A heating device, method and nucleic acid amplification system for nucleic acid amplification

Abstract

The application discloses a heating device, a method and a nucleic acid amplification system for nucleic acid amplification, which comprises a bottom plate, a heating assembly and a control device, the heating assembly and the control device are connected to the bottom plate; the heating assembly comprises a temperature measuring electrode and a heating electrode, the temperature measuring electrode is used for measuring the real-time temperature of a reaction assembly, the reaction assembly is used for nucleic acid amplification, and the heating electrode is used for heating the reaction assembly; the control device is connected with the heating assembly and is used for controlling the heating assembly to heat. The application controls the heating assembly through the control device, controls the heating electrode to heat according to the real-time temperature measured by the temperature measuring electrode, and makes the reaction assembly quickly change temperature to reach the required temperature condition in different nucleic acid amplification reaction stages, so that fast and accurate temperature control is provided for the nucleic acid detection process, the sensitivity is high, the accuracy is high, the heating device is simple in structure, high in miniaturization degree, high in integration degree and low in cost.

Description

Technical Field

[0001] This application relates to the field of nucleic acid amplification technology, and in particular to a heating device, method and nucleic acid amplification system for nucleic acid amplification. Background Technology

[0002] PCR technology has been widely used in the detection of microbial pathogens, but traditional PCR reactions are time-consuming, the reaction and instrument are highly complex, the detection equipment is not portable, and the detection cost is high, making it difficult to promote PCR technology as a favorable method for on-site real-time detection. However, existing PCR-based detection equipment can only achieve a constant temperature detection process, and the detection efficiency is still low and the sensitivity is insufficient. Summary of the Invention

[0003] To address the problems existing in the prior art, this application provides a heating device, method, and nucleic acid amplification system for nucleic acid amplification, providing rapid and accurate temperature control for nucleic acid amplification during nucleic acid detection. It features high integration, high sensitivity, and low cost. The technical solution is as follows:

[0004] This application provides a heating device for nucleic acid amplification, including a base plate, a heating assembly, and a control device, wherein the heating assembly and the control device are connected to the base plate;

[0005] The heating component includes a temperature measuring electrode and a heating electrode. The temperature measuring electrode is used to measure the real-time temperature of the reaction component, which is used for nucleic acid amplification. The heating electrode is used to heat the reaction component.

[0006] The controller is connected to the heating assembly and is used to control the heating assembly to perform heating.

[0007] Furthermore, the heating assembly includes a substrate, an adhesive layer, and an electrode layer. The substrate is connected to the base plate, and the electrode layer is connected to the substrate through the adhesive layer. The electrode layer includes the temperature measuring electrode and the heating electrode.

[0008] Furthermore, the thickness of the adhesive layer is 40–60 nm, and the thickness of the electrode layer is 140–160 nm.

[0009] Furthermore, the substrate includes a heating region, and the electrode layer is disposed in the heating region.

[0010] Furthermore, the length of the heating area is 1–30 mm and the width is 1–30 mm.

[0011] Furthermore, the linewidth of the temperature measuring electrode is 10–50 μm, and the linewidth of the heating electrode is 1–5 mm.

[0012] Furthermore, the base plate is provided with a temperature measuring circuit and a temperature control circuit. The controller is connected to the temperature measuring electrode through the temperature measuring circuit, and the controller is connected to the heating electrode through the temperature control circuit.

[0013] Furthermore, the controller is used to output a temperature control signal to control the heating component to heat; the temperature control signal includes a first heating signal and a second heating signal, the first heating signal is used to control the temperature control circuit to be in a conducting state, and the second heating signal is used to control the temperature control circuit to be in a closed state.

[0014] This application also provides a heating method for nucleic acid amplification, wherein heating is performed based on the heating device described in any of the preceding claims, comprising:

[0015] The real-time temperature of the reaction component is obtained through a temperature measuring electrode.

[0016] The preset temperature data and the real-time temperature are processed to obtain a temperature control signal, which is then output. The temperature control signal is used to control the heating component to heat the components.

[0017] This application also provides a nucleic acid amplification system, including a reaction assembly and a heating device as described in any of the preceding claims, the heating device being used to heat the reaction assembly.

[0018] Implementing this application will have the following beneficial effects:

[0019] 1. This application controls the heating component through a controller device, and controls the heating electrode to heat according to the real-time temperature measured by the temperature measuring electrode. This enables the reaction component to rapidly change temperature to reach the required temperature conditions at different nucleic acid amplification reaction stages, providing rapid and accurate temperature control for the nucleic acid detection process. It has high sensitivity and high accuracy. Moreover, the heating device has a simple structure, high miniaturization, and high integration, and can take into account the temperature control efficiency, sensitivity, and accuracy of miniaturized heating devices, while also being low in cost.

[0020] 2. This application enhances heat transfer efficiency by using a miniaturized heating component with a preset size. On the one hand, it can greatly enhance the sensitivity of the temperature measuring electrode and the heating electrode and increase the temperature change rate. On the other hand, the heating component and the control device work together to greatly improve the real-time performance and accuracy of the heating component in temperature control, and avoid the adverse effects of excessive temperature change of the heating electrode on the nucleic acid amplification reaction when the response speed is too fast.

[0021] 3. Based on the preset temperature data of each reaction stage in the PCR amplification reaction and the real-time temperature measured by the temperature measuring electrode, this application controls the on / off of the temperature control circuit through the controller to realize the heating of the heating electrode. The feedback control is fast and sensitive, and can quickly change the temperature and maintain a good dynamic stability. It has good real-time performance, high accuracy, and high degree of automation. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in the embodiments will be briefly described below, wherein the same components are represented by the same reference numerals. Obviously, the drawings described below are merely some embodiments of this application, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0023] Figure 1 A schematic diagram of a heating device for nucleic acid amplification provided in this application;

[0024] Figure 2 for Figure 1 Exploded view of the heating device in the middle;

[0025] Figure 3 This is a schematic diagram of the connection relationship of the electrode layers in one optional embodiment provided in this application;

[0026] Figure 4 This is a schematic diagram of the electrode structure in the heating region in one optional embodiment provided in this application;

[0027] Figure 5 This is a schematic diagram of the structure of the temperature measuring electrode in one optional embodiment provided in this application;

[0028] Figure 6 A circuit diagram of the temperature measuring circuit in one optional embodiment provided in this application;

[0029] Figure 7 A circuit diagram of the temperature control circuit in one optional embodiment provided in this application;

[0030] Figure 8 A flowchart of a heating process based on a heating device is provided in this application;

[0031] Figure 9 A schematic diagram of a feedback processing algorithm provided in this application;

[0032] Figure 10 A schematic diagram of the signal output principle of the temperature control signal in one optional embodiment provided in this application;

[0033] Figure 11 This is a schematic diagram of the structure of a fluorescence detection device in one optional embodiment of this application.

[0034] The corresponding reference numerals in the attached drawings are as follows: 1-base plate, 101-single gain amplifier, 102-current limiting resistor, 111-external power supply, 112-optocoupler switch, 113-field-effect transistor, 2-heating component, 21-substrate, 210-support area, 211-cutout area, 212-heating area, 22-adhesive layer, 23-electrode layer, 230-temperature measuring electrode, 231-heating electrode, 24-thermal conductive layer, 25-substrate, 3-control device, 4-display device, 5-fixed bracket, 51-first mounting part, 52-second mounting part, 6-excitation light emitting element, 7-filter component, 71-first filter, 72-second filter, 73-focusing lens. Detailed Implementation

[0035] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments, and therefore should not be construed as limiting this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] It should be noted that the terms "first," "second," etc., used in the specification, claims, and drawings of this application 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 embodiments of this application can be implemented in orders other than those illustrated or described below. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or server that includes 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 these processes, methods, products, or devices.

[0037] Example

[0038] This application provides a heating device for nucleic acid amplification, such as... Figure 1 As shown, the heating device includes a base plate 1, a heating assembly 2, and a control device 3, such as... Figure 2As shown, both the heating component 2 and the control device 3 are connected to the base plate 1. The heating component 2 is used to heat the reaction component (not shown), which is used for nucleic acid amplification. In application, the reaction component is placed in the area corresponding to the heating component 2. The reaction component provides a place for the nucleic acid amplification reaction (i.e., PCR amplification reaction) to occur. The heating component 2 is used to heat the reaction component so that the nucleic acid amplification reaction in the reaction component can occur under the required temperature conditions. The control device 3 is connected to the heating component 2 and is used to output a temperature control signal to control the heating component 2 to heat. All components in this heating device are integrated on the base plate 1, with a high degree of integration and good miniaturization. The length and width of the heating device are less than or equal to 10cm, the overall structure is compact, occupies little space, and is easy to carry. It can be conveniently applied to on-site real-time detection. Furthermore, miniaturization helps to improve heat transfer efficiency, improve temperature uniformity, and facilitate the real-time performance of temperature control. At the same time, the control accuracy is also greatly improved, and the temperature can be quickly and accurately controlled to achieve a good dynamic stability.

[0039] In one optional embodiment, the reaction component includes either a microcentrifuge tube or a microplate, used to load reaction reagents for PCR amplification to facilitate the reaction; in a preferred embodiment, the reaction component is a microfluidic component, which is highly automated and easy to control; in a specific embodiment, the reaction component is a microfluidic chip, which is compact, highly integrated, and has good heat transfer, facilitating temperature regulation by the heating component 2; in another specific embodiment, the reaction component is a digital microfluidic chip.

[0040] Specifically, such as Figure 2 and Figure 3 As shown, the heating assembly 2 includes a substrate 21, an adhesive layer 22, and an electrode layer 23. The substrate 21 is connected to the base plate 1 and supports the adhesive layer 22 and the electrode layer 23. The electrode layer 23 is connected to the substrate 21 through the adhesive layer 22 and includes a temperature measuring electrode 230 and a heating electrode 231. Optionally, the material of the substrate 21 includes any one of silicon, silicon dioxide, silicon / silicon dioxide, and indium tin oxide glass. Optionally, the electrode material of the electrode layer 23 includes any one of gold, silver, copper, nickel, platinum, and palladium. Preferably, the electrode layer 23 is a platinum layer, and correspondingly, the adhesive layer 22 is a titanium layer, which is easy to prepare and has low cost.

[0041] Optionally, the thickness of the substrate 21 is less than or equal to 600 nm, which has good thermal conductivity; alternatively, the thickness of the substrate 21 is less than or equal to 500 nm, which is beneficial to improving the heat transfer effect, thereby improving the real-time temperature measurement of the temperature measuring electrode 230 and the response speed of the heating electrode 231, improving the temperature control efficiency and temperature control accuracy, and achieving a highly efficient and precise dynamic temperature control effect.

[0042] In one optional embodiment, the thickness of the adhesive layer 22 is 30–70 nm, and the thickness of the electrode layer 23 is 120–180 nm, resulting in good thermal conductivity. In another optional embodiment, the thickness of the adhesive layer 22 is 40–60 nm, and the thickness of the electrode layer 23 is 140–160 nm, which helps to improve the sensitivity of the electrode layer 23 to temperature changes, improve temperature control efficiency and accuracy, avoid excessive heating or cooling, and also helps to reduce costs. Optionally, the thickness of the electrode layer 23 is 145–155 nm, and the thickness of the adhesive layer 22 is 45–55 nm. Further optionally, the thickness of the electrode layer 23 is 145–150 nm, and the thickness of the adhesive layer 22 is 45–50 nm.

[0043] Specifically, such as Figure 2 As shown, the substrate 21 includes a support region 210, a hollow region 211, and a heating region 212. The support region 210 is located outside the hollow region 211 and is used to support the structure of the substrate 21, thereby improving the structural strength. The heating region 212 is located inside the hollow region 211. The electrode layer 23 and the adhesive layer 22 are disposed on the heating region 212. That is, the heating region 212, the adhesive layer 22, and the electrode layer 23 constitute the heat plate of the heating assembly 2. The temperature measuring electrode 230 and the heating electrode 231 are disposed on the heating region 212. Domain 211 extends through the substrate 21 in a direction perpendicular to the plane of the substrate 21. In a direction parallel to the plane of the substrate 21, the hollowed-out area 211 is located between the heating area 212 and the support area 210, which is used to isolate the heating area 212 from the support area 210 for effective heat insulation. On the one hand, it avoids the influence of external temperature on the heating area 212, improves the temperature measurement accuracy, and is also conducive to heat preservation. On the other hand, in a direction perpendicular to the plane of the substrate 21, it is conducive to the effective heat transfer of the heating electrode 231, resulting in high heating efficiency and good accuracy.

[0044] Optionally, such as Figure 2 As shown, the substrate 21 includes a heating area 212, that is, the substrate 21 is provided with a hot plate. The hot plate, which is composed of the heating area 212, the adhesive layer 22 and the electrode layer 23, is provided with a temperature measuring electrode 230 and a heating electrode 231 on the electrode layer 23, so as to perform positioning temperature measurement and positioning temperature control on the corresponding area of ​​the heating area 212.

[0045] In one optional embodiment, the heating region 212 has a length of 6–30 mm and a width of 6–30 mm, which can be applied to a nucleic acid amplification reaction system of 5–20 μL, occupying a small area and having a high degree of integration; in another optional embodiment, the heating region 212 has a length of 6–25 mm and a width of 6–25 mm; in other optional embodiments, the heating region 212 has a length of 10–25 mm and a width of 10–25 mm; alternatively, the heating region 212 has a length of 10–20 mm and a width of 10–20 mm; for example, in one specific embodiment, the size of the heating region 212 is 15 mm × 15 mm, and in another specific embodiment, the size of the heating region 212 is 18 mm × 18 mm.

[0046] Specifically, such as Figure 4 As shown, the heating component 2 includes a temperature measuring electrode 230 and a heating electrode 231. The temperature measuring electrode 230 is used to measure the real-time temperature of the reaction component. In this embodiment, the resistance value of the temperature measuring electrode 230 is positively correlated with the temperature. Preferably, the resistance value of the temperature measuring electrode 230 is basically linearly correlated with the temperature or linearly correlated with the temperature, which makes temperature measurement convenient and quick, and helps to improve the accuracy and real-time performance of the real-time temperature. The heating electrode 231 can generate heat and is used to heat the reaction component. The controller 3 obtains the real-time temperature measured by the temperature measuring electrode 230, outputs a temperature control signal after logic operation, and controls the heating electrode 231 to generate heat according to the temperature control signal, thereby realizing the control of the temperature of the reaction component. It has a high degree of automation, high sensitivity, and high control accuracy.

[0047] Optionally, the linewidth of the temperature sensing electrode 230 is 10–50 μm to improve temperature sensing sensitivity; alternatively, the linewidth of the temperature sensing electrode 230 is 15–40 μm; alternatively, the linewidth of the temperature sensing electrode 230 is 15–30 μm; in one specific embodiment, the linewidth of the temperature sensing electrode 230 is 15 μm, resulting in high sensitivity; in another specific embodiment, the linewidth of the temperature sensing electrode 230 is 25 μm; in other specific embodiments, the linewidth of the temperature sensing electrode 230 is 30 μm.

[0048] Specifically, such as Figure 5 As shown, the temperature sensing electrode 230 is arranged in a U-shape, increasing the overall length of the temperature sensing electrode 230 to increase its total resistance, thereby improving its temperature response and sensitivity. Optionally, the area occupied by the temperature sensing electrode 230 on the heating area 212 is 0.04–2 mm². 2 Alternatively, the area occupied by the temperature sensing electrode 230 in the heating area 212 is 0.04–1.5 mm². 2 For example, in one specific embodiment, the temperature sensing electrode 230 occupies an area of ​​0.64 mm² on the heating region 212. 2.

[0049] Specifically, the heating component 2 is provided with at least one temperature measuring electrode 230; optionally, the heating area 212 is provided with one temperature measuring electrode 230, which saves electrode material, is easy to process, and has high temperature measurement accuracy; alternatively, the heating area 212 is provided with multiple temperature measuring electrodes 230, which are evenly distributed in the heating area 212. Correspondingly, the controller 3 can obtain the real-time temperature measured by multiple temperature measuring electrodes 230, which improves the accuracy and reliability of real-time temperature and is also conducive to improving the accuracy of temperature control.

[0050] Specifically, the heating electrode 231 is arranged in a ring on the heating component 2, that is, the heating electrode 231 is arranged in a ring on the heating area 212, which facilitates heating of the entire heating area 212, resulting in high heat transfer efficiency and improved uniformity of heating temperature.

[0051] Specifically, the linewidth of the heating electrode 231 is 1-5 mm, which allows for rapid and sensitive temperature rise, while also improving the accuracy of temperature control and preventing over-temperature control. Optionally, the linewidth of the heating electrode 231 is 1-4 mm; alternatively, the linewidth of the heating electrode 231 is 1-3 mm. For example, in a specific embodiment, the linewidth of the heating electrode 231 is 3 mm. On the one hand, this results in high heat transfer efficiency, high temperature control sensitivity, rapid response, and fast heating or cooling speed, which can greatly reduce the side reactions caused by temperature changes. On the other hand, it improves the temperature control accuracy of the miniaturized heating device, enabling it to quickly and accurately reach the preset temperature and maintain a good dynamic stability.

[0052] Specifically, the heating component 2 can be optionally prepared by a metal stripping process (abbreviated as lift-off process). After coating a photoresist on the substrate, exposing and developing it, the required metal is evaporated with the photoresist film having a certain pattern as a mask. Then, while removing the photoresist, the metal on the film is also stripped off cleanly, leaving only the metal with the original pattern on the substrate.

[0053] For example, in one specific embodiment, the heating component 2 is obtained through the following steps:

[0054] Double-sided polished silicon dioxide wafers were selected as substrates for substrate pretreatment. The substrates were placed at 150-200℃ to remove surface moisture and then subjected to plasma surface treatment after natural cooling.

[0055] Apply release adhesive to the substrate surface, dry, and then cool.

[0056] Photoresist is coated on the substrate surface coated with release adhesive, covering 2 / 3 of the substrate surface, and then dried and cooled; wherein, the 2 / 3 of the substrate surface includes the heating area 212 of the substrate 21 after processing, that is, the width of the photoresist coating area is greater than the width of the heating area 212, and the length of the photoresist coating area is greater than the length of the heating area 212, effectively ensuring the integrity of the electrode pattern.

[0057] The substrate is exposed and developed using a photomask, and the patterned areas corresponding to the electrodes are washed away to complete the photolithography.

[0058] Metal sputtering is performed on the photolithographically etched substrate to sputter a platinum-titanium thin layer onto the substrate;

[0059] The photoresist on the substrate surface is washed away using a stripping solution, leaving the platinum electrode, which is then dried after cleaning.

[0060] The substrate corresponding to the hollowed-out area 211 is removed by laser cutting, leaving the central heating area 212 and the surrounding area. The heating component is separated from the substrate, cleaned and dried to obtain the heating component 2, which includes the substrate 21, the adhesive layer 22 and the electrode layer 23.

[0061] Specifically, such as Figure 2 As shown, the heating assembly 2 also includes a heat-conducting layer 24 and a substrate 25. The substrate 21 is connected to the substrate 25 through the heat-conducting layer 24, so that the electrode layer 23 is fixed on the substrate 25. The substrate 25 is also connected to the base plate 1, so that the heating assembly 2 is fixed on the base plate 1 as a whole. Optionally, the heat-conducting layer 24 is thermally conductive silicone, which has good thermal conductivity, greatly improves the heating and cooling speed of the heating plate, and has low cost.

[0062] Specifically, the base plate 1 is equipped with a temperature measuring circuit and a temperature control circuit. The controller 3 is connected to the temperature measuring electrode 230 through the temperature measuring circuit, that is, the temperature measuring electrode 230 is connected to the temperature measuring circuit. The temperature measuring circuit is used to generate a temperature measuring signal based on the temperature measuring electrode 230 and transmit it to the controller 3. The controller 3 is connected to the heating electrode 231 through the temperature control circuit. The controller 3 obtains a temperature control signal based on the received temperature measuring signal. The temperature control circuit is used to turn on or off the external power supply 111 and the heating electrode 231 based on the received temperature control signal, so that the heating electrode 231 heats up or does not heat up. By controlling the heat generated by the heating electrode 231, the heating electrode 231 is heated up, cooled down or maintained at the temperature. The temperature measuring and temperature control processes are both fast and accurate.

[0063] In one optional embodiment, the temperature sensing electrode 230, temperature sensing circuit, controller 3, temperature control circuit, and heating electrode 231 of the heating device can form a closed-loop structure. During the PCR reaction, the temperature sensing electrode 230 senses the real-time temperature of the hot plate (i.e., the real-time temperature of the reaction components measured by the temperature sensing electrode 230). The resistance value of the temperature sensing electrode 230 corresponds to the current real-time temperature of the hot plate, and the resistance value of the temperature sensing electrode 230 changes with the current real-time temperature of the hot plate. The temperature sensing circuit generates a corresponding temperature sensing signal based on this resistance value, and this temperature sensing signal is transmitted to the current real-time temperature of the hot plate. The controller 3 receives this temperature sensing signal. The signal can be used to obtain the real-time temperature of the hot plate. The controller 3 converts the received temperature signal to obtain a digital signal corresponding to the real-time temperature of the hot plate. After logical operation, the real-time temperature data of the hot plate is obtained, and a temperature control signal is generated and output. The temperature control signal is connected or disconnected through the temperature control circuit between the external power supply 111 and the heating electrode 231 to make the heating electrode 231 heat up or cool down. It has good real-time performance, fast and accurate response and control. The heating electrode 231 can quickly and accurately reach the preset temperature and maintain good dynamic stability in different reaction stages of nucleic acid amplification reaction.

[0064] Specifically, the resistance value of the temperature sensing electrode 230 is basically linearly related to the temperature, and the temperature sensing circuit is a constant current source circuit, which can effectively inherit this basic linear correlation, so that the real-time temperature measured by the temperature sensing electrode 230 is also basically linearly related to the temperature sensing voltage across the temperature sensing electrode 230; for example Figure 6 As shown, the constant current source circuit includes a power supply, a single-gain amplifier 101, an input resistor, a current-limiting resistor 102, and a passive RC filter circuit. In one optional embodiment, the power supply is a 5V power supply via USB. The power supply powers the single-gain amplifier 101. The current-limiting resistor 102 is connected between the temperature sensing electrode 230 and the output terminal of the single-gain amplifier 101. The passive RC filter circuit is connected to the rear end of the temperature sensing electrode 230 to improve the stability of the output signal. It has a simple structure and good stability. After passing through this constant current source circuit, the temperature sensing voltage V at the front end of the temperature sensing electrode 230 is reduced. m for:

[0065]

[0066] Where VDD is the voltage provided by the power supply, R s R is the resistance value of the current-limiting resistor 102. t denoted as , where is the resistance value of the temperature measuring electrode at the real-time temperature, and k is a coefficient related to the connected resistance.

[0067] The relationship between the connected resistors satisfies the following formula:

[0068]

[0069] R1, R2, R3, and R4 represent the resistance values ​​of each resistor connected in the constant current source circuit.

[0070] Furthermore, the temperature of the temperature-sensing electrode 230 is linearly related to its resistance value, that is...

[0071] R t =R0·(1+a·T)

[0072] Where R0 is the resistance value of the temperature measuring electrode at 0℃, a is the temperature coefficient, and T is the real-time temperature in ℃.

[0073] Then the temperature measuring voltage V m The final result is:

[0074]

[0075] The corresponding temperature measuring voltage V on the temperature measuring electrode 230 m After removing high-frequency interference signals using a first-order passive RC filter circuit, a low-frequency output signal V is obtained. out The temperature measurement signal transmitted to the controller 3 after passing through the temperature measurement circuit is the low-frequency output signal V. out It is a continuous voltage signal. By acquiring the low-frequency output signal, the controller 3 calculates the real-time temperature data, performs logic processing to obtain the temperature control signal, and controls the heating electrode 231 to heat.

[0076] Specifically, the controller 3 is used to output a temperature control signal to control the heating component 2 to heat; the temperature control signal includes a first heating signal and a second heating signal. The first heating signal is used to control the temperature control circuit to be in a conducting state. At this time, the heating electrode 231 is connected to the external power supply 111, and current flows through the heating electrode 231, causing the heating electrode 231 to heat up. The second heating signal is used to control the temperature control circuit to be in a disconnected state. At this time, the heating electrode 231 is disconnected from the external power supply 111, there is no current in the circuit where the heating electrode 231 is located, and the heating electrode 231 does not heat up.

[0077] In an optional embodiment, the controller 3 is used to obtain the signal duty cycle information based on the temperature measurement signal, generate a temperature control signal based on the signal duty cycle information and output it, wherein the signal duty cycle information represents the signal duty cycle corresponding to the temperature control signal; in this embodiment, the signal duty cycle is equal to the time ratio occupied by the first heating signal in one cycle. The controller 3 processes the real-time temperature data to obtain the signal duty cycle information of the temperature control signal, determines the digital waveform of the temperature control signal based on the signal duty cycle information, and outputs it to the temperature control circuit to adjust the voltage power applied across the heating electrode 231.

[0078] In one optional embodiment, the temperature control signal output is a pulse-adjustable waveform signal (i.e., a PWM signal), output in the form of a digital waveform. This pulse-adjustable waveform signal is a control signal with a variable duty cycle to meet the temperature change and heat preservation requirements of different PCR amplification processes, providing good flexibility. In a specific embodiment, the first heating signal output is a high level of the PWM signal, and the second heating signal output is a low level of the PWM signal. The PWM duty cycle is equal to the time ratio of the high level in one cycle, enabling the heating electrode 231 to heat flexibly.

[0079] In this embodiment, the controller 3 is built based on an STM32 microcontroller. The control function of the controller 3 is implemented based on the STM32 microcontroller, and the reference voltage of the STM32 microcontroller is 3.3V. The temperature control circuit is a power amplifier circuit, which is used to enhance the heating power of the heating electrode 231 in conjunction with the temperature control signal, so as to effectively control the heating electrode 231 to heat up and improve the effectiveness and reliability of the heating device.

[0080] Specifically, such as Figure 7 As shown, the temperature control circuit includes an external power supply 111, an optocoupler switch 112, and a field-effect transistor 113. The output terminal of the controller 3 is connected to the input terminal of the optocoupler switch 112, and the field-effect transistor 113 is connected to the output terminal of the optocoupler switch 112. The field-effect transistor 113 is correspondingly positioned with the heating electrode 231. The external power supply V1 and the heating electrode R... h Connected in series with the field-effect transistor 113, the controller 3 is used to control the opening and closing of the optocoupler switch 112 and the field-effect transistor 113. The field-effect transistor 113 is used to control the heating electrode 231 to be turned on or off, thereby precisely controlling the heating electrode 231 to be heated by controlling the field-effect transistor 113. The temperature control efficiency is fast and the dynamic temperature control accuracy is high.

[0081] When the controller 3 outputs the first heating signal, the optocoupler switch 112 is turned on, and a voltage is applied between the gate terminal and the source terminal of the field-effect transistor 113. The drain of the field-effect transistor 113 generates current to achieve conduction. At this time, the external power supply 111 and the heating electrode 231 form a closed-loop path through the field-effect transistor 113. The external power supply 111 provides current to the heating electrode 231 to generate a thermal effect and thus heat the heating plate. When the controller 3 outputs the second heating signal, the voltage is too low to turn on the optocoupler switch 112, and the field-effect transistor 113 is also turned off. The heating electrode 231 is in an open circuit and cannot generate a thermal effect, so heating stops. By changing the first heating signal and the second heating signal in the temperature control signal, the external DC power supply 111 is equivalently driven to supply power to the heating electrode 231. By opening and closing the optocoupler switch 112 or the field-effect transistor 113 a limited number of times within a single sampling period, the digital signal within the period is converted into an equivalent analog signal, and the temperature control signal is amplified, so that the heating electrode 231 can effectively rise and fall in temperature, and the temperature control reliability is good.

[0082] Optionally, the field-effect transistor 113 includes either a junction field-effect transistor (JFET) or an insulated-gate field-effect transistor (MOS transistor); preferably, the field-effect transistor 113 is an insulated-gate field-effect transistor (MOS transistor).

[0083] Specifically, the heating device, by setting a heating component 2 and its hot plate of a preset size, and in conjunction with the control device 3, can achieve a heating rate of 21-40℃ / s and a cooling rate of 6-20℃ / s. This ensures high temperature change efficiency while avoiding excessive sensitivity that could lead to over-temperature and inability to stabilize the temperature in time, greatly improving the accuracy, stability, and reliability of temperature control. Optionally, the heating device can achieve a heating rate of 25-35℃ / s and a cooling rate of 6-15℃ / s, further improving the control accuracy of the temperature change rate, which is beneficial to improving the accuracy and reliability of temperature change. For example, in a specific embodiment, the heating device can achieve a heating rate of 30℃ / s and a cooling rate of 10℃ / s. The temperature change rate is moderate, which can meet the temperature change requirements of nucleic acid amplification reaction. Moreover, the control difficulty is low, and it is not easy to over-respond. It has good accuracy and achieves a balance between temperature control efficiency, sensitivity, and precision.

[0084] Specifically, such as Figure 8 As shown, the controller 3 includes an information transmission module, which is used to receive preset temperature data and temperature measurement signals obtained through the temperature measuring electrode 230 and the temperature measuring circuit. The preset temperature data can be optionally pre-configured in the controller 3 or configured in the host computer; preferably, the preset temperature data is configured in the host computer, and the information transmission module is used to connect to the host computer to obtain the preset temperature data; the information transmission module is also connected to the temperature control circuit and is used to output temperature control signals to the temperature control circuit.

[0085] Optionally, the information transmission module includes a first transmission module, a second transmission module, and a third transmission module. The first transmission module is used to transmit preset temperature data. In one specific embodiment, the first transmission module is a serial communication module used to connect with a host computer to obtain the preset temperature data. The second transmission module is used for the temperature measurement signal of the structural temperature measurement circuit. In this embodiment, the second transmission module can be selected as the 12-bit ADC module built into the STM32 microcontroller for signal acquisition. The low-frequency output signal readout resolution of this ADC module is 0.806mV. The third transmission module is used to output the temperature control signal to the temperature control circuit so as to control the current flowing through the heating electrode through the temperature control circuit, effectively and accurately control the heating electrode to heat, and improve the heating efficiency and heating accuracy of the miniaturized heating device.

[0086] Specifically, such as Figure 8 As shown, the controller 3 includes a control module, which is used to obtain a temperature control signal based on preset temperature data and real-time temperature. The real-time temperature is obtained through the temperature measuring electrode 230. Specifically, the real-time temperature of the reaction component is basically linearly related to or linearly related to the resistance value of the temperature measuring electrode 230. The resistance value is converted into a continuous temperature measurement signal, such as a voltage signal, by the temperature measuring circuit. The information transmission module of the controller 3 receives the temperature measurement signal, converts it into a digital signal, and transmits the digital signal to the control module. The control module is used to perform feedback processing based on the preset temperature data and the digital signal corresponding to the temperature measurement signal to obtain a temperature control signal and output it to control the heating electrode. In an optional embodiment, the controller 3 is a miniaturized MCU development board, which is easy to operate and control, and has a small size, convenient assembly, and low cost.

[0087] Specifically, a PCR nucleic acid amplification reaction includes a pre-denaturation stage and a certain number of thermal cycling stages. The thermal cycling stages include a denaturation stage, an annealing stage, and an extension stage. This miniaturized heating device can significantly shorten the waiting time between different reaction stages, as well as the time required for temperature changes between different reaction stages, avoiding potential side reactions caused by temperature changes, improving product purity, and enhancing the accuracy of the nucleic acid amplification reaction and detection. In addition, it can effectively maintain the dynamic stability of temperature control in each reaction stage, avoiding excessive control that could cause temperature fluctuations that affect the reaction accuracy of that stage, further improving the accuracy and reliability of the nucleic acid amplification reaction. Optionally, before the pre-denaturation stage, the PCR nucleic acid amplification reaction also includes a reverse transcription stage. Correspondingly, the preset heating data includes the time parameters and temperature parameters corresponding to each reaction stage in the PCR nucleic acid amplification reaction, and also includes heating rate parameters, cooling rate parameters, and the number of thermal cycles.

[0088] In one specific embodiment, the time and temperature parameters of the PCR nucleic acid amplification reaction satisfy a preset function, which is a function of time. The temperature parameter value at a given time can be directly calculated based on the time value. The control module can obtain the preset heating data and calculate the time of a single thermal cycle based on the preset heating data. It can also obtain the temperature value corresponding to each moment in the entire PCR amplification reaction and obtain a two-dimensional array corresponding to the preset heating data. That is, the preset heating data is a set of two-dimensional arrays composed of time parameters and temperature parameters. The two-dimensional array is used to store the time parameters and temperature parameters.

[0089] In one optional embodiment, the preset heating data includes pre-denaturation time and temperature parameters, denaturation time and temperature parameters, annealing time and temperature parameters, and extension time and temperature parameters; in another optional embodiment, the preset heating data includes reverse transcription time and temperature parameters, pre-denaturation time and temperature parameters, denaturation time and temperature parameters, annealing time and temperature parameters, and extension time and temperature parameters; in still other optional embodiments, the preset heating data includes reverse transcription time and temperature parameters, pre-denaturation time and temperature parameters, denaturation time and temperature parameters, annealing time and temperature parameters, extension time and temperature parameters, heating rate parameters, cooling rate parameters, and the number of thermal cycles.

[0090] Specifically, the control module includes a digital-to-analog conversion module and a feedback control module, such as... Figure 8 As shown, the analog-to-digital converter (ADC) is connected to the information transmission module and receives the temperature measurement signal transmitted from the information transmission module. The ADC is used to convert and process the temperature measurement signal to obtain the real-time temperature data corresponding to the temperature measuring electrode 230. The feedback control module receives the real-time temperature data from the ADC and also receives preset heating data through the information transmission module. The feedback control module is used to perform feedback processing based on the preset heating data and the real-time temperature data to obtain and output a temperature control signal. In a preferred embodiment, the temperature control signal is a PWM signal including a PWM duty cycle.

[0091] Among them, such as Figure 9As shown, the feedback control module uses a PID control algorithm to improve the speed and accuracy of the temperature control signal in regulating the voltage applied to the heating electrode, thereby improving the overall temperature control efficiency, sensitivity, and accuracy of the miniaturized heating device. In an optional embodiment, the feedback control module includes a discretization submodule, used to discretize the preset heating data and temperature measurement signal to effectively control the reliability and accuracy of the temperature control signal. Assuming a sampling period of T, the discrete time of the sampling point is used to replace the continuous time of the analog PID control algorithm, resulting in the mathematical expression of the PID control algorithm:

[0092]

[0093] Where u(k) is the output of the PID control algorithm, and k p T is the proportionality coefficient. i T d Let be the integral time constant and the derivative time constant, respectively, and let e(i) be the error between the set value and the output value at time i.

[0094] In the adjustment process of this PID control algorithm, P, I, and D represent proportional control, integral control, and derivative control, respectively; proportional control is the process of multiplying the input deviation of the controller by the proportional coefficient and then outputting the result. p The magnitude of the value determines the strength of the control, and it will respond immediately when a deviation occurs in the system, featuring simplicity and speed. Integral control records all deviations generated during system operation and eliminates system errors. Derivative control is used to reduce overshoot, overcome the oscillation phenomenon caused by excessive overshoot due to strong integral action, make the entire controller more stable, and accelerate the dynamic response speed of the controller, improve adjustment efficiency, and thus improve the dynamic performance of the controller. Combining the proportional coefficient, integral time constant, and derivative time constant in the PID algorithm, it can effectively handle deviations, steady-state errors, and oscillation fluctuations generated during the control process, thereby meeting the good temperature control effect required for rapid PCR amplification reaction.

[0095] Specifically, in one optional embodiment, the controller includes a timer capable of outputting a high-precision temperature control signal. The controller also includes multiple registers, including a first register and a second register. The first register determines the frequency of the temperature control signal, and the second register determines the duty cycle of the temperature control signal. Based on the timer and registers, the first heating signal and the second heating signal can be switched, improving the temperature control accuracy of the heating device. When the timer counter is less than the value of the second register, the second heating signal is output. When the timer counter is greater than or equal to the value of the second register and less than the value of the first register, the first heating signal is output. When the timer counter is greater than or equal to the value of the first register, the timer overflows and starts counting again, at which point the second heating signal is output. This cycle repeats until one temperature control signal cycle is completed.

[0096] In one specific embodiment, the temperature control signal obtained through PID feedback processing is output in the form of a PWM waveform signal, and the timer is capable of outputting a high-precision PWM waveform signal; such as Figure 10 As shown, the first register is used to determine the frequency of the PWM signal, and the second register is used to determine the duty cycle of the PWM signal. The high and low levels are toggled according to the timer and the register. When the timer counter reaches the value of the second register, the effective level of the corresponding channel will be changed to achieve the toggle from low to high level and continue counting. When the counter value reaches the value of the first register, the timer overflows and starts counting upward again. This counting process will complete one PWM cycle.

[0097] Optionally, the first register is the autoload register (TIMx_ARR), and the second register is the capture / compare register (TIMx_CCRx).

[0098] Specifically, such as Figure 1 As shown, the heating device also includes a display device 4, which is connected to the control device 3 and is used to display various real-time data during nucleic acid amplification. The real-time data reflects the current reaction stage of the nucleic acid amplification reaction in the current reaction component and the corresponding real-time environmental conditions, so as to provide information on the PCR amplification reaction in a convenient and intuitive way. Optionally, the real-time data includes real-time temperature, time, and real-time thermal cycle number. The real-time data may also optionally include the temperature parameter corresponding to the time. In addition, in an optional embodiment, the display device 4 is an organic electro-laser display device (OLED).

[0099] This embodiment also provides a heating method for nucleic acid amplification, which uses the heating device described above for heating, including:

[0100] The real-time temperature of the reaction components is obtained; the real-time temperature is obtained through temperature measuring electrodes.

[0101] The preset temperature data and real-time temperature are processed to obtain a temperature control signal, which is then output. The temperature control signal is used to control the heating element to heat the components.

[0102] Specifically, obtaining the real-time temperature of the reaction components includes:

[0103] Acquire the temperature measurement signal corresponding to the temperature measuring electrode in the temperature measuring circuit; the temperature measurement signal includes information about the temperature measuring voltage applied to the temperature measuring electrode, and is a voltage signal;

[0104] The real-time temperature of the temperature measuring electrode is obtained based on the voltage signal; this real-time temperature includes information about the real-time temperature value measured by the temperature measuring electrode.

[0105] The temperature measuring circuit is a constant current source circuit. The voltage at the upper end of the temperature measuring electrode is basically linearly related to the resistance value of the temperature measuring electrode at the current temperature. Furthermore, the resistance value of the temperature measuring electrode at each temperature is basically linearly related to the real-time temperature at which the temperature measuring electrode is located. Therefore, the real-time temperature value measured by the temperature measuring electrode can be obtained by acquiring and analyzing the voltage signal.

[0106] When acquiring real-time temperature, the controller simultaneously acquires the current time corresponding to that real-time temperature, i.e., the sampling time. Based on the current time, the reaction stage of the PCR amplification reaction is determined. The controller then combines the time and temperature parameters of the corresponding reaction stage in the preset temperature data for judgment. When the real-time temperature value is less than the temperature parameter of the corresponding reaction stage, the controller outputs a first heating signal, which turns the temperature control circuit on and heats the heating electrode. When the real-time temperature value is greater than or equal to the temperature parameter of the corresponding reaction stage, the controller outputs a second heating signal, which turns the temperature control circuit off and the heating electrode does not heat.

[0107] This embodiment also provides an application of the heating device described above in nucleic acid amplification. The nucleic acid amplification reaction includes multiple different reaction stages, and the preset temperature parameters corresponding to some reaction stages are different. The heating device can control the heating component 2 to reach the temperature corresponding to different preset temperature parameters, so that the nucleic acid amplification reaction in each reaction stage is carried out under the required temperature conditions, and the temperature-switching amplification reaction proceeds smoothly. The heating device can be applied to nucleic acid detection based on isothermal amplification method and also to nucleic acid detection based on temperature-switching amplification method, and has a wide range of applications.

[0108] Specifically, this application provides a nucleic acid amplification system for performing nucleic acid amplification. The nucleic acid amplification system includes a reaction component and the aforementioned heating device. The heating device is used to heat the reaction component, which is placed on the heating component 2. The cross-sectional area of ​​the reaction component can optionally be less than or equal to the cross-sectional area of ​​the heating component 2, resulting in good miniaturization and improved temperature control accuracy. In an optional embodiment, the reaction component can be used for a nucleic acid amplification reaction system of 5–20 μL. Compared to typical small-scale system reactions, the reaction volume of this reaction component is greatly increased. The heating device can cooperate with the reaction component for positioning and temperature control, resulting in high temperature control efficiency and significantly improved temperature control accuracy. This enhances the reaction efficiency, sensitivity, and precision of the nucleic acid amplification reaction in the miniaturized nucleic acid amplification system.

[0109] Optionally, the nucleic acid amplification system also includes a host computer, which is used to acquire multiple preset heating data during PCR amplification. In one specific embodiment, the host computer provides an input control panel, which allows operators to adjust the parameter values ​​of the preset heating data according to different nucleic acid amplification reactions. The host computer is connected to the controller 3 and is used to send the preset heating data to the controller. The information transmission module in the controller is also used to send the data to the host computer in string form via a serial port. The host computer receives and parses the data from the controller at a preset frequency, performs real-time monitoring of the rapid PCR amplification reaction, and displays real-time parameter data during the PCR amplification process, including the current time (Time), preset temperature (Temp_Set), real-time temperature measured by the temperature measuring electrode (Temp_Measure), the duty cycle of the temperature control signal, i.e., the duty cycle of the PWM waveform output of the PID algorithm (Duty), and the current cycle number (Current_Cycle). In addition, the host computer can also plot and display temperature-time curves based on the real-time parameter data, so that users can more intuitively read the temperature information during the rapid PCR reaction process. It has good real-time performance, high accuracy, and good visualization.

[0110] Taking a specific embodiment as an example, the nucleic acid amplification reaction in the reaction assembly is started. Based on the temperature parameters of the reverse transcription stage and the real-time temperature measured by the temperature measuring electrode, a temperature control signal is obtained and output. This temperature control signal applies voltage to the heating electrode through the temperature control circuit to heat it, so that the real-time temperature in the reaction assembly corresponding to the heating electrode reaches the temperature value corresponding to the preset temperature parameter of the reverse transcription stage. The above steps are repeated according to the time parameters of the pre-denaturation stage, the denaturation stage, the annealing stage, and the extension stage, so that the real-time temperature in the reaction assembly reaches the temperature value corresponding to the preset temperature parameter of each reaction stage, and the temperature control is fast and accurate.

[0111] Furthermore, during the reaction process, a sampling period is set, and the real-time temperature of the reaction components is sampled through a temperature measuring electrode. Based on the sampling time point, real-time temperature, and heating data, a first heating signal is output, and a voltage is applied to the heating electrode through the temperature control circuit to raise the temperature. When the real-time temperature measured by the temperature measuring electrode is greater than or equal to the preset temperature parameter corresponding to that moment in the preset heating data, a second heating signal is output. At this time, the heating electrode in the temperature control circuit is in an open circuit state, and the heating electrode stops or remains in a non-heating state.

[0112] In an optional embodiment, the heating device is also provided for use in nucleic acid detection, i.e., the nucleic acid amplification system can also be used for nucleic acid detection; preferably, this embodiment also provides a nucleic acid detection system, which includes the above-mentioned nucleic acid amplification system and a fluorescence detection device. The fluorescence detection device is used to perform fluorescence detection on the PCR amplification reaction occurring in the reaction component; the fluorescence detection device has a length of less than or equal to 10 cm, a width of less than or equal to 10 cm, and a height of less than or equal to 15 cm. It is small in size, easy to carry, and can be widely used in various point-of-care testing environments. It can perform on-site sampling and testing, with high detection efficiency and accurate detection results.

[0113] Specifically, the reaction assembly is used for fluorescent PCR amplification, which involves adding a fluorescent group to the PCR amplification reaction system to obtain a product carrying the fluorescent group. As PCR amplification proceeds, the product accumulates and the fluorescence signal intensity gradually increases. The amount of product can be obtained based on the fluorescence signal intensity. In this embodiment, the reaction volume corresponding to the reaction assembly is 5-20 μL, which allows for more sampling compared to general micro-devices. The detection volume is more than 10 times that of general micro-devices, greatly reducing the possibility of missed detections and significantly improving detection sensitivity.

[0114] Specifically, such as Figure 11 As shown, the fluorescence detection device includes a fixed bracket 5 and an excitation light emitting element 6. The excitation light emitting element 6 is used to emit an emission beam in the direction of the heating device to excite the fluorescent group to emit fluorescence. In an optional embodiment, the excitation light emitting element is an LED lamp, resulting in a miniaturized fluorescence detection device based on an LED light source, which has high integration, convenient control, and low cost. In a specific embodiment, the excitation light emitting element is an LED lamp with a center wavelength of 480nm, which can effectively excite fluorescence and produce clear fluorescence imaging.

[0115] When the reaction assembly is placed on the heating assembly 2, the emitted light beam can excite the fluorescent groups in the reaction assembly to emit fluorescence. The fluorescence signal image can be acquired by an imaging device, such as a smartphone or detection camera, which is convenient, fast, and produces clear and distinct images. Preferably, the imaging device is perpendicular to the surface of the reaction assembly and has a detection field diameter of 25-35 mm, covering the entire reaction assembly. It can directly acquire the fluorescence signal image on the entire chip without the need for complex mechanical movement structures to switch fluorescence channels or move the reaction assembly, which greatly shortens the detection time and further improves the efficiency of the entire nucleic acid detection process.

[0116] Specifically, the fixed bracket 5 includes a first mounting part 51 and a second mounting part 52. The first mounting part 51 is used to place the heating device for nucleic acid amplification. Optionally, the first mounting part 51 is a support plane, on which the heating device can be directly placed, which is convenient to use and allows for flexible adjustment of the position of the heating device. Alternatively, the first mounting part 51 is a snap-fit ​​structure, which matches and connects with the base plate 1, ensuring accurate positioning and a reliable connection, thereby improving the accuracy and reliability of detection.

[0117] Specifically, the second mounting portion 52 is used to connect with the excitation light emitting element 6, and the second mounting portion 52 is inclined so that the emitted light beam emitted by the excitation light emitting element 6 can be obliquely projected onto the surface of the heating assembly 2; optionally, the included angle formed between the first mounting portion 51 and the second mounting portion 52 is 15 to 75°, that is, the included angle formed between the emission direction of the excitation light emitting element 6 and the surface of the heating assembly 2 is 15 to 75°; alternatively, the included angle formed between the emission direction of the excitation light emitting element 6 and the surface of the heating assembly 2 is 30 to 60°; preferably, the included angle formed between the emission direction of the excitation light emitting element 6 and the surface of the heating assembly 2 is 45°.

[0118] Optionally, the mounting bracket 5 includes two second mounting portions 52, and correspondingly, the fluorescence detection device includes two excitation light emitting elements 6, and the two second mounting portions 52 (or the two excitation light emitting elements 6) are symmetrically arranged to effectively excite the fluorescent group to emit a fluorescence signal.

[0119] Specifically, the fluorescence detection device includes a filter assembly 7, which includes a first filter element 71. The first filter element 71 is disposed between the excitation light emitting element 6 and the first mounting portion 51, and is located in the optical path between the emitted beam and the heating assembly 2. The first filter element 71 is used to separate and select the characteristic wavelength spectrum of the excitation light of the fluorescent group and the emitted beam, and to filter out the excitation light from the emitted beam to effectively excite the fluorescent group to emit fluorescence. Optionally, the passing wavelength of the first filter element 71 is 450-490 nm; or alternatively, the passing wavelength of the first filter element 71 is 455-485 nm. For example, in a specific embodiment, the first filter element 71 is an excitation light filter with a center wavelength (i.e., passing wavelength) of 470 nm, so that excitation light with a wavelength of 470 nm can pass through, and light of other wavelengths is reflected, so that the excitation light is stably projected onto the reaction assembly to excite fluorescence.

[0120] Specifically, the light filtering assembly 7 further includes a second light filter 72, which is disposed between the imaging device and the first mounting part 51, i.e., in the optical path between the reaction assembly and the imaging device. The second light filter 72 is used to select and transmit the fluorescence characteristics emitted by the fluorescent group in the reaction assembly and to block light of other wavelengths. The wavelength of the second light filter 72 is greater than that of the first light filter 71. Optionally, the wavelength of the second light filter 72 is 500-550 nm; alternatively, the wavelength of the second light filter 72 is 515-535 nm.

[0121] Optionally, the filter assembly 7 includes a focusing lens 73, at least part of which is located between the excitation light emitting element 6 and the first filter 71 to increase the flux of the emitted light beam. Alternatively, at least part of the focusing lens 73 is located on the side of the second filter 72 away from the first mounting portion 51 to expand the detection area and improve detection sensitivity and accuracy. This filter assembly 7 does not require the introduction of complex optical components, avoiding the influence of optical components such as beam splitters or dichroic mirrors on the fluorescence generated by the excitation of fluorescent groups, thus greatly improving detection accuracy.

[0122] In this embodiment, both the excitation light emitting element 6 and the filter assembly 7 are detachably connected to the fixed bracket 5, allowing the excitation light emitting element and the filter assembly 7 to be replaced according to different fluorescent groups and different detection requirements, so as to adapt to the detection of a variety of different substances and improve applicability.

[0123] Furthermore, this fluorescence detection device can be used for endpoint detection or real-time fluorescence detection. When used for endpoint detection, fluorescence signal images are acquired during the extension phase of the 0th and last (typically 40) thermal cycles in a rapid PCR reaction. When used for real-time fluorescence detection, fluorescence signal images are acquired during the extension phase of each thermal cycle in the rapid PCR reaction. By processing and analyzing the acquired fluorescence signal images, the fluorescence intensity can be used to quantitatively characterize the reaction results within the reaction components. This device offers high flexibility and can handle different types of test samples, targets, or methods, catering to various detection needs, thus having a wide range of applications.

[0124] This nucleic acid amplification system boasts a high degree of integration, miniaturization, and portability. This miniaturized system enables ultra-fast PCR amplification reactions, completing the entire process in just 15 minutes, and in some cases as little as 10 minutes. Compared to conventional nucleic acid amplification devices, this represents a 6-8 fold reduction in time, significantly saving time. Furthermore, this miniaturized system exhibits high sensitivity, rapid response, and excellent real-time performance, effectively improving temperature control efficiency and the speed of the nucleic acid amplification reaction. In addition, the synergistic effect of the heating plate and control components within the system enhances the efficiency and sensitivity of the miniaturized device while maintaining precise temperature control to prevent excessive temperature fluctuations. It allows for rapid temperature changes while simultaneously maintaining the required real-time temperature at each stage of the reaction. In terms of temperature, this system improves the dynamic stability and reliability of temperature control, thereby enhancing the accuracy of nucleic acid amplification reactions while maintaining the efficiency, sensitivity, and precision of the miniaturized nucleic acid amplification system. This system can be used with a simple, miniaturized fluorescence detection device to simultaneously detect up to three pathogens, with a detection sensitivity as low as 10 copies / reaction, effectively avoiding missed detections. Furthermore, the specific viral load can be calculated based on the fluorescence signal intensity at the endpoint for quantitative detection, with a dynamic range of up to four orders of magnitude for pathogen DNA concentration detection. This nucleic acid amplification system features high integration, high sensitivity, high precision, high throughput, multi-channel operation, speed, ease of operation, and low cost. Its portable structure provides numerous advantages for rapid, multiplex, on-site pathogen detection, enabling both qualitative and quantitative detection, and significantly improving the applicability of PCR technology in the field of nucleic acid molecular detection.

[0125] The above description is merely some embodiments of this application and is not intended to limit this application. Those skilled in the art should understand that various changes and improvements may be made to this application, and any modifications, equivalent substitutions and improvements made in accordance with this application fall within the scope of protection claimed by this application.

Claims

1. A heating device for nucleic acid amplification, characterized by, The heating assembly (2) and the control device (3) are connected to the bottom plate (1); The heating assembly (2) includes a substrate (21), an adhesion layer (22) and an electrode layer (23), the substrate (21) is connected to the bottom plate (1), the electrode layer (23) is connected to the substrate (21) through the adhesion layer (22), the electrode layer (23) includes a temperature measuring electrode (230) and a heating electrode (231), the temperature measuring electrode (230) is used for measuring the real-time temperature of a reaction assembly, the reaction assembly is used for nucleic acid amplification, the reaction assembly is a microfluidic assembly, and the heating electrode (231) is used for heating the reaction assembly; The substrate (21) includes a support area (210), a hollow area (211) and a heating area (212), the support area (210) is located outside the hollow area (211), the heating area (212) is located inside the hollow area (211), and the temperature measuring electrode (230) and the heating electrode (231) are arranged on the heating area (212); the length of the heating area (212) is 6-30mm, the width is 6-30mm, and it is used for 5-20μL nucleic acid amplification reaction system; The control device (3) is connected with the heating assembly (2) and is used for controlling the heating assembly (2) to heat.

2. The heating device of claim 1, wherein The thickness of the adhesion layer (22) is 30-70nm, and the thickness of the electrode layer (23) is 120-180nm.

3. The heating device of claim 1, wherein, The line width of the temperature measuring electrode (230) is 10-50μm, and the line width of the heating electrode (231) is 1-5mm.

4. The heating device of claim 1, wherein, The bottom plate (1) is provided with a temperature measuring circuit and a temperature control circuit, the control device (3) is connected with the temperature measuring electrode (230) through the temperature measuring circuit, and the control device (3) is connected with the heating electrode (231) through the temperature control circuit.

5. The heating device of claim 4, wherein, The control device (3) is used for outputting a temperature control signal to control the heating assembly (2) to heat; the temperature control signal includes a first heating signal and a second heating signal, the first heating signal is used for controlling the temperature control circuit to be in a conduction state, and the second heating signal is used for controlling the temperature control circuit to be in a cut-off state.

6. A heating method for nucleic acid amplification based on the heating device as claimed in any one of claims 1 5. The heating method according to any one of claims 1 to 4, wherein the heating device is used. The method comprises: Obtaining the real-time temperature corresponding to the reaction assembly, the real-time temperature being obtained through the temperature measuring electrode; According to the preset temperature data and the real-time temperature, the temperature control signal is obtained and outputted through feedback processing; the temperature control signal is used for controlling the heating assembly to heat.

7. A nucleic acid amplification system, characterized by, comprising a reaction assembly and a heating device as claimed in any one of claims 1 5 The heating device of any one of claims 1 to 4, for heating the reaction assembly.

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