阅读说明：本技术 低成本广适应离心式数字液滴发生方法及装置 (Low-cost widely-adaptive centrifugal digital liquid drop generation method and device ) 是由 何赛灵 程潇羽 张文耀 贺泽玮 纪佳丽 纪长 于 2020-10-28 设计创作，主要内容包括：本发明公开了低成本广适应离心式数字液滴发生方法及装置,将上层水相离心单元内嵌套在下层油相离心单元,上层水相离心单元的下部设有微米管,在离心力的作用下,上层水相克服表面张力作用通过微米管通道生成的液滴进入到下层油相中,形成尺寸均一的油包水液滴,通过调节微米管的直径和/或离心力的大小调节油包水液滴的尺寸。所述的油相中可添加有互溶激光诱导固化剂,诱导源的作用下使油相快速交联固化；应用于核酸等温扩增,实现数字化核酸扩增。本发明可生成大小均一、高质量油包水液滴。微升级的水相溶液在几分钟内即可快速产生高通量的油包水液滴,与核酸的等温扩增结合,同时可借助固化平台实现液滴的超快固化。(The invention discloses a low-cost widely-adaptive centrifugal digital liquid drop generation method and a device, wherein an upper-layer water phase centrifugal unit is nested in a lower-layer oil phase centrifugal unit, a micron tube is arranged at the lower part of the upper-layer water phase centrifugal unit, liquid drops generated by an upper-layer water phase overcoming the surface tension effect through a micron tube channel enter a lower-layer oil phase under the action of centrifugal force to form water-in-oil liquid drops with uniform size, and the size of the water-in-oil liquid drops is adjusted by adjusting the diameter of the micron tube and/or the size of the centrifugal force. The oil phase can be added with a mutual-soluble laser induction curing agent, and the oil phase is rapidly crosslinked and cured under the action of an induction source; the method is applied to nucleic acid isothermal amplification to realize digital nucleic acid amplification. The invention can generate high-quality water-in-oil droplets with uniform size. The micro-upgraded aqueous phase solution can quickly generate high-flux water-in-oil droplets within a few minutes, is combined with isothermal amplification of nucleic acid, and can realize ultra-fast solidification of the droplets by virtue of a solidification platform.)

1. A low-cost widely-adaptive centrifugal digital liquid drop generation method is characterized by comprising the following steps: the upper-layer water phase centrifugal unit is internally nested in the lower-layer oil phase centrifugal unit, the lower part of the upper-layer water phase centrifugal unit is provided with a micron tube, under the action of centrifugal force, the upper-layer water phase overcomes the action of surface tension and enters the lower-layer oil phase through liquid drops generated by the micron tube channel to form water-in-oil liquid drops with uniform size, and the size of the water-in-oil liquid drops is adjusted by adjusting the diameter of the micron tube and/or the size of the centrifugal force.

2. The method of claim 1, wherein: the oil phase is added with a mutual-soluble laser induction curing agent, and the oil phase is rapidly crosslinked and cured under the action of an induction source; the method is applied to nucleic acid isothermal amplification to realize digital nucleic acid amplification.

3. The method of claim 2, wherein: the water-in-oil liquid drop is tiled on a glass slide, the glass slide is placed on a three-dimensional stepping displacement platform, ultrafast laser emitted by a femtosecond laser is collimated through a lens group, a diffraction light spot or a pattern is emitted through an SLM projection module, the SLM projection module is positioned below the displacement platform and controlled by a controller, light beams are converged through an inverted microscope and focusing system to reach threshold energy for oil phase solidification and reduce the light spot, and finally the light beams are emitted to a specific position in an oil phase, the oil phase can be instantly solidified due to multiphoton absorption polymerization of a curing agent component, the three-dimensional stepping displacement platform scans and controls the position of a focusing point from a parallel direction and a vertical direction in sequence, the external oil phase of the liquid drop on the plane is scanned layer by layer, and the rapid.

4. An apparatus employing the method of claim 1, wherein:

the centrifugal device comprises an upper-layer water phase centrifugal unit and a lower-layer oil phase centrifugal unit, wherein the upper-layer water phase centrifugal unit is embedded in the lower-layer oil phase centrifugal unit, a micron tube is arranged at the lower part of the upper-layer water phase centrifugal unit, under the action of centrifugal force, liquid drops generated by an upper-layer water phase overcoming the action of surface tension through a micron tube channel enter the lower-layer oil phase to form water-in-oil liquid drops with uniform size, and the size of the water-in-oil liquid drops is adjusted by adjusting the diameter of the micron tube and/or the size of centrifugal.

5. The apparatus of claim 4, wherein: the upper-layer water phase centrifugal unit comprises an inner pipetting gun suction head and an outer pipetting gun suction head, and the inner pipetting gun suction head is assembled on the outer pipetting gun suction head through an O-shaped rubber ring; the suction head of the inner pipetting gun is obtained by refitting the suction head of the pipetting gun with the specification of 200 mu L: the lower part 1/3 part of the pipette tip with the specification of 200 mu L is cut flatly and then sealed by high molecular polymer, the center of the high molecular polymer is penetrated and fixed in the pipette tip by a micron tube to be used as the only downward channel of the upper water phase, and the diameter of the micron tube is 100-200 mu m; the external pipette tip is obtained by modifying a pipette tip with the specification of 1000 mu L: flatly cutting the lower part of a pipette tip with the specification of 1000 mu L, and then leveling the pipette tip with a micron tube of the assembled pipette tip; the lower oil phase centrifugal unit adopts a 1.5ml specification centrifugal tube.

6. The apparatus of claim 4, wherein: the micron tube adopts a sample application capillary tube.

7. The apparatus of claim 4, wherein: the high molecular polymer comprises one or more of PDMS glue and epoxy resin.

8. The apparatus of claim 4, wherein: the oil phase comprises one or more of long-chain grease and emulsifier.

9. The apparatus of claim 4, wherein: the oil phase is added with a mutual-soluble laser induction curing agent, and the oil phase is rapidly crosslinked and cured under the action of an induction source.

10. The apparatus of claim 4, wherein: the aqueous phase sample comprises one or more of nucleic acid, protein, cell and nanoparticle.

Technical Field

The invention belongs to the field of analysis and sensing, and mainly relates to a low-cost widely-adaptive centrifugal digital droplet generation method and device, which can be used for single-molecule nucleic acid detection and the like.

Background

In recent years, digital nucleic acid detection techniques have been used in fields such as biological detection, chemical sensing, and medical diagnosis. 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.

How efficiently to produce high throughput, uniform size, stable droplets will affect the accuracy and sensitivity of the final assay. Conventional microfluidic technology can produce monodisperse droplets but requires specialized instruments such as syringe pumps and custom microfabricated chips. Among droplet generating apparatuses studied in recent years, CN109536590A proposes a method for detecting a single cell gene using a microwell chip, but such a multilayer chip is complicated to manufacture and complicated in processing steps. CN110841734A proposes a single pump droplet generation system, however syringe pumps introduce a risk of mixed contamination.

In addition, the liquid drops in the continuous oil phase have the tendency of aggregation in thermodynamics, the expansion and contraction are more likely to occur in PCR thermal cycling, the high precision of the experiment is not facilitated, and the existing solution is to add a surfactant such as ABIL EM90 in the oil component and add an emulsifier in the liquid drops to resist the aggregation collision among the liquid drops so as to locally relieve the problem of the expansion of the liquid drops. The Murray project group in 2018 and 2020 respectively provides a scheme of thermal curing and ultraviolet curing, effectively relieves the problem of liquid drop expansion, but also has the problem of overlong curing time.

In summary, the presently disclosed droplet-type digital PCR for nucleic acid detection has the following problems:

1) the structure of the liquid drop generating device is generally complex and is not easy to popularize on a large scale;

2) pumps in microfluidic chips introduce a risk of external contamination;

3) the droplets may have problems of flowing, agglomerating, or curing for too long a time during heating or ultraviolet light irradiation.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a low-cost and widely adaptable centrifugal digital droplet generation method and apparatus.

A low-cost widely-adaptive centrifugal digital liquid drop generation method is characterized in that an upper-layer water phase centrifugal unit is embedded in a lower-layer oil phase centrifugal unit, a micron tube is arranged at the lower part of the upper-layer water phase centrifugal unit, liquid drops generated by the upper-layer water phase overcoming the surface tension effect and passing through the micron tube channel enter the lower-layer oil phase under the action of centrifugal force to form water-in-oil liquid drops with uniform size, and the size of the water-in-oil liquid drops is adjusted by adjusting the diameter of the micron tube and/or the size of the centrifugal force.

The oil phase is added with a mutual-soluble laser induction curing agent, and the oil phase is rapidly crosslinked and cured under the action of an induction source; the method is applied to nucleic acid isothermal amplification to realize digital nucleic acid amplification.

The water-in-oil liquid drop is tiled on a glass slide, the glass slide is placed on a three-dimensional stepping displacement platform, ultrafast laser emitted by a femtosecond laser is collimated through a lens group, a diffraction light spot or a pattern is emitted through an SLM projection module, the SLM projection module is positioned below the displacement platform and controlled by a controller, light beams are converged through an inverted microscope and focusing system to reach threshold energy for oil phase solidification and reduce the light spot, and finally the light beams are emitted to a specific position in an oil phase, the oil phase can be instantly solidified due to multiphoton absorption polymerization of a curing agent component, the three-dimensional stepping displacement platform scans and controls the position of a focusing point from a parallel direction and a vertical direction in sequence, the external oil phase of the liquid drop on the plane is scanned layer by layer, and the rapid.

The device adopting the method comprises an upper-layer water phase centrifugal unit and a lower-layer oil phase centrifugal unit, wherein the upper-layer water phase centrifugal unit is internally nested in the lower-layer oil phase centrifugal unit, a micron tube is arranged at the lower part of the upper-layer water phase centrifugal unit, under the action of centrifugal force, the upper-layer water phase overcomes the action of surface tension and enters the lower-layer oil phase through liquid drops generated by a micron tube channel to form water-in-oil liquid drops with uniform size, and the size of the water-in-oil liquid drops is adjusted by adjusting the diameter of the micron tube and/or the size of centrifugal force.

The upper-layer water phase centrifugal unit comprises an inner pipetting gun suction head and an outer pipetting gun suction head, and the inner pipetting gun suction head is assembled on the outer pipetting gun suction head through an O-shaped rubber ring; the suction head of the inner pipetting gun is obtained by refitting the suction head of the pipetting gun with the specification of 200 mu L: the lower part 1/3 part of the pipette tip with the specification of 200 mu L is cut flatly and then sealed by high molecular polymer, the center of the high molecular polymer is penetrated and fixed in the pipette tip by a micron tube to be used as the only downward channel of the upper water phase, and the diameter of the micron tube is 100-200 mu m; the external pipette tip is obtained by modifying a pipette tip with the specification of 1000 mu L: flatly cutting the lower part of a pipette tip with the specification of 1000 mu L, and then leveling the pipette tip with a micron tube of the assembled pipette tip; the lower oil phase centrifugal unit adopts a 1.5ml specification centrifugal tube.

The micron tube adopts a sample application capillary tube.

The high molecular polymer comprises one or more of PDMS glue and epoxy resin.

The oil phase comprises one or more of long-chain grease and emulsifier.

The oil phase is added with a mutual-soluble laser induction curing agent, and the oil phase is rapidly crosslinked and cured under the action of an induction source.

The aqueous phase sample comprises one or more of nucleic acid, protein, cell and nanoparticle.

The invention has the beneficial effects that:

the invention utilizes the common consumables of chemical laboratory such as pipette, sample application capillary, macromolecule polymer to finish the preparation of low-cost centrifugal droplet generator, can produce the liquid droplet effectively, compared with the liquid droplet generating method or apparatus using special apparatus such as microfluid channel, etc. at present, the invention is relatively easy and all manufacturing processes can be carried on the ordinary laboratory work bench.

In addition, ultra-fast curing of the droplets on the plane can be achieved: for example, in a droplet solidification platform based on ultrafast laser, under the action of strong pulse laser, some substances can simultaneously absorb two or more photons, so that one electron energy level generates transition to generate a two-photon effect. The specific soluble photoresist containing the photoinitiator with the multiphoton absorption characteristic is added into the oil component, and the effect of quickly curing liquid drops on a plane can be realized by matching the action of inducing and curing the oil phase by ultrafast laser.

The invention provides a simple preparation method of the liquid drop generating device, and provides a thought and a method for assisting the ultra-fast solidification of the liquid drop while checking the application of the liquid drop generating device.

(1) The invention can generate high-quality water-in-oil droplets with uniform size. The micro-upgraded aqueous phase solution can quickly generate high-flux water-in-oil droplets within a few minutes, is combined with isothermal amplification of nucleic acid, and can realize ultra-fast solidification of the droplets by virtue of a solidification platform.

(2) The invention can be used for packaging and distributing samples such as nucleic acid, cells, protein, nano particles and the like, and further completes detection application by combining isothermal amplification.

(3) The invention provides an idea method for ultrafast curing of liquid drops, for example, based on ultrafast laser multiphoton absorption polymerization curing, which realizes on-chip fixation of liquid drop samples, can reduce pollution, simultaneously prevent liquid drops from flowing, and is suitable for fluorescence imaging observation.

Drawings

Fig. 1 is a schematic structural diagram of a low-cost widely-adaptable centrifugal digital droplet generator according to the present invention.

FIG. 2 is a schematic diagram of an embodiment of the present invention in which droplets are generated by centrifugal force.

FIG. 3 shows microdroplets generated by the device of the present invention.

FIG. 4 is a graph showing the droplet size distribution of the apparatus of the present invention at a centrifugal force of 6000 RCF.

FIG. 5 is a diagram of the experiment of the present invention applied to isothermal amplification of nucleic acid of Staphylococcus aureus.

FIG. 6 is an ultrafast droplet solidification platform of the present invention.

Description of reference numerals: the device comprises an O-shaped rubber ring 1, an inner pipetting gun suction head 2, a high polymer 3, a microtube 4, an outer pipetting gun suction head 5, a 1.5ml specification centrifuge tube 6, a water phase sample 7, an oil phase 8, a water-in-oil droplet 9, a femtosecond laser 10, an optical lens group 11, an SLM projection module 12, a controller 13, a focusing lens group and inverted microscope system 14, a three-dimensional stepping displacement platform 15 and a glass slide 16.

Detailed Description

The invention is further elucidated with reference to the drawing.

Example 1

The invention discloses an implementation mode of assembling a centrifugal liquid drop generating device, which comprises 1 and 200 mu L of O-shaped rubber rings

The suction head 2 of the inner pipetting gun, a high molecular polymer (such as PDMS) 3, a microtube 4 (a sample application capillary), a suction head 5 of an outer pipetting gun with the volume of 1000 mu L, a centrifuge tube 6 with the volume of 1.5mL, and a water phase sample 7 (which can be a pre-amplified nucleic acid mixed solution, a cell suspension and the like) are stored in the suction head of the modified pipetting gun which encapsulates the PDMS and the capillary, the water phase sample is communicated with the capillary, and the size of the capillary is 100 and 200 mu m, under the size, the water phase sample cannot leak out due to the action of surface tension. The inner pipetting gun suction head 2 and the outer pipetting gun suction head 5 are assembled together through an O-shaped rubber ring, the combined device and a centrifuge tube are matched in size and clamped on a 1.5mL centrifuge tube 6, an oil phase 8 is filled in the centrifuge tube, and the oil phase component at the lower layer can be mineral oil, ester oil and the like and is used for generating water-in-oil droplets 9 at the next step.

As shown in FIG. 2, under the action of centrifugation, the water phase sample 7 has enough centrifugal force to overcome the surface tension from the capillary wall, and passes through the capillary and enters the lower oil phase 8 in the form of small droplets to form water-in-oil droplets 9 (micro-droplets), and the magnitude of the centrifugal force can be 2000 RCF-10000 RCF. Finally, the micro-droplets with uniform size are generated, as shown in FIG. 3, and the size range is adjustable between 40 and 100 μm. The sample of 10 μ L can generate 2 w-3 w micro-droplets, and the size distribution of the sampling experiment is shown in FIG. 4.

Example 2

The micro-droplets can be combined with nucleic acid isothermal amplification to realize digital nucleic acid amplification, and as shown in FIG. 5, the micro-droplet-based nucleic acid loop-mediated isothermal amplification application of Staphylococcus aureus is provided, in which the aqueous phase components include target nucleic acid, enzyme, and buffer solution (containing MgCl)₂And CaCl₂) The primers, dNTPs and calcein are used as a fluorescent indicator, a sample is distributed into liquid drops, then isothermal amplification is carried out, and after the isothermal amplification is finished, the calcein and Mn are contained in negative liquid drops²⁺Bind and fluoresce dimly; in the positive droplets, the reaction by-product pyrophosphate abstracts the Mn bound to calcein²⁺Binding so as to generate free calcein molecules and Mg²⁺Binding and emission of strong fluorescence is shown in figure 5. By counting the positive droplets and the total droplets, and according to the poisson distribution, the quantitative detection of the sample nucleic acid can be completed.

Example 3

Example 3 is one example of an implementation of a droplet ultrafast curing method, in which ultrafast laser assisted droplet curing may be used, and an embodiment of this example is described in further detail below with reference to fig. 6.

As shown in fig. 6, the water-in-oil droplet 9 is tiled on a slide 16, the slide is placed on a three-dimensional stepping displacement platform 15, ultrafast laser emitted by a femtosecond laser 10 is collimated by a lens group 11 and emits diffraction spots or patterns through an SLM 12, the SLM module is positioned below the displacement platform and controlled by a controller 13, light beams are converged by an inverted microscope and focusing system 14 to reach threshold energy for oil phase solidification and reduce the spots, and finally enter a specific position in an oil phase, the oil phase can be instantaneously solidified due to multiphoton absorption polymerization of a curing agent component, the three-dimensional stepping displacement platform 15 scans and controls the position of a focusing point from a parallel direction and a vertical direction in sequence, scans the external oil phase of the droplet on a plane layer by layer, and finally realizes rapid fixation of the droplet.

The embodiments in the above description can be further combined or replaced, and the embodiments are only described as preferred embodiments 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.