CN111185248A - Polymerase chain reaction chip containing bubble elimination structure and treatment method of liquid drop emulsion - Google Patents
Polymerase chain reaction chip containing bubble elimination structure and treatment method of liquid drop emulsion Download PDFInfo
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- CN111185248A CN111185248A CN202010060128.2A CN202010060128A CN111185248A CN 111185248 A CN111185248 A CN 111185248A CN 202010060128 A CN202010060128 A CN 202010060128A CN 111185248 A CN111185248 A CN 111185248A
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The application discloses a polymerase chain reaction chip containing a bubble elimination structure and a processing method of a liquid drop emulsion, which comprises a sample introduction structure 100, a micro-flow channel 200, a bubble floating structure 300 and a liquid drop storage structure 400 which are arranged on the same surface of the polymerase chain reaction chip. Since the bubble floating chamber 302 in the bubble floating structure 300 can be used for bubble elimination treatment of the droplet emulsion, the introduction of bubbles can be reduced or even eliminated, so that the subsequent amplification reaction can be smoothly performed.
Description
Technical Field
The application relates to the field of sample analysis micro-fluidic chips, in particular to a polymerase chain reaction chip containing a bubble elimination structure and a processing method of a liquid drop emulsion.
Background
Nucleic acids are one of the most basic substances of life, and evidence can be found at the nucleic acid level for any disease. Nucleic acid detection is currently widely used in the fields of disease research, in vitro diagnostics, veterinary medicine, food safety, biosafety, agriculture, and the like.
There are currently three methods for the quantitative detection of nucleic acid molecules: photometry, real-time fluorescent quantitative Polymerase Chain Reaction (PCR), and digital PCR techniques. Real-time fluorescence quantitative PCR belongs to semi-quantitative detection technology. The digital PCR technology is a nucleic acid quantification technology based on a single-molecule PCR method for counting, is a nucleic acid molecule amplification and absolute quantification technology, and disperses a diluted nucleic acid solution into a large number of microreactors, wherein the number of nucleic acid templates of each microreactor is less than or equal to 1. Thus, after PCR thermal cycling, a reactor with a nucleic acid molecule template will exhibit a fluorescent signal, while a reactor without a template will exhibit no fluorescent signal. During the reading process of the result, the nucleic acid concentration of the original solution can be calculated according to the relative proportion of the fluorescence signals of the micro reaction cavity.
The droplet digital PCR based on the droplet microfluidic technology has attracted much attention because of its advantages such as easy provision of a microreactor with a small volume and a high flux. The liquid drop digital PCR reaction is divided into three links: droplet preparation, amplification and droplet counting statistics. Currently, there are 3 representative droplet digital PCR systems, QX-100/QX-200 from Bio-Rad, Rain Drop from Raindane, and Naica systems from Stilla technologies, respectively. QX-200TM and Rain Drop TM do not adopt integrated chip technology, so that the operation is complicated and pollution is easy, and the integrated chip can effectively solve the problems.
The technical route of the integrated chip is mainly characterized in that the liquid drop generation structure and the liquid drop storage and amplification cavity are directly connected and integrated in the same chip, and bubbles introduced in the liquid drop generation link directly flow into the liquid drop storage cavity 402. These bubbles cause serious problems during PCR thermal cycling as follows: when bubbles are present in the amplification chamber, the bubbles expand in volume upon heating; the volume of the bubble shrinks when the temperature is reduced. Thus, the bubbles form a source of turbulence during thermal cycling of the PCR, perturb and break down the droplets, create fine, fragmented droplets, and induce droplet fusion (DropletcaAlcesence), which in turn leads to failure of the digital PCR assay. Thus, the bubble problem is an inherent difficulty of such integrated chips.
Disclosure of Invention
The application provides a polymerase chain reaction chip containing a bubble elimination structure and a treatment method of a liquid drop emulsion, which are used for reducing and even eliminating the introduction of bubbles in a liquid drop generation link so as to facilitate the smooth proceeding of the subsequent amplification reaction.
In one aspect, the embodiment of the present application provides a pcr chip with a bubble elimination structure, which includes a sample injection structure 100, a microfluidic channel 200, a bubble floating structure 300, and a droplet storage structure 400 disposed on the same side of the pcr chip;
the sample injection structure 100 comprises a sample injection cavity 101 and an oil phase injection cavity 102, wherein the sample injection cavity 101 and the oil phase injection cavity 102 are respectively communicated with the microfluidic channel 200; the microfluidic channel 200 is used for merging the sample from the sample injection cavity 101 and the oil phase from the oil phase injection cavity 102 to prepare a droplet emulsion;
the bubble floating structure 300 comprises a dropping emulsion inlet 301, a bubble floating cavity 302 and a dropping emulsion outlet 303; the dropping emulsion inlet 301 is communicated with the microfluidic channel 200, and the bubble floating cavity 302 is used for carrying out bubble elimination treatment on the dropping emulsion;
the droplet storage structure 400 includes a droplet storage inlet 401 and a droplet storage chamber 402; the drip emulsion outlet 303 is communicated with the drip storage inlet 401; the droplet storage chamber 402 is used to store the bubble depleted droplet emulsion exiting from the droplet emulsion outlet 303 and the droplet storage inlet 401.
Optionally, the polymerase chain reaction chip according to claim 1, wherein the bubble floating structure 300 further comprises a top sealing layer 304; the top sealing layer 304 covers the top of the bubble floating cavity 302, and the thickness of the bubble floating cavity 302 is not less than 1 micron.
Alternatively, the polymerase chain reaction chip according to claim 1, wherein the bubble floating chamber 302 is any one of a cylinder, a cube and a polyhedron; the horizontal size and the vertical size of the bubble floating cavity 302 are not less than 1 millimeter, and the volume of the bubble floating cavity 302 is not less than 1 microliter.
Optionally, the PCR chip according to claim 1, wherein the dropping emulsion inlet 301 is disposed at a middle position of the bubble floating chamber 302, and the dropping emulsion outlet 303 is disposed at a bottom of the bubble floating chamber 302.
Optionally, the pcr chip of claim 1, wherein the sample injection cavity 101 and the oil phase injection cavity 102 have the same recess depth, for example, the recess depths of the sample injection cavity 101 and the oil phase injection cavity 102 are both 2 mm.
Optionally, the PCR chip according to claim 1, wherein the bottom of the sample injection cavity 101, the bottom of the oil phase injection cavity 102 and the bottom of the microfluidic channel 200 are at the same level.
Optionally, the PCR chip according to claim 1, wherein the bottom of the sample injection cavity 101, the bottom of the oil phase injection cavity 102 and the top of the microfluidic channel 200 are at the same level.
Optionally, the pcr chip according to claim 1, wherein the sample injection structure 100 further comprises at least one first branch 103 and at least one second branch 104;
one end of the first shunting branch 103 is communicated with the oil phase sample injection cavity 102, and the other end of the first shunting branch 103 is communicated with the microfluidic channel 200;
one end of the second branch 104 is communicated with the sample injection cavity 101, and the other end of the second branch 104 is communicated with the microfluidic channel 200; the second flow bifurcating leg 104 includes a bent configuration.
Optionally, the polymerase chain reaction chip of claim 1, wherein the droplet storage structure 400 further comprises a droplet storage chamber outlet 403.
Optionally, the polymerase chain reaction chip of claim 1, wherein the droplet storage chamber 402 is located at the bottom of the bubble floatation chamber 302; the depth of the droplet storage chamber 402 is 0.5 to 2 times the diameter of the droplets in the droplet emulsion.
On the other hand, the method for processing the droplet emulsion based on the polymerase chain reaction chip with the bubble elimination structure is provided, and is characterized in that the polymerase chain reaction chip comprises a sample introduction structure 100, a microfluidic channel 200, a bubble floating structure 300 and a droplet storage structure 400 which are arranged on the same surface of the polymerase chain reaction chip, and the processing method comprises the following steps:
the microfluidic channel 200 is used for merging a sample from the sample injection cavity 101 in the sample injection structure 100 and an oil phase from the oil phase sample injection cavity 102 in the sample injection structure 100 to prepare a droplet emulsion;
performing bubble elimination treatment on the droplet emulsion through the bubble floating structure 300;
the liquid drop emulsion after the bubble elimination treatment is tiled through the liquid drop storage structure 400;
performing a seal breaking operation on the bubble floating structure 300, and performing an amplification reaction on the droplet emulsion;
and identifying and counting the droplet emulsion to obtain the copy number of the nucleic acid in the sample.
The embodiment of the application provides a polymerase chain reaction chip with a bubble elimination structure and a processing method of a liquid drop emulsion, which are used for reducing and even eliminating the introduction of bubbles in a liquid drop generation link so as to facilitate the smooth proceeding of the subsequent amplification reaction.
Drawings
In order to more clearly illustrate the technical solutions and advantages of the embodiments of the present application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of a PCR chip with bubble elimination structure according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a microfluidic channel according to an embodiment of the present disclosure;
FIG. 3 is a schematic diagram of a microfluidic channel according to an embodiment of the present disclosure;
FIG. 4 is a flow diagram of an internal flow path schematic provided by an embodiment of the present application;
FIG. 5 is a partially enlarged schematic view of a bubble floating structure provided in an embodiment of the present application;
FIG. 6 is a schematic diagram of a liquid drop floating chamber for eliminating bubbles according to an embodiment of the present application;
FIG. 7 is a schematic diagram of a liquid drop floating chamber for eliminating bubbles according to an embodiment of the present application;
FIG. 8 is a schematic diagram of a droplet storage structure according to an embodiment of the present disclosure
FIG. 9a is a layout of droplets in a droplet storage chamber prior to thermal amplification according to an embodiment of the present disclosure;
FIG. 9b is a schematic diagram of a thermally amplified droplet arrangement in a droplet storage chamber according to an embodiment of the present disclosure;
FIG. 9c is a fluorescence image of a thermally amplified droplet in a droplet storage chamber according to an embodiment of the present application;
FIG. 10a is a prior art drop placement pattern for a thermal amplification in a drop storage chamber according to an embodiment of the present application;
FIG. 10b is a prior art drop placement pattern for thermal amplification in a de drop storage chamber according to embodiments of the present application
FIG. 11 is a schematic flow chart of a method for processing a droplet emulsion based on a PCR chip with a bubble elimination structure according to an embodiment of the present disclosure.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the 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.
It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data sets so used are interchangeable under appropriate circumstances such that the embodiments of the application described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or server that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
Referring to fig. 1, fig. 1 is a schematic structural diagram of a pcr chip including a bubble elimination structure according to an embodiment of the present disclosure, the pcr chip includes a sample introduction structure 100, a micro flow channel 200, a bubble floating structure 300, and a droplet storage structure 400 disposed on the same plane, and the chip shown in fig. 1 includes an M plane and an N plane, where the sample introduction structure 100, the micro flow channel 200, the bubble floating structure 300, and the droplet storage structure 400 may be disposed on the M plane.
The sample injection structure 100 includes a sample injection cavity 101 and an oil phase injection cavity 102, and the sample injection cavity 101 and the oil phase injection cavity 102 are respectively communicated with the microfluidic channel 200. The microfluidic channel 200 is used to merge the sample from the sample injection cavity 101 and the oil phase from the oil phase injection cavity 102 to prepare a droplet emulsion. The bubble floating structure 300 comprises a dropping emulsion inlet 301, a bubble floating cavity 302 and a dropping emulsion outlet 303, wherein the dropping emulsion inlet 301 is communicated with the microfluidic channel 200, and the bubble floating cavity 302 is used for performing bubble elimination treatment on the dropping emulsion. The droplet storage structure 400 includes a droplet storage inlet 401 and a droplet storage chamber 402, the droplet emulsion outlet 303 is in communication with the droplet storage inlet 401, and the droplet storage chamber 402 is used for storing the droplet emulsion after the bubble removal treatment from the droplet emulsion outlet 303 and the droplet storage inlet 401. Therefore, the introduction of bubbles can be reduced or even eliminated in the droplet generation link, so that the subsequent amplification reaction can be smoothly carried out.
In the examples of the present application, mineral oil and surfactant EM90, Triton X-100, Span80, etc. are used as the oil phase, and the selection of oil phase and surfactant in the examples is merely illustrative, and other types of oil phase and surfactant may be used.
In an alternative embodiment, the sample injection structure 100 includes a sample injection cavity 101 and an oil phase injection cavity 102 both recessed from the M-plane to the N-plane, and the recessed depths of the sample injection cavity 101 and the oil phase injection cavity 102 may be the same, for example, both 2 mm, and both do not penetrate to the N-plane of the chip.
Optionally, as shown in fig. 2, in order to avoid residual liquid in the sample injection cavity 101 and the oil phase injection cavity 102, the bottom of the microfluidic channel 200 communicating the sample injection cavity 101 and the oil phase injection cavity 102, the bottom of the sample injection cavity 101, and the bottom of the oil phase injection cavity 102 are on the same horizontal plane. That is, the microfluidic channel 200 may be recessed upward from the bottom of the sample injection cavity 101 and the oil phase injection cavity 102 to form a flow channel, wherein the upward recess may be up to 50 micrometers.
Alternatively, as shown in fig. 3, the bottom of the sample injection cavity 101, the bottom of the oil phase injection cavity 102, and the top of the microfluidic channel 200 are at the same level. That is, the microfluidic channel 200 may be recessed downward from the bottoms of the sample injection cavity 101 and the oil phase injection cavity 102 to form a flow channel.
In this embodiment, the sample injection structure 100 further includes at least one first branch 103 and at least one second branch 104, wherein one end of the first branch 103 is communicated with the oil phase sample injection cavity 102, and the other end of the first branch 103 is communicated with the microfluidic channel 200. One end of the second branch 104 is communicated with the sample injection cavity 101, and the other end of the second branch 104 is communicated with the microfluidic channel 200; the second flow bifurcating leg 104 includes a bent configuration. As shown in fig. 4, the sample injection structure 100 includes 2 first branch paths 103 and 1 second branch path 104 with a bent structure, two ends of the 2 first branch paths 103 are respectively communicated with the oil phase sample injection cavity 102 and the microfluidic channel 200, and two ends of the 1 second branch paths 104 are respectively communicated with the sample injection cavity 101 and the microfluidic channel 200. The bent structure can control the flow resistance of the sample and adjust the flow ratio of the water phase and the oil phase of the generated liquid drops. Alternatively, the generation rate of the droplets may be controlled to 50 per second.
In this way, the sample from the sample injection cavity 101 and the oil phase from the oil phase injection cavity 102 can merge within the microfluidic channel 200 to prepare a droplet emulsion. Alternatively, the size of the droplets can be adjusted by means of the specific size of the liquid-oil cross opening or/and the specific flow rate of the liquid oil, so that the diameter of the droplets in the droplet emulsion can reach 90 micrometers.
In the embodiment of the present application, the bubble floating structure 300 comprises a dropping lotion inlet 301, a bubble floating chamber 302 and a dropping lotion outlet 303, and further comprises a top sealing layer 304 as shown in fig. 5, wherein the top sealing layer 304 covers the top of the bubble floating chamber 302. The number of the drop emulsion inlets 301 may be determined according to the number of the communication ports of the microfluidic channel 200 and the bubble floating structure 300, for example, as shown in fig. 5, the microfluidic channel 200 includes 2 microfluidic branches 200a and 200b, and then the drop emulsion inlets 301 may include 301a and 301 b. Optionally, the thickness of the bubble flotation chamber 302 is not less than 1 micron.
In an alternative embodiment, the bubble-floating chamber 302 may be any one of a cylinder, a cube and a polygon, the horizontal and vertical dimensions of the bubble-floating chamber 302 are not less than 1 mm, and the volume of the bubble-floating chamber 302 is not less than 1 μ l. For example, the bubble-floating chamber 302 is a cylinder with a diameter of 3 mm and a height of 2 mm.
In the embodiment of the present application, as shown in fig. 6, the dropping emulsion inlet 301 of the bubble floating structure 300 may be disposed at a middle position of the bubble floating chamber 302, and the dropping emulsion outlet 303 may be disposed at a bottom of the bubble floating chamber 302. So, from micro-fluidic channel 200's liquid drop emulsion from the liquid drop emulsion import 301 flow in bubble floating cavity 302 after, because density is poor, the bubble is at the internal spontaneous come-up of cavity, and the liquid drop emulsion of having eliminated the bubble then flows out from the liquid drop emulsion export 303 of bubble floating cavity 302 bottom under the effect of gravity, gets into liquid drop storage structure 400. Alternatively, as shown in fig. 7, the dropping emulsion inlet 301 of the bubble floating structure 300 may be disposed at the bottom of the bubble floating chamber 302, and the dropping emulsion outlet 303 may be disposed at the bottom of the bubble floating chamber 302, that is, the dropping emulsion inlet 301 and the dropping emulsion outlet 303 exist on the same horizontal plane, which can also be implemented: the bubbles are screened out from the bubble floating cavity 302, so that no bubble residue is left in the liquid drop storage cavity 402, and the effectiveness of the digital PCR chip is improved.
As shown in fig. 9, the droplet storage structure 400 further comprises at least one droplet storage chamber outlet 403(403a and 403b), the droplet storage chamber 402 being located at the bottom of the bubble flotation chamber 302, and the droplet storage inlet 401 may coincide with the droplet emulsion outlet 303. The drip emulsion from the bubble flotation chamber 302 flows through the drip emulsion outlet 303 and the droplet storage inlet 401 into the droplet storage chamber 402 where it is spread. Optionally, the depth of the droplet storage chamber 402 is 0.5-2 times the diameter of the droplets in the droplet emulsion. For example, the depth of the droplet storage chamber 402 is 100 μm and the diameter of the droplet is about 90 μm, so that the droplet emulsion can be in a monolayer arrangement under the limitation of physical size. The droplet storage chamber 402 is generally sized to hold more than 4 thousand droplets, and preferably more than 5 thousand droplets. The air in the droplet storage chamber 402 can be released through the droplet storage chamber outlet 403 to facilitate the smooth introduction of the droplet emulsion into the droplet storage chamber 402. In some cases, some biochemical detection experiments require amplified droplet emulsion for subsequent detection, and the droplet emulsion can be sucked out through the outlet 403 of the droplet storage chamber.
After the liquid drop preparation and tiling link is stopped, the top sealing layer 304 of the bubble floating cavity 302 is punctured by using a 16G needle, and the operation aims to destroy the sealing structure of the bubble floating cavity 302, exhaust air in the bubble floating cavity 302 and maintain the stability of the pressure in the cavity of the bubble floating cavity 302 in the thermal circulation process. It should be noted that the 16G needle used in this embodiment is only illustrative for piercing the top closed layer 304 of the bubble floating chamber 302, and laser ablation or the like can also be used for breaking the top closed layer 304 of the bubble floating chamber 302. It should be noted that the droplet emulsion flowing into the droplet storage chamber 402 is bubble free due to the presence of the bubble flotation chamber 302, and thus the droplet storage chamber 402 is bubble free. Thus, the stability of the liquid drop in the PCR thermal cycling process is effectively ensured.
After the integrated digital PCR chip in the present embodiment employs the bubble floating chamber 302, the droplet emulsion will be spread in a layer in the droplet storage chamber 402, and the bright field before thermal amplification is shown in fig. 9a, and the bright field after PCR thermal amplification is shown in fig. 9b and the fluorescence fig. 9 c. Since no bubble is contained in the droplet storage chamber 402 during PCR thermal cycling, the droplet is stably survived and well preserved. Meanwhile, because the liquid drops are tiled into a layer, the number of the fluorescent liquid drops and the number of all the liquid drops can be counted by recognition through photographing in a liquid drop counting link, and the number of the target nucleic acid in the nucleic acid sample can be calculated through the two parameters.
On the other hand, if the integrated digital PCR chip does not use the bubble floating chamber 302, the droplet emulsion directly flows from the microfluidic channel 200 into the droplet storage chamber 402, in which case the thermal amplification front field diagram of the droplet storage chamber 402 refers to fig. 10a, and the thermal amplification rear field diagram refers to fig. 10 b. Since no bubble flotation chamber 302 is employed, bubbles in the droplet emulsion will pass directly into the droplet storage chamber 402. During PCR thermal cycling, the bubbles in the droplet storage chamber 402 will continue to expand, contract, disturb and break up the droplets, making digital PCR difficult to achieve.
The present application also provides a method for processing a droplet emulsion based on a pcr chip with bubble elimination structure, the method shown in fig. 11 includes:
s1101: a droplet emulsion is prepared by a microfluidic channel 200 for merging the sample from the sample injection chamber 101 and the oil phase from the oil phase injection chamber 102.
And adding a sample from the sample injection cavity 101, adding an oil phase from the oil phase injection cavity 102, and converging the sample and the sample in the microfluidic channel 200 to obtain a droplet emulsion.
S1102: the droplet emulsion is subjected to a bubble elimination process by the bubble floating structure 300.
The liquid drop emulsion enters the bubble floating cavity 302 through a liquid drop emulsion inlet 301 communicated with the microfluidic channel 200, bubbles are removed, and the liquid drop emulsion flows out from a liquid drop emulsion outlet 303.
S1103: the droplet emulsion after the bubble removal process is tiled by the droplet storage structure 400.
Tiling the droplet emulsion flowing into the droplet storage chamber 402.
S1104: performing a seal breaking operation on the bubble floating structure 300, and performing an amplification reaction on the droplet emulsion;
after the droplet emulsion is generated, the top sealing layer 304 of the droplet bubble floating cavity 302 is punctured, and PCR thermal cycle is performed on the droplet storage cavity 402 to complete PCR amplification reaction.
S1105: and identifying and counting the droplet emulsion to obtain the copy number of the nucleic acid in the sample.
It should be noted that: the sequence of the embodiments of the present application is only for description, and does not represent the advantages and disadvantages of the embodiments. And specific embodiments thereof have been described above. Other embodiments are within the scope of the following claims. In some cases, the actions or steps recited in the claims may be performed in a different order than in the embodiments and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In some embodiments, multitasking and parallel processing may also be possible or may be advantageous.
The embodiments in the present specification are described in a progressive manner, and the same and similar parts among the embodiments are referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the apparatus embodiment, since it is substantially similar to the method embodiment, the description is relatively simple, and for the relevant points, reference may be made to the partial description of the method embodiment.
It will be understood by those skilled in the art that all or part of the steps for implementing the above embodiments may be implemented by hardware, or may be implemented by a program instructing relevant hardware, where the program may be stored in a computer-readable storage medium, and the above-mentioned storage medium may be a read-only memory, a magnetic disk or an optical disk, etc.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (11)
1. A polymerase chain reaction chip containing a bubble elimination structure is characterized by comprising a sample introduction structure 100, a micro-flow channel 200, a bubble floating structure 300 and a liquid drop storage structure 400 which are arranged on the same surface of the polymerase chain reaction chip;
the sample injection structure 100 comprises a sample injection cavity 101 and an oil phase injection cavity 102, and the sample injection cavity 101 and the oil phase injection cavity 102 are respectively communicated with the microfluidic channel 200; the microfluidic channel 200 is used for merging the sample from the sample injection cavity 101 and the oil phase from the oil phase injection cavity 102 to prepare a droplet emulsion;
the bubble floating structure 300 comprises a dropping emulsion inlet 301, a bubble floating cavity 302 and a dropping emulsion outlet 303; the dropping emulsion inlet 301 is communicated with the microfluidic channel 200, and the bubble floating cavity 302 is used for performing bubble elimination treatment on the dropping emulsion;
the drop storage structure 400 includes a drop storage inlet 401 and a drop storage chamber 402; the drip emulsion outlet 303 is in communication with the drip storage inlet 401; the droplet storage chamber 402 is used to store the droplet emulsion after the bubble removal process from the droplet emulsion outlet 303 and the droplet storage inlet 401.
2. The polymerase chain reaction chip of claim 1, wherein the bubble floating structure 300 further comprises a top sealing layer 304;
the top sealing layer 304 covers the top of the bubble floating cavity 302, and the thickness of the bubble floating cavity 302 is not less than 1 micron.
3. The PCR chip of claim 1, wherein the bubble-floating chamber 302 is any one of a cylinder, a cube and a polyhedron;
the horizontal size and the vertical size of the bubble floating cavity 302 are not less than 1 millimeter, and the volume of the bubble floating cavity 302 is not less than 1 microliter.
4. The PCR chip as set forth in claim 1, wherein the dropping emulsion inlet 301 is disposed at a middle position of the bubble floating chamber 302, and the dropping emulsion outlet 303 is disposed at a bottom of the bubble floating chamber 302.
5. The PCR chip of claim 1, wherein the sample injection cavity 101 and the oil phase injection cavity 102 have the same recess depth.
6. The PCR chip of claim 1, wherein the bottom of the sample injection cavity 101, the bottom of the oil phase injection cavity 102 and the bottom of the microfluidic channel 200 are at the same level.
7. The PCR chip of claim 1, wherein the bottom of the sample injection cavity 101, the bottom of the oil phase injection cavity 102 and the top of the microfluidic channel 200 are at the same level.
8. The PCR chip of claim 1, wherein the sample injection structure 100 further comprises at least one first branch 103 and at least one second branch 104;
one end of the first shunting branch 103 is communicated with the oil phase sample feeding cavity 102, and the other end of the first shunting branch 103 is communicated with the microfluidic channel 200;
one end of the second branch 104 is communicated with the sample injection cavity 101, and the other end of the second branch 104 is communicated with the microfluidic channel 200; the second flow-splitting leg 104 includes a bell crank configuration.
9. The polymerase chain reaction chip of claim 1 wherein the droplet storage structure 400 further comprises a droplet storage chamber outlet 403.
10. The polymerase chain reaction chip of claim 1 wherein the droplet storage chamber 402 is located at the bottom of the bubble flotation chamber 302; the depth of the droplet storage chamber 402 is 0.5 to 2 times the diameter of the droplets in the droplet emulsion.
11. A method for processing a droplet emulsion based on a polymerase chain reaction chip with a bubble elimination structure is characterized in that the polymerase chain reaction chip comprises a sample introduction structure 100, a microfluidic channel 200, a bubble floating structure 300 and a droplet storage structure 400 which are arranged on the same surface of the polymerase chain reaction chip, and the method comprises the following steps:
the microfluidic channel 200 is used for merging the sample from the sample injection cavity 101 in the sample injection structure 100 and the oil phase from the oil phase sample injection cavity 102 in the sample injection structure 100 to prepare a droplet emulsion;
subjecting the droplet emulsion to a bubble elimination process by the bubble floating structure 300;
tiling the droplet emulsion after bubble elimination treatment by the droplet storage structure 400;
performing a seal breaking operation on the bubble floating structure 300 and performing an amplification reaction on the droplet emulsion;
and identifying and counting the droplet emulsion to obtain the copy number of the nucleic acid in the sample.
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Cited By (3)
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| CN112275338A (en) * | 2020-10-29 | 2021-01-29 | 王晓冬 | Droplet single-layer tiled nucleic acid detection chip and preparation method thereof |
| WO2022247121A1 (en) * | 2021-05-27 | 2022-12-01 | 京东方科技集团股份有限公司 | Micro-fluidic chip centrifugal structural piece and centrifuge |
| CN115487879A (en) * | 2021-06-17 | 2022-12-20 | 湖南乐准智芯生物科技有限公司 | Structure and method for reducing vacuum sample injection bubbles and biochip |
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