CN114058495A - Industrial-grade asymmetric liquid drop generating device and digital nucleic acid amplification detection system - Google Patents
Industrial-grade asymmetric liquid drop generating device and digital nucleic acid amplification detection system Download PDFInfo
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Abstract
The invention discloses an industrial-grade asymmetric liquid drop generating device and a digital nucleic acid amplification detection system. The industrial-grade asymmetric liquid drop generating device comprises an upper-layer water phase unit and a lower-layer oil phase unit, wherein the upper-layer water phase unit is fixed on the lower-layer oil phase unit, the upper-layer water phase unit is provided with a drainage tube with a large upper part and a small lower part, and an upper-layer water phase in the drainage tube enters the lower-layer oil phase under the action of external force to form water-in-oil liquid drops with uniform sizes. The drainage tube is formed by tapering a capillary tube at the end part. A digital nucleic acid amplification detection system, comprising: a high repetition rate asymmetric droplet generator for generating a plurality of uniform droplets; the nucleic acid amplification temperature control device is used for carrying out nucleic acid amplification reaction in the liquid drops generated by the liquid drop generating device; and a product signal acquisition device for acquiring a product signal after the nucleic acid amplification reaction in the liquid droplet. The invention has the obvious advantages of controllable cost, high repetition rate and the like.
Description
Technical Field
The invention belongs to the field of analysis and detection, and mainly relates to an industrial-grade asymmetric liquid drop generating device and a digital nucleic acid amplification detection system.
Background
The liquid drop (Microdroplet) is used as a micro reactor, can greatly accelerate the biochemical reaction rate, and is widely applied to the fields of analysis and detection, chemical sensing, medical detection, chemical synthesis and the like. Conventional methods of droplet generation are often based on chip microfluidics, and such droplet generation devices typically require expensive chip fabrication and sophisticated peristaltic pumps to regulate the flow rates of the two phases, water and oil, to control the droplet generation size. The main problems with the use of pumps: inconvenient operation, high cost and easy reagent waste.
The digital nucleic acid detection technology plays a great role in the fields of biological detection, chemical sensing, medical diagnosis and the like. As a third generation nucleic acid amplification method, a sample is diluted to a single molecule level by distributing the sample into tens of millions of units by using the technical idea of micro-droplets. Each droplet contains or does not contain nucleic acid, and after amplification, the droplets containing the nucleic acid have a fluorescence signal, and the fluorescence droplets quantitatively conform to a Poisson distribution. By counting, absolute quantitative detection of a sample such as a target nucleic acid can be achieved. Therefore, the micro-droplet generating device is very important for the digital PCR technology.
Among droplet generating apparatuses proposed in recent years, CN112522374A "method and apparatus for generating digital droplets in a centrifugal type at low cost and wide adaptability" proposes a centrifugal type droplet generating apparatus which is simple in operation, low in cost and wide adaptability. However, the sealing of the micron tube with uniform size by the high molecular polymer such as PDMS paste used in this device may cause the following problems: (1) if the high molecular polymer is not sealed tightly, the upper water phase is easy to enter the lower oil phase through a gap between the high molecular polymer and the suction head of the inner pipetting gun, and the micron tube is not the only channel leading to the lower oil phase, so that the generated liquid drops are not uniform; (2) if the high molecular polymer is improperly treated during sealing, the superfine micron tubes can be blocked, and the upper-layer water phase cannot be centrifuged into the lower-layer oil phase, so that the separation rate of uniform liquid drops is reduced; (3) when the high molecular polymer is sealed with the micron tube, if the upper end of the micron tube and the upper layer of the solidified high molecular polymer are not controlled on the same horizontal plane, the micro-scale upper water phase cannot be completely centrifuged into the lower oil phase, so that the sample is wasted.
The above reasons lead to the insufficient repetition rate of the invention CN112522374A or similar technologies, and can not reach the industrial production standard.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide an industrial-grade asymmetric droplet generation device and a digital nucleic acid amplification detection system, which utilize an asymmetric structure to guide flow, but not a symmetric structure with the same size at two ends to generate droplets, thereby realizing high repetition rate.
The utility model provides an asymmetric liquid drop generating device of industrial grade, includes upper water phase unit and lower floor oil phase unit, and upper water phase unit is fixed on lower floor oil phase unit, and upper water phase unit is equipped with big-end-up's drainage tube, and under the effect of external force, the upper water phase in the drainage tube enters into lower floor oil phase, forms the water-in-oil liquid drop of size homogeneity.
The drainage tube is formed by tapering a capillary tube at the end part.
The cone tip of the capillary is cut under a microscope with a blade.
The external force is centrifugal force.
The lower oil phase unit adopts a plastic centrifuge tube.
The upper water phase unit comprises a fixing frame and a drainage tube, the fixing frame is used for fixing the drainage tube, and the fixing frame comprises one or more nested pipette suction heads.
The pipette tip in the fixing frame has a caliber which is cut flatly or is additionally provided with an O-shaped rubber ring, so that the drainage tube is convenient to fix, or the upper water phase unit is convenient to fix on the lower oil phase unit.
The size of the generated liquid drops is adjusted by controlling the inner diameter and the opening size of the drainage tube.
The droplet generating device is applied to nucleic acid dPCR and dLamp amplification to realize digital nucleic acid detection.
A digital nucleic acid amplification detection system based on the liquid drop generating device comprises: a high repetition rate asymmetric droplet generator for generating a plurality of uniform droplets; the nucleic acid amplification temperature control device is used for carrying out nucleic acid amplification reaction in the liquid drops generated by the liquid drop generating device; and a product signal acquisition device for acquiring a product signal after the nucleic acid amplification reaction in the liquid droplet.
The invention has the beneficial effects that:
the invention breaks through the bottlenecks of high production cost, difficult use and the like of a micro-fluidic chip required by the production of water-in-oil droplets in the existing droplet generating device, and achieves the aim of forming a large amount of high-quality droplets by using conventional centrifugal equipment or similar technologies.
According to the invention, the tip of the capillary tube is tapered, and an asymmetric hollow drainage structure is adopted, so that the liquid drop generating device capable of realizing industrial-grade mass production is constructed, and the liquid drop generating device has the characteristics of controllable cost, high repetition rate, simplicity in operation and the like.
In addition, the industrial-grade asymmetric droplet generation device is combined with nucleic acid amplification, and a trace amount of aqueous phase solution can quickly generate high-flux uniform water-in-oil droplets within a few minutes, so that the quantitative detection of the digital PCR nucleic acid is realized.
The invention can be used for packaging and distributing samples such as nucleic acid, cells, protein, nanoparticles and the like, and further can be used for completing detection by combining nucleic acid amplification and the like.
Drawings
FIG. 1 is a schematic structural diagram of a droplet generator according to the present invention.
FIG. 2 is an enlarged view showing the asymmetric conical structure of the capillary in the droplet generator of the present invention.
FIG. 3is an enlarged view of a tapered capillary tip configuration in a droplet generator according to the present invention.
FIG. 4 is a schematic view of a uniform droplet generated by the droplet generator of the present invention.
FIG. 5 is a graph showing the relationship between the outer diameter of the tip of a tapered capillary used in the droplet generator of the present invention and the outer diameter of a droplet to be uniformly formed.
FIG. 6 is a diagram showing an experiment in which the droplet generating apparatus of the present invention is used for amplifying a nucleic acid Lamp of a novel coronavirus.
FIG. 7 is the experimental diagram of fluorescent quantitative PCR amplification and other digital PCR amplification of the new coronavirus.
FIG. 8 is an experimental diagram of the droplet generator of the present invention for PCR amplification of new coronavirus nucleic acid.
Description of reference numerals: drainage tube 1, upper water phase unit 2, mount 3, centrifuging tube 4, lower floor oil phase unit 5.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described with reference to the accompanying drawings.
Example 1
The invention constructs an industrial-grade asymmetric liquid drop generating device.
The device can be assembled by using a micropipette head (or a structure similar to scale production) and a centrifugal tube which are common in a biochemical laboratory, a conical capillary tube is fixed in the micropipette head, an upper-layer water phase with a micro-scale enters a lower-layer oil phase to generate water-in-oil droplets with uniform size and high quality under the action of high-speed centrifugation, and the size of the generated water-in-oil droplets can be changed by adjusting the outer diameter of the tip of the conical capillary tube.
As shown in figure 1, the device comprises an upper water phase unit 2 and a lower oil phase unit 5, wherein the upper water phase unit comprises a fixing frame 3 and a drainage tube 1 (a capillary tube in the case), the fixing frame 3is used for fixing the drainage tube 1, and the fixing frame 3 comprises one or more nested pipette tips. The upper water phase unit is formed by assembling three micropipette heads and capillaries, the outermost pipetting gun head is formed by cutting and modifying a 1000-microliter pipetting gun head at a gun head projection, the middle pipetting gun head is formed by cutting and modifying a 200-microliter pipetting gun head at the gun head projection and is fixed on the inner side of the outermost pipetting gun head by using an O-shaped collar, the innermost pipetting gun head is formed by cutting and modifying a 10-microliter pipetting gun head at a position 0.5cm away from the lowest end, the micropipette heads of three different specifications are mutually nested, and the capillary with the inner diameter of 690 mu m is just fixed in the innermost micropipette head. The lower oil phase unit 5 consists of a 1.5ml centrifuge tube 4. The outermost pipette tip in the upper water phase unit 2 can be fixed in a 1.5ml centrifuge tube of the lower oil phase system and jointly placed in a common centrifuge of a laboratory to generate water-in-oil droplets. In this example, the pipette tips were of the following type, 1000. mu.l pipette tips: axygen T-1000-B; 200 μ l pipette tip: axygen T-200-Y; 10 μ l pipette tip: axygen T-300. The centrifuge tube can use a common centrifuge tube and is matched with a liquid-transfer gun head of the upper water phase. It will be apparent to those skilled in the art that other micropipette tips commonly found in biochemical laboratories may be used, or similar structures and products on the market may be used.
The capillary used in the device is a common capillary with uniform inner diameter, and the tip is tapered by methods such as a microelectrode pin drawing instrument and the like, so that the tip forms a tapered structure. The use of this structure significantly improves the rate of formation of uniform-sized, high-quality water-in-oil droplets. The capillary used in this example has an inner diameter of 690 μm (outer diameter of 1200 μm) and a length of 10cm, and after the capillary is pulled by a capillary needle puller or other instrument, the tip of the capillary forms a tapered structure (fig. 2). Thus, after the capillary has been tapered, the capillary tip is cut under a microscope using a blade or the like to determine the outer diameter of the tip to ensure that the size of the centrifuged droplet can be controlled. For example: a capillary having a tip with an outer diameter of 10 μm (FIG. 3) was centrifuged to obtain uniform droplets having a diameter of about 50 μm (FIG. 4).
When the device is used for loading the upper-layer water phase, instruments such as a trace loading needle and the like are used for directly adding a sample into the capillary, so that the phenomenon that uneven liquid drops are generated due to the liquid leakage caused by poor sealing is completely avoided; meanwhile, the upper-layer water phase injected into the capillary can be completely centrifuged to generate liquid drops, so that the generation quantity of the liquid drops is increased, and the waste of reagents is avoided. For example, 10. mu.l of the upper aqueous phase can produce more than 7 ten thousand uniform droplets.
The successful generation rate results of uniform droplets of 10 droplet generators and 10 droplet generators of CN112522374A in the present invention are shown in table 1 below, and only 1 droplet generator of CN112522374A successfully generated uniform droplets with a success rate of 10%, while 8 droplet generators successfully generated uniform droplets with a success rate of 80%. If the cut-out device is excluded, the repetition rate is 100%.
The device of the present invention can change the size of the generated water-in-oil droplet by adjusting the outer diameter of the tapered capillary tip. The invention cuts 52 conical capillaries with different tip external diameters to generate uniform liquid drops, and the conditions are kept consistent except the tip external diameters. The resulting relationship of the tapered capillary tip outer diameter to the diameter of the resulting uniform droplet is shown in FIG. 5. The outer diameter of the tip of the tapered capillary is in the range of 1.833-17.277 μm, and the diameter of the generated uniform liquid drop is in the range of 15.169-52.038 μm.
Compared with the centrifugal liquid drop generating device of CN112522374A (shown in Table 1), the device of the invention can be assembled by using a micropipette head (or a structure similar to scale production), a centrifugal tube and a tapered capillary tube which are common in a biochemical laboratory, and improves the separation rate of uniform liquid drops, the generation quantity of the liquid drops and the waste of a micro-level upper-layer water phase by using the tapered capillary tube.
TABLE 1
Example 2
The industrial-grade asymmetric droplet generation device constructed by the invention is combined with the nucleic acid lamp to realize digital lamp nucleic acid detection.
Selection of a sample: new coronavirus positive standard sample. The cells were diluted to 1-100 cp/. mu.l with RNA-free-water.
Reverse transcription: the reverse transcription was performed using the HiFiScript cDNA Synthesis Kit of Shanghai kang, a century Biotechnology Ltd, according to the instructions, and the DNA obtained by reverse transcription was stored at-20 ℃ for a long period of time at-80 ℃. Lamp system: a nucleic acid detection kit for a New crown constant temperature fluorescence probe method used by New Haerbin Gene detection Limited company is adopted in the Lamp reaction, and a 25 mul Lamp system comprises: 5 μ l of a positive standard of DNA of the new coronavirus, 10 μ l of nCoV-23 IsoAmp Solution A and 10 μ l of nCoV-23 IsoAmp Solution B in the kit. Micro-droplet generation: in the same way as in example 1, when the upper aqueous phase of the device of the present invention is loaded, a prepared sample of the lamp system is directly added into the capillary by using an instrument such as a micro loading needle. The lower oil phase system consisted of 1.5ml centrifuge tubes (isopropyl palmitate with surfactant EM 180). The outermost pipette tip in the upper aqueous phase system can be fixed in a 1.5ml centrifuge tube of the lower oil phase system and jointly placed in a common centrifuge (6000 rcf, 3 min) in a laboratory to generate water-in-oil droplets.
Lamp amplification: the generated droplets were transferred to a PCR tube and placed in a fluorescent quantitative PCR apparatus, and the procedure was set at 65 ℃ for 10s and 65 ℃ for 50s as a cycle unit, and cycled 35 times.
And (4) detecting a result: after lamp amplification was complete, the droplets were carefully transferred with a pipette tip to a glass coverslip or 48-well plate for fluorescent microscopy. The results are shown in FIG. 6.
Furthermore, the same sample (1-100 cp/. mu.l of new coronavirus DNA) was used for detection by the fluorescent quantitative PCR system and the commercial digital PCR system (DropDx-2044 digital PCR system), respectively, and the results are shown in FIG. 7.
Example 3
The industrial-grade asymmetric droplet generation device constructed by the invention is combined with nucleic acid PCR amplification, and a trace amount of aqueous phase solution can quickly generate high-flux uniform water-in-oil droplets within a few minutes, so that digital PCR nucleic acid detection is realized.
Selection of a sample: new coronavirus positive standard DNA sample.
And (3) PCR system: 20 μ l of PCR bodyThe system includes: 10 μ l SYBR Green I PCR Mix, 2 μ l New coronavirus DNA positive standard, 1 μ l upstream and downstream primers, 5 μ l glycerol and 1 μ l ddH2O。
Micro-droplet generation: as in example 1, when the upper aqueous phase of the apparatus of the present invention is loaded, a prepared sample of the PCR system is directly added into the capillary by using an instrument such as a micro loading needle. The lower oil phase system consisted of 1.5ml centrifuge tubes (containing ePCR droplet generation oil). The outermost pipette tip in the upper aqueous phase system can be fixed in a 1.5ml centrifuge tube of the lower oil phase system and jointly placed in a common centrifuge (5000 rcf, 3 min) in a laboratory to generate water-in-oil droplets.
And (3) PCR amplification: transferring the generated liquid drop into a PCR tube, placing the PCR tube into a fluorescent quantitative PCR instrument, and carrying out PCR reaction with the reaction procedures of 94 ℃, 3 min, 10min, 95 ℃, 20 s, 60 ℃, 30s, 72 ℃, 30s, 20 cycle and 72 ℃, 3 min.
And (4) detecting a result: after PCR amplification was complete, the droplets were carefully transferred with a pipette tip to a glass coverslip or 48-well plate for fluorescent microscopy. The results are shown in FIG. 8.
The embodiments in the above description can be further combined or replaced, and the embodiments are only described as preferred examples of the present invention, and do not limit the concept and scope of the present invention, and various changes and modifications made to the technical solution of the present invention by those skilled in the art without departing from the design concept of the present invention belong to the protection scope of the present invention. The scope of the invention is given by the appended claims and any equivalents thereof.
Claims (10)
1. An industrial-grade asymmetric droplet generation device, characterized in that:
the device comprises an upper water phase unit and a lower oil phase unit, wherein the upper water phase unit is fixed on the lower oil phase unit, the upper water phase unit is provided with a drainage tube with a large upper part and a small lower part, and an upper water phase in the drainage tube enters into the lower oil phase under the action of external force to form water-in-oil droplets with uniform size.
2. A droplet generator according to claim 1, wherein: the drainage tube is formed by tapering a capillary tube at the end part.
3. A droplet generator according to claim 2, wherein: the cone tip of the capillary is cut under a microscope with a blade.
4. A droplet generator according to claim 1, wherein: the external force is centrifugal force.
5. A droplet generator according to claim 1, wherein: the lower oil phase unit adopts a plastic centrifuge tube.
6. A droplet generator according to claim 1, wherein: the upper water phase unit comprises a fixing frame and a drainage tube, the fixing frame is used for fixing the drainage tube, and the fixing frame comprises one or more nested pipette suction heads.
7. A droplet generator according to claim 6, wherein: the pipette tip in the fixing frame has a caliber which is cut flatly or is additionally provided with an O-shaped rubber ring, so that the drainage tube is convenient to fix, or the upper water phase unit is convenient to fix on the lower oil phase unit.
8. A droplet generator according to claim 1, wherein: the size of the generated liquid drops is adjusted by controlling the inner diameter and the opening size of the drainage tube.
9. A droplet generator according to claim 1, wherein: the method is applied to nucleic acid dPCR and dLamp amplification to realize digital nucleic acid detection.
10. A digital nucleic acid amplification detection system based on the droplet generation apparatus of claim 1, wherein: the method comprises the following steps:
a high repetition rate asymmetric droplet generator for generating a plurality of uniform droplets;
the nucleic acid amplification temperature control device is used for carrying out nucleic acid amplification reaction in the liquid drops generated by the liquid drop generating device; and
and the product signal acquisition device is used for acquiring a product signal after the nucleic acid amplification reaction in the liquid drop.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450891A (en) * | 2014-11-17 | 2015-03-25 | 中国科学院微生物研究所 | Method and system for digital quantitative analysis of nucleic acid amplification based on micro-droplet |
| EP3187252A1 (en) * | 2014-08-25 | 2017-07-05 | Universidad De Sevilla | Method and device for producing simple and compound micrometre-sized emulsions |
| CN107511189A (en) * | 2017-08-02 | 2017-12-26 | 肇庆市华师大光电产业研究院 | A kind of preparation method of the single dispersing microlayer model based on capillary |
| CN108159976A (en) * | 2018-01-03 | 2018-06-15 | 西南交通大学 | A kind of Water-In-Oil Bao Shui（W/W/O）Monodisperse double emulsion preparation method and its micro fluidic device |
| CN109908986A (en) * | 2019-02-21 | 2019-06-21 | 浙江大学 | A kind of drop formation system and application method based on the asymmetric capillary in outlet |
| CN110841734A (en) * | 2019-12-06 | 2020-02-28 | 中国科学院长春光学精密机械与物理研究所 | Digital PCR device and single-pump liquid drop generating system thereof |
| US20200101454A1 (en) * | 2018-10-01 | 2020-04-02 | Lifeng XIAO | Micro-pipette tip for forming micro-droplets |
| CN112439470A (en) * | 2019-08-30 | 2021-03-05 | 北京达微生物科技有限公司 | Sample adding needle for preparing micro-droplets and preparation method of micro-droplets |
| CN112522374A (en) * | 2020-10-28 | 2021-03-19 | 浙江大学 | Low-cost widely-adaptive centrifugal digital liquid drop generation method and device |
| CN216688147U (en) * | 2021-10-29 | 2022-06-07 | 浙江大学 | Industrial-grade asymmetric liquid drop generating device and digital nucleic acid amplification detection system |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140193857A1 (en) * | 2012-09-12 | 2014-07-10 | Cypho, Inc. | Centrifuge tube droplet generator |
| EP3117897A4 (en) * | 2014-03-11 | 2017-10-18 | Toppan Printing Co., Ltd. | Droplet producing device, droplet producing method, liposome producing method, fixture, and droplet producing kit |
| WO2016078339A1 (en) * | 2014-11-17 | 2016-05-26 | 中国科学院微生物研究所 | Apparatus, system, and method for generating micro liquid droplets and single-cell/single-molecule analysis apparatus |
| CN106076443B (en) * | 2016-06-12 | 2019-05-24 | 北京天天极因科技有限公司 | A kind of preparation method of micro channel array plate, the device and drop forming method for using it to obtain drop |
| CN105854965B (en) * | 2016-06-12 | 2019-04-12 | 北京天天极因科技有限公司 | A kind of oil phase composition generating Water-In-Oil drop for centrifugal process |
| CN110872550B (en) * | 2018-08-31 | 2022-10-04 | 北京致雨生物科技有限公司 | Method for generating liquid drops with uniform size and digital PCR detection method |
-
2021
- 2021-10-29 CN CN202111274923.2A patent/CN114058495A/en active Pending
- 2021-11-30 WO PCT/CN2021/134672 patent/WO2023070820A1/en active Application Filing
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3187252A1 (en) * | 2014-08-25 | 2017-07-05 | Universidad De Sevilla | Method and device for producing simple and compound micrometre-sized emulsions |
| CN104450891A (en) * | 2014-11-17 | 2015-03-25 | 中国科学院微生物研究所 | Method and system for digital quantitative analysis of nucleic acid amplification based on micro-droplet |
| CN107511189A (en) * | 2017-08-02 | 2017-12-26 | 肇庆市华师大光电产业研究院 | A kind of preparation method of the single dispersing microlayer model based on capillary |
| CN108159976A (en) * | 2018-01-03 | 2018-06-15 | 西南交通大学 | A kind of Water-In-Oil Bao Shui（W/W/O）Monodisperse double emulsion preparation method and its micro fluidic device |
| US20200101454A1 (en) * | 2018-10-01 | 2020-04-02 | Lifeng XIAO | Micro-pipette tip for forming micro-droplets |
| CN109908986A (en) * | 2019-02-21 | 2019-06-21 | 浙江大学 | A kind of drop formation system and application method based on the asymmetric capillary in outlet |
| CN112439470A (en) * | 2019-08-30 | 2021-03-05 | 北京达微生物科技有限公司 | Sample adding needle for preparing micro-droplets and preparation method of micro-droplets |
| CN110841734A (en) * | 2019-12-06 | 2020-02-28 | 中国科学院长春光学精密机械与物理研究所 | Digital PCR device and single-pump liquid drop generating system thereof |
| CN112522374A (en) * | 2020-10-28 | 2021-03-19 | 浙江大学 | Low-cost widely-adaptive centrifugal digital liquid drop generation method and device |
| CN216688147U (en) * | 2021-10-29 | 2022-06-07 | 浙江大学 | Industrial-grade asymmetric liquid drop generating device and digital nucleic acid amplification detection system |
Non-Patent Citations (1)
| Title |
|---|
| MASAMUNE MORITA等: "Droplet-Shooting and Size-Filtration (DSSF) Method for Synthesis of Cell-Sized Liposomes with Controlled Lipid Compositions", CHEMBIOCHEM, 17 August 2015 (2015-08-17) * |
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