CN108587862B - Sample droplet generator - Google Patents
Sample droplet generator Download PDFInfo
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- CN108587862B CN108587862B CN201810381994.4A CN201810381994A CN108587862B CN 108587862 B CN108587862 B CN 108587862B CN 201810381994 A CN201810381994 A CN 201810381994A CN 108587862 B CN108587862 B CN 108587862B
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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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a sample liquid drop generator, which comprises a sample cup body with openings at two ends, a sealing cover arranged at the upper end of the sample cup body, an upper piece connected with the lower end of the sample cup body and a lower piece connected with the upper piece, wherein the upper piece is provided with a sealing ring; the upper surface of the lower sheet is provided with a plurality of bonding ribs which are distributed annularly around the sample cup body, the upper surface of the lower sheet outside each bonding rib is provided with a buffer pool, a main flow channel is formed between adjacent bonding ribs, the buffer pool outside each main flow channel is internally provided with a buffer rib, the edge of the upper surface of the lower sheet is provided with a plurality of rectangular lugs, a sub-flow channel is formed between adjacent rectangular lugs, and each sub-flow channel is provided with a tension forming structure. The invention has the characteristics of excellent stability and reproducibility, ultrahigh sensitivity and extreme accuracy.
Description
Technical Field
The invention relates to the technical field of gene molecular diagnostic reagents, in particular to a sample liquid drop generator which has excellent stability and reproducibility, ultrahigh sensitivity and extreme accuracy.
Background
At present, fluorescent Quantitative PCR (Quantitative Polymerase Chain Reaction, qPCR) has been developed into a key conventional technology in the field of in vitro molecular diagnosis, and the development of life science in the field of health care is greatly promoted. However, the quantitative PCR quantification is only a relative quantification, and the accuracy and reproducibility thereof still cannot meet the development requirements of the current in vitro molecular diagnostic system and the research field of biological genetics. However, in many projects such as liquid biopsy and NIPT, some technical bottlenecks are faced, for example, the content of tumor circulating DNA in blood is extremely low, and the sensitivity and precision of each conventional detection means cannot meet the detection requirements. Although the currently common second-generation sequencing technology can be used for liquid biopsy detection, the sample preparation is complex, the sequencing sample library building is complex, the sequencing reagent cost is high, the sequencing time is long, the accuracy is low, the operation of professional personnel is required, the data analysis is very complex, and a professional doctor of non-biological information cannot directly read the data. These technical bottlenecks greatly limit the application and popularization in hospital clinics.
The detection sensitivity of the digital PCR multiple mutation site liquid biopsy diagnosis technology can reach one ten thousandth, the accuracy is 1000 times of that of a sequencer, and a revolutionary breakthrough can be provided for liquid biopsy. Compared with sequencing detection, the detection sensitivity is only 30% of the sequencing technology, the operation is simple, any student of high school and higher school can apply the operation, the detection result is directly given, and the detection method is clear at a glance and does not need professional data analysts. The site of digital PCR detection can cover the common clinical diagnosis requirement of liquid biopsy, so the method is suitable for large-scale popularization in hospitals.
In addition, due to the inhibition effect of the PCR amplification product on the enzyme catalysis reaction, the gene variation detection method of the existing qPCR technology often has no effect on low-abundance tumor gene variation in somatic cells.
Disclosure of Invention
The invention aims to overcome the defects that the qPCR technology in the prior art cannot identify low-abundance tumor gene variation in somatic cells and maternal fetal gene targets, and provides a sample droplet generator with excellent stability and reproducibility, ultrahigh sensitivity and extreme accuracy.
In order to achieve the purpose, the invention adopts the following technical scheme:
a sample droplet generator comprises a sample cup body with openings at two ends, a sealing cover or a sealing film arranged at the upper end of the sample cup body, an upper sheet connected with the lower end of the sample cup body and a lower sheet connected with the upper sheet; the upper surface of the lower sheet is provided with a plurality of bonding ribs annularly distributed around the sample cup body, the upper surface of the lower sheet outside each bonding rib is provided with a sunken buffer pool, a main flow channel is formed between adjacent bonding ribs, the buffer pool outside each main flow channel is internally provided with a buffer rib, the edge of the upper surface of the lower sheet is provided with a plurality of rectangular lugs, micro-flow channels are formed between adjacent rectangular lugs, and each micro-flow channel is provided with a tension forming structure;
the tension forming structure is a flared opening which is arranged on the outer edge of the micro-channel and is opened from inside to outside, and the opening angle of the flared opening is 60-120 degrees.
The invention is formed by combining an upper wafer substrate and a lower wafer substrate (or more than two wafer substrates) (or si, sio2 and the like similar to the wafers) or any two substrates which can be bonded, wherein a cup body mounting opening is arranged in the middle of any wafer substrate, and a sample cup body is arranged on the cup body mounting opening.
The buffer pool with sample fluid etched on the lower surface of the upper piece or the upper surface of the lower piece, a plurality of main flow channels (more than 2) for leading in the sample fluid are etched on the periphery of the through hole, one end of the main flow channel is communicated with the buffer pool, a plurality of bonding ribs (more than 2) are arranged on the periphery of the mounting hole of the cup body, the buffer ribs for weakening the impulse force and direction of the fluid flow direction are arranged in the buffer pool, the pressure borne by the fluid in the buffer pool is forced to be balanced, the pressure dispersed to all micro flow channels is balanced, the problem that the signal collection is influenced by the uneven size of the generated liquid drops due to the uneven pressure on some micro flow channels is avoided, meanwhile, the air bubbles can be generated in the unobstructed flow channels due to the uneven pressure, the air bubbles are merged into the liquid drop carrier oil, and unstable factors are caused to the generated liquid drop particles due to the large difference of thermal expansion coefficients of the gas and the fluid when the sample liquid drops are circularly amplified, causing the sample droplet to break up, resulting in a failure or distortion of the result.
A large number of micro-channels are formed between the upper plate and the lower plate, the cross section of each micro-channel tends to be square or round, the cross section area of each micro-channel is larger than 300 square microns, one end of each micro-channel is communicated with the buffer pool, the other end of each closed-loop channel is positioned on the extension of the base material and presents a horn mouth from inside to outside, when sample fluid flows out through the channels, the sample fluid is gathered at the horn mouths under the action of the horn mouths and the surface tension of the fluid, liquid drops are formed step by step, and when the self weight of the liquid drops is larger than the adhesive force of the sample fluid, the liquid drops separate from the horn mouths and fall into the oil phase carrier to form independent sample liquid drops.
Preferably, each of the keyed ribs and each of the cushion ribs are in the shape of an outwardly arched arc, or each of the keyed ribs and each of the cushion ribs are shaped to promote a swirling effect in the fluid.
Preferably, the sealing cover is provided with a cross-shaped sealed groove, the lower end of the sample cup body is provided with an annular boss matched with the sample cup body mounting opening of the upper piece, and the lower end of the annular boss is flush with the lower surface of the upper piece, or the lower end of the annular boss is higher than the lower surface of the upper piece.
Preferably, the sealing cover is made of elastic silica gel and rubber materials with stable molecular structures, and the sealing film is made of plastic-paper composite materials.
Preferably, the edge of the sealing cover is provided with a bent edge which extends downwards and then bends inwards, and the upper end of the sample cup body is provided with an annular sealing boss which extends outwards.
Preferably, the lower surface of the upper plate is provided with a plurality of bonding ribs annularly distributed around the sample cup body, the lower surface of the upper plate outside each bonding rib is provided with a concave buffer pool, a main flow channel is formed between adjacent bonding ribs, the buffer pool outside each main flow channel is internally provided with a buffer rib, the edge of the lower surface of the upper plate is provided with a plurality of rectangular lugs, and a micro-flow channel is formed between adjacent rectangular lugs.
Preferably, the upper piece and the lower piece are both rectangular or circular; the upper sheet and the lower sheet are made of materials with stable molecular structures, and the materials with the stable molecular structures comprise metal, silicon materials and hard plastics.
Preferably, the micro flow channel is replaced by an embedded capillary, and the embedded capillary is made of rigid plastics or other materials with stable molecular structures.
Preferably, the sample cup body is detachably connected with the upper sheet, or the sample cup body and the upper sheet are integrally formed.
Therefore, the invention has the following beneficial effects: the method has the advantages that liquid drops with uniform sizes are formed, the collection amount of samples is not limited intentionally, all collected and extracted sample templates can produce liquid drops, the size of the liquid drops is adjusted randomly according to the size of the actual verification requirement, the diameter of the liquid drops meeting the verification requirement is adjusted randomly, so that the quantity of the liquid drops of the samples is not limited by the collection amount of the samples, but is caused by the size and the total quantity of the diameter of the liquid drops, the utilization rate of the collected samples is extremely high, and no dead volume exists; the small integrated processing of being convenient for of chip volume, great improvement sample flux detects, and the internal structure size of chip is single is convenient for process, ensures the machining precision and reaches the rate, reduces the defective rate, reduce cost, but the realizability and the large-scale production of being convenient for.
Drawings
FIG. 1 is a schematic diagram of an embodiment of the present invention;
FIG. 2 is a schematic view of one configuration of a sample cup of the present invention;
FIG. 3 is a schematic view of a configuration of the lower sheet upper surface of the present invention;
FIG. 4 is another schematic view of the structure of the lower sheet upper surface of the present invention.
In the figure: the sample cup comprises a sample cup body 1, a sealing cover 2, an upper sheet 3, a lower sheet 4, a bonding rib 5, a buffer pool 6, a sample cup body mounting port 7, a main flow channel 8, a buffer rib 9, a rectangular lug 10, a micro flow channel 11, a tension forming structure 111, a cross-shaped sealed groove 21, an annular boss 101, a bent edge 22 and an annular sealing boss 102.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
Example 1
The embodiment shown in fig. 1, 2, 3, and 4 is a sample droplet generator, which includes a sample cup 1 with openings at both ends, a sealing cap 2 disposed at the upper end of the sample cup, an upper plate 3 connected to the lower end of the sample cup, and a lower plate 4 connected to the upper plate; the upper surface of the lower sheet is provided with 4 bonding ribs 5 which are distributed annularly around the sample cup body, the upper surface of the lower sheet outside each bonding rib is provided with a buffer pool 6, a main flow channel 8 is formed between adjacent bonding ribs, the buffer pool outside each main flow channel is internally provided with a buffer rib 9, the edge of the upper surface of the lower sheet is provided with 40 rectangular lugs 10, micro flow channels 11 are formed between adjacent rectangular lugs, and each micro flow channel is provided with a tension forming structure 111. Each of the key ribs and each of the cushion ribs has an outwardly arched circular arc shape.
As shown in fig. 2, a cross-shaped closed groove 21 is formed in the sealing cover, an annular boss 101 matched with the sample cup mounting opening 7 of the upper piece is arranged at the lower end of the sample cup, and the lower end of the annular boss is flush with the lower surface of the upper piece, or the lower end of the annular boss is higher than the lower surface of the upper piece.
The sealing cover is made of elastic silica gel and rubber materials with stable molecular structures.
The edge of the sealing cover is provided with a bent edge 22 which extends downwards and then bends inwards, and the upper end of the sample cup body is provided with an annular sealing boss 102 which extends outwards.
As shown in fig. 3, the tension forming structure is a bell mouth opened from the inside to the outside provided on the outer edge of the microchannel, and the optimum angle of opening of the bell mouth is 68 °. The upper piece and the lower piece are both rectangular, and the upper piece and the lower piece are both wafers. The sample cup body is detachably connected with the upper piece.
The invention is formed by bonding an upper wafer and a lower wafer, wherein a cup body mounting port is arranged in the middle of the upper wafer, a sample cup body is arranged on the cup body mounting port, a buffer pool of sample fluid is etched on the upper surface of the lower wafer, 4 main flow channels for introducing the sample fluid are etched on the periphery of the cup body mounting port, one end of each main flow channel is communicated with the buffer pool, 4 bonding ribs are arranged on the periphery of the cup body mounting port, the buffer ribs for weakening the impulse force and direction of the fluid flowing direction are arranged in the buffer pool, the pressure of the fluid in the buffer pool is forced to be balanced, the pressure dispersed to all micro flow channels is balanced, the problem that the signal collection is influenced by the uneven pressure on some micro flow channels due to the uneven pressure is avoided, meanwhile, the dead volume is generated on the inner flow channels due to the pressure, the smooth flow channels can generate bubbles and are merged into a liquid drop oil carrier, and unstable factors caused by the large difference of thermal expansion coefficients of gas and fluid when the sample liquid drops are circularly amplified Causing the sample droplet to break up, leading to failure of the result.
40 micro-channels are formed between the upper plate and the lower plate, the cross section of each micro-channel tends to be square, the cross section area of each micro-channel is larger than 300 square microns, one end of each micro-channel is communicated with the buffer pool, the other end of each micro-channel is positioned on the extension of the upper plate and presents a horn mouth from inside to outside, the angle of each horn mouth is 90 degrees, when sample fluid flows out through the channels, the sample fluid is gathered at the horn mouths under the action of the horn mouths and the surface tension of the fluid, liquid drops are formed gradually, and when the self weight of the liquid drops is larger than the adhesive force of the sample fluid, the liquid drops fall into liquid drop carrier oil from the horn mouths to form independent sample liquid drops.
The upper piece, the lower piece and the sample cup body are required to be subjected to hydrophobic surface modification treatment, a generator main body structure and an internal flow channel structure are firstly completed, the sample cup body is combined, and the sealing cover is mounted at last to form a complete liquid drop generation chip.
Example 2
It should be understood that this example is for illustrative purposes only and is not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Claims (9)
1. A sample liquid drop generator is characterized by comprising a sample cup body (1) with openings at two ends, a sealing cover (2) or a sealing film arranged at the upper end of the sample cup body, an upper piece (3) connected with the lower end of the sample cup body and a lower piece (4) connected with the upper piece; the upper surface of the lower sheet is provided with a plurality of bonding ribs (5) which are distributed annularly around the sample cup body, the upper surface of the lower sheet outside each bonding rib is provided with a buffer pool (6), a main flow channel (8) is formed between adjacent bonding ribs, a buffer rib (9) is arranged in the buffer pool outside each main flow channel, the edge of the upper surface of the lower sheet is provided with a plurality of rectangular lugs (10), a micro flow channel (11) is formed between adjacent rectangular lugs, and each micro flow channel is provided with a tension forming structure (111);
the tension forming structure is a flared opening which is arranged on the outer edge of the micro-channel and is opened from inside to outside, and the opening angle of the flared opening is 60-120 degrees.
2. The sample droplet generator of claim 1, wherein each of the bonding ribs and each of the buffer ribs are in the shape of an outwardly arched arc, or each of the bonding ribs and each of the buffer ribs are shaped to promote a vortex effect in the fluid.
3. The sample droplet generator according to claim 1, wherein the sealing cover is provided with a cross-shaped sealing groove (21), the lower end of the sample cup body is provided with an annular boss (101) matched with the sample cup body mounting opening (7) of the upper sheet, and the lower end of the annular boss is flush with the lower surface of the upper sheet or the lower end of the annular boss is higher than the lower surface of the upper sheet.
4. The sample droplet generator of claim 1, wherein the sealing cover is made of a silicone rubber and rubber material having elasticity and stable molecular structure, and the sealing film is made of a plastic-paper composite material.
5. The sample droplet generator of claim 1, wherein the edge of the sealing cover is provided with a bent edge (22) extending downwards and then bending inwards, and the upper end of the sample cup is provided with an annular sealing boss (102) extending outwards.
6. The sample droplet generator of claim 1, wherein the lower surface of the upper plate has a plurality of bonding ribs annularly distributed around the sample cup, the lower surface of the upper plate outside each bonding rib has a concave buffer pool, main flow channels are formed between adjacent bonding ribs, a buffer rib is formed in the buffer pool outside each main flow channel, a plurality of rectangular projections are formed on the edge of the lower surface of the upper plate, and micro flow channels are formed between adjacent rectangular projections.
7. The sample droplet generator of claim 1, wherein the upper and lower pieces are rectangular or circular; the upper sheet and the lower sheet are made of materials with stable molecular structures, and the materials with the stable molecular structures comprise metal, silicon materials and hard plastics.
8. The sample droplet generator of claim 1, wherein the micro flow channel is replaced with an embedded capillary, and the embedded capillary is made of rigid plastic or other materials with stable molecular structures.
9. The sample droplet generator of claim 1, 2, 3, 4, 5, 6, 7, or 8, wherein the sample cup is removably attached to the top piece, or the sample cup and the top piece are integrally formed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810381994.4A CN108587862B (en) | 2018-04-20 | 2018-04-20 | Sample droplet generator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810381994.4A CN108587862B (en) | 2018-04-20 | 2018-04-20 | Sample droplet generator |
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| CN108587862A CN108587862A (en) | 2018-09-28 |
| CN108587862B true CN108587862B (en) | 2022-02-22 |
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| CN201810381994.4A Active CN108587862B (en) | 2018-04-20 | 2018-04-20 | Sample droplet generator |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999001688A1 (en) * | 1997-07-03 | 1999-01-14 | The Regents Of The University Of Michigan | Thermal microvalves |
| US6155710A (en) * | 1996-02-20 | 2000-12-05 | Japan, As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries | Method and device for producing emulsions |
| CN104741158A (en) * | 2015-04-03 | 2015-07-01 | 北京大学 | Device and method for generating microdroplets by inertial force |
| CN106635777A (en) * | 2016-11-15 | 2017-05-10 | 杭州凯基科技有限公司 | Gene micro-drop granule chip making device and gene micro-drop granule chip making method |
| CN107674819A (en) * | 2017-09-07 | 2018-02-09 | 杭州凯基科技有限公司 | A kind of sample droplets particle-forming apparatus |
-
2018
- 2018-04-20 CN CN201810381994.4A patent/CN108587862B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6155710A (en) * | 1996-02-20 | 2000-12-05 | Japan, As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries | Method and device for producing emulsions |
| WO1999001688A1 (en) * | 1997-07-03 | 1999-01-14 | The Regents Of The University Of Michigan | Thermal microvalves |
| CN104741158A (en) * | 2015-04-03 | 2015-07-01 | 北京大学 | Device and method for generating microdroplets by inertial force |
| CN106635777A (en) * | 2016-11-15 | 2017-05-10 | 杭州凯基科技有限公司 | Gene micro-drop granule chip making device and gene micro-drop granule chip making method |
| CN107674819A (en) * | 2017-09-07 | 2018-02-09 | 杭州凯基科技有限公司 | A kind of sample droplets particle-forming apparatus |
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| CN108587862A (en) | 2018-09-28 |
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Effective date of registration: 20190807 Address after: 311100 Room 101, 1st Floor, Building 2, 2636 Yuhangtang Road, Cangqian Street, Yuhang District, Hangzhou City, Zhejiang Province Applicant after: Hangzhou Xinghaohui Biotechnology Co.,Ltd. Address before: 311100 No. 307C, Taiji Road, Yuhang Economic Development Zone, Hangzhou City, Zhejiang Province Applicant before: HANGZHOU KAIJI TECHNOLOGY Co.,Ltd. |
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Effective date of registration: 20221216 Address after: 518000 12th floor, building 1, Baiwang R & D building, 5158 xilishahe West Road, Nanshan District, Shenzhen City, Guangdong Province Patentee after: Shenzhen maiketian Biomedical Technology Co.,Ltd. Address before: 311100 Room 101, 1 / F, building 2, 2636 yuhangtang Road, Cangqian street, Yuhang District, Hangzhou City, Zhejiang Province Patentee before: Hangzhou Xinghaohui Biotechnology Co.,Ltd. |