CN110616143A - Micro-fluidic chip for rapid detection of nucleic acid and application method thereof - Google Patents
Micro-fluidic chip for rapid detection of nucleic acid and application method thereof Download PDFInfo
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- CN110616143A CN110616143A CN201810684327.3A CN201810684327A CN110616143A CN 110616143 A CN110616143 A CN 110616143A CN 201810684327 A CN201810684327 A CN 201810684327A CN 110616143 A CN110616143 A CN 110616143A
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Abstract
The invention relates to the technical field of nucleic acid detection, in particular to a micro-fluidic chip integrating nucleic acid extraction, amplification and detection and a using method thereof. The chip consists of an outer functional area and an inner functional area, wherein the outer functional area comprises a purification reagent storage unit, a waste liquid unit and an amplification detection unit; the inner functional region comprises a connecting unit and a nucleic acid extracting unit, and a sealing gasket is arranged between the outer functional region and the inner functional region. The chip can realize the rapid extraction, amplification and detection processes of sample nucleic acid. The chip does not need a complex structure and a micro valve, so that the cost and the difficulty of batch production are greatly reduced, and the complexity of an external control system is reduced. Nucleic acid purification, amplification and detection reagents are pre-loaded in the chip, and the chip is stored at normal temperature and operated in a closed manner, so that sample cross contamination and aerosol contamination of amplification products can be avoided.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a micro-fluidic chip integrating nucleic acid extraction, amplification and detection.
Background
Nucleic acid is the most basic genetic material of life and is widely present in all animal and plant cells and microbial cells. With the rapid development of nucleic acid analysis technology, more and more fields of discipline take purification, detection and analysis of nucleic acid as main technical means. Such as the prevention, control and diagnosis of infectious diseases, identification of bioterrorism agents, food-borne pathogenic agents and mutated genetic components, port hygiene and pest defense quarantine. The common nucleic acid extracting and purifying methods comprise phenol chloroform extraction method, centrifugal column method and magnetic bead method. The extraction process is complex, the operation is complicated, the time consumption is long, toxic reagents can be used, and the yield and the purity of the purified nucleic acid and the safety, the consistency and the repeatability of the operation are reduced.
Nucleic acid amplification analysis is one of the main detection techniques after nucleic acid purification, and comprises temperature-variable amplification and isothermal amplification. Temperature-variable amplification, also known as Polymerase Chain Reaction (PCR), is the earliest technique for nucleic acid amplification, and obtains exponentially amplified DNA fragments by temperature cycling to achieve denaturation-renaturation between specific primer probes and template DNA. The isothermal amplification technology can realize exponential amplification of DNA molecules at a constant temperature, gets rid of the dependence on excellent equipment, and shows good application prospects in clinical and on-site rapid diagnosis. However, due to the complexity of the nucleic acid extraction process and the sensitivity of the amplification detection technique, cross-contamination, especially aerosol contamination, is prone to occur, directly leading to erroneous detection results.
The microfluidic chip technology is also called as a Lab-on-chip (Lab-on-chip), and is used for realizing a multi-step biochemical reaction process by utilizing a highly integrated functional unit in a microscale space, reducing manual operation, realizing sample input-output (sampling-Answer out), and providing an automatic solution for the fields of biomedical diagnosis, analytical chemistry, life science and the like. The microfluidic chip has the advantages of reducing the consumption of a reaction system and required reagents, greatly shortening the sample processing time, avoiding cross contamination of the totally-enclosed chip-reagent system, reducing the manual operation process through automatic control and improving the operation consistency. The integration of processes such as nucleic acid sample extraction and detection analysis by using a microfluidic chip is a hot point of research in recent years. The existing micro-fluidic chip research mainly focuses on the scientific research field, the integration level is high, a large number of micro valves or precise structures are designed on the chip, the complexity of the chip manufacturing process is increased, the batch production is difficult, the cost is high, and an external control system is complex.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a micro-fluidic chip for quickly detecting nucleic acid, which integrates the functions of extracting, amplifying and detecting nucleic acid. The chip design is simplified, a complex structure and a micro valve are not needed, the processing difficulty is reduced, and mass production is easy to realize. The liquid flow on the chip is realized by negative pressure, and an external control system is simplified. Meanwhile, a use method of the nucleic acid detection chip is also provided.
The invention provides a micro-fluidic chip for rapid detection of nucleic acid.
The chip is composed of an outer functional area and an inner functional area, a reaction reagent capable of being stored at normal temperature is preset on the chip, and the specific structure is as follows:
the outer functional area consists of a cover plate and a bin body layer;
the outer functional area is provided with 3 positioning grooves for fixing the chip laminated structure in the manufacturing process and fixing the outer functional area in use;
the bin body layer of the external functional area comprises a purified reagent storage unit, a waste liquid unit and an amplification detection unit;
wherein, the purifying reagent storage unit comprises a pretreatment liquid chamber, a sample chamber, a washing liquid I chamber, a washing liquid II chamber, a dealcoholization liquid chamber and an elution liquid chamber;
the waste liquid unit comprises a waste liquid chamber, and a water absorbing material is contained in the waste liquid chamber;
the amplification detection unit comprises a freeze-drying reagent chamber, a drying agent chamber, an amplification reaction chamber and a communication pipeline;
the inner functional area is circular and comprises a nucleic acid extraction unit and an elastic buckle;
wherein the nucleic acid extraction unit comprises an inlet, an outlet, a purification chamber and a communicating structure;
the outer functional area and the inner functional area are connected through an elastic buckle;
a sealing gasket is arranged at the joint of the outer functional area and the inner functional area;
the outer functional area and the inner functional area can rotate relatively under the action of external force, the rotating mode comprises that the outer functional area is fixed, the inner functional area is rotated and fixed, and the outer functional area is rotated and the outer functional area and the inner functional area are rotated reversely and simultaneously.
Preferably, the sealing gasket material at the joint of the outer functional area and the inner functional area comprises silica gel and rubber.
Preferably, the reaction reagents required by nucleic acid purification and amplification detection are preset on the microfluidic chip; specifically, the reagent types and compositions were as follows:
1) the pretreatment liquid chamber contains pretreatment liquid with the pH of 6.0 ~ 7.5.5, and the components comprise 4 ~ 6M guanidine hydrochloride, 10 ~ 100mM Tris-HCl, 1 ~ 10mM EDTA, 0.01 ~ 1% (v/v) Triton-X100 and 0.01-0.5% (v/v) Tween 20;
2) the pH of the lysate is 6.0 ~ 7.5.5, and the components comprise 4 ~ 6M guanidine hydrochloride, 10 ~ 100mM Tris-HCl, 1 ~ 10mM EDTA, 0.1 ~ 1.5.5M sodium acetate, 0.01 ~ 1% (v/v) Triton-X100, 0.01-0.5% (v/v) Tween 20;
3) the washing solution I chamber contains washing solution I with the component of 50% ~ 80% (v/v) isopropanol;
4) the washing liquid II chamber contains washing liquid II, and the component of the washing liquid II is 50% ~ 80% (v/v) ethanol;
5) the dealcoholization chamber contains dealcoholization solution with the pH of 6.0 ~ 7.5.5, and the components comprise 1 ~ 10mM Tris-HCl and 0.1 ~ 1mM EDTA;
6) the eluent chamber contains an eluent with the pH of 8.0 ~ 9.0.0, and the components comprise 10mM Tris-HCl and 1mM EDTA;
7) the freeze-drying reagent chamber contains a nucleic acid amplification reaction freeze-drying reagent which can be stored at normal temperature;
8) the purification chamber of the nucleic acid extraction unit is internally provided with super-paramagnetic beads.
Preferably, the water-absorbing material in the waste liquid chamber can prevent the waste liquid from flowing backwards, and comprises sponge and water-absorbing resin.
Preferably, the amplification detection unit can perform reactions including PCR amplification, RT-PCR amplification and isothermal amplification.
Preferably, the waste liquid chamber on the chip is connected with a syringe outside the chip, the liquid driving mode is negative pressure, and the reagents in the purification reagent storage unit are sequentially sucked into the purification unit by the suction of the syringe to react, and finally reach the waste liquid chamber.
Preferably, the chip material is one or more of glass, silicon, PMMA, epoxy resin, PTFE, PS, PC, COC, COP and PDMS.
Another aspect of the present invention provides a method for using a microfluidic chip for rapid detection of nucleic acids, comprising:
1) adding an equal amount of sample into the treatment solution, uniformly mixing, and injecting into a sample chamber of the chip;
2) placing the chip on a matched device; initializing equipment, wherein an inner functional area is connected with a pretreatment liquid chamber and a waste liquid chamber, a purification chamber and a corresponding pipeline of a nucleic acid extraction unit are infiltrated by the pretreatment liquid, dry powder-shaped superparamagnetic beads are redissolved, and after an external magnetic field is started to adsorb the beads, the pretreatment liquid is sucked into the waste liquid chamber;
3) the motor rotates the inner functional area and is communicated with the sample chamber, the purification chamber and the waste liquid chamber;
4) the processing solution containing the sample is pumped by the injector to flow through the purification chamber at a constant speed, and the nucleic acid is adsorbed by the magnetic beads;
5) the motor rotates the inner functional area, connect washing liquid I room, purification room and waste liquid room;
6) the injector sucks the washing liquid I to flow through the purification chamber at a constant speed to remove residual impurities;
7) the motor rotates the inner functional area, connect washing liquid II room, purification room and waste liquid room;
8) the injector pumps the washing liquid II to flow through the purification chamber at a constant speed, so as to further remove residual impurities and residual washing liquid I;
9) the motor rotates the inner functional area, connect dealcoholize room, purified room and waste liquid room;
10) the ethanol removal liquid is pumped by a syringe and rapidly flows through a purification chamber to remove the residual washing liquid II;
11) the motor rotates the inner functional area and is communicated with the eluent chamber, the purification chamber and the waste liquid chamber;
12) the injector sucks the eluent to flow through the purification chamber at a constant speed, and the eluted target nucleic acid flows through the freeze-drying reagent chamber and finally reaches the amplification reaction chamber;
13) the motor rotates the inner functional area to an initial position to seal all the cavity pipelines;
14) performing nucleic acid amplification reaction, and reading fluorescence signals in real time.
The micro-fluidic chip for rapid detection of nucleic acid integrates nucleic acid extraction, amplification and detection, can realize the automatic extraction, amplification and detection processes of sample nucleic acid, and has the advantages that:
a) the design of the micro-fluidic chip is simplified, a complex structure and a micro valve are not needed, and the cost and the difficulty of batch production are reduced;
b) the waste liquid chamber of the micro-fluidic chip is connected with an external injector through a waste liquid chamber communicating hole, and liquid path control is carried out through the negative pressure and the rotation of the inner functional area, so that the complexity of an external control system is reduced, and the automation is easy to realize;
c) nucleic acid purification, amplification and detection reagents are pre-loaded in the chip, and are stored at normal temperature and operated in a closed manner, so that sample cross contamination and aerosol contamination of amplification products can be avoided, and the reliability and stability of detection results are improved.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip for rapid detection of nucleic acids according to the present invention.
FIG. 2 is an exploded view of a microfluidic chip structure for rapid detection of nucleic acids according to the present invention.
FIG. 3 is a schematic diagram of functional regions in a microfluidic chip for rapid detection of nucleic acids according to the present invention.
FIG. 4 shows the detection result of a microfluidic chip for rapid detection of nucleic acid according to the present invention.
Wherein the reference numbers: 1. the kit comprises a cover plate, 2, a cover plate positioning groove, 3, a bin body layer, 4, a sealing gasket, 5, a connecting unit, 6, a nucleic acid extraction unit, 7, a connecting unit limiting hole, 8, a nucleic acid extraction unit limiting hole, 9, an elastic buckle, 10, a waste liquid chamber, 11, an amplification reaction chamber, 12, a bin body layer positioning groove, 13, a reaction chamber pipeline B, 14, a freeze-drying reagent chamber, 15, a drying agent chamber, 16, a waste liquid chamber communication hole, 17, an inner functional region clamping groove, 18, a purification chamber inlet, 19, a waste liquid chamber communication pipe, 20, an elution liquid chamber, 21, a dealcoholization chamber, 22, a washing liquid II, 23, a washing liquid I chamber, 24, a sample chamber, 25, a pretreatment liquid chamber, 26, a snake-shaped purification chamber, 27, a purification chamber outlet, 28, a reaction chamber pipeline B outlet, 29 and a.
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 exemplary and in no way limit the present invention and its applications or uses.
Example 1.
The micro-fluidic chip consists of an outer functional area and an inner functional area, and a reaction reagent which can be stored at normal temperature is preset on the chip.
The outer functional area consists of a cover plate (1) and a storehouse body layer (3).
The outer functional area is provided with 3 constant head tanks, is located the corresponding position on cover plate (1) and storehouse layer (3) respectively. Its function is to secure the chip stack during fabrication and the external functional area in use.
The storehouse body layer (3) of the outer functional area comprises a purification reagent storage unit, a waste liquid unit and an amplification detection unit.
Wherein, the purifying reagent storage unit comprises a pretreatment liquid chamber (25), a sample chamber (24), a washing liquid I chamber (23), a washing liquid II chamber (22), a dealcoholization liquid chamber (21) and an elution liquid chamber (20).
The liquid storage chamber of the purification reagent storage unit is sequentially filled with nucleic acid purification reagents, the pretreatment liquid chamber (25) contains pretreatment liquid, the sample chamber (24) is used for storing lysis solution containing samples, the washing liquid I chamber (23) contains washing liquid I, the washing liquid II chamber (22) contains washing liquid II, the dealcoholization liquid chamber (21) contains dealcoholization liquid, and the elution liquid chamber (20) contains elution liquid.
The waste liquid unit comprises a waste liquid chamber (10), wherein sponge is arranged in the waste liquid chamber to prevent the waste liquid from flowing back/overflowing; the waste chamber contains a plurality of channels that can communicate with the serpentine purification chamber (26) for introducing liquid. In addition, the waste liquid chamber (10) contains a waste liquid chamber communication hole (16) which can be externally connected with a syringe to drive liquid flow by negative pressure.
The amplification detection unit comprises a freeze-drying reagent chamber (14), a drying agent chamber (15), an amplification reaction chamber (11) and communication pipelines (13 and 29).
The inner functional area is circular and comprises a nucleic acid extraction unit (6) and an elastic buckle (9), wherein the elastic buckle (9) is used for connecting the inner functional area and the outer functional area.
Wherein the nucleic acid extraction unit (6) comprises an inlet (18), an outlet (27), a serpentine purification chamber (26) and a waste chamber communicating tube (19).
The joint of the outer functional area and the inner functional area is provided with a sealing gasket (4) made of rubber.
This scheme is fixed with outer functional area constant head tank for, and the two relative rotation is realized to rotatory circular shape interior functional area.
The microfluidic chip is preset with reaction reagents required by nucleic acid purification and amplification detection, and the types and components of the reagents are as follows:
1) the pretreatment liquid chamber (25) contains a pretreatment liquid having a pH of 6.5, and the components include 6M guanidine hydrochloride, 10mM Tris-HCl, 1mM EDTA, 1% (v/v) Triton-X100, and 0.1% (v/v) Tween 20;
2) the pH of the lysate is 6.5, and the components comprise 6M guanidine hydrochloride, 10mM Tris-HCl, 1mM EDTA, 0.3M sodium acetate, 1% (v/v) Triton-X100 and 0.1% (v/v) Tween 20;
3) the washing solution I chamber (23) contains washing solution I, and the component is 80% (v/v) isopropanol;
4) the washing liquid II chamber (22) contains washing liquid II, and the component is 70% (v/v) ethanol;
5) the dealcoholization chamber (21) contains a dealcoholization solution having a pH of 6.5 and containing 10mM Tris-HCl and 1mM EDTA;
6) the eluent chamber (20) contains an eluent with pH of 8.5, and the components comprise 10mM Tris-HCl and 1mM EDTA;
7) the nucleic acid amplification reaction freeze-drying reagent in the freeze-drying reagent chamber (14) is self-made by the company;
8) 200nm superparamagnetic beads are arranged in a purification chamber of the nucleic acid extraction unit. The pre-setting method is that 200nm monodisperse superparamagnetic beads are washed for 3 times by using 0.2M trehalose, 10mM Tris, 1mM EDTA and ph7.5 buffer solution; using buffer solution to dissolve the superparamagnetic beads back to a final concentration of 25 mg/ml; 50ul of magnetic bead solution is injected into a snake-shaped purification chamber (26); oven drying at 65 deg.C, and storing at room temperature.
The purification principle of the reagent system is as follows: reagents required by nucleic acid purification are pre-loaded in a reagent chamber of a purification reagent storage unit of the microfluidic chip, and superparamagnetic beads are pre-loaded in a snake-shaped purification chamber of the nucleic acid extraction unit (6). The rotation of the inner functional area is controlled to switch between different reagent chambers. When the sample flows through the serpentine purification chamber (26), the nucleic acid is adsorbed, and impurities and waste liquid directly enter the waste liquid unit (10); washing impurities as wash solution I/II flows through serpentine purification chamber (26); residual ethanol in washing solution II can be removed when the dealcoholized liquid flows through the serpentine purification chamber (26); when eluent flows through the serpentine purification chamber (26), nucleic acid is eluted and then injected into a freeze-drying reagent chamber (14) of the amplification detection unit to dissolve nucleic acid amplification reaction freeze-drying reagent therein, and then enters the amplification reaction chamber (11), and the key of the step is that an outlet (28) of a reaction chamber pipeline B is connected with a waste liquid chamber (10) through a waste liquid chamber communicating pipe (19) to realize liquid flow driving.
The microfluidic chip material is PC, and the manufacturing process is briefly as follows:
all structural parts of the chip are formed by injection molding of a high-precision mold. The chamber and the pipeline which are included by the chamber layer (3) of the outer functional area and the nucleic acid extraction unit (6) of the inner functional area on the chip are subjected to surface hydrophilic treatment by using a plasma technology. The sealing gasket is made of rubber and is formed by injection molding of a high-precision mold. And the outer functional area and the inner functional area of the chip are independently bonded, and a hot-press bonding technology is selected. Firstly, put into special design's fixture after cover plate (1) and storehouse body layer (3) with outer functional area through cover plate constant head tank (2) and storehouse body layer constant head tank (12) alignment, have 3 archs on the fixture, can accurately match the three constant head tank of outer functional area. The PC hot pressing conditions were as follows: and cooling to room temperature after lasting for 15 minutes at a temperature of 160 ℃, a vacuum degree of <0.1bar and a pressure of 1 MPa. Secondly, after aligning the limiting hole (8) of the nucleic acid extraction unit (6) in the inner functional area with the limiting hole (7) of the connection unit (5), inserting a limiting sheath, and putting the limiting sheath into a specially designed fixture for hot-press bonding under the same conditions as those of the outer functional area.
The application method of the microfluidic chip comprises the following steps:
1) adding an equal amount of sample into the treatment fluid, uniformly mixing, and injecting into a sample chamber (24) of the microfluidic chip;
2) placing the chip on a matched portable device; initializing equipment, connecting an inner functional area with a pretreatment liquid chamber (25) and a waste liquid chamber (10), infiltrating a snake-shaped purification chamber (26) of a nucleic acid extraction unit (6) with the pretreatment liquid, redissolving dry powder-shaped superparamagnetic nano magnetic beads, starting an external magnetic field to adsorb the magnetic beads, and then pumping the pretreatment liquid into the waste liquid chamber (10);
3) the motor rotates the inner functional area, communicate sample room (24), snakelike purified room (26) and waste liquid room (10);
4) the processing solution containing the sample is pumped by the injector to flow through the snakelike purification chamber (26) at a constant speed, and the nucleic acid is adsorbed by the magnetic beads;
5) the motor rotates the inner functional area, connect washing liquid I room (23), snakelike purified room (26) and waste liquid room (10);
6) the injector sucks washing liquid I to flow through the snakelike purification chamber (26) at a constant speed to remove residual impurities;
7) the motor rotates the inner functional area, connect washing liquid II room (22), snakelike purified room (26) and waste liquid room (10);
8) the injector sucks washing liquid II to flow through the snakelike purification chamber (26) at a constant speed, and further residual impurities and residual washing liquid I are removed;
9) the motor rotates the inner functional area, connect and remove the alcoholic solution room (21), snakelike purified room (26) and waste liquor room (10);
10) the injector sucks dealcoholization liquid to rapidly flow through a snakelike purification chamber (26) to remove residual washing liquid II;
11) the motor rotates the inner functional area, and communicates the eluent chamber (20), the snake-shaped purification chamber (26) and the waste liquid chamber (10);
12) the eluent pumped by the injector flows through the snakelike purification chamber (26) at a constant speed, and flows through the freeze-drying reagent chamber (14) after eluting the target nucleic acid and finally reaches the amplification reaction chamber (11);
13) the motor rotates the inner functional area to an initial position to seal all the cavity pipelines;
14) and performing PCR amplification reaction and reading a fluorescent signal in real time, wherein the PCR amplification reaction conditions are 95, 5 minutes and 40 cycles, and each cycle comprises 95, 15, 60 and 30 seconds.
The fluorescence detection results are shown in FIG. 4.
Example 2.
The structure design and the types and components of the purification reagents of the microfluidic chip in this example are the same as those of example 1.
PDMS is selected as the material of the microfluidic chip, and the manufacturing process is briefly described as follows:
all structural parts of the chip are formed by injection molding of a high-precision mold. And (3) carrying out surface treatment on the PDMS structural member by using an oxygen plasma technology, aligning and laminating the treated cover plate (1) of the outer functional region and the treated bin body layer (3) through a positioning groove, putting the aligned and laminated cover plate and the bin body layer into a fixture, pressurizing at 1MPa, and keeping the temperature for 100 ℃ for one hour to realize permanent sealing. After the limiting hole (8) of the nucleic acid extraction unit (6) of the inner functional area and the limiting hole (7) of the connecting unit (5) are aligned, a limiting sheath is inserted, and other processing methods are the same as those of the outer functional area. And adhering the injection-molded sealing gasket (4) to the corresponding position on the back surface of the outer functional area bin body layer (3). And (3) fixing the elastic buckle (9) of the inner functional area after the plasma bonding on the bonded outer functional area after passing through the center of the outer functional area.
The application method of the microfluidic chip comprises the following steps:
1) adding an equal amount of sample into the treatment solution, uniformly mixing, and injecting into a sample chamber (24) of the chip;
2) placing the chip on a matched portable device; initializing equipment, connecting an inner functional area with a pretreatment liquid chamber (25) and a waste liquid chamber (10), infiltrating a snake-shaped purification chamber (26) of a nucleic acid extraction unit (6) with the pretreatment liquid, redissolving dry powder-shaped superparamagnetic nano magnetic beads, starting an external magnetic field to adsorb the magnetic beads, and then pumping the pretreatment liquid into the waste liquid chamber (10);
3) the motor rotates the inner functional area, communicate sample room (24), snakelike purified room (26) and waste liquid room (10);
4) the processing solution containing the sample is pumped by the injector to flow through the snakelike purification chamber (26) at a constant speed, and the nucleic acid is adsorbed by the magnetic beads;
5) the motor rotates the inner functional area, connect washing liquid I room (23), snakelike purified room (26) and waste liquid room (10);
6) the injector sucks washing liquid I to flow through the snakelike purification chamber (26) at a constant speed to remove residual impurities;
7) the motor rotates the inner functional area, connect washing liquid II room (22), snakelike purified room (26) and waste liquid room (10);
8) the injector sucks washing liquid II to flow through the snakelike purification chamber (26) at a constant speed, and further residual impurities and residual washing liquid I are removed;
9) the motor rotates the inner functional area, connect and remove the alcoholic solution room (21), snakelike purified room (26) and waste liquor room (10);
10) the injector sucks dealcoholization liquid to rapidly flow through a snakelike purification chamber (26) to remove residual washing liquid II;
11) the motor rotates the inner functional area, and communicates the eluent chamber (20), the snake-shaped purification chamber (26) and the waste liquid chamber (10);
12) the eluent pumped by the injector flows through the snakelike purification chamber (26) at a constant speed, and flows through the freeze-drying reagent chamber (14) after eluting the target nucleic acid and finally reaches the amplification reaction chamber (11);
13) the motor rotates the inner functional area to an initial position to seal all the cavity pipelines;
14) performing isothermal amplification reaction and reading fluorescence signals in real time at 65 ℃.
It is within the scope of the invention to cover such minor variations within the spirit and scope of the invention as defined by the appended claims. Such as the material, shape and size of the microfluidic chip, the shape and size of the chamber, the shape and size of various functional and connective channels, etc.
Claims (8)
1. A micro-fluidic chip for rapid detection of nucleic acid is characterized in that the chip consists of an outer functional area and an inner functional area; a reaction reagent capable of being stored at normal temperature is preset on the chip; more specifically:
the outer functional area consists of a cover plate and a bin body layer; the outer functional area is provided with 3 positioning grooves; the bin body layer of the outer functional zone comprises a purified reagent storage unit, a waste liquid unit and an amplification detection unit;
the purifying reagent storage unit comprises a pretreatment liquid chamber, a sample chamber, a washing liquid I chamber, a washing liquid II chamber, a dealcoholization liquid chamber and an elution liquid chamber;
the waste liquid unit comprises a waste liquid chamber, and the waste liquid chamber contains a water absorbing material;
the amplification detection unit comprises a freeze-drying reagent chamber, a drying agent chamber, an amplification reaction chamber and a communication pipeline;
the inner functional area is circular and comprises a nucleic acid extraction unit and an elastic buckle;
the nucleic acid extraction unit comprises an inlet, an outlet, a purification chamber and a communicating structure;
the outer functional area and the inner functional area are connected through an elastic buckle;
a sealing gasket is arranged at the joint of the outer functional area and the inner functional area;
the outer functional area and the inner functional area can rotate relatively under the action of external force;
the relative rotation comprises the fixation of an outer functional area, the rotation of an inner functional area, the fixation of the inner functional area, the rotation of the outer functional area and the reverse simultaneous rotation of the outer functional area and the inner functional area.
2. The microfluidic chip for rapid detection of nucleic acid according to claim 1, wherein the material of the sealing pad comprises silica gel and rubber.
3. The microfluidic chip for rapid detection of nucleic acid according to claim 1, wherein the chip is pre-loaded with reagents required for nucleic acid purification and amplification detection; specifically, the reagent types and compositions were as follows:
1) the pretreatment liquid chamber contains pretreatment liquid with the pH of 6.0 ~ 7.5.5, and the components comprise 4 ~ 6M guanidine hydrochloride, 10 ~ 100mM Tris-HCl, 1 ~ 10mM EDTA, 0.01 ~ 1% (v/v) Triton-X100 and 0.01-0.5% (v/v) Tween 20;
2) the pH of the lysate is 6.0 ~ 7.5.5, and the components comprise 4 ~ 6M guanidine hydrochloride, 10 ~ 100mM Tris-HCl, 1 ~ 10mM EDTA, 0.1 ~ 1.5.5M sodium acetate, 0.01 ~ 1% (v/v) Triton-X100, 0.01-0.5% (v/v) Tween 20;
3) the washing solution I chamber contains washing solution I with the component of 50% ~ 80% (v/v) isopropanol;
4) the washing liquid II chamber contains washing liquid II, and the component of the washing liquid II is 50% ~ 80% (v/v) ethanol;
5) the dealcoholization chamber contains dealcoholization solution with the pH of 6.0 ~ 7.5.5, and the components comprise 1 ~ 10mM Tris-HCl and 0.1 ~ 1mM EDTA;
6) the eluent chamber contains an eluent with the pH of 8.0 ~ 9.0.0, and the components comprise 10mM Tris-HCl and 1mM EDTA;
7) the freeze-drying reagent chamber contains a nucleic acid amplification reaction freeze-drying reagent which can be stored at normal temperature;
8) the purification chamber of the nucleic acid extraction unit is internally provided with super-paramagnetic beads.
4. The microfluidic chip for rapid detection of nucleic acid according to claim 1, wherein the water-absorbent material in the waste liquid chamber comprises sponge and water-absorbent resin.
5. The microfluidic chip for rapid detection of nucleic acid according to claim 1, wherein the amplification detection unit can perform reactions including PCR amplification, RT-PCR amplification, and isothermal amplification.
6. The microfluidic chip for rapid detection of nucleic acid according to claim 1, wherein the waste liquid chamber is connected to a syringe outside the chip, and the liquid driving manner is a negative pressure.
7. The microfluidic chip for rapid detection of nucleic acid according to claim 1, wherein the chip material is selected from one or more of glass, silicon, PMMA, epoxy, PTFE, PS, PC, COC, COP and PDMS.
8. A use method of a microfluidic chip for rapid detection of nucleic acid comprises the following steps:
1) adding a proper amount of sample into the lysate, uniformly mixing, and injecting into a sample chamber of the chip;
2) placing the chip on a matched device;
3) the injector drives the lysate containing the sample to flow through the nucleic acid extraction unit at a constant speed, and the nucleic acid is adsorbed by the magnetic beads;
4) the motor drives the nucleic acid extraction unit to rotate, and the inlet is communicated to the next chamber;
5) the injector drives the washing solution I to flow through the nucleic acid extraction unit at a constant speed to wash residual impurities;
6) the motor drives the nucleic acid extraction unit to rotate, and the inlet is communicated to the next chamber;
7) the injector drives the washing liquid II to flow through the nucleic acid extraction unit at a constant speed, so as to wash residual impurities and residual washing liquid I;
8) the motor drives the nucleic acid extraction unit to rotate, and the inlet is communicated to the next chamber;
9) the injector drives the dealcoholization liquid to flow through the nucleic acid extraction unit at a constant speed, and residual impurities and residual washing liquid II are further washed;
10) the motor drives the nucleic acid extraction unit to rotate, and the inlet is communicated to the next chamber;
11) the injector drives the eluent to flow through the nucleic acid extraction unit at a constant speed, elutes the target nucleic acid and directly injects the target nucleic acid into the amplification detection unit;
12) when the eluent containing the target nucleic acid flows through the freeze-drying reagent chamber, the freeze-drying reagent for the nucleic acid amplification reaction is dissolved and is directly injected into the amplification reaction chamber;
13) the motor drives the nucleic acid extraction unit to rotate to an initial position, and the amplification reaction chamber is sealed;
14) the amplification reaction was performed while reading the fluorescence data.
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