CN115770627A

CN115770627A - Liquid drop reading chip and using method thereof

Info

Publication number: CN115770627A
Application number: CN202111038501.5A
Authority: CN
Inventors: 周洪波; 宋汝渊; 许潇楠
Original assignee: Zhejiang Dapu Biotechnology Co ltd
Current assignee: Zhejiang Dapu Biotechnology Co ltd
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2023-03-10

Abstract

The invention relates to the field of droplet microfluidics, in particular to a droplet reading chip and a using method thereof. The liquid drop reading chip comprises a microcavity and a sample sucking needle, wherein the microcavity is used for flatly laying liquid drops, and the sample sucking needle is used for sucking the liquid drops into the microcavity. The chip can simultaneously tile a large number of liquid drops, and the requirement of high-throughput detection of the liquid drops is met. In addition, the chip of the invention can ensure that the liquid drop is in a relatively closed space during detection, thereby reducing the pollution risk.

Description

Liquid drop reading chip and using method thereof

Technical Field

The invention relates to the field of droplet microfluidics, in particular to a droplet reading chip and a using method thereof.

Background

Microfluidics refers to a technique for manipulating fluids in a microscale space, which can scale down the basic functions of chemical, biological, etc. laboratories to a few square centimeters on a chip, and is therefore also referred to as "lab-on-a-chip". The droplet microfluidics is an important branch of a recently developed microfluidic platform, is a discontinuous flow microfluidic technology for performing experimental operation by using dispersed micro droplets generated by two mutually incompatible liquids, and has the advantages of reducing reagent consumption, reducing pollution risk, improving reaction precision, shortening reaction time and the like. Wherein, the liquid drop is used as a 'microreactor' and has the characteristics of regionalization, miniaturization, controllability, high flux and the like.

Droplet microfluidic technology includes droplet generation, droplet manipulation, and droplet detection. Among them, the reading of high-throughput droplet signals is an important link of droplet detection technology. In the prior art, researches on droplet generation, droplet operation, detector performance, chip manufacturing, application and the like in the droplet microfluidic technology are mostly focused, and how the droplets enter the detector and how the droplets are arranged are rarely and independently researched. In addition, the liquid droplets are often in an open state in contact with the outside atmosphere at the time of signal reading, and contamination is easily caused.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the liquid drop reading chip and the use method thereof, the chip has low cost, convenient use and small pollution risk in the use process, and meets the detection requirement of high-flux liquid drops.

The invention aims to solve the problems by the following technical scheme:

a liquid drop reading chip is characterized by comprising a microcavity and a sample sucking needle; the micro-cavity is used for flatly paving liquid drops; the sample sucking needle is used for sucking liquid drops into the micro-cavity. The sample suction needle is communicated with the micro-cavity and can exchange fluid. The micro-cavity can be a closed cavity, and the solution containing the liquid drops is pressurized to enable the liquid drops to enter the micro-cavity through the sample sucking needle. An orifice may also be provided in the microcavity, and a droplet may be drawn into the microcavity through the pick-up needle by suction at the orifice.

Preferably, the chip comprises a channel communicated with the outside, and the channel is not communicated with the microcavity.

Further, when the channel and the sample sucking needle are connected with the same closed space, the channel can enable the solution containing liquid drops in the closed space to enter the microcavity through the sample sucking needle through the circulation with the outside air.

Further, the channels are capable of venting gases within the enclosed space and microcavities. The air can be pumped through the channel, so that the air in the micro-cavity and the closed space is exhausted, and negative pressure with the same pressure is formed.

Further, gas is input into the closed space through the channel, the pressure of the closed space is increased, negative pressure is kept constant in the microcavity, a pressure difference is formed between the microcavity and the closed space, and under the action of the pressure difference, solution containing liquid drops in the closed space enters the microcavity through the sample sucking needle.

Further, in the gas discharging process, one end of the sample sucking needle is immersed into the solution containing the liquid drops in the closed space, and one end of the channel is not immersed into the liquid containing the liquid drops.

Furthermore, the other end of the sample sucking needle is communicated with the microcavity, and the other end of the channel is communicated with the outside atmosphere.

Furthermore, the closed space is formed by a sealed sample tube, the sample tube is filled with a solution containing liquid drops, and a gas area is arranged above the solution. The sample tube can be a container for holding liquid drops, such as a PCR tube, a 96-well plate, a 384-well plate, a centrifuge tube, a liquid phase vial and the like. The gas area is a blank space for gas

Preferably, a filter sieve is arranged in the channel, and the filter sieve is of a net structure and is used for preventing aerosol pollution; the filter sieve may further be a microporous membrane structure with a smaller pore size, which allows gas to pass through but does not allow microorganisms such as bacteria to pass through, for example, a 0.22 μm microporous membrane, which may reduce the risk of contamination more effectively.

Preferably, the chip comprises a snake-shaped cavity, the microcavity is connected with the snake-shaped cavity, and the snake-shaped cavity is generally arranged between the microcavity and the sample sucking needle and used for trapping possible bubbles and preventing the bubbles from entering the microcavity.

Preferably, the chip comprises a liquid storage cavity, the microcavity is connected with the liquid storage cavity, and the liquid storage cavity is used for storing continuous phases such as an oil phase or a water phase in the solution containing the liquid drops.

Preferably, the chip comprises a transparent upper cover plate and a microfluidic plate; the transparent upper cover plate and the microfluidic plate are sealed to form the micro-cavity. The transparent upper cover plate and the microfluidic plate are provided with notches, so that the transparent upper cover plate and the microfluidic plate can be conveniently embedded and sealed.

Preferably, the microfluidic plate comprises a serpentine channel sealed with the transparent upper cover plate to form the serpentine cavity.

Preferably, the microfluidic plate comprises a liquid storage tank, and the liquid storage tank and the transparent upper cover plate are sealed to form a liquid storage cavity.

Further, the sample sucking needle comprises a base and a needle, and the base is used for being embedded with the microfluidic plate.

Furthermore, the transparent upper cover plate, the microfluidic plate and the base are all provided with through holes for forming the channel after combination.

Preferably, the microcavity limits the liquid drops in height, and the height of the microcavity is 0.5-1.2 times of the diameter of the liquid drops, so that the liquid drops are tiled into a layer, the acquisition of subsequent liquid drop signals is facilitated, and the mutual interference is reduced. The microcavity height is optimally the droplet diameter.

Further, the number of the microcavities and the number of the sample sucking needles are not less than 1. The micro-cavities and the sample sucking needles can be independent systems corresponding to one another, and the number of the micro-cavities and the number of the sample sucking needles can be adjusted according to use requirements, so that signals of a plurality of liquid drop samples can be read at the same time in the subsequent process.

Preferably, the draw needle comprises a tapered hollow needle to facilitate access to the sample solution.

Preferably, the chip includes an auxiliary sealing member for sealing the hermetic space.

Preferably, the auxiliary seal is provided with a seal groove and a tear groove. The sealing groove is used for sealing the sample tube to ensure the tightness of the sealed space; the tear groove can be generally made thinner or of a softer material to facilitate penetration by the lancet.

Further, the auxiliary sealing member is a silica gel cap. The auxiliary seal may be made of other elastic material such as rubber, resin, or plastic. In order to ensure sealing, an aluminum foil gland and the like can be further arranged at the periphery.

Referring to fig. 1, a method for using a droplet reading chip includes the following steps:

step (1): the method comprises the following steps of (1) enabling a solution containing liquid drops to be located in a sealed sample tube to form a sealed space, wherein the upper end of the sealed space is a gas zone which is marked as a zone B, the zone B is communicated with a channel, one end of the channel is communicated with the zone B, the other end of the channel is communicated with the outside, and gas exchange between the sealed space and the outside can be carried out through the channel; the sample aspirating needle enters the solution containing the liquid drop.

Step (2): gas in the B area is discharged through the channel, if gas exists in the microcavity, the gas can be discharged through the sample sucking needle, the B area and the channel, and at the moment, the pressure of the microcavity is recorded as P _aim . The gas in the B area can be exhausted by pumping at one end of the channel communicated with the outside.

And (3): introducing gas into the zone B through the passage, wherein the gas pressure in the zone B is P ₀ ；

And (4): the P is ₀ Higher than said P _aim Forming negative pressure in the micro-cavity, and enabling the solution containing the liquid drops to enter the micro-cavity through the sample sucking needle until the air pressure of the B area is equal to that of the micro-cavity;

and (5): and the liquid drop is flatly laid in the microcavity, the chip is placed in an optical detection system, and the liquid drop signal in the microcavity is read.

Further, by controlling said P ₀ And P _aim A specific negative pressure is created in the microcavity, thereby controlling the amount of the solution containing droplets entering the microcavity.

Further, the passage may be opened to the outside atmosphere in the step (3) to allow air to enter the region B, at which time P is present ₀ Is at atmospheric pressure; it is also possible to fill the region B with gas through the channels to a certain gas pressure.

Further, the gas in step (3) may be air, or inert gases such as nitrogen, helium, and argon may also be used, so that the solution containing the liquid droplets is prevented from contacting with air, the risk of contamination is further reduced, but the cost is increased.

Further, the channel may be disposed on the droplet reading chip, such as through the droplet reading chip to communicate one end with the region B; or on a sealed sample tube, such as an auxiliary sealing device for sealing the sample tube; or can be directly arranged on the sample tube.

Preferably, a means is provided at the end of the passage communicating with the outside to place the end of the passage in an open or closed state. The end is in a closed state when the channel is not subjected to gas exchange, so that the communication between the closed space and the outside can be further reduced, and the pollution risk is reduced. The device can be a piston or a switching valve structure.

The invention has the advantages that:

the invention provides a liquid drop reading chip and a using method thereof, and provides a feasible scheme for high-throughput detection of liquid drops. The chip provided by the invention provides a relatively sealed system, so that the pollution risk is reduced, and the chip provided by the invention is simple in structure and convenient to use, and can reduce the detection cost of liquid drops.

Drawings

Features, advantages and technical effects of exemplary embodiments of the present invention will be described below with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of a droplet reading chip and a method for using the same according to an embodiment of the present invention

FIG. 2 is a view of a droplet reading chip according to an embodiment of the present invention

FIG. 3 is an exploded view of a droplet reading chip according to an embodiment of the present invention

FIG. 4 is a top view of the drop reading chip of FIG. 3

FIG. 5 is a front view of the drop reading chip of FIG. 3

FIG. 6 is a schematic view of an auxiliary seal silicone cap and its application according to an embodiment of the present invention

In the figure: 8-auxiliary sealing element, 9-closed space, 10-sample tube, 71-transparent upper cover plate, 72-microfluidic plate, 73-sample suction needle, 74-oil storage cavity, 75-microcavity, 76-serpentine cavity, 77-channel, 78-filter sieve, 711-pore channel, 712-notch, 721-oil storage pool, 722-liquid level tiling groove, 723-serpentine channel, 724-sample inlet, 731-sample outlet, 732-needle, 733-needle seat, 81-PCR tube groove, 82-tearing groove, 91-sample solution, 911-liquid drop and 912-continuous phase.

Detailed Description

The following detailed description should be read with reference to the drawings, in which like elements in different drawings are numbered the same. The drawings, which are not necessarily to scale, depict illustrative embodiments and are not intended to limit the scope of the invention.

Fig. 1 shows a droplet reading chip according to the invention. As shown in fig. 1a, the chip comprises a micro-cavity 75, a sample-aspirating needle 73, a reservoir 74, a channel 77 and an auxiliary seal 8. The pipette needle 73 and the channel 77 are connected to the same sealed space 9. The sealed space 9 is formed by sealing the auxiliary seal 8 and the sample tube 10. The closed space 9 contains a sample solution 91 and a gas zone B (fig. 1B) above the sample solution 91. The pipette needle 73 penetrates the auxiliary seal 8 into the bottom of the sample solution 91, thereby communicating with the closed space 9. The channel 77 is connected to the region B of the closed space 9 through the auxiliary seal 8. Sample solution 91 includes droplets 911 and continuous phase 912.

In addition, fig. 1 shows a method of using the chip, which includes the following processes:

(1) In FIG. 1a, droplet 911 has a volume V _drop Continuous phase 912 volume is V _oil . The other end of the channel 77 is open to the atmosphere.

(2) As shown in FIG. 1B, a negative pressure is formed by pumping at the end of the channel 77 which is communicated with the atmosphere, and the gas in the B area is exhausted to generate a gas pressure Paim with a specified vacuum degree. At this time, if the pressure in the area A formed by the communication of the micro-cavity 75, the liquid storage cavity 74 and the sample suction needle 73 is higher than that in the area B, the gas in the area A can be discharged through the area B and then through the channel 77 until the pressure in the area A and the pressure in the area P are equal _aim And are equal. And stopping pumping.

(3) As shown in FIG. 1c, the channel 77 is again vented to atmosphere and air enters zone B, which is at ambient pressure P ₀ And are equal.

(4) In FIG. 1d, the gas pressure P in the area B ₀ Greater than region A P _aim In aUnder the action of the pressure difference, the sample solution 91 enters the micro-cavity 75 through the sample suction needle 73, and the entering process of the sample solution 91 is finished when the air pressure in the area A is equal to the air pressure in the area B.

(5) As shown in fig. 1e, the continuous phase 912 is ideally fully compressed into the reservoir 74 and the droplets 911 are fully spread in the microcavity 75 to form a layer of spread droplets. The upper surface of the microcavity 75 is transparent, allowing light to pass through. And starting an optical detection system to perform signal reading and the like on the tiled liquid drops.

In this process, the volume of solution V = (Vcc + Vcd + Vcn) (P) pressed into zone a ₀ -P _aim )/P ₀ . Wherein Vcc is the volume of the liquid storage cavity 74, vcd is the volume of the microcavity 75, and Vcn is the volume of the sample sucking needle 73. Thus, by controlling P _aim The amount of solution drawn into the chip can be effectively controlled.

Fig. 2 shows a droplet reading chip according to the invention, differing from the chip of fig. 1a in that a filter sieve 78 is added at the channel 77. The filter screen 78 is in a mesh structure for preventing aerosol contamination; it may further be a microporous filter membrane structure with a smaller pore size, which allows gas to pass through but does not allow microorganisms such as bacteria to pass through, for example, a 0.22 μm microporous filter membrane, which may be more effective in reducing the risk of contamination.

Fig. 3-5 illustrate a drop reading chip according to the present invention. As shown in FIG. 3, the chip is composed of three parts, namely a transparent upper cover plate 71, a microfluidic plate 72 and a sample sucking needle 73. The transparent upper cover 71 is made of a transparent material, and allows signals such as light to pass therethrough. The micro-fluidic plate 72 is provided with a liquid storage tank 721, a liquid level tiling groove 722 and a serpentine channel 723. The transparent upper cover plate 71 and the microfluidic plate 72 are provided with notches 712 to facilitate the embedding of the two, and the two are sealed to form the liquid storage cavity 74, the micro-cavity 75 and the serpentine cavity 76 (fig. 4) after being embedded. During use, reservoir chamber 74 is used to store the continuous phase; the microcavity 75 is used for tiling the liquid droplets, and limits the liquid droplets on the height, so that the liquid droplets are tiled into a layer, wherein the height of the microcavity is 0.5-1.2 times of the diameter of the liquid droplets, and the microcavity is optimally the same as the diameter of the liquid droplets; the serpentine cavity 76 is curved and located at the front end of the microcavity 75 to buffer any incoming bubbles and trap them in the channel to prevent them from entering the microcavity 75. The pipetting needle 73 includes a base 733 and a needle 732. The pipette needle 73 is mounted below the microfluidic plate 72. The sample outlet 731 of the needle 732 is connected with the sample inlet 724 of the microfluidic plate 72, and is used for allowing the sample solution to enter the snake-shaped cavity 76 through the sample sucking needle; further, the droplets in the sample solution enter the micro-cavity 75 and the continuous phase enters the reservoir 74. Needle 732 may be a tapered hollow needle to facilitate puncturing of the secondary seal into the bottom of the enclosed space. The transparent upper cover plate 71, the microfluidic plate 72 and the sample sucking needle 73 are all provided with a through hole 711, and the three are combined to form a channel 77.

It should be noted that, after the transparent upper cover plate 71, the microfluidic plate 72 and the sample sucking needle 73 of the chip in fig. 3 to 5 are combined, 8 independent channels (composed of a needle, a snake-shaped cavity, a microcavity and a liquid storage cavity) are formed, which can be used for detecting 8 sample solutions at the same time, and the number of the channels can be adjusted according to the needs in the actual use process.

Fig. 6 shows an auxiliary seal-silicone cap according to one embodiment of the present invention. The silica gel cap is designed for a PCR tube. As shown in FIG. 6a, the silica gel cap can be hooped at the opening of the PCR tube to seal the PCR tube. As shown in FIG. 6b, the silicone cap 8 comprises a PCR tube slot 25 and a tearing slot 26, the PCR tube slot 26 is used for clamping the PCR tube; the tearing groove 26 is formed in a relatively thin silicone mold, for example, 100 μm, so that the pipette needle can easily enter the PCR tube.

In addition to the drawing of the liquid drop by the negative pressure control shown in fig. 1, the method of the present invention may also be used to directly press the liquid drop sample into the micro-cavity 75 by pressurization, or connect a suction device to the micro-cavity 75 to suck the liquid drop sample into the micro-cavity 75 through the sample sucking needle 73. The above-mentioned channel 77 for gas ingress and egress may be provided on the device of the present invention, or on the device for holding a droplet sample or other auxiliary devices. The sample tube 10 may be a container for containing a solution, such as a PCR tube, a 96-well plate, a 384-well plate, a centrifuge tube, or a liquid phase vial.

It is to be understood that those skilled in the art can appropriately change or modify the above-described embodiments without departing from the spirit and scope of the present invention. For example: the relative positions of the reservoir 74, the microcavity 75, and the serpentine cavity 76 can be adjusted as appropriate, for example, the reservoir 74 can be located at the top of the microcavity 75. The liquid drops can be of an O/W type, and the liquid storage cavity 74 is used for storing oil phase; the droplets may also be of the W/O type, in which case reservoir 74 is used to store the aqueous phase.

The above description is only for the purpose of illustrating the technical ideas and features of the present invention, and the technical ideas and features of the present invention are not limited thereto. The technology not related to the invention can be realized by the prior art.

Claims

1. A liquid drop reading chip is characterized by comprising a microcavity and a sample sucking needle; the micro-cavity is used for flatly paving liquid drops; the sample sucking needle is used for sucking liquid drops into the micro-cavity.

2. A droplet reading chip according to claim 1, wherein the pipetting needle is in fluid communication with the microcavity and is capable of fluid communication.

3. A droplet reading chip according to claim 1 or 2, wherein the chip comprises a channel in communication with the environment, the channel not being in communication with the microcavity.

4. The chip of claim 3, wherein when the channel and the sample-sucking needle are connected to the same enclosed space, the channel can communicate with the outside air to allow the solution containing the liquid drop in the enclosed space to enter the micro-cavity through the sample-sucking needle.

5. A liquid droplet reading chip according to claim 4 wherein the channel is capable of venting gas from the enclosed space and the microcavity.

6. The liquid drop reading chip of claim 5, wherein a pressure difference is formed between the micro-cavity and the enclosed space by introducing a gas into the enclosed space through the channel, and under the action of the pressure difference, the solution containing liquid drops in the enclosed space enters the micro-cavity through the sample sucking needle.

7. A droplet reading chip according to claim 4, wherein one end of the pipetting needle is immersed in the solution containing the droplet and one end of the channel is not immersed in the solution containing the droplet.

8. The liquid drop reading chip of claim 7, wherein the other end of the sample sucking needle is communicated with the micro-cavity, and the other end of the channel is communicated with the outside.

9. A droplet reading chip according to claim 4, wherein the enclosed space is formed by a sealed sample tube containing the droplet solution and having a gas zone above the solution.

10. A droplet reading chip according to claim 3, wherein the channel is provided with a filter screen.

11. A droplet reading chip according to claim 1, wherein the chip comprises a serpentine cavity, and the microcavity is connected to the serpentine cavity.

12. The droplet reading chip of claim 1, wherein the chip comprises a reservoir cavity, and the microcavity is coupled to the reservoir cavity.

13. A droplet reading chip according to claim 1, wherein the chip comprises a transparent upper cover plate and a microfluidic plate; the transparent upper cover plate and the microfluidic plate are sealed to form the micro-cavity.

14. A droplet reading chip according to claims 11 and 13, wherein the microfluidic plate comprises a serpentine channel sealed to the transparent top cover plate to form the serpentine cavity.

15. A droplet reading chip according to claims 12 and 13, wherein the microfluidic plate comprises a reservoir sealed with the transparent top cover plate to form a reservoir chamber.

16. The droplet reading chip of claim 1, wherein the pipetting needle comprises a base and a needle, the base for engaging with the microfluidic plate.

17. A liquid droplet reading chip according to claims 3, 13 and 16, wherein the transparent top cover plate, the microfluidic plate and the base are provided with channels for forming the channels when combined.

18. A droplet reading chip according to claim 1, wherein the micro-cavity allows the droplets to be tiled in a layer.

19. A droplet reading chip according to claim 1, wherein the number of the micro-cavities and the number of the pipette tips are not less than 1.

20. A droplet reading chip according to claim 1, wherein the pipetting needle comprises a tapered hollow needle.

21. A droplet reading chip according to claim 4, wherein the chip comprises an auxiliary seal for sealing the closed space.

22. A drop reading chip as claimed in claim 21, wherein said secondary seal is provided with a seal groove and a tear groove.

23. A drop reading chip according to claim 21, wherein said auxiliary seal is a silicone cap.

24. The method of using a droplet reading chip according to claim 1, comprising the steps of:

step (1): the method comprises the following steps of (1) enabling a solution containing liquid drops to be located in a closed sample tube to form a closed space, wherein the upper end of the closed space is a section of gas area marked as an area B, the area B is communicated with a channel, one end of the channel is communicated with the area B, the other end of the channel is communicated with the outside, and gas exchange between the closed space and the outside can be carried out through the channel; the sample sucking needle enters the solution containing the liquid drops;

step (2): gas in the B area is discharged through the channel, if gas exists in the microcavity, the gas can be discharged through the sample sucking needle, the B area and the channel, and at the moment, the pressure of the microcavity is recorded as P _aim ；

And (3): gas enters the B area through the channel, and the gas pressure in the B area is P ₀ ；

And (4): said P is ₀ Higher than said P _aim Forming negative pressure in the microcavity, and enabling the solution containing the liquid drops to enter the microcavity through the sample sucking needle until the air pressure of the B area is equal to that of the microcavity;

and (5): and placing the chip in an optical detection system, and reading the droplet signals in the microcavity.

25. The method of claim 24, wherein said P is controlled by controlling said P ₀ And P _aim Causing a specified negative pressure to develop in the microcavity, thereby controlling the amount of the solution containing the droplet entering the microcavity.

26. The method of claim 24 or 25, wherein P is selected from the group consisting of ₀ Is at atmospheric pressure.

27. The method of claim 24, wherein the gas in step (3) comprises air, nitrogen, helium, and argon.