Micro-droplet sampling device used in digital PCR imaging detection

Abstract

The present invention provides a micro-droplet sampling device used in digital PCR imaging detection, the micro-droplet sampling device comprises a micro-droplet floating module and a micro-droplet storage module, the two are sealed and connected; the surface of the micro-droplet floating module is provided with a micro-droplet tiling chamber, and the micro-droplets in the micro-droplet storage module enter the micro-droplet tiling chamber in batches to realize micro-droplet single-layer tiling and imaging detection. The present invention realizes batch sampling of a micro-droplet sample, improves the sensitivity and reliability of digital PCR detection, and improves the throughput of micro-droplet detection.

Description

Technical Field

[0001] The invention relates to the field of microfluidics technology, and in particular to a micro-droplet sampling device used in digital PCR imaging detection. Background Art

[0002] Droplet digital PCR (ddPCR) is a new PCR technology that has emerged in recent years. Based on the original PCR technology, this technology has been greatly innovated. Compared with the classic fluorescence quantitative PCR, ddPCR can analyze DNA or RNA molecules in an absolutely quantitative manner without the need for standard curve auxiliary analysis. This technology divides each sample reaction system in the traditional PCR into several parts, and the sample is only encapsulated in the microdroplet, and then the PCR reaction is carried out. After the PCR reaction is completed, the detection equipment is used to read the fluorescence signal value of each microdroplet, and then each droplet is distinguished according to the threshold of the fluorescence signal value. The microdroplet with a fluorescence signal higher than the threshold is called a positive droplet, and the microdroplet with a fluorescence signal lower than the threshold is called a negative droplet. According to the Poisson distribution principle and the number and proportion of negative droplets, the starting copy number of the sample target molecule is calculated. This technology has been widely used in the discovery of cancer molecular markers, infectious disease research, gene structure variation analysis, gene expression analysis and other fields.

[0003] With the increasing progress of microfluidics and microfabrication technology, the emergence of microfluidics chips has greatly improved the performance of digital PCR and brought many possibilities for the generation and detection of droplets. When performing fluorescence detection on droplets after PCR, imaging methods are often used. The existing imaging methods first lay all the droplets to be detected in a single layer on a plane, and then use CCD or CMOS to perform fluorescence imaging and analyze their positive and negative properties.

[0004] In traditional micro-droplet imaging detection, the micro-droplets in the sample are often all spread out in the area to be detected at once. Due to the limitation of the chip area, the number of droplets that can be spread out in a single layer is limited, which limits the total number of micro-droplets in each sample. Summary of the invention

[0005] In order to solve the above problems, in one embodiment, the present invention provides a micro-droplet sampling device used in digital PCR imaging detection, wherein the micro-droplet sampling device includes a micro-droplet floating module and a micro-droplet storage module, which are sealed and connected with each other; a micro-droplet tiling chamber is provided on the surface of the micro-droplet floating module, and the micro-droplets in the micro-droplet storage module enter the micro-droplet tiling chamber in batches to realize micro-droplet single-layer tiling and imaging detection.

[0006] In one embodiment, the micro-droplet floating module also includes a floating agent pipeline, a micro-droplet sampling pipeline, a cleaning agent pipeline and a waste liquid tank. The floating agent enters the micro-droplet storage module through the floating agent pipeline, so that the micro-droplets float and enter the micro-droplet tiling chamber through the micro-droplet sampling pipeline; after the micro-droplets realize imaging detection in the tiling chamber, the cleaning agent enters the micro-droplet tiling chamber through the cleaning agent pipeline to clean the micro-droplet tiling chamber, so that the detected micro-droplets enter the waste liquid tank.

[0007] In one embodiment, one end of the cleaning agent pipeline is located on the outer surface of the micro-droplet floating module and is connected to the cleaning agent injection device, and the other end is located on the micro-droplet tiling chamber. The cleaning agent pipeline is used to inject the cleaning agent into the micro-droplet tiling chamber through the injection device.

[0008] In one embodiment, one end of the cleaning agent pipeline is located on the side of the micro-droplet floating module and is connected to the cleaning agent injection device, and the other end is connected to the micro-droplet injection pipeline. The two share a pipeline after the connection and lead to the micro-droplet tiling chamber.

[0009] In one embodiment, the micro-droplet storage module is an eight-tube row, a centrifuge tube, or a chip chamber.

[0010] In one embodiment, the micro-droplet floating module and the micro-droplet storage module are sealed by a screw cap structure, a thread or external pressure, or the two are sealed by an airtight material, and the airtight material is preferably rubber or silicone.

[0011] In one embodiment, the micro-droplet tiling chamber has a transparent top cover above, which can transmit the excitation light and emission light required for the fluorescence detection of the droplet sample; the micro-droplet tiling chamber has a bottom surface below, which is transparent or opaque; preferably, the bottom surface has a reflective layer. The reflective layer can increase the sensitivity of micro-droplet detection.

[0012] In one embodiment, the top cover is a stepped structure, and the top cover is fixedly connected to the micro-droplet tiling platform by a clamp, and partially contacts with the micro-droplet tiling platform, and the non-contact part of the two forms the micro-droplet tiling chamber; or, the top cover is a flat plate structure, and the top cover is connected to the droplet tiling platform as a whole, and the micro-droplet tiling chamber is formed between the two. The top cover can form a spatially continuous whole with other parts of the micro-droplet sampling device, or it can be an independent part, which is fixed to the top of the micro-droplet sampling device by auxiliary clamps, gluing, welding, threaded connection, etc.

[0013] In one embodiment, the micro-droplet tiling chamber, the micro-droplet injection pipeline and / or the inner surface of the storage module in the micro-droplet floating module and the micro-droplet storage module of the micro-droplet sampling device are all treated with hydrophobic or hydrophilic treatment.

[0014] In one embodiment, the present invention provides a digital PCR imaging detection system, wherein the detection system comprises any one of the above-mentioned micro-droplet sampling devices.

[0015] In traditional micro-droplet imaging detection, the micro-droplets in the sample are often all spread out in the area to be detected at once. Due to the limitation of the chip area, the number of droplets that can be spread out in a single layer is limited, which limits the total number of micro-droplets in each sample. The present invention realizes batch sampling of a micro-droplet sample, so that a large number of droplets can be spread out in batches in a very small area, breaking the upper limit of the number of sample droplets in the imaging method and improving the sensitivity and reliability of digital PCR detection. This method also reduces the area required to spread out all the micro-droplets in a sample. When the imaging area of ​​the detector is limited, more samples can be detected in one imaging, thereby improving the throughput of droplet detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 It is a schematic structural diagram of the micro-droplet sampling device of the present invention;

[0018] Figure 2 A half-section view of a floating module in the present invention;

[0019] Figure 3 A half-section view of another floating module in the present invention;

[0020] Figure 4 It is a schematic diagram of the structure of two micro-droplet sampling devices connected in parallel according to the present invention;

[0021] Figure 5 A top view of a flat-laying chamber formed by one-time injection molding according to the present invention;

[0022] Figure 6 A top view of a tiling chamber formed by connecting a bottom surface and a top cover by double-sided adhesive tape; and

[0023] Figure 7 for Figure 6 A cross-sectional view of a tiled chamber. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions in the present application, the present invention will be further described below in conjunction with the following embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should belong to the scope of protection of the present application. The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0025] Example: Micro-droplet sampling device used in digital PCR imaging detection of the present invention

[0026] In the present invention, the overall structure diagram of the micro-droplet sampling device used in the digital PCR imaging method detection is as follows Figure 1 As shown, it includes two parts: a micro-droplet floating module 01 and a micro-droplet storage module 02.

[0027] In one embodiment, a half-section view of the micro-droplet floating module 01 is as follows: Figure 2 As shown, it includes a floating agent pipeline 011, a cleaning agent pipeline 012, a micro-droplet sampling pipeline 013, a paving chamber 014, a waste liquid pool 015 and a threaded connection groove 016. The micro-droplet storage module 02 is a hollow cylindrical cavity, and the sample is stored inside the micro-droplet storage module 02. The outer surface of the cavity is provided with threads, and the micro-droplet storage module 02 can be fixed on the threaded connection groove 016 of the micro-droplet floating module 01 by threads, and the two are tightly connected.

[0028] The sample in the micro-droplet storage module 02 is generally a water-in-oil droplet with a diameter of 80 microns to 110 microns. In some embodiments, the sample can be a micro-droplet of oil-in-water droplets.

[0029] The floatation agent is usually an oil phase liquid with a density greater than that of the sample. The floatation agent enters the micro-droplet storage module 02 through the floatation agent pipeline 011, so that the micro-droplets in the micro-droplet storage module 02 float.

[0030] In one embodiment, one end of the flotation agent pipeline 011 is located on the side of the micro-droplet flotation module 01 and is connected to the sampling device, and the other end is located on the threaded connection groove 016. The flotation agent pipeline 011 is used to inject the flotation agent from the side of the micro-droplet flotation module 01 into the micro-droplet storage module 02 through the micro-droplet sampling device.

[0031] In one embodiment, one end of the cleaning agent pipeline 012 is located on the side of the micro-droplet floating module 01 and connected to the cleaning agent injection device, and the other end is located on the tiling chamber 014. The cleaning agent pipeline 012 is used to inject the cleaning agent into the tiling chamber 014 through the injection device. The cleaning agent is usually an oil phase liquid with a higher density than the sample.

[0032] One end of the micro-droplet injection pipe 013 is located on the threaded connection groove 016 , and the other end is located on the flattening chamber 014 . The droplets in the micro-droplet storage module 02 can float to the flattening chamber 014 through the micro-droplet injection pipe 013 .

[0033] In one embodiment, the tiling chamber 014 is located at the top of the micro-droplet floating module 01, and is a rectangular space with a height of 100 microns, a length of 10 mm, and a width of 8 mm. The tiling chamber 014 has openings on both sides to connect the tiling chamber 014 to the atmosphere. The top of the tiling chamber 014 is made of transparent material, and light inside and outside the tiling chamber 014 can pass through the top of the tiling chamber 014. Due to the height limit of the tiling chamber 014, the droplets can only be tiled in a single layer in the tiling chamber 014.

[0034] In one embodiment, the waste liquid pool 015 is located on the upper surface of the micro-droplet floating module 01, and is an annular groove surrounding the flat chamber 014. The substances flowing out of the openings on both sides of the flat chamber 014 will flow into the waste liquid pool 015 by gravity. The waste liquid pool 015 is used to collect micro-droplet samples, cleaning agents, etc. flowing out of the flat chamber 014.

[0035] In one embodiment, the threaded connection groove 016 is located at the bottom end of the micro-droplet floating module 01 and is a circular groove. The inner surface of the groove has threads that match the outer surface of the micro-droplet storage module 02, and the two are tightly connected through the threads.

[0036] In one embodiment, Figure 3 As shown, one end of the cleaning agent pipeline 012 is located on the side of the micro-droplet floating module 01 and is connected to the sampling device, and the other end is connected to the micro-droplet sampling pipeline 013. The two have a common area at the upper end and lead to the tiling chamber 014. The cleaning agent pipeline 012 is used to inject the cleaning agent into the tiling chamber 014 through the sampling device.

[0037] In some embodiments, the inner surfaces of the tiling chamber 014, the micro-droplet injection pipe 013 and the micro-droplet storage module 02 are all subjected to hydrophobic treatment, and the specific steps are as follows:

[0038] (1) Ultrasonic cleaning of the tiled structure using 100% ethanol for 1 hour;

[0039] (2) Use nitrogen to blow away the ethanol remaining on the surface;

[0040] (3) ultrasonically cleaning the tiled structure with 100% acetone for 1 hour;

[0041] (4) Use nitrogen to blow away the residual acetone on the surface;

[0042] (5) using ultrapure water to ultrasonically clean the tiled structure for 1 hour;

[0043] (6) Use nitrogen to blow dry the ultrapure water remaining on the surface;

[0044] (7) Place the cleaned tile structure in supersaturated silane vapor for 6 hours.

[0045] In some embodiments, if the sample is an oil-in-water droplet, the inner surfaces of the tiling chamber 014, the micro-droplet injection channel 013, and the micro-droplet storage module 02 are all treated with hydrophobicity accordingly.

[0046] The micro-droplet floating module 01 and the micro-droplet storage module 02 are both made of transparent polycarbonate material.

[0047] When using the device to perform step-by-step sampling of samples for digital PCR imaging detection, the method of using the device is as follows:

[0048] (1) Connecting a sample injection device containing a cleaning agent to a cleaning agent pipeline 012 port on the side of the micro-droplet floating module 01;

[0049] (2) Connecting the sampling device containing the floating agent to the floating agent pipeline 011 port on the side of the micro-droplet floating module 01;

[0050] (3) Using a sample injection device, the cleaning agent is injected into the paving chamber 014 through the cleaning agent pipeline 012, so that the cleaning agent fills the paving chamber 014;

[0051] (4) Using a sampling device, a flotation agent is injected into the storage container through the flotation agent pipeline 011. The droplets in the storage container begin to float due to the buoyancy. The floating droplets enter the micro-droplet sampling pipeline 013 and are transferred to the tiling chamber 014. The droplets form a single layer in the tiling chamber 014.

[0052] (5) When the droplets are about to fill the flattening chamber 014, the injection of the floating agent is stopped, the droplets stop floating, and the droplets in the flattening chamber 014 are stationary relative to the device. At this time, the droplets in the flattening chamber 014 are imaged and detected by an external detection device;

[0053] (6) After the test is completed, the cleaning agent is injected into the tiling chamber 014 through the cleaning agent pipeline 012 by the sample injection device. Due to the injection of the cleaning agent, the original droplets and other substances in the cleaning agent pipeline 012 and the tiling chamber 014 are flushed into the waste liquid pool 015;

[0054] (7) Repeat steps (4) to (6) until all droplets in the micro-droplet storage module 02 are detected.

[0055] In one embodiment, if Figure 4 As shown, the device contains two identical micro-droplet floating modules 01 and two identical micro-droplet storage modules 02, each micro-droplet floating module 01 works independently, and the above method is used to implement independent step-by-step sampling of samples in the two micro-droplet storage modules 02. It can be understood that the device contains eight identical micro-droplet floating modules 01 and eight identical micro-droplet storage modules 02, each micro-droplet floating module 01 works independently, and the above method is used to implement independent step-by-step sampling of samples in the eight micro-droplet storage modules 02; wherein the floating module 01 is an eight-row tube.

[0056] Embodiment 2 Micro-droplet tiling chamber of the present invention

[0057] In one embodiment, if Figure 5 As shown, the structure around the tiling chamber is formed by one-time injection molding, and the space in the middle forms a tiling chamber 014. The tiling chamber is a cylindrical space with a height of 100 microns; the cross section of the tiling chamber is a circle with a radius of 4 mm, and the outer side of the circle has two channels 0141 that can allow waste liquid to flow into the waste liquid tank, and the circle has two channel openings 0142 and 0143, the channel opening 0142 is connected to the droplet injection channel, and the other channel opening 0143 is connected to the cleaning agent channel.

[0058] In one embodiment, if Figure 6 , 7 As shown, the tiling chamber is a cavity formed by connecting the bottom surface 0145 and the top cover 0146 through two double-sided adhesive tapes 0147. The bottom surface 0145 is located at the top of the micro-droplet sampling device. The surface of the bottom surface 0145 is evenly covered with 100 nanometers thick metal aluminum. The top cover 0146 is a rectangular polycarbonate with a length of 8 mm, a width of 4 mm, and a height of 1 mm. The double-sided adhesive tape 0147 is 8 mm long, 0.5 mm wide, and 100 microns high. The tiling chamber is 100 microns high, and the top view is as shown in FIG. Figure 6 The cross section of the tiling chamber is a rectangle with a length of 7 mm and a width of 4 mm. There are two channel openings 0412 and 0413 in the middle of the rectangle. The channel opening 0412 is connected to the droplet injection channel, and the channel opening 0413 is connected to the cleaning agent channel. The cross-sectional view of the tiling chamber is shown in FIG. Figure 7 As shown, two double-sided tapes 0147 are located on the left and right sides of the flattening chamber, tightly connecting the bottom surface 0145 and the top cover 0146. There are no double-sided tapes 0147 on the front and back sides of the flattening chamber and they are connected to the atmosphere, and waste liquid can flow into the waste liquid pool through the front and back sides.

[0059] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0060] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A micro-droplet sampling device used in digital PCR imaging detection, characterized in that: The micro-droplet sampling device comprises a micro-droplet floating module and a micro-droplet storage module, which are sealed and connected with each other; a micro-droplet tiling chamber is arranged on the surface of the micro-droplet floating module, and the micro-droplets in the micro-droplet storage module enter the micro-droplet tiling chamber in batches to realize micro-droplet single-layer tiling and imaging detection; the micro-droplet floating module also comprises a floating agent pipeline, a micro-droplet sampling pipeline, a cleaning agent pipeline and a waste liquid pool, and the floating agent enters the micro-droplet storage module through the floating agent pipeline, so that the micro-droplet The droplets float up and enter the micro-droplet tiling chamber through the micro-droplet injection pipeline; after the micro-droplets realize imaging detection in the tiling chamber, the cleaning agent enters the micro-droplet tiling chamber through the cleaning agent pipeline to clean the micro-droplet tiling chamber, so that the detected micro-droplets enter the waste liquid pool; the floating agent and the cleaning agent are alternately input into the micro-droplet tiling chamber until all the droplets in the micro-droplet storage module are detected; the micro-droplet storage module is an eight-row tube, a centrifuge tube, or a chip chamber.

2. The micro-droplet sampling device according to claim 1, characterized in that: One end of the cleaning agent pipeline is located on the outer surface of the micro-droplet floating module and is connected to the cleaning agent injection device, and the other end is located on the micro-droplet tiling chamber. The cleaning agent pipeline is used to inject the cleaning agent into the micro-droplet tiling chamber through the injection device.

3. The micro-droplet sampling device according to claim 1, characterized in that: One end of the cleaning agent pipeline is located at the side of the micro-droplet floating module and is connected to the cleaning agent injection device, and the other end is connected to the micro-droplet injection pipeline. The two share a pipeline after the connection and lead to the micro-droplet tiling chamber.

4. The micro-droplet sampling device according to claim 1, characterized in that: The micro-droplet floating module and the micro-droplet storage module are sealed and connected by a screw cap structure, a thread or external pressure, or the two are sealed and connected by an airtight material.

5. The micro-droplet sampling device according to claim 4, characterized in that: The airtight material is rubber or silicone.

6. The micro-droplet sampling device according to claim 1, characterized in that: The top of the micro-droplet tiling chamber is a transparent top cover, which can transmit the excitation light and the emission light required for the fluorescence detection of the droplet sample; the bottom of the micro-droplet tiling chamber is a bottom surface, which is transparent or opaque.

7. The micro-droplet sampling device according to claim 6, characterized in that: The bottom surface has a reflective layer.

8. The micro-droplet sampling device according to claim 6, characterized in that: The top cover is a stepped structure, and is fixedly connected to the top of the micro-droplet tiling platform by a clamp, and is in partial contact with the micro-droplet tiling platform, and the non-contact part of the two forms the micro-droplet tiling chamber; or, the top cover is a flat plate structure, and the top cover is connected to the droplet tiling platform as a whole, and the micro-droplet tiling chamber is formed between the two.

9. The micro-droplet sampling device according to any one of claims 1 to 8, characterized in that: The micro-droplet tiling chamber, the micro-droplet sampling pipeline and / or the inner surface of the storage module in the micro-droplet floating module and the micro-droplet storage module of the micro-droplet sampling device are all subjected to hydrophobic or hydrophilic treatment.

10. A digital PCR imaging detection system, characterized in that: The detection system comprises the micro-droplet sampling device according to any one of claims 1-9.

Images