CN216808822U - Integrated micro-droplet chip - Google Patents

Abstract

The utility model provides an integrated micro-droplet chip which comprises a chip body, wherein the chip body is provided with a reaction cavity, a sample adding cavity, a micro-droplet generating structure, an oil interface, a gas-liquid interface and a fluorescence detection area; when liquid drops are generated, pressure differences are formed between the sample adding cavity and the gas-liquid interface and between the oil liquid interface and the gas-liquid interface, and the pressure differences respectively drive a sample in the sample adding cavity and generated oil of the oil liquid interface to enter the micro liquid drop generating structure to generate micro liquid drops to enter the reaction cavity; when the liquid drop is detected, external pressure drives detection to push oil to enter the reaction cavity, so that the micro liquid drops flow out of the reaction cavity to the micro liquid drop generating structure, external pressure drives detection separation oil to enter the micro liquid drop generating structure, and the detection separation oil separates the micro liquid drops flowing out of the reaction cavity to the micro liquid drop generating structure to form a queue and enters the fluorescence detection area. The utility model integrates the generation, amplification and detection of the liquid drop into one chip by time-sharing multiplexing of the micro-liquid drop generation structure, thereby realizing the fully integrated and totally closed digital PCR process.

Description

Integrated micro-droplet chip

Technical Field

The utility model belongs to the technical field of digital PCR analyzers, and particularly relates to an integrated micro-droplet chip.

Background

Droplet-based microfluidics (droplet-based microfluidics) is a technical platform for controlling micro-volume liquid developed on a microfluidic chip in recent years, and the principle of the platform is as follows: two mutually insoluble liquids are taken as an example, one of the liquids is an oil phase, and the other one is a water phase, after the oil phase and the water phase simultaneously enter the micro-channel, the water phase is distributed in the oil phase in the form of micro volume units under the action of the micro-channel, and a series of discrete micro liquid drops are formed. Each droplet acts as a microreactor to accomplish a set of chemical or biological reactions.

The digital PCR technology is known as the third generation PCR technology, has the advantages of absolute quantification and single molecule detection sensitivity, and has important application prospect in the field of molecular diagnosis. A mainstream technical route of the digital PCR technology adopts a droplet microfluidic chip to divide a reaction system into tens of thousands or even millions of droplets with uniform size, complete generation, amplification and fluorescence detection, and calculate the accurate copy number of target molecules in a sample by using a mathematical model according to a fluorescence detection result.

In the droplet digital PCR technology, a structure in a droplet generation chip is often used to complete droplet generation, then droplets are transferred to a reaction tube for amplification, and finally a droplet queue with a certain interval is formed on the droplets by using a structure in a droplet detection chip, and each droplet passes through a fluorescence detection region in turn to excite and detect a fluorescence signal in the droplet. The method for realizing digital PCR by utilizing droplet microfluidics has the advantages of uniform droplet size, difficulty in limiting the number of droplets, high signal-to-noise ratio of fluorescence detection and the like, but also has the defects of complex chip structure, low integration level, difficulty in automation and the like, and generation and detection are finished in different chips.

SUMMERY OF THE UTILITY MODEL

Therefore, the technical problem to be solved by the present invention is to provide an integrated micro droplet chip to overcome the disadvantages of low integration level and low automation level in the prior art, in which droplet generation and detection are completed in different droplet chips.

In order to solve the problems, the utility model provides an integrated micro-droplet chip, which comprises a chip body, wherein the chip body is provided with a reaction cavity and a sample adding cavity, a micro-droplet generation structure, an oil liquid interface, a gas-liquid interface and a fluorescence detection area are constructed in the chip body, the gas-liquid interface is communicated with the reaction cavity, the sample adding cavity is communicated with the micro-droplet generation structure, and the oil liquid interface is communicated with the micro-droplet generation structure;

when liquid drops are generated, a first pressure difference is formed between the sample adding cavity and the gas-liquid interface, a second pressure difference is formed between the oil liquid interface and the gas-liquid interface, the sample in the sample adding cavity and the generated oil of the oil liquid interface are respectively driven by the first pressure difference and the second pressure difference to enter the micro liquid drop generating structure, and the generated micro liquid drops enter and are stored in the reaction cavity;

when the liquid drops are detected, external pressure drives detection to push oil to enter the reaction cavity from the gas-liquid interface, so that micro liquid drops in the reaction cavity flow out of the reaction cavity to the micro liquid drop generating structure, external pressure drives detection separation oil to enter the micro liquid drop generating structure from the oil liquid interface, and the detection separation oil separates the micro liquid drops flowing out of the reaction cavity to the micro liquid drop generating structure to form a queue and enters the fluorescence detection area.

Preferably, the micro-droplet generation structure comprises an oil liquid pipeline and a communication pipeline, the oil liquid pipeline and the communication pipeline are crossed, the communication pipeline comprises a first pipeline which is positioned at the cross point and communicated with the reaction cavity and a second pipeline which is positioned at the second side of the cross point and communicated with the sample adding cavity, and the oil liquid interface is communicated with the oil liquid pipeline.

Preferably, with reference to the first side surface of the chip body being in the horizontal position, the reaction chamber and the sample injection chamber are located on the first side surface, and a connection interface between the reaction chamber and the first side surface of the chip body extends upward and forms a horn mouth with a small lower part and a large upper part.

Preferably, a gas-liquid pipeline extending from bottom to top is further configured in the reaction chamber, a lower opening of the gas-liquid pipeline is communicated with the gas-liquid interface, and an upper opening of the gas-liquid pipeline is higher than an upper opening of the connecting interface.

Preferably, a microdroplet observation area is arranged between the first pipeline and the connecting interface.

Preferably, the sample application cavity comprises an open cavity and a sealing cover which is connected to the opening of the open cavity in a sealing manner.

Preferably, the sample application cavity is provided with a filter membrane or a vent hole.

Preferably, the fluorescent detection zone is on the second conduit.

Preferably, the sample application cavity is located on the first side surface, the reaction cavity is located on the second side surface, and the second side surface and the first side surface are opposite sides of the chip body.

Preferably, before the micro-droplets enter the reaction cavity, light oil is preset in the reaction cavity.

The integrated micro-droplet chip provided by the utility model is characterized in that the chip body is integrated with a sample adding cavity, a reaction cavity, a micro-droplet generating structure and a fluorescence detection area, and the generation, amplification and detection of droplets are integrated in one chip through time-sharing multiplexing of the micro-droplet generating structure, so that a fully integrated and totally closed digital PCR process is realized.

Drawings

Fig. 1 is a schematic perspective view of an integrated micro droplet chip according to an embodiment of the present invention;

FIG. 2 is a schematic structural diagram of a droplet generation structure in an integrated droplet chip according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of the internal structure of a reaction chamber in the integrated micro droplet chip according to an embodiment of the present invention;

FIG. 4 is a schematic of a microdroplet generation process;

FIG. 5 is a schematic view of the micro-droplets being generated and then stored in the reaction chamber;

FIG. 6 is a schematic view of the state in the reaction chamber after the integrated micro-droplet chip is turned over by 180 degrees;

FIG. 7 is a schematic view of the state of FIG. 6 after oil is introduced into the reaction chamber;

FIG. 8 is a schematic view of the micro-droplets after being forced out of the reaction chamber (arrows in the figure show the micro-droplets and the flow direction of the oil liquid);

fig. 9 is a schematic perspective view of an integrated micro droplet chip according to another embodiment of the utility model.

The reference numerals are represented as:

1. a chip body; 11. a reaction chamber; 111. connecting an interface; 112. a gas-liquid pipeline; 12. a sample application cavity; 121. an open cavity; 122. a sealing cover; 21. an oil pipe; 22. a first conduit; 23. a second conduit; 31. an oil interface; 32. a gas-liquid interface; 33. a fluorescent detection zone; 34. a microdroplet observation area; 4. micro-droplets; 5. the pusher oil is detected.

Detailed Description

With reference to fig. 1 to 9, according to an embodiment of the present invention, an integrated micro droplet chip is provided, including a chip body 1, where the chip body 1 has a reaction cavity 11 and a sample adding cavity 12, a micro droplet generating structure, an oil interface 31, an air-liquid interface 32, and a fluorescence detection region 33 are configured in the chip body 1, the air-liquid interface 32 is communicated with the reaction cavity 11, the sample adding cavity 12 is communicated with the micro droplet generating structure, and the oil interface 31 is communicated with the micro droplet generating structure;

when liquid drops are generated, a first pressure difference is formed between the sample adding cavity 12 and the gas-liquid interface 32, a second pressure difference is formed between the oil liquid interface 31 and the gas-liquid interface 32, the sample in the sample adding cavity 12 and the generated oil in the oil liquid interface 31 are respectively driven by the first pressure difference and the second pressure difference to enter the micro liquid drop generating structure, and the generated micro liquid drops 4 enter and are stored in the reaction cavity 11;

when liquid drops are detected, external pressure drives detection pushing oil 5 to enter the reaction cavity 11 from the gas-liquid interface 32, so that micro liquid drops 4 in the reaction cavity 11 flow out of the reaction cavity 11 to the micro liquid drop generating structure, external pressure drives detection separation oil to enter the micro liquid drop generating structure from the oil interface 31, and the detection separation oil separates the micro liquid drops 4 flowing out of the reaction cavity 11 to the micro liquid drop generating structure to form a queue and enters the fluorescence detection area 33.

In the technical scheme, the chip body 1 is integrated with a sample adding cavity 12, a reaction cavity 11, a micro-droplet generating structure and a fluorescence detection area 33, so that the generation and detection of the micro-droplets are completed on the same chip, and the integration level and the automation degree can be improved; more importantly, through time-sharing multiplexing of the micro-droplet generation structure (taking the front and the back of the turnover of the integrated micro-droplet chip as time-sharing boundaries), the generation, amplification and detection of droplets are integrated in one chip, so that the fully-integrated and totally-enclosed digital PCR process is realized, the advantages of uniform droplet size, difficulty in limiting droplet quantity, high fluorescence detection signal-to-noise ratio and the like are inherited, the difficulties of complex structure, low integration level, difficulty in automation and the like of the original chip are overcome, and the method is an important technical breakthrough in the field of digital PCR.

As a specific implementation manner of the micro droplet generating structure, the micro droplet generating structure includes an oil pipe 21 and a communicating pipe, the oil pipe 21 and the communicating pipe cross, the communicating pipe includes a first pipe 22 located on a first side of the cross and communicating with the reaction chamber 11, and a second pipe 23 located on a second side of the cross and communicating with the sample adding chamber 12, and the oil interface 31 is communicated with the oil pipe 21.

In some embodiments, referring to fig. 1, with the first side surface of the chip body 1 in a horizontal position as a reference, the reaction chamber 11 and the sample application chamber 12 are located on the first side surface, the connection interface 111 of the reaction chamber 11 and the first side surface of the chip body 1 extends upward and is a bell mouth with a small lower part and a large upper part, and the connection interface 111 forming the bell mouth can facilitate the micro droplets 4 to enter the reaction chamber 11 from the first channel 22 and also facilitate the micro droplets 4 to enter the first channel 22 from the reaction chamber 11, so as to prevent the micro droplets 4 from being retained. It should be noted that, at this time, the reaction chamber 11 and the sample application chamber 12 are both located at a first side surface (specifically, a top surface) of the chip body 1, and the micro droplets 4 entering the reaction chamber 11 are all gathered at the connection interface 111, and when performing PCR amplification, the chip body 1 needs to be inverted, that is, turned over 180 °, so that the micro droplets 4 can be located in the reaction region of the reaction chamber 11.

In some embodiments, a gas-liquid pipe 112 extending from bottom to top is further configured in the reaction chamber 11, a lower opening of the gas-liquid pipe 112 is communicated with the gas-liquid interface 32, and an upper opening of the gas-liquid pipe 112 is higher than an upper opening of the connection interface 111, so that when negative pressure is applied in the reaction chamber 11, the micro-droplets 4 generated by the micro-droplet generating structure can be prevented from further flowing out of the gas-liquid pipe 112 after entering the reaction chamber 11.

In some embodiments, a micro-droplet observation area 34 is provided between the first pipe 22 and the connection interface 111, and a flow area of the micro-droplet observation area 34 is much larger than a flow area of the first pipe 22, that is, the micro-droplet observation area 34 is an enlarged area (with a larger width) on the first pipe 22, so that a flow velocity of the micro-droplets 4 entering the area is reduced, which is convenient for an external camera to image, record droplet shapes, and determine whether the state of the droplet generation process is normal.

As a specific implementation manner, the sample adding cavity 12 comprises an open cavity 121 and a sealing cover 122 connected to the opening of the open cavity 121 in a sealing manner, so that an operator can add a sample into the sample adding cavity 12 conveniently. Furthermore, the sample adding cavity 12 is provided with a filter membrane or an exhaust hole, so that when the sample adding cavity 12 becomes a waste liquid pool (namely when the liquid drop chip is turned upside down), certain air is removed, and pressure accumulation in the sample adding cavity 12 is prevented.

In one embodiment, the fluorescence detection region 33 is located on the second pipe 23, and the micro-droplets 4 flowing out of the reaction chamber 11 on the second pipe 23 can be separated into droplet arrays with proper intervals under the action of the detection oil in the oil pipe 21 when passing through the cross point, so that the fluorescence detection is completed under the action of an external system.

As shown in fig. 9, another implementation manner of the integrated micro droplet chip is provided, which is different from the integrated micro droplet chip shown in fig. 1 in that the reaction chamber 11 and the sample application chamber 12 are respectively located on two opposite sides of the chip body 1, specifically, the sample application chamber 12 is located on the first side, the reaction chamber 11 is located on the second side, and the second side and the first side are opposite sides of the chip body 1, at this time, the working principle and the flow of the integrated micro droplet chip are substantially the same as those of the previous integrated micro droplet chip, except that, in the droplet generation process, since the reaction chamber 11 is located on the bottom side of the chip body 1 (the sample application chamber 12 is located on the top side), the micro droplet 4 directly falls to the reaction region at the bottom of the reaction chamber 11 when entering the reaction chamber 11, and the micro-droplet chip is collected in the reaction area, so that the integral micro-droplet chip can directly enter a subsequent amplification link without turning 180 degrees after the generation of the droplets is finished.

In some embodiments, before the micro-droplets 4 enter the reaction chamber 11, light oil (i.e., oil with a relatively low density) is preset in the reaction chamber 11, so that the light oil can be always located at the top of the micro-droplets 4 in the reaction chamber 11, thereby solving the problem of evaporation of the micro-droplets during amplification and realizing hot-cap-free PCR.

According to the embodiment of the present invention, there is also provided a digital PCR method for an integrated micro droplet chip, the integrated micro droplet chip comprises a chip body 1, the chip body 1 has a reaction chamber 11 and a sample adding chamber 12, a micro droplet generation structure, an oil interface 31, an air-liquid interface 32 and a fluorescence detection region 33 are configured in the chip body 1, the air-liquid interface 32 is communicated with the reaction chamber 11, the sample adding chamber 12 is communicated with the micro droplet generation structure, the oil interface 31 is communicated with the micro droplet generation structure,

the digital PCR method comprises the following steps:

a micro-droplet segmentation and generation step, in which a first pressure difference is formed between the sample adding cavity 12 and the gas-liquid interface 32, and a second pressure difference is formed between the oil interface 31 and the gas-liquid interface 32, so that the first pressure difference and the second pressure difference respectively drive the sample in the sample adding cavity 12 and the generated oil at the oil interface 31 to enter the micro-droplet generation structure, and the generated micro-droplets 4 enter and are stored in the reaction cavity 11, specifically, oil is provided to the oil interface 31, negative pressure is provided to the gas-liquid interface 32, under the action of the negative pressure, the sample in the sample adding cavity 12 and the oil at the oil interface 31 are respectively driven to be converged at the intersection of the micro-droplet generation structure along the second pipeline 23 and the oil pipeline 21, and under the action of the shearing force and the surface tension of the oil fluid, forming micro-droplets 4 (water-in-oil droplets) with uniform size, and finally, under the action of the negative pressure, the micro-droplets 4 finally enter the reaction chamber 11 through the first pipeline 22 to be stored, it should be noted that when the micro-droplets enter the micro-droplet observation area 34, the flow velocity of the micro-droplets 4 forms a dense droplet community, which is convenient for camera imaging recording;

an amplification reaction step, in which the reaction chamber 11 is placed in a heating module (not shown in the figure) to heat and amplify according to a preset cycle, and the heating module can be an existing heating module;

a droplet detection step of controlling external pressure to drive detection push oil 5 to enter the reaction chamber 11 from the gas-liquid interface 32, so that the droplets 4 in the reaction chamber 11 flow out of the reaction chamber 11 to the droplet generation structure, and external pressure to drive detection separation oil to enter the droplet generation structure from the oil interface 31, where the detection separation oil separates the droplets 4 flowing out of the reaction chamber 11 to the droplet generation structure to form a queue, and enters the fluorescence detection region 33, so as to complete fluorescence detection, specifically, providing oil (i.e. detection push oil 5, also called as floating oil) to the gas-liquid interface 32, floating the droplets 4 after the buoyancy amplification reaction in the reaction chamber 11 by the oil, and enabling the droplets 4 to flow out of the reaction chamber 11 through the connection interface 111 and enter the first pipeline 22 under the buoyancy of the oil, and flows through the microdroplet observation area 34 to enter the intersection, and enters the second channel 23 to be detected in the fluorescence detection area 33, and then finally enters the sample adding cavity 12, wherein the sample adding cavity 12 is a waste liquid pool.

According to the technical scheme, the generation, amplification and detection of the droplets are integrated in one chip through time division multiplexing of the micro-droplet generation structure (the time division boundary is formed before and after the integral micro-droplet chip is turned over), so that the fully integrated and totally closed digital PCR process is realized, the advantages of uniform droplet size, difficulty in limiting the droplet quantity, high fluorescence detection signal-to-noise ratio and the like are inherited, the difficulties that the original chip structure is complex, the generation and the detection are completed in different chips, the integration level is low, the automation is difficult and the like are overcome, and the method is an important technical breakthrough in the field of digital PCR.

In some embodiments, the reaction chamber 11 and the sample application chamber 12 are located on the first side of the chip body 1 with reference to the first side being in a horizontal orientation, and before the amplification reaction step, after the micro-droplet splitting generation step, the method further includes: and a chip overturning step, namely controlling the chip body 1 to overturn up and down by 180 degrees, at the moment, enabling the micro liquid drops 4 in the reaction cavity 11 to be arranged on one side far away from the connecting interface 111 from the side close to the connecting interface 111, at the moment, enabling the position of the reaction cavity 11 corresponding to the micro liquid drops 4 to be a reaction region of the reaction cavity 11, and enabling the reaction region to be in contact with the heating module to adjust the temperature so as to realize the temperature adjustment reaction of the sample.

The operation flow of the present invention using the integrated micro droplet chip is further described with reference to fig. 1 to 8 as follows:

first, 30. mu.l of a PCR system (i.e., the aforementioned sample) containing 10. mu.l of ddPCR Supermix for Probes, GJB2 gene upstream and downstream primer reagents and 5. mu.l of a template containing 1ng of genomic DNA was loaded into the loading chamber 12. The whole chip is shown in fig. 1, one chip comprises 8 parallel independent droplet chip structures, and each structure comprises a sample adding cavity 12, an oil interface 31, an air-liquid interface 32, a micro droplet generating structure and a reaction cavity 11.

Then, the sealing lid 122 is tightly covered or adhered to seal the sample application chamber 12, and the sample application chamber 12 preferably has a filter membrane or a vent hole with a small aperture.

Then, the Bio-Rad Generation Oil required for droplet Generation is supplied to the Oil interface 31, and the generated Oil contains a surfactant capable of stabilizing the droplets; and negative pressure is provided for the gas-liquid interface 32, and the pressure is-200 mBar, so that a pressure difference is formed between the gas-liquid interface and the sample adding cavity 12 and the oil interface 31. The sample adding cavity 12 is connected with the second pipeline 23 in the micro-droplet generating structure, the oil interface 31 is connected with the oil pipeline 21 in the micro-droplet generating structure, the first pipeline 22 and the gas-liquid interface 32 are both connected with the reaction cavity 11, wherein the first pipeline 22 is connected with the connecting interface 111, and the gas-liquid interface 32 is connected with the gas-liquid pipeline 112. Each part of the droplet generating structure is shown in fig. 2, the oil pipe 21 has two branches, which are respectively located at two sides of the second pipe 23 and the first pipe 22, and are connected to the oil interface 31. The first pipeline 22 can contain a micro-droplet observation area 34, the observation area pipeline is widened, the flow velocity is reduced after the droplets enter, an external camera can conveniently image, the droplet form is recorded, and whether the state of the droplet generation process is normal or not is judged.

Driven by pressure difference, the reaction system enters the second pipeline 23, the generated oil enters the oil pipeline 21 and meets at the cross structure (namely the cross intersection), and water-in-oil micro-droplets 4 with uniform size are formed under the action of fluid shearing force and surface tension. The depth of the channel at the cross was about 70 microns, the width was 80 microns, and the droplet size was about 100 microns. The micro-droplets 4 enter the first pipeline 22, and after entering the micro-droplet observation area 34, the flow rate is reduced, so that a dense droplet community is formed, which is convenient for camera imaging recording, and a schematic diagram of a droplet generation process is shown in fig. 4.

The generated droplets flow through the first pipe 22 and reach the connection port 111 of the reaction chamber 11. The bottom of the connection interface 111 has a slope structure (i.e. the aforementioned flare), and the slope bottom is communicated with the first pipe 22. At the end of the droplet generation process, the pressure difference applied at the chip interface is removed, and the droplet is still located below the gas-liquid pipe 112.

Thereafter, the chip is turned upside down, so that the liquid drops are transferred from the connection interface 111 to the reaction region (i.e., away from the connection interface 111), as shown in FIG. 5. The structure of the reaction zone should adopt the design that the heat transfer efficiency is high, for example adopt the design of the flat that degree of depth is high, thickness is thin, let the external system (also be heating module) heat and refrigerate for the reaction zone from the left and right sides, not only guarantee that the distance of temperature conduction is short, still guarantee great area of contact to realize efficient heat transfer. In this example, the temperature cycle procedure was a pre-denaturation at 95 ℃ for 10 min, followed by 40 temperature cycles of 5 sec at 95 ℃ and 15 sec at 60 ℃ in each cycle, and finally a 4 ℃ incubation. To reduce evaporation, 30. mu.l of a low-density anti-volatilization reagent may be placed in advance in the reaction chamber 11.

After the temperature cycle is finished, the amplification reaction in the droplet containing the template is correspondingly finished, and the fluorescence detection link of the droplet needs to be entered. The gas-liquid interface 32 is filled with the detection oil (i.e., the detection pusher oil 5) to continuously fill the reaction chamber 11. During this process, the droplet level rises and is then directed through the ramp of the connection interface 111 into the first conduit 22. This process is illustrated in fig. 7.

The droplet may also be brightfield imaged by the camera as it passes through the observation region in the first conduit 22 to assess the state of the droplet after the amplification reaction. Meanwhile, the detection oil is injected into the oil interface 31, joins the droplet queue at the cross pipe via the oil pipe 21, and separates the droplets arranged closely into droplet queues having appropriate intervals. The queue of droplets passes in turn through a fluorescence detection zone 33 located in the second conduit 23 as shown in figure 8. The fluorescence detection region 33 corresponds to a position that is a fluorescence detection focus of an external system. An external system focuses excitation light, such as laser light with wavelengths of 488nm and 532nm or LED narrow-band light, to a detection focus. In the process that the liquid drops sequentially pass through the detection focus, fluorescence excited in the liquid drops is received by a collecting light path of an external system, and therefore fluorescence information of each liquid drop is obtained. And (3) defining a signal threshold value by using fluorescence information of the liquid drop, distinguishing the negative and positive of the liquid drop, and calculating the copy number of the target molecule in the sample by using a Poisson distribution model.

Finally, the liquid drops which finish the fluorescence detection enter the sample adding cavity 12, and the sample adding cavity 12 is sealed by the sealing cover 122, so that the liquid drops cannot contact with the environment outside the chip, the possibility of aerosol pollution is avoided, and the totally-closed digital PCR process is realized.

In this embodiment, a method of time-multiplexing the droplet generation structures is pioneered. When the liquid drop is generated, the liquid drop generation is realized by utilizing the micro-liquid drop generation structure. And when the liquid drop fluorescence is detected, the micro-liquid drop generating structure completes the separation of the liquid drop queue, and the detection of the liquid drop fluorescence signal is ensured. By adopting the time-sharing multiplexing method, the digital PCR process of completing droplet generation, amplification and detection by one chip on the analog flow type digital PCR technical route is realized for the first time, and the method is an important technical breakthrough in the field of digital PCR.

In another embodiment, when the droplets pass through the observation area in the first conduit 22, the droplets are subjected to fluorescence imaging by a camera, and the imaged picture is analyzed to obtain fluorescence intensity information of each droplet in the picture, thereby completing droplet fluorescence signal detection.

It is readily understood by a person skilled in the art that the advantageous ways described above can be freely combined, superimposed without conflict.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention. The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (10)

1. An integrated micro-droplet chip is characterized by comprising a chip body (1), wherein the chip body (1) is provided with a reaction cavity (11) and a sample adding cavity (12), a micro-droplet generation structure, an oil interface (31), a gas-liquid interface (32) and a fluorescence detection area (33) are constructed in the chip body (1), the gas-liquid interface (32) is communicated with the reaction cavity (11), the sample adding cavity (12) is communicated with the micro-droplet generation structure, and the oil interface (31) is communicated with the micro-droplet generation structure;

when liquid drops are generated, a first pressure difference is formed between the sample adding cavity (12) and the gas-liquid interface (32), a second pressure difference is formed between the oil liquid interface (31) and the gas-liquid interface (32), the sample in the sample adding cavity (12) and the generated oil of the oil liquid interface (31) are respectively driven by the first pressure difference and the second pressure difference to enter the micro liquid drop generating structure, and the generated micro liquid drops (4) enter and are stored in the reaction cavity (11);

when the droplets are detected, external pressure drives and detects to push oil (5) to enter the reaction cavity (11) from the gas-liquid interface (32), so that the micro droplets (4) in the reaction cavity (11) flow out of the reaction cavity (11) to the micro droplet generation structure, external pressure drives and detects to separate oil to enter the micro droplet generation structure from the oil interface (31), and the detected and separated oil separates the micro droplets (4) flowing out of the reaction cavity (11) to the micro droplet generation structure to form a queue and enters the fluorescence detection area (33).

2. The integrated micro droplet chip of claim 1, wherein the micro droplet generation structure comprises an oil pipe (21) and a communication pipe, the oil pipe (21) crosses the communication pipe, the communication pipe comprises a first pipe (22) at a first side of the cross point and communicating with the reaction chamber (11), and a second pipe (23) at a second side of the cross point and communicating with the sample application chamber (12), and the oil interface (31) communicates with the oil pipe (21).

3. The integrated micro droplet chip of claim 2, wherein the reaction chamber (11) and the sample application chamber (12) are located on the first side of the chip body (1) with reference to the first side of the chip body being in a horizontal orientation, and the connection interface (111) of the reaction chamber (11) and the first side of the chip body (1) extends upward and is a horn mouth with a smaller bottom and a larger top.

4. The integrated micro droplet chip of claim 3, wherein a gas-liquid pipe (112) extending from bottom to top is further configured in the reaction chamber (11), a lower opening of the gas-liquid pipe (112) is communicated with the gas-liquid interface (32), and an upper opening of the gas-liquid pipe (112) is higher than an upper opening of the connection interface (111).

5. The integral microfluidic chip according to claim 4, wherein there is a microfluidic observation zone (34) between the first conduit (22) and the connection interface (111).

6. The integrated micro droplet chip of claim 1, wherein the sample application chamber (12) comprises an open chamber (121) and a sealing cover (122) hermetically connected to an opening of the open chamber (121).

7. The integrated micro-droplet chip of claim 1, wherein the sample application chamber (12) is provided with a filter or a vent.

8. The integral micro droplet chip according to claim 2 wherein the fluorescence detection zone (33) is on the second conduit (23).

9. The integrated micro droplet chip of claim 3, wherein the sample application cavity (12) is on the first side, the reaction cavity (11) is on a second side, and the second side and the first side are opposite sides of the chip body (1).

10. The integral micro droplet chip according to claim 1, wherein light oil is pre-filled in the reaction chamber (11) before the micro droplets (4) enter the reaction chamber (11).

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