Photothermal RT-LAMP Chip and Its Fabrication and Detection Methods

Abstract

A photothermal RT-LAMP chip and its manufacturing and detection methods, which relate to the technical field of nucleic acid detection. To solve the technical problems in the prior art that the existing centralized diagnostic systems have limitations and require a long detection time, the technical solution provided by the present invention is as follows: a photothermal RT-LAMP chip, the chip includes: a top cover plate and a polypropylene double-sided adhesive film that form the upper closed structure of the chip; a sample loading groove, a diversion layer and a filtration layer for sample loading and fluid guiding; a polyethersulfone filter membrane and Fusion5 filter paper for separating impurities and fixing target molecules; paraffin particles for assisting in sample analysis; a reaction chamber for carrying reaction reagents and performing photothermal RT-LAMP reactions; an absorption pad for absorbing the reaction solution; a bottom cover plate, a PDMS film, a gold film and a glass substrate that form the bottom support and the photothermal reaction base of the chip. It can be applied to disease diagnosis work, especially for early, rapid and accurate disease diagnosis in resource-limited environments.

Description

Technical Field

[0001] It relates to the field of nucleic acid detection technology. Background Art

[0002] Early, rapid, and accurate disease diagnosis in resource-limited settings is crucial for controlling virus transmission and improving treatment outcomes. Existing centralized diagnostic systems (where test samples are sent to hospitals or testing institutions and operated by professionals) have limitations and require long testing times, especially in areas lacking expensive medical equipment, making them more vulnerable to infectious disease outbreaks. Therefore, there is an urgent need to develop a POCT platform that is easy to operate, fast in detection, low in cost, high in sensitivity, and strong in specificity. Unfortunately, diagnostic platforms with such capabilities are not common. Lateral flow immunochromatographic test strips are fast and inexpensive, but their sensitivity and specificity need to be improved. Polymerase chain reaction (PCR) is usually the gold standard for virus diagnosis, but the accuracy of this method depends on large analytical equipment and professional technicians. As an alternative, loop-mediated isothermal amplification (LAMP) has high specificity due to the use of 6 primers, strong tolerance to impurities, does not require expensive detection equipment, and has a short detection time.

[0003] Although LAMP has many advantages, there are still many aspects that need to be improved for POCT. To achieve the goal of user-friendliness, complex sample handling and detection operations, such as nucleic acid extraction and multiplexed sample addition operations, must be avoided. Traditional nucleic acid extraction methods such as magnetic bead method and adsorption column method inevitably sacrifice sensitivity due to the limitation of elution volume. Therefore, there is an urgent need to develop a super-sensitive multi-target detection scheme suitable for POCT. Summary of the Invention

[0004] To solve the technical problem in the prior art that existing centralized diagnostic systems have limitations and require long testing times, the technical solution provided by the present invention is as follows:

[0005] A photothermal RT-LAMP chip, the chip comprising:

[0006] A top cover plate and a polypropylene double-sided adhesive film constituting the upper closed structure of the chip;

[0007] A sample addition slot, a diversion layer, and a filtration layer for sample addition and fluid guiding;

[0008] A polyethersulfone filter membrane and Fusion5 filter paper for separating impurities and immobilizing target molecules;

[0009] Paraffin particles for assisting in sample analysis;

[0010] A reaction chamber for carrying reaction reagents and performing photothermal RT-LAMP reactions;

[0011] Absorbent pad for absorbing reaction liquid;

[0012] The bottom cover plate, PDMS film, gold film and glass substrate that constitute the bottom support of the chip and the bottom of the photothermal reaction base.

[0013] Furthermore, a preferred embodiment is provided, where the top cover plate and the polypropylene double-sided adhesive film are located on the top layer of the photothermal RT-LAMP chip;

[0014] The sample addition groove is arranged above the diversion layer and the filtration layer and below the top cover plate and the polypropylene double-sided adhesive film;

[0015] The polyethersulfone filter membrane and the Fusion 5 filter paper are arranged on the sample inlet hole of the filtration layer;

[0016] The paraffin particles are arranged in the sample inlet hole of the filtration layer;

[0017] The reaction chamber is arranged between the bottom cover plate and the PDMS film;

[0018] The absorbent pad is arranged at the bottom of the reaction chamber;

[0019] The PDMS film is arranged between the bottom cover plate and the gold film;

[0020] The gold film is arranged on the glass substrate;

[0021] The glass substrate serves as the fixed base of the photothermal RT-LAMP chip.

[0022] Method for fabricating a photothermal RT-LAMP chip, used for fabricating the photothermal RT-LAMP chip described above, the method comprising:

[0023] Steps for fabricating a reaction chamber mold;

[0024] Steps for fabricating a reaction chamber according to the reaction chamber mold;

[0025] Steps for fabricating a gold film;

[0026] Steps for modifying the Fusion 5 filter paper;

[0027] Steps for assembling the photothermal RT-LAMP chip.

[0028] Furthermore, a preferred embodiment is provided, where the reaction chamber mold is fabricated by laser cutting.

[0029] Furthermore, a preferred embodiment is provided, where PDMS is poured into the mold and the reaction holes and partition strips are processed according to preset requirements to obtain the reaction chamber.

[0030] Further, a preferred embodiment is provided, in which the gold thin film is fabricated on the glass substrate by photolithography and evaporation methods.

[0031] Further, a preferred embodiment is provided, in which the Fusion5 filter paper is chemically treated until its surface has a preset affinity.

[0032] Further, a preferred embodiment is provided, and the method further includes a step of encapsulating and storing the assembled photothermal RT-LAMP chip.

[0033] The photothermal RT-LAMP chip detection method is implemented by the above-mentioned photothermal RT-LAMP chip, and the method includes:

[0034] A step of adding the sample to be tested mixed with the lysis solution into the sample loading groove so that the liquid level of the sample to be tested mixed with the lysis solution is aligned with the reaction chamber;

[0035] A step of exciting the photothermal reaction of the gold thin film;

[0036] A step of judging the reaction result according to the color change of the sample to be tested mixed with the lysis solution.

[0037] Further, a preferred embodiment is provided, in which the liquid level of the sample to be tested mixed with the lysis solution is aligned with the reaction chamber by rotation.

[0038] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows:

[0039] The photothermal RT-LAMP chip detection method provided by the present invention realizes on-chip nucleic acid amplification through photothermal conversion driven by sunlight focusing or LED irradiation. This method saves energy, improves the heating speed, and is applicable to resource-poor areas.

[0040] Compared with the prior art, traditional detection methods usually rely on large-scale analytical equipment and professional technicians, while this solution realizes rapid and convenient nucleic acid amplification through a simple photothermal conversion method, avoiding complex sample processing and detection operations.

[0041] Multiple reaction cells can be designed to simultaneously detect multiple diseases. By pre-loading different gelation reagents, simultaneous detection of multiple diseases such as hepatitis B, hepatitis C, AIDS, and influenza A can be achieved.

[0042] Compared with the prior art, traditional detection methods usually can only detect a single pathogen, and multiple detections are required to obtain comprehensive results. While this solution can simultaneously detect multiple diseases through one detection, improving the detection efficiency.

[0043] The photothermal conversion method used and the design of simultaneous multi-target detection make the photothermal RT-LAMP chip fast, convenient, and efficient. It is suitable for resource-scarce areas and has important practical application value. Compared with the existing technology, this solution has obvious advantages in detection efficiency and operation convenience.

[0044] It can be applied to disease diagnosis work, especially for early, rapid, and accurate disease diagnosis in resource-limited environments. It can detect multiple diseases simultaneously. Brief Description of the Drawings

[0045] Figure 1 It is an overall exploded schematic diagram of the photothermal RT-LAMP chip;

[0046] Figure 2 It is a combined schematic diagram of the photothermal RT-LAMP chip;

[0047] Figure 3 It is a schematic diagram of the diversion layer of the photothermal RT-LAMP chip;

[0048] Figure 4 It is a schematic diagram of the filter layer of the photothermal RT-LAMP chip;

[0049] Figure 5 It is a schematic diagram of the reaction chamber of the photothermal RT-LAMP chip;

[0050] Figure 6 It is a schematic diagram of the gold thin film mask of the photothermal RT-LAMP chip;

[0051] Figure 7 It is a schematic diagram of the reaction chamber mold of the photothermal RT-LAMP chip.

[0052] Among them, 1 represents the top cover plate, 2 represents the polypropylene double-sided adhesive film, 3 represents the sample addition groove, 4 represents the diversion layer, 5 represents the polyethersulfone filter membrane, 6 represents the filter layer, 7 represents the paraffin particles, 8 represents the Fusion 5 filter paper, 9 represents the reaction chamber, 10 represents the absorption pad, 11 represents the bottom cover plate, 12 represents the PDMS thin film, 13 represents the gold thin film, and 14 represents the glass substrate. Detailed Embodiments

[0053] To make the advantages and beneficial effects of the technical solution provided by the present invention more clearly manifested, the technical solution provided by the present invention will now be further described in detail with reference to the accompanying drawings. Specifically:

[0054] Embodiment 1. This embodiment provides a photothermal RT-LAMP chip, and the chip includes:

[0055] The top cover plate 1 and the polypropylene double-sided adhesive film 2 that constitute the upper closed structure of the chip;

[0056] A sample loading groove 3, a diversion layer 4, and a filter layer 6 for sample loading and fluid guiding;

[0057] A polyethersulfone filter membrane 5 and Fusion 5 filter paper for separating impurities and immobilizing target molecules;

[0058] Paraffin particles 7 for assisting in sample analysis;

[0059] A reaction chamber 9 for carrying reaction reagents and performing photothermal RT-LAMP reaction;

[0060] An absorption pad 10 for absorbing the reaction solution;

[0061] A bottom cover plate 11, a PDMS film 12, a gold film 13, and a glass substrate 14 that constitute the bottom support of the chip and the base of the photothermal reaction;

[0062] Embodiment 2: This embodiment further defines the photothermal RT-LAMP chip provided in Embodiment 1. The top cover plate 1 and the polypropylene double-sided adhesive film 2 are located on the top layer of the photothermal RT-LAMP chip;

[0063] The sample loading groove 3 is arranged above the diversion layer 4 and the filter layer 6 and below the top cover plate 1 and the polypropylene double-sided adhesive film 2;

[0064] The polyethersulfone filter membrane 5 and the Fusion 5 filter paper 8 are arranged on the sample inlet hole of the filter layer 6;

[0065] The paraffin particles 7 are arranged in the sample inlet hole of the filter layer 6;

[0066] The reaction chamber 9 is arranged between the bottom cover plate 11 and the PDMS film 12;

[0067] The absorption pad 10 is arranged at the bottom of the reaction chamber 9;

[0068] The PDMS film 12 is arranged between the bottom cover plate 11 and the gold film 13;

[0069] The gold film 13 is arranged on the glass substrate 14;

[0070] The glass substrate 14 serves as the fixed base of the photothermal RT-LAMP chip.

[0071] Embodiment 3: This embodiment provides a method for manufacturing a photothermal RT-LAMP chip for manufacturing the photothermal RT-LAMP chip according to claim 1. The method includes:

[0072] Steps for manufacturing the mold of the reaction chamber 9;

[0073] Steps for fabricating the reaction chamber 9 according to the mold of the reaction chamber 9;

[0074] Steps for fabricating the gold thin film 13;

[0075] Steps for modifying the Fusion5 filter paper;

[0076] Steps for assembling the optothermal RT-LAMP chip;

[0077] Embodiment 4: This embodiment further limits the method for fabricating the optothermal RT-LAMP chip provided in Embodiment 3, and the mold of the reaction chamber 9 is fabricated by laser cutting.

[0078] Embodiment 5: This embodiment further limits the method for fabricating the optothermal RT-LAMP chip provided in Embodiment 3. PDMS is poured into the mold, and reaction holes and partition bars are processed according to preset requirements to obtain the reaction chamber 9.

[0079] Embodiment 6: This embodiment further limits the method for fabricating the optothermal RT-LAMP chip provided in Embodiment 3. The gold thin film 13 is fabricated on the glass substrate 14 by photolithography and evaporation.

[0080] Embodiment 7: This embodiment further limits the method for fabricating the optothermal RT-LAMP chip provided in Embodiment 3. The Fusion5 filter paper is chemically treated until its surface has a preset affinity.

[0081] Embodiment 8: This embodiment further limits the method for fabricating the optothermal RT-LAMP chip provided in Embodiment 3. The method further includes steps for encapsulating and storing the assembled optothermal RT-LAMP chip.

[0082] Embodiment 9: This embodiment provides a method for detecting an optothermal RT-LAMP chip, which is implemented by the optothermal RT-LAMP chip according to Claim 1. The method includes:

[0083] Steps of adding the test sample mixed with the lysis solution into the sample loading groove 3 to align the liquid level of the test sample mixed with the lysis solution with the reaction chamber 9;

[0084] Steps of exciting the optothermal reaction of the gold thin film 13;

[0085] Steps of judging the reaction result according to the color change of the test sample mixed with the lysis solution.

[0086] Embodiment 10: This embodiment further limits the method for detecting the optothermal RT-LAMP chip provided in Embodiment 9. The liquid level of the test sample mixed with the lysis solution is aligned with the reaction chamber 9 by rotation.

[0087] Embodiment XI. Combination Figures 1-7 To describe this embodiment, this embodiment further describes the above-provided technical solution in detail and completely. Specifically:

[0088] As Figures 1-6 shown. The optothermal RT-LAMP chip includes a top cover plate 1, a polypropylene double-sided adhesive film 2, a sample addition groove 3, a diversion layer 4, a polyethersulfone (PES) filter membrane 5, a filtration layer 6, paraffin particles 7, a Fusion 5 filter paper 8, a reaction chamber 9, an absorption pad 10, a bottom cover plate 11, a PDMS film 12, a gold film 13, and a glass substrate 14 that are sequentially bonded from top to bottom. The top cover plate 1 is made of PMMA. The top cover plate 1 and the polypropylene double-sided adhesive film 2 are annular structures with the same outer diameter, and the inner diameter of the top cover plate 1 is smaller than the inner diameter of the polypropylene double-sided adhesive film 2. Sampling holes 4-1 and 6-1 are provided on both the diversion layer 4 and the filtration layer 6, and are respectively pasted on both sides of the sampling hole of the filtration layer with the polyethersulfone filter membrane 5 and the Fusion 5 filter paper 6. The paraffin particles 7 are placed in the sampling hole 6-1 of the filtration layer. The reaction chamber 9 is also an annular structure, cast from PDMS. The sample addition groove 3, the diversion layer 4, the filtration layer 6, and the absorption pad 10 are placed inside the annular structure. The upper side is bonded to the top cover plate 1 through the polypropylene double-sided adhesive film 2, and the lower side is bonded to the bottom cover plate 11. The absorption pad 10 is made by cutting cotton paper. The gold film 13 is a patterned structure and is fixed on the glass substrate 14 by evaporation coating.

[0089] The sample addition groove 3 can be manually rotated, so it is designed as a cross-shaped structure. By rotating the sample addition groove 3, the relative positions of the sampling holes 4-1 and 6-1 on the diversion layer 4 and the filtration layer 6 and the reaction chamber 9 can be changed. The diameters of the sampling holes on the diversion layer 4 and the filtration layer 6 are both 2 mm, and the diameter of the paraffin particles 7 is 200 μm. The filtration pore diameter of the polyethersulfone filter membrane 5 is 2 μm, and its shape is a circular thin sheet with a diameter of 3 mm. The Fusion 5 filter paper 6 is a circular thin sheet with a diameter of 6 mm, and its surface is modified with chitosan oligosaccharide for specifically adsorbing nucleic acid molecules. Five reaction grooves 9-1 with a diameter of 4 mm are provided on the reaction chamber 9, and the internal pre-filled reaction reagents are gelified. The central sample addition groove is used for negative control, and the other four sample addition grooves are used for detection. The PDMS film 12 is a circular thin sheet with the same outer diameter as the reaction chamber 9 and a thickness of 100 μm.

[0090] To separate each reaction chamber, partition strips 9-2 are provided on the reaction chamber, and the width of each partition strip is 4 mm. The absorption pad 10 is in a fan-shaped structure with a thickness of 1.5 mm.

[0091] The gold thin film 13 is composed of five squares formed by S-shaped curves. The gold thin film near the bottom of the glass substrate 14 is led out to two pads for measuring the resistance of the gold thin film.

[0092] A method for fabricating a photothermal RT-LAMP chip includes the following steps:

[0093] Step 1: Preparation of the reaction chamber mold: The outer circle 16 of the mold, the central circle 17 of the mold, and the sector-shaped absorption pad 18 are processed by laser cutting, and each part is fixed on the glass plate 15 with UV glue in the manner shown in the figure. The thickness of the outer circle 16 of the mold is 3 mm, and the thicknesses of the central circle 17 of the mold and the sector-shaped absorption pad 18 are 1.5 mm, as Figure 7 shown.

[0094] Step 2: Preparation of the reaction chamber 9: The fabricated mold is subjected to silanization treatment to facilitate the subsequent peeling of the PDMS reaction chamber. Weigh the PDMS prepolymer and the curing agent, mix the two evenly according to a weight ratio of 10:1, add 15% mineral oil to the mixture, and stir evenly again; pour the mixture into the mold, and place it in a vacuum desiccator and let it stand for 30 min to remove air bubbles; place a glass cover plate above the mold and fix it with a dovetail clip; put the mold into an 80 °C oven and heat it for 2 h. After the PDMS is completely cured, carefully peel the PDMS from the mold. Use a 4-mm punch to punch holes at the specified positions to form reaction holes, and fill the corresponding gelling reagent in the reaction holes. The gelling reagent includes Bst DNA polymerase, reverse transcriptase, MgCl2, dNTP, primers, phenol red, 0.5% low-melting-point agarose, and buffer solution.

[0095] Step 3: Preparation of the gold thin film 13: Spin-coat 10 μm of photoresist on the glass substrate 14, perform photolithography using the Figure 6 mask shown, and obtain the shape of the gold thin film through wet etching; put the glass substrate into a vacuum evaporation instrument for surface evaporation. First, evaporate a 5-nm chromium thin film, and then evaporate a 120-nm gold thin film. Take out the glass substrate, soak it in an acetone solution for 2 h to remove the photoresist on the surface, wash it with alcohol and water, and then dry it with nitrogen.

[0096] Step 4. Modification of Fusion 5 filter paper 8: First, prepare a methanol solution containing 2.5% GPTMS (3-glycidoxypropyltrimethoxysilane); subsequently, prepare a 0.1% acetic acid solution, dissolve 0.1% chitosan oligosaccharide in the 0.1% acetic acid, and adjust the pH of the solution to 6 using 1M NaOH. Place the Fusion 5 filter paper in a plasma cleaner for plasma treatment, then soak it in the GPTMS solution for 1 h. After taking it out, place it in the chitosan oligosaccharide solution and incubate it overnight on a shaker; after taking it out, wash it three times with deionized water, dry it in an oven at 50 °C for 1 h, and punch it into circular pieces with a diameter of 6 mm for standby.

[0097] Step 5. Chip assembly and storage: Paste the polyethersulfone filter membrane 5 above the sample injection hole 6-1 of the filter layer 6, and perform thermocompression bonding on the sample addition groove 3, the diversion layer 4, and the filter layer 6; add 6 mg of paraffin particles 7 to the sample injection hole 6-1 of the filter layer 6, and then paste the Fusion 5 filter paper 6 at the bottom of the sample injection hole 6-1 to seal the sample injection hole. After bonding the bottom cover plate 11 to the reaction chamber 9, place the absorbent pad 10 and the sample addition groove 3 in sequence; paste and perform plasma treatment on the polypropylene double-sided adhesive film 2 and the top cover plate 1, and then bond them to the top of the reaction chamber 9. Bond the gold-plated glass substrate 14 to the PDMS film 12, align the reaction chamber 9 and place it on the gold-plated glass substrate 14, and place it in a vacuum bag and evacuate it for standby.

[0098] The detection method of the photothermal RT-LAMP chip for simultaneous detection of multiple diseases includes the following steps:

[0099] Step 1: Take out the photothermal RT-LAMP chip from the vacuum bag and ensure that the sample injection hole on the sample addition groove is aligned with the absorbent pad; after mixing the test sample (whole blood, serum, plasma or saliva) with the lysis solution, take 1.4 mL and add it to the sample addition groove; after the solution has drained, add 0.6 mL of deionized water to the sample addition groove; after the solution has drained, rotate the sample addition groove to align the sample injection hole with the reaction hole on the reaction chamber.

[0100] Step 2: Focus sunlight or use an LED to irradiate the gold film. Utilize the light absorption characteristics of the gold film to generate hot electrons on its surface, thereby realizing photothermal conversion; the gelled reagent becomes liquid when heated, eluting the nucleic acid molecules from the Fusion5 filter paper and performing loop-mediated isothermal amplification (LAMP) in the reaction chamber.

[0101] Step 3: After the amplification is completed, the color of the solution can be observed with the naked eye. Red is negative and yellow is positive.

[0102] The photothermal RT-LAMP chip and detection method provided by this embodiment can be used to simultaneously detect four diseases, namely hepatitis B, hepatitis C, AIDS, and influenza A. The specific steps are as follows: During the fabrication of the photothermal RT-LAMP chip, the gelling reagents corresponding to the four diseases are pre-added to four sample loading slots respectively. Collect venous blood samples from patients, obtain serum after centrifugation, aspirate 512 μL of serum into a centrifuge tube, mix it with 853 μL of lysis buffer and 35 μL of proteinase K, let it stand at room temperature for 5 min, and then add the solution to the sample loading slots of the chip to complete the enrichment and filtration of nucleic acids. Subsequently, add 600 μL of deionized water to the sample loading slots to remove the impurities remaining on the filter paper. After the reagent injection is completed, rotate the sample loading slots to align the filter paper with the reaction chambers, place the chip under blue light with a wavelength of 465 nm, and monitor the temperature of the reaction chambers in real time by detecting the resistance of the gold thin film. Adjust the light intensity to keep the temperature of the reaction chambers at 63°C for 30 min. After taking out the chip, rotate the sample loading slots back to their original positions, and observe the color change of each reaction chamber with the naked eye. If the solution in the reaction chamber is yellow, it indicates that the patient has the disease corresponding to this reaction chamber.

[0103] This embodiment has the following advantages:

[0104] (1) During the sample loading process, high-efficiency enrichment (about 350x) of nucleic acids and reagent aliquoting can be completed synchronously, omitting complex manual operation steps and shortening the sample loading time;

[0105] (2) On-chip nucleic acid elution and amplification are achieved through simple solar light focusing or LED irradiation-driven photothermal conversion, saving energy, increasing the heating speed, and being applicable to resource-poor areas;

[0106] (3) An on-chip gold thin film temperature sensor is integrated, and the temperature of the reaction chamber can be monitored in real time during the heating process;

[0107] (4) The design size can be flexibly changed as needed, and on-chip processing and amplification of 2, 4, or 6 samples can be carried out simultaneously;

[0108] (5) The sensitivity can be guaranteed not to decline within four weeks through pre-gelled reagents and self-sealing bags for preservation.

[0109] In addition, this embodiment has the characteristics of low cost, low energy consumption, high detection sensitivity, and batch manufacturing, and has important practical application value.

[0110] The above further describes the technical solutions provided by the present invention through several specific embodiments to highlight the advantages and beneficial effects of the technical solutions provided by the present invention. However, the several specific embodiments described above are not used as a limitation to the present invention. Any reasonable modifications and improvements to the present invention, combinations of embodiments, equivalent replacements, etc. within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. Photothermal RT-LAMP chip, characterized in that: The chip comprises: A top cover plate and a polypropylene double-sided adhesive film constituting the upper closed structure of the chip; Sample loading grooves, flow guide layers and filter layers for sample loading and fluid guidance; Polyethersulfone membrane and Fusion5 filter paper for separating impurities and immobilizing target molecules; Paraffin particles to aid in sample analysis; A reaction chamber for carrying reaction reagents and performing photothermal RT-LAMP reaction; an absorbent pad for absorbing the reaction solution; The bottom cover plate, PDMS film, gold film and glass substrate constitute the bottom support of the chip and the photothermal reaction base; the top cover plate and polypropylene double-sided adhesive film are located on the top layer of the photothermal RT-LAMP chip; The sample loading slot is arranged above the guide layer and the filter layer, and below the top cover plate and the polypropylene double-sided adhesive film; The polyethersulfone filter membrane and Fusion 5 filter paper are arranged on the injection hole of the filter layer; The paraffin particles are arranged in the injection hole of the filter layer; The reaction chamber is arranged between the bottom cover plate and the PDMS film; The absorption pad is arranged at the bottom of the reaction chamber; The PDMS film is arranged between the bottom cover plate and the gold film; The gold thin film is disposed on the glass substrate; The glass substrate serves as a fixed base for the photothermal RT-LAMP chip.

2. A method for manufacturing a photothermal RT-LAMP chip, for manufacturing the photothermal RT-LAMP chip according to claim 1, characterized in that: The method comprises: Steps for making a reaction chamber mold; A step of manufacturing a reaction chamber according to the reaction chamber mold; Steps for making gold thin film; Modification steps for Fusion5 filter paper; The step of assembling the photothermal RT-LAMP chip.

3. The method for preparing a photothermal RT-LAMP chip according to claim 2, characterized in that: The reaction chamber mold is manufactured by laser cutting.

4. The method for preparing a photothermal RT-LAMP chip according to claim 2, characterized in that: The PDMS is poured into a mold, and reaction holes and separation bars are processed according to preset requirements to obtain the reaction chamber.

5. The method for preparing a photothermal RT-LAMP chip according to claim 2, characterized in that: The gold thin film is produced on a glass substrate by photolithography and evaporation methods.

6. The method for preparing a photothermal RT-LAMP chip according to claim 2, characterized in that: The Fusion5 filter paper is chemically treated until its surface has a preset affinity.

7. The method for preparing a photothermal RT-LAMP chip according to claim 2, characterized in that: The method also includes the step of packaging and preserving the assembled photothermal RT-LAMP chip.

8. A photothermal RT-LAMP chip detection method, implemented by the photothermal RT-LAMP chip according to claim 1, the method comprising: The step of mixing the sample to be tested with the lysate and adding it to the sample adding tank, so that the liquid level of the sample to be tested mixed with the lysate is aligned with the reaction chamber; A step of stimulating a photothermal reaction of the gold film; The step of judging the reaction result according to the color change of the mixed lysate of the sample to be tested.

9. The photothermal RT-LAMP chip detection method according to claim 8, wherein the liquid surface of the mixed lysate of the sample to be tested is aligned with the reaction chamber by rotation.

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