An ultrafast PCR combined with paper-based detection platform based on nanoparticle photothermal effect

Abstract

The utility model discloses a kind of superfast PCR combined paper-based detection platform based on nanoparticle photo-thermal effect, belong to PCR chip technical field, including laser and PCR chip, the PCR chip includes sequentially bonded polymethyl methacrylate bottom layer from bottom to top, reaction and microchannel chamber layer and top layer closed layer;The reaction and microchannel chamber layer surface is equipped with sample adding area, one end of the sample adding area is provided with a sample adding side microchannel, the other end is provided with a paper-based side microchannel, and photo-thermal PCR amplification reaction chamber is equipped between the sample adding side microchannel and paper-based side microchannel;Lateral flow test strip is embedded in the paper-based side microchannel, the lateral flow test strip is filled with nanoparticles, solve the existing technical problems of the operation contamination and long time consumption of the existing PCR chip structure.

Description

Technical Field

[0001] This invention belongs to the field of PCR chip technology, specifically relating to an ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles. Background Technology

[0002] Photothermal ultrafast PCR technology provides a rapid method for nucleic acid amplification and has been widely accepted by medical professionals and researchers. As an essential consumable material for photothermal ultrafast PCR equipment, the compatibility, ease of use, and cost-effectiveness of PCR chips are of great concern. Generally, PCR chips only have a basic nucleic acid amplification reaction chamber. In actual operation, the reacted nucleic acid mixture needs to be removed and analyzed by gel electrophoresis to determine the positive or negative result. Extracting the amplified reagent mixture can easily cause contamination, leading to false positives. Furthermore, it is time-consuming and inconvenient for practical testing. Reducing operational contamination and shortening result reading time would significantly improve the ease of use of the product.

[0003] The result reading mechanism of paper-based biosensors can be based on various principles, including colorimetry, electrochemical methods, and fluorescence methods. Among them, colorimetry is widely used because it requires no other equipment and can be read directly by the naked eye. Lateralflow nucleic acid biosensors (LFNABs) possess all the advantages of paper-based biosensors, including ease of use, low cost, fast on-site detection speed, short detection time, and visual visibility. Therefore, they are widely used in primary diagnostics, environmental pollutant detection, and food safety testing.

[0004] However, the detection areas of common LFNABs are all open, meaning that the C-line and T-line are directly exposed to and in direct contact with the air. If the detection involves a pre-amplification step, open LFNABs are prone to aerosol contamination, resulting in false positive results. Therefore, it is urgent to develop a composite microfluidic chip to improve the problems of liquid evaporation and aerosol contamination in LFNABs.

[0005] In recent years, microfluidic systems based on polymer materials have been widely developed, with commonly used polymer materials including ABS, PLA, and polymethyl methacrylate. However, polymer microfluidic chips rely on pump drive and valve control, which not only increases detection costs but also makes the complex pump and valve setup unsuitable for use in resource-limited areas. Chinese patent CN117046530A discloses a paper-based microfluidic chip and its manufacturing and usage methods. It employs a stepped substrate layer and flow layer, controlling liquid flow through hydrophobic walls and channels. Combined with recombinant polymerase amplification technology and a CRISPR / Cas12a system, it achieves automated detection, reducing operational barriers and costs. However, the diced CRISPR reaction paper in this chip structure is exposed to an open environment, fluorescence detection requires external equipment, and there is a high risk of aerosol contamination. Utility Model Content

[0006] In order to overcome the shortcomings of the prior art, the purpose of this utility model is to provide an ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles, so as to solve the technical problem of aerosol contamination in the detection process of existing chip structures.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles, including a laser and a PCR chip. The PCR chip includes, from bottom to top, a polymethyl methacrylate (PMMA) bottom layer, a reaction and microchannel chamber layer, and a top sealing layer. The surface of the reaction and microchannel chamber layer is provided with a sample application area. One end of the sample application area has a sample application side microchannel, and the other end has a paper-based side microchannel. A photothermal PCR amplification reaction chamber is provided between the sample application side microchannel and the paper-based side microchannel. A side-flow test strip is embedded in the paper-based side microchannel, and the side-flow test strip is filled with nanoparticles.

[0008] Preferably, the polymethyl methacrylate (PMMA) bottom layer, reaction and microchannel chamber layer, and top sealing layer are all 65 mm in length and 15-25 mm in width; the diameter of the photothermal PCR amplification reaction chamber is 6 mm-10 mm; the length of the sample loading side microchannel is 8 mm-10 mm, and the length of the paper base side microchannel is 3 mm-7 mm.

[0009] Preferably, the side-flow test strip is provided with a conjugate pad, and a detection line and a quality control line are provided below the conjugate pad.

[0010] Preferably, a groove is formed on the top sealing layer, and the surface of the groove is covered with quartz glass of the same size.

[0011] Preferably, the groove has a size of 15mm x 15mm.

[0012] Preferably, the nanoparticles are photothermal nanoparticles.

[0013] Preferably, the photothermal nanoparticles are gold nanorods coated with silicon, and the optical density value of the gold nanorods coated with silicon is 1-10 OD.

[0014] Preferably, the length of the side-flow test strip is 35mm-45mm.

[0015] Preferably, the thickness of the polymethyl methacrylate bottom layer and the top sealing layer is 1 mm, and the material is polymethyl methacrylate.

[0016] Preferably, the laser wavelength of the laser is 750-850nm.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an ultrafast PCR combined with a paper-based detection platform based on the photothermal effect of nanoparticles. By setting up a polymethyl methacrylate (PMMA) bottom layer + reaction layer + top sealing layer, a physically sealed space is formed. The sample mixture is detected through a path from the sample application area → PCR amplification reaction chamber → paper-based side microfluidic channel → side-flow test strip. This path is connected by built-in microfluidic channels, allowing amplification products to directly enter the detection stage within the sealed channels, completely eliminating the need for opening the lid. The PCR amplification reaction chamber and the side-flow test strip are internally connected via the paper-based side microfluidic channel, eliminating manual transfer steps, preventing the generation of contamination sources, effectively reducing the risk of contamination, and shortening operation time.

[0018] Furthermore, the sample loading side microchannel is 8-10 mm long, forming sufficient capillary force to drive the fluid automatically to the photothermal PCR amplification reaction chamber; the paper-based side microchannel is 3-7 mm long, reducing fluid retention time and preventing evaporation of amplification products during transfer. The dimensions of the sample loading and paper-based side microchannels ensure rapid entry of amplification products into the test strip, reducing the aerosol generation window. The photothermal PCR amplification reaction chamber has a diameter of 6-10 mm: suitable for 10-30 μL reaction systems, providing better thermal uniformity.

[0019] Furthermore, the binding pad is pre-loaded with a marker to capture amplification products and form a complex. The test line and control line facilitate observation of the final test results.

[0020] Furthermore, the recess, measuring 15×15mm, covers the entire interface between the photothermal PCR amplification reaction chamber and the microfluidic channel, providing a panoramic observation window. The recess is covered with quartz glass, which, on the one hand, allows for real-time fluorescence monitoring due to its high light transmittance, eliminating the need to open the window to verify amplification results; on the other hand, its thermal stability matches the temperature-dependent requirements of photothermal PCR, preventing thermal stress-induced seal failure.

[0021] Furthermore, gold nanorods coated with silicon are selected as photothermal nanoparticles because of their high photothermal conversion efficiency, which shortens the amplification time and thus significantly reduces the total amount of aerosols generated. In addition, it can prevent the gold rods from directly contacting the reaction solution, eliminating microbubbles (aerosol precursors) generated by metal ion-catalyzed DNA hydrolysis.

[0022] With an optical density of 1-10 OD, it achieves efficient heating, reduces the amount of nanoparticles used, and lowers the complexity of the reaction system, while avoiding the formation of eddies induced by nanoparticle aggregation.

[0023] Furthermore, the 35-45mm long side-flow test strips are matched with the length of the microchannels on the paper base to ensure no waste liquid residue and eliminate the risk of secondary contamination. This also guarantees chromatography time and target capture rate; test strips that are too short will not develop sufficient color, while strips that are too long will increase background interference.

[0024] Furthermore, the use of polymethyl methacrylate (PMMA) material enables both the bottom and top sealing layers to be hydrophobic, thereby inhibiting droplet splashing and aerosol formation; on the other hand, electrostatic dissipation prevents the electrostatic adsorption of environmental pollutants. A thickness of 1mm allows for a seamless structure between the PMMA bottom and top sealing layers, enhancing sealing strength.

[0025] Furthermore, the 750-850nm laser wavelength can penetrate quartz glass to a depth of >5mm, enabling non-contact heating and avoiding steam jets caused by the thermal inertia of traditional metal heating blocks. Moreover, this laser wavelength can match the LSPR absorption peak of gold nanorods, achieving directional conversion of light energy and thus eliminating micro-boiling phenomena caused by uneven heating. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the ultrafast PCR combined with paper-based detection platform of this utility model; Figure 2 This is a schematic diagram of the side-flow test strip structure of this utility model; Figure 3 This is a physical structural diagram of the ultrafast PCR combined with paper-based detection platform of this utility model; Among them, 1-polymethyl methacrylate bottom layer; 2-reaction and microchannel chamber layer; 3-top sealing layer; 4-sample application area; 5-sample application side microchannel; 6-photothermal PCR amplification reaction chamber; 7-binding pad; 8-detection line; 9-control line; 10-paper base side microchannel; 11-sideflow test strip. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the protection scope of the present invention.

[0028] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this utility model are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the utility model described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0029] The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles provided by this utility model has a PCR amplification reaction chamber 6 at the top, which provides a platform and basis for nucleic acid amplification; and a flow test strip 11 at the bottom, which can directly read the results, reducing the time for reading gel electrophoresis results, shortening the experimental and result reading time for medical staff and researchers, reducing contamination and helping to reduce operation steps.

[0030] This invention relates to an ultrafast PCR combined with a paper-based detection platform that integrates paper-based biosensors and polymer microfluidic chips to construct a composite microfluidic chip. This chip can utilize the capillary action of the paper-based chip as a driving force for one-step "sample in - result out" detection, avoiding the need for pumps, valves, and result reading devices. Furthermore, the polymer chip can form a sealed chamber, preventing solution evaporation and aerosol contamination caused by nucleic acid amplification during the detection process. This facilitates the miniaturization and portability of the chip, enabling real-time, highly sensitive detection of ultrafast photothermal PCR amplification.

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments: Example 1 This invention provides an ultrafast PCR combined with a paper-based detection platform based on the photothermal effect of nanoparticles, such as... Figures 1 to 3 As shown, the device includes a laser and a PCR chip. The PCR chip comprises, from bottom to top, a polymethyl methacrylate (PMMA) bottom layer 1, a reaction and microchannel chamber layer 2, and a top sealing layer 3 bonded together. The surface of the reaction and microchannel chamber layer 2 is provided with a sample application area 4. One end of the sample application area 4 has a sample application side microchannel 5, and the other end has a paper-based side microchannel 10. A photothermal PCR amplification reaction chamber 6 is provided between the sample application side microchannel 5 and the paper-based side microchannel 10. A sideflow test strip 11 is embedded in the paper-based side microchannel 10, and the sideflow test strip 11 is filled with nanoparticles.

[0032] Preferably, the polymethyl methacrylate bottom layer 1, the reaction and microchannel chamber layer 2, and the top sealing layer 3 are all 65 mm in length and 15-25 mm in width; the photothermal PCR amplification reaction chamber 6 has a diameter of 6 mm-10 mm; the sample loading side microchannel 5 has a length of 8 mm-10 mm; and the paper base side microchannel 10 has a length of 3 mm-7 mm.

[0033] More preferably, the flow channels between the sample loading side microchannel 5 and the photothermal PCR amplification reaction chamber 6, and between the photothermal PCR amplification reaction chamber 6 and the paper-based side microchannel 10, are 0.3 mm. This ensures the flow of liquid and reduces the evaporation of liquid in the reaction chamber to a certain extent.

[0034] Preferably, the side-flow test strip 11 is provided with a conjugate pad 7, and a detection line 8 and a quality control line 9 are provided below the conjugate pad 7.

[0035] Preferably, a groove is formed on the top sealing layer 3, and the surface of the groove is covered with quartz glass of the same size. Quartz glass has better light transmittance, which facilitates the transmission of laser light through the PCR chip.

[0036] Preferably, the groove has a size of 15mm x 15mm.

[0037] In a further preferred embodiment, the gap between the quartz glass and the reaction and microchannel chamber layer 2 is filled with UV adhesive, and then irradiated with a UV lamp for 30 seconds to prevent liquid from flowing in and causing liquid loss.

[0038] Preferably, the nanoparticles are photothermal nanoparticles.

[0039] Preferably, the photothermal nanoparticles are gold nanorods coated with silicon, and the optical density value of the gold nanorods coated with silicon is 1-10 OD.

[0040] Preferably, the length of the side-flow test strip 11 is 35mm-45mm.

[0041] Preferably, the thickness of the polymethyl methacrylate bottom layer 1 and the top sealing layer 3 is 1 mm, and the material is polymethyl methacrylate.

[0042] Preferably, the laser wavelength of the laser is 750 nm - 850 nm.

[0043] The working principle of this novel ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles includes: adding a prepared sample mixture in the sample application area 4, which then passes through the sample application side microfluidic channel 5 and reaches the PCR amplification reaction chamber 6. The sample mixture undergoes photothermal nucleic acid amplification in the PCR amplification reaction chamber 6. After amplification, the mixture flows through the paper-based side microfluidic channel 10 to the side-flow test strip 11. When flowing through the binding pad 7, the mixture binds to the silicon-coated gold nanorods already conjugated with antibodies on the binding pad 7, forming a target gene conjugated structure. Then, using capillary force, the mixture flows through the detection line 8 (C line) and the control line 9 (T line) of the side-flow test strip 11. When only the control line 9 (T line) shows color, it indicates that the added sample does not contain the target gene or the target gene concentration is too low, and the result is negative. When both the detection line 8 (C line) and the control line (T line) show color, it indicates that the added sample contains the target gene, and the result is positive.

[0044] The above content is only for illustrating the technical concept of this utility model and should not be construed as limiting the scope of protection of this utility model. Any modifications made to the technical solution based on the technical concept proposed in this utility model shall fall within the scope of protection of the claims of this utility model.

Claims

1. An ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles, characterized in that, The device includes a laser and a PCR chip. The PCR chip comprises, from bottom to top, a polymethyl methacrylate (PMMA) bottom layer (1), a reaction and microchannel chamber layer (2), and a top sealing layer (3). The surface of the reaction and microchannel chamber layer (2) is provided with a sample application area (4). One end of the sample application area (4) is provided with a sample application side microchannel (5), and the other end is provided with a paper-based side microchannel (10). A photothermal PCR amplification reaction chamber (6) is provided between the sample application side microchannel (5) and the paper-based side microchannel (10). A side-flow test strip (11) is embedded in the paper-based side microchannel (10), and the side-flow test strip (11) is filled with nanoparticles.

2. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 1, characterized in that, The polymethyl methacrylate bottom layer (1), reaction and microchannel chamber layer (2) and top sealing layer (3) are all 65 mm in length and 15-25 mm in width; the diameter of the photothermal PCR amplification reaction chamber (6) is 6 mm-10 mm; the length of the sample loading side microchannel (5) is 8 mm-10 mm; and the length of the paper-based side microchannel (10) is 3 mm-7 mm.

3. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 1, characterized in that, The side-flow test strip (11) is provided with a conjugate pad (7), and a detection line (8) and a quality control line (9) are provided below the conjugate pad (7).

4. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 1, characterized in that, A groove is formed on the top sealing layer (3), and the surface of the groove is covered with quartz glass of the same size.

5. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 4, characterized in that, The groove measures 15mm x 15mm.

6. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 1, characterized in that, The nanoparticles are photothermal nanoparticles.

7. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 6, characterized in that, The photothermal nanoparticles are gold nanorods coated with silicon, and the optical density value of the gold nanorods coated with silicon is 1-10 OD.

8. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 1, characterized in that, The length of the side-flow test strip (11) is 35mm-45mm.

9. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 1, characterized in that, The thickness of the polymethyl methacrylate bottom layer (1) and the top sealing layer (3) is 1 mm, and the material is polymethyl methacrylate.

10. The ultrafast PCR combined with paper-based detection platform based on the photothermal effect of nanoparticles according to claim 1, characterized in that, The laser wavelength of the laser is 750nm-850nm.

Images