A device and method for three-dimensional digital droplet rapid nucleic acid amplification and detection

Abstract

The present invention discloses a device and method for three-dimensional digital droplet rapid nucleic acid amplification and detection, wherein a droplet dispersion storage device is involved, including: an outer tube with an opening at one end and an inner tube sleeved in the outer tube, a slit for dispersing droplets is formed between the outer tube and the inner tube, the width of the slit matches the diameter of the droplet, and a heating cylinder is sleeved on the outer side of the outer tube. The droplet dispersion storage device of the present invention can heat the droplets evenly, shorten the equilibrium time between the heating module and the sample temperature in the droplet, reduce the time required for nucleic acid amplification, and achieve the purpose of rapid nucleic acid amplification. At the same time, an endoscope probe droplet fluorescence detection module is coupled to achieve high-throughput rapid droplet imaging.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology auxiliary equipment, and in particular to a method and a device for rapid uniform heating and detection of three-dimensional digital droplets. Background Art

[0002] At present, many methods have been developed to quantify the amount of nucleic acid as accurately as possible, and PCR-based nucleic acid quantification technology has always been favored. Real-time quantitative PCR (qPCR) has long been the preferred method for quantifying the amount of nucleic acid and is also considered a routine experiment in biomedical laboratories. With the vigorous development of next-generation sequencing technology and single-cell analysis technology, people's interest in nucleic acid quantification has reached an unprecedented single-molecule level. This has led to the prosperity of digital droplet amplification technology. Digital droplet amplification technology is to distribute samples into many reaction chambers or droplets so that samples are amplified in independent but identical systems, and the all-or-nothing detection results of each reaction follow a Poisson distribution. After calculating the sum of the positive reactions, through Poisson correction, not only the sample nucleic acid concentration can be obtained, but also the absolute number of target molecules can be obtained.

[0003] Generally speaking, the traditional droplet nucleic acid amplification process includes three main steps: distribution, amplification and counting. The distribution process can be achieved through droplets or chip microchambers. The chip microchamber method is mostly used for constant temperature rapid nucleic acid amplification due to the difficulty in coupling variable temperature modules and easy evaporation of samples. Droplet PCR is mostly performed in a PCR instrument. However, the actual time required for PCR is much longer than the time required for the reaction itself. Therefore, using other more effective methods to shorten the PCR time and improve the efficiency of digital droplet PCR has always been a goal pursued by people.

[0004] In droplet fluorescence detection, the droplet plane imaging equipment is simple, high-throughput, and fast. It is not only suitable for flowable droplets, but also very compatible with nucleic acid amplification based on chip microchambers. However, this method currently has a series of problems such as droplet loss and contamination. Therefore, how to optimize the droplet plane imaging method to achieve a higher throughput imaging method, avoid droplet loss and contamination, and reduce experimental costs is currently a top priority for research. Summary of the invention

[0005] In order to solve the problem of uneven heating of droplets in PCR tubes in the prior art, as well as the problem of high cost of droplet flat imaging and complex image processing requirements.

[0006] The present invention provides a device for dispersing and storing droplets, comprising: an outer tube with an opening at one end and an inner tube sleeved in the outer tube, a slit for dispersing droplets is formed between the outer tube and the inner tube, the width of the slit matches the diameter of the droplets, and a heating cylinder is sleeved on the outer side of the outer tube. The width of the slit matches the diameter of the droplets, which means that the width of the slit should be less than twice the diameter of the droplets.

[0007] Furthermore, the outer tube and the inner tube have the same shape but different sizes, and the outer tube and the inner tube are concentrically arranged.

[0008] Furthermore, the outer tube and the inner tube are both circular tubes, and the difference between the inner diameter of the outer tube and the outer diameter of the inner tube is less than 4 times the diameter of the droplet.

[0009] Furthermore, the outer tube and the inner tube are both square tubes, and the difference between the inner diameter side length of the outer tube and the outer diameter side length of the inner tube is less than 4 times the droplet diameter.

[0010] Furthermore, the outer tube and the inner tube are both amorphous tubes, and the distance between the outer wall of the inner tube and the inner wall of the outer tube is less than 2 times the diameter of the droplet.

[0011] Furthermore, the top of the slit is connected to the external atmosphere.

[0012] Furthermore, the inner surface of the outer tube and the outer surface of the inner tube are hydrophobic and lipophilic surfaces.

[0013] The hydrophobic and oleophilic surface is obtained by performing hydrophobic treatment on the inner surface of the outer tube and the outer surface of the inner tube, and the hydrophobic treatment method is one of the following: hydrophobic treatment of the material itself, Teflon solution treatment, silanization agent treatment or fluorosilane agent treatment.

[0014] The outer tube and the inner tube are made of one of PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), quartz, borosilicate glass, single crystal silicon, calcium fluoride or high molecular polymer.

[0015] When in use, the slit is perpendicular to the horizontal plane or forms a certain angle with the horizontal plane, that is, the slit cannot be parallel to the horizontal plane.

[0016] Furthermore, a heating rod is detachably connected to the interior of the inner tube.

[0017] Furthermore, an endoscope-type droplet imaging device is detachably connected to the interior of the inner tube.

[0018] Furthermore, the droplet dispersion storage device of the present invention may be a device for three-dimensional digital droplet rapid nucleic acid amplification and detection.

[0019] Furthermore, the droplet dispersion storage device of the present invention can be used in conjunction with an external droplet detection system as a device for three-dimensional digital droplet rapid nucleic acid amplification and detection, and is used to achieve droplet observation, droplet counting, fluorescence signal collection, etc. The droplet detection system includes but is not limited to one or more of an optical microscopy module, a fluorescence module, a Raman module, an ultraviolet module, an infrared module, etc. The device for three-dimensional digital droplet rapid nucleic acid amplification and detection is also provided with a fixing and rotating device, and a height adjustment device, which is manually adjusted or electrically adjusted, and a slider that can move up and down is provided on the slide rail to adjust the height of the droplet single layer paving device.

[0020] Furthermore, the droplet dispersion storage device of the present invention can be used alone or in multiple devices at the same time. When multiple devices are used at the same time, high-throughput reactions and detections can be achieved.

[0021] The application of the droplet dispersion storage device in nucleic acid amplification and detection should also be within the protection scope of the present invention.

[0022] The present invention also provides a method for rapid nucleic acid amplification and detection by three-dimensional digital droplets, using the device for rapid nucleic acid amplification and detection by three-dimensional digital droplets, specifically comprising the following steps:

[0023] 1) Prepare a premix containing nucleic acid samples, primers, enzymes, etc., shake and mix, and use it as an aqueous solution to generate droplets in the inner tube 2. Appropriate pressure is applied to make it flow into the slit 3 to achieve a single layer of flat. Cover the top of the droplet with oil, or seal the top of the slit 3 to prevent the droplet from evaporating.

[0024] 2) Insert a heating rod into the inner tube 2, which may contain a heat-conducting medium, and place the entire device in an external heating cylinder, which may contain a heat-conducting medium. Set the heating temperature to perform a constant temperature PCR or a variable temperature PCR reaction.

[0025] 3) After the reaction, the heating rod in the inner tube 2 is taken out, and the endoscope probe type detection device is installed in the inner tube 2. There may be a medium in the inner tube 2 to eliminate the phase difference, adjust the rotation and height, and detect all the droplets in the slit.

[0026] The droplet dispersion storage device of the present invention can heat the droplets evenly, shorten the equilibration time between the heating module and the sample temperature in the droplets, reduce the time required for nucleic acid amplification, and achieve the purpose of rapid nucleic acid amplification. At the same time, it is coupled with an endoscope-type droplet fluorescence detection module to achieve high-throughput rapid droplet imaging. It can be used in the fields of digital droplet PCR, digital droplet nucleic acid amplification, and can also be used in the fields of rapid cultivation of microbial droplets. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1It is a schematic diagram of the structure of the droplet dispersion storage device (installed with a heating rod), wherein the left figure is a side view and the right figure is a top view;

[0028] Figure 2 It is a schematic diagram of the structure of a droplet dispersion storage device (endoscopic probe type droplet imaging device), wherein the left figure is a side view and the right figure is a top view;

[0029] Figure 3 This is the imaging result of a single layer of droplets;

[0030] Figure 4 A schematic diagram of a high-throughput (single-row) droplet monolayer tiling and detection device;

[0031] Figure 5 A schematic diagram of a high-throughput (array) droplet monolayer tiling and detection device;

[0032] Main reference numerals: outer tube 1 , inner tube 2 , slit 3 , heating device 4 (heating rod 41 , heating cylinder 42 ), endoscope probe type droplet imaging device 5 . DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0035] The technical field provided by the present invention is digital droplet nucleic acid amplification. This technology can make the droplets heated evenly, shorten the equilibrium time between the heating module and the sample temperature in the droplet, reduce the time required for nucleic acid amplification, and achieve the purpose of rapid nucleic acid amplification. At the same time, an endoscope-type droplet fluorescence detection module is coupled to achieve high-throughput rapid droplet imaging. It is applied to the fields of digital droplet PCR, digital droplet constant temperature amplification, and can also be used in the fields of rapid cultivation of microbial droplets.

[0036] Example 1

[0037] As an embodiment of the present invention, Figure 1-2As shown, the present invention relates to a droplet dispersion storage device, comprising two hydrophobic circular tubes, namely an outer tube 1 and an inner tube 2. The outer tube 1 is hollow, sealed at the bottom, and its inner diameter is larger than the outer diameter of the inner tube 2, and the size difference between the former and the latter should be less than 4 times the droplet diameter. The inner tube 2 is a hollow tube. The outer tube 1 is sleeved on the outside of the inner tube 2, and is adjusted to be concentric by a circular tube fixing device, and a slit is formed between the inner tube 2 and the outer tube 1. The bottom of the inner tube 2 may be provided with a through hole connected to the slit 3, so that when pressure is applied to the inside of the inner tube 2, the liquid inside the inner tube 2 can be pressed into the slit 3; the inside of the inner tube 2 may also be disconnected from the slit 3. When in use, the liquid is directly input into the slit through the opening at the top of the slit 3 to form a droplet surface. The droplets are injected and dispersed in the slit 3. When standing, the droplets are naturally stratified in the slit 3 by using the density difference between water and oil. If the density of the oil phase is greater than the density of the water phase, the droplets are on the top and the oil phase is on the bottom, and the bubbles are discharged from the top of the slit 3. If the density of the oil phase is less than that of the water phase, the liquid droplets are at the bottom and the oil phase is at the top, and the bubbles are discharged from the top of the slit 3. The heating device 4 is divided into two parts, the heating rod 41 is placed inside the inner tube, and the heating tube 42 is placed outside the outer tube, and there may be a heat-conducting medium between the heating device and the tube. The heating rod and the inner tube are detachable. When liquid imaging is required, the heating rod is removed from the inner tube after the droplet reaction is completed. The endoscope probe droplet imaging device is placed in the inner tube 2, and there may be a medium in the inner tube 2 to eliminate the phase difference, so as to perform droplet imaging detection.

[0038] For example, the hollow circular tube in the device of this embodiment is made of quartz. The inner diameter of the outer tube 1 is 4 mm and the outer diameter is 6 mm. The inner tube 2 is a hollow tube with an outer diameter of 3.7 mm and an inner diameter of 1.5 mm, and the corresponding slit size is 0.15 mm. The distance between the outer tube and the bottom of the inner tube is about 1 mm. (The width of the slit can match the size of the droplet and can be adjusted according to the droplet size of different liquids, and its unit can be from microns to millimeters)

[0039] The material of the structure of the inner tube and the outer tube is selected from but not limited to PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), quartz, borosilicate glass, single crystal silicon, calcium fluoride, and high molecular polymer.

[0040] The surface of the slit structure is a hydrophobic and lipophilic surface.

[0041] In another preferred embodiment, the hydrophobic treatment method is selected from but not limited to the hydrophobicity of the material itself, Teflon solution treatment, silanization agent treatment, and fluorosilane agent treatment.

[0042] The nucleic acid amplification and detection using the three-dimensional digital droplet rapid nucleic acid amplification and detection device of this embodiment specifically includes the following steps:

[0043] 1) Droplet generation

[0044] The premix containing nucleic acid samples, primers, enzymes, etc. is mixed and shaken to form droplets as the aqueous phase solution in the inner tube 2, and appropriately pressurized to make it flow into the slit 3 (droplets can also be generated by other methods, and then the droplets (through the inner tube 2 or other methods) are placed into the slit) to achieve single-layer paving. Cover the top of the droplet with oil, or seal the top of the slit 3 to prevent the droplet from evaporating.

[0045] 2) Droplet nucleic acid amplification

[0046] Insert a temperature control rod into the inner tube 2, and place the entire device in an external temperature control module. Set the temperature of the temperature control module to perform a constant temperature PCR or a variable temperature PCR reaction.

[0047] 3) Droplet fluorescence detection

[0048] After the reaction, the droplet paving device is taken out from the temperature control module, and the endoscope probe type detection device is installed in the inner tube 2, and the rotation and height are adjusted to detect all the droplets in the slit.

[0049] Example 2: Single-layer tiling and imaging of droplets

[0050] Using the droplet dispersion storage device described in Example 1, the droplet monolayer tiling and imaging are achieved through the following steps:

[0051] ① Place the droplet dispersion storage device vertically.

[0052] ② Inject the droplets (about 100 mm in diameter) through the inner tube 2, and make them flow into the bottom of the inner tube 2 by appropriate pressure, and then disperse them in the slit 3 (the size of the slit is equivalent to 1.5 times the diameter of the droplets).

[0053] ③Stand still, the droplets are naturally layered in the slit 3 by the density difference between water and oil, and the bubbles are discharged from the top of the slit 3. The oil phase in the droplets in step ③ uses fluorocarbon oil, which has a greater density than the water phase, and the droplets are tightly spread on it. If the density of the oil phase is less than that of the water phase, the droplets are tightly spread below it.

[0054] Whether the droplets can spread out as a single layer is determined by the density of the water-oil phase, the width of the slit 3, and the hydrophilic and hydrophobic properties of the tube wall.

[0055] The circular tube used in this embodiment is placed vertically, and the droplet layer is perpendicular to the horizontal plane. An endoscope probe microscope is inserted into the inner tube 2 to observe the droplet imaging. Fluorocarbon oil exists in the inner tube 2 to eliminate phase difference. There is also a microscope rotation and height adjustment device to achieve imaging of all droplets, such as Figure 3 shown.

[0056] Example 3: Cell culture response in droplets

[0057] The device described in Example 1 was used to perform cell culture reactions in droplets and analyze cell metabolic activity.

[0058] ① Prepare a premixed solution with cells and culture medium, shake and mix evenly, use it as the aqueous phase solution to generate droplets (droplet diameter is about 80 mm).

[0059] ② Inject the droplet into the inner tube 2 and apply appropriate pressure to make it flow into the slit 3 (the size of the slit is equivalent to 1.87 times the diameter of the droplet) to achieve single-layer paving.

[0060] ③ Cover the top of the droplet with oil, or seal the top of slit 3 to prevent the droplet from evaporating.

[0061] ④ Insert the temperature control module into the inner tube 2, and place the entire device in the external temperature control module. There may be a heat conducting medium between the temperature control module and the tube.

[0062] ⑤Set the temperature of the temperature control module for cell culture.

[0063] ⑥ After the culture, the droplet paving device is taken out from the temperature control module, and the cells are observed and detected using an endoscope microscope. A medium may be provided in the inner tube 2 to eliminate the phase difference.

[0064] Example 4: High-throughput reactions in droplets

[0065] Multiple devices shown in Example 1 are placed inside a temperature control plate, and multiple droplet generation modules are coupled with a movable endoscope-type microscope to achieve large-scale high-throughput droplet reaction and analysis, such as Figure 4 or Figure 5 For each droplet, the imaging is as follows:

[0066] ① Multiple aqueous phase samples are simultaneously and sequentially placed in the corresponding inner tubes 2 to generate droplets (the droplet diameter is about 100 mm).

[0067] ② Apply appropriate pressure to make the droplet flow into the slit 3 (the size of the slit is equivalent to 1.5 times the diameter of the droplet) to achieve single-layer paving.

[0068] ③ Cover the top of the droplet with oil, or seal the top of slit 3 to prevent the droplet from evaporating.

[0069] ④Insert the temperature control rod into the inner tube 2.

[0070] ⑤ Set the temperature of the temperature control module to react, and there may be a heat conducting medium between the temperature control module and the tube.

[0071] ⑥ After the reaction, the temperature control rod is taken out, and the droplets are observed and detected in sequence using an endoscope-type microscope. A medium may be provided in the inner tube 2 to eliminate the phase difference, and the endoscope-type microscope is provided with a moving platform.

[0072] Example 5

[0073] As a deformation of the structure of the droplet dispersion storage device in Example 1, the outer tube and the inner tube can both be square tubes, and the difference between the inner diameter side length of the outer tube and the outer diameter side length of the inner tube is less than 4 times the droplet diameter.

[0074] Furthermore, the outer tube and the inner tube may both be amorphous tubes, and the distance between the outer wall of the inner tube and the inner wall of the outer tube is less than twice the diameter of the droplet.

[0075] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A droplet dispersion storage device, characterized in that: include: An outer tube with an opening at one end and an inner tube sleeved in the outer tube, a slit for dispersing droplets is formed between the outer tube and the inner tube, and the width of the slit matches the diameter of the droplets; a heating cylinder is sleeved on the outer side of the outer tube; the outer tube and the inner tube have the same shape but different sizes, and the outer tube and the inner tube are concentrically arranged; the width of the slit is less than twice the diameter of the droplets.

2. The droplet dispersion storage device according to claim 1, characterized in that: The outer tube and the inner tube are both circular tubes, and the difference between the inner diameter of the outer tube and the outer diameter of the inner tube is less than 4 times the diameter of the droplet; Alternatively, the outer tube and the inner tube are both square tubes, and the difference between the inner diameter side length of the outer tube and the outer diameter side length of the inner tube is less than 4 times the droplet diameter; Alternatively, both the outer tube and the inner tube are amorphous tubes, and the distance between the outer wall of the inner tube and the inner wall of the outer tube is less than 2 times the diameter of the droplet.

3. The droplet dispersion storage device according to claim 1, characterized in that: The top of the slit is communicated with the external atmosphere.

4. The droplet dispersion storage device according to claim 3, characterized in that: The inner surface of the outer tube and the outer surface of the inner tube are hydrophobic and lipophilic surfaces.

5. The droplet dispersion storage device according to claim 4, characterized in that: The heating tube sleeved on the outer side of the outer tube is replaced with a heating rod detachably connected to the inside of the inner tube; or a heating rod is detachably connected to the inside of the inner tube.

6. The droplet dispersion storage device according to claim 4, characterized in that: An endoscope-type droplet imaging device is detachably connected to the interior of the inner tube.

7. The droplet dispersion storage device according to claim 6, characterized in that: The droplet dispersion storage device is a device used for three-dimensional digital droplet rapid nucleic acid amplification and detection.

8. Use of the droplet dispersion storage device according to any one of claims 1 to 7 in nucleic acid amplification and detection.

9. A three-dimensional digital droplet rapid nucleic acid amplification and detection method, characterized in that: Use the droplet dispersion storage device described in any one of claims 1 to 7.

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