CN210886049U - Micro-droplet sampling device applied to detection by digital PCR imaging method and system thereof - Google Patents
Micro-droplet sampling device applied to detection by digital PCR imaging method and system thereof Download PDFInfo
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- CN210886049U CN210886049U CN201921335660.XU CN201921335660U CN210886049U CN 210886049 U CN210886049 U CN 210886049U CN 201921335660 U CN201921335660 U CN 201921335660U CN 210886049 U CN210886049 U CN 210886049U
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Abstract
The utility model provides a micro-droplet sampling device and a system thereof applied in the detection of a digital PCR imaging method, wherein the micro-droplet sampling device comprises a micro-droplet floating module and a micro-droplet storage module which are connected in a sealing way; the surface of the micro-droplet floating module is provided with a micro-droplet tiling chamber, and 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 utility model discloses a to the sample of in batches of a little liquid drop sample, improved the sensitivity and the reliability that digital PCR detected and improved the flux that little liquid drop detected.
Description
Technical Field
The utility model relates to a micro-fluidic technology field, concretely relates to little liquid drop sampling device and system of using in digital PCR imaging method detects.
Background
Droplet digital PCR (ddPCR) is a PCR technology that has recently emerged. Based on the original PCR technology, the technology is greatly improved. Compared with the classical fluorescent quantitative PCR, ddPCR can analyze DNA or RNA molecules in an absolute quantitative mode without the need of standard curve auxiliary analysis. The technology equally divides each sample reaction system in the traditional PCR into a plurality of parts, the samples are only wrapped in the micro-droplets, and then the PCR reaction is carried out. After the PCR reaction is finished, reading the fluorescence signal value of each micro-droplet by using a detection device, distinguishing each droplet according to the threshold value of the fluorescence signal value, wherein the micro-droplets with the fluorescence signals higher than the threshold value are called positive droplets, the micro-droplets with the fluorescence signals lower than the threshold value become negative droplets, and calculating the initial copy number of the sample target molecules according to the Poisson distribution principle and the number and proportion of the negative droplets. The technology is widely applied to the fields of discovery of cancer molecular markers, infectious disease research, gene structure variation analysis, gene expression analysis and the like.
With the increasing progress of the microfluidic technology and the improvement of the micro-processing technology, the appearance of the microfluidic chip greatly improves the performance of the digital PCR and brings many possibilities for generating and detecting the liquid drops. When the droplets after PCR are subjected to fluorescence detection, an imaging method is often used, and the existing imaging method firstly lays all the droplets to be detected on a plane in a single layer mode, and then utilizes CCD or CMOS to perform fluorescence imaging on the droplets and analyze the positivity and negativity of the droplets.
In the traditional detection of the micro-droplet imaging method, micro-droplets in a sample are usually laid in a detection area at one time, and due to the limitation of the area of a chip, the number of droplets capable of being laid in a single layer is limited, so that the total number of the micro-droplets in each sample is limited.
SUMMERY OF THE UTILITY MODEL
In order to solve the above problem, in one embodiment, the present invention provides a droplet sampling device for use in digital PCR imaging detection, the droplet sampling device includes a droplet floating module and a droplet storage module, which are connected in a sealing manner; the surface of the micro-droplet floating module is provided with a micro-droplet tiling chamber, and 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.
In one embodiment, the micro-droplet floating module further comprises a floating agent pipeline, a micro-droplet sampling pipeline, a cleaning agent pipeline and a waste liquid pool, wherein a floating agent enters the micro-droplet storage module through the floating agent pipeline, so that the micro-droplets float upwards and enter the micro-droplet tiling chamber through the micro-droplet sampling pipeline; after the micro-droplets are imaged and detected in the tiling chamber, a 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.
In one embodiment, one end of the cleaning agent pipeline is located on the outer surface of the micro-droplet floating module and connected with a cleaning agent sampling device, the other end of the cleaning agent pipeline is located on the micro-droplet tiling chamber, and the cleaning agent pipeline is used for injecting a cleaning agent into the micro-droplet tiling chamber through the sampling device.
In one embodiment, one end of the cleaning agent pipeline is positioned on the side surface of the micro-droplet floating module and connected with a cleaning agent sampling device, the other end of the cleaning agent pipeline is connected with the micro-droplet sampling pipeline, and the cleaning agent pipeline and the micro-droplet sampling pipeline share a pipeline behind the connection part and are communicated with the micro-droplet tiling chamber.
In one embodiment, the micro-droplet storage module is an eight-row tube, a centrifuge tube, or a chip chamber.
In one embodiment, the micro-droplet floating module and the micro-droplet storage module are hermetically connected through a screw cap structure, a thread or an external pressure, or are hermetically connected through an airtight material, and the airtight material is preferably rubber or silica gel.
In one embodiment, a transparent top cover is arranged above the micro-droplet tiling chamber and can transmit excitation light and emission light required by fluorescence detection of the droplet sample; the bottom surface is arranged below the micro-droplet tiling chamber and is transparent or opaque; preferably the bottom surface has a reflective layer. The reflective layer may increase the sensitivity of the microdroplet detection.
In one embodiment, the top cover is of a stepped structure, the top cover is fixedly connected above the droplet tiling platform through a clamp and is partially contacted with the droplet tiling platform, and the non-contact parts of the top cover and the droplet tiling platform form the droplet tiling chamber; or the top cover is of a plane flat plate structure, the top cover and the liquid drop tiling platform are connected into a whole, and the micro liquid drop tiling chamber is formed between the top cover and the liquid drop tiling platform. The top cover can form a spatially continuous whole with other parts of the micro-droplet sampling device, and can also be an independent part which is fixed at the top end of the micro-droplet sampling device in the modes of auxiliary fixture, bonding, welding, threaded connection and the like.
In one embodiment, the inner surfaces of the micro-droplet tiling chamber, the micro-droplet sampling pipeline and/or the storage module in the micro-droplet floating module and the micro-droplet storage module of the micro-droplet sampling device are subjected to hydrophobic or hydrophilic treatment.
In one embodiment, the present invention provides a digital PCR imaging detection system, which comprises any one of the above-mentioned micro-droplet sampling devices.
In the traditional detection of the micro-droplet imaging method, micro-droplets in a sample are usually laid in a detection area at one time, and due to the limitation of the area of a chip, the number of droplets capable of being laid in a single layer is limited, so that the total number of the micro-droplets in each sample is limited. The utility model discloses a sample to the sample of in batches of a little liquid drop sample for can tile a large amount of liquid drops in batches in a very little region, break the upper limit of sample liquid drop number in the imaging method, improve the sensitivity and the reliability that digital PCR detected. The method also reduces the area required by tiling all micro-droplets in one sample, enables one-time imaging to detect more samples under the condition of limited imaging area of the detector, and improves the flux of droplet detection.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
Fig. 1 is a schematic structural view of a micro-droplet sampling device according to the present invention;
fig. 2 is a half sectional view of a floating module according to the present invention;
fig. 3 is a half sectional view of another floating module of the present invention;
fig. 4 is a schematic structural diagram of two micro-droplet sampling devices connected in parallel according to the present invention;
fig. 5 is a top view of the flat laying chamber formed by one-time injection molding according to the present invention;
FIG. 6 is a top view of the flat laying chamber formed by the bottom and the top covers connected by the double-sided adhesive tape; and
fig. 7 is a cross-sectional view of the tiling chamber of fig. 6.
Detailed Description
In order to make the technical solutions in the present application better understood by those skilled in the art, the present invention will be further described with reference to the following embodiments, and it is apparent that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application. The present invention will be further described with reference to the accompanying drawings and examples.
Example (b): the utility model discloses little liquid drop sampling device who uses in digital PCR imaging method detects
The utility model discloses in, the overall structure sketch map of the little liquid drop sampling device who uses in the digital PCR imaging method detects is shown in figure 1, including little liquid drop come-up module 01 and little liquid drop storage module 02 two parts.
In one embodiment, a half-sectional view of the micro droplet floating module 01 is shown in fig. 2, and includes a floating agent pipeline 011, a cleaning agent pipeline 012, a micro droplet sampling pipeline 013, a tiling chamber 014, a waste liquid pool 015, and a threaded connection groove 016 structure. The micro-droplet storage module 02 is a hollow cylindrical cavity, a sample is stored in the micro-droplet storage module 02, threads are arranged on the outer surface of the cavity, and the micro-droplet storage module 02 can be fixed on the thread connecting groove 016 of the micro-droplet floating module 01 through threads and is tightly connected with the micro-droplet floating module 01.
The sample in the microdroplet storage module 02 is typically water-in-oil droplets with a diameter of 80 to 110 microns. In some embodiments, the sample may be oil-in-water droplet microdroplets.
The floating agent is generally oil phase liquid with the density larger than that of the sample, and the floating agent enters the micro-droplet storage module 02 through the floating agent pipeline 011, so that micro-droplets in the micro-droplet storage module 02 float upwards.
In one embodiment, the floating agent pipeline 011 is located at one end of the micro-droplet floating module 01 side and connected with a sample injection device, and the other end of the floating agent pipeline 011 is located on the threaded connecting groove 016, and the floating agent pipeline 011 is used for injecting the floating agent into the micro-droplet storage module 02 from the micro-droplet floating module 01 side through the micro-droplet sample injection device.
In an embodiment, cleaning agent pipeline 012 one end is located micro-droplet upward-floating module 01 side and links to each other with cleaning agent sampling device, and the other end is located tiling chamber 014, cleaning agent pipeline 012 is used for injecting the cleaning agent into tiling chamber 014 through sampling device. The cleaning agent is typically an oil phase liquid having a density greater than the sample.
One end of the micro-droplet sample injection pipeline 013 is located on the threaded connection groove 016, the other end of the micro-droplet sample injection pipeline 013 is located on the flat laying chamber 014, and droplets in the micro-droplet storage module 02 can float upwards into the flat laying chamber 014 through the micro-droplet sample injection pipeline 013.
In one embodiment, the flat chamber 014 is located at the top end of the droplet floating module 01, and is a rectangular solid space with a height of 100 microns, a length of 10 mm and a width of 8 mm, and openings are formed on both sides of the flat chamber 014 to connect the flat chamber 014 with the atmosphere. The top of the flat laying chamber 014 is made of transparent material, so that light inside and outside the flat laying chamber 014 can penetrate through the top of the flat laying chamber 014. Due to the height limitation of the tile chamber 014, the droplets can only be tiled in a single layer in the tile chamber 014.
In one embodiment, the waste liquid pool 015 is located on the upper surface of the droplet floating module 01 and is an annular groove surrounding the flat chamber 014, and substances flowing out of openings at both sides of the flat chamber 014 flow into the waste liquid pool 015 by gravity. The waste liquid pool 015 is used to collect the micro-droplet sample, the cleaning agent, etc. flowing out from the flat chamber 014.
In one embodiment, the screw connecting groove 016 is located at the bottom end of the microdroplet floating module 01 and is a circular groove, the inner surface of the groove is provided with a screw thread matching with the outer surface of the microdroplet storage module 02, and the two are tightly connected through the screw thread.
In one embodiment, as shown in fig. 3, the cleaning agent line 012 is connected to the sample injection device at one end of the module 01 and connected to the sample injection device at the other end of the module 013, and both have a common area at the upper end and jointly open into the flat chamber 014. The cleaning agent pipe 012 is used to inject the cleaning agent into the laying chamber 014 through the sample injection device.
In some embodiments, the interior surfaces of the tiling chamber 014, the micro-droplet injection conduit 013, and the micro-droplet storage module 02 are all subjected to hydrophobic treatment by the specific steps of:
(1) ultrasonically cleaning the tiled structure for 1 hour by using 100% ethanol;
(2) drying the residual ethanol on the surface by using nitrogen;
(3) ultrasonically cleaning the tiled structure for 1 hour by using 100% acetone;
(4) blowing the acetone remained on the surface by using nitrogen;
(5) ultrasonically cleaning the tiled structure for 1 hour by using ultrapure water;
(6) blowing the ultrapure water remained on the surface by using nitrogen;
(7) the cleaned tile was placed in supersaturated silane vapor for 6 hours.
In some embodiments, if the sample is an oil-in-water droplet, the interior surfaces of the tile chamber 014, the micro-droplet injection conduit 013, and the micro-droplet storage module 02, respectively, are all hydrophobically treated.
The micro-droplet floating module 01 and the micro-droplet storage module 02 are both made of transparent polycarbonate materials.
When the device is used for sampling samples step by step and carrying out digital PCR imaging detection, the using method of the device is as follows:
(1) connecting a sample introduction device containing a cleaning agent to a port of a cleaning agent pipeline 012 on the side surface of the micro-droplet floating module 01;
(2) connecting a sample introduction device containing a floating agent to a 011 port of a floating agent pipeline on the side surface of the micro-droplet floating module 01;
(3) injecting a cleaning agent into the flat laying chamber 014 through a cleaning agent pipeline 012 by using a sample injection device to fill the flat laying chamber 014 with the cleaning agent;
(4) injecting a floating agent into a storage container through a floating agent pipeline 011 by using a sample injection device, wherein liquid drops in the storage container start floating under the influence of buoyancy, the floating liquid drops enter a micro-liquid drop sample injection pipeline 013 and are transferred into a flat laying chamber 014, and the liquid drops form single-layer flat laying in the flat laying chamber 014;
(5) when the liquid drops are about to fill the flat chamber 014, stopping injecting the floating agent, stopping floating the liquid drops, and making the liquid drops in the flat chamber 014 static relative to the device, and then performing imaging detection on the liquid drops in the flat chamber 014 through an external detection device;
(6) after the detection is finished, the cleaning agent is injected into the flat laying chamber 014 through the cleaning agent pipeline 012 by the sample injection equipment, and because the cleaning agent is injected, original liquid drops and other substances in the cleaning agent pipeline 012 and the flat laying chamber 014 are flushed into the waste liquid pool 015;
(7) and (5) repeating the steps (4) to (6) until all the droplets in the micro-droplet storage module 02 are detected.
In one embodiment, as shown in fig. 4, the apparatus comprises two identical microdroplet flotation modules 01 and two identical microdroplet storage modules 02, each microdroplet flotation module 01 works independently, and independent fractional sampling of samples in the two microdroplet storage modules 02 is realized by the method. It can be understood that the device comprises eight identical micro-droplet floating modules 01 and eight identical micro-droplet storage modules 02, each micro-droplet floating module 01 works independently, and independent step-by-step sampling of samples in the eight micro-droplet storage modules 02 is realized by the method; wherein the upward floating module 01 is an eight-row pipe.
Second embodiment the utility model discloses a little liquid drop tiling room
In one embodiment, as shown in fig. 5, the structure around the tiled chamber is injection molded in one shot, with the intervening space forming the tiled chamber 014. The flat laying chamber is a cylindrical space, and the height of the flat laying chamber is 100 micrometers; the cross section of the flat laying chamber is a circle with the radius of 4 mm, two channels 0141 enabling waste liquid to flow into a waste liquid pool are arranged on the outer side of the circle, two channel ports 0142 and 0143 are arranged in the circle, the channel port 0142 is connected with a liquid drop sampling channel, and the other channel port 0143 is connected with a cleaning agent channel.
In one embodiment, as shown in fig. 6 and 7, the tiling compartment is a cavity formed by a bottom surface 0145 and a top cover 0146 connected by two double-sided tapes 0147. Bottom surface 0145 is located micro-droplet sampling device top, and the even metallic aluminum that covers 100 nanometers thick in bottom surface 0145 surface, cap 0146 is long 8 millimeters, and wide 4 millimeters, high cuboid polycarbonate of 1 millimeter, double faced adhesive tape 0147 is long 8 millimeters, and wide 0.5 millimeter, high 100 microns. The tiling chamber is 100 microns high and the top view is shown in figure 6. The cross section of the flat laying chamber is a rectangle with the length of 7 mm and the width of 4 mm, two channel ports 0412 and 0413 are arranged in the middle of the rectangle, the channel port 0412 is connected with the droplet sampling channel, and the channel port 0413 is connected with the cleaning agent channel. The cross-sectional view of the flat chamber is shown in fig. 7, two double-sided tapes 0147 are provided on the left and right sides of the flat chamber, the bottom surface 0145 is tightly connected to the top cover 0146, the front and rear sides of the flat chamber are not provided with the double-sided tapes 0147 and are in communication with the atmosphere, and waste liquid can flow into the waste liquid tank through the front and rear sides.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
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 intended to be encompassed by the following claims.
Claims (12)
1. The micro-droplet sampling device applied to the detection of the digital PCR imaging method is characterized by comprising a micro-droplet floating module and a micro-droplet storage module which are hermetically connected; the surface of the micro-droplet floating module is provided with a micro-droplet tiling chamber, and 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.
2. The micro-droplet sampling device of claim 1, wherein the micro-droplet floating module further comprises a floating agent pipeline, a micro-droplet sampling pipeline, a cleaning agent pipeline and a waste liquid pool, wherein a floating agent enters the micro-droplet storage module through the floating agent pipeline, so that the micro-droplets float upwards and enter the micro-droplet tiling chamber through the micro-droplet sampling pipeline; after the micro-droplets are imaged and detected in the tiling chamber, a 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.
3. The micro-droplet sampling device of claim 2, wherein one end of the cleaning agent pipeline is located on the outer surface of the micro-droplet floating module and connected with a cleaning agent sampling device, the other end of the cleaning agent pipeline is located on the micro-droplet tiling chamber, and the cleaning agent pipeline is used for injecting a cleaning agent into the micro-droplet tiling chamber through the sampling device.
4. The micro-droplet sampling device of claim 2, wherein one end of the cleaning agent pipeline is located on the side surface of the micro-droplet floating module and connected with a cleaning agent sampling device, and the other end of the cleaning agent pipeline is connected with the micro-droplet sampling pipeline, and the cleaning agent pipeline and the micro-droplet sampling pipeline share a pipeline behind the connection position and lead to the micro-droplet tiling chamber.
5. The micro-droplet sampling device of claim 1, wherein the micro-droplet storage module is an eight-row tube, a centrifuge tube, or a chip chamber.
6. The micro-droplet sampling device of claim 1, wherein the micro-droplet flotation module and the micro-droplet storage module are hermetically connected by a screw cap structure, a thread, or an externally applied pressure, or are hermetically connected by a gas-tight material.
7. The micro droplet sampling device of claim 6, wherein the gas-tight material is rubber or silicone.
8. The micro-droplet sampling device of claim 1, wherein a transparent top cover is disposed above the micro-droplet tiling chamber, and the excitation light and the emission light required for fluorescence detection of the micro-droplet sample can be transmitted through the top cover; the bottom surface is arranged below the micro-droplet tiling chamber and is transparent or opaque.
9. The micro-droplet sampling device of claim 8, wherein the bottom surface has a reflective layer.
10. The micro-droplet sampling device of claim 8, wherein the top cover is a step-shaped structure, the top cover is fixedly connected above the micro-droplet laying platform through a clamp and is in contact with a part of the micro-droplet laying platform, and the non-contact part of the top cover and the part of the top cover form the micro-droplet laying chamber; or the top cover is of a plane flat plate structure, the top cover and the liquid drop tiling platform are connected into a whole, and the micro liquid drop tiling chamber is formed between the top cover and the liquid drop tiling platform.
11. The micro-droplet sampling device according to any one of claims 1 to 10, wherein the inner surfaces of the micro-droplet tiling chamber, the micro-droplet sampling pipe and/or the storage module in the micro-droplet floating module and the micro-droplet storage module of the micro-droplet sampling device are subjected to hydrophobic or hydrophilic treatment.
12. A digital PCR imaging detection system, characterized in that it comprises a microdroplet sampling device according to any one of claims 1-11.
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| CN110358676A (en) * | 2019-08-17 | 2019-10-22 | 清华大学 | The microlayer model sampler applied in the detection of digital pcr imaging method |
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| CN110358676A (en) * | 2019-08-17 | 2019-10-22 | 清华大学 | The microlayer model sampler applied in the detection of digital pcr imaging method |
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| GR01 | Patent grant | ||
| GR01 | Patent grant |