Centrifugal microfluidic isothermal amplification chip

Abstract

The application discloses a centrifugal microfluidic isothermal amplification chip, and relates to the technical field of in-vitro diagnosis. The centrifugal microfluidic isothermal amplification chip is a layered structure with a center expanding towards a periphery. The layered structure comprises, from the center to the periphery, a fixed hole, an isolation layer, a nucleic acid extraction layer and a liquid separation reaction layer. The layers are connected through interlayer channels, and fluid is driven to flow through each functional area in sequence through centrifugal force. At least part of the interlayer channels or each functional area is provided with a phase change material to form a phase change valve. The application has the technical effects of high efficiency, low pollution and easy control.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic technology, and in particular to a centrifugal microfluidic isothermal amplification chip. Background Technology

[0002] In the field of nucleic acid testing technology, the rapid detection of pathogenic microorganisms is increasingly attracting widespread attention. With rising public health demands, the requirements for the timeliness and accuracy of nucleic acid testing in scenarios such as clinical diagnosis and food safety monitoring are rapidly increasing. Traditional testing methods are not only cumbersome and time-consuming, but also fail to meet the needs of rapid on-site testing.

[0003] To improve this situation, researchers have been exploring new nucleic acid amplification technologies. However, existing isothermal amplification chips have many shortcomings, such as low integration, susceptibility to aerosol contamination, and complex rotation speed control, which makes it difficult to improve detection efficiency and reliability.

[0004] Therefore, developing a high-efficiency, low-pollution, and easily controllable centrifugal microfluidic isothermal amplification chip has become an urgent task. Summary of the Invention

[0005] This application provides a centrifugal microfluidic isothermal amplification chip, which has the technical advantages of more efficient detection, lower pollution, and easier control.

[0006] This application provides a centrifugal microfluidic isothermal amplification chip, which is a layered structure that expands from the center to the periphery. The layered structure includes, from the center outward, a fixing hole, an isolation layer, a nucleic acid extraction layer and a liquid separation reaction layer. Each layer is connected by interlayer channels, and fluid flows through each functional area in sequence by centrifugal force. In this process, phase change materials are provided in at least a portion of the interlayer channels or functional areas to form phase change valves.

[0007] In some examples, the phase change material is a temperature-controlled phase change material; or, the phase change material is a pressure-controlled phase change material.

[0008] In some examples, the isolation layer is disposed around the fixing hole, and the functional areas on the isolation layer include interconnected closed holes, injection inlets, isolation cavities and injection outlets; The sealing hole is set on the isolation chamber to seal any openings or potential leakage points on the isolation chamber other than the preset injection channel, so as to ensure the sealing of the isolation chamber, prevent the injection material from leaking from unexpected paths during the injection process, and maintain the injection pressure and injection fullness. The injection port is connected to the isolation chamber. The injection port serves as the starting point for the injection material to enter the isolation chamber and is used to connect with external injection equipment or material supply system to deliver the injection material into the isolation chamber. The injection outlet is located on the isolation chamber and is used to expel the air or other media that were originally in the isolation chamber at the beginning of the injection. During the injection process, the isolation chamber is judged to be completely filled by observing whether the injection material flows out smoothly. The injection outlet is located at the other end of the isolation chamber or at a preset position. When a continuous, bubble-free injection material flows out of the injection outlet, it indicates that the isolation chamber is full. The location and structure of the injection port are configured to ensure that the injection material is evenly distributed within the isolation chamber and to reduce the generation of air bubbles. The sealing hole, injection inlet, isolation chamber, and injection outlet together constitute a complete injection system. The sealing hole provides a basic guarantee for the injection to have a sealed environment, the injection inlet is the material input channel, the isolation chamber is the area where the material is filled and acts, and the injection outlet is the area for process control and result confirmation.

[0009] In some examples, the nucleic acid extraction layer is located outside the isolation layer, and the functional areas on the nucleic acid extraction layer include interconnected sample loading ports, lysis chambers, washing chambers, and clearance chambers; The outlet of the sample dispensing port is connected to the inlet of the lysis chamber to quantitatively transfer the raw sample received at the sample dispensing port to the lysis chamber; The outlet of the pyrolysis chamber is connected to the inlet of the cleaning chamber, so that the sample that releases the target analyte after pyrolysis can enter the cleaning chamber for purification. The outlet of the washing chamber is connected to the inlet of the scavenging chamber so that the sample after impurities have been removed can be transferred to the scavenging chamber for separation and acquisition of the target analyte; The sample loading port, lysis chamber, washing chamber, and elution chamber are connected in series according to the sample processing flow, forming a complete processing path from the input of the original sample to the output of the target analyte solution.

[0010] In some examples, the inner diameter of the sample inlet is 1mm~2mm, and the depth is 3mm~5mm; or, The lysis chamber has a volume of 50 μL to 100 μL, and an array of micropillars with a diameter of 50 μm to 100 μm and a spacing of 100 μm to 200 μm is arranged inside the lysis chamber; or, The cleaning chamber is equipped with a first outlet and a second outlet, and each outlet is provided with solid paraffin to form a phase change valve.

[0011] In some examples, the liquid separation reaction layer is located outside the nucleic acid extraction layer, and the functional areas on the liquid separation reaction layer include interconnected recovery tank, liquid channel, liquid separation tank, air valve, reaction chamber, waste liquid chamber, air channel and filter chamber; The recovery tank is connected to the distribution tank via a liquid channel to transport the liquid collected in the recovery tank to the distribution tank. The liquid channels connect the recovery tank to the distribution tank, the distribution tank to the reaction chamber, and the distribution tank to the waste liquid chamber, forming a path for the liquid to flow between the recovery tank, the distribution tank, the reaction chamber, and the waste liquid chamber. The separating tank is connected to the reaction chamber and the waste liquid chamber through different liquid channels to transport the separated liquid components to the reaction chamber or the waste liquid chamber respectively. An air valve is installed on the air passage, which is connected to the reaction chamber. By adjusting the air valve, the flow and flow of gas in the air passage are controlled, thereby changing the gas pressure in the reaction chamber. The reaction chamber is connected to the liquid separator via a liquid channel to receive liquid, and is connected to the air valve and filter chamber via a gas channel to allow gas to enter or exit. The waste liquid chamber is connected to the distribution tank via a liquid channel to receive and store the waste liquid transported from the distribution tank. The air passage connects the reaction chamber, the filter chamber, and the air valve, and is used to deliver gas to the reaction chamber and to deliver the gas generated in the reaction chamber to the filter chamber. The filter chamber is installed at the end of the airway and is connected to the reaction chamber through the airway to receive and filter the gas discharged from the reaction chamber.

[0012] In some examples, at least part of the fluid channel is arc-shaped, and its diameter gradually increases from the starting point to the ending point; or, The reaction chambers consist of 4 to 8 chambers arranged in a ring array, each with a volume of 10 μL to 20 μL, and contain lyophilized enzyme reagents; or... The filtration chamber includes a solid paraffin particle layer, a filter membrane isolation layer, and an activated carbon layer. The activated carbon filling amount is 10mg~20mg, and it is connected to the isolation chamber through the airway.

[0013] In some examples, the isolation chamber is connected to the filtration chamber, the cleaning chamber is connected to the inlet of the liquid channel, the outlet of the liquid channel is connected to the separator, and the separator is connected to the waste liquid chamber.

[0014] In some examples, the centrifugal microfluidic isothermal amplification chip includes a chip body, a front sealing film covering a first side of the chip body, and a back sealing film covering a second side of the chip body; The front sealing film has a rotating shaft hole, a sample dispensing opening, and a breathable membrane. The rotating shaft hole corresponds to the fixing hole, the sample dispensing opening corresponds to the sample dispensing port, and the breathable membrane corresponds to the filter chamber.

[0015] The aforementioned structure features a layered configuration from the center outwards, consisting of a fixing hole, an isolation layer, a nucleic acid extraction layer, and a liquid-liquid reaction layer. Centrifugal force drives fluid to flow sequentially through each functional area, enabling fully automated operation from sample introduction to nucleic acid extraction, liquid-liquid separation, and isothermal amplification. This approach significantly reduces human intervention, avoids the risk of cross-contamination that can occur in traditional methods, and substantially improves the efficiency and standardization of experimental procedures, making the entire detection process simpler, faster, and more reliable.

[0016] Phase change valves, formed by phase change materials and installed in at least part of the interlayer channels or functional areas, play a crucial role in fluid control. During centrifugation, by precisely controlling the temperature, the phase change material can transform between a solid and a liquid state, thereby achieving precise timing control of fluid flow. When it is necessary to stop fluid flow, the phase change material remains solid, effectively blocking the channel; while when a specific temperature is reached, the phase change material melts into a liquid state, opening the channel and allowing the fluid to continue flowing to the next functional area under the action of centrifugal force.

[0017] This valve control mechanism under phase change conditions not only ensures the strict timing and independence between each step, avoiding interference from the mixing of reagents or products in different reaction stages, but also allows for flexible adjustment of the fluid flow path and time according to experimental needs, further enhancing the chip's integration and multifunctionality, and providing strong technical support for realizing complex biochemical reactions and rapid detection. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this application or the prior art, the drawings used in the examples or prior art description will be briefly introduced below. Obviously, the drawings described below are only some examples of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the main body of the chip in a centrifugal microfluidic isothermal amplification chip in one example of this application; Figure 2 This is a top view of the chip body region division in a centrifugal microfluidic isothermal amplification chip example of this application; Figure 3 This is a top view of the main body of the chip in a centrifugal microfluidic isothermal amplification chip in one example of this application; Figure 4 This is a schematic diagram of the back side of the chip body in a centrifugal microfluidic isothermal amplification chip in one example of this application; Figure 5 This is a schematic diagram of the filter cavity in a centrifugal microfluidic isothermal amplification chip in one example of this application; Figure 6 This is a schematic diagram of the structure explosion of a centrifugal microfluidic isothermal amplification chip in one example of this application; Figure 7 This is a schematic diagram of the fluid position in the initial flow stage of a centrifugal microfluidic isothermal amplification chip in one example of this application; Figure 8 This is a schematic diagram of the fluid position of a centrifugal microfluidic isothermal amplification chip in an example of this application during the filling of the dispensing tank. Figure 9 This is a schematic diagram of the fluid position in the reaction chamber stage of a centrifugal microfluidic isothermal amplification chip in one example of this application; Figure 10 This is a schematic diagram of the effect of a centrifugal microfluidic isothermal amplification chip on oil-to-liquid separation in an example of this application. Figure 11 This is a schematic diagram showing the results of detecting Mycoplasma pneumoniae using a conventional chip-based comparative reagent in existing technology. Figure 12 This is a schematic diagram showing the results of detection using a centrifuged microfluidic isothermal amplification chip in one example of this application. Figure 13 This is a schematic diagram showing the test results of the reagent leakage prevention performance of the centrifugal microfluidic isothermal amplification chip in one example of this application.

[0020] Figure label: 1. Chip body; 11. Fixing hole; 12. Isolation layer; 13. Nucleic acid extraction layer; 14. Liquid-liquid reaction layer; 121. Sealing hole; 122. Injection port; 123. Isolation chamber; 124. Injection outlet; 131. Sample loading port; 132. Lysis chamber; 133. Lysis chamber outlet; 134. Injection hole; 135. Washing chamber; 136. Washing chamber outlet; 138. De-icing chamber; 139. First outlet; 1310. Second outlet; 141. Recovery tank; 142. Liquid channel; 143. Liquid-liquid separator; 144. Air valve; 145. Reaction chamber; 146. Waste liquid chamber; 147. Gas channel; 148. Filter chamber; 149. Solid paraffin particle layer; 1410. Activated carbon layer; 2. Front sealing film; 21. Rotary shaft hole; 22. Sample loading opening; 23. Gas permeable membrane; 3. Back sealing film. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and examples. It should be understood that the specific examples described herein are merely illustrative and not intended to limit the scope of this application.

[0022] Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification technique proposed by Notomi et al. in 2000, has been widely applied in fields such as pathogen detection and infectious disease diagnosis. Stumpf et al. proposed a centrifugal microfluidic isothermal amplification chip based on LAMP technology. However, this method relies on the extrusion release of externally placed reaction solutions for nucleic acid extraction, resulting in low chip integration. Furthermore, while LAMP amplification efficiency is extremely high, an amplified LAMP product often consists of repetitive sequences of the same fragment. Therefore, if the laboratory is contaminated with aerosols, the false positive rate will be high and difficult to remove. LAMP chips require strict prevention of leakage and cross-contamination of amplification products.

[0023] Furthermore, the flow of liquid in each step of centrifugal microfluidics relies on the control of different rotational speeds. To accurately control the reaction at each step and avoid erroneous liquid flow, a certain gradient spacing needs to be maintained between rotational speeds. The more steps there are, the greater the gradient in rotational speed, and the higher the driving speed becomes. This increases the difficulty and cost of control on the one hand, and the complexity of chip design on the other, reducing the reliability of the chip's response on the other.

[0024] In specific applications of nucleic acid testing technologies, rapid detection of pathogenic microorganisms suffers from low efficiency and susceptibility to contamination, failing to meet the needs of on-site real-time diagnosis and hindering automated detection. There is an urgent need for a high-efficiency, low-contamination, and easily controllable centrifugal microfluidic isothermal amplification chip.

[0025] To solve the above technical problems, please refer to Figures 1-5 As shown, the first aspect of this application proposes a centrifugal microfluidic isothermal amplification chip. The centrifugal microfluidic isothermal amplification chip of this application has the characteristics of high efficiency amplification, low risk of contamination, and easy operation and control. The centrifugal microfluidic isothermal amplification chip not only helps to improve the efficiency and accuracy of pathogen detection, but also realizes automated detection process, reduces false positive rate caused by aerosol contamination, and meets the needs of on-site real-time diagnosis.

[0026] The technical advantages and improvements of this application are mainly reflected in the following aspects: This centrifugal microfluidic isothermal amplification chip innovatively employs a synergistic mechanism between a phase-change valve and an isolation chamber. By precisely controlling the opening of the phase-change valve, the pre-placed oil phase medium within the isolation chamber can be orderly filled into the microfluidic channel system, forming an effective fluid barrier. This design not only significantly reduces the risk of cross-contamination between different reaction chambers but also prevents accidental leakage of biological samples and reagents. Simultaneously, the integration of a phase-change valve and an activated carbon adsorption layer in the filtration chamber further blocks potential leakage pathways of amplification products, thereby greatly improving the operational safety and reliability of the chip in sensitive biological experiments.

[0027] The centrifugal microfluidic isothermal amplification chip employs oil-phase-filled channel technology, effectively reducing surface tension and flow resistance effects when liquid flows within the microchannels. This improvement allows for precise liquid transfer driven by only two different gradients of centrifugal force, simplifying fluid manipulation. Compared to traditional multi-stage pressure or complex valve control systems, this design significantly reduces the complexity of chip structure design, improves manufacturing feasibility, and enhances process stability and reaction repeatability under long-term use and various operating conditions.

[0028] The nucleic acid extraction module breaks away from the conventional serial layout, adopting a radially parallel arrangement. This design fully fits the circular geometry of the chip, achieving high-density integration of functional units within a limited area and significantly reducing the module size. During centrifugation, each unit operates independently, eliminating inter-step interference caused by liquid cross-flow, thereby improving the efficiency and stability of nucleic acid extraction and enhancing the overall coordination and functionality of the chip structure.

[0029] The isolation chamber structure employs a sealing material system composed of liquid and solid paraffin, achieving reliable phase isolation between the oil-water and gas-liquid phases. This composite isolation layer effectively resists the high-concentration alcohol components commonly encountered in the magnetic bead-based nucleic acid extraction process, preventing material swelling, deformation, or failure. This improves the chip's chemical compatibility and long-term storage stability, thereby ensuring the chip's performance under complex biochemical environments.

[0030] Reference Figures 1 to 4 In some examples of this application, the centrifugal microfluidic isothermal amplification chip is a layered structure that expands from the center to the periphery. The layered structure includes, from the center outward, a fixing hole 11, an isolation layer 12, a nucleic acid extraction layer 13, and a liquid separation reaction layer 14. Each layer is connected through interlayer channels and the fluid flows through each functional area in sequence by centrifugal force. In this process, phase change materials are provided in at least a portion of the interlayer channels or functional areas to form phase change valves.

[0031] The aforementioned centrifugal microfluidic isothermal amplification chip achieves certain effects and functions through the application of layered structure and phase change valve.

[0032] The device features a layered structure arranged sequentially from the center outwards, consisting of a fixing hole 11, an isolation layer 12, a nucleic acid extraction layer 13, and a liquid-liquid reaction layer 14. Centrifugal force drives fluid to flow sequentially through each functional area, enabling fully automated operation from sample introduction to nucleic acid extraction, liquid-liquid separation, and isothermal amplification reaction. This method significantly reduces human intervention, avoids the risk of cross-contamination that may occur in traditional operations, and significantly improves the efficiency and standardization of experimental operations, making the entire detection process simpler, faster, and more reliable.

[0033] Phase change valves, formed by phase change materials and installed in at least part of the interlayer channels or functional areas, play a crucial role in fluid control. During centrifugation, by precisely controlling the temperature, the phase change material can transform between a solid and a liquid state, thereby achieving precise timing control of fluid flow. When it is necessary to stop fluid flow, the phase change material remains solid, effectively blocking the channel; while when a specific temperature is reached, the phase change material melts into a liquid state, opening the channel and allowing the fluid to continue flowing to the next functional area under the action of centrifugal force.

[0034] This valve control mechanism under phase change conditions not only ensures the strict timing and independence between each step, avoiding interference from the mixing of reagents or products in different reaction stages, but also allows for flexible adjustment of the fluid flow path and time according to experimental needs, further enhancing the chip's integration and multifunctionality, and providing strong technical support for realizing complex biochemical reactions and rapid detection.

[0035] In some examples, the phase change material is a temperature-controlled phase change material; or, the phase change material is a pressure-controlled phase change material.

[0036] Temperature-controlled phase change materials can undergo a phase change at a specific temperature point according to preset temperature range parameters, thereby precisely controlling the timing of fluid flow within the chip. For example, in the nucleic acid extraction process, after the lysis step is completed, the phase change material is melted by raising the temperature, opening the channel to the cleaning chamber 135, allowing the lysed sample to smoothly enter the cleaning chamber 135 for purification.

[0037] Pressure phase change materials can undergo phase change according to preset pressure range parameters to control fluid flow. In scenarios requiring precise control of liquid distribution, pressure phase change materials can open or close channels based on pressure changes to ensure that liquids of different components are accurately distributed into reaction chamber 145 or waste liquid chamber 146.

[0038] Reference Figure 3 In some examples, the isolation layer 12 is disposed around the fixing hole 11, and the functional areas on the isolation layer 12 include interconnected closed holes 121, injection inlet 122, isolation cavity 123 and injection outlet 124; A sealing hole 121 is provided on the isolation chamber 123 to seal any openings or potential leakage points on the isolation chamber 123 other than the preset injection channel. The sealing hole 121 can ensure the sealing of the isolation chamber 123, prevent the injection material from leaking from unexpected paths during the injection process, and maintain the injection pressure and injection fullness. The injection port 122 is connected to the isolation chamber 123. The injection port 122 serves as the starting point of the channel for the injection material to enter the isolation chamber 123. It is used to connect with external injection equipment or material supply system to deliver the injection material into the isolation chamber 123. The injection outlet 124 is provided on the isolation chamber 123 to discharge the air or other media originally in the isolation chamber 123 at the initial stage of injection, and to determine whether the isolation chamber 123 has been completely filled by observing whether the injection material flows out smoothly during the injection process. The injection outlet 124 is located at the other end of the isolation chamber 123 or at a preset position. When continuous, bubble-free injection material flows out of the injection outlet 124, it indicates that the isolation chamber 123 is full. The position and structure of the injection port 122 are configured to ensure that the injection material is evenly distributed within the isolation chamber 123 and to reduce the generation of air bubbles. The sealing hole 121, the injection inlet 122, the isolation chamber 123, and the injection outlet 124 together constitute a complete injection system. The sealing hole 121 provides a basic guarantee for the injection to have a sealed environment, the injection inlet 122 is the material input channel, the isolation chamber 123 is the area for material filling and action, and the injection outlet 124 is the area for process control and result confirmation.

[0039] The aforementioned structure ensures the airtightness of the isolation chamber 123. Specifically, the sealing hole 121 effectively seals any openings or potential leakage points on the isolation chamber 123 except for the pre-set injection channel, creating a reliable sealed environment. This design fundamentally prevents the injection material from leaking through unexpected paths during the injection process, ensuring that the injection pressure can be maintained stably, while also ensuring that the injection material can fully fill the isolation chamber 123, significantly improving the injection fullness.

[0040] It also enables efficient delivery and precise control of the injection material. Specifically, the injection port 122 serves as the clear starting point for the injection material to enter the isolation chamber 123. Its design facilitates reliable connection with external injection equipment or material supply systems, ensuring that the injection material can be stably and continuously delivered into the isolation chamber 123. Simultaneously, the optimized position and structure of the injection port 122 guides the injection material to achieve uniform distribution within the isolation chamber 123 and effectively reduces the generation of air bubbles during the injection process, laying the foundation for high-quality injection.

[0041] It can also provide a monitoring and result confirmation mechanism for the injection process. Specifically, the injection outlet 124 is set at the other end of the isolation chamber 123 or at a preset position. In the initial stage of injection, it can efficiently expel the air or other media originally in the isolation chamber 123, avoiding the influence of these substances on the injection quality. During the injection process, by observing whether the injection material flows out smoothly from the injection outlet 124, especially when there is a continuous flow of injection material without air bubbles, it is possible to intuitively and accurately determine whether the isolation chamber 123 has been completely filled, thereby achieving effective monitoring of the injection process and reliable confirmation of the final injection result.

[0042] A complete and coordinated injection system can also be constructed. Specifically, the sealing orifice 121, injection inlet 122, isolation chamber 123, and injection outlet 124 together constitute a fully functional and collaborative injection system. The sealing orifice 121 provides the fundamental guarantee of a sealed environment, providing the prerequisite for the entire injection process; the injection inlet 122 serves as the material input channel, ensuring a stable material supply; the isolation chamber 123, as the core area for material filling and function, is the key location for achieving the injection objective; and the injection outlet 124 undertakes the important functions of process control and result confirmation. Each component has a clear division of labor and cooperates with each other, jointly ensuring the smooth progress of the injection process and the achievement of the final injection effect.

[0043] In practical applications, the design of this complete infusion system is crucial for ensuring the performance stability and reliability of centrifugal microfluidic isothermal amplification chips. The sealed environment is fundamentally guaranteed by the sealing orifice 121, effectively preventing interference from external impurities or gases and ensuring the purity of the environment within the isolation chamber 123. The infusion port 122, serving as the material input channel, is optimized in its position and structure, allowing the infusion material to be rapidly and evenly distributed within the isolation chamber 123, while reducing the generation of air bubbles and avoiding problems such as uneven infusion or infusion failure caused by air bubbles.

[0044] The isolation chamber 123 serves as the area for material filling and function. Its size and shape are customized according to specific application requirements to ensure sufficient space to accommodate the infused material and allow it to function effectively. During the infusion process, by observing whether there is a continuous, bubble-free flow of infused material from the infusion outlet 124, it is possible to accurately determine whether the isolation chamber 123 has been completely filled, thereby avoiding experimental errors or equipment damage caused by insufficient or excessive infusion.

[0045] Furthermore, this complete perfusion system offers high flexibility and scalability. Depending on different experimental needs or application scenarios, components such as the sealing orifice 121, perfusion inlet 122, isolation chamber 123, and perfusion outlet 124 can be adjusted and optimized to adapt to different perfusion materials and processes. Simultaneously, this system can be integrated with other functional areas or components to form a more complex and efficient centrifugal microfluidic isothermal amplification chip system, providing more convenient and reliable experimental tools for biomedical research, clinical diagnosis, and other fields.

[0046] In some examples, the nucleic acid extraction layer 13 is disposed around the isolation layer 12, and the functional areas on the nucleic acid extraction layer 13 include a connected sample dispensing port 131, a lysis chamber 132, a washing chamber 135, and a clearance chamber 138. The outlet of the sample loading port 131 is connected to the inlet of the lysis chamber 132 so as to quantitatively transfer the original sample received by the sample loading port 131 to the lysis chamber 132. The outlet of the pyrolysis chamber 132 is connected to the inlet of the cleaning chamber 135, so that the sample that releases the target analyte after pyrolysis can enter the cleaning chamber 135 for purification. The outlet of the washing chamber 135 is connected to the inlet of the cleaning chamber 138 so that the sample after impurities are removed can be transferred to the cleaning chamber 138 for separation and acquisition of the target analyte; The sample loading port 131, lysis chamber 132, washing chamber 135 and scavenging chamber 138 are connected in series according to the sample processing flow to form a complete processing path from the input of the original sample to the output of the target analyte solution.

[0047] This sequential, cascaded structure, taking nucleic acid detection as an example, allows the nucleic acid extraction process to proceed in an orderly manner according to a predetermined procedure, ensuring the independence and accuracy of each processing step. From the accurate introduction of the original sample into the sample loading port 131, through the lysis chamber 132 to effectively lyse cells or tissues and release the target analyte, to the washing chamber 135 to deeply purify the lysed sample, removing any impurities and interfering substances, and finally, in the elution chamber 138, the target analyte is separated from the sample, forming a pure target analyte solution. The entire process is tightly linked and interconnected.

[0048] As the starting point of the nucleic acid extraction process, the sample loading port 131 must not only accurately receive the raw sample but also have the ability to quantitatively transfer the sample. This ensures that the amount of sample entering the lysis chamber 132 remains consistent each time, thereby guaranteeing the repeatability and reliability of experimental results. The sample loading port 131 typically employs a sophisticated microfluidic design, achieving quantitative sample transfer by precisely controlling the fluid flow rate and volume. Simultaneously, the sample loading port 131 may also be equipped with sample identification and detection functions, enabling preliminary detection of parameters such as sample type and concentration, providing basic data support for subsequent processing.

[0049] The lysis chamber 132 is a crucial step in the nucleic acid extraction process. Its function is to lyse the incoming raw sample, disrupting the structure of cells or tissues and releasing the target analytes. The lysis chamber 132 is typically equipped with specific lysis reagents and conditions, such as temperature and pH, to achieve efficient lysis of different types of samples. During lysis, the lysis reagents react chemically with the cells or tissues in the sample, disrupting their cell membranes and cell walls, thus releasing the target analytes. The design of the lysis chamber 132 also needs to consider the stability of the lysed products and the convenience of subsequent processing, ensuring that the lysed sample can smoothly enter the washing chamber 135 for further processing.

[0050] The primary function of the washing chamber 135 is to purify the lysed sample, removing any impurities and interfering substances, such as proteins, polysaccharides, and salts. If these impurities and interfering substances are not thoroughly removed, they can severely impact the subsequent separation and detection of target analytes, leading to inaccurate results or even the inability to detect the target analyte. The washing chamber 135 typically employs a multi-stage washing process, gradually removing impurities and interfering substances by continuously replacing the washing solution. The choice of washing solution is also crucial; it must be rationally selected based on the sample type and the properties of the target analyte to ensure that impurities are removed without causing loss or damage to the target analyte.

[0051] The elution chamber 138 is the final step in the nucleic acid extraction process. Its function is to separate the target analyte from the cleaned sample, forming a pure target analyte solution. The elution chamber 138 is typically equipped with specific elution reagents and elution conditions, such as temperature and elution time, to achieve efficient elution of the target analyte. During elution, the elution reagent specifically binds to or chemically reacts with the target analyte in the sample, separating it from the sample. The eluted target analyte solution can be collected using a collection device for subsequent detection and analysis. The design of the elution chamber 138 also needs to consider elution efficiency and the purity of the eluted product to ensure the acquisition of a high-quality target analyte solution.

[0052] In some examples, the inner diameter of the sample inlet 131 is 1mm~2mm, and the depth is 3mm~5mm; or, The lysis chamber 132 has a volume of 50 μL to 100 μL. The interior of the lysis chamber 132 contains a micropillar array structure with a diameter of 50 μm to 100 μm and a spacing of 100 μm to 200 μm; or... The cleaning chamber 138 is provided with a first outlet 139 and a second outlet 1310, and each outlet is provided with solid paraffin to form a phase change valve.

[0053] The sample inlet 131 described above has a large inner diameter and a suitable depth. This size design facilitates accurate sample introduction, accommodates samples of different types and viscosities, and reduces clogging or residue problems during sample addition. Furthermore, the suitable depth ensures a stable amount of sample can be accommodated during quantitative sample transfer, guaranteeing good repeatability of sample volume for each transfer. For example, when processing blood samples, the large inner diameter prevents blood cells from clogging the sample inlet 131, while the suitable depth ensures the accuracy of each sampling.

[0054] The lysis chamber 132 is configured with a specific volume and an internal micropillar array structure. The volume is determined based on the amount of reagent and reaction space required for the lysis reaction; a volume range of 50 μL to 100 μL can meet the lysis needs of most common samples, ensuring sufficient contact between the lysis reagent and the sample while avoiding waste of both. The internal micropillar array structure serves multiple purposes. The micropillars increase the surface area inside the lysis chamber 132, thus increasing the contact area between the lysis reagent and the sample, thereby improving lysis efficiency. Simultaneously, the spacing between the micropillars facilitates sample flow and mixing, resulting in a more uniform and thorough lysis reaction. For example, when lysing cell samples, the micropillar array structure can better disrupt the cell structure, releasing the target analyte.

[0055] The elution chamber 138 is equipped with a first outlet 139 and a second outlet 1310, each with a solid paraffin phase change valve. This design provides a more flexible and precise control method for the elution process. The first outlet 139 and the second outlet 1310 can be selected according to different experimental needs. For example, when it is necessary to collect target analyte solutions of different purities or at different stages, this can be achieved by controlling the opening of different outlets. The solid paraffin phase change valve can control the opening and closing of the outlets according to preset temperature conditions. During the elution process, when a specific temperature is reached, the paraffin melts, the outlet opens, and the eluted target analyte solution flows out; under other temperature conditions, the paraffin remains solid, the outlet closes, and it prevents premature solution outflow or the entry of external impurities. This setup improves the controllability and accuracy of the elution process, enabling the acquisition of higher quality target analyte solutions.

[0056] In some examples, the liquid separation reaction layer 14 is disposed around the nucleic acid extraction layer 13, and the functional areas on the liquid separation reaction layer 14 include a connected recovery tank 141, a liquid channel 142, a liquid separation tank 143, an air valve 144, a reaction chamber 145, a waste liquid chamber 146, an air channel 147, and a filter chamber 148. The recovery tank 141 is connected to the distribution tank 143 via the liquid channel 142 to transport the liquid collected in the recovery tank 141 to the distribution tank 143. Liquid channels 142 are connected to the recovery tank 141 and the distribution tank 143, the distribution tank 143 and the reaction chamber 145, and the distribution tank 143 and the waste liquid chamber 146 respectively, forming a path for liquid to flow between the recovery tank 141, the distribution tank 143, the reaction chamber 145 and the waste liquid chamber 146. The separating tank 143 is connected to the reaction chamber 145 and the waste liquid chamber 146 respectively through different liquid channels 142, so as to transport the different component liquids after separation to the reaction chamber 145 or the waste liquid chamber 146 respectively. An air valve 144 is installed on an air passage 147, and the air passage 147 is connected to a reaction chamber 145. By adjusting the air valve 144, the flow and flow of gas in the air passage 147 are controlled, thereby changing the gas pressure in the reaction chamber 145. The reaction chamber 145 is connected to the liquid separator 143 through the liquid channel 142 to receive liquid, and is connected to the air valve 144 and the filter chamber 148 through the gas channel 147 to allow gas to enter or exit. Waste liquid chamber 146 is connected to liquid distribution tank 143 via liquid channel 142 to receive and store waste liquid transported from liquid distribution tank 143; The air passage 147 connects the reaction chamber 145, the filter chamber 148 and the air valve 144, and is used to deliver gas to the reaction chamber 145 and to deliver the gas generated in the reaction chamber 145 to the filter chamber 148. The filter chamber 148 is installed at the end of the air passage 147 and is connected to the reaction chamber 145 through the air passage 147 to receive and filter the gas discharged from the reaction chamber 145.

[0057] The structure of the liquid-liquid reaction layer 14 described above, taking nucleic acid detection reaction as an example, ensures the high efficiency and accuracy of the reaction process. Starting from the recovery tank 141, liquids flowing out from the nucleic acid extraction layer 13 or other upstream processing stages are collected. These liquids may contain target analytes, reaction reagents, and some impurities. The liquid in the recovery tank 141 is stably transported to the liquid-liquid separator 143 through the liquid channel 142.

[0058] Separating tank 143 can precisely separate liquids according to their different components and subsequent reaction requirements. Through different liquid channels 142, the separated liquid components are guided to either reaction chamber 145 or waste liquid chamber 146. This separation mechanism ensures that only the target analyte and necessary reaction reagents can enter reaction chamber 145, while waste liquid is safely stored in waste liquid chamber 146, avoiding cross-contamination and interference with the reaction.

[0059] Air valve 144, installed on gas passage 147, is a key component for controlling the gas pressure within reaction chamber 145. By adjusting air valve 144, the on / off state and flow rate of gas within gas passage 147 can be precisely controlled, thereby altering the gas pressure within reaction chamber 145. This pressure regulation is crucial for the smooth progress of the reaction. For example, in some reactions, a certain gas pressure needs to be maintained to ensure thorough mixing of reactants and reaction efficiency; while in other cases, the gas pressure needs to be reduced to prevent the reaction from becoming too vigorous or generating bubbles.

[0060] The reaction chamber 145 is the core area of ​​the liquid-liquid reaction layer 14, used to receive liquid from the liquid-liquid tank 143, and connected to the air valve 144 and the filter chamber 148 via the gas channel 147. Within the reaction chamber 145, the target analyte undergoes a specific chemical reaction with the reaction reagents to generate the desired product. Simultaneously, the gas generated during the reaction is transported through the gas channel 147 to the filter chamber 148 for filtration to ensure that the emitted gas meets environmental protection requirements and avoids pollution of the experimental environment.

[0061] Waste liquid chamber 146 is responsible for receiving and storing waste liquids transported from separatory tank 143. These waste liquids may contain unreacted reagents, impurities, and byproducts generated during the reaction. By safely storing these waste liquids, their adverse effects on subsequent experiments or the environment can be prevented.

[0062] The filter chamber 148, installed at the end of the gas duct 147, is the final barrier for processing the gas discharged from the reaction chamber 145. It receives the gas from the reaction chamber 145 and filters out harmful substances and particulate matter through its internal filter material, ensuring that the discharged gas is clean and harmless. This filtration mechanism not only protects the health of laboratory personnel but also complies with environmental regulations.

[0063] In some examples, at least a portion of the fluid channel 142 is arc-shaped, and its diameter gradually increases from the starting point to the ending point; or, The reaction chambers 145 consist of 4 to 8 chambers arranged in a ring array, each with a volume of 10 μL to 20 μL, and contain lyophilized enzyme reagents; or... The filter chamber 148 includes a solid paraffin particle layer, a filter membrane isolation layer 12, and an activated carbon layer 1410. The activated carbon filling amount is 10mg~20mg, and it is connected to the isolation chamber 123 through the airway 147.

[0064] At least a portion of the liquid channel 142 is designed as an arc shape with a diameter that gradually increases from the starting point to the ending point. This unique shape design offers several advantages. The arc-shaped liquid channel 142 reduces resistance during liquid flow, allowing for smoother flow and minimizing eddies and energy losses caused by the irregular shape of the liquid channel 142. Simultaneously, the gradually increasing diameter design provides suitable flow channel space at different locations based on the liquid's flow characteristics, ensuring a stable flow rate and volume during transport and preventing liquid accumulation or impeded flow. For example, in the process of transporting liquid from the recovery tank 141 to the distribution tank 143, this liquid channel 142 design ensures that the liquid is transported smoothly and efficiently.

[0065] The number of reaction chambers 145 is set to 4 to 8, arranged in a circular array. This layout fully considers the diversity and efficiency of experiments. Multiple reaction chambers 145 can simultaneously perform different reactions or process different samples of the same reaction, greatly improving the parallel processing capability of the experiment and saving experimental time. The circular array distribution ensures that the reaction chambers 145 are evenly distributed in space, facilitating the connection of the gas channels 147 and the uniform distribution of gas, ensuring that each reaction chamber 145 can obtain stable gas pressure conditions. The volume of each reaction chamber 145 is 10μL to 20μL. This volume range is determined based on the reagent usage and reaction requirements of common nucleic acid detection reactions, which can meet the requirements of sufficient contact and reaction between the reaction reagents and the target analyte, while avoiding waste of reagents and samples. The built-in lyophilized enzyme reagent facilitates experimental operation. The lyophilized enzyme reagent has the advantages of good stability, easy storage and transportation. When using it, only an appropriate amount of liquid needs to be added to restore its activity and participate in the reaction.

[0066] The filter chamber 148 includes a solid paraffin granule layer 149 and an activated carbon layer 1410. A filter membrane isolation layer is disposed between the solid paraffin granule layer 149 and the activated carbon layer 1410. The activated carbon filling amount of the activated carbon layer 1410 is 10mg to 20mg, and it is connected to the isolation chamber 123 through the gas passage 147. This multi-layer filtration structure can achieve efficient filtration of the gas discharged from the reaction chamber 145. The solid paraffin granule layer can play a preliminary filtration role, intercepting larger particles and droplets in the gas and preventing them from entering subsequent filtration layers. The filter membrane isolation layer 12 can further filter out small particles and some harmful substances in the gas, improving the filtration accuracy. The activated carbon layer 1410 is the core filtration part of the filter chamber 148. Activated carbon has a strong adsorption capacity and can adsorb harmful gases, odor substances, and some small particulate matter in the gas, ensuring that the discharged gas is clean and harmless. The activated carbon loading amount is between 10mg and 20mg, determined based on the volume of the filter chamber 148 and the filtration requirements. This ensures sufficient adsorption capacity while preventing excessive activated carbon loading from clogging the gas passage 147 or affecting gas flow. Through this multi-layer filtration structure, the filter chamber 148 can effectively purify the gas discharged from the reaction chamber 145, protecting the experimental environment and the health of the personnel.

[0067] In some examples, the isolation chamber 123 is connected to the filter chamber 148, the cleaning chamber 138 is connected to the inlet of the liquid channel 142, the outlet of the liquid channel 142 is connected to the separator 143, and the separator 143 is connected to the waste liquid chamber 146.

[0068] This connection method constructs a complete and orderly liquid and gas flow system. The isolation chamber 123 is connected to the filter chamber 148, allowing the gas in the isolation chamber 123 to smoothly enter the filter chamber 148 for purification, ensuring the cleanliness of the gas circulation within the entire device and preventing harmful gases from accumulating and interfering with the experiment. The elution chamber 138 is connected to the inlet of the liquid channel 142, meaning that the target analyte solution after elution can be transported through the liquid channel 142, providing a channel for further processing in the separation reaction layer 14. The outlet of the liquid channel 142 is connected to the separation tank 143, allowing the liquid flowing from the elution chamber 138 to accurately enter the separation tank 143, enabling precise separation based on the different components of the liquid and subsequent reaction requirements. The separation tank 143 is connected to the waste liquid chamber 146, ensuring that the separated waste liquid can be stored in the waste liquid chamber 146 in a timely and safe manner, preventing the waste liquid from adversely affecting other experimental steps or the environment. The entire interconnected design works closely with the functions of each area, enabling the nucleic acid extraction and detection process to be carried out efficiently and accurately, thus improving the reliability and stability of the experiment.

[0069] In some examples, the centrifugal microfluidic isothermal amplification chip includes a chip body 1, a front sealing film 2 covering a first side of the chip body 1, and a back sealing film 3 covering a second side of the chip body 1; The front sealing film 2 has a rotating shaft hole 21, a sample feeding opening 22 and a breathable membrane 23. The rotating shaft hole 21 corresponds to the fixing hole 11, the sample feeding opening 22 corresponds to the sample feeding port 131, and the breathable membrane 23 corresponds to the filter chamber 148.

[0070] The chip body 1 serves as the supporting structure for the entire centrifugal microfluidic isothermal amplification chip. It is typically made of high-precision, chemically stable materials to ensure structural integrity and performance stability under various experimental conditions. A front sealing film 2 covers the first side of the chip body 1. The spindle hole 21 is designed to cooperate with specific fixing devices, allowing the chip to rotate stably during centrifugation and other operations, ensuring normal liquid flow and smooth reaction within the chip. The sample loading opening 22 corresponds to the sample loading port 131, facilitating accurate sample addition into the chip. Its size and shape are matched to the design of the sample loading port 131 to ensure smooth sample addition and minimize leakage. A permeable membrane 23 corresponds to the filter chamber 148. It has specific permeability properties, allowing gas within the filter chamber 148 to escape smoothly while preventing external impurities from entering the chip and contaminating the experiment. The back sealing film 3 covers the second side of the chip body 1 and works together with the front sealing film 2 to seal the chip body 1, forming a relatively independent and stable reaction space to prevent liquid leakage and interference from external factors, providing reliable protection for various reactions inside the chip.

[0071] On the other hand, this application provides a nucleic acid detection device based on a centrifugal microfluidic isothermal amplification chip, including the centrifugal microfluidic isothermal amplification chip and the reaction control device as described above; The centrifugal microfluidic isothermal amplification chip includes a lysis chamber 132, a washing chamber 135, a cleaning chamber 138, a dispensing tank 143, a reaction chamber 145, and a filter chamber 148. The sample dispensing port 131 is sealed with an adhesive tape, liquid paraffin is used as an isolation medium, and an air valve 144 is set to achieve liquid homogenization. The reaction control device includes a heating stage, a magnet module driven by a swing arm motor, and a lifting mechanism driven by a cam motor. The heating stage is designed with an outer ring heating area adapted to the multi-reaction chamber 145 and a fan-shaped heating area adapted to the nucleic acid extraction area, and is provided with a swing arm through hole.

[0072] This nucleic acid detection device based on a centrifugal microfluidic isothermal amplification chip achieves efficient and accurate nucleic acid detection through the coordinated operation of its components. The lysis chamber 132 within the centrifugal microfluidic isothermal amplification chip is responsible for lysis of the sample, releasing the target nucleic acid. Its specific internal volume and micropillar array structure ensure the efficiency and uniformity of the lysis process. The washing chamber 135 cleans the lysed sample, removing impurities and interfering substances to improve detection accuracy. The elution chamber 138 efficiently elutes the target nucleic acid from the sample using specific elution reagents and conditions, and the design of the outlet and phase-change valve allows for flexible control of the elution process. The separating tank 143 precisely separates the liquid according to its different components and subsequent reaction requirements, guiding it to the reaction chamber 145 or the waste chamber 146, avoiding cross-contamination and interference with the reaction. The reaction chamber 145 is the core area for nucleic acid amplification and detection. The built-in lyophilized enzyme reagent facilitates experimental operation, while multiple reaction chambers 145 arranged in a ring array improve the parallel processing capability of the experiment. The filter chamber 148 efficiently filters the gas discharged from the reaction chamber 145, protecting the experimental environment and the health of the experimental personnel.

[0073] The heating stage in the reaction control device provides a stable temperature environment for the chip. Its design, with an outer ring heating zone adapted to the multi-reaction chamber 145 and a fan-shaped heating zone adapted to the nucleic acid extraction zone, ensures that different areas can obtain suitable temperature conditions to meet the needs of nucleic acid extraction and amplification. The magnet module driven by the swing arm motor and the lifting mechanism driven by the cam motor enable precise control of the chip, such as the adsorption and release of magnetic beads and the lifting of the chip, further improving the automation and accuracy of the experiment. The design of the swing arm through-hole avoids the influence of the swing arm movement on the temperature field of the heating stage, ensuring the stability of temperature control. The entire nucleic acid detection device, through the close cooperation between the centrifuged microfluidic isothermal amplification chip and the reaction control device, realizes full automation from sample processing to nucleic acid amplification and detection, greatly improving the efficiency and accuracy of nucleic acid detection, and providing strong technical support for epidemic prevention and control and disease diagnosis.

[0074] In some examples, the magnet module is driven to rotate in a horizontal plane by a swing arm motor. The magnet module includes magnets that manipulate magnetic beads to move between the chip's pyrolysis chamber 132, cleaning chamber 135 and desorption chamber 138 by magnetic force. A cam motor drives the heating stage to rise and fall, enabling the centrifugal microfluidic isothermal amplification chip to either adhere to the heating stage to form a metal bath or separate to form an air bath. When the heating stage rises and comes into contact with the chip, the centrifugal microfluidic isothermal amplification chip is heated by a metal bath through the outer ring heating zone or the fan-shaped heating zone. When the heating stage descends and separates from the chip, the centrifuged microfluidic isothermal amplification chip is in an air bath state for oscillation or centrifugation.

[0075] The magnet module rotates on the horizontal plane under the drive of the swing arm motor. The magnets in the magnet module can accurately control the orderly movement of magnetic beads among the lysis chamber 132, washing chamber 135 and elution chamber 138 of the chip凭借强大的磁力,精准地操控磁珠在芯片的裂解室132、清洗室135与清脱室138之间有序运动。在裂解室132中,磁珠可以协助裂解过程,使目标核酸更充分地释放;进入清洗室135后,磁珠能帮助去除杂质和干扰物质,提升后续检测的准确性;而在清脱室138,磁珠又能促进目标核酸的高效洗脱。这种通过磁力操控磁珠的方式,实现了样品在各个处理环节的自动化转移,减少了人工操作可能带来的误差和污染,大大提高了实验的可靠性和稳定性。

[0076]

[0076] The function of the cam motor to drive the heating stage to rise or fall brings great flexibility to the entire nucleic acid detection device. When the cam motor drives the heating stage to rise and closely fit with the centrifugal microfluidic isothermal amplification chip, the outer ring heating zone or the fan-shaped heating zone starts to function and heats the chip by metal bath. Metal bath heating can provide a stable and uniform temperature environment, which is crucial for nucleic acid extraction and amplification reactions, because a suitable and stable temperature is the key factor to ensure the smooth progress of the reaction and obtain accurate results. Different regions obtain appropriate temperatures through the outer ring heating zone and the fan-shaped heating zone according to their functional requirements. For example, the nucleic acid extraction region may require a specific temperature to promote the lysis and washing processes, while the reaction chamber 145 region requires precise temperature control to achieve nucleic acid amplification.

[0077] When the cam motor drives the heating stage to lower and separate it from the chip, the centrifugal microfluidic isothermal amplification chip is in the air bath state. In this state, the chip can perform shaking or centrifugation operations. The shaking operation helps to make the components in the sample mix more evenly and improve the reaction efficiency; while the centrifugation operation can separate various components in the sample according to the density differences of different substances and further purify the target nucleic acid. This flexible switching between metal bath and air bath enables the entire nucleic acid detection device to provide the most suitable environmental conditions according to different experimental steps and requirements, thus ensuring the high efficiency and accuracy of the nucleic acid detection process.

[0078] This application also provides a method for controlling the flow inside a centrifugal microfluidic isothermal amplification chip, including the following stages: Initial flow stage: When the centrifugal force F acting on the liquid satisfies F1 < F < F2, the liquid overcomes the first resistance F2 and flows out and enters the liquid separation groove 143, where F2 is the resistance of the air valve 144; Filling the dispensing tank 143 stage: When the liquid fills the first few dispensing tanks 143, the liquid flow needs to overcome two surface tensions F1 and F2, and the surface of F1 becomes smaller, which leads to an increase in surface tension. At this time, a centrifugal force F>F1+F2 is required in real time to make the sample in the liquid channel 142 flow into the dispensing tank 143. Entering the reaction chamber 145 stage: When the liquid fills all the dispensing tanks 143, a centrifugal force F > F3 is required for the liquid to enter the reaction chamber 145; In order to accurately control the liquid flow, three significant gradients F1, F1+F2, and F3 are set. The liquid must overcome these three gradients in order to complete the above three stages of flow. Furthermore, an oil phase is introduced into the liquid channel 142 to reduce the centrifugal force that needs to be overcome in the filling stage of the separatory tank 143, so that the flow of liquid can be accurately controlled with only two speed gradients.

[0079] Reference Figure 7 In the initial flow stage, precisely controlling the centrifugal force F within the critical range between F1 and F2 is fundamental to ensuring the smooth initiation of liquid flow and accurate entry into the separatory tank 143. Success in this stage relies on the precise measurement and control of the resistance F2 of the air valve 144, as well as the precise setting of the centrifuge speed. By meticulously adjusting these parameters, it can be ensured that after overcoming the initial resistance, the liquid flows into the separatory tank 143 at a stable and controllable speed, laying a solid foundation for subsequent steps.

[0080] Reference Figure 8 During the filling stage of the distribution tanks 143, as the liquid gradually fills the first few distribution tanks 143, the challenges to liquid flow gradually increase. At this point, in addition to continuing to overcome the resistance F2 of the air valve 144, it is also necessary to cope with the increased surface tension F1 due to the smaller liquid surface area. This change requires real-time adjustment of the centrifugal force F to ensure that it is greater than the sum of F1 and F2 in order to maintain continuous liquid flow. Precise control in this stage is crucial to avoiding uneven distribution or stagnation of liquid among the distribution tanks 143, directly affecting the filling effect of the subsequent reaction chamber 145 and the accuracy of experimental results.

[0081] Reference Figure 9 Entering the reaction chamber 145 stage, after all the dispensing tanks 143 are filled with liquid, the next goal of the liquid flow is to enter the reaction chamber 145. This stage requires the centrifugal force F to exceed a new threshold F3 to overcome the additional resistance at the inlet of the reaction chamber 145, ensuring that the liquid can enter smoothly and begin the reaction. The setting of F3 needs to take into account the structural characteristics of the reaction chamber 145, the properties of the liquid, and the experimental requirements to ensure that the liquid can be rapidly and uniformly distributed after entering the reaction chamber 145, providing ideal conditions for subsequent reactions such as nucleic acid amplification.

[0082] To accurately control the liquid flow in the three stages mentioned above, setting three significant gradients F1, F1+F2, and F3 is crucial. These three gradients not only provide clear path guidance for the liquid flow but also ensure that the liquid flows at the appropriate speed and manner in each stage through progressively increasing resistance requirements. By monitoring and adjusting the centrifugal force F in real time to overcome these three gradients, precise control of the liquid flow can be achieved, thereby ensuring the smoothness and accuracy of the liquid flow within the entire centrifugal microfluidic isothermal amplification chip.

[0083] Introducing an oil phase into channel 142 is an innovative measure to reduce the centrifugal force required to overcome during the filling and separating tank stage 143. The introduction of the oil phase effectively reduces the frictional resistance of the liquid flowing in channel 142, particularly reducing the additional resistance caused by changes in surface tension. This change allows for precise control of the liquid flow during the filling and separating tank stage 143, requiring only adjustments to the centrifuge to two speed gradients. This simplification not only improves the convenience of experimental operations but also enhances the stability and controllability of the liquid flow, providing more reliable technical support for the application of centrifugal microfluidic isothermal amplification chips in fields such as nucleic acid detection.

[0084] In other examples, this application also provides a centrifugal microfluidic isothermal amplification chip reaction control device for controlling the chip's sample lysis, cleaning, elution, liquid separation, amplification reaction and fluorescence detection processes, as well as anti-contamination control, comprising: a drive motor, a heating stage, a fluorescence detection module and a magnet; The drive motor is connected to the chip holder via a rotating shaft, providing power for the rotation of the chip to control the speed, direction, and position of the chip body 1; the chip body 1 is mounted on the chip holder, which supports and drives the chip to rotate horizontally. A heating platform is provided below the chip body 1. The heating platform provides heat to the chip body 1 and controls the heating temperature. The heating platform is fixed to the connector below. The connector is fixed to the slider. The slider and the heating platform are restricted to moving up and down on the slide rail. The up and down movement of the heating platform is controlled by a cam motor. The heating stage is provided with a fluorescence detection hole, and a fluorescence detection module is provided below it. The fluorescence detection module emits excitation light to detect the fluorescence emitted by the chip body 1 during the reaction process. The swing arm motor is connected to the swing arm via a rotating shaft. The other end of the swing arm is equipped with a magnet. The magnet rotates and moves on the horizontal plane under the drive of the swing arm motor to operate the movement of the magnetic beads in the chip. The heating stage is also equipped with a swing arm through hole to avoid affecting the movement of the swing arm when the heating stage moves up and down.

[0085] The heating stage is provided with an outer ring heating area, a first sector heating area, a second sector heating area, and a fixing hole 11. The heating stage is connected to the connector through the fixing hole 11. The outer ring heating area is annular and is used to heat multiple reaction chambers 145 of the chip. The first sector heating area and the second sector heating area are used to heat the nucleic acid extraction area of ​​the chip.

[0086] The magnet is fixed on the swing arm, which is driven by a servo motor to move in the swing arm direction, thereby manipulating the movement of the magnetic beads in the microfluidic chip through magnetic force.

[0087] In other examples, this application also provides a reaction control method for a centrifugal microfluidic isothermal amplification chip, employing the above-mentioned centrifugal microfluidic isothermal amplification chip reaction control device, including the following steps: Sample addition: Remove the adhesive label from the chip, add the liquid sample into the lysis chamber 132 through the sample addition port 131, seal the sample addition opening 22 with a new adhesive label, and place the chip on the chip holder of the reaction control device. Sample lysis: The cam motor rotates to lower the heating stage, and the drive motor rotates the chip to rotate the lysis chamber 132 to the first sector heating area. The cam motor rotates to raise the heating stage to fit the chip, raising the temperature of the chip lysis chamber 132 to 50°C. Then the heating stage lowers to switch to an air bath. The chip is rotated to perform reciprocating motion to oscillate the chip and achieve sample lysis, allowing the magnetic beads in the lysis chamber 132 to bind with nucleic acids. The temperature of the lysis chamber 132 is further increased to 60°C to melt the solid paraffin at the lysis chamber outlet 133. The swing arm motor is driven to move, and a magnet is used to attract the magnetic beads in the lysis chamber 132 to move out of the lysis chamber outlet 133 and into the isolation chamber 123 filled with liquid paraffin. Sample cleaning: The magnetic beads are transferred from the oil phase to the aqueous phase of the cleaning chamber 135 through the cleaning chamber outlet 136. The chip is rotated and the swing arm motor is driven to move the magnet away, leaving the magnetic beads in the cleaning chamber 135. The chip is rotated by the drive motor to make the chip reciprocate to clean the magnetic beads. The swing arm motor is then driven to move the magnet to attract the magnetic beads to move out of the cleaning chamber outlet 136, pass through the oil phase and enter the isolation chamber 123. The rotating swing arm and chip transfer the magnetic beads from the oil phase to the liquid phase of the cleaning chamber 138. Sample elution: The drive arm motor moves to move the magnet away, leaving the magnetic beads in the elution chamber 138. The drive motor rotates the chip to reciprocate and oscillate the chip to elute the magnetic beads. The rotating chip moves the elution chamber 138 to the first sector heating zone and the sealing hole 121 to the second sector heating zone, raising the temperature to 70 degrees Celsius to melt the solid paraffin in the second outlet 1310 of the elution chamber 138 and the sealing hole 121. The drive arm motor moves to move the magnet to attract the magnetic beads from the first outlet 139 and move them to the washing chamber 135, leaving the nucleic acid extraction product in the elution chamber 138. Sample separation: The heating platform descends and switches to an air bath. At a speed of 600 rpm, the melted paraffin at the second outlet 1310 enters the recovery tank 141 under centrifugal force. The nucleic acid extraction product flows out from the second outlet 1310 and into the liquid channel 142, and then into the separation tank 143. Due to the obstruction of the air valve 144, the nucleic acid extraction product flows into the separation tank 143 sequentially and is evenly distributed. Excess liquid flows into the waste liquid chamber 146, while some of the liquid paraffin in the isolation chamber 123 flows into the liquid channel 142. The speed is further increased to 1200 rpm, so that the liquid in the separation tank 143 overcomes the resistance of the air valve 144 and enters the reaction chamber 145. Excess liquid paraffin flows into the separation tank 143 through the liquid channel 142 to seal the reaction chamber 145. The rotating chip rotates the filter chamber 148 to the first sector heating area. The heating platform rises and switches to a metal bath to heat the chip to 70 degrees, so that the solid paraffin in the filter chamber 148 melts to seal the filter chamber 148. Amplification reaction: After the nucleic acid extraction product enters the reaction chamber 145, the lyophilized bulbs are melted and mixed with enzymes and dNTPs. The reaction chamber is heated to 65°C through the outer ring heating zone to carry out the isothermal amplification reaction. The fluorescence detection module excites and detects the fluorescence signal emitted by the reaction chamber 145. The heating stage switches to an air bath at fixed intervals and then rotates at fixed angles in sequence. Each reaction chamber 145 is detected in sequence by the fluorescence detection module. After the detection is completed, it switches to a metal bath to continue heating.

[0088] The reaction chamber 145 is isolated by liquid paraffin, and the activated carbon in the filter chamber 148 is used to adsorb leaked nucleic acid in order to achieve contamination control.

[0089] In other examples, this application also provides a leak-proof detection method for centrifuged microfluidic isothermal amplification chips, comprising the following steps: dividing 12 chips that test positive for Mycoplasma pneumoniae into two groups, with 6 chips placed at room temperature for 15 days and the other 6 chips placed in a 40°C oven for 15 days; using 6 tubes of Mycoplasma pneumoniae amplification positive samples placed in a 40°C oven for 15 days as a positive control group; taking out the chips and positive control group samples after the above treatment, and testing the positive and negative results of the samples with lyophilized microspheres of Mycoplasma pneumoniae amplification; the results show that no leakage was detected in the positive chips after treatment at room temperature and 40°C, while the positive control group showed normal positive results.

[0090] In other examples, the chip radiates outward from a circle, consisting of: a fixing hole 11, an isolation layer 12, a nucleic acid extraction layer 13, and a liquid-liquid reaction layer 14.

[0091] The fixing hole 11 in the center of the chip is used to cooperate with the chip bracket and drive the chip body 1 to rotate through the drive motor.

[0092] The chip's isolation layer 12 includes: a sealing hole 121, a filling inlet 122, an isolation cavity 123, and a filling outlet 124. The sealing hole 121, located at the pyrolysis chamber outlet 133, is used to fill the chip with a temperature-controlled phase-change material and to block the pyrolysis chamber outlet 133, thus isolating it from the pyrolysis chamber 132. The isolation cavity 123 communicates with the filling inlet 122, the pyrolysis chamber outlet 133, the cleaning chamber outlet 136, and the first outlet 139. The isolation cavity 123 is used to fill and fill with an oil phase, maintaining the fluidity of the cleaning chamber 135 and the aqueous phase liquid within it, while simultaneously preventing liquid mixing. The filling outlet 124 allows the oil phase to flow in a specific direction, allowing excess oil phase to flow out. Sealing the filling outlet 124 confines the oil phase within the isolation cavity 123.

[0093] The nucleic acid extraction layer 13 of the chip includes: a sample inlet 131, a lysis chamber 132, a lysis chamber outlet 133, a perfusion port 134, a washing chamber 135, a washing chamber outlet 136, a perfusion port 134, a cleansing chamber 138, a first outlet 139, a sealing port 121, and a second outlet 1310. The sample inlet 131, located in a corner of the lysis chamber 132, is used to add liquid samples to the lysis chamber 132 within the chip, allowing them to react with the lysed components within the lysis chamber 132. The washing chamber 135 has a perfusion port 134 and a washing chamber outlet 136. The perfusion port 134 is used to inject washing solution into the washing chamber 135 to wash the lysed sample; the washing chamber outlet 136 allows magnetic beads to move between the oil phase in the isolation chamber and the aqueous phase in the washing chamber 135. The cleansing chamber 138 has a perfusion port 134, a first outlet 139, and a second outlet 1310. The injection port 134 is used to inject eluent into the cleansing chamber 138 for elution of nucleic acid samples. The first outlet 139 allows magnetic beads to move between the oil phase in the isolation chamber and the aqueous phase in the cleansing chamber 138. The sealing orifice 121 is used to inject a temperature-controlled phase-change material into the chip to block the second outlet 1310, preventing eluent from flowing out of the room-temperature second outlet 1310.

[0094] The chip's liquid separation reaction layer 14 includes: a recovery tank 141, a liquid channel 142, a separation tank 143, an air valve 144, a reaction chamber 145, a waste liquid chamber 146, an air channel 147, and a filter chamber 148. One side of the recovery tank 141 is connected to the second outlet 1310, and the other side is connected to the liquid channel 142. It is used to recover melted solid paraffin flowing out of the second outlet 1310, preventing paraffin from flowing into the liquid channel 142. The liquid channel 142 is an arc shape, with its diameter gradually increasing from the starting point to the ending point. The liquid channel 142 is connected to multiple separation tanks 143. It is used to separate the eluent into the multiple separation tanks 143 by centrifugal force. The air valve 144 connects to the separation tank 143 and the reaction chamber 145. The waste liquid chamber 146 is connected to the liquid channel 142 and is used to collect excess liquid into the waste liquid chamber 146. Meanwhile, the waste liquid chamber 146 is also connected to the gas duct 147 for gas exhaust. One side of the gas duct 147 is connected to the filter chamber 148 for gas exhaust. The other side of the gas duct 147 is also connected to the sealing port. The filter chamber 148 is provided with a layer of solid paraffin particles, and an activated carbon layer on top. A filter membrane isolation layer 12 is provided between the two layers. When heated, the solid paraffin particles indirectly block one side of the gas duct 147. The activated carbon can filter the gas leaking from the gas duct 147.

[0095] The chip has a structure and function that includes triple amplification product leakage and contamination: The role of oil in preventing cross-contamination in reaction chamber 145 When the second outlet 1310 and the sealed hole 121 connected to the gas channel 147 are heated, the paraffin wax in the second outlet and the sealed hole 121 melts and opens. The nucleic acid extraction product will first enter the liquid channel 142 and the separating tank 143 under the action of centrifugal force. Finally, the oil phase in the isolation chamber 123 will also enter the liquid channel 142, thus isolating each reaction chamber 145. This avoids cross-contamination between the reaction chambers 145.

[0096] Leak-proof function of paraffin in filter chamber 148 After the amplification reaction occurs in the reaction chamber 145, there is a possibility of nucleic acid aerosol leakage inside the chip. Before chip amplification, heating the filter chamber 148 will cause the solid paraffin inside the chamber to melt and block the gas passage 147 on one side of the filter chamber 148 to prevent aerosol leakage.

[0097] The leak-proof function of activated carbon The activated carbon in the filter chamber 148 can absorb aerosols that may accidentally leak through the airway 147.

[0098] In other examples, the preparation of the amplification reagents may include: This example uses a Mycoplasma pneumoniae detection reagent as an example. The isothermal amplification reagent for detecting Mycoplasma pneumoniae is prepared into lyophilized pellets through solution preparation, dropping, and lyophilization.

[0099] The reagents required for isothermal amplification were prepared according to a certain volume, as shown in Table 1.

[0100] Table 1. Amplification reagent formulation Using a dispensing apparatus, droplets were generated at a rate of 20 μL / drop and rapidly frozen into ice balls in liquid nitrogen. The ice balls were then placed in a freeze dryer and freeze-dried according to the procedure shown in Table 2. After freeze-drying, the freeze-dried balls were stored in a sealed container containing a desiccant for later use.

[0101] Table 2. Freeze-drying process In other examples, the preparation method of the nucleic acid extraction reagent may include: This example illustrates the preparation and formulation of nucleic acid extraction reagents. Following the formulas shown in Table 3, prepare the necessary lysis buffer, washing buffer, and elution buffer for nucleic acid extraction. The magnetic beads can be placed in either the washing buffer or the lysis buffer. Store the prepared solutions at room temperature for later use.

[0102] Table 3. Preparation of nucleic acid extraction reagents In other examples, the reagent encapsulation method includes encapsulating the prepared lyophilized pellets and nucleic acid extraction reagents into the aforementioned chip. The reagent encapsulation process is as follows: Encapsulation of the lyophilized beads: Place the lyophilized beads inside the reaction chamber 145, and seal the chip with the front sealing film 2, thus encapsulating the lyophilized beads inside the chip. For example... Figure 6 As shown. The front sealing film 2 includes: a pivot hole 21, a sample dispensing opening 22, and a breathable membrane 23, with the breathable membrane 23 corresponding to the position of the filter chamber 148. The breathable membrane 23 is a 0.22µm microporous filter membrane bonded to the front sealing film 2 with sealant. The pivot hole 21 is located at the center of the membrane. The sample dispensing opening 22 corresponds to the position of the sample dispensing port 131 on the chip.

[0103] The perfusion of nucleic acid extraction reagents includes perfusion of solid paraffin, liquid perfusion, and chip encapsulation, as detailed below: Injection of solid paraffin: Molten solid paraffin is used to plug the pyrolysis chamber outlet 133 through the injection sealing port, and then the second outlet 1310 is plugged through the injection sealing hole 121.

[0104] Liquid perfusion: Pour liquid paraffin into the perfusion inlet 122. At the same time, pour the cleaning liquid into the perfusion hole 134 in the cleaning chamber 135. When the cleaning liquid reaches the cleaning chamber outlet 136, stop pouring the cleaning liquid. Then pour the eluent from the perfusion hole 134. When the elution chamber 138 is filled with the eluent, stop pouring the eluent. When the isolation chamber 123 is filled with liquid paraffin, stop pouring the liquid paraffin. Put the lysis solution, or the lyophilized lysis solution, into the lysis chamber 132.

[0105] Chip encapsulation Put the solid paraffin particles into the filtration chamber 148, then place a layer of filter membrane as the isolation layer 12 on the solid paraffin, and then fill the filtration chamber 148 with activated carbon. Seal the back of the chip with the back sealing film 3, thereby sealing the perfusion inlet 122, the perfusion outlet 124, the sealing hole 121, and the perfusion hole 134 of the chip. Finally, seal the sample loading opening 22 and the air vent hole with a sticker.

[0106] In some other examples, refer to Figure 10 , the liquid separation control method of the chip includes: Through experimental research and analysis of this application, it is found that: in order to flow from the sample processing chamber to the liquid channel 142, then to the liquid separation tank 143, and finally to the reaction chamber 145 at one time. Due to the effect of surface tension, three resistances F1, F2, and F3 need to be overcome, as Figures 7 to 9 shown. These three resistances are controlled by centrifugal force to enable the liquid to flow out of the sample processing chamber to the reaction chamber 145. The following stages will be experienced: Stage of liquid entering the flow channel. When the liquid flows out of the sample processing chamber into the liquid channel 142, since the surface area of F1 is very large, the centrifugal force F needs to satisfy F1 < F < F2. At this time, only the resistance F2 needs to be overcome to allow the liquid to flow out and enter the liquid separation tank 143, but it is blocked by the resistance F2 of the air valve 144.

[0107] Stage of liquid filling the liquid separation tank 143. When the previous several liquid separation tanks 143 are filled with liquid, due to the certain volume of the liquid channel 142, the flow of the liquid needs to overcome two surface tensions F1 and F2 at this time. At this time, compared with the previous stage, the surface area of F1 becomes smaller and the surface tension becomes larger. The two surfaces of the liquid are in such a state that the sample in the liquid channel 142 can flow into the liquid separation tank 143. When the real-time centrifugal force F > F1 + F2, it can flow forward.

[0108] Stage of liquid entering the reaction chamber 145. When all the liquid separation tanks 143 are filled with liquid, when the centrifugal force F > F3, the liquid can enter the reaction chamber 145.

[0109] To accurately control the flow of a liquid, three significant gradients are required: F1, F1 + F2, and F3. Only by overcoming each of these three gradients can the flow of the liquid be controlled.

[0110] To compare the relationship between the liquid flow and rotational speed in the three stages mentioned above, with and without the oil phase flowing into liquid channel 142, two sets of experiments were conducted. In set 1, the isolation chamber 123 was left empty, while in set 2, the isolation chamber 123 was filled with liquid paraffin. Each experiment was repeated three times. The results show that the inflow of the oil phase into liquid channel 142 significantly reduces the centrifugal force in the second stage. Therefore, only two rotational speed gradients are needed to accurately control the liquid flow.

[0111] In other examples, reaction control devices based on centrifugal microfluidic isothermal amplification chips. A centrifugal microfluidic isothermal amplification chip reaction control device is used to control the following processes of the chip: sample lysis, washing, elution, liquid separation, amplification reaction, and fluorescence detection, as well as contamination prevention. This centrifugal microfluidic isothermal amplification chip reaction control device includes: a drive motor, a heating stage, a fluorescence detection module, and a magnet.

[0112] The drive motor, connected to the chip carrier via a rotating shaft, provides power for the chip's rotation and controls the speed, direction, and position of the chip body 1. The chip body 1 is mounted on the chip carrier, which supports and drives the chip to rotate horizontally. Below the chip body 1 is a heating stage, which provides heat to the chip body 1 and controls its heating temperature. The heating stage is fixed to a connector below it. The connector is fixed to a slider. The slider and heating stage are confined to a slide rail, allowing only up-and-down movement. The up-and-down movement of the heating stage is controlled by a cam motor. A fluorescence detection hole is provided on the heating stage, and a fluorescence detection module is located below it. The fluorescence module emits excitation light to detect the fluorescence emitted during the reaction process of the chip body 1. A swing arm motor is connected to a swing arm via a rotating shaft, and a magnet is mounted on the other end of the swing arm. Driven by the swing arm motor, the magnet rotates and moves on a horizontal plane to manipulate the movement of the magnetic beads within the chip. The heating stage also has a swing arm through-hole, ensuring that the up-and-down movement of the heating stage does not affect the movement of the swing arm.

[0113] The heating stage includes an outer ring heating area, a first sector-shaped heating area, and a fixing hole 11. The heating stage connects to a connector via the fixing hole 11. The outer ring heating area is annular and used to heat multiple reaction chambers 145 of the chip. The first and second sector-shaped heating areas are used to heat the nucleic acid extraction area of ​​the chip. The heating stage has a swing arm through-hole to provide space for the swing arm to rotate, allowing the magnetic beads to operate on the magnetic beads inside the chip. Driven by a cam motor, the heating stage can move up and down, thereby changing the distance between the heating stage and the chip, enabling the chip to be attached to or separated from the heating stage, and facilitating switching between a metal bath and an air bath.

[0114] The magnet is fixed to the swing arm, which is driven by a servo motor and moves in the swing arm direction. The magnetic force is used to manipulate the movement of the magnetic beads in the microfluidic chip.

[0115] In other examples, this example illustrates the use of a packaged microfluidic chip, including: sample loading, sample lysis, sample washing, sample elution, sample separation, and amplification reaction.

[0116] Adding samples Remove the adhesive label from the chip and add 200 μL of liquid sample into the lysis chamber 132 through the sample application port 131. Seal the sample application port 131 with a new adhesive label attached to the sample application opening 22. Place the chip onto the chip holder of the reaction control device.

[0117] Sample lysis A cam motor rotates, lowering the heating stage and separating the chip from it. A drive motor rotates the chip, moving the lysis chamber 132 to the first sector-shaped heating area. The cam motor then rotates, raising the heating stage and bringing the chip into contact with it. The temperature of the lysis chamber 132 rises to 50°C. The heating stage then lowers, switching to an air bath, and reciprocates, vibrating the chip and causing it to lyse within the lysis chamber 132, allowing the magnetic beads inside to bind to the nucleic acid. The temperature of the lysis chamber 132 is further increased to 60°C, melting the solid paraffin at the lysis chamber outlet 133. A drive arm motor moves, using magnets to attract the magnetic beads from the lysis chamber outlet 133, moving them into the isolation chamber 123 filled with liquid paraffin.

[0118] Sample cleaning Magnetic beads enter the aqueous phase of the cleaning chamber 135 from the oil phase through the cleaning chamber outlet 136. Rotating the chip drives a swing arm motor to move the magnet away, leaving the magnetic beads inside the cleaning chamber 135. The motor rotates the chip reciprocatingly, vibrating it and cleaning the nucleic acid-containing magnetic beads within the cleaning chamber 135 to remove impurities. The swing arm motor then re-energizes the magnet, attracting the magnetic beads out of the cleaning chamber 135 through the outlet 136, passing through the oil phase, and entering the isolation chamber 123. Rotating the swing arm and chip again transfers the magnetic beads from the oil phase back to the liquid phase of the cleansing chamber 138.

[0119] Sample elution The drive arm motor moves the magnet away, leaving the magnetic beads inside the elution chamber 138. The drive motor rotates the chip, causing it to reciprocate and vibrate, eluting the nucleic acid-laden magnetic beads in the elution chamber 138. The rotating chip moves from the elution chamber 138 to the first sector-shaped heating zone, and from the sealed orifice 121 to the second sector-shaped heating zone, heating to 70 degrees Celsius, melting the solid paraffin at the second outlet 1310 of the elution chamber 138. The solid paraffin in the sealed orifice 121 also melts. The drive arm motor then moves the magnet to attract the magnetic beads, removing them from the first outlet 139 and moving them to the washing chamber 135. At this point, the magnetic beads are removed, and the extracted nucleic acid product remains in the elution chamber 138.

[0120] Sample separation The heating platform descends, switching to an air bath. As the solid paraffin in the second outlet 1310 melts, it is centrifugally drawn into the recovery tank 141 at a rotation speed of 600 rpm. Meanwhile, the nucleic acid extraction product flows out of the second outlet 1310 and, driven by centrifugal force, enters the liquid channel 142. The nucleic acid extraction product flows into the separating tank 143 through the liquid channel 142. Due to the obstruction of the air valve 144, the nucleic acid extraction product flows sequentially into the separating tank 143 but not into the reaction chamber 145. Since the separating tank 143 has a uniform volume, the nucleic acid extraction product is evenly distributed. Excess liquid flows into the waste liquid chamber 146. Because the cleaning chamber 138 is connected to the isolation chamber 123, and the isolation chamber 123 is connected to the filter chamber 148 via the air channel 147, some of the liquid paraffin in the isolation chamber 123 flows into the liquid channel 142.

[0121] The rotation speed is further increased to 1200 rpm. At this point, the liquid in the separating tank 143 overcomes the resistance of the air valve 144 and enters the reaction chamber 145. Excess liquid paraffin flows into the separating tank 143 through the liquid channel 142, sealing the reaction chamber 145. The chip is rotated, moving the chip's filter chamber 148 to the first sector heating zone. The heating platform rises, switching to metal bath heating of the chip to 70 degrees Celsius, melting the solid paraffin in the filter chamber 148 and thus sealing the filter chamber 148.

[0122] Amplification reaction After the nucleic acid extraction product enters reaction chamber 145, the liquid melts the lyophilized bulbs, at which point the nucleic acid extraction product mixes with the melted enzymes and dNTPs. The outer ring of the chip is heated to 65°C, and isothermal amplification occurs within the reaction zone. If the sample contains the nucleic acid sequence to be detected, the reaction strong reaction will be excited by the fluorescence detection module and emit fluorescence. The fluorescence signal is detected by the fluorescence detection module. To detect signals in multiple reaction chambers 145 in turn, the heating stage switches to an air bath at fixed intervals, such as 1 minute, and then rotates at fixed angles sequentially, detecting each reaction chamber 145 sequentially through the fluorescence detection module. After each detection, the heating stage switches to a metal bath, heating the chip through contact with the heating zone.

[0123] Because the LAMP reaction generates a large amount of amplification products, the reaction chamber 145 is isolated by liquid paraffin, making it difficult to generate aerosols. Furthermore, the filter chamber 148 of the chip is sealed by melted paraffin, reducing the possibility of amplification product leakage. Finally, to prevent incomplete sealing by paraffin, activated carbon is placed inside the filter chamber 148 to adsorb any leaked nucleic acids, further reducing the risk of leakage. This achieves triple protection, preventing leakage of amplification products.

[0124] To verify the performance of this chip in the detection of Mycoplasma pneumoniae, Mycoplasma pneumoniae culture was used as a standard. The chip and detection reagent of this application were compared with those of a commercially available Mycoplasma pneumoniae nucleic acid detection kit (PCR-fluorescence probe, medical device registration number 20223401366), by serially diluting the Mycoplasma pneumoniae culture tenfold (10x, 100x, 1000x, 10000x) to compare the detection limits. Results were compared with those of the reagents. Figure 11 And the test results of this application Figure 12 It can be seen that after the positive sample was diluted to 10³ and 10⁴, the control reagent could not detect it, while this method could still detect the positive result. Furthermore, the detection time was significantly shortened.

[0125] Reference Figure 13 In other examples, three sets of experiments were set up to test the leakage prevention performance of the chips, as shown in Table 4. Chips that might leak were placed in a container containing ddH2O. Twelve chips that tested positive for Mycoplasma pneumoniae were divided into two groups. Six chips were placed at room temperature for 15 days, and the other group was placed in a 40°C oven for 15 days. As a positive control group, six tubes of Mycoplasma pneumoniae amplification-positive samples were placed in a 40°C oven for 15 days. Finally, 15 samples were taken, and their positivity / negativeness was tested using lyophilized microspheres containing Mycoplasma pneumoniae amplification.

[0126] Table 4 Comparative Experiment Groups As can be seen from the above, no leakage was detected in the positive chip, whether it was processed at room temperature or in a 40°C oven. The control group, however, produced normal positive results.

[0127] In the accompanying drawings of this application, the same or similar reference numerals correspond to the same or similar components. In the description of this application, it should be understood that if terms such as "upper," "lower," "left," "right," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, they are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the accompanying drawings are only for illustrative purposes and should not be construed as limiting this patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0128] The above are merely preferred examples of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application shall be included within the scope of protection of this application.

Claims

1. A centrifugal microfluidic isothermal amplification chip, characterized in that, The centrifugal microfluidic isothermal amplification chip is a layered structure that expands from the center to the periphery. The layered structure includes, from the center outward, a fixing hole, an isolation layer, a nucleic acid extraction layer, and a liquid separation reaction layer. Each layer is connected through interlayer channels, and fluid flows through each functional area in sequence by centrifugal force. In this embodiment, at least a portion of the interlayer channel or each functional area is provided with phase change material to form a phase change valve.

2. The centrifugal microfluidic isothermal amplification chip according to claim 1, characterized in that, The phase change material is a temperature-controlled phase change material; or, the phase change material is a pressure-controlled phase change material.

3. The centrifugal microfluidic isothermal amplification chip according to claim 1, characterized in that, The isolation layer is disposed around the fixing hole, and the functional areas on the isolation layer include interconnected closed holes, injection inlets, isolation chambers and injection outlets; The sealing hole is provided on the isolation cavity to seal any openings or potential leakage points on the isolation cavity except for the preset injection channel, so as to ensure the sealing of the isolation cavity, prevent the injection material from leaking from unexpected paths during the injection process, and maintain the injection pressure and injection fullness. The injection port is connected to the isolation chamber. The injection port serves as the starting point for the injection material to enter the isolation chamber and is used to connect with external injection equipment or material supply system to deliver the injection material into the isolation chamber. The infusion outlet is located on the isolation chamber and is used to discharge the air or other media originally in the isolation chamber at the initial stage of infusion, and to determine whether the isolation chamber has been completely filled by observing whether the infusion material flows out smoothly during the infusion process. The injection outlet is located at the other end of the isolation chamber or at a preset position. When continuous, bubble-free injection material flows out of the injection outlet, it indicates that the isolation chamber is full. The location and structure of the injection port are configured to ensure that the injection material is evenly distributed within the isolation cavity and to reduce the generation of air bubbles. The sealing hole, the injection inlet, the isolation chamber, and the injection outlet together constitute a complete injection system. The sealing hole provides a basic guarantee for the injection to have a sealed environment, the injection inlet is a material input channel, the isolation chamber is the area for material filling and action, and the injection outlet is the area for process control and result confirmation.

4. The centrifugal microfluidic isothermal amplification chip according to claim 3, characterized in that, The nucleic acid extraction layer is disposed around the isolation layer, and the functional areas on the nucleic acid extraction layer include a connected sample dispensing port, a lysis chamber, a washing chamber, and a elution chamber; The outlet of the sample dispensing port is connected to the inlet of the lysis chamber to quantitatively transfer the original sample received by the sample dispensing port to the lysis chamber; The outlet of the pyrolysis chamber is connected to the inlet of the cleaning chamber, so that the sample that releases the target analyte after pyrolysis can enter the cleaning chamber for purification. The outlet of the washing chamber is connected to the inlet of the scavenging chamber so that the sample after impurities have been removed can be transferred to the scavenging chamber for separation and acquisition of the target analyte. The sample loading port, the lysis chamber, the washing chamber, and the scavenging chamber are connected in series according to the sample processing flow, forming a complete processing path from the input of the original sample to the output of the target analyte solution.

5. The centrifugal microfluidic isothermal amplification chip according to claim 4, characterized in that, The sample dispensing port has an inner diameter of 1mm~2mm and a depth of 3mm~5mm; or, The lysis chamber has a volume of 50 μL to 100 μL, and a micropillar array structure is provided inside the lysis chamber. The diameter of the micropillars is 50 μm to 100 μm, and the spacing is 100 μm to 200 μm. or, The cleaning chamber is provided with a first outlet and a second outlet, and each outlet is provided with solid paraffin to form the phase change valve.

6. The centrifugal microfluidic isothermal amplification chip according to claim 4, characterized in that, The liquid separation reaction layer is disposed around the nucleic acid extraction layer. The functional areas on the liquid separation reaction layer include a connected recovery tank, liquid channel, liquid separation tank, air valve, reaction chamber, waste liquid chamber, air channel and filter chamber. The recovery tank is connected to the distribution tank via a liquid channel to transport the liquid collected in the recovery tank to the distribution tank. The liquid channels are respectively connected to the recovery tank and the distribution tank, the distribution tank and the reaction chamber, and the distribution tank and the waste liquid chamber, forming a path for the liquid to flow between the recovery tank, the distribution tank, the reaction chamber and the waste liquid chamber; The separating tank is connected to the reaction chamber and the waste liquid chamber through different liquid channels, so as to transport the separated different component liquids to the reaction chamber or the waste liquid chamber respectively; The air valve is installed on the air passage, and the air passage is connected to the reaction chamber. By adjusting the air valve, the on / off state and flow rate of the gas in the air passage are controlled, thereby changing the gas pressure in the reaction chamber. The reaction chamber is connected to the liquid distribution tank through the liquid channel to receive liquid, and is connected to the air valve and the filter chamber through the gas channel to allow gas to enter or exit. The waste liquid chamber is connected to the liquid distribution tank through the liquid channel to receive and store the waste liquid transported from the liquid distribution tank; The air passage connects the reaction chamber, the filter chamber, and the air valve, and is used to deliver gas to the reaction chamber and deliver the gas generated in the reaction chamber to the filter chamber. The filter chamber is installed at the end of the air passage and is connected to the reaction chamber through the air passage to receive and filter the gas discharged from the reaction chamber.

7. The centrifugal microfluidic isothermal amplification chip according to claim 6, characterized in that, At least part of the liquid channel is arc-shaped, and its diameter gradually increases from the starting point to the ending point.

8. The centrifugal microfluidic isothermal amplification chip according to claim 6, characterized in that, The number of reaction chambers is 4 to 8, arranged in a ring array, and the volume of each reaction chamber is 10 μL to 20 μL. The reaction chamber contains lyophilized enzyme reagent. The filter chamber includes a solid paraffin particle layer and an activated carbon layer, with a filter membrane isolation layer disposed between the solid paraffin particle layer and the activated carbon layer. The activated carbon filling amount is 10mg~20mg, and it is connected to the isolation chamber through an air passage.

9. The centrifugal microfluidic isothermal amplification chip according to claim 6, characterized in that, The isolation chamber is connected to the filtration chamber, the cleaning chamber is connected to the inlet of the liquid channel, the outlet of the liquid channel is connected to the separating tank, and the separating tank is connected to the waste liquid chamber.

10. The centrifugal microfluidic isothermal amplification chip according to any one of claims 1 to 9, characterized in that, The centrifugal microfluidic isothermal amplification chip includes a chip body, a front sealing film covering a first side of the chip body, and a back sealing film covering a second side of the chip body. The front sealing film has a rotating shaft hole, a sample dispensing opening, and a breathable membrane. The rotating shaft hole corresponds to the fixing hole, the sample dispensing opening corresponds to the sample dispensing port, and the breathable membrane corresponds to the filter chamber.

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