Amplification chip and amplification analyzer

Abstract

The invention discloses an amplification chip and an amplification analysis device, and belongs to the field of nucleic acid detection, the amplification chip comprises a chip main body provided with a flow channel, the amplification chip also comprises a temperature control assembly, the temperature control assembly comprises at least one heating member, the at least one heating member heats a part of the flow channel, so that the flow channel forms at least two areas with different temperatures, and the heating member is used for heating the flow channel. The reagent reciprocates in two regions with different temperatures, so that the reaction time of the reagent is shortened, and the detection sensitivity of the amplification chip is improved.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid amplification, and in particular to amplification chips and amplification analysis apparatus including amplification chips. Background Technology

[0002] Nucleic acid amplification detection technology achieves precise quantification of nucleic acids by monitoring signal changes during the rapid and massive replication of specific DNA or RNA fragments, and is one of the important technologies in the field of molecular biology. Currently, mainstream nucleic acid amplification is broadly divided into two categories: thermal cycling amplification and isothermal amplification, with thermal cycling amplification being the most typical and commonly used.

[0003] Rapid nucleic acid amplification and analysis is one of the important development directions in this field. Conventional qPCR platforms use the temperature-changing capability of semiconductor Peltiers to achieve multiple temperature gradients to amplify nucleic acids. However, due to limitations in temperature load and Peltier power, the temperature change rate is usually 2℃ / s - 3℃ / s. A few advanced platforms can achieve a temperature change capability of 6℃ / s - 7℃ / s, thereby controlling the entire nucleic acid quantification process within 25 minutes.

[0004] To further shorten the nucleic acid amplification analysis time, a method has been proposed that sets up multiple constant temperature zones and alternately places the sample in multiple temperature zones to achieve the purpose of temperature change and thus realize nucleic acid amplification. However, it is usually difficult to balance the reaction sample volume and reaction time. When the reaction system volume is too small, it seriously affects the sensitivity of nucleic acid detection. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide an amplification chip with short reaction time and high detection sensitivity.

[0006] To overcome the shortcomings of the prior art, the second objective of this invention is to provide an amplification analysis device with short reaction time and high detection sensitivity.

[0007] One of the objectives of this invention is achieved through the following technical solution: An amplification chip includes a chip body with a flow channel. The amplification chip also includes a temperature control component, which includes at least one heating element. The at least one heating element heats a portion of the flow channel, causing the flow channel to form at least two regions with different temperatures.

[0008] Furthermore, there are two heating elements, which heat different positions of the flow channel, thereby creating two regions with different temperatures in the flow channel.

[0009] Furthermore, the two regions with different temperatures are the denaturation region and the annealing region, respectively, with the temperature of the denaturation region being higher than that of the annealing region.

[0010] Furthermore, the two heating elements are spaced apart.

[0011] Furthermore, the temperature control component also includes a heat insulation element located between the two heating elements.

[0012] Furthermore, the chip body is also provided with an inlet and an outlet, both of which are connected to the flow channel, and at least two regions with different temperatures are located between the inlet and the outlet.

[0013] The second objective of this invention is achieved by the following technical solution: An amplification analysis device includes a support, a liquid-driven structure, and any one of the aforementioned amplification chips. The amplification chip and the liquid-driven structure are mounted on the support. The liquid-driven structure includes a pump tube, a conduit, and a drive pump. The drive pump drives the pump tube through the conduit. The pump tube injects liquid into the flow channel through a pipette. The drive pump drives the liquid to reciprocate in at least two regions with different temperatures.

[0014] Furthermore, the liquid drive structure also includes a second drive member, a mounting plate, and a third drive member. The mounting plate is mounted on the output end of the second drive member, the third drive member is fixed to the mounting plate, and the pump tube is mounted on the output end of the third drive member. The second drive member drives the pump tube to move along a first direction, and the third drive member drives the pump tube to move along a second direction perpendicular to the first direction.

[0015] Furthermore, the amplification analysis device includes an imaging structure, which includes a camera, and the camera acquires images of the flow channel.

[0016] Furthermore, the amplification analysis device also includes a driving structure mounted on the support. The driving structure includes a driving component and a moving component. The moving component is connected to the driving component in a transmission manner. The chip body is fixedly connected to the moving component. The driving component drives the amplification chip to move through the moving component.

[0017] Compared with the prior art, the amplification chip of the present invention includes a chip body with a flow channel. The amplification chip is characterized in that: the amplification chip also includes a temperature control component, which includes at least one heating element. The at least one heating element heats part of the flow channel, so that the flow channel forms at least two regions with different temperatures. By causing the reagent to move back and forth between the two regions with different temperatures, the reaction time of the reagent is shortened and the detection sensitivity of the amplification chip is improved. Attached Figure Description

[0018] Figure 1 This is a three-dimensional view of the amplification analysis device of the present invention; Figure 2for Figure 1 A three-dimensional view of the amplification chip of the amplification analysis device; Figure 3 for Figure 2 An exploded view of the amplification chip; Figure 4 for Figure 3 Internal structure diagram of the amplification chip; Figure 5 for Figure 1 A partial three-dimensional view of the amplification analysis device; Figure 6 for Figure 1 Another partial three-dimensional view of the amplification analysis device; Figure 7 for Figure 1 A three-dimensional image of the imaging structure of the amplification analysis device; Figure 8 The temperature change curve of the sample inside the flow channel; Figure 9 This is a graph showing the change in fluorescence imaging results with cycle number; Figure 10 Nucleic acid amplification curves of standard concentration plasmid samples at different dilution levels.

[0019] In the diagram: 10. Bracket; 11. Base plate; 12. Column; 13. Panel; 20. Amplification chip; 21. Chip body; 210. Main body; 211. Top layer; 212. Bottom layer; 213. Inlet; 214. Outlet; 215. Flow channel; 22. Temperature control component; 220. Mounting plate; 2201. First placement hole; 221. First heating element; 222. Second heating element; 223. Heat insulation element; 224. Mounting block; 22 40. Second placement hole; 30. Drive structure; 31. First drive component; 32. Mounting bracket; 33. Limit switch; 34. Moving component; 35. Linkage rod; 40. Liquid drive structure; 41. Second drive component; 42. Mounting plate; 43. Third drive component; 44. Pump pipe; 45. Pipeline; 46. Drive pump; 50. Imaging structure; 51. Light source; 52. Excitation light filter assembly; 53. Emission light filter assembly; 54. Camera. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or it can be fixed through another intermediate component. When a component is said to be "connected to" another component, it can be directly connected to the other component or it may be fixed through another intermediate component. When a component is said to be "set on" another component, it can be set directly on the other component or it may be set through another intermediate component. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] Please see Figure 1 The amplification analysis device of this application includes a support 10, an amplification chip 20, a driving structure 30, a liquid driving structure 40, and an imaging structure 50.

[0024] The bracket 10 includes a base plate 11, a column 12, and a panel 13. The two ends of the column 12 are fixedly connected to the base plate 11 and the panel 13, respectively. The column 12 is perpendicular to the base plate 11, and the panel 13 is parallel to the base plate 11. The base plate 11, column 12, and panel 13 together form an installation space, in which the light source 51 and the excitation light filter assembly 52 are installed. The panel 13 has an opening for the camera 54 to acquire images of the amplification chip 20 mounted at the bottom of the panel 13.

[0025] Please continue reading. Figure 2 , Figure 3 as well as Figure 4 The amplification chip 20 includes a chip body 21 and a temperature control component 22. The temperature control component 22 causes the flow channel 215 of the chip body 21 to form at least two regions with different temperatures, so that the reactants can react repeatedly in at least two regions with different temperatures.

[0026] The chip body 21 includes a main body 210, which includes a top layer 211 and a bottom layer 212. The top layer 211 and the bottom layer 212 are fixedly connected, and a flow channel 215 is formed between them. In this embodiment, the thickness of the top layer 211 is greater than that of the bottom layer 212, and the bottom layer 212 is thinner to ensure rapid heat transfer. The top layer 211 is injection molded from PDMS or plastic material. The plastic material can be any one of PC, PMMA, COC, or COP. The thickness of the bottom layer 212 is 1μm-1mm, that is, the thickness of the bottom layer 212 can be 1μm, 500μm, or 1mm. The top layer 211 and the bottom layer 212 are connected by any one of physical bonding, ultrasonic bonding, thermo-press bonding, or chemical bonding to form a whole. In other embodiments, the main body 210 can also be a three-layer structure.

[0027] The main body 210 has an internal flow channel 215, which is S-shaped. The flow channel 215 is used for the flow of reactants and the reaction. In this application, the temperature control component 22 creates different temperatures in different parts of the flow channel 215, allowing the reagent to move back and forth in different temperature regions, shortening the reaction time of the reagent and improving the detection sensitivity of the amplification chip. In this embodiment, there are two regions with different temperatures: a high-temperature denaturation region and a low-temperature annealing extension region. The temperature of the high-temperature denaturation region is 90-100 degrees Celsius, and the temperature of the low-temperature annealing extension region is 60-70 degrees Celsius. Specifically, the length of the flow channel 215 is 1 mm-500 mm, the inner wall diameter is 0.1 mm-2 mm, and it can hold a sample volume of 20 μL-1.5 mL. The main body 210 also has an inlet 213 and an outlet 214. The inlet 213 is connected to one end of the flow channel 215, and the outlet 214 is connected to the other end of the flow channel 215. Inlet 213 is used for reagent entry, and outlet 214 is used for reactant exit. In this embodiment, there are two inlets 213, one for sample injection and the other for oil injection. The specific injection sequence is: oil-sample-oil-air, thereby achieving sample sealing in channel 215 to prevent evaporation. The final air section drives the oil-sample-oil system to move within channel 215. Outlet 214 is connected to the atmosphere for pressure balance, ensuring smooth movement of the oil-sample-oil system within channel 215.

[0028] The temperature control assembly 22 includes a mounting plate 220, heating elements, and a mounting block 224. The heating elements are installed inside the mounting plate 220 to heat the flow channel 215, creating temperature-different zones within the flow channel 215. Specifically, the mounting plate 220 has a first placement hole 2201 for placing the sample loading nozzle. There are two heating elements: a first heating element 221 and a second heating element 222, spaced apart to prevent interference. The heating elements are made of a high-transmittance and conductive material to facilitate subsequent fluorescence excitation and image acquisition. Specifically, the heating elements are made of glass coated with indium tin oxide (ITO), gallium-doped zinc oxide (GZO), fluorine-doped tin oxide (FTO), aluminum-doped zinc oxide (AZO), or other small-molecule conductive organic polymer materials. In this embodiment, the first heating element 221 and the second heating element 222 are insulated from each other by a heat insulation element 223. The heat insulation element 223 is made of a material with low thermal conductivity, such as polytetrafluoroethylene (PTFE). Mounting block 224 is fixed to mounting plate 220. Mounting block 224 is provided with second placement hole 2240 for placing test tubes. In this embodiment, there are two second placement holes 2240, which are used to place samples and sealing oil, respectively.

[0029] Please continue reading. Figure 6 The driving structure 30 includes a first driving member 31, a mounting bracket 32, a limit switch 33, a moving member 34, and a connecting rod 35. The mounting bracket 32 ​​is fixed to the bottom of the panel 13, and the first driving member 31 is mounted on the mounting bracket 32. The limit switch 33 is fixed to the mounting bracket 32 ​​and is used to detect the position of the amplification chip 20. The moving member 34 is connected to the first driving member 31, converting the rotational motion of the first driving member 31 into linear motion. In other embodiments, the moving member 34 and the first driving member 31 can also be cylinders. The connecting rod 35 is fixed to the moving member 34 and fixedly connected to the amplification chip 20. The driving structure 30 drives the amplification chip 20 to move on the panel 13, enabling the amplification chip 20 to switch between the sample application position and the image acquisition position.

[0030] Please continue reading. Figure 5The liquid-driven structure 40 includes a second driving member 41, a mounting plate 42, a third driving member 43, a pump tube 44, a conduit 45, and a driving pump 46. The second driving member 41 is fixed to the panel 13 and drives the mounting plate 42, the third driving member 43, and the pump tube 44 to move along a first direction. In this embodiment, the first direction is parallel to the panel 13. The mounting plate 42 is fixed to the output end of the second driving member 41, the third driving member 43 is fixed to the mounting plate 42, and the pump tube 44 is mounted to the output end of the third driving member 43. The third driving member 43 drives the pump tube 44 to move along a second direction perpendicular to the first direction; in this embodiment, the second direction is vertical. The pump tube 44 is connected to the driving pump 46 through the conduit 45, and the driving pump 46 drives the pump tube 44 to draw in or expel liquid through the conduit 45.

[0031] Please continue reading. Figure 7 The imaging structure 50 includes a light source 51, an excitation light filter assembly 52, an emission light filter assembly 53, and a camera 54. The light source 51 and the excitation light filter assembly 52 are mounted on the bottom of the panel 13. The light source 51 can be selected as a monochromatic or mixed-color light source. The excitation light emitted by the light source 51 is filtered by the excitation light filter assembly 52 and then shines into the flow channel 215 of the chip body 21. The reagents in the flow channel 215 generate fluorescence under the action of the excitation light, and the fluorescence reaches the camera 54 after passing through the emission light filter assembly 53.

[0032] The following example uses a nucleic acid amplification reaction to illustrate the working principle of the amplification analysis device: a. Equipment startup: Select the test item. The first heating element 221 and the second heating element 222 on the temperature control panel will automatically heat up to the appropriate low temperature (around 65°C) and high temperature (around 95°C) according to the configuration file, and the liquid drive structure 40 will extend. b. Preparation of reaction system: Add the prepared nucleic acid reaction system (usually including buffer, premix, forward primer, reverse primer, probe, PCR additive, and nuclease-free water for PCR reaction) into the ep tube and place it in the second placement well 2240 on the left. Place the second placement well 2240 on the right with sealing oil (such as high temperature fluorinated oil FC150). c. Consumable preparation: Place the two 100μL pipette tips in the first placement hole 2240 respectively, and operate the liquid drive structure 40 to inject the sample; d. Drive pump 46 to the middle position to ensure that 25 μL of sample can be aspirated while ensuring that air can continue to drive the flow of the sample in the flow channel 215; f. The second drive unit 41 and the third drive unit 43 push the pump tube 44 to the first placement hole 2240, and after the pump tube 44 is lowered to install the gun head, it rises. g. The second driving member 41 and the third driving member 43 push the pump tube 44 to the second placement hole 2240, and independently draw 25 μL of the reaction system and 20 μL of the sealing oil, respectively. h. After injecting about 3μL of sealing oil into the flow channel 215 through one pump pipe 44 (actually considering the volume of the branch flow), the oil seal at the head end of the reaction system is achieved. Then, the reaction liquid is injected into the flow channel 215 through another pump pipe 44, and about 3μL of oil is injected (actually considering the volume of the branch flow) to achieve the oil seal at the tail end. i. Determine the dwell time in the high-temperature and low-temperature regions according to the amplification reaction protocol, such as... Figure 8 As shown, the steps of reverse transcription of nucleic acid, hot-start enzyme, and nucleic acid amplification are respectively implemented; j. While driving the sample to the low-temperature annealing extension region, use camera 54 to record single-channel or multi-channel fluorescence patterns for fluorescence imaging, such as... Figure 9 As shown, the fluorescence intensity was calculated.

[0033] k. Plot the cycle number-fluorescence value curve of the tested sample, such as... Figure 10 As shown, calculate the nucleic acid concentration.

[0034] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These are all equivalent modifications and improvements made to the above embodiments based on the essential technology of the present invention, and all of these fall within the protection scope of the present invention.

Claims

1. An amplification chip, comprising a chip body, wherein the chip body is provided with flow channels, characterized in that: The amplification chip also includes a temperature control component, which includes at least one heating element. The at least one heating element heats a portion of the flow channel, causing the flow channel to form at least two regions with different temperatures.

2. The amplification chip according to claim 1, characterized in that: The number of heating elements is two, and the two heating elements heat different positions of the flow channel, so that the flow channel forms two regions with different temperatures.

3. The amplification chip according to claim 2, characterized in that: The two regions with different temperatures are the denaturation region and the annealing region, respectively, with the temperature of the denaturation region being higher than that of the annealing region.

4. The amplification chip according to claim 2, characterized in that: The two heating elements are spaced apart.

5. The amplification chip according to claim 4, characterized in that: The temperature control component also includes a heat insulation element, which is located between the two heating elements.

6. The amplification chip according to claim 1, characterized in that: The chip body is also provided with an inlet and an outlet, both of which are connected to the flow channel, and at least two regions with different temperatures are located between the inlet and the outlet.

7. An amplification analysis device, comprising a support, characterized in that: The amplification analysis device further includes a liquid-driven structure and an amplification chip as described in any one of claims 1-6. The amplification chip and the liquid-driven structure are mounted on the support. The liquid-driven structure includes a pump tube, a conduit, and a drive pump. The drive pump drives the pump tube through the conduit. The pump tube injects liquid into the flow channel through a pipette. The drive pump drives the liquid to reciprocate in at least two regions with different temperatures.

8. The amplification analysis apparatus according to claim 7, characterized in that: The liquid drive structure further includes a second drive component, a mounting plate, and a third drive component. The mounting plate is installed at the output end of the second drive component, the third drive component is fixed to the mounting plate, and the pump tube is installed at the output end of the third drive component. The second drive component drives the pump tube to move along a first direction, and the third drive component drives the pump tube to move along a second direction perpendicular to the first direction.

9. The amplification analysis apparatus according to claim 7, characterized in that: The amplification analysis device includes an imaging structure, which includes a camera, and the camera acquires images of the flow channel.

10. The amplification analysis apparatus according to claim 7, characterized in that: The amplification analysis device further includes a driving structure mounted on the support. The driving structure includes a driving component and a moving component. The moving component is connected to the driving component in a transmission manner. The chip body is fixedly connected to the moving component. The driving component drives the amplification chip to move through the moving component.

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