CN115747318A - Portable liquid drop PCR device, method and application thereof - Google Patents

Abstract

The invention discloses a portable droplet PCR method, device and application thereof, including a sandwich chamber, a droplet storage and temperature control area disposed in the sandwich chamber, and discloses its control mode; the sandwich chamber includes parallel first A flat plate and a second flat plate, a first support member and a second support member for supporting the sides of the first flat plate and the second flat plate, a photothermal layer is provided in the droplet temperature control area, and the black photothermal layer is pasted on the The water-in-oil droplets on the first flat plate are loaded and stored in the photothermal interlayer, and the photothermal interlayer is periodically and rapidly heated and rapidly cooled by the program-controlled infrared light frequency to realize the rapid cycle amplification and detection of the spreading droplets; further After the cycle is over, the droplet classification area of the gradient sandwich plate assembly with stepped slit channels can meet the droplet uniformity requirements of digital quantification and other applications. The invention transforms the traditional three-dimensional sample heating into two-dimensional plane heating without complex design of the chip, completely gets rid of the complex temperature change and expensive equipment control limitations of traditional PCR, and effectively solves the miniaturization of digital ultra-fast droplet PCR , Portability issues.

Description

A portable droplet PCR device, method and application thereof

technical field

The invention relates to the field of biochemical detection, in particular to a portable droplet PCR device, method and application thereof.

Background technique

Efficient control of temperature ramping programs is the key to many reaction processes. For example, polymerase chain reaction (PCR) is a rapid amplification technology of DNA, and it is a necessary link in molecular diagnosis such as real-time fluorescence quantitative PCR, gene sequencing and gene chip. A complete PCR temperature control process can be: 1) heating the sample to 90-96°C for denaturation; cooling the sample to 55°C-60°C for annealing; 3) heating the sample to 70°C-75°C for extension; 4) Repeat steps 1 to 3 25 to 40 times. It can be seen that the PCR temperature control process is a relatively complicated process, which requires repeated switching of temperature zones.

The traditional PCR reaction is carried out in a small test tube, and the temperature control speed of the reaction process is slow, which generally takes 80 minutes to 2 hours to complete. The time required to complete the PCR reaction on the PCR instrument depends on the following factors: 1. Module heating and cooling time 2. Temperature equilibrium time between the module and the PCR tube 3. Extension time 4. How to improve the heating and cooling process and reduce PCR The equilibration time of the solution in the tube has always been the core issue of ultrafast PCR.

Microfluidic chips have the characteristics of small size, large specific surface area, high integration, fast reaction speed, and fast heat transfer, and have been widely used. According to the difference of the chip sample chamber, PCR can be divided into static chamber PCR and dynamic continuous flow PCR. Static chamber PCR is the miniaturization of traditional PCR. The reaction mixture is fixed in the micro-reaction tank, and the temperature of the temperature zone itself is changed through temperature control devices such as Peltier and PI film heaters to reach the set temperature. The temperature control target is used for temperature control. Because the temperature change of the temperature zone itself depends on the temperature rise and fall speed, it can generally only reach 2-5°C/s, and its temperature is continuously repeated; or use a fluid with a suitable temperature to directly enter the temperature control. Replacing the original fluid in the cavity is to achieve rapid temperature control. However, it takes a certain amount of time (usually a few seconds) to replace the original fluid through the fluid at a suitable temperature into the temperature-controlled cavity, and it is difficult to guarantee the uniformity and efficiency of fluid replacement, which can easily lead to time-consuming growth or decrease in temperature control accuracy.

Dynamic continuous flow PCR uses microfluidic technology to flow the PCR reaction solution between different temperature zones. This method does not have the process of heating and cooling, which can reduce the time for digestion. However, this solution also has some defects: the sample is constantly flowing in the chip, so the control of variables such as reaction time is mainly realized by designing the structure of the microchannel on the chip. Usually the structure is relatively more complicated and requires a relatively large space. And the corresponding driving mechanism for driving the continuous flow of the sample in the chip will also be more complicated; under the pressure driving condition, the velocity distribution of the cross-section of the microchannel is a parabola or similar shape, the middle velocity is the largest and the velocity on both sides is the smallest near the wall ( close to 0), so PCR samples at different positions in the cross-section will experience different reaction times. More importantly, it is difficult to realize the quantitative analysis of the process for the flowing liquid, and the quantification is difficult. The current methods, whether static or dynamic, often have a heating module, temperature control, and chip design that are very complicated, difficult to control, and high in cost.

To achieve rapid heating and cooling, there are still some methods that use the photothermal effect for rapid surface heating. It has been reported that infrared rays act on the surface of nanoparticles or gold nano-films to rapidly heat up the surface of the object to 10-100°C per second. However, due to the large reaction volume (hundreds of nanoliters to tens of microliters), the exothermic surface heating reaction system will transfer heat slowly, and in practical applications there will be problems such as low reaction efficiency and insufficient products, which are difficult to determine. Its Ct value is difficult to carry out quantitative analysis. Microfluidic water-in-oil microdroplets have the advantages of small size, high flux, internal stability, and absolute quantification, and are widely used in biological and chemical detection and analysis, especially in nucleic acid and biological large-scale applications such as digital PCR and single-molecule non-amplification. Molecular absolute quantitative POCT detection. Dispersed small droplets (pL~nL) are smaller in volume and faster in heat transfer than traditional reaction methods, and may be more suitable for improving the heating and cooling process. However, the traditional droplet generation is based on microfluidic chips. This kind of droplet generation requires a precision syringe pump to strictly control the flow rate of the oil phase and water phase fluid in the channel to generate droplets. Its operation requires precise control and is expensive. Based on the unavoidable shortcomings of complex structure or difficult manipulation of the existing droplet generation methods, whether the driving force is based on micro-oscillation or centrifugal force, it severely restricts the need for simplicity and portability in droplet POCT detection. In short, there is currently no coupled droplet technology that can achieve efficient and portable heating and cooling, and a PCR reaction system that can achieve absolute quantitative characteristics.

Contents of the invention

In view of this, the present application provides a portable droplet PCR device, method and application thereof, without the need for complex design of the chip, completely getting rid of the complicated temperature change program and expensive equipment control restrictions of traditional PCR, and effectively solving the problem of high-throughput ultra-fast Miniaturization and portability of droplet PCR.

In order to achieve the above-mentioned technical purpose, the application adopts the following technical solutions:

In a first aspect, the present application provides a portable droplet digital PCR control method, comprising the following steps:

S1. Use the droplet dispersing component to disperse the reaction solution into water-in-oil droplets, transfer and spread them to the surface of the photothermal layer in the droplet temperature control zone in the portable droplet digital PCR device;

S2. Periodically perform infrared irradiation heating and air-cooling cooling operations on the water-in-oil droplets in the droplet temperature control zone, so that the water-in-oil droplets can be cyclically amplified, that is, the target droplets are obtained;

S3. Squeeze the air into the first plate and the second plate corresponding to the droplet temperature control zone, or raise the top of the first plate corresponding to the droplet temperature control zone to form an angle gradient to drive the target liquid The droplet is transferred to the droplet grading area, and the target droplet is automatically assembled in a gradient dispersion arrangement, and is to be tested and observed.

Preferably, in step S3, the test observation is detected by direct signal detection, fluorescence microscope or photodiode.

Preferably, in step S3, the time and number of repetitions of the infrared irradiation and air-cooling cooling operations in step S2 are based on the PCR reaction program.

Preferably, in step S3, it also includes loading the plurality of interlayer cavities into a centrifugal turntable for high-throughput transfer of reaction droplets into a classification area for observation and quantification.

Preferably, the number of loaded interlayer cavities is greater than or equal to 1, and the loading angle is 0-70°.

In the second aspect, the present application provides a droplet digital PCR device for portable droplet digital PCR control, including a sandwich chamber, a droplet temperature control area and a droplet classification area arranged in the sandwich chamber, for The droplet dispersing component that transfers the dispersed droplets to the droplet temperature control zone, the droplet temperature control zone is connected to the droplet classification zone; the interlayer cavity includes a first flat plate and a second flat plate in parallel . A first support and a second support for supporting the first flat side and the second flat side, the first flat, the first support, the second flat, and the second support connected end to end in sequence , the droplet temperature control area is provided with a photothermal layer, the photothermal layer is attached to the first plate, and the droplet classification area is provided with a gradient sandwich plate assembly with a stepped slit channel, the said The slit channel width of the gradient sandwich plate assembly decreases from close to the droplet temperature control zone to away from the droplet temperature control zone.

Preferably, the photothermal layer includes a photothermal layer and a thermal conduction layer covering the heat-generating layer.

Preferably, the gradient sandwich panel assembly includes a flat sheet, a bent sheet, a first limiting member, and a second limiting member, one end of the flat sheet is attached to one end of the bent sheet, and the The first limiting member is used to fix the other end of the flat plate and the other end of the obtuse angle bending piece, and the second limiting member is used to fix the middle part of the flat plate and the obtuse angle bending piece.

Preferably, a collection container is provided at the end of the gradient sandwich panel assembly.

Preferably, the photothermal layer is not in contact with either the first support or the second support.

The beneficial effects of the application are as follows:

1. Disperse the reaction solution into high-flux droplets, and spread them on a two-dimensional plane, which greatly improves the efficiency of heat transfer, and at the same time gets rid of the limitation of the solution volume, and can meet the requirements of ul~ml level Droplet making;

2. Heating by black photothermal film, the heating method is low cost, easy to produce, low power, and high heating efficiency;

3. Good heating stability; once the film material and heating power used are determined, the temperature control of the reaction process is stable;

4. It can be mass-produced, with simple structure, convenient operation and low cost; in contrast, microfluidic chips require MEMS process production, bonding, packaging, complex process and high cost;

5. The detection chip has independent components, flexible assembly, and portability. This method can be applied to the detection of a single PCR sample, and multiple reaction positions can also be constructed on the disk. Through the action of centrifugal force, the transfer, distribution and finalization of the sample can be completed. The collection of products has a compact structure, low cost, and wide adaptability, which is very suitable for POCT detection applications;

6. Absolute quantification: through gradient sandwich plate components, high-throughput droplets can be quickly and large-scale hierarchically assembled, which greatly overcomes the problem of relatively large variation in the size of the generated droplets, and can arrange droplets in a single layer The cloth is relatively uniform, which can meet the requirements of numerical quantification such as biochemical detection.

Instructions attached

Fig. 1 is a structural side view of a portable droplet PCR device;

Fig. 2 is the top view of the structure of the portable droplet PCR device;

Fig. 3 is a schematic diagram of the assembly of the portable droplet PCR device and the centrifugal turntable.

In the figure: 1. In the interlayer cavity; 2. Droplet temperature control area; 3. Droplet classification area; 11. First flat plate; 12. Second flat plate; 13. First support member; 14. Second support member; 21. Photothermal layer; 31. Gradient interlayer plate assembly; 311. Flat sheet; 312. Bending sheet; 313. First stopper; 314. Second stopper.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The present invention aims at the problems of low heating and cooling efficiency in the existing traditional PCR reaction process, the liquid droplet method based on the microfluidic chip is complex in structure, difficult to be portable, the heating principle is complicated to control, and the production cost is high. The ultra-fast PCR device and method for portable droplets, by efficiently dispersing the reaction solution into high-throughput water-in-oil droplets, and then transferring them to the droplet temperature control zone 2 in the interlayer chamber 1 with a photothermal layer 21 attached inside, Through infrared light irradiation, the photothermal layer 21 quickly generates heat after absorbing infrared rays and quickly transfers it to the high-flux droplets to realize efficient heating of the droplets. After reaching the preset temperature, stop the infrared irradiation and start the fan system to share The opened droplet cools down quickly, and the cycle is repeated to achieve ultra-fast temperature control, which is used for temperature-controlled reactions such as PCR; finally, the high-throughput droplet is transferred to the gradient sandwich plate assembly 31, and the droplet self-assembles in the sandwich plate. Cloth, can be used for observation and quantitative detection, the device does not need complex design of the chip, completely get rid of the traditional PCR complex temperature change program and expensive equipment control restrictions, effectively solve the miniaturization and portability of high-throughput ultra-fast droplet PCR problem.

Technical scheme of the present invention is as follows:

As shown in Figure 3, the application provides a portable droplet PCR method, comprising the following steps:

S1. After the reaction solution is efficiently dispersed into high-throughput water-in-oil droplets by using the droplet dispersing component, it is transferred to the droplet temperature control zone 2 with the photothermal layer 21 in the interlayer cavity 1 and flattened. Control zone 2 is used for droplet storage and implements temperature control on the droplet in this area;

S2. Infrared irradiation and air-cooling cooling operations are performed on the water-in-oil droplets in the droplet temperature control zone 2 to obtain target droplets. In this process, through infrared light irradiation, the photothermal layer 21 quickly generates heat after absorbing infrared rays and quickly The high-flux water-in-oil droplets are transferred to the high-flux water-in-oil droplets to achieve efficient heating of the droplets. After reaching the preset temperature, the infrared radiation is stopped and the fan system is started, the droplets cool down rapidly, and the cycle is repeated to achieve ultra-fast temperature control; this scheme Using the low-temperature fan channel system for cooling can simultaneously cool the interlayer cavity and the photothermal layer 21; this step can periodically and rapidly heat and cool the photothermal interlayer through the program-controlled infrared light frequency, so as to realize the rapid cooling of the spreading droplets. Cycle amplification, and transform traditional three-dimensional sample heating into two-dimensional plane heating;

S3. After the cycle is over, squeeze the air into the first plate and the second plate corresponding to the droplet temperature control zone, or raise the top of the first plate corresponding to the droplet temperature control zone to form an angle gradient to drive The target droplets are transferred to the droplet grading area, and the target droplets are automatically assembled in a gradient dispersion arrangement for observation and quantitative detection. Dispersed liquid droplets converge on the other end of the glass slide through the gradient slit in the droplet temperature control zone 2, and then naturally flow into the gradient sandwich plate assembly 31 for droplet grading, which meets the requirements for droplet uniformity such as digital quantification.

The result of the temperature-controlled reaction in step S2 can be directly observed or detected, or the droplets in the droplet temperature-controlled zone 2 can be transferred to the gradient sandwich plate assembly 31, and the results can be obtained after the droplets are dispersed and arranged through a fluorescence microscope or a photodiode. detection.

This scheme implements infrared light irradiation through an infrared LED light source array, and its emission wavelength is preferably 850nm, and its power is 5W-500W; its trigger and termination are set and controlled accurately to 0.1 second level through a relay;

In step S2, the time and number of repetitions for cyclically performing infrared irradiation and air cooling operations are based on the PCR reaction program.

This application provides an application of a portable droplet PCR device. The portable PCR droplet pretreatment device is loaded into the centrifugal turntable. After the temperature control reaction is completed, the liquid in the droplet temperature control zone 2 is transferred to the gradient sandwich plate by low-speed centrifugal force. Efficient tiling and observation and detection are realized in the component 31, and high-throughput sample processing capabilities are realized; a collection container is further added at the end of the gradient sandwich plate component 31, and the reacted product can be centrifuged at a higher speed and collected for other downstream Analysis; the loading quantity of the portable droplet PCR device is greater than or equal to 1 to achieve high-throughput analysis.

In the above method, the portable droplet PCR device applied is shown in Fig. 1 and Fig. 2, including a interlayer cavity, a droplet temperature control zone 2 and a droplet classification zone 3 arranged in the interlayer cavity 1, for The droplet is transferred to the droplet dispersing component of the droplet temperature control zone 2, the droplet temperature control zone 2 is connected with the droplet classification zone 3, and the interlayer cavity includes a first flat plate 11 and a second flat plate 12 parallel to each other. On the first support 13 and the second support 14 supporting the side of the first flat plate 11 and the side of the second flat 12, the first flat 11, the first support 13, the second flat 12, and the second support 14 end to end Connected to form a cuboid cavity with a communication channel. The droplet temperature control zone 2 is provided with a photothermal layer 21. The black photothermal layer 21 is attached to the first plate 11, and the water-in-oil droplets are loaded and stored in the corresponding interlayer. In the chamber, the droplet classification area 3 is provided with a gradient sandwich plate assembly 31 with stepped slit channels, and the width of the slit channel of the gradient sandwich plate assembly 31 becomes smaller as it is farther away from the droplet temperature control area 2 .

In some embodiments, the first flat plate 11 and the second flat plate 12 of the interlayer cavity are high-temperature-resistant plates, such as injection molded plates or glass sheets, with a thickness of 0.05-0.4mm and an interval of 0.01-1mm. 13. The second support 14 is a support plate or double-sided adhesive tape. The first flat plate, the second flat plate, the first support 13, and the second support 14 are connected by bonding. The droplet temperature control area 2 is located at one end of the interlayer cavity, and the droplet classification area 3 is located at the other end of the interlayer cavity; The support member 14 is integrally formed by injection molding;

The droplet dispersing component can be various microfluidic droplet generating systems or instruments and equipment with oscillation function. Preferably, the droplet dispersing component of this solution includes a channel piece, a compressed sponge loaded in the channel piece, and is used to fix A fixing member that compresses the sponge body in the channel member. In some embodiments, the droplet dispersing component is a gun head filled with polyester sponge. Specifically, the sponge is cut into cubes of 1 to 3 mm and squeezed in the channel The size is about 0.5-1mm, and it is fixed at the front end of the channel; the tip is connected with a syringe or a pipette gun, and is used to suck liquid; the pumped liquid includes oil phase and water phase, and the oil phase is mineral oil containing surfactant , fluorinated oil or aliphatic hydrocarbon polyester oil phase; by first pumping the oil phase, occupying the sponge medium, and then absorbing the water phase, when the water phase passes through the pores of the sponge, it is efficiently dispersed into a high-flux water-in-oil liquid Droplets can be efficiently prepared by repeating this process twice.

The present invention disperses the droplets into water-in-oil droplets through the droplet dispersing component and distributes them in the droplet temperature control zone 2 in the interlayer cavity 1, effectively reducing the volume of the reaction solution and the thickness of the heat transfer in millimeters Change to tens of microns, so that the heat exchange process occurs quickly, and then solve the problem of low reaction temperature and heat transfer efficiency in heating and cooling; through infrared radiation on the droplet temperature control zone 2, due to the high-efficiency heat generation of the photothermal layer 21, the realization of Low-cost, easy-to-process heating module customization; at the same time, the gradient sandwich plate assembly 31 can automatically classify droplets of different sizes formed in various ways under the action of capillary force, realizing high-throughput and uniform digitalization of droplet reactions Quantitative needs.

The photothermal layer 21 includes a photothermal layer and a thermally conductive layer covering the photothermal layer. The photothermal layer is a black electrical insulating cloth with a thickness of 0.01-0.5 mm. The insulating cloth is covered with a layer of thermally conductive layer. Ensure rapid and uniform heating of droplets, which can further improve the uniformity of heating.

The gradient sandwich panel assembly 31 includes a flat sheet 311, a bent sheet 312, a first stopper 313, and a second stopper 314. One end of the flat sheet 311 is attached to one end of the bent sheet 312, and the first stopper The piece 313 is used to fix the other end of the flat plate 311 and the other end of the obtuse angle bent piece 312 , and the second limiting piece 314 is used to fix the middle part of the flat plate 311 and the obtuse angle bent piece 312 . The flat sheet 311 and the bent sheet 312 can be made of PC plastic or glass sheet with a certain hardness or made by 3D printing technology; the first limiting part 313 and the second limiting part 314 can The gradient sandwich plate assembly 31 is prepared by integral injection molding; the first limiting member 313 and the second limiting member 314, the flat sheet 311, and the obtuse angle bending sheet 312 can also be discrete structures, and can be bonded and pressed together. , pressing and other methods to form the gradient sandwich panel assembly 31 .

Preferably, the gradient sandwich panel assembly 31 provides a structure including:

1. The first limiting part 313 and the second limiting part 314 are adhesive parts with different thicknesses. At this time, the adhesive part has the functions of limiting, bonding and supporting, and the first limiting part 313 corresponds to the adhesive plate The left side of the sheet 311 and the left side of the obtuse angle bending sheet 312, the second stopper 314 is correspondingly bonded to the middle side of the flat sheet 311 and the middle side of the obtuse angle bending sheet 312, and the thickness of the adhesive can be different Double-sided adhesive, the thickness and position of the double-sided adhesive directly determine the separation and distribution position of the droplets in the slit, and the right end is closely fitted to form a stepped slit channel for the hierarchical arrangement of the droplets. The measured droplets are transferred to the interlayer plate with a height gradient, and the droplets of different sizes are arranged in stages in the interlayer plate, which is used for digital absolute quantitative observation and detection with high uniformity requirements. In some embodiments, the flat sheet 311, obtuse angle The bending pieces 312 are all glass slides, the thickness of the first stopper 313 is 50-500mm, the thickness of the second stopper 314 is 0.01mm, the double-sided adhesive area is about 1cm distributed on both sides of the glass slide, and the middle is capillary Force channel, under the capillary force of the oil phase liquid, the droplets of different sizes are fixed at specific positions, so as to realize the rapid arrangement of the droplets;

2. The first limiting part 313 is an adhesive part, the second limiting part 314 is an elastic fastener, and the first limiting part 313 corresponds to the left side of the adhesive plate 311 and the left side of the obtuse angle bending piece 312 Side, the thickness of the first stopper 313 is 50-500mm, the second stopper 314 is wrapped around the middle part of the flat sheet 311 and the middle part of the obtuse angle bending sheet 312 and fixed by pressing so that the middle part of the flat sheet 311 and the middle part of the obtuse angle bending sheet 312 Form a 0.01mm slit between them. For example, an elastic rubber band can be used to fix the splint in the middle. The elastic force of the rubber can press the splint to form a similar segmental stepped gap in the middle.

In some embodiments, the flat plate 311 of the gradient sandwich plate assembly 31 overlaps or shares the same plate with the second flat plate 12 of the sandwich cavity, and the left end of the obtuse angle bending piece 312 of the gradient sandwich plate assembly 31 is attached to the first flat plate 11. Close, the right end of the obtuse angle bending piece 312 is attached to the right end of the flat plate piece 311 of the gradient sandwich panel assembly 31.

A collection container is provided at the end of the gradient sandwich plate assembly 31 .

The photothermal layer 21 is not in contact with the first support member 13 and the second support member 14, and the photothermal layer 21 is pasted on the first flat plate 11, keeping a certain distance from the first support member 13 and the second support member 14 to prevent oil Phase overflow and communication cause droplet loss, and the volume that it can hold liquid can hold 1-100 microliters of solution according to the area of the photothermal film.

As above, the method of manufacturing and implementing the portable droplet PCR device of this program is as follows:

Production of droplets: The solution is prepared into droplets, which can be prepared by methods such as microfluidics, but the current methods often have problems such as time-consuming and expensive equipment. In some embodiments, the channel member is filled with a polyester-based sponge medium. The porous structure of the sponge can become dense micro-channels with specific pore distribution under proper extrusion to form a droplet dispersion component. When the sponge is filled with oil After the phase liquid, when the water phase is introduced, the water can be efficiently cut into specific water-in-oil droplets. It is also possible to use sponges with different soft and hard conditions to control the tightness of compression, construct media with different sizes of pores, and then adjust the size of the generated droplets. The size of the sponge used for compression is preferably spherical or cubic in the range of 1 to 3 mm. shape; the channel member is a pipeline for fixing and compressing the sponge, pumping and storing the first and second phase liquids, and the oil phase used to prepare the droplets is preferably mineral oil, fluorinated oil or aliphatic hydrocarbon polyester oil containing surfactants Mutually;

Provide heating conditions for droplet temperature control zone 2: choose infrared 850nm LED light source, power 50W, choose black electrical insulation cloth as the heating film material of photothermal layer 21, its thickness is about 0.02mm, and paste it on the inner side of the second plate 12 , the LED light source is arranged above or below the second plate 12;

Droplet loading process: Use a syringe or a pipette holster to fix the tip loaded with sponge, pump the oil phase to fill the porous medium, then move the tip to the oil-water interface or the aqueous solution, and continue to pump, the aqueous solution is in the oil phase Under the protection of the pore medium, the droplets are dispersed into high-flux droplets, and then the droplets are slowly loaded on the end of the interlayer chamber, which is close to the droplet temperature control zone 2, and can also be passed through The first plate 11 is loaded with an inlet, and the oil phase is automatically pulled into the droplet temperature control zone ² under the action of the capillary force and gravity of the interlayer cavity, and about 25 Microliter The volume of the oil phase containing the droplets.

Control of heating conditions: set the delay switch of the infrared LED through a relay. In some embodiments, a 50W LED lamp can be used to irradiate for 5 seconds to quickly heat the droplet temperature control zone 2 containing water-in-oil droplets from 60°C to 95°C. ℃, after turning off the LED light, the heat will drop to 60 ℃ through the natural cooling process in about 20 seconds; in order to accelerate the cooling process, a fan cooling system is set up, which can drop from 95 ℃ to 60 ℃ in 5 seconds; through the relay, these The heating and air cooling process is cyclically operated to realize the PCR process of droplets. In addition, for some long-chain PCR products, it is necessary to maintain a specific temperature for a long time for extension. For example, PCR needs to be extended for 10s at 72°C. By setting high-frequency pulse infrared heating, the droplet temperature can be kept stable at 72°C. ±1°C, to meet the needs of long-segment nucleic acid amplification.

Observation of the droplets after the temperature control treatment in the droplet temperature control zone 2: the fluorescence signal is detected and observed directly through a fluorescence microscope or a photodiode, but due to the large particle size change of the droplets, the results of quantitative statistics are affected . In order to eliminate the inhomogeneity of droplet observation, it is also possible to further use the gradient interlayer assembly to transfer and sort the generated droplets, press the first plate 11 and the second plate 12 corresponding to the droplet temperature control zone 2, and the droplet temperature The dispersed water-in-oil droplets in the control area 2 flow into the gradient interlayer assembly at the other end. When the droplets of different sizes pass through the slits of different heights, they are fixed at specific positions, thereby realizing the sorting of the droplets. The gradient interlayer assembly can simplify The high-throughput droplet observation method does not need to provide additional power for droplet transport.

As shown in Figure 3, this solution also applies a portable droplet PCR device to form a high-throughput droplet PCR integrated system: multiple interlayer cavities are fixed on a rotating plate at a certain angle of 0-70°; through the rotation of the turntable, Realize the control of the frequency of infrared light heating; after the reaction is completed, the reaction liquid in the droplet temperature control zone 2 is centrifugally thrown into the gradient sandwich plate assembly 31 by the appropriate rotating centrifugal force of the turntable after the reaction is completed, and the droplet can be rapidly classified and assembled. Form a uniform and neat array of droplets for absolute quantitative statistics. In addition, increasing the centrifugal force can centrifuge the reaction solution in the interlayer chamber and collect it into a PCR tube for downstream analysis of the product, etc.

The present invention does not require complex equipment and precise introduction of a syringe pump, greatly overcomes the professionalism of the droplet generation process, and is easy to operate. The heating process is often an extremely complicated control process of PCR, especially the heating and cooling process is difficult to be fast. We disperse the solution into droplets, and flatten it in the interlayer cavity, and skillfully heat it through the photothermal layer 21 thin film , directly heating and cooling the droplets, which completely relieved the heat generation and heat transfer limitations of PCR in the past; further, we realized the hierarchical assembly of droplets through the gradient splint assembly, which can be effectively different by the flow of the oil phase. Classified collection and observation of droplet size, low system cost, simple and efficient functions, suitable for quantitative needs such as POCT.

The solution will be further described below through specific embodiments.

Example 1

As shown in Figures 1 and 2, a portable droplet PCR device includes a sandwich cavity, a droplet temperature control zone 2 and a droplet classification zone 3 located in the sandwich cavity 1, and is used to transfer the droplet to the liquid droplet. The droplet dispersing assembly in the droplet temperature control zone 2, the droplet temperature control zone 2 is connected with the droplet classification zone 3, and the interlayer cavity includes a first flat plate 11 and a second flat plate 12 in parallel, for supporting the first flat plate 11 The first support member 13 and the second support member 14 of the side and the second flat plate 12 side, the first flat plate 11, the first support member 13, the second flat plate 12, the second support member 14 are connected end to end, forming a channel with Cuboid cavity, droplet temperature control area 2 is provided with a photothermal layer 21 attached to the first plate 11, the first plate 11 is located below the second plate 12, and the droplet classification area 3 is provided with a stepped slit channel The gradient sandwich plate assembly 31, the width of the stepped slit channel close to the droplet temperature control zone 2 is larger than the width away from the droplet temperature control zone 2; the droplet dispersion component includes a channel piece, a compressed sponge loaded in the channel piece , a fixing member for fixing the compressed sponge body in the channel member; the gradient sandwich plate assembly 31 includes a flat sheet 311, a bent sheet 312, a first limiting member 313, a second limiting member 314, and an end of the flat sheet 311 Attached to one end of the bent piece 312, the first stopper 313 is used to fix the left end side of the flat plate 311 and the left end side of the obtuse angle bent piece 312, and the second stopper 314 is used to fix the middle of the flat plate 311 The side and the middle side of the obtuse-angle bending piece 312 and the end of the gradient sandwich plate assembly 31 are provided with a collection container;

In this embodiment, the photothermal layer 21 includes an insulating layer and a heat conduction layer covering the insulating layer. The insulating layer is an ultra-thin electrical insulating glue with a thickness of 0.02mm and an area of 1.2*1.5CM. The interlayer board adopts a commonly used rectangular cover glass The size of the sheet is 5CM*2CM*0.5mm. Double-sided adhesive tape is used as the first support 13 and the second support 14 between the layers, and the dispersed droplets are loaded on the sandwich plate with the black film.

A portable droplet PCR method, comprising the following steps:

S1. Use the PCR system (including KAPA 2G Fast Multiplex Mix fast polymerase, the template is 100bp-cDNA) as the reaction solution, use the droplet dispersion component to efficiently disperse the reaction solution into high-throughput water-in-oil droplets, and transfer to the interlayer The droplet temperature control zone 2 with the photothermal layer 21 in the cavity 1 is flattened;

Wherein, the preparation method of the liquid droplet is as follows: the medium density (30KG/m ³ ) sponge unfilled material is used to make the liquid droplet. The channel part is selected as a 0.25ml dispensing gun head. Cut the sponge into a 3mm cube and fill it into the top of the gun head. It should not be over-extruded, just to the bottom, because the bottom is a funnel-shaped interface with a cross-sectional diameter of about 1mm. At this time, the sponge Squeeze about 1 mm in size, take 30 microliters of liquid in a PCR tube, and then take 80 microliters of 7% EM180 ethyl laurate oil phase to cover. Use the tip of the pipette to absorb the oil phase first, then the water phase, and the water phase passes through the pore medium filled with the oil phase to complete the preparation of droplets. In order to overcome the difference in suction techniques, the suction process can be repeated twice to complete the preparation of droplets .

S2. Infrared irradiation and air-cooling cooling operations are performed on the water-in-oil droplets in the droplet temperature control zone 2 to obtain target droplets. In this process, through infrared light irradiation, the photothermal layer 21 quickly generates heat after absorbing infrared rays and quickly The high-flux water-in-oil droplets are transferred to the high-flux water-in-oil droplets to achieve efficient heating of the droplets. After reaching the preset temperature, stop the infrared irradiation and start the fan system, the droplets cool down rapidly, and the cycle is repeated to achieve ultra-fast temperature control;

The specific temperature control process is: through the relay, control the circulating temperature to 65-94°C, set the infrared radiation for 4 seconds, cool down for 5 seconds, 40 cycles, a total of 6 minutes; then use the low-temperature fan channel system to cool down, which can be simultaneously Droplet interlayer and infrared LED panel cooling;

In order to better detect the temperature change during the heating process, a sheet-shaped (0.05mm) K-type temperature measuring line is used to monitor the temperature change in real time; the infrared light source is a 50W LED array, and the switch of the light source is controlled in milliseconds by a time relay , When cooling down, start the fan cooling system, and when the temperature drops to 70°C, the fan stops. The actual measurement results show that the initial temperature is 25°C, and after five seconds of heating, the temperature rises rapidly to 65°C, then, the temperature rises from 65°C to 95°C in 5 seconds, and the cooling process takes 6 seconds to complete 30 cycles. The time is 5.6 minutes.

S3. Squeeze the first flat plate 11 and the second flat plate 12 corresponding to the droplet temperature control zone 2, transfer the target droplet to the droplet classification zone 3, and the target droplet is automatically arranged and assembled in gradient dispersion.

After the amplification, the reaction droplets were collected and the product was detected, which was consistent with the target band, indicating that the rapid amplification was successfully achieved.

In the method of the present invention, the reaction system is dispersed with high-throughput droplets and flattened in the droplet temperature control zone 2 of the interlayer cavity, and the high-efficiency heat production characteristics of the photothermal layer 21 film absorbing infrared rays are used to skillfully improve the solution At the same time, the high-throughput droplet reactor can realize digital absolute quantification, etc., and can provide low-cost, portable temperature control and detection components for micro-nano synthesis reactions, biochemical analysis and detection, and other fields.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. All should be covered within the protection scope of the present invention.

Claims (10)

1. A portable droplet digital PCR control method, is characterized in that, comprises the following steps: S1. Use the droplet dispersing component to disperse the reaction solution into water-in-oil droplets, transfer and spread them to the surface of the photothermal layer (21) in the droplet temperature control zone (2) in the portable droplet digital PCR device; S2. Periodically perform infrared irradiation heating and air-cooling cooling operations on the water-in-oil droplets in the droplet temperature control zone (2), so that the water-in-oil droplets can be cyclically amplified, that is, the target droplets are obtained; S3. Squeeze the air into the first plate (11) and the second plate (12) corresponding to the droplet temperature control zone (2), or raise the first plate corresponding to the droplet temperature control zone (2) (11) At the top, an included angle gradient is formed to drive the target droplet to transfer to the droplet classification area (3), and the target droplet is automatically assembled in a gradient dispersion arrangement, to be tested and observed. 2. The portable droplet digital PCR control method according to claim 1, characterized in that, in step S2 or S3, the test observation is detected by direct signal detection, fluorescence microscope or photodiode. 3. The portable droplet digital PCR control method according to claim 1, characterized in that, in step S3, the time and repetition times for cyclically performing infrared irradiation and air cooling operations in step S2 are based on the PCR reaction program. 4. The portable droplet digital PCR control method according to claim 1, characterized in that, in step S3, it also includes loading the plurality of interlayer cavities (1) into a centrifugal turntable, using centrifugal force to realize high-pass Observation of quantitative response results. 5. The portable droplet digital PCR control method according to claim 4, characterized in that, the number of said loading interlayer chambers (1) is greater than or equal to 1, and the loading angle is 0-70°. 6. A droplet digital PCR device for portable droplet digital PCR control, characterized in that it comprises a sandwich cavity (1), a droplet temperature control zone (2) located in the sandwich cavity (1) ) and a droplet classification area (3), a droplet dispersion component for transferring dispersed droplets to the droplet temperature control area (2), the droplet temperature control area (2) and the droplet classification area ( 3) communicated; the interlayer cavity includes a first flat plate (11) and a second flat plate (12) parallel to each other, for supporting the side of the first flat plate (11) and the side of the second flat plate (12) The first support member (13) and the second support member (14), the first flat plate (11), the first support member (13), the second flat plate (12), the second support member (14) head to tail in sequence The droplet temperature control area (2) is provided with a photothermal layer (21), and the photothermal layer (21) is attached to the first plate (11), and the droplet classification area (3) A gradient sandwich plate assembly (31) with a stepped slit channel is provided, and the width of the slit channel of the gradient sandwich plate assembly (31) is from close to the droplet temperature control area (2) to far away from the droplet temperature. The control area (2) is reduced. 7. The droplet digital PCR device according to claim 6, characterized in that, the photothermal layer (21) comprises a photothermal layer and a heat conduction layer covering the photothermal layer. 8. The droplet digital PCR device according to claim 6, characterized in that, the gradient sandwich plate assembly (31) comprises a flat sheet (311), a bent sheet (312), a first stopper (313) , the second limiting part (314), one end of the flat plate (311) is attached to one end of the bending piece (312), and the first limiting part (313) is used to fix the The other end of the flat plate (311) and the other end of the obtuse angle bending piece (312), the second limiting member (314) is used to fix the middle part of the flat plate (311) and the obtuse angle bending piece (312) Middle. 9. The droplet digital PCR device according to claim 6, characterized in that a collection container is provided at the end of the gradient sandwich plate assembly (31). 10. The droplet digital PCR device according to claim 6, characterized in that, the photothermal layer (21) is not in contact with the first support (13) and the second support (14).

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