Portable nucleic acid isothermal amplification device and method using flexible thin film heating element

Abstract

The application discloses a portable nucleic acid isothermal amplification device and method using a flexible film heating element, comprising a heating unit and a control unit; the heating unit comprises a heat preservation shell and a top cover matched with the heat preservation shell, a plurality of reaction cavities are arranged between the heat preservation shell and the top cover, a flexible film heating element, a heat conduction fixed sleeve and a reaction tube are coaxially and sequentially sleeved from outside to inside in each reaction cavity; during assembly, the flexible film heating element is wound into a cylindrical shape and is sleeved on the outer wall of the heat conduction fixed sleeve; the flexible film heating element comprises a PI base film, an electrothermal circuit formed on the surface of the PI base film and a resistance temperature sensor integrally prepared with the electrothermal circuit; the control unit comprises a circuit board, the resistance temperature sensor is electrically connected with the resistance temperature sensor through sensor electrodes of the resistance temperature sensor, the electrothermal circuit is electrically connected with the electrothermal circuit through circuit electrodes of the electrothermal circuit, and the heating power of the electrothermal circuit is controlled through a PWM pulse width regulation mode.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a portable nucleic acid isothermal amplification device and method using a flexible thin-film heating element. Background Technology

[0002] Nucleic acid isothermal amplification technologies (such as LAMP and RPA) do not require temperature cycling and can complete nucleic acid amplification at a constant temperature. They have the advantages of simple operation and short time consumption, and have important application value in scenarios such as on-site testing.

[0003] Existing nucleic acid isothermal amplification instruments have the following main shortcomings: traditional heating modules (such as metal heating blocks and semiconductor cooling chips) are large in size and have slow thermal response speed, making it difficult to achieve miniaturization and portability of the device; the contact between the heating element and the reaction tube is mostly planar contact, which is not close enough and affects the heat transfer efficiency and temperature uniformity; existing heating elements and temperature sensors are independent of each other and have low integration, resulting in limited temperature control accuracy.

[0004] Therefore, it is necessary to provide a portable nucleic acid isothermal amplification device with high integration, excellent heat transfer efficiency, and precise temperature control. Summary of the Invention

[0005] The purpose of this invention is to provide a portable nucleic acid isothermal amplification device and method using a flexible thin-film heating element, which is suitable for rapid nucleic acid amplification and detection in point-of-care testing (POCT) scenarios, in order to overcome the defects of the existing technology. This invention can achieve miniaturization and weight reduction of the device, while improving heating uniformity and temperature control accuracy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A portable nucleic acid isothermal amplification device employing a flexible thin-film heating element includes a heating unit and a control unit; The heating unit includes an insulation shell and an upper cover that cooperates with the insulation shell. Several reaction chambers are provided between the insulation shell and the upper cover. In each reaction chamber, a flexible thin film heating element, a heat-conducting fixing sleeve and a reaction tube are coaxially sleeved from the outside to the inside. During assembly, the flexible thin film heating element is wound into a cylindrical shape and sleeved on the outer wall of the heat-conducting fixing sleeve. The flexible thin-film heating element includes a PI substrate film, an electrothermal circuit formed on the surface of the PI substrate film, and a resistive temperature sensor integrally formed with the electrothermal circuit. The control unit includes a circuit board, which is electrically connected to the resistive temperature sensor through the sensor electrode of the resistive temperature sensor, and electrically connected to the electric heating circuit through the circuit electrode of the electric heating circuit, and controls the heating power of the electric heating circuit through PWM pulse width modulation.

[0007] Furthermore, the reaction chamber is provided with four chambers, and each reaction chamber is independently temperature-controlled.

[0008] Furthermore, both the insulation shell and the top cover are made of polylactic acid material, and the control unit is provided with an outer shell, which is fixedly connected to the insulation shell as a whole.

[0009] Furthermore, the heat-conducting fixing sleeve is made of aluminum alloy material using 3D printing technology, and its inner wall matches the outer wall of the reaction tube, while its outer wall matches the inner wall of the flexible thin film heating element after it is wound.

[0010] Furthermore, the electrothermal circuit is composed of a titanium layer, a copper layer, a nickel layer, and a gold layer sequentially deposited on the surface of the PI substrate film. The titanium layer has a thickness of 50 nm, the copper layer has a thickness of 800 nm, the nickel layer has a thickness of 20 nm, and the gold layer has a thickness of 30 nm. The heating wire of the electrothermal circuit has a linewidth of 1000 μm, and there is no portion between adjacent heating wires where a resistive temperature sensor is embedded. The spacing between adjacent heating wires is 1000 μm.

[0011] Furthermore, the resistive temperature sensor and the electrothermal circuit are fabricated using the same photolithographic patterning and magnetron sputtering process; The heating circuit is arranged in a serpentine manner, with its two ends connected to the circuit electrodes respectively; the resistive temperature sensor is arranged in a serpentine manner in the gap between adjacent folds of the heating circuit, with its two ends connected to the sensor electrodes respectively. The resistive temperature sensor has a sensing wire width of 100μm, an adjacent sensing wire spacing of 300μm, and a sensing wire spacing of 400μm from an adjacent heating wire.

[0012] Furthermore, the circuit board integrates a signal acquisition module, a digital-to-analog converter module, a microcontroller, a PWM drive module, and a power management module; The microcontroller is programmed with a PID temperature control program. This program converts the acquired resistance value into a temperature value based on a pre-calibrated resistance-temperature curve, and outputs a PWM signal with a corresponding duty cycle to the PWM drive module based on the deviation between the measured temperature and the preset target temperature. The signal acquisition module is electrically connected to the sensor electrode of the resistive temperature sensor and is used to acquire the real-time resistance value of the resistive temperature sensor. The digital-to-analog converter is electrically connected to the signal acquisition module and is used to convert the analog temperature signal output by the signal acquisition module into a digital signal and transmit it to the microcontroller. The PWM drive module is electrically connected to the microcontroller and is used to receive the PWM control signal output by the microcontroller and adjust the heating power of the flexible thin-film heating element by controlling the on / off state of the heating circuit of the heating circuit containing the flexible thin-film heating element.

[0013] Furthermore, the power management module is used to convert the externally input 48V DC power into 12V, 5V and 3.3V DC power outputs, respectively, for the DC voltage power supply of the PWM drive module, the digital-to-analog conversion module, the signal acquisition module and the microcontroller.

[0014] Furthermore, the reaction tube is a 0.2 mL standard plastic centrifuge tube.

[0015] A portable isothermal nucleic acid amplification method using a flexible thin-film heating element includes the following steps: Step 1: Prepare the reaction reagents according to actual needs, and then dispense them into reaction tubes. Step 2: Open the top cover and vertically insert the sealed reaction tube into the corresponding heat-conducting fixing sleeve of the reaction chamber. After loading, close the top cover to form a closed heat-insulating space in the reaction chamber. Step 3: After powering on, set the target amplification temperature and amplification time for each reaction chamber in the temperature control program. After starting the device, the microcontroller on the circuit board drives the PWM drive module to output a PWM signal. The electric heating circuit in the flexible thin film heating element is energized to generate Joule heat. The heat is conducted sequentially through the heat-conducting fixing sleeve to the reaction tube and the reaction reagents therein, and the device enters the rapid heating stage. Step 4: The resistive temperature sensor continuously collects real-time temperature signals, which are then processed by the signal acquisition module and the digital-to-analog converter module and transmitted to the microcontroller. The microcontroller runs a PID control program and dynamically adjusts the PWM duty cycle according to the deviation between the measured temperature and the preset target temperature. This precisely controls the output power of the flexible thin-film heating element and keeps the reaction chamber temperature stable within the allowable error range of the preset target amplification temperature. The amplification reaction is then carried out under this constant temperature condition. Step 5: After amplification is complete, remove the reaction tube and add the reaction reagent to the loading area of ​​the lateral flow chromatography detection card that matches the amplified nucleic acid. After chromatographic development, read the detection result.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects: First, the flexible heating element integrates heating and temperature measurement functions, achieving precise in-situ temperature control. The resistive temperature sensor and the heating circuit are integrated onto the PI substrate thin film surface using the same photolithography patterning and magnetron sputtering process. This allows the temperature sensor to be directly embedded within the heating element, enabling real-time in-situ monitoring of the reaction chamber temperature and eliminating measurement errors introduced by independently installed sensors due to positional deviations and thermal conduction lags. The acquired real-time temperature signal is directly input to the microcontroller, forming a high-frequency closed-loop feedback loop with the built-in PID temperature control program. This effectively overcomes system thermal inertia, enabling the reaction chamber temperature to quickly establish and stably maintain within the preset target temperature range of ±0.5℃. This significantly reduces the risk of enzyme inactivation due to temperature fluctuations, providing stable and reliable isothermal boundary conditions for nucleic acid isothermal amplification reactions.

[0017] Secondly, the structural design of this invention optimizes the heat conduction path, improves the system's energy utilization efficiency, and achieves miniaturization and weight reduction of the device. The streamlined electrothermal circuit is transformed from a two-dimensional planar configuration to a three-dimensional cylindrical configuration through winding, precisely fitting the outer wall of the 3D-printed aluminum alloy thermally conductive fixing sleeve; the inner wall of the thermally conductive fixing sleeve is also tightly fitted to the outer wall of the reaction tube. The above structure eliminates the gap thermal resistance at the heat transfer interface, realizing uniform 360° radial enveloping heat conduction to the reaction tube. While reducing the input power required to maintain a constant temperature, it also reduces the structural thickness required for the polylactic acid (PLA) insulation shell and top cover to meet the heat-locking requirements, thereby achieving weight reduction and miniaturization of the device while ensuring thermal insulation performance. In addition, the low thermal conductivity of PLA material effectively shields the heating unit from thermal radiation interference to the circuit board below, ensuring the stable operation of the control circuit.

[0018] Finally, the independent temperature control design of each reaction chamber effectively suppresses thermal crosstalk between channels and supports parallel detection of multiple samples. This invention features four structurally independent reaction chambers. Each chamber contains a flexible thin-film heating element and a resistive temperature sensor, forming an independent heating and temperature measurement unit. Each channel is individually temperature-controlled in a closed loop. A low-thermal-conductivity PLA insulation shell circumferentially encloses the four reaction chambers, structurally creating mutually isolated thermal boundaries for each channel and effectively suppressing thermal crosstalk between channels. This design enables each reaction chamber to simultaneously perform independent detection of different targets at different target temperatures, improving sample screening throughput and application flexibility in real-time detection scenarios. Attached Figure Description

[0019] The accompanying drawings are provided to further understand the invention and constitute a part of this invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 This is an overall structural view of the portable nucleic acid isothermal amplification device of the present invention; Figure 2 This is a cross-sectional view of the portable nucleic acid isothermal amplification device of the present invention; Figure 3 This is a schematic diagram of the flexible thin-film heating element in this invention; Figure 4 This is a structural unfolded view of the flexible thin-film heating element in this invention; Figure 5 This is a schematic diagram of the temperature control system in this invention.

[0021] Among them, 1 is the top cover; 2 is the heat-insulating shell; 3 is the shell; 4 is the circuit board; 5 is the flexible thin film heating element; 6 is the thermally conductive fixing sleeve; 7 is the reaction tube; 8 is the reaction reagent; 9 is the resistance temperature sensor; 10 is the electric heating circuit; 11 is the sensor electrode; and 12 is the circuit electrode. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0023] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0024] Example 1 This embodiment provides a portable nucleic acid isothermal amplification device using a flexible thin-film heating element. The device is divided into two functional units: the upper part is the heating unit, and the lower part is the control unit, which are integrated into one unit through a tight snap-fit ​​mechanism. The portable nucleic acid isothermal amplification device has a compact structure, measuring 100mm in length, 100mm in width, and 75mm in height, making it easy to carry and use in the field.

[0025] See Figure 1 The heating unit is externally formed by a top cover 1 and an insulation shell 2, creating a sealed cavity. The top cover 1, made of PLA material, is located at the top of the device and can be opened and closed vertically to seal the upper opening of the reaction chamber, preventing heat loss from the top of the reaction chamber. The insulation shell 2, also made of PLA material, has low thermal conductivity and is lightweight, providing circumferential coverage for the four reaction chambers of the heating unit, effectively reducing lateral heat loss and ensuring the constant temperature environment required for isothermal amplification. The control unit is encapsulated in a shell 3, housing a circuit board 4 and related control circuits. The shell 3 and the insulation shell 2 are fixed together by snap-fit ​​or threaded connection, achieving a high degree of integration between the heating unit and the control unit.

[0026] See Figure 2 The heating unit has four identical and independent reaction chambers, which can simultaneously heat four reaction tubes 7, supporting parallel detection of multiple samples. Each reaction chamber consists of a flexible thin-film heating element 5, a thermally conductive fixing sleeve 6, and a reaction tube 7 coaxially fitted from the outside to the inside. The flexible thin-film heating element 5 is wound into a cylindrical shape and tightly fitted onto the outer wall of the thermally conductive fixing sleeve 6, forming a ring-shaped enveloping heating contact; the inner wall of the thermally conductive fixing sleeve 6 precisely matches the outer wall of the reaction tube 7 to ensure sufficient thermal contact; the reaction tube 7 contains reaction reagents 8 containing nucleic acids.

[0027] The thermally conductive fixing sleeve 6 is manufactured using aluminum alloy through 3D printing. Aluminum alloy has high thermal conductivity, enabling efficient axial heat conduction. Its inner wall contour is precisely adapted to the shape of the outer wall of the reaction tube 7, and the outer wall contour matches the inner wall of the flexible thin-film heating element 5 wound into a cylinder, ensuring sufficient thermal contact area on both the inner and outer sides and reducing interfacial thermal resistance. The reaction tube 7 uses a commercially available 0.2mL standard plastic centrifuge tube, which is compatible with general nucleic acid amplification consumables, facilitating operation and result detection.

[0028] See Figure 3 and Figure 4The flexible thin-film heating element 5 uses a PI substrate film with a thickness of 50 μm as the flexible substrate. A 50 nm thick titanium layer, an 800 nm thick copper layer, a 20 nm thick nickel layer, and a 30 nm thick gold layer are sequentially deposited on the surface of the PI substrate film using photolithography patterning combined with magnetron sputtering to form a streamlined electrothermal circuit 10. The wire width of the electrothermal circuit 10 is 1000 μm, and the spacing between adjacent heating wires is 1000 μm in areas where no sensing wires are inserted. The titanium layer serves as an adhesion layer, enhancing the bonding force between the metal film and the PI substrate; the copper layer has low resistivity and excellent conductivity, serving as the main resistive heating layer; the nickel layer, located between the copper and gold layers, acts as a diffusion barrier layer, effectively suppressing the mutual diffusion of copper and gold atoms under thermal stress and electric field. The gold layer, located on the outermost side, is chemically stable, further enhancing the corrosion resistance of the element surface and effectively reducing electrode contact resistance, thus improving the reliability of the electrical connection. The streamlined wiring design ensures that the heating power is evenly distributed on the unfolded surface of the component. After being wound into a cylindrical shape and attached to the outer wall of the heat-conducting fixing sleeve 6, it can form a uniform 360° annular heating of the heat-conducting fixing sleeve 6, and the thermal uniformity is significantly better than that of the planar contact heating method.

[0029] In the fabrication process of the flexible thin-film heating element 5, the resistive temperature sensor 9 and the electrothermal circuit 10 are integrated onto a PI substrate thin film through the same photolithography patterning and magnetron sputtering process, achieving integrated fabrication of heating and temperature measurement functions. The resistive temperature sensor 9 has a sensing wire linewidth of 100 μm, a spacing of 300 μm between two adjacent sensing wires, and a spacing of 400 μm between the sensing wire and the adjacent heating wire. The resistive temperature sensor 9 measures temperature based on the principle that the resistance of a metal thin film increases linearly with temperature. The sensor electrode 11 of the resistive temperature sensor 9 is connected to the signal acquisition module on the circuit board 4 to output a temperature signal in real time. The electrothermal circuit 10 is connected to a 48V DC power supply via its circuit electrode 12. A switching device is provided in the connected circuit. The PWM drive module on the circuit board 4 controls the switching device to turn on and off, thereby adjusting the input power of the electrothermal circuit 10. The aforementioned integrated approach places the temperature sensor inside the heating element, enabling in-situ real-time monitoring of the reaction chamber temperature. This eliminates measurement errors caused by positional deviations in independently installed sensors, significantly improving the accuracy and response speed of temperature measurement.

[0030] The circuit board 4 integrates a signal acquisition module, a digital-to-analog converter module, a microcontroller, a PWM drive module, and a power management module.

[0031] The signal acquisition module is electrically connected to the sensor electrode 11 of the resistive temperature sensor 9, and is used to acquire the real-time resistance value of the resistive temperature sensor 9. The digital-to-analog conversion module is electrically connected to the signal acquisition module, and is used to convert the analog temperature signal output by the signal acquisition module into a digital signal and transmit it to the microcontroller. The PWM drive module is electrically connected to the microcontroller, and is used to receive the PWM control signal output by the microcontroller, and adjust the heating power of the flexible thin-film heating element by controlling the on / off state of the electrothermal circuit where the flexible thin-film heating element is located.

[0032] The microcontroller is programmed with a PID temperature control program. It receives and collects resistance values, converts them into corresponding temperature values ​​based on a pre-calibrated resistance-temperature curve, and runs the PID temperature control program based on the deviation between the measured temperature and the preset target temperature. Then, it compares the measured temperature value with the preset target temperature to obtain the temperature deviation. And calculate the control output according to the following control law.

[0033]

[0034] in, The control output at time t; The deviation between the measured temperature and the target temperature at time t; This is a proportionality coefficient used to quickly adjust the heating power based on the current temperature deviation; The integral coefficient is used to eliminate steady-state errors in the sampling system and ensure that the measured temperature accurately converges to the target temperature. The differential coefficient is used to suppress temperature overshoot in advance based on the changing trend of temperature deviation. The above three parameters are pre-tuned according to the thermal characteristics of the reaction chamber to achieve optimal control effect. The PID temperature control module converts the calculated control output u(t) into a PWM signal with a corresponding duty cycle, and outputs it to the switching device between the heating circuit and the input 48V DC power supply via the PWM drive module. The switching device periodically controls the on / off state of the heating circuit according to the duty cycle of the PWM signal, thereby adjusting the actual input power of the flexible thin film heating element, so that the temperature of the reaction chamber rises rapidly and is stably maintained at the preset target temperature to meet the strict requirements of temperature consistency and stability for nucleic acid isothermal amplification reaction.

[0035] The power management module is electrically connected to the microcontroller, signal acquisition module, digital-to-analog converter module, PWM drive module, and flexible thin-film heating element, respectively. It can step down the 48V DC input to 12V, 5V, and 3.3V through different DC-DC circuits. The PWM drive module requires a 12V DC power supply, the digital-to-analog converter module and the signal acquisition module require a 5V DC power supply, and the microcontroller requires a 3.3V DC power supply.

[0036] Example 2 To further verify the applicability of this device in actual nucleic acid testing scenarios, the complete testing process of this device is explained using LAMP combined with Lateral Flow Assay (LFA) visualization detection as an example.

[0037] Step 1, Reaction System Preparation: Based on the target nucleic acid sequence, six specific LAMP primers were designed using primer design software, including outer primers (F3 / B3), inner primers (FIP / BIP), and loop primers (LF / LB). To enable visualization of subsequent lateral flow chromatography results, biotin and digoxigenin were introduced onto the inner primers FIP and BIP, respectively. The primers were then mixed with Bst DNA polymerase, dNTPs, appropriate buffer solutions, and the sample to be tested to prepare reaction reagent 8, which was then aliquoted into reaction tubes 7.

[0038] Step 2, loading the sample: Open the top cover 1 and vertically insert the sealed reaction tube 7 into the corresponding thermally conductive fixing sleeve 6 of the reaction chamber; 1 to 4 reaction tubes can be placed at the same time, and each chamber is independently temperature-controlled without interference. After loading, close the top cover 1 to form a closed, insulated space in the reaction chamber.

[0039] Step 3, Parameter Setting and Start-up: After powering on, set the target amplification temperature of each reaction chamber to 60~65℃ and the amplification time to 30~60 minutes in the temperature control program. The specific amplification temperature and amplification time need to be determined based on the specific target nucleic acid. After starting the device, the microcontroller on circuit board 4 drives the PWM drive module to output a PWM signal. The electrothermal circuit 10 in the flexible thin-film heating element 5 is energized to generate Joule heat. The heat is sequentially conducted through the thermally conductive fixing sleeve 6 to the reaction tube 7 and the reaction reagent 8 therein, and the device enters the rapid heating stage.

[0040] Step four, isothermal amplification: The resistive temperature sensor 9 continuously collects real-time temperature signals, which are processed by the signal acquisition module and the digital-to-analog converter module and then transmitted to the microcontroller. The microcontroller runs a PID control program, dynamically adjusting the PWM duty cycle according to the deviation between the measured temperature and the preset target temperature, precisely controlling the output power of the flexible thin-film heating element 5, and stabilizing the reaction chamber temperature within the preset target temperature ±0.5℃ range. Under this constant temperature condition, Bst DNA polymerase continuously catalyzes the strand displacement amplification reaction, and the target nucleic acid sequence is exponentially amplified under primer-mediated conditions.

[0041] Step 5: After amplification is complete, remove reaction tube 7 and add the reaction reagent to the sample loading area of ​​the lateral flow chromatography detection card. After chromatographic development, the test results can be visually interpreted within a few minutes. No additional instruments are required, making it suitable for on-the-fly detection scenarios.

[0042] Verification results show that the LAMP amplification results obtained using this device are consistent with those obtained using commercial nucleic acid amplification instruments, proving that the device of this invention can meet the requirements of temperature uniformity and stability for isothermal nucleic acid amplification detection and has practical application feasibility. Furthermore, the system of this invention has significant advantages in terms of device size, energy consumption, and detection cost, making it more suitable for miniaturized, portable, and point-of-care testing applications.

[0043] It should be noted that this embodiment uses LAMP amplification as an example for illustration. The device of the present invention is also applicable to other isothermal amplification technologies such as recombinase polymerase amplification (RPA, target temperature 37℃~42℃) and nucleic acid sequence-dependent amplification (NASBA, target temperature 41℃). Only the target temperature parameters and reaction time need to be modified in the control interface. There is no need to change the hardware structure of the device, and it has a wide range of applications.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit its scope of protection. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that after reading the present invention, they can still make various changes, modifications or equivalent substitutions to the specific implementation of the invention, but these changes, modifications or equivalent substitutions are all within the scope of protection of the pending claims of the invention.

Claims

1. A portable nucleic acid isothermal amplification device employing a flexible thin-film heating element, characterized in that, Includes a heating unit and a control unit; The heating unit includes a heat-insulating shell (2) and an upper cover (1) that cooperates with the heat-insulating shell (2). A number of reaction chambers are provided between the heat-insulating shell (2) and the upper cover (1). In each reaction chamber, a flexible thin film heating element (5), a heat-conducting fixing sleeve (6) and a reaction tube (7) are coaxially sleeved from the outside to the inside. During assembly, the flexible thin film heating element (5) is wound into a cylindrical shape and sleeved on the outer wall of the heat-conducting fixing sleeve (6). The flexible thin-film heating element (5) includes a PI substrate film, an electrothermal circuit (10) formed on the surface of the PI substrate film, and a resistance temperature sensor (9) integrally formed with the electrothermal circuit (10). The control unit includes a circuit board (4), which is electrically connected to the resistive temperature sensor (9) through the sensor electrode (11) of the resistive temperature sensor (9), and electrically connected to the electric heating circuit (10) through the circuit electrode (12) of the electric heating circuit (10), and controls the heating power of the electric heating circuit (10) through PWM pulse width adjustment.

2. The portable nucleic acid isothermal amplification device using a flexible thin-film heating element according to claim 1, characterized in that, The reaction chamber is provided with four chambers, and each chamber is independently temperature-controlled.

3. The portable nucleic acid isothermal amplification device using a flexible thin-film heating element according to claim 1, characterized in that, The heat insulation shell (2) and the top cover (1) are both made of polylactic acid material. The control unit is provided with a shell (3) on the outside. The shell (3) is fixedly connected to the heat insulation shell (2) as a whole.

4. The portable nucleic acid isothermal amplification device using a flexible thin-film heating element according to claim 1, characterized in that, The heat-conducting fixing sleeve (6) is made of aluminum alloy material by 3D printing process. Its inner wall matches the outer wall of the reaction tube (7), and its outer wall matches the inner wall of the flexible thin film heating element (5) after it is wound.

5. The portable nucleic acid isothermal amplification device using a flexible thin-film heating element according to claim 1, characterized in that, The electrothermal circuit (10) is composed of a titanium layer, a copper layer, a nickel layer and a gold layer sequentially deposited on the surface of the PI substrate film. The titanium layer has a thickness of 50 nm, the copper layer has a thickness of 800 nm, the nickel layer has a thickness of 20 nm and the gold layer has a thickness of 30 nm. The heating wire of the electrothermal circuit (10) has a line width of 1000 μm. There is no part between adjacent heating wires where a resistive temperature sensor (9) is embedded, and the distance between adjacent heating wires is 1000 μm.

6. The portable nucleic acid isothermal amplification device employing a flexible thin-film heating element according to claim 5, characterized in that, The resistive temperature sensor (9) and the electrothermal circuit (10) are fabricated using the same photolithography patterning and magnetron sputtering process; The electric heating circuit (10) is arranged in a serpentine manner, and its two ends are respectively connected to the circuit electrode (12); the resistive temperature sensor (9) is arranged in a serpentine manner in the gap between adjacent folds of the electric heating circuit, and its two ends are respectively connected to the sensor electrode (11). The resistive temperature sensor (9) has a sensing wire width of 100μm, an adjacent sensing wire spacing of 300μm, and a sensing wire spacing of 400μm with the adjacent heating wire.

7. The portable nucleic acid isothermal amplification device using a flexible thin-film heating element according to claim 1, characterized in that, The circuit board (4) integrates a signal acquisition module, a digital-to-analog conversion module, a microcontroller, a PWM drive module, and a power management module; The microcontroller is programmed with a PID temperature control program. The PID temperature control program converts the collected resistance value into a temperature value according to a pre-calibrated resistance-temperature relationship curve, and outputs a PWM signal with a corresponding duty cycle to the PWM drive module based on the deviation between the measured temperature and the preset target temperature. The signal acquisition module is electrically connected to the sensor electrode (11) of the resistive temperature sensor (9) and is used to acquire the real-time resistance value of the resistive temperature sensor (11). The digital-to-analog conversion module is electrically connected to the signal acquisition module and is used to convert the analog temperature signal output by the signal acquisition module into a digital signal and transmit it to the microcontroller. The PWM drive module is electrically connected to the microcontroller and is used to receive the PWM control signal output by the microcontroller and adjust the heating power of the flexible thin film heating element by controlling the on / off state of the heating circuit of the heating circuit where the flexible thin film heating element is located.

8. The portable nucleic acid isothermal amplification device employing a flexible thin-film heating element according to claim 7, characterized in that, The power management module is used to convert the externally input 48V DC power into 12V, 5V and 3.3V DC power outputs, which are used to power the DC voltage of the PWM drive module, the digital-to-analog converter module, the signal acquisition module and the microcontroller, respectively.

9. The portable nucleic acid isothermal amplification device using a flexible thin-film heating element according to claim 1, characterized in that, The reaction tube (7) is a 0.2 mL standard plastic centrifuge tube.

10. A portable nucleic acid isothermal amplification method using a flexible thin-film heating element, based on the portable nucleic acid isothermal amplification device using a flexible thin-film heating element as described in any one of claims 1-9, characterized in that, Includes the following steps: Step 1: Prepare the reaction reagent (8) according to actual needs, and dispense the prepared reaction reagent (8) into the reaction tube (7); Step 2: Open the top cover (1), vertically insert the sealed reaction tube (7) into the corresponding heat-conducting fixing sleeve (6) of the reaction chamber, and close the top cover (1) after loading to form a closed heat-insulating space in the reaction chamber; Step 3: After the power is turned on, the target amplification temperature and amplification time of each reaction chamber are set in the temperature control program. After the device is started, the microcontroller on the circuit board (4) drives the PWM drive module to output the PWM signal. The electric heating circuit (10) in the flexible thin film heating element (5) is powered on to generate Joule heat. The heat is conducted to the reaction tube (7) and the reaction reagent (8) in sequence through the heat-conducting fixed sleeve (6). The device enters the rapid heating stage. Step 4: The resistive temperature sensor (9) continuously collects real-time temperature signals, which are then processed by the signal acquisition module and the digital-to-analog conversion module and transmitted to the microcontroller. The microcontroller runs a PID control program and dynamically adjusts the PWM duty cycle according to the deviation between the measured temperature and the preset target temperature. It precisely controls the output power of the flexible thin film heating element (5) and keeps the reaction chamber temperature stable within the range of the preset target amplification temperature allowable error. The amplification reaction is carried out under this constant temperature condition. Step 5: After amplification is complete, remove the reaction tube (7), add the reaction reagent to the loading area of ​​the lateral flow chromatography detection card that matches the amplified nucleic acid, and read the detection result after chromatography and color development.

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