, and the device is of a disposable design and is disposable.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.