Capillary cartridge reactor for ultrafast PCR

Abstract

The application belongs to the technical field of molecular biology detection, and particularly relates to a capillary type cassette reactor for ultrafast PCR, which comprises a cassette body, a transparent detection window, capillaries, independent sample pools, a first oil storage groove and a light-heat conversion layer. The cassette body is made of 3D printing or injection molding process, the transparent detection window is arranged in the middle of the cassette body, multiple capillaries are arranged side by side in the transparent detection window, one end of the capillary is communicated with the independent sample pool, the other end is communicated with the first oil storage groove, and the capillary is of a transparent structure; the light-heat conversion layer is fixed with the cassette body and is correspondingly arranged with the transparent detection window, and the multiple capillaries are located between the light-heat conversion layer and the transparent detection window. The application has the advantages of compact structure, fast heat response speed, good temperature uniformity, effective inhibition of sample evaporation, real-time fluorescence signal acquisition, simple operation, low cost, and suitability for ultrafast PCR nucleic acid amplification and on-site nucleic acid detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, and particularly relates to a capillary cartridge reactor for ultra-fast PCR. Background Technology

[0002] Polymerase chain reaction (PCR) and real-time fluorescence PCR are core technologies in the field of nucleic acid detection, and are widely used in clinical diagnosis, infectious disease screening and gene analysis.

[0003] With the increasing demand for on-site rapid testing, ultra-rapid PCR has become an important direction for technological development. It requires a heating / cooling rate of no less than 10-20℃ / s to complete nucleic acid amplification in a short time. Most existing mainstream PCR equipment uses semiconductor cooling pads for contact heating, with a temperature heating / cooling rate of only 1-2℃ / s. This results in high system thermal inertia, a single test taking more than 1 hour, and the equipment is complex, bulky, and expensive, making it difficult to adapt to rapid on-site testing.

[0004] Traditional PCR tubes or plate-type reaction wells suffer from problems such as thick walls, high thermal resistance, and large thermal inertia of the reaction system. They also exhibit poor temperature consistency across multiple channels, leading to severe sample evaporation under high-speed thermal cycling conditions, and lack a dedicated real-time fluorescence detection window. While microfluidic PCR chips are compact, they rely on complex processes such as photolithography and bonding, resulting in high manufacturing costs. They require specialized dispensing equipment and are prone to air bubbles and channel blockage during use. Conventional capillary consumables, although suitable for rapid thermal cycling, still suffer from inconvenient sample addition, insufficient evaporation control, difficulties in array integration, and a lack of optical detection windows, failing to reliably meet the application requirements of ultra-fast PCR.

[0005] Therefore, there is an urgent need for a capillary cartridge reactor for ultra-fast PCR. Summary of the Invention

[0006] The purpose of this invention is to provide a capillary cartridge reactor for ultrafast PCR to solve the above-mentioned problems.

[0007] To achieve the above objectives, the present invention provides the following solution: A capillary cartridge reactor for ultrafast PCR, comprising: The card holder itself is manufactured using 3D printing or injection molding processes; A transparent detection window is provided in the middle of the cartridge body. Multiple capillaries are arranged side by side in the transparent detection window. One end of each capillary is connected to an independent sample cell, and the other end of each capillary is connected to a first oil storage tank. The capillary is transparent; It also includes a photothermal conversion layer fixed to the card holder body, which is correspondingly disposed to the transparent detection window, and a plurality of capillaries are located between the photothermal conversion layer and the transparent detection window.

[0008] Optionally, the photothermal conversion layer is composed of a high light-absorbing and thermally conductive material made of any one or a combination of black metal thin film, graphite thin film, graphene coating, carbon black coating or metal oxide light-absorbing layer.

[0009] Optionally, a transparent section is provided in the middle of the capillary, and the transparent section is provided in correspondence with the transparent detection window.

[0010] Optionally, the capillary is made of glass, fused silica, borosilicate glass, or a transparent polymer material.

[0011] Optionally, the cartridge body is further provided with a second oil storage tank, and the liquid inlet ends of the multiple independent sample cells are connected to the same second oil storage tank.

[0012] Optionally, the liquid outlet ends of the multiple capillaries are connected to the same first oil storage tank.

[0013] Optionally, two adjacent independent sample cells are staggered.

[0014] Optionally, the card holder body is made of black PLA material.

[0015] Optionally, slots for inserting the capillary tube are provided on both sides of the transparent detection window.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention utilizes capillaries as the reaction carrier, resulting in a small reaction system with low thermal inertia, enabling rapid heating and cooling to meet the temperature cycling requirements of ultrafast PCR. The corresponding arrangement of capillaries and transparent detection windows ensures effective excitation light incidence and stable fluorescence signal transmission, improving the signal-to-noise ratio for real-time detection. Independent sample cells and oil reservoirs work together to form a stable evaporation barrier during the reaction, reducing sample evaporation loss under high-speed thermal cycling. The staggered arrangement of adjacent independent sample cells makes the capillary arrangement more compact, and combined with uniform heating from the photothermal conversion layer, improves the temperature consistency and amplification parallelism of multi-channel reactions. The cartridge body is made of black light-shielding material, reducing background light interference and further improving detection accuracy. The integrated design simplifies assembly, eliminating the need for specialized liquid injection equipment for sample addition, resulting in lower material and manufacturing costs, making it suitable for single-use and large-scale application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 This is a schematic diagram of the structure of the present invention; The components include: 1. Card body; 2. Capillary tube; 3. Independent sample cell; 4. First oil storage tank; 5. Transparent detection window; 6. Photothermal conversion layer. Detailed Implementation

[0018] 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.

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Reference Figure 1 This invention discloses a capillary cartridge reactor for ultrafast PCR, comprising: The card box body 1 is manufactured by 3D printing or injection molding. A transparent detection window 5 is set in the middle of the card body 1. Multiple capillaries 2 are arranged side by side in the transparent detection window 5. One end of the capillaries 2 is connected to an independent sample cell 3, and the other end of the capillaries 2 is connected to the first oil storage tank 4. Capillary tube 2 is set to be transparent; It also includes a photothermal conversion layer 6 fixed to the card holder body 1, which is correspondingly set to the transparent detection window 5, and multiple capillaries 2 are located between the photothermal conversion layer 6 and the transparent detection window 5.

[0021] In use, the cartridge body 1 is made by 3D printing or injection molding. The transparent detection window 5 is located in the middle of the cartridge body 1. Multiple capillaries 2 are placed side by side in the transparent detection window 5. One end of the capillaries 2 is connected to the independent sample cell 3, and the other end is connected to the first oil storage tank 4. The capillaries 2 are kept transparent. The photothermal conversion layer 6 is fixed to the cartridge body 1 and corresponds to the transparent detection window 5. Multiple capillaries 2 are located between the photothermal conversion layer 6 and the transparent detection window 5. The whole system realizes sample injection, capillary reaction, rapid photothermal heating and cooling, and real-time fluorescence detection. The structure is compact and suitable for ultra-fast PCR.

[0022] As an optional implementation, the photothermal conversion layer 6 is composed of a high light-absorbing and thermally conductive material made of any one or a combination of black metal thin film, graphite thin film, graphene coating, carbon black coating or metal oxide light-absorbing layer.

[0023] The photothermal conversion layer 6 is made of one or more high light-absorbing and thermally conductive materials selected from black metal film, graphite film, graphene coating, carbon black coating or metal oxide light-absorbing layer. It can efficiently absorb light energy and quickly convert it into heat, thereby increasing the heating and cooling rate of capillary 2 and meeting the thermal cycling requirements of ultra-fast PCR.

[0024] As an optional implementation, a transparent section is provided in the middle of the capillary 2, and the transparent section is provided in correspondence with the transparent detection window 5.

[0025] A transparent section is provided in the middle of the capillary 2, which corresponds to the position of the transparent detection window 5. This ensures that the excitation light can smoothly enter the interior of the capillary 2 and that the fluorescence signal can be stably transmitted, thereby improving the signal-to-noise ratio of real-time detection.

[0026] As an alternative implementation, the capillary 2 is made of glass, fused silica, borosilicate glass, or a transparent polymer material.

[0027] The capillary tube 2 is made of glass, fused silica, borosilicate glass or transparent polymer material, and has good light transmittance and thermal conductivity, making it suitable for ultra-fast thermal cycling and fluorescence signal acquisition.

[0028] As an optional implementation, the cartridge body 1 is also provided with a second oil storage tank, and the liquid inlet end of multiple independent sample cells 3 is connected to the same second oil storage tank.

[0029] The cartridge body 1 is equipped with a second oil storage tank. The liquid inlet ends of multiple independent sample cells 3 are all connected to the same second oil storage tank, which can uniformly perform oil sealing treatment on the independent sample cells 3, further reducing the risk of sample evaporation.

[0030] As an optional implementation, the liquid outlet ends of multiple capillaries 2 are connected to the same first oil storage tank 4.

[0031] The liquid outlets of multiple capillary tubes 2 are all connected to the same first oil storage tank 4. The sealing oil in the first oil storage tank 4 can simultaneously provide an evaporation barrier for all capillary tubes 2, simplifying the structure and improving the anti-evaporation effect.

[0032] As an optional implementation, two adjacent independent sample cells 3 are staggered.

[0033] The two adjacent independent sample cells 3 are arranged in an alternating manner, which makes the capillary 2 more compact, the temperature of the heating area more uniform, and improves the consistency of multi-channel amplification.

[0034] As an optional implementation, the card holder body 1 is made of black PLA material.

[0035] The card holder body 1 is made of black PLA material, which can effectively reduce background light interference and improve the fluorescence detection accuracy of the transparent detection window 5.

[0036] As an optional implementation, slots for inserting capillary tubes 2 are provided on both sides of the transparent detection window 5.

[0037] Slots are provided on both sides of the transparent detection window 5. The capillary tube 2 is inserted and installed through the slots, which facilitates the positioning and fixation of the capillary tube 2 and ensures that the position of the capillary tube 2 is stable and neatly arranged.

[0038] Specifically, the present invention comprises a cartridge body 1, multiple capillary tubes arranged side by side 2, multiple independent sample cells 3, a first oil storage tank 4, a transparent detection window 5, and a photothermal conversion layer 6. The structure and parameters of each component are as follows: The capillary 2 is made of glass, fused silica, borosilicate glass or transparent polymer, with an outer diameter of 200–800µm, an inner diameter of 100–600µm and a length of 10–30mm. The capillary 2 is arranged in parallel in the alignment groove inside the cartridge body 1, and the number of array channels is 1–96. Different throughput cartridge structures can be selected according to the detection requirements.

[0039] An independent sample cell 3 is connected to the inlet end of the capillary tube 2. The number of independent sample cells 3 matches the number of capillary tubes 2, and automatic liquid aspiration can be achieved through the capillary force of the capillary tube 2.

[0040] Multiple independent sample cells 3 are staggered along the longitudinal direction, which enables the capillary tubes 2 to be arranged compactly and improves the temperature uniformity of the heating area.

[0041] The first oil storage tank 4 is located at the outlet end of the capillary tube 2 and is filled with mineral oil or silicone oil, so that the outlet end of the capillary tube 2 is completely immersed in the oil layer, forming a stable evaporation barrier.

[0042] A transparent detection window 5 is set in the detection area of ​​capillary 2 for real-time fluorescence signal acquisition. The transparent detection window 5 is made by laser etching to remove the outer coating of capillary 2.

[0043] The photothermal conversion layer 6 is located below the capillary 2 and is made of any one or a combination of black metal film, graphite film, graphene coating, carbon black coating or metal oxide light-absorbing layer to achieve rapid heating and cooling.

[0044] The cartridge body 1 is manufactured by injection molding or 3D printing. The internal structure is integrated with a capillary tube 2 alignment groove, an independent sample cell 3, a first oil storage tank 4, a photothermal layer mounting position, and a detection window opening. The overall structure is compact and suitable for single use.

[0045] Taking a 24-channel capillary PCR cartridge reactor as an example, its preparation method and sample addition and usage are as follows.

[0046] Capillary 2 is made of polyimide-coated fused silica capillary with an outer diameter of 450µm, an inner diameter of 320µm, and a length of 20–22mm. After the capillary 2 is cut to the required length, it is placed horizontally in the 24 parallel alignment slots inside the custom 3D printed card box body 1 and fixed with UV curing adhesive to ensure that the capillary 2 maintains a consistent height and spacing in the heating area.

[0047] The polyimide coating on the outside of the detection area of ​​capillary 2 is removed by laser etching to form a transparent detection area without coating, so that the excitation light can enter the interior of capillary 2 and the fluorescence signal can be smoothly transmitted.

[0048] Twenty-four independent sample cells 3 are set on the top of the cartridge body 1. The cells are 2 mm in diameter, 1 mm in depth, and have a volume of about 3 µL. Users can directly add samples to the cells using a pipette. The samples automatically enter the capillary tube 2 under the action of capillary force.

[0049] Add 3µL of PCR reaction solution to each independent sample cell 3, of which about 0.4µL of the liquid column with a length of about 5mm is located in the heating area, which is the actual PCR reaction volume; the inlet end of the capillary 2 is sealed with a small amount of mineral oil by filling the second oil tank with mineral oil, and the outlet end is completely immersed in the mineral oil in the first oil tank 4, ensuring that the outlet end of the capillary 2 is completely immersed under the oil layer, forming a stable evaporation barrier to prevent evaporation during the ultra-fast thermal cycling process.

[0050] A photothermal conversion layer 6 is installed in the photothermal layer mounting position below multiple capillaries 2. In this embodiment, a black light-absorbing aluminum foil with a size of 55mm×5mm×0.11mm and a mass of 45mg is used. As a broadband light absorption layer, the aluminum foil can uniformly conduct the heat generated by laser irradiation to the capillaries 2 to achieve rapid heating. The back of the card box is completely covered with the black aluminum foil to enhance the photothermal efficiency.

[0051] The cartridge body 1 is made of black PLA material and is prepared by 3D printing. The black material can reduce background light interference and improve the signal-to-noise ratio of fluorescence detection. The cartridge body 1 is integrated with a capillary tube 2 alignment groove, an independent sample cell 3, a first oil storage tank 4, a photothermal layer mounting groove and a detection window opening. The overall structure is compact and suitable for single use.

[0052] The specific usage steps are as follows: ① The user adds 3µL of PCR reaction solution to the independent sample cell 3; ②The reaction solution automatically enters the interior of capillary tube 2 under the action of capillary force, forming a stable liquid column; ③ Place the cartridge on the heating platform of the photothermal PCR device to complete the positioning; ④ Laser irradiation of the photothermal conversion layer 6 enables ultra-rapid heating and cooling within the capillary 2 through photothermal conduction, meeting the temperature cycling requirements of PCR. ⑤ Real-time acquisition of fluorescence signals through transparent detection window 5 enables monitoring of the PCR amplification process; ⑥ Throughout the entire thermal cycle, the oil layer in the first oil storage tank 4 and the oil seal layer at the inlet end work together to prevent the reaction liquid from evaporating.

[0053] The total material cost of the 24-channel disposable cartridge in this embodiment is less than $1, which is significantly lower than the manufacturing cost of microfluidic chips and has advantages for large-scale application.

[0054] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0055] The above embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A capillary cartridge reactor for ultrafast PCR, characterized in that, include: The card box body (1) is manufactured by 3D printing or injection molding process; A transparent detection window (5) is provided in the middle of the card body (1). Multiple capillaries (2) are arranged side by side in the transparent detection window (5). One end of the capillaries (2) is connected to an independent sample cell (3), and the other end of the capillaries (2) is connected to a first oil storage tank (4). The capillary tube (2) is transparent; It also includes a photothermal conversion layer (6) fixed to the card body (1), which is correspondingly arranged with the transparent detection window (5), and a plurality of capillaries (2) are located between the photothermal conversion layer (6) and the transparent detection window (5).

2. The capillary cartridge reactor for ultrafast PCR according to claim 1, characterized in that, The photothermal conversion layer (6) is composed of a high light-absorbing and thermally conductive material made of any one or a combination of black metal film, graphite film, graphene coating, carbon black coating or metal oxide light-absorbing layer.

3. A capillary cartridge reactor for ultrafast PCR according to claim 1, characterized in that, A transparent section is provided in the middle of the capillary (2), and the transparent section is provided in correspondence with the transparent detection window (5).

4. A capillary cartridge reactor for ultrafast PCR according to claim 1, characterized in that, The capillary (2) is made of glass, fused silica, borosilicate glass or transparent polymer material.

5. A capillary cartridge reactor for ultrafast PCR according to claim 1, characterized in that, The cartridge body (1) is also provided with a second oil storage tank, and the liquid inlet end of the multiple independent sample pools (3) is connected to the same second oil storage tank.

6. A capillary cartridge reactor for ultrafast PCR according to claim 1, characterized in that, The liquid outlet ends of the multiple capillaries (2) are connected to the same first oil storage tank (4).

7. A capillary cartridge reactor for ultrafast PCR according to claim 1, characterized in that, The two adjacent independent sample cells (3) are staggered.

8. A capillary cartridge reactor for ultrafast PCR according to claim 1, characterized in that, The card box body (1) is made of black PLA material.

9. A capillary cartridge reactor for ultrafast PCR according to claim 1, characterized in that, Slots for inserting the capillary tube (2) are respectively opened on both sides of the transparent detection window (5).

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