A nucleic acid amplification reaction tube based on a light-heat conversion material and a use method

Abstract

The application discloses a nucleic acid amplification reaction tube based on a light-heat conversion material and a use method thereof. A lower end of a first light guide body is inserted into a bottom of a first outer tube, and a first inner tube is inserted into a groove on an upper surface of the first light guide body. An outer wall of the first inner tube is sprayed with a light-heat conversion layer mixed with a color-changing paint except for a contact position with the first light guide body, and the first outer tube is in a vacuum state. A second outer tube carries a light-heat conversion liquid, a support is away from a bottom of the second outer tube, the light-heat conversion liquid passes through a liquid exchange groove of the support and is above an upper surface of the support, a second inner tube is inserted into a groove on the upper surface of the support, a first through hole for placing a temperature detection needle is formed in a tube cover of the second outer tube, and a second light guide body in contact with an outer wall of the second inner tube and an inner wall of the second outer tube is inserted into a second through hole of the support. Tube openings of the first and second inner tubes are sealed with transparent tube covers, outer walls of the first and second outer tubes are transparent, and the first and second inner tubes are made of a heat-conducting material.

Description

Technical Field

[0001] This invention relates to the technical field of medical devices, specifically to a nucleic acid amplification reaction tube based on photothermal conversion material and its usage method. Background Technology

[0002] In molecular biology detection, fluorescence detection at specific temperatures is required, and this is particularly common in nucleic acid amplification detection. Nucleic acid amplification detection refers to the process where, under certain temperature conditions and with pre-prepared reagents, the target nucleic acid undergoes massive amplification under the action of primers and related working enzymes, resulting in a rapid increase in fluorescence intensity. By detecting the fluorescence intensity in real time, the presence or absence of the target nucleic acid can be analyzed.

[0003] Currently, nucleic acid amplification reaction control devices suitable for fluorescence detection are large, power-consuming, and complex in structure, resulting in a narrow range of applications and demanding operating conditions. For example, a real-time fluorescence nucleic acid detector using semiconductor technology for nucleic acid amplification reaction temperature control has dimensions exceeding 350mm × 500mm × 500mm and power consumption exceeding 800W, directly limiting its use in areas with insufficient resources. Therefore, existing technologies cannot solve the problems of size and power consumption of such control devices.

[0004] Photothermal conversion is a phenomenon where photothermal conversion materials absorb light energy (especially near-infrared light), convert it into the kinetic energy of electrons or holes through resonance or the energy generated by electron transitions via localized surface plasmon resonance, and then transfer this vibrational energy to the surrounding environment through lattice scattering, thereby increasing the ambient temperature. Therefore, theoretically, photothermal conversion materials can be used to control the temperature during nucleic acid amplification reactions. However, currently, these materials are mainly used for photothermal killing therapy targeting tumor cells. There are also reports of foreign researchers using unmodified magnetic beads to investigate microbial cell disruption, but because photothermal conversion materials are generally dark in color and have high absorbance, they often affect fluorescence in nucleic acid amplification reactions. Summary of the Invention

[0005] The purpose of this invention is to provide a nucleic acid amplification reaction tube based on photothermal conversion material and its usage method. The photothermal conversion material is used to control the temperature of the nucleic acid amplification reaction, which facilitates the incubation of nucleic acid and increases the concentration of the target nucleic acid through amplification, making it easier to perform fluorescence detection.

[0006] This invention is achieved through the following technical solution:

[0007] A nucleic acid amplification reaction tube based on a photothermal conversion layer includes a first inner tube, a first outer tube, a first light guide, and a photothermal conversion layer;

[0008] The lower end of the first light guide is inserted into the bottom of the first outer tube along the height direction of the first outer tube. The shape of the lower end of the first light guide is the same as the shape of the area corresponding to the bottom of the first outer tube. The upper surface of the first light guide is provided with a groove with the same shape as the bottom of the outer wall of the first inner tube. The first inner tube is inserted into the groove.

[0009] The outer wall of the first inner tube is coated with a photothermal conversion layer except at the contact position with the first light guide. The opening of the first inner tube is sealed inside the opening of the first outer tube, and the opening of the first inner tube is sealed with a transparent tube cap.

[0010] The outer wall of the first outer tube is transparent, the first inner tube is made of thermally conductive material, the photothermal conversion layer contains color-changing coating, and the first outer tube is in a vacuum state.

[0011] Preferably, the material of the first light guide is acrylic or silicon dioxide.

[0012] Preferably, the photothermal conversion layer is a gold nanorod layer, a Prussian blue nanoparticle layer, a polydopamine particle layer, or a porphyrin ring particle layer.

[0013] A method for using a nucleic acid amplification reaction tube based on a photothermal conversion layer, using any one of the above-described nucleic acid amplification reaction tubes based on a photothermal conversion layer, includes the following steps:

[0014] S1, the required nucleic acid amplification reaction system is carried out in the first inner tube. When heating is required, near-infrared light is used to irradiate the outside of the first outer tube corresponding to the photothermal conversion layer. The near-infrared light passes through the corresponding outer wall of the first outer tube to reach the photothermal conversion layer.

[0015] S2, after the photothermal conversion layer is heated, the temperature of the reaction system inside the first inner tube rises, and the photothermal conversion layer displays the color of the photothermal conversion material itself. Then, it is intermittently irradiated to maintain the color of the photothermal conversion material itself, and then proceeds according to S31 or S32.

[0016] S31, below the first outer tube, the bottom of the first outer tube is irradiated with visible light. The visible light is guided to the first inner tube through the light guide channel of the first light guide body, which excites the dye in the fluorescent probe in the first inner tube. The fluorescence is emitted from the tube cap of the first inner tube.

[0017] S32, above the first outer tube, the cap of the first inner tube is irradiated with visible light. The visible light enters the first inner tube and excites the dye in the fluorescent probe in the first inner tube. The fluorescence is emitted from the bottom of the first outer tube through the light guide channel of the first light guide.

[0018] A nucleic acid amplification reaction tube based on photothermal conversion fluid includes a second inner tube, a second outer tube, a second light guide, and a support component;

[0019] The second outer tube contains a photothermal conversion fluid, and a support is fixedly inserted into the second outer tube. The support is far from the bottom of the second outer tube. The support has a liquid exchange tank that connects the upper and lower surfaces of the support along its height. The photothermal conversion fluid passes through the liquid exchange tank and covers the upper surface of the support. The upper surface of the support has a groove that can accommodate the bottom of the second inner tube, and the second inner tube is inserted into the groove.

[0020] The outer wall of the second outer tube is transparent, and the second inner tube is made of thermally conductive material. The cap of the second outer tube is fitted into the opening of the second outer tube. The cap of the second outer tube has a first through hole for placing a temperature detection needle. The bottom of the temperature detection needle is supported on the upper surface of the support member. The outer wall of the support member has a second through hole that communicates with the groove from the outside to the inside. A second light guide that contacts the outer wall of the second inner tube and the inner wall of the second outer tube is inserted into the second through hole. The opening of the second inner tube is sealed with a transparent cap, and the second inner tube is set higher than the opening of the second outer tube.

[0021] Preferably, the device further includes a placeholder plug, which has the same shape as the bottom of the second outer tube. The bottom of the placeholder plug is in contact with the bottom of the second outer tube. A gap is left between the support member and the placeholder plug, and the photothermal conversion fluid is located on the upper surface of the placeholder plug.

[0022] Preferably, the photothermal conversion fluid is a dispersion of Prussian blue nanoparticles in deionized water, and the ratio of Prussian blue nanoparticles to deionized water is (1.5-2.5) mg.

[0023] (450~550)μl.

[0024] Preferably, the main structure of the support member is cylindrical, the diameter of the support member is the same as the inner diameter of the second outer tube, and the support member is located at the middle of the height of the second outer tube.

[0025] Furthermore, the liquid exchange tanks are located on the outside of the support and are evenly distributed in 3 to 5 places. The number of second through holes is the same as the number of liquid exchange tanks, and each second through hole is located between two liquid exchange tanks.

[0026] A method for using a nucleic acid amplification reaction tube based on a photothermal conversion solution, using the nucleic acid amplification reaction tube based on the photothermal conversion solution described in any one of the above-mentioned methods, includes the following steps:

[0027] S1. First, place a set volume of photothermal conversion fluid in the second outer tube, and then carry out the required nucleic acid amplification reaction system in the second inner tube. When heating is required, irradiate the outside of the second outer tube corresponding to the photothermal conversion fluid located below the support with near-infrared light. The near-infrared light passes through the corresponding outer wall of the second outer tube to reach the photothermal conversion fluid.

[0028] S2, after the photothermal conversion liquid is heated, the temperature of the reaction system in the second inner tube rises, and the temperature value of the temperature detection needle reaches the temperature required for the reaction. Then, it is intermittently irradiated to maintain the temperature.

[0029] S3, on the outside of the second outer tube corresponding to the second light guide, visible light is used to illuminate both sides of the second outer tube. The visible light is guided to the second inner tube through the light guide channel of the second light guide, exciting the dye in the fluorescent probe in the second inner tube, and the fluorescence is emitted from the tube cap of the second inner tube.

[0030] Compared with the prior art, the present invention has the following beneficial technical effects:

[0031] This invention discloses a nucleic acid amplification reaction tube based on a photothermal conversion layer. The transparent outer wall of the first outer tube facilitates the reach of near-infrared light to the photothermal conversion layer, thereby heating the layer. The vacuum state of the first outer tube ensures that the first inner tube maintains the temperature of the reaction system. Temperature control of the nucleic acid amplification reaction is achieved using the photothermal conversion material, facilitating nucleic acid incubation and increasing the target nucleic acid concentration through amplification. The photothermal conversion layer contains a color-changing coating; when the desired reaction temperature is reached, the photothermal conversion layer displays its own color, facilitating temperature monitoring and nucleic acid incubation. The lower end of the first light guide is in contact with the bottom of the first outer tube, and the first inner tube is in contact with the first light guide. When visible light is irradiated at the bottom of the first outer tube from below, the visible light is guided through the light guide channel of the first light guide to the first inner tube, exciting the dye in the fluorescent probe within the first inner tube. The fluorescence then exits from the cap of the first inner tube. Furthermore, since the cap of the first inner tube is transparent, visible light can be shone onto the cap of the first inner tube from above the first outer tube. This allows the visible light to directly enter the first inner tube, exciting the dye in the fluorescent probe within it. The fluorescence then exits from the bottom of the first outer tube through the light-guiding channel of the first light guide. Collecting fluorescence from different locations facilitates fluorescence detection, and the system features low power consumption, rapid heating, and a simple structure.

[0032] This invention discloses a method for using a nucleic acid amplification reaction tube based on a photothermal conversion layer. Nucleic acid amplification reactions can be performed in the first inner tube as needed. When heating is required, near-infrared light can be irradiated on the outside of the first outer tube. The near-infrared light passes through the outer wall of the first outer tube and reaches the photothermal conversion layer, thus heating the photothermal conversion layer. The temperature of the reaction system in the first inner tube gradually rises, and the photothermal conversion layer displays its own color, facilitating nucleic acid incubation. Visible light can be used to irradiate the bottom of the first outer tube. The visible light is guided through the light guide channel of the first light guide to the first inner tube, exciting the dye in the first inner tube. Fluorescence is then emitted from the cap of the first inner tube. Alternatively, visible light can be used to irradiate the cap of the first inner tube from above the first outer tube. The visible light directly enters the first inner tube, exciting the dye. Fluorescence is emitted from the bottom of the first outer tube via the light guide channel of the first light guide. Collecting fluorescence from different locations facilitates fluorescence detection. This method features low power consumption and rapid heating.

[0033] This invention discloses a nucleic acid amplification reaction tube based on a photothermal conversion solution. Near-infrared light is used to heat the photothermal conversion solution in the second outer tube, facilitating temperature control of the nucleic acid amplification reaction in the second inner tube and simplifying nucleic acid incubation. This allows for increasing the target nucleic acid concentration through amplification. A support member supports the second inner tube and also houses a second light guide, ensuring it contacts the outer wall of both the second inner and outer tubes. This facilitates the guidance of near-infrared light from outside the second outer tube into the second inner tube via its light-guiding channel. A temperature sensing needle is placed in the cap of the second outer tube, allowing it to be immersed in the photothermal conversion solution for easy monitoring of the reaction system temperature. The structural design of this nucleic acid amplification reaction tube based on the photothermal conversion solution ensures low power consumption, rapid heating, and a simple structure.

[0034] This invention discloses a method for using a nucleic acid amplification reaction tube based on a photothermal conversion solution. The photothermal conversion solution required for the nucleic acid amplification reaction is placed in a second outer tube. The specific reaction is carried out in a second inner tube as needed. When heating is required, the outer side of the second outer tube corresponding to the photothermal conversion solution located below the support is selected, and near-infrared light is used for irradiation. The light passes through the outer wall to reach the photothermal conversion solution. Heating the photothermal conversion solution increases the temperature of the system inside the second inner tube, and the temperature reading on the temperature detection needle gradually reaches the reaction temperature. Intermittent irradiation can maintain this temperature. The outer side of the second outer tube corresponding to the second light guide is then irradiated with visible light. The light passes through the light guide channel of the second light guide to the second inner tube, exciting the dye in the second inner tube. Fluorescence is emitted from the tube cap of the second inner tube, facilitating subsequent fluorescence detection. This method features low power consumption and fast heating speed. Attached Figure Description

[0035] Figure 1This is a front view schematic diagram of the nucleic acid amplification reaction tube based on the photothermal conversion layer described in this invention.

[0036] Figure 2 This is a schematic diagram of the nucleic acid amplification reaction tube based on the photothermal conversion layer described in this invention from another perspective.

[0037] Figure 3 This is a schematic cross-sectional view of the nucleic acid amplification reaction tube based on the photothermal conversion layer described in this invention.

[0038] Figure 4 This is a schematic diagram of the optical path of the nucleic acid amplification reaction tube based on the photothermal conversion layer described in this invention.

[0039] Figure 5 This is a front view schematic diagram of the nucleic acid amplification reaction tube based on photothermal conversion solution described in this invention.

[0040] Figure 6 This is a schematic diagram of the nucleic acid amplification reaction tube based on photothermal conversion solution described in this invention from another perspective.

[0041] Figure 7 This is a cross-sectional schematic diagram of the nucleic acid amplification reaction tube based on photothermal conversion solution described in this invention.

[0042] Figure 8 This is a schematic diagram of the support structure of the nucleic acid amplification reaction tube based on photothermal conversion fluid according to the present invention.

[0043] Figure 9 yes Figure 8 The main view.

[0044] Figure 10 This is a schematic diagram of the optical path of the nucleic acid amplification reaction tube based on photothermal conversion solution described in this invention.

[0045] In the figure: 101-first inner tube, 102-first outer tube, 103-first light guide, 104-photothermal conversion layer; 111-second inner tube, 112-second light guide, 113-support member, 114-second outer tube, 105-first through hole, 106-placeholder plug, 201-liquid exchange tank, 202-second through hole, 203-groove. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments. These descriptions are for illustrative purposes only and are not intended to limit the scope of the invention.

[0047] Implementation Plan 1

[0048] This invention provides a nucleic acid amplification reaction tube based on a photothermal conversion layer, such as... Figure 1 and Figure 2As shown, it includes a first inner tube 101, a first outer tube 102, a first light guide 103, and a photothermal conversion layer 104. The first inner tube 101 is made of a thermally conductive material, which is polypropylene in this invention. The outer wall of the first outer tube 102 is transparent. The first light guide 103 is made of acrylic or silicon dioxide. The photothermal conversion layer 104 is specifically a gold nanorod layer, a Prussian blue nanoparticle layer, a polydopamine particle layer, or a porphyrin ring particle layer.

[0049] from Figure 3 It is known that a T-shaped cap is inserted into the opening of the first inner tube 101. Since the diameter of the main body of the cap is slightly larger than the diameter of the opening of the first inner tube 101, a sealing effect is achieved. The cap of the first inner tube 101 is transparent. The first light guide 103 is elongated, with its lower end inserted into the bottom of the first outer tube 102 along its height direction. The shape of the lower end of the first light guide 103 is the same as the shape of the corresponding area at the bottom of the first outer tube 102, ensuring contact between the lower end of the first light guide 103 and the bottom of the first outer tube 102. A groove with the same shape as the bottom of the outer wall of the first inner tube 101 is provided on the upper surface of the first light guide 103, and the first inner tube 101 is inserted into this groove. The outer wall of the first inner tube 101, except for the contact area with the first light guide 103, is coated with a photothermal conversion layer 104. Figure 4 As shown, by irradiating the first outer tube 102 with near-infrared light in the horizontal direction on both sides of the first outer tube 102, the photothermal conversion layer 104 can release heat, heating the reaction system of the first inner tube 101. The photothermal conversion layer 104 is mixed with a color-changing coating that facilitates temperature detection. As an example, the present invention selects thermochromic powder, which is obtained by spraying a uniformly mixed photothermal conversion material (gold nanorods, Prussian blue nanoparticles, polydopamine particles or porphyrin ring particles) and color-changing coating onto the outer wall of the first inner tube 101. After reaching the target temperature, it becomes transparent, and the color of the photothermal conversion material itself is displayed. Specifically, the thermochromic powder that changes in the corresponding temperature range can be selected according to different reaction systems. The first outer tube 102 is evacuated to ensure that the reaction system can achieve the heat preservation effect.

[0050] The first inner tube 101 and the first outer tube 102 selected in this invention have the same shape, both being conventional shapes, that is, the bottom is a hemispherical shape and a frustum shape from bottom to top, and the top is cylindrical. The opening of the first outer tube 102 extends horizontally outward, and the extension and the cylindrical part form an L-shaped structure. The opening of the first inner tube 101 also extends horizontally outward, and the extension and the cylindrical part also form an L-shaped structure, with the extension flush with the size of the tube cap. The openings of the first inner tube 101 and the first outer tube 102 are sealed by the tube cap of the first outer tube 102, that is, the tube cap of the first outer tube 102 is inserted at the junction of the openings of the first inner tube 101 and the first outer tube 102. The cap of the first outer tube 102 is a hollow structure with a T-shaped cross-section. The cap of the first outer tube 102 overlaps the opening of the first outer tube 102, and the L-shaped structure of the first inner tube 101 overlaps the hollow part of the cap of the first outer tube 102. Since the wall thickness of the cap of the first outer tube 102 is slightly greater than the gap between the opening of the first inner tube 101 and the opening of the first outer tube 102, a sealing effect can be achieved. The contact position between the cap of the first inner tube 101 and the cap of the first outer tube 102 may also lack the photothermal conversion layer 104.

[0051] The first light guide 103 can form a light guiding channel, and its function can be selected in two ways. One way is to guide the visible light emitted by the external laser diode set below to the first inner tube 101 through the first outer tube 102, and excite the dye in the fluorescent probe in the first inner tube 101. The fluorescence intensity of the dye is positively correlated with the concentration of the target nucleic acid. The fluorescence is emitted from the cap of the first inner tube 101, and the fluorescence intensity is detected above the cap of the first inner tube 101. The other way is to emit visible light from the external laser diode set above into the first inner tube 101 through the cap of the first inner tube 101, and excite the dye in the fluorescent probe in the first inner tube 101. The fluorescence is emitted from the bottom of the first inner tube 101 and is exported through the light guiding channel of the first light guide 103, and the fluorescence intensity is detected.

[0052] The first inner tube 101 contains several different reaction systems, including real-time fluorescent polymerase chain reaction (RT-PCR), real-time fluorescent loop-mediated isothermal amplification reaction (RT-LAMP), real-time fluorescent recombinase polymerase amplification reaction (RT-RPA), real-time fluorescent chain substitution amplification reaction (RT-SDA), and real-time fluorescent helicase-dependent amplification reaction (RT-HDA).

[0053] Therefore, based on the above description of the nucleic acid amplification reaction tube, the method of using the nucleic acid amplification reaction tube based on the photothermal conversion layer of the present invention can be summarized as follows:

[0054] Step 1: In the first inner tube 101, the corresponding nucleic acid amplification reaction system is carried out according to the requirements. When the reaction requires heating, near-infrared light is used to irradiate both sides of the first outer tube 102. At this time, the near-infrared light passes through the outer wall of the first outer tube 102 and reaches the photothermal conversion layer 104.

[0055] Step 2, the photothermal conversion layer 104 is heated as a result, so the temperature of the reaction system in the first inner tube 101 will gradually rise, and finally the photothermal conversion layer 104 will show the color of the photothermal conversion material itself. Then, it is intermittently irradiated to maintain the color of the photothermal conversion material itself.

[0056] Step 3: Illuminate the bottom of the first outer tube 102 with visible light below the first outer tube 102. At this time, the visible light is guided to the first inner tube 101 through the light guide channel of the first light guide 103, which will excite the dye in the fluorescent probe of the first inner tube 101. At this time, the fluorescence will be emitted from the tube cap of the first inner tube 101.

[0057] Alternatively, the cap of the first inner tube 101 can be irradiated with visible light above the first outer tube 102. The visible light will directly enter the first inner tube 101, thereby exciting the dye in the fluorescent probe of the first inner tube 101. The fluorescence is emitted from the bottom of the first outer tube 102 through the light guiding channel of the first light guide 103.

[0058] Implementation Plan 2

[0059] This invention provides a nucleic acid amplification reaction tube based on a photothermal conversion solution, such as... Figure 5 and Figure 6 As shown, it mainly includes a second inner tube 111, a second outer tube 114, a second light guide 112, and a support 113. The second outer tube 114 is used to carry the photothermal conversion fluid, which is a dispersion of Prussian blue nanoparticles in deionized water. During preparation, the ratio of Prussian blue nanoparticles to deionized water is (1.5~2.5) mg: (450~550) μl. The outer wall of the second outer tube 114 is transparent and made of PC plastic. The second inner tube 111 is made of thermally conductive material. The opening of the second inner tube 111 is sealed with a transparent cap. The material of the second light guide 112 is the same as that of the first light guide 103, which is acrylic or silicon dioxide.

[0060] like Figure 7 As shown, a cylindrical support member 113 is fixedly inserted into the second outer tube 114. The diameter of the support member 113 is the same as the inner diameter of the second outer tube 114. The support member 113 is located away from the bottom of the second outer tube 114 and can be positioned at the middle of the height of the second outer tube 114. Figure 8 and Figure 9As shown, the support member 113 has a liquid exchange tank 201 along its height direction, connecting the upper and lower surfaces of the support member 113. The liquid exchange tank 201 is located on the outside of the support member 113 and is evenly distributed in 3 to 5 sections. The lower end of the support member 113 is a small cylinder with a smaller diameter and lower profile, which forms a transition section with the cylindrical main structure, forming a frustum structure. Therefore, the liquid exchange tank 201 also passes through this section. The photothermal conversion fluid passes through the liquid exchange tank 201 and covers the upper surface of the support member 113. The upper surface of the support member 113 has a groove 203 that can accommodate the bottom of the second inner tube 111, so the second inner tube 111 is inserted into the groove 203.

[0061] The cap of the second outer tube 114 is engaged with the opening of the second outer tube 114. A first through hole 105 is provided on the cap of the second outer tube 114. A temperature detection needle is placed in the first through hole 105, and its bottom is supported on the upper surface of the support member 113. Therefore, the temperature detection needle is immersed in the photothermal conversion liquid, which facilitates monitoring the temperature of the reaction system. A second through hole 202 communicating with the groove 203 is provided on the outer wall of the support member 113 from the outside to the inside. The second through hole 202 facilitates the installation of the second light guide 112. The second light guide 112 is inserted into the second through hole 202 and contacts the outer wall of the second inner tube 111 and the inner wall of the second outer tube 114. The number of second through holes 202 is the same as the number of liquid exchange tanks 201. Each second through hole 202 is located between two liquid exchange tanks 201. The second inner tube 111 is higher than the opening of the second outer tube 114.

[0062] In addition, this solution also includes a placeholder plug 106 with the same shape as the bottom of the second outer tube 114. Its bottom contacts the bottom of the second outer tube 114. Therefore, the support member 113 and the placeholder plug 106 have a certain gap, which facilitates the carrying of photothermal conversion fluid, thereby reducing the amount of photothermal conversion fluid used and improving the temperature control effect. The photothermal conversion fluid is located on the upper surface of the placeholder plug 106.

[0063] It should be noted that in this design, the second inner tube 111 and the first inner tube 101 have the same shape. The main structure of the second outer tube 114 has the same shape as the main structure of the first outer tube 102. The difference is that the cap of the second outer tube 114 and the main structure below it are integrated, and the two are set to open and close on the right side. The lower left end of the cap of the second outer tube 114 has a baffle of one large and one small integrally formed. The distance between the inner walls of the two baffles is slightly less than the wall thickness at the corresponding position of the opening of the second outer tube 114, thus facilitating the snap-fit ​​and installation of the cap of the second outer tube 114.

[0064] The second inner tube 111 contains several different reaction systems, including real-time fluorescent polymerase chain reaction (RT-PCR), real-time fluorescent loop-mediated isothermal amplification reaction (RT-LAMP), real-time fluorescent recombinase polymerase amplification reaction (RT-RPA), real-time fluorescent chain substitution amplification reaction (RT-SDA), and real-time fluorescent helicase-dependent amplification reaction (RT-HDA).

[0065] Based on the above description of the nucleic acid amplification reaction tube, the method of using the nucleic acid amplification reaction tube based on the photothermal conversion solution of the present invention can be summarized as follows:

[0066] Step 1: Place the photothermal conversion solution required for nucleic acid amplification reaction in the second outer tube 114. Separately, conduct the specific reaction as needed in the second inner tube 111. If heating is required, such as... Figure 10 As shown, the outer position of the second outer tube 114 corresponding to the photothermal conversion fluid located below the support 113 is selected and irradiated with near-infrared light, and the light passes through the outer wall to reach the photothermal conversion fluid;

[0067] Step 2: After being heated, the photothermal conversion liquid will increase the system temperature in the second inner tube 111. The temperature value of the temperature detection needle will gradually reach the reaction temperature. Then, intermittent irradiation can maintain this temperature.

[0068] Step 3: Select the outer side of the second outer tube 114 corresponding to the second light guide 112, and then irradiate both sides of the second outer tube 114 with visible light. The light is guided to the second inner tube 111 through the light guide channel of the second light guide 112. At this time, the dye in the second inner tube 111 is excited, and the fluorescence is emitted from the tube cap of the second inner tube 111.

[0069] Example 1

[0070] When performing RT-RPA, take the first inner tube 101 with pre-placed protease (pre-prepared lyophilized powder), add 25 μl of RPA reaction buffer, 2.1 μl each of forward and reverse primers, 0.6 μl of fluorescent probe, 15.2 μl of water, and 2 μl of detection target.

[0071] Add 3 μl of initiator through the cap of the first inner tube 101, followed by centrifugation to initiate the reaction. Irradiate the Prussian blue nanoparticle layer on the outer wall of the first inner tube 101 using infrared light with a wavelength of 808 nm through the first outer tube 102. The thermochromic powder, mixed in a 1:5 mass ratio (thermochromic powder:Prussian blue nanoparticles), facilitates temperature detection. Upon reaching 37°C, the nanoparticles become transparent, revealing their color. After the reaction system temperature in the first inner tube 101 reaches 37°C, intermittent irradiation is used to maintain the temperature between 37°C and 39°C, ensuring the Prussian blue nanoparticles remain visible throughout the process. This temperature range is suitable for nucleic acid incubation. Visible light emitted by the laser diode positioned below is guided through the first outer tube 102 and the first light guide 103 into the first inner tube 101, exciting the fluorescent dye in the reaction system. The fluorescence is collected above the first inner tube 101 and detected at a fixed interval of 5 seconds. The final result of the test was that the fluorescence intensity gradually increased within 30 minutes, and the curve showed a clear upward trend. The fluorescence intensity value was 8 times the initial background fluorescence intensity value, thus proving that the target nucleic acid was present in the detected target.

[0072] Example 2

[0073] When performing RT-RPA, place the relevant enzymes, primers, probes, reaction gain agent, water, and detection target in the second inner tube 111. The specific system configuration is as follows:

[0074] One part of protease (pre-prepared lyophilized powder), 25 μl of RPA reaction buffer, 2.1 μl each of forward and reverse primers, 0.6 μl of fluorescent probe, 15.2 μl of water, and 2 μl of detection target.

[0075] 500 μl of water was injected into the second outer tube 114 and shaken thoroughly to uniformly disperse 2 mg of Prussian blue nanoparticles, thus obtaining the photothermal conversion fluid.

[0076] The cap of the second inner tube 111 was opened, 3 μl of initiator was added, and then centrifuged to initiate the reaction. A temperature detection needle was inserted into the second outer tube 114 through the first through-hole 105. Infrared heating light with a wavelength of 808 nm was used to irradiate the photothermal conversion solution inside the second outer tube 114, raising the temperature of the reaction system in the second inner tube 111 to 37°C. Irradiation was then intermittent to maintain the temperature of the reaction system at no lower than 37°C and no higher than 39°C, facilitating nucleic acid incubation. Visible light emitted by the laser diode was directed through the second light guide 112, exciting the fluorescence of the system in the second inner tube 111. The fluorescence was collected from above the second inner tube 111 and detected at a fixed detection interval of 5 seconds. The final result showed that the fluorescence intensity gradually increased over 30 minutes, with a clear upward trend. The fluorescence intensity was 10 times the initial background fluorescence intensity, thus proving that the target nucleic acid was present in the detected sample.

Claims

1. A photothermal conversion layer-based nucleic acid amplification reaction tube, characterized by, It includes a first inner tube (101), a first outer tube (102), a first light guide (103), and a photothermal conversion layer (104); The lower end of the first light guide (103) is inserted into the bottom of the first outer tube (102) along the height direction of the first outer tube (102). The shape of the lower end of the first light guide (103) is the same as the shape of the area corresponding to the bottom of the first outer tube (102). The upper surface of the first light guide (103) is provided with a groove with the same shape as the bottom of the outer wall of the first inner tube (101). The first inner tube (101) is inserted into the groove. The outer wall of the first inner tube (101) is coated with a photothermal conversion layer (104) except at the contact position with the first light guide (103). The opening of the first inner tube (101) is sealed in the opening of the first outer tube (102). The opening of the first inner tube (101) is sealed with a transparent tube cap. The outer wall of the first outer tube (102) is transparent, the first inner tube (101) is made of thermally conductive material, the photothermal conversion layer (104) contains color-changing paint, the first outer tube (102) is irradiated by near-infrared light in the horizontal direction on both sides, the photothermal conversion layer (104) releases heat, and when the target temperature is reached, the color-changing paint turns transparent, the photothermal conversion layer (104) displays its own color, and the first outer tube (102) is in a vacuum state.

2. The photothermal conversion layer-based nucleic acid amplification reaction tube according to claim 1, wherein The first light guide (103) is made of acrylic or silicon dioxide.

3. The photothermal conversion layer-based nucleic acid amplification reaction tube according to claim 1, wherein The photothermal conversion layer (104) is a gold nanorod layer, a Prussian blue nanoparticle layer, a polydopamine particle layer, or a porphyrin ring particle layer.

4. A method for using a photothermal conversion layer-based nucleic acid amplification reaction tube, characterized by, The nucleic acid amplification reaction tube based on the photothermal conversion layer according to any one of claims 1 to 3 includes the following steps: S1, the required nucleic acid amplification reaction system is prepared in the first inner tube (101) and the reaction is carried out. When heating is required, near-infrared light is used to irradiate the outside of the first outer tube (102) corresponding to the photothermal conversion layer (104). The near-infrared light passes through the corresponding outer wall of the first outer tube (102) to reach the photothermal conversion layer (104). S2, after the photothermal conversion layer (104) is heated, the temperature of the reaction system inside the first inner tube (101) rises, and the photothermal conversion layer (104) displays the color of the photothermal conversion material itself. Then, it is intermittently irradiated to maintain the color of the photothermal conversion material itself, and then proceeds according to S31 or S32. S31, below the first outer tube (102), the bottom of the first outer tube (102) is irradiated with visible light. The visible light is guided to the first inner tube (101) through the light guide channel of the first light guide (103), which excites the dye in the fluorescent probe in the first inner tube (101). The fluorescence is emitted from the tube cap of the first inner tube (101). S32, above the first outer tube (102), the cap of the first inner tube (101) is irradiated with visible light. The visible light enters the first inner tube (101) and excites the dye in the fluorescent probe in the first inner tube (101). The fluorescence is emitted from the bottom of the first outer tube (102) through the light guide channel of the first light guide (103).

5. A nucleic acid amplification reaction tube based on a photothermal conversion liquid, characterized by It includes a second inner tube (111), a second outer tube (114), a second light guide (112), and a support member (113). The second outer tube (114) carries a photothermal conversion fluid. A support member (113) is fixedly inserted into the second outer tube (114). The support member (113) is far from the bottom of the second outer tube (114). The support member (113) is provided with a liquid exchange tank (201) along its height direction, which connects the upper surface and the lower surface of the support member (113). The photothermal conversion fluid passes through the liquid exchange tank (201) and covers the upper surface of the support member (113). The upper surface of the support member (113) is provided with a groove (203) that can accommodate the bottom of the second inner tube (111). The second inner tube (111) is inserted into the groove (203). The outer wall of the second outer tube (114) is transparent, the second inner tube (111) is made of thermally conductive material, the cap of the second outer tube (114) is fitted into the opening of the second outer tube (114), the cap of the second outer tube (114) is provided with a first through hole (105) for placing a temperature detection needle, the bottom of the temperature detection needle is supported on the upper surface of the support member (113), the outer wall of the support member (113) is provided with a second through hole (202) communicating with the groove (203) from the outside to the inside, a second light guide (112) is inserted into the second through hole (202) and contacts the outer wall of the second inner tube (111) and the inner wall of the second outer tube (114), the opening of the second inner tube (111) is sealed with a transparent cap, and the second inner tube (111) is set higher than the opening of the second outer tube (114).

6. The photothermally convertible liquid-based nucleic acid amplification reaction tube according to claim 5, wherein It also includes a placeholder plug (106), which has the same shape as the bottom of the second outer tube (114). The bottom of the placeholder plug (106) is in contact with the bottom of the second outer tube (114). The support member (113) and the placeholder plug (106) have a gap. The photothermal conversion fluid is located on the upper surface of the placeholder plug (106).

7. The photothermally convertible liquid-based nucleic acid amplification reaction tube according to claim 5, wherein The photothermal conversion fluid is a dispersion of Prussian blue nanoparticles in deionized water, with a ratio of (1.5~2.5) mg to (450~550) μl between the Prussian blue nanoparticles and the deionized water.

8. The photothermally convertible liquid-based nucleic acid amplification reaction tube according to claim 5, wherein The main structure of the support member (113) is cylindrical. The diameter of the support member (113) is the same as the inner diameter of the second outer tube (114). The support member (113) is located at the middle of the height of the second outer tube (114).

9. The photothermally convertible liquid-based nucleic acid amplification reaction tube according to claim 8, wherein The liquid exchange tank (201) is located on the outside of the support (113) and there are 3 to 5 of them evenly distributed. The number of second through holes (202) is the same as that of liquid exchange tanks (201), and each second through hole (202) is located between two liquid exchange tanks (201).

10. A method for using a nucleic acid amplification reaction tube based on a photothermal conversion liquid, characterized by, The nucleic acid amplification reaction tube based on photothermal conversion solution according to any one of claims 5 to 9 is characterized by comprising the following steps: S1, first place a set volume of photothermal conversion liquid in the second outer tube (114), then prepare the required nucleic acid amplification reaction system in the second inner tube (111) for reaction, when heating is required, irradiate the outside of the second outer tube (114) corresponding to the photothermal conversion liquid located below the support (113) with near-infrared light, and the near-infrared light passes through the corresponding outer wall of the second outer tube (114) to reach the photothermal conversion liquid; S2, after the photothermal conversion liquid is heated, the temperature of the reaction system in the second inner tube (111) rises, the temperature value of the temperature detection needle reaches the temperature required for the reaction, and then it is intermittently irradiated to maintain the temperature. S3, on the outside of the second outer tube (114) corresponding to the second light guide (112), visible light is used to irradiate both sides of the second outer tube (114). The visible light is guided through the light guide channel of the second light guide (112) to the inside of the second inner tube (111), which excites the dye in the fluorescent probe in the second inner tube (111). The fluorescence is emitted from the tube cap of the second inner tube (111).

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