Portable droplet PCR device, method and application thereof

Abstract

This invention discloses a portable droplet PCR method, apparatus, and its application, including a sandwich cavity, a droplet storage and temperature control zone disposed within the sandwich cavity, and its control method. The sandwich cavity includes a first plate and a second plate parallel to each other, a first support member and a second support member for supporting the sides of the first plate and the second plate, respectively. The droplet temperature control zone is provided with a photothermal layer. The black photothermal layer is attached to the first plate, and water-in-oil droplets are loaded and stored in the photothermal sandwich. The photothermal sandwich is periodically and rapidly heated and cooled by a program-controlled infrared light frequency to achieve rapid cyclic amplification and detection of the spread droplets. Furthermore, after the cycle, a gradient sandwich plate assembly with stepped slit channels is used to meet the droplet uniformity requirements for applications such as digital quantification in a droplet grading zone. This invention transforms traditional three-dimensional sample heating into two-dimensional planar heating, eliminating the need for complex chip design and completely overcoming the limitations of traditional PCR's complex, slow temperature changes and expensive equipment control. It effectively solves the miniaturization and portability problems of digital ultrafast droplet PCR.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection, and more particularly to a portable droplet PCR device, method, and application thereof. Background Technology

[0002] Efficient temperature control is crucial for many reaction processes. For example, polymerase chain reaction (PCR), a rapid DNA amplification technique, is an essential step in molecular diagnostics such as real-time quantitative PCR, gene sequencing, and gene chips. A complete PCR temperature control process can be summarized as follows: 1) Heating the sample to 90-96℃ for denaturation; 2) Cooling the sample to 55℃-60℃ for annealing; 3) Heating the sample to 70℃-75℃ for extension; 4) Repeating steps one through three 25-40 times. As can be seen, PCR temperature control is a relatively complex process requiring repeated switching between temperature zones.

[0003] Traditional PCR reactions are carried out in small test tubes, and the reaction process is slow, generally requiring 80 minutes to 2 hours to complete. The time required to complete a PCR reaction on a PCR instrument depends on the following factors: 1. Module heating and cooling time; 2. Temperature equilibration time between the module and the PCR tube; 3. Extension time; 4. Number of cycles. How to improve the heating and cooling process and reduce the equilibration time of the solution in the PCR tube has always been the core issue of ultrafast PCR.

[0004] Microfluidic chips, with their small size, large specific surface area, high integration, fast reaction speed, and rapid heat transfer, have been widely used. Based on the different sample chambers on the chip, PCR can be divided into static chamber PCR and dynamic continuous flow PCR. Static chamber PCR is a miniaturization of traditional PCR. The reaction mixture is fixed in a microreactor. Temperature control devices, such as Peltiers or PI film heating plates, first cause the temperature of the temperature zone itself to change until the set temperature is reached. Then, the temperature of the target temperature is controlled. Since the temperature change of the temperature zone itself depends on the heating and cooling rate, it can generally only reach 2-5℃ / s, and the temperature continuously cycles. Alternatively, a fluid with a suitable temperature can be directly introduced into the temperature control chamber to replace the original fluid, achieving rapid temperature control. However, this process of introducing a suitable fluid into the temperature control chamber to replace the original fluid also consumes a certain amount of time (generally several seconds), and the uniformity and efficiency of fluid replacement are difficult to guarantee, easily leading to increased processing time or decreased temperature control accuracy.

[0005] Dynamic continuous flow PCR uses microfluidics to flow the PCR reaction solution between different temperature zones. This method eliminates the need for heating and cooling, reducing digestion time. However, this approach also has some drawbacks: the sample flows continuously within the chip, so controlling variables such as reaction time is primarily achieved through the design of the microchannel structure on the chip. This structure is typically more complex and requires more space, and the corresponding actuation mechanism for driving the continuous flow of the sample within the chip is also more complex. Under pressure-driven conditions, the velocity distribution across the microchannel cross-section is parabolic or similar, with the highest velocity in the center and the lowest (close to zero) velocity near the sides. Therefore, PCR samples at different locations in the cross-section will experience different reaction times. More importantly, the flowing liquid makes quantitative analysis of the process difficult, leading to quantification challenges. Current methods, whether static or dynamic, often involve highly complex heating modules, heating and cooling controls, and chip designs, resulting in high costs and difficulties in control.

[0006] To achieve rapid heating and cooling, some methods currently employ photothermal effects for rapid surface heating. Reports indicate that applying infrared radiation to the surface of nanoparticles or gold nanofilms can rapidly raise the surface temperature to 10–100°C per second. However, due to the large reaction volume (hundreds of nanoliters to tens of microliters), the surface-heated reaction system exhibits slow heat transfer, leading to low reaction efficiency, incomplete product production, and difficulty in determining the Ct value for quantitative analysis. Microfluidic oil-in-water microdroplets, with their advantages of small size, high throughput, internal stability, and absolute quantification, are widely used in biological and chemical detection and analysis, particularly in digital PCR and single-molecule amplification-free point-of-care testing (POCT) for absolute quantification of nucleic acids and biomacromolecules. Dispersed small droplets (pL–nL) have a smaller reaction volume and faster heat transfer rate compared to traditional methods, potentially making them more suitable for improving heating and cooling processes. However, traditional droplet generation is based on microfluidic chips, requiring precise injection pumps to strictly control the flow rates of the oil and water phases in the channels, resulting in high costs and demanding operation. Due to the inherent structural complexity or difficulty in manipulation of existing droplet generation methods, both micro-oscillation and centrifugal force-based driving forces severely limit the need for simplicity and portability in droplet-based point-of-care testing. In summary, no droplet-coupled technology currently exists that can achieve efficient and portable heating and cooling, and enable PCR reaction systems with absolute quantitative characteristics. Summary of the Invention

[0007] In view of this, this application provides a portable droplet PCR device, method and application, which does not require complex chip design, completely gets rid of the limitations of complex temperature change program and expensive equipment control in traditional PCR, and effectively solves the miniaturization and portability problems of high-throughput ultrafast droplet PCR.

[0008] To achieve the above technical objectives, this application adopts the following technical solution:

[0009] In a first aspect, this application provides a portable droplet digital PCR control method, comprising the following steps:

[0010] S1. The reaction solution is dispersed into water-in-oil droplets using a droplet dispersion component, and then transferred and spread evenly onto the photothermal layer surface of the droplet temperature control zone in the portable droplet digital PCR device;

[0011] S2. Periodically subject the water-in-oil droplets in the droplet temperature control zone to infrared irradiation heating and air cooling to allow the water-in-oil droplets to cyclically expand, thereby obtaining the target droplet;

[0012] S3. Compress air into the first and second plates 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, so as to drive the target droplet to be transferred to the droplet grading zone. The target droplet is automatically gradient dispersed and assembled for testing and observation.

[0013] Preferably, in step S3, the test observation is performed by direct signal detection, fluorescence microscopy, or photodiode.

[0014] Preferably, in step S3, the time and number of repetitions of the infrared irradiation and air-cooling operations in step S2 are based on the PCR reaction program.

[0015] Preferably, step S3 further includes loading the plurality of sandwich cavities into a centrifuge turntable for high-throughput transfer of reaction droplets into the fractionation zone for observation and quantification.

[0016] Preferably, the number of loading interlayer cavities is greater than or equal to 1, and the loading angle is 0-70°.

[0017] Secondly, this application provides a droplet digital PCR device for portable droplet digital PCR control, comprising a sandwich cavity, a droplet temperature control zone and a droplet grading zone disposed within the sandwich cavity, and a droplet dispersion component for transferring dispersed droplets to the droplet temperature control zone, wherein the droplet temperature control zone and the droplet grading zone are connected; the sandwich cavity includes a first plate and a second plate parallel to each other, a first support member and a second support member for supporting the sides of the first plate and the second plate, wherein the first plate, the first support member, the second plate, and the second support member are connected end to end in sequence; the droplet temperature control zone is provided with a photothermal layer attached to the first plate; the droplet grading zone is provided with a gradient sandwich plate assembly having a stepped slit channel, wherein the width of the slit channel of the gradient sandwich plate assembly decreases from near the droplet temperature control zone to far away from the droplet temperature control zone.

[0018] Preferably, the photothermal layer includes a photothermal layer and a heat-conducting layer covering the heated layer.

[0019] 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. The first limiting member is used to fix the other end of the flat sheet and the other end of the obtuse-angled bent sheet. The second limiting member is used to fix the middle part of the flat sheet and the middle part of the obtuse-angled bent sheet.

[0020] Preferably, the gradient sandwich panel assembly has a collection container at its end.

[0021] Preferably, the photothermal layer does not contact either the first support member or the second support member.

[0022] The beneficial effects of this application are as follows:

[0023] 1. The reaction solution is dispersed into high-throughput droplets and spread evenly on a two-dimensional plane, which greatly improves the heat transfer efficiency and also eliminates the limitation of solution volume, enabling the production of droplets at the μL to ml level.

[0024] 2. Heating is achieved through a black photothermal film, a low-cost, easy-to-produce, low-power, and high-efficiency heating method;

[0025] 3. Good heating stability; once the thin film material and heating power are determined, the temperature control of the reaction process is stable;

[0026] 4. It can be mass-produced, has a simple structure, is easy to operate, and has a low cost; in contrast, microfluidic chips require MEMS processes for production, bonding, and packaging, which are complex and costly.

[0027] 5. The detection chip is an independent component that can be flexibly assembled and is portable. This method can be applied to the detection of a single PCR sample or multiple reaction sites can be constructed on the disc. The transfer, distribution and collection of the final product are completed by centrifugal force. The structure is small, the cost is low and the adaptability is wide, making it very suitable for POCT detection applications.

[0028] 6. Absolute Quantification: Through the gradient sandwich panel assembly, high-throughput droplets can be rapidly and massively graded and assembled, greatly overcoming the problem of large relative variation in droplet size. It can arrange droplets in a single layer, which is relatively uniform and can meet the requirements of numerical quantification such as biochemical detection.

[0029] Instruction manual illustrations

[0030] Figure 1 A side view of the structure of a portable droplet PCR device;

[0031] Figure 2This is a top view of the portable droplet PCR device.

[0032] Figure 3 This is a schematic diagram of the assembly of a portable droplet PCR device with a centrifuge disc.

[0033] In the figure: 1. Inside the sandwich cavity; 2. Droplet temperature control zone; 3. Droplet classification zone; 11. First plate; 12. Second plate; 13. First support; 14. Second support; 21. Photothermal layer; 31. Gradient sandwich plate assembly; 311. Flat plate; 312. Bending plate; 313. First limiting member; 314. Second limiting member. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] This invention addresses the problems of low heating and cooling efficiency in existing traditional PCR reactions, the complex structure and difficulty in portability of droplet methods based on microfluidic chips, and the complex heating principle control and high production costs. It proposes an ultrafast PCR device and method based on portable droplets. The reaction solution is efficiently dispersed into high-throughput water-in-oil droplets, which are then transferred to the droplet temperature control zone 2 within a sandwich cavity 1 with an inner photothermal layer 21. Upon infrared light irradiation, the photothermal layer 21 absorbs the infrared radiation, rapidly generates heat, and quickly transfers it to the high-throughput droplets. The device achieves efficient heating of the droplets. Once the preset temperature is reached, infrared irradiation is stopped and the fan system is activated, causing the dispersed droplets to cool down rapidly. This cycle is repeated to achieve ultra-fast temperature control, which is used for temperature-controlled reactions such as PCR. Finally, the high-throughput droplets are transferred to the gradient sandwich plate assembly 31, where the droplets self-assemble and arrange themselves for observation and quantitative detection. This device eliminates the need for complex chip design, completely eliminating the limitations of complex temperature-changing procedures and expensive equipment control in traditional PCR, and effectively solving the miniaturization and portability problems of high-throughput ultra-fast droplet PCR.

[0036] The technical solution of the present invention is as follows:

[0037] like Figure 3 As shown, this application provides a portable droplet PCR method, including the following steps:

[0038] S1. After the reaction solution is efficiently dispersed into high-throughput water-in-oil droplets using a droplet dispersion component, the droplet temperature control area 2 with a photothermal layer 21 attached to the jacket cavity 1 is transferred and flattened. The droplet temperature control area 2 is used for droplet storage and temperature control of the droplets is implemented in this area.

[0039] S2. The water-in-oil emulsion droplets in the droplet temperature control zone 2 are circulated and subjected to infrared irradiation and air cooling to obtain the target droplets. In this process, the photothermal layer 21 absorbs infrared light and quickly generates heat, which is then rapidly transferred to the high-throughput water-in-oil emulsion droplets, achieving efficient heating of the droplets. After reaching the preset temperature, infrared irradiation is stopped and the fan system is started, and the droplets are rapidly cooled. This cycle is repeated to achieve ultra-fast temperature control. This scheme uses a low-temperature fan channel system for cooling, which 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 irradiation frequency, achieving rapid cyclic expansion of the spread droplets and transforming traditional three-dimensional sample heating into two-dimensional planar heating.

[0040] S3. After the cycle is completed, compressed air is introduced into the first and second plates corresponding to the droplet temperature control zone, or the top of the first plate corresponding to the droplet temperature control zone is raised to form an angle gradient, so as to drive the target droplet to the droplet classification zone. The target droplet is automatically gradient-dispersed and assembled for observation and quantitative detection. By squeezing one end of the rectangular glass slide, the dispersed droplet liquid stored in the droplet temperature control zone 2 is gathered at the other end of the glass slide through the gradient slit of the droplet temperature control zone 2, and then flows naturally into the gradient sandwich plate assembly 31 for droplet classification, satisfying the droplet uniformity requirements such as digital quantification.

[0041] The temperature-controlled reaction results in step S2 can be observed or detected directly, or the droplets in the temperature-controlled droplet zone 2 can be transferred to the gradient sandwich panel assembly 31, and the results can be detected by fluorescence microscopy or photodiode after the droplets are dispersed.

[0042] This solution uses an infrared LED light source array to irradiate infrared light, with a preferred emission wavelength of 850nm and a power of 5W-500W; its triggering and termination are set and controlled by relays with an accuracy of 0.1 seconds.

[0043] The time and number of repetitions for the infrared irradiation and air-cooling operations in step S2 are based on the PCR reaction program.

[0044] This application provides an application of a portable droplet PCR device. The portable PCR droplet pretreatment device is loaded into a centrifuge turntable. After the temperature-controlled reaction is complete, the liquid in the temperature-controlled droplet zone 2 is transferred into a gradient sandwich panel assembly 31 using low-speed centrifugation to achieve efficient spreading and observation, thus realizing high-throughput sample processing capabilities. Furthermore, a collection container is added to the end of the gradient sandwich panel assembly 31, allowing for higher-speed centrifugation of the reaction products for collection and subsequent downstream analysis. The number of portable droplet PCR devices loaded is greater than or equal to one to achieve high-throughput analysis.

[0045] The portable droplet PCR device used in the above method is as follows: Figure 1 , Figure 2 As shown, the device includes a sandwich cavity, a droplet temperature control zone 2 and a droplet classification zone 3 disposed within the sandwich cavity 1, and a droplet dispersion assembly for transferring droplets to the droplet temperature control zone 2. The droplet temperature control zone 2 and the droplet classification zone 3 are connected. The sandwich cavity includes a first plate 11 and a second plate 12 that are parallel to each other, a first support member 13 and a second support member 14 for supporting the sides of the first plate 11 and the second plate 12. The first plate 11, the first support member 13, the second plate 12, and the second support member 14 are connected end to end to form a cuboid cavity with a communicating 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. Water-in-oil droplets are loaded and stored in the corresponding sandwich cavity. The droplet classification zone 3 is provided with a gradient sandwich plate assembly 31 with a stepped slit channel. The width of the slit channel of the gradient sandwich plate assembly 31 decreases as it is further away from the droplet temperature control zone 2.

[0046] In some embodiments, the first plate 11 and the second plate 12 of the sandwich cavity are high-temperature resistant plates, such as injection-molded plates or glass sheets, with a thickness of 0.05-0.4 mm and a spacing of 0.01-1 mm. The first support member 13 and the second support member 14 are both support plates or double-sided adhesive. The first flat plate, the second flat plate, the first support member 13, and the second support member 14 are connected by adhesive bonding. The droplet temperature control zone 2 is located at one end of the sandwich cavity, and the droplet classification zone 3 is located at the other end of the sandwich cavity. In other embodiments, the first plate 11 and the second plate 12, the first support member 13, and the second support member 14 of the sandwich cavity are integrally formed by injection molding.

[0047] The droplet dispersion component can be any microfluidic droplet generation system or an instrument with oscillation function. Preferably, the droplet dispersion component of this solution includes a channel, a compressed sponge loaded in the channel, and a fixing component for fixing the compressed sponge in the channel. In some embodiments, the droplet dispersion component is a nozzle filled with polyester sponge. Specifically, the sponge is cut into cubes of 1-3 mm, compressed into a size of about 0.5-1 mm in the channel, and fixed at the front end of the channel. The nozzle is connected to a syringe or pipette for aspirating liquid. The aspirated liquid includes an oil phase and an aqueous phase, wherein the oil phase is a mineral oil, fluorinated oil, or aliphatic hydrocarbon polyester oil phase containing surfactants. By first aspirating the oil phase to occupy the sponge medium, and then aspirating the aqueous phase, the aqueous phase is efficiently dispersed into high-throughput water-in-oil droplets when it passes through the pores of the sponge. This process can be repeated twice to complete the efficient preparation of droplets.

[0048] This invention disperses droplets into water-in-oil droplets using a droplet dispersion component and distributes them within the droplet temperature control zone 2 of the sandwich cavity 1. This effectively reduces the volume of the reaction solution and the thickness of the heat transfer layer from millimeters to tens of micrometers, allowing the heat exchange process to occur rapidly. This solves the problems of temperature changes and low heat transfer efficiency during heating and cooling. By irradiating the droplet temperature control zone 2 with infrared light, the photothermal layer 21 generates heat efficiently, enabling the customization of a low-cost and easily manufactured heating module. At the same time, the gradient sandwich plate component 31 allows droplets of different sizes formed in various ways to be automatically graded under capillary force, achieving the digital quantitative requirements for high throughput and uniformity of droplet reactions.

[0049] 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 0.01-0.5mm. A thermally conductive layer is covered on the insulating cloth. Through heat transfer, the droplets can be heated quickly and uniformly, which can further improve the uniformity of heating.

[0050] The gradient sandwich panel assembly 31 includes a flat sheet 311, a bent sheet 312, a first limiting member 313, and a second limiting member 314. One end of the flat sheet 311 is attached to one end of the bent sheet 312. The first limiting member 313 is used to fix the other end of the flat sheet 311 and the other end of the obtuse-angled bent sheet 312. The second limiting member 314 is used to fix the middle part of the flat sheet 311 and the middle part of the obtuse-angled bent sheet 312. The flat sheet 311 and the bent sheet 312 can be made of PC plastic or glass sheet with a certain hardness, or they can be made by 3D printing technology. The first limiting member 313 and the second limiting member 314, together with the flat sheet 311 and the obtuse-angled bent sheet 312, can be integrally injection molded to form the gradient sandwich panel assembly 31. Alternatively, the first limiting member 313 and the second limiting member 314, together with the flat sheet 311 and the obtuse-angled bent sheet 312, can also be separate structures and assembled into the gradient sandwich panel assembly 31 by bonding, pressing, or compression.

[0051] Preferably, the gradient sandwich panel assembly 31, depending on the different limiting members, is provided in this solution with the following structure:

[0052] 1. The first limiting member 313 and the second limiting member 314 are adhesive members with different thicknesses. In this case, the adhesive members serve to limit, adhere, and support. The first limiting member 313 is bonded to the left side of the flat plate 311 and the left side of the obtuse-angled bent piece 312, respectively. The second limiting member 314 is bonded to the middle side of the flat plate 311 and the middle side of the obtuse-angled bent piece 312. The adhesive members can be double-sided tape of different thicknesses. The thickness and position of the double-sided tape directly determine the separation and distribution position of the droplets in the slit. The right end is tightly adhered, forming a stepped slit channel for the graded arrangement of droplets. High-throughput droplets are transferred into a sandwich plate with a high gradient. Droplets of different sizes are arranged in a graded manner in the sandwich plate for digital absolute quantitative observation and detection with high uniformity requirements. In some embodiments, the flat plate 311 and the obtuse-angled bent plate 312 are both glass slides. The thickness of the first limiting member 313 is 50-500mm, the thickness of the second limiting member 314 is 0.01mm, and the double-sided adhesive with an area of ​​about 1cm is distributed on both sides of the glass slide. The middle is a capillary channel. Under the traction of the capillary force, the oil phase liquid is fixed in a specific position for droplets of different sizes, thereby realizing the rapid arrangement of droplets.

[0053] 2. The first limiting member 313 is an adhesive component, and the second limiting member 314 is an elastic fastener. The first limiting member 313 corresponds to the left side of the adhesive flat plate 311 and the left side of the obtuse angle bent piece 312. The thickness of the first limiting member 313 is 50-500mm. The second limiting member 314 wraps around the middle of the flat plate 311 and the middle of the obtuse angle bent piece 312 and presses it to fix it so that a 0.01mm slit is formed between the middle of the flat plate 311 and the middle of the obtuse angle bent piece 312. For example, an elastic rubber band can be used to fix it in the middle of the clamp. The elasticity of the rubber band can press the clamp to form a similar segmented stepped slit in the middle.

[0054] In some embodiments, the flat plate 311 of the gradient sandwich panel assembly 31 overlaps with or shares the same plate with the second flat plate 12 of the sandwich cavity, the left end of the obtuse angle bent piece 312 of the gradient sandwich panel assembly 31 is attached to the first flat plate 11, and the right end of the obtuse angle bent piece 312 is attached to the right end of the flat plate 311 of the gradient sandwich panel assembly 31.

[0055] A collection container is provided at the end of the gradient sandwich panel assembly 31.

[0056] The photothermal layer 21 is not in contact with the first support member 13 and the second support member 14. The photothermal layer 21 is attached to the first plate 11 and maintains a certain distance from the first support member 13 and the second support member 14 to prevent the oil phase from overflowing and connecting, causing droplet loss. The volume of liquid it can hold can be 1-100 microliters of solution depending on the area of ​​the photothermal film.

[0057] As described above, the manufacturing and implementation methods of the portable droplet PCR device in this scheme are as follows:

[0058] Droplet fabrication: Dropletization of a solution can be achieved using microfluidics and other methods, but these methods often suffer from time consumption and expensive equipment. In some embodiments, a channel component is filled with a polyester sponge medium. The sponge's porous structure, under appropriate compression, transforms into dense microchannels with a specific pore distribution, forming a droplet dispersion component. When the sponge is filled with an oil phase liquid, the introduction of an aqueous phase efficiently cuts the water into specific water-in-oil droplets. Different sponges with varying hardness can control the degree of compression, creating media with different pore sizes, thereby regulating the size of the generated droplets. The sponge used for compression is preferably spherical or cubical, with a size of 1–3 mm. The channel component serves as a conduit for fixing the compressed sponge, drawing in and storing the first and second phase liquids. The oil phase used for droplet preparation is preferably mineral oil, fluorinated oil, or an aliphatic hydrocarbon polyester oil phase containing surfactants.

[0059] Heating conditions are provided for the droplet temperature control zone 2: an infrared 850nm LED light source with a power of 50W is selected, and the heating film material of the photothermal layer 21 is selected as black electrical insulating cloth with a thickness of about 0.02mm, which is attached to one side of the inside of the second plate 12. The LED light source is set above or below the second plate 12.

[0060] Droplet loading process: Using a syringe or pipette tip secured with a sponge, the oil phase is aspirated to fill the porous medium. The pipette tip is then moved to the oil-water interface or aqueous solution, and aspiration continues. Protected by the oil phase, the aqueous solution penetrates the porous medium and is dispersed into high-flux droplets. These droplets are then slowly loaded onto the end of the interlayer cavity, with the inlet located near the droplet temperature control zone 2. Alternatively, the droplets can be loaded by opening an inlet on the first plate 11. Under the influence of capillary force and gravity within the interlayer cavity, the oil phase is automatically drawn into the droplet temperature control zone 2, at a flow rate of 1 cm. 2 The film interlayer of this size can hold approximately 25 microliters of oil phase containing droplets.

[0061] Heating conditions are controlled by a relay-controlled delay switch for the infrared LED. In some embodiments, a 50W LED lamp irradiates the droplet temperature control zone 2 (containing water-in-oil emulsion) from 60°C to 95°C for 5 seconds. After the LED lamp is turned off, the temperature drops to 60°C naturally in about 20 seconds. To accelerate the cooling process, a fan cooling system is used, which can reduce the temperature from 95°C to 60°C within 5 seconds. The relay cycles these heating and cooling processes to achieve the PCR process of the droplet. Furthermore, for some long-chain PCR products, a specific temperature needs to be maintained for an extended period. For example, PCR at 72°C requires an extension of 10 seconds. High-frequency pulsed infrared heating can maintain the droplet temperature stable at 72±1°C, meeting the requirements for long-fragment nucleic acid amplification.

[0062] The droplets after temperature control treatment in droplet temperature control zone 2 are observed directly using a fluorescence microscope or photodiode to detect fluorescence signals. However, the large droplet size variation affects the quantitative statistical results. To eliminate the inhomogeneity of droplet observation, a gradient sandwich assembly can be used to transfer and sort the generated droplets. By pressing the first plate 11 and the second plate 12 corresponding to droplet temperature control zone 2, the dispersed water-in-oil droplets in droplet temperature control zone 2 flow into the gradient sandwich assembly at the other end. Droplets of different sizes are fixed at specific positions when passing through slits of different heights, thereby achieving droplet sorting. This gradient sandwich assembly simplifies the observation of high-throughput droplets without requiring additional power for droplet transport.

[0063] like Figure 3 As shown, this scheme also applies a portable droplet PCR device to form a high-throughput droplet PCR integrated system: multiple sandwich cavities are fixed at a certain angle of 0-70° on a rotating tray; the frequency of infrared light heating is controlled by rotating the turntable; after the reaction is completed, the reaction liquid in the droplet temperature control zone 2 is centrifuged and thrown into the gradient sandwich plate assembly 31 by appropriate centrifugal force of the turntable for rapid droplet fractionation and assembly, which can quickly form a uniform and neat droplet array for absolute quantitative statistics. In addition, increasing the centrifugal force can centrifuge and collect the reaction solution in the sandwich cavity into PCR tubes for downstream analysis of the product, etc.

[0064] This invention eliminates the need for complex equipment and precise injection pumps, greatly overcoming the specialized nature of droplet generation and simplifying operation. Heating is often a highly complex process in PCR, particularly the difficulty in rapidly increasing and decreasing temperature. By dispersing the solution into droplets and flattening them within a sandwich cavity, and cleverly using a photothermal layer 21 film for direct heating and cooling, we completely overcome the limitations of traditional PCR heat generation and transfer. Furthermore, we achieve graded droplet assembly through a gradient clamp assembly, enabling efficient collection and observation of droplets of different sizes via oil phase flow. The system is low-cost, simple, and highly efficient, suitable for quantitative needs such as point-of-care testing (POCT).

[0065] The following detailed implementation method further illustrates this solution.

[0066] Example 1

[0067] like Figure 1 , Figure 2 As shown, a portable droplet PCR device includes a sandwich cavity, a droplet temperature control zone 2 and a droplet grading zone 3 disposed within the sandwich cavity 1, and a droplet dispersion assembly for transferring droplets to the droplet temperature control zone 2. The droplet temperature control zone 2 is connected to the droplet grading zone 3. The sandwich cavity includes a first plate 11 and a second plate 12 that are parallel to each other, a first support member 13 and a second support member 14 for supporting the sides of the first plate 11 and the second plate 12. The first plate 11, the first support member 13, the second plate 12, and the second support member 14 are connected end to end to form a cuboid cavity with channels. The droplet temperature control zone 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. The droplet grading zone 3 is provided with a gradient sandwich plate assembly 3 having stepped slit channels. 1. The width of the stepped slit channel near 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 component, a compressed sponge loaded in the channel component, and a fixing component for fixing the compressed sponge in the channel component; the gradient sandwich panel assembly 31 includes a flat plate 311, a bent plate 312, a first limiting component 313, and a second limiting component 314. One end of the flat plate 311 is attached to one end of the bent plate 312. The first limiting component 313 is used to fix the left side of the flat plate 311 and the left side of the obtuse angle bent plate 312. The second limiting component 314 is used to fix the middle side of the flat plate 311 and the middle side of the obtuse angle bent plate 312. A collection container is provided at the end of the gradient sandwich panel assembly 31; the photothermal layer 21 does not contact the first support component 13 and the second support component 14.

[0068] In this embodiment, the photothermal layer 21 includes an insulating layer and a thermally conductive layer covering the insulating layer. The insulating layer is an ultra-thin electrical insulating adhesive with a thickness of 0.02 mm and an area of ​​1.2*1.5 cm. The sandwich panel uses a commonly used rectangular cover glass with dimensions of 5 cm*2 cm*0.5 mm. Double-sided adhesive is used as the first support 13 and the second support 14 between the layers to load the dispersed droplets into the sandwich panel covered with a black film.

[0069] A portable droplet PCR method includes the following steps:

[0070] S1. Using a PCR system (containing KAPA 2G Fast Multiplex Mix fast polymerase, template is 100bp-cDNA) as the reaction solution, the reaction solution is efficiently dispersed into high-throughput water-in-oil droplets using a droplet dispersion component, and then transferred to the droplet temperature control area 2 with photothermal layer 21 attached in the interlayer cavity 1 and spread out.

[0071] The droplet preparation method is as follows: using medium density (30KG / m³) droplets... 3 To prepare droplets, use an unfilled sponge as the dispensing medium. Select a 0.25ml dispensing nozzle tip. Cut the sponge into 3mm cubes and fill the top of the nozzle tip, being careful not to over-squeeze; just enough to reach the bottom, as the bottom is a funnel-shaped section with a diameter of approximately 1mm. Take 30µL of liquid in a PCR tube, then cover it with 80µL of 7% EM180 ethyl lauryl ester oil phase. Use the nozzle tip to first draw the oil phase, then the aqueous phase. The aqueous phase passes through the porous medium filled with the oil phase, completing the droplet preparation. To overcome differences in aspiration technique, the aspiration process can be repeated twice to complete the droplet preparation.

[0072] S2. The water-in-oil emulsion droplets in the droplet temperature control zone 2 are circulated and subjected to infrared irradiation and air cooling to obtain the target droplets. In this process, the photothermal layer 21 absorbs the infrared light and quickly generates heat, which is then rapidly transferred to the high-throughput water-in-oil emulsion droplets to achieve efficient heating of the droplets. After the preset temperature is reached, the infrared irradiation is stopped and the fan system is started to cool the droplets rapidly. This cycle is repeated to achieve ultra-fast temperature control.

[0073] The specific temperature control process is as follows: the circulating temperature is controlled at 65-94℃ via a relay, infrared irradiation is set for 4 seconds, cooling for 5 seconds, 40 cycles, for a total time of 6 minutes; then a low-temperature fan channel system is used for cooling, which can simultaneously cool the droplet interlayer and the infrared LED panel.

[0074] To better detect temperature changes during the heating process, a 0.05mm K-type temperature sensing wire was used to monitor temperature changes in real time. The infrared light source was a 50W LED array, and the switching on and off of the light source was controlled in milliseconds via a time relay. During cooling, the fan cooling system was activated, and the fan stopped when the temperature dropped to 70℃. Actual test results showed that, starting at 25℃, the temperature rapidly rose to 65℃ after five seconds of heating, then rose from 65℃ to 95℃ in another five seconds, with the cooling process taking six seconds. The time to complete thirty cycles was 5.6 minutes.

[0075] S3. The first plate 11 and the second plate 12 corresponding to the droplet temperature control zone 2 are squeezed to transfer the target droplet to the droplet classification zone 3, and the target droplet is automatically arranged and assembled in a gradient.

[0076] After amplification, the reaction droplets were collected and the product was detected. The result was consistent with the target band, indicating that rapid amplification was successfully achieved.

[0077] The method of this invention disperses the reaction system into high-throughput droplets and spreads them out in the droplet temperature control zone 2 of the sandwich cavity. It also utilizes the high-efficiency heat generation characteristics of the photothermal layer 21 thin film absorbing infrared rays to ingeniously improve the efficiency of the solution heating and cooling process. 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.

[0078] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A droplet digital PCR device for portable droplet digital PCR control, characterized in that, The device includes a sandwich cavity (1), a droplet temperature control zone (2) and a droplet classification zone (3) disposed within the sandwich cavity (1), and a droplet dispersion assembly for transferring dispersed droplets to the droplet temperature control zone (2). The droplet temperature control zone (2) is connected to the droplet classification zone (3). The sandwich cavity includes a first plate (11) and a second plate (12) that are parallel to each other, a first support member (13) and a second support member (14) for supporting the sides of the first plate (11) and the second plate (12). The first plate (11), the first support member (13), the second plate (12), and the second support member (14) are connected end to end in sequence. The droplet temperature control zone (2) is provided with a photothermal layer (21), which is attached to the first plate (11). The droplet grading zone (3) is provided with a gradient sandwich panel assembly (31) having a stepped slit channel. The width of the slit channel of the gradient sandwich panel assembly (31) decreases from near the droplet temperature control zone (2) to far away from the droplet temperature control zone (2). The photothermal layer (21) includes a photothermal layer and a thermally conductive layer covering the photothermal layer. The end of the gradient sandwich panel assembly (31) is provided with a collection container. The photothermal layer (21) does not contact the first support member (13) or the second support member (14). The droplet dispersion assembly includes a channel component, a compressed sponge loaded within the channel component, and a fixing component for fixing the compressed sponge within the channel component; The droplet dispersion component is a nozzle filled with polyester sponge. The polyester sponge is cut into cubes with a side length of 1 to 3 mm, squeezed to a size of 0.5 to 1 mm in the channel, and fixed at the front end of the channel. The nozzle is connected to a syringe or pipette for aspirating liquid, which includes oil and water phases.

2. A portable droplet digital PCR control method using the device of claim 1, characterized in that, Includes the following steps: S1. The reaction solution is dispersed into water-in-oil droplets using a droplet dispersion component, and then transferred and spread evenly onto the surface of the photothermal layer (21) of the droplet temperature control zone (2) in the portable droplet digital PCR device; the droplet dispersion component includes a channel component, a compressed sponge loaded in the channel component, and a fixing component for fixing the compressed sponge in the channel component; Step S1 further includes: filling the channel component with a polyester sponge medium. Under appropriate compression, the porous structure of the sponge medium becomes a dense microchannel with a specific pore distribution, forming a droplet dispersion component. When the sponge medium is filled with an oil phase liquid, the water phase is introduced, which efficiently cuts the water into specific water-in-oil droplets. By using sponge media with different hardness and softness conditions, the degree of compression is controlled to construct media with different pore sizes, thereby regulating the size of the generated droplets. S2. The water-in-oil droplets in the droplet temperature control zone (2) are periodically heated by infrared irradiation and cooled by air cooling so that the water-in-oil droplets can be cyclically expanded to obtain the target droplet; S3. Squeeze air into the first plate (11) and the second plate (12) corresponding to the droplet temperature control zone (2), or raise the top of the first plate (11) corresponding to the droplet temperature control zone (2) to form an angle gradient, so as to drive the target droplet to transfer to the droplet classification zone (3). The target droplet is automatically gradient dispersed and assembled for testing and observation. The time and number of repetitions for the periodic infrared irradiation and air-cooling operations in step S2 are based on the PCR reaction program.

3. The portable droplet digital PCR control method according to claim 2, characterized in that, In step S3, the test observation is performed by direct signal detection, fluorescence microscopy, or photodiode.

4. The portable droplet digital PCR control method according to claim 2, characterized in that, Step S3 also includes loading the plurality of sandwich cavities (1) into a centrifugal turntable and using centrifugal force to observe the high-throughput reaction results.

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