CN110724632A

CN110724632A - Digital PCR (polymerase chain reaction) liquid drop forming method

Info

Publication number: CN110724632A
Application number: CN201911065986.XA
Authority: CN
Inventors: 吴炫烨; 关一民
Original assignee: Shanghai Industrial Utechnology Research Institute
Current assignee: Shanghai Industrial Utechnology Research Institute
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2020-01-24

Abstract

The invention provides a digital PCR liquid drop forming method, which comprises the following steps: injecting a digital PCR solution into the PCR reagent chamber, and enabling the digital PCR solution to enter a liquid drop spray orifice communicated with the PCR reagent chamber to form a digital PCR solution liquid; adding liquid drop forming oil into a liquid drop forming cavity, wherein the liquid drop forming cavity is defined by a heat conducting plate, a cover plate and an inverted U-shaped step arranged on the side surface of the cover plate; vaporizing a portion of the digital PCR solution liquid with a vaporizing part and rapidly pushing the remaining digital PCR solution liquid into the droplet-forming oil in the droplet-forming chamber to form the digital PCR droplets. The invention uses the hot bubble technology to form high-speed digital PCR liquid drops, has high-efficiency digital PCR oil utilization rate, and can carry out in-situ PCR temperature control and in-situ digital PCR signal collection.

Description

Digital PCR (polymerase chain reaction) liquid drop forming method

Technical Field

The invention belongs to the field of biomedicine, particularly relates to the field of disease detection, and relates to an integrated in-situ digital PCR system and a liquid drop forming method.

Background

Polymerase Chain Reaction (PCR) has been proposed for 20 years, during which PCR has developed into a key technology and a conventional technology in the field of molecular biology, and has greatly promoted the development of various fields of life science. Particularly, in the later 90 s, real-time fluorescent quantitative PCR (qPCR) technology and related products proposed by ABI company in America developed PCR from in vitro synthesis and qualitative/semi-quantitative detection technology into a gene analysis technology with high sensitivity, high specificity and accurate quantification.

Although the qPCR technique has been used for diagnosis of all diseases except for trauma and nutritional deficiency through rapid development over a period of ten years, there are many factors affecting the amplification efficiency during PCR amplification, and it cannot be guaranteed that the amplification efficiency remains the same during reaction and that the amplification efficiency is the same between the actual sample and the standard sample as well as between different samples, thereby leading to the basis on which its quantitative analysis depends-the Cycle Threshold (CT) is not constant. Therefore, qPCR is only "relative quantitative", and the accuracy and reproducibility thereof still cannot meet the requirements of molecular biological quantitative analysis.

Vogelstein et al proposed the concept of digital PCR (digital PCR) by dividing a sample into tens to tens of thousands of portions, assigning them to different reaction units, each containing one or more copies of a target molecule (DNA template), performing PCR amplification of the target molecule in each reaction unit, and performing statistical analysis of the fluorescent signals of the reaction units after amplification is complete. Different from qPCR, digital PCR does not depend on CT value, so that the method is not influenced by amplification efficiency, the average concentration (content) of each reaction unit is calculated by direct counting or a Poisson distribution formula after amplification is finished, the error can be controlled within 5%, and absolute quantitative analysis can be realized by digital PCR without reference to a standard sample and a standard curve.

Digital PCR (also known as single molecule PCR) generally involves two parts, PCR amplification and fluorescence signal analysis. In the PCR amplification stage, unlike the conventional art, digital PCR generally requires that a sample be diluted to a single molecule level and equally distributed into several tens to several tens of thousands of units for reaction. Unlike the method of real-time fluorescence measurement for each cycle by qPCR, the digital PCR technique is to collect the fluorescence signal of each reaction unit after amplification is completed. And finally, calculating to obtain the original concentration or content of the sample through direct counting or a Poisson distribution formula.

Since digital PCR is an absolute nucleic acid molecule quantification technique, compared to qPCR, the number of DNA molecules can be directly counted, which is an absolute quantification of the starting sample, and thus it is particularly suitable for application fields that cannot be well resolved depending on CT values, such as copy number variation, mutation detection, gene relative expression studies (e.g., allele imbalance expression), second-generation sequencing result verification, miRNA expression analysis, single-cell gene expression analysis, and the like.

There are three major types of digital PCR technology currently on the market. One is to form droplets by cutting off the PCR solution of the aqueous phase with flowing oil in a specific instrument and then to perform PCR and detection in two other instruments; one is to distribute PCR solution on a hollowed silicon chip, and then carry out PCR in a specific instrument and carry out detection in another instrument; the last one is to inject the liquid into the chamber through a narrow channel on one instrument to form droplets, and to perform PCR, and then to perform detection in another instrument. However, there are limitations to the drop formation speed or throughput of the current three methods. In addition, the three techniques mentioned above, without exception, rely on multiple large instruments. This not only increases the cost of purchase of the instrument, but also limits the widespread use of digital PCR; but also increases the complexity of the experimental operation.

Therefore, how to provide a high-speed digital PCR droplet formation technology that forms more than 1000 droplets per second, an in-situ PCR technology that integrates droplet formation with a PCR temperature control and detection instrument, and an efficient method for digital PCR oil utilization rate becomes an important technical problem to be solved urgently by those skilled in the art.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention provides an integrated in-situ digital PCR system and a droplet formation method, which are used to solve the problems of slow droplet formation speed, low throughput, complex operation and low PCR oil utilization rate in the prior art.

To achieve the above and other related objects, the present invention provides a digital PCR system, comprising:

the liquid drop forming assembly comprises a heat conducting plate and a cover plate, wherein at least one inverted U-shaped step is arranged on one side surface of the cover plate, and the heat conducting plate, the cover plate and the inverted U-shaped step form a liquid drop forming cavity with an opening at the bottom through enclosing together;

and the liquid drop spray orifice component is connected below the liquid drop forming component and comprises a plurality of liquid drop spray orifices, the liquid drop spray orifices are opened from the upper surface of the liquid drop spray orifice component and extend towards the lower surface of the liquid drop spray orifice component but do not penetrate through the lower surface of the liquid drop spray orifice component, the liquid drop spray orifices are communicated with the liquid drop forming cavity, and a vaporization part is arranged in the liquid drop spray orifices and is used for vaporizing part of digital PCR solution liquid in the liquid drop spray orifices and rapidly pushing the residual digital PCR solution liquid into liquid drop forming oil in the liquid drop forming cavity so as to form digital PCR liquid drops.

Optionally, the droplet ejection orifice assembly comprises a thermal bubble print chip.

Optionally, the height of the inverted U-shaped step is less than 2 times the diameter of the digital PCR droplet to be formed, such that the resulting digital PCR droplet is tiled into a layered structure within the droplet-forming chamber.

Optionally, a side surface of the heat conducting plate facing the cover plate is provided with a boss arranged along the outer edge of the inverted U-shaped step.

Optionally, the heat conducting plate gradually extends outwards near the opening of the droplet forming chamber to form a slope, so as to enlarge the opening size of the droplet forming chamber.

Optionally, the droplet forming assembly further comprises at least one droplet forming oil injection hole passing through the thermally conductive plate in communication with the droplet forming chamber.

Optionally, the drop formation assembly further comprises at least one drop formation chamber vent hole passing through the thermally conductive plate in communication with the drop formation chamber.

Optionally, the vaporization member is disposed at a bottom surface or a sidewall of the droplet ejection orifice.

Optionally, the opening shape of the droplet ejection orifice includes any one of a circle, an ellipse, and a polygon.

Optionally, the vaporizing part comprises a heating part that partially vaporizes the digital PCR solution liquid by heating it.

Optionally, the heating component comprises at least one metal layer.

Optionally, the PCR system further includes at least one PCR reagent chamber for storing a digital PCR solution, a flow channel is disposed in the droplet ejection orifice assembly, and the droplet ejection orifice is communicated with the PCR reagent chamber through the flow channel.

Optionally, the flow channel includes at least one main flow channel and a plurality of branch flow channels connected to the main flow channel, and each of the droplet ejection holes is connected to one of the branch flow channels.

Optionally, the digital PCR system further includes a base, the PCR reagent chamber extends from the opening on the upper surface of the base toward the lower surface of the base but does not penetrate through the lower surface of the base, and the droplet ejection hole assembly is connected to the upper surface of the base and covers the opening of the PCR reagent chamber.

Optionally, the lower surface of the base is provided with at least one digital PCR solution injection hole, and the digital PCR solution injection hole is communicated with the PCR reagent chamber.

Optionally, the lower surface of the base is provided with at least one PCR reagent chamber vent hole, and the PCR reagent chamber vent hole is communicated with the PCR reagent chamber.

Optionally, the digital PCR system further includes a flexible printed circuit board, the flexible printed circuit board is connected above the base, a through hole for accommodating the droplet ejection orifice assembly is formed in the flexible printed circuit board, a plurality of first connection pads and a plurality of second connection pads are formed on the surface of the flexible printed circuit board, and the droplet ejection orifice assembly is connected to the first connection pads through wires.

Optionally, the flexible printed circuit board is connected to the base by gluing.

Optionally, the surface of the base is provided with at least one channel for preventing glue from flowing to the droplet ejection orifice assembly, and the channel is distributed on the periphery of the droplet ejection orifice assembly.

Optionally, at least two positioning through holes are formed in the flexible printed circuit board, and positioning protrusions corresponding to the positioning through holes are formed on the surface of the base.

Optionally, the digital PCR system further includes a controller, the controller includes a controller housing and a controller circuit board located in the controller housing, the controller housing has a carrying portion for placing the base, the surface of the carrying portion is provided with a plurality of circuit connection conductive pins connected to the controller circuit connection board, and the circuit connection conductive pins correspond to the second connection pads.

Optionally, one end of the base is provided with at least one limiting groove, and the controller housing is provided with at least one limiting piece corresponding to the limiting groove.

Optionally, the base is provided with a limiting through hole, the limiting through hole penetrates through the front surface and the back surface of the base, and the controller shell is provided with a limiting part corresponding to the limiting through hole.

Optionally, the controller further comprises a cover coupled to the controller housing for covering the base.

Optionally, the digital PCR system further comprises an external semiconductor refrigerator for heating or cooling the droplet formation chamber.

Optionally, the external semiconductor cooler has a fan.

Optionally, the digital PCR system further comprises an external temperature sensor for testing the temperature of the droplet formation chamber.

Optionally, the digital PCR system is further configured with an optical detection system for performing PCR signal collection detection without transferring the sample.

Optionally, the cover plate is made of a transparent material.

The invention also provides a digital PCR liquid drop forming method, which comprises the following steps:

injecting a digital PCR solution into the PCR reagent chamber, and enabling the digital PCR solution to enter a liquid drop spray orifice communicated with the PCR reagent chamber to form a digital PCR solution liquid;

adding liquid drop forming oil into a liquid drop forming cavity, wherein the liquid drop forming cavity is defined by a heat conducting plate, a cover plate and an inverted U-shaped step arranged on one side face of the cover plate;

vaporizing a portion of the digital PCR solution liquid with a vaporizing part and rapidly pushing the remaining digital PCR solution liquid into the droplet-forming oil in the droplet-forming chamber to form the digital PCR droplets.

Optionally, the forming speed of the digital PCR liquid drop is controlled by controlling the heating time, the heating times and the heating interval time of the heating component.

Optionally, the thickness of the digital PCR solution liquid is in a range of 0.2nm to 300000 nm.

Optionally, the thickness of the droplet-forming chamber is less than 2 times the diameter of the digital PCR droplet to be formed, such that the resulting digital PCR droplet is laid down in a layered structure within the droplet-forming chamber.

Optionally, after the digital PCR solution in the PCR reagent chamber is completely pushed into the droplet formation chamber to form a digital PCR droplet, the PCR reagent chamber is filled with droplet formation oil.

Optionally, the droplet forming chamber is warmed or cooled using an external semiconductor refrigerator.

Optionally, the digital PCR droplets are formed at a rate greater than 1000/sec.

As described above, the digital PCR system and the digital PCR droplet forming method according to the present invention have the following advantageous effects:

(1) the invention uses the hot bubble technology to form high-speed digital PCR liquid drops, the rapid formation of the liquid drops depends on the instant partial vaporization of the liquid with the nanometer thickness by the vaporization part in the liquid drop spray orifice, so that the residual digital PCR solution in the liquid drop spray orifice is rapidly pushed into the liquid drop forming oil to form the digital PCR liquid drops, and compared with the forming speed of 100 liquid drops per second on the market, the liquid drop forming technology in the invention can realize the forming speed of more than 1000 liquid drops per second.

(2) Compared with a method for generating liquid drops by the joint movement of the oil phase and the water phase, the oil phase in the technical scheme of the invention is static, so that the consumption of the oil phase is greatly reduced, and about 50 percent of the oil phase consumption is reduced.

(3) The external semiconductor refrigerator can be utilized to accurately control the temperature of the liquid drop forming cavity, so that the in-situ temperature control PCR is realized. And the integrated optical system can perform detection without transferring the sample. This both reduces the operating time and improves the accuracy of the detection by reducing human error.

(4) The in situ digital PCR droplets can be tiled into a layered structure.

Drawings

FIG. 1 is a schematic perspective view of a digital PCR system according to the present invention.

FIG. 2 shows a top view of the digital PCR system of the present invention.

Fig. 3 shows a bottom view of the digital PCR system of the present invention.

FIGS. 4-7 show side views of a digital PCR system of the present invention.

FIG. 8 is an exploded view of the digital PCR system of the present invention.

FIG. 9 is a schematic perspective view of a cover plate of the digital PCR system of the present invention.

Fig. 10 is a perspective view of a thermal conductive plate in the digital PCR system of the present invention.

FIG. 11 is a schematic perspective view of a droplet formation module of the digital PCR system of the present invention.

FIG. 12 shows a top view of a droplet formation assembly in a digital PCR system of the present invention.

FIG. 13 is a bottom view of a droplet forming assembly in a digital PCR system of the present invention.

FIGS. 14-17 show side views of a droplet formation assembly in a digital PCR system of the present invention.

FIG. 18 is a schematic front perspective view of a combination of a droplet ejection orifice assembly and a flexible printed circuit board in a digital PCR system according to the present invention.

FIG. 19 is a top view of the combination of a droplet ejection orifice assembly and a flexible printed circuit board in a digital PCR system according to the present invention.

FIG. 20 is a bottom view of the assembly of the droplet ejection orifice assembly and the flexible printed circuit board in the digital PCR system of the present invention.

FIGS. 21-24 are side views of the combination of a droplet ejection orifice assembly and a flexible circuit board in a digital PCR system of the present invention.

FIG. 25 is a schematic perspective view of a droplet ejection orifice assembly of the digital PCR system of the present invention.

FIG. 26 is a partial cross-sectional view of a droplet ejection orifice assembly in a digital PCR system of the present invention.

FIG. 27 is a front perspective view of a base of the digital PCR system of the present invention.

FIG. 28 shows a top view of a base in the digital PCR system of the present invention.

FIG. 29 is a bottom view of a base in the digital PCR system of the present invention.

FIGS. 30-33 show side views of a base in a digital PCR system of the present invention.

Fig. 34 is a schematic perspective view of a controller in a digital PCR system according to the present invention.

FIG. 35 is a top view of the controller shown in the digital PCR system of the present invention with the cover removed.

FIG. 36 shows a bottom view of the controller shown after the controller housing bottom plate has been removed in the digital PCR system of the present invention.

FIG. 37 is a schematic diagram of the external semiconductor refrigerator disposed in the controller housing of the digital PCR system according to the present invention.

FIG. 38 shows an optical microscope image of a digital PCR droplet formed using the digital PCR system of the present invention.

FIG. 39 shows a fluorescence plot of a digital PCR droplet formed using the digital PCR system of the present invention.

Description of the element reference numerals

1 droplet forming assembly

2 Heat-conducting plate

3 cover plate

4 inverted U-shaped step

5 droplet ejection orifice assembly

6 liquid drop nozzle

7 vaporization part

8 boss

9 slope

10 droplet forming oil injection orifice

11 droplet formation chamber vent

12 PCR reagent chamber

13 main flow passage

14 flow passages

15 base

16 digital PCR solution injection hole

17 PCR reagent chamber vent hole

18 flexible circuit board

19 through hole

20 second connection pad

21 channel

22 positioning perforation

23 positioning projection

24 controller

25 controller shell

26 bearing part

27 circuit connection conductive pin

28 limiting groove

29. 31 stopper

30 limit through hole

32 machine cover

33 external semiconductor refrigerator

34 Fan

35 formula platform that sinks

36 convex

37 circuit board connection point

38 casing supporting structure

39 air vent

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Please refer to fig. 1 to 39. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the present invention, and the components related to the present invention are only shown in the drawings rather than drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of the components in actual implementation may be changed freely, and the layout of the components may be more complicated.

Example one

Referring to fig. 1 to 7, fig. 1 is a schematic perspective view of the digital PCR system, fig. 2 is a top view of the digital PCR system, fig. 3 is a bottom view of the digital PCR system, and fig. 4, 5, 6, and 7 are side views of the digital PCR system in four directions.

Referring to fig. 8, which is an exploded schematic view of the digital PCR system, it can be seen that the digital PCR system includes a droplet forming assembly 1 and a droplet ejection orifice assembly 5, wherein the droplet ejection orifice assembly 5 is connected below the droplet forming assembly 1.

Specifically, the droplet forming assembly 1 includes a heat conducting plate 2 and a cover plate 3. Referring to fig. 9 and 10, fig. 9 is a schematic perspective view of the cover plate 3, and fig. 10 is a schematic perspective view of the heat conducting plate 2. As shown in fig. 9, at least one inverted U-shaped step 4 is provided on one side of the cover plate 3.

Referring to fig. 11 to 17, fig. 11 is a schematic perspective view of the droplet forming assembly 1 composed of the heat conducting plate 2 and the cover plate 3, fig. 12 is a top view of the droplet forming assembly 1, fig. 13 is a bottom view of the droplet forming assembly 1, and fig. 14, 15, 16 and 17 are side views of the droplet forming assembly 1 in four directions. As can be seen, the heat conducting plate 2, the cover plate 3 and the inverted U-shaped step 4 together enclose a droplet forming chamber with an opening at the bottom. In this embodiment, the number of the inverted U-shaped steps 4 is one, and correspondingly, the number of the droplet forming chambers is also one. In other embodiments, the number of the inverted U-shaped steps 4 may be plural to construct a plurality of droplet forming chambers.

Specifically, the height of the inverted U-shaped step 4 is less than 2 times the diameter of the digital PCR droplet to be formed, so that the obtained digital PCR droplet is spread into a layer structure in the droplet forming chamber.

As an example, as shown in fig. 9, the inverted U-shaped step 4 is formed by connecting three straight steps, in other embodiments, each section of the inverted U-shaped step 4 may also not be limited to a straight line, for example, the inverted U-shaped step 4 may be formed by combining a straight step and a curved step, and the protection scope of the present invention should not be limited too here.

As an example, the inverted U-shaped step 4 may be obtained by performing processes such as photolithography and etching on the cover plate 3, or may be a double-sided adhesive tape with an appropriate thickness. The inverted U-shaped step 4 and the outer edge of the cover plate 3 are arranged at a preset distance, if the cover plate 3 and the heat conducting plate 2 are in adhesive connection, the peripheral area of the inverted U-shaped step 4 can be used for dispensing glue.

Specifically, the material of the cover plate 3 includes, but is not limited to, any one of transparent or opaque plastic and glass, and the cover plate 3 may also be made of metal. In this embodiment, the cover plate 3 is preferably made of a transparent material.

As an example, as shown in fig. 10, a side surface of the heat conducting plate 2 facing the cover plate 3 is provided with a boss 8 arranged along the outer edge of the inverted U-shaped step 4, and the boss 8 plays a role in positioning the bonding of the cover plate 3. In this embodiment, the height of the boss 8 is equal to the thickness of the cover plate 3, and the absolute height of the boss 8 is not critical and can be adjusted as needed.

As an example, as shown in fig. 10, the heat conducting plate 2 gradually extends outward near the opening of the droplet forming chamber to form a slope 9 to enlarge the opening size of the droplet forming chamber. The slope 9 can reserve space for the ejected liquid drops, and prevent the liquid drops ejected at high speed from colliding and fusing with the formed liquid drops.

As an example, as shown in fig. 10, the droplet forming assembly further includes at least one droplet-forming oil injection hole 10, and the droplet-forming oil injection hole 10 penetrates the heat conductive plate 2 to communicate with the droplet-forming chamber. In this embodiment, the droplet-forming oil injection hole 10 is preferably provided at the slope 9.

As an example, as shown in fig. 10, the droplet forming assembly further includes at least one droplet forming chamber vent hole 11, the droplet forming chamber vent hole 11 penetrating the heat conductive plate 2 to communicate with the droplet forming chamber.

By way of example, the drop ejection orifice assembly 5 may comprise a thermal bubble print chip. Thermal bubble printing is a major technique in the field of printers, and its basic principle is to eject ink droplets by heating. In the present invention, the droplet ejection orifice assembly 5 may be an existing thermal bubble printing chip.

In this embodiment, the droplet ejection orifice assembly 5 is connected to a flexible printed circuit 18. Referring to fig. 18 to 24, fig. 18 is a schematic front perspective view of the combination of the droplet ejection orifice assembly 5 and the flexible printed circuit 18, fig. 19 is a top view of the combination of the droplet ejection orifice assembly 5 and the flexible printed circuit 18, fig. 20 is a bottom view of the combination of the droplet ejection orifice assembly 5 and the flexible printed circuit 18, and fig. 21, 22, 23 and 24 are side views of the combination of the droplet ejection orifice assembly 5 and the flexible printed circuit 18 in four directions.

Specifically, the flexible printed circuit 18 is provided with a through hole 19 for accommodating the droplet ejection orifice assembly 5, the surface of the flexible printed circuit 18 is provided with a plurality of first connection pads (not shown) and a plurality of second connection pads 20, and the droplet ejection orifice assembly 5 is connected to the first connection pads through wires, so that the droplet ejection orifice assembly 5 is connected to an external controller through the flexible printed circuit 18. The droplet ejection orifice assembly 5 may be connected to the first connection pad through a standard Wire Bond (Wire Bond) process.

Referring to fig. 25 and 26, fig. 25 is a schematic perspective view of the droplet ejection orifice assembly 5, and fig. 26 is a partial cross-sectional view of the droplet ejection orifice assembly 5. As shown in fig. 25, the droplet ejection orifice assembly 5 includes a plurality of droplet ejection orifices 6, the droplet ejection orifices 6 communicating with the droplet forming chamber. In this embodiment, the droplet ejection holes 6 are arranged in two rows, and the droplet ejection holes in each row are uniformly distributed. In other embodiments, the drop ejection openings 6 may be arranged in other ways, and the scope of the present invention should not be limited to this. As shown in fig. 26, the droplet ejection orifice 6 opens from the upper surface of the droplet ejection orifice assembly 5, extends in the direction of the lower surface of the droplet ejection orifice assembly 5, and does not penetrate the lower surface of the droplet ejection orifice assembly 5. The opening shape of the droplet ejection orifice 5 includes, but is not limited to, any one of a circle, an ellipse, and a polygon, as an example.

Specifically, as shown in fig. 26, a vaporizing part 7 is provided in the droplet ejection hole 6, and is configured to vaporize a part of the digital PCR solution liquid in the droplet ejection hole 6 and rapidly push the rest of the digital PCR solution liquid into the droplet formation oil in the droplet formation chamber to form a digital PCR droplet. Wherein the volume of the drop ejection orifice 6 determines the volume of the digital PCR drop to be formed.

As an example, the vaporizing part 7 is disposed at a bottom surface of the droplet ejection hole 6, and the vaporizing part 7 may employ a heating part that partially vaporizes the digital PCR solution liquid by heating it. In this embodiment, the heating component includes a heating sheet, and the heating sheet may be a single metal layer or a composite multilayer metal layer. The vaporizing part 7 has a shape including, but not limited to, a circle or a square, and an area of 0.5 to 2 times of an area of a bottom of the droplet ejection hole 6. In other embodiments, the vaporization part 7 may be disposed on the sidewall of the droplet ejection orifice 6, and the scope of the present invention should not be limited too much.

As shown in fig. 8, the PCR system further includes at least one PCR reagent chamber 12 for storing digital PCR solutions. As shown in fig. 25, a flow channel is provided in the droplet ejection hole assembly 5, and the droplet ejection hole 6 communicates with the PCR reagent chamber 12 through the flow channel.

As an example, the flow channel includes at least one main flow channel 13 and a plurality of branch flow channels 14 connected to the main flow channel, and each of the droplet ejection orifices 6 is connected to one of the branch flow channels 14. In fig. 20 and 25, the droplet ejection orifice assembly 5 includes one main flow channel 13, and in other embodiments, when the number of the droplet forming chambers is plural, the number of the main flow channels 13 may be plural accordingly.

By way of example, the material from which the flow channel and the drop ejection orifice 6 are constructed includes, but is not limited to, silicon, polymer, photoresist, and the like.

Specifically, as shown in fig. 8, the digital PCR system further includes a base 15, and the PCR reagent chamber 12 is disposed in the base 15. By way of example, the base 15 may be made of any one of transparent or opaque plastic and glass, and the base 15 may also be made of metal.

Referring to fig. 27 to 33, fig. 27 is a schematic front perspective view of the base, fig. 28 is a top view of the base, fig. 29 is a bottom view of the base, and fig. 30, 31, 32 and 33 are side views of the base in four directions.

Specifically, the PCR reagent chamber 12 is opened from the upper surface of the base 15, extends toward the lower surface of the base 15, but does not penetrate the lower surface of the base 15, and the droplet ejection orifice assembly 5 is connected to the upper surface of the base 15 and covers the opening of the PCR reagent chamber 12.

Specifically, the lower surface of the base 15 is provided with at least one digital PCR solution injection hole 16, and the digital PCR solution injection hole 16 is communicated with the PCR reagent chamber. The lower surface of the base 15 is provided with at least one PCR reagent chamber vent hole 17, and the PCR reagent chamber vent hole 17 is communicated with the PCR reagent chamber.

Specifically, the flexible printed circuit 18 is connected above the base 15. The flexible printed circuit 18 is connected to the base 15 by gluing, for example. As shown in fig. 27 and 28, in this embodiment, the surface of the base 15 is further provided with at least one channel 21 for preventing glue from flowing to the droplet ejection orifice assembly 5, and the channel 21 is distributed on the periphery of the droplet ejection orifice assembly 5.

In this embodiment, the surface of the base 15 has a sunken platform 35 for accommodating the flexible printed circuit board, four corners of the sunken platform 35 have arc-shaped extending spaces, and the protrusions 36 around the sunken platform 35 play a role in positioning when the flexible printed circuit board is adhered to the surface of the sunken platform 35.

As shown in fig. 19, at least two positioning through holes 22 are formed in the flexible printed circuit 18, and as shown in fig. 28, a positioning protrusion 23 corresponding to the positioning through hole 22 is formed on the surface of the base 15.

Specifically, the digital PCR system further includes a controller 24, please refer to fig. 34, which is a schematic perspective view of the controller 24, and the controller 24 includes a controller housing 25 and a controller circuit board (not shown) located in the controller housing 25. In this embodiment, the controller 24 further comprises a cover 32, and the cover 32 is connected to the controller housing 25 and is used for covering the base 15 and providing a light-shielding environment for the PCR reaction.

Referring to fig. 35, which is a top view of the controller after the cover 32 is removed, it can be seen that the controller housing 25 has a carrying portion 26 for placing the base, a plurality of conductive pins 27 (also referred to as pins) for circuit connection are disposed on the surface of the carrying portion 26, and the conductive pins 27 for circuit connection are connected to the circuit connection board of the controller, and the positions of the conductive pins 27 for circuit connection correspond to the positions of the second connection pads 20.

Referring to fig. 36, a bottom view of the controller in the digital PCR system is shown after the bottom plate of the controller housing is removed, wherein a plurality of circuit board connection points 37 corresponding to the circuit connection conductive pins 27 are disposed on the back surface of the supporting portion 26, and the controller circuit board can output signals to the circuit connection conductive pins 27 through the circuit board connection points 37.

Specifically, as shown in fig. 27, one end of the base 15 is provided with at least one limiting groove 28, and as shown in fig. 35, the controller housing 25 is provided with at least one limiting member 29 corresponding to the limiting groove 28. The stop 29 may be a spring plunger.

Specifically, as shown in fig. 27, the base 15 is provided with a limiting through hole 30, the limiting through hole 30 penetrates through the front surface and the back surface of the base 15, and as shown in fig. 35, the controller housing 25 is provided with a limiting member 31 corresponding to the limiting through hole 30. The stopper 31 may be a spring plunger.

Specifically, the digital PCR system further comprises an external semiconductor refrigerator for heating or cooling the droplet formation chamber to provide reaction conditions of a specific temperature. Semiconductor refrigerators (Thermo Electric Cooler, TEC for short) are made using the peltier effect of semiconductor materials. The peltier effect is a phenomenon in which when a direct current passes through a couple composed of two semiconductor materials, one end absorbs heat and the other end releases heat. The heavily doped N-type and P-type bismuth telluride are mainly used as semiconductor materials of TEC, and the bismuth telluride elements are electrically connected in series and generate heat in parallel. The TEC comprises a number of P-type and N-type pairs (sets) connected together by electrodes and sandwiched between two ceramic electrodes; when current flows through the TEC, heat generated by the current is transferred from one side of the TEC to the other side of the TEC, and a "hot" side and a "cold" side are generated on the TEC, which is the heating and cooling principle of the TEC.

By way of example, referring to fig. 37, the external semiconductor cooler 33 is disposed in the controller housing 25 and on the side of the thermal plate 2 of the drop forming assembly 1. In this embodiment, the external semiconductor refrigerator further has a fan 34, wherein the fan 34 is used for cooling the "hot" side when the external semiconductor refrigerator is cooling, and is used for heating the "cold" side when the external semiconductor refrigerator is heating. As shown in fig. 37, a vent 34 is further provided beside the fan 34. Also shown in fig. 37 is the housing support structure 16.

Specifically, the digital PCR system further comprises an external temperature sensor for testing the temperature of the liquid drop formation chamber. As an example, the external temperature sensor is disposed on a surface of the external semiconductor cooler in contact with the heat conducting plate.

Further, the digital PCR system is also provided with an optical detection system for PCR signal collection detection without sample transfer. The main parts of the optical system include: the device comprises a fluorescent light source, a bright field light source, a control circuit, an optical amplification lens group, a fluorescent filter, a CCD camera, a sliding table system for moving a lens and a shell for avoiding light. The photographing area of the optical system is the entire area of the cover plate. Such taking may be one imaging or taking and stitching pictures multiple times.

The digital PCR system can be used for forming the digital PCR liquid drops, the rapid formation of the liquid drops depends on the instant partial vaporization of the liquid with the nanometer-scale thickness by the vaporization component in the liquid drop spray orifice, so that the digital PCR solution in the liquid drop spray orifice is rapidly pushed into the liquid drop forming oil to form the digital PCR liquid drops, and compared with the forming speed of 100 liquid drops per second on the market, the liquid drop forming technology can realize the forming speed of more than 1000 liquid drops per second. Compared with a method for generating liquid drops by the joint movement of the oil phase and the water phase, the oil phase in the technical scheme of the invention is static, so that the consumption of the oil phase is greatly reduced, and about 50 percent of the oil phase consumption is reduced. The external semiconductor refrigerator can be utilized to accurately control the temperature of the liquid drop forming cavity, so that the in-situ temperature control PCR is realized. And the integrated optical system can perform detection without transferring the sample. This both reduces the operating time and improves the accuracy of the detection by reducing human error. The in situ digital PCR droplets can be tiled into a layered structure.

Example two

The invention also provides a digital PCR liquid drop forming method, which comprises the following steps: a vaporizing part is used to vaporize a portion of the digital PCR solution liquid and to rapidly push the remaining digital PCR solution liquid into the droplet forming oil to form digital PCR droplets.

As an example, high-speed digital PCR droplet formation is performed using a thermal bubble technique, and the vaporizing part includes a heating part that partially vaporizes the digital PCR solution liquid by heating it.

Specifically, the forming speed of the digital PCR liquid drops is controlled by controlling the heating time, the heating times and the heating interval time of the heating parts. The digital PCR droplet forming method can achieve the digital PCR droplet forming speed of more than 1000/s.

As an example, the digital PCR droplet formation method comprises the steps of:

s1: injecting a digital PCR solution into the PCR reagent chamber, and enabling the digital PCR solution to enter a liquid drop spray orifice communicated with the PCR reagent chamber to form a digital PCR solution liquid;

s2: adding liquid drop forming oil into a liquid drop forming cavity, wherein the liquid drop forming cavity is defined by a heat conducting plate, a cover plate and an inverted U-shaped step arranged on one side face of the cover plate;

s3: vaporizing a portion of the digital PCR solution liquid with a vaporizing member and rapidly pushing into the droplet-forming oil in the droplet-forming chamber to form the digital PCR droplets.

Specifically, the thickness of the digital PCR solution liquid is nano-scale and greater than 0.2nm, and in this embodiment, the thickness of the digital PCR solution liquid is preferably in a range of 0.2nm to 300000 nm.

Specifically, the thickness of the droplet formation chamber is less than 2 times the diameter of the digital PCR droplet to be formed, so that the resulting digital PCR droplet is laid down in a layered structure within the droplet formation chamber.

Specifically, after the droplet forming oil is added to the droplet forming chamber, and before the droplet ejection orifice assembly (e.g., a thermal bubble printing chip) is placed in the controller, the droplet forming oil injection hole formed in the droplet forming chamber wall is sealed by a sealing member such as a rubber plug or a sealing film.

Specifically, after the digital PCR solution in the PCR reagent chamber is completely pushed into the droplet forming chamber to form the digital PCR droplets, the PCR reagent chamber is filled with droplet forming oil, so that the PCR reagent chamber is in a filling state, and the formed droplets are prevented from flowing back to the PCR reagent chamber. The droplet-forming chamber vent hole disposed on the droplet-forming chamber wall, the digital PCR solution injection hole disposed on the PCR reagent chamber wall, and the PCR reagent chamber vent hole may then be sealed with a seal. The sealing member includes, but is not limited to, a rubber plug, a sealing film, a rubber ring, a sealing cushion, etc. The sealing element can be made of soft plastics such as rubber, PDMS and the like.

Specifically, after the sealing, the liquid drop forming chamber is heated or cooled by an external semiconductor refrigerator, and the liquid drop forming chamber is controlled at a temperature required for performing PCR, so that in-situ temperature control PCR is realized.

In particular, an integrated optical system may also be used to allow PCR signal collection detection without sample transfer.

Referring to FIG. 38, which is an optical microscope photograph of the digital PCR droplets formed by the digital PCR system of the present invention, it can be seen that the formed digital PCR droplets are symmetrical and uniform in shape.

After the droplets were formed by using standard digital PCR, positive droplets with fluorescent signals were visible after 40 cycles by in situ conventional PCR temperature control reaction. FIG. 39 is a fluorescence image of a digital PCR droplet formed using the digital PCR system of the present invention.

The digital PCR system and the digital PCR liquid drop forming method can meet the use requirements of all digital PCR biochemical reagents. Because the concentration of a plurality of biomarker molecules in blood is very low (for example, the circulating tumor DNA has only 3 DNA molecules in every 2ml of blood), the digital PCR system and the digital PCR drop forming method have the characteristics that the drop forming quantity is not limited by the oil quantity and the high speed is realized, so that the detection is possible to be applied to the digital PCR.

In summary, the digital PCR system and the digital PCR droplet forming method of the present invention use the thermal bubble technique to perform high-speed digital PCR droplet formation, and the rapid formation of the droplet depends on the instant partial vaporization of the nano-sized liquid by the vaporization component in the droplet ejection hole, so as to rapidly push the remaining digital PCR solution in the droplet ejection hole into the droplet formation oil to form the digital PCR droplet, compared with the formation speed of 100 droplets per second on the market, the droplet formation speed of more than 1000 droplets per second can be achieved by the droplet formation technique of the present invention. Compared with a method for generating liquid drops by the joint movement of the oil phase and the water phase, the oil phase in the technical scheme of the invention is static, so that the consumption of the oil phase is greatly reduced, and about 50 percent of the oil phase consumption is reduced. The external semiconductor refrigerator can be utilized to accurately control the temperature of the liquid drop forming cavity, so that the in-situ temperature control PCR is realized. And the integrated optical system can perform detection without transferring the sample. This both reduces the operating time and improves the accuracy of the detection by reducing human error. The in situ digital PCR droplets can be tiled into a layered structure. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A digital PCR droplet formation method, comprising the steps of:

2. The digital PCR droplet formation method of claim 1, wherein: the vaporizing part includes a heating part that partially vaporizes the digital PCR solution liquid by heating it.

3. The digital PCR droplet forming method of claim 2, wherein: and controlling the forming speed of the digital PCR liquid drop by controlling the heating time, the heating times and the heating interval time of the heating part.

4. The digital PCR droplet formation method of claim 1, wherein: the thickness range of the digital PCR solution liquid is 0.2 nm-300000 nm.

5. The digital PCR droplet formation method of claim 1, wherein: the thickness of the droplet-forming chamber is less than 2 times the diameter of the digital PCR droplet to be formed, such that the resulting digital PCR droplet lays down into a layered structure within the droplet-forming chamber.

6. The digital PCR droplet formation method of claim 1, wherein: and filling the PCR reagent chamber with droplet forming oil after the digital PCR solution in the PCR reagent chamber is completely pushed into the droplet forming chamber to form digital PCR droplets.

7. The digital PCR droplet formation method of claim 1, wherein: and heating or cooling the liquid drop forming cavity by using an external semiconductor refrigerator.

8. The digital PCR droplet formation method of claim 1, wherein: the formation rate of the digital PCR droplets is greater than 1000/sec.