A low-cost hbv virus nucleic acid rapid detection method based on functionalized modified paper base

Abstract

The application discloses a low-cost hepatitis B (HBV) virus nucleic acid rapid detection method based on a functionally modified paper base, and can realize rapid detection of HBV virus nucleic acid.The method comprises the following steps: (1) pre-amplifying HBV virus nucleic acid to obtain Texas red-labeled nucleic acid; (2) capturing and enriching the Texas red-labeled nucleic acid by using a functionally modified paper base; and (3) performing signal detection.The functionally modified paper base is modified with a fusion protein containing a TR512 polypeptide.The protein functionally modified paper base is simple in preparation, fast in nucleic acid detection time and low in detection cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of viral nucleic acid detection, and particularly relates to a paper base functionally modified with a fusion protein containing a TR512 polypeptide for rapid detection of HBV viral nucleic acid. BACKGROUND

[0002] Sensitive and rapid viral nucleic acid detection is crucial in disease diagnosis, point-of-care testing (POCT), environmental pathogen detection, and genotyping, among other applications.

[0003] In terms of viral detection, real-time quantitative polymerase chain reaction (RT-PCR) is the gold standard for viral nucleic acid detection. However, RT-PCR relies on expensive qPCR instruments and trained professionals. To shorten the amplification time, isothermal recombinase polymerase amplification (RPA), recombinase assisted amplification (RAA), and loop-mediated isothermal amplification (LAMP) techniques have gradually emerged. In recent years, the development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has combined with other amplification steps to produce CRISPR-based diagnostic tools (CRISPR-Dx), such as the SHERLOCK and DETECTR detection platforms. However, these techniques still have non-specific amplification and require expensive fluorescence detection modules for detection. Therefore, there is an urgent need for a rapid, accurate, low-cost, and convenient detection strategy for viral nucleic acid detection. SUMMARY

[0004] Based on the above, the present application develops a low-cost viral nucleic acid rapid detection method and device based on functionally modified paper base. The present application selects hepatitis B (HBV) virus for detection, and the paper base functionally modified with GST-TR512-6xHis protein can capture the Texas red fluorophore-labeled nucleic acid produced after pre-amplification. The detection method greatly shortens the detection period, has low detection cost, and is simple to operate.

[0005] To provide a low-cost rapid detection method for HBV viral nucleic acid, the present application provides a paper base functionally modified with GST-TR512-6xHis protein, which is simple to prepare, can specifically capture and enrich Texas red modified nucleic acid, and realizes rapid detection of HBV viral nucleic acid.

[0006] According to one aspect of the present application, a paper base functionally modified for HBV viral nucleic acid detection is provided, comprising the following steps:

[0007] (1) Pre-amplifying HBV viral nucleic acid to obtain Texas red-labeled nucleic acid;

[0008] (2) using the functionalized paper substrate to capture and enrich Texas red-labeled nucleic acids;

[0009] (3) detecting the signal;

[0010] The functionalized paper substrate is immobilized with a fusion protein containing a TR512 polypeptide.

[0011] Optionally, the fusion protein containing the TR512 polypeptide is immobilized on the paper substrate by sol-gel; the fusion protein containing the TR512 polypeptide and the sol-gel are combined with each other by electrostatic interaction.

[0012] The sol-gel has a positive charge after hydrolysis.

[0013] Optionally, the immobilization amount of the fusion protein containing the TR512 polypeptide is 1×10 -10 mol~5×10 -10 mol.

[0014] The sol-gel is 3-aminopropyltriethoxysilane sol-gel.

[0015] Optionally, the fusion protein containing the TR512 polypeptide is a GST-TR512-6×His fusion protein.

[0016] Optionally, the preparation method of the functionalized paper substrate comprises: adding solution I containing the sol-gel to the detection area of the paper substrate, adding solution II containing the fusion protein to the detection area of the paper substrate after drying, and drying to obtain the functionalized paper substrate.

[0017] Optionally, the volume ratio of the solution I to the solution II is 1:100~1:500.

[0018] In the solution I, the concentration of the sol-gel is 0.61mM~5mM.

[0019] In the solution II, the concentration of the fusion protein is 1μM~20μM.

[0020] Optionally, the pre-amplification technical means are selected from one of polymerase chain reaction (PCR), recombinase aided amplification (RAA), or clustered regularly interspaced short palindromic repeats (CRISPR).

[0021] Optionally, in step (1), a heating device is used for pre-amplification.

[0022] Optionally, in step (3), a handheld fluorescence detector is used for signal detection; the handheld fluorescence detector comprises LED excitation light, filter I, filter II, and CCD camera

[0023] In this application, the GST-TR512-6xHis protein is combined with the APTES sol gel by electrostatic interaction, and the GST-TR512-6xHis protein is immobilized on the paper base, wherein the TR512 polypeptide serves as the main functional group and can be non-covalently and highly affinity combined with Texas red-labeled nucleic acid; glutathione S-transferase (GST, PDB ID: IM99) is a highly soluble protein and is widely used in the expression of various fusion proteins; the 6xHis at the tail end of the protein is mainly used for protein purification purposes.

[0024] As an embodiment of the present application, a set of low-cost HBV viral nucleic acid rapid detection methods based on functionalized paper base is provided, which uses a nucleic acid heating device to amplify viral nucleic acid for a short time, and then uses the GST-TR512-6xHis fusion protein functionalized paper base prepared by the above method to capture and enrich Texas red-labeled nucleic acid, and finally uses a handheld fluorescence detection instrument for signal detection.

[0025] Optionally, the method comprises:

[0026] S1: using a heating device to pre-amplify viral nucleic acid, the core component of the heating device is a heating block, which is simple to construct and low in cost

[0027] S2: using a GST-TR512-6xHis fusion protein functionalized paper base to capture and enrich Texas red-labeled nucleic acid, the protein functionalized paper base is simple to prepare and low in cost

[0028] S3: using a handheld fluorescence detection instrument for signal detection, the handheld fluorescence detection instrument is low in cost, and the important components include excitation light, two filters, and a camera, and the subsequent data processing is completed by Adobe Photoshop using the red channel value R in RGB

[0029] S4: pre-amplification methods include but are not limited to polymerase chain reaction (PCR), recombinase assisted amplification (RAA), and clustered regularly interspaced short palindromic repeat (CRISPR) amplification methods

[0030] S5: the solution gradient is added to the GST-TR512-6xHis fusion protein functionalized paper base, and then the fluorescence intensity value of the positive group is significantly enhanced and the fluorescence intensity ratio with the negative control group is increasingly larger on the handheld fluorescence detection instrument.

[0031] Specifically, the application includes: (1) the design and preparation of paper-based: the pattern consists of square hydrophobic regions (13.5 mm x 13.5 mm) and circular hydrophilic detection regions (diameter 5.0 mm) designed by Adobe Photoshop software, printed on Whatman no. 41 quantitative filter paper using a wax printer. The printed paper is heated in an oven at 200°C for 4 minutes to allow the wax to penetrate the paper. Finally, the test paper is cooled and kept at room temperature; (2) the design, synthesis and purification of recombinant proteins: the gene sequence of GST-TR512-6xHis fusion protein is designed and synthesized by GenScript, and then subcloned into the pET-29b(+) vector. The synthesis and purification of GST-TR512-6xHis fusion protein is completed by conventional methods. Finally, according to the SDS-PAGE gel, no obvious impurities are identified, and the purity is 98%, meeting the purity requirements. (3) Preparation of APTES sol-gel solution: 200 μL of APTES is reacted with 200 μL of tetramethylammonium hydroxide (25% aqueous solution) and 1 mL of ethanol in an ice water bath for 1 hour. (4) Add 1 μL of APTES sol-gel to the detection area of the paper-based, and let it dry naturally, seal and store in the dark. (5) Add GST-TR512-6xHis fusion protein (1 μM, 100 μL) to the detection area of the paper-based, and let it dry naturally for about 10 minutes, ready for detection; (6) Rapid detection of viral nucleic acid: heat the device to amplify the HBV viral nucleic acid for a short time, then capture and enrich the Texas red-labeled nucleic acid with the paper-based functionalized with GST-TR512-6xHis protein, and finally detect the signal with a handheld fluorescence detector.

[0032] Alternatively, the GST-TR512-6xHis fusion protein functionalized paper-based is used in the detection method of different pre-amplification methods of HBV virus. The GST-TR512-6xHis fusion protein functionalized paper-based prepared is combined with traditional amplification methods (including polymerase chain reaction (PCR), recombinase assisted amplification (RAA) and clustered regularly interspaced short palindromic repeat (CRISPR) technology). The capture and enrichment function of the functionalized paper-based for Texas red-labeled HBV nucleic acid greatly shortens the pre-amplification time, reduces the probability of false positives, and finally the three methods achieve good differentiation of positive and negative signals. Compared with traditional nucleic acid detection methods, the detection cost of the functionalized paper-based is greatly reduced.

[0033] In this application, the sequence of GST-TR512-6xHis fusion protein is shown in Table 1.

[0034] Table 1. Sequence of GST-TR512-6xHis protein

[0035]

[0036] The application produces beneficial effects, including:

[0037] 1) The GST-TR512-6xHis fusion protein functionalized paper base is constructed, which fully maintains the binding activity of the protein and realizes the enrichment of Texas red-labeled nucleic acids.

[0038] 2) The functionalized paper base is constructed for rapid detection of viral nucleic acids. The complete detection device is composed of a heating device, a protein functionalized paper base, and a handheld fluorescence detector, which is simple to operate and low in cost.

[0039] 3) The functionalized paper base is constructed for detection of viral nucleic acid detection, which greatly shortens the pre-amplification time and reduces the probability of false positives. It greatly saves the detection cost and ensures the practicability and superiority of the detection device.

[0040] 4) The functionalized paper base has the potential to detect different viral nucleic acids and combine different pre-amplification methods, so as to ensure the universality of the method. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 A schematic diagram of the rapid HBV virus detection based on the functionalized paper base of the present application.

[0042] Figure 2 A structural schematic diagram of the functionalized paper base used in the present application.

[0043] Figure 3 A schematic diagram of the construction of the functionalized paper base used in the present application.

[0044] Figure 4 A structural schematic diagram of the heating device and the handheld fluorescence detector.

[0045] Figure 5 A flow chart of the virus detection device constructed in the present application.

[0046] Figure 6 A process diagram of the functionalized paper base used in Example 4 for HBV PCR detection.

[0047] Figure 7 A process diagram of the functionalized paper base used in Example 5 for HBV RAA detection.

[0048] Figure 8 A process diagram of the functionalized paper base used in Example 6 for HBV CRISPR detection. DETAILED DESCRIPTION

[0049] The application will be described in detail below with reference to the examples, but the application is not limited to these examples.

[0050] The endpoints of the ranges and any values disclosed herein are not limited to the precise values. The endpoints of the ranges and any values are provided as a separate point or an additional range alternately. Any numerical value, however, can inherently contain certain errors necessarily resulting from the round-off or measurement of its components. Accordingly, it is the stated value plus or minus ten percent (10%) unless otherwise indicated.

[0051] Unless otherwise specified, the raw materials in the examples of the present application are purchased through commercial channels.

[0052] The instrument used for the fluorescence spectrum measurement of the pre-amplification solution in the examples of the present application is Tecan Spark Fluorescence Plate Reader, the excitation wavelength is selected as 580 nm, and the emission wavelength is selected as 605 nm.

[0053] The heating device in the examples of the present application is shown in FIG. 1 (a), and the core component is a heating block, which realizes the shortening of the pre-amplification time. Figure 4

[0054] The handheld fluorescence detector in the examples of the present application is shown in FIG. 1 (b), and the components include LED excitation light, filter I of 570 nm (under the lamp), filter II of 610 nm (at the top of the dark box), and a CCD camera. Figure 4

[0055] Example 1

[0056] This example shows the design, synthesis and purification method of the GST-TR512-6xHis fusion protein, which includes the following steps:

[0057] (1) The gene sequence of the GST-TR512-6xHis fusion protein (as shown in SEQ ID NO: 2) is designed and synthesized by GenScript, and then subcloned into the pET-29b(+) vector, wherein the sequence of the TR512 polypeptide is as shown in SEQ ID NO: 1.

[0058] (2) The GST-TR512-6xHis fusion protein plasmid is transformed into BL21 DE3 E. coli cells, and when the OD600 value of the cells is in the range of 0.6-0.8, IPTG (0.5 mM) is used to induce at 30°C for 4 hours. The cells are collected and resuspended in resuspension buffer (50 mM Tris•HCl, 100 mM NaCl, pH=8.0); then the cells are ultrasonically dissolved at 4°C (12000 rpm, 30 minutes).

[0059] ​​(3) The supernatant was then collected and applied to a 10 mL Ni-NTA chromatography column, which was washed with buffer (50 mM Tris-HCl, 100 mM NaCl, pH=8.0) and then with an imidazole buffer (50 mM Tris-HCl, 100 mM NaCl, 500 mM imidazole, pH=8) to increase the gradient elution of the protein. The purity was then analyzed by SDS-PAGE gel, and the protein was further purified by an SEC column with a buffer (10 mM sodium phosphate, 100 mM potassium chloride, 1 mM EDTA, acidified with hydrochloric acid to pH=7.40), and the purity was analyzed by SDS-PAGE gel. No obvious impurities were identified, and the purity was 98%, meeting the purity requirement.

[0060] Example 2

[0061] This example illustrates the preparation process of a paper-based functionalized modification of GST-TR512-6xHis protein, which comprises the following steps:

[0062] (1) Obtain a paper-based material with a 5 mm diameter circular detection zone;

[0063] (2) Obtain an APTES sol-gel solution;

[0064] (3) Add 1 μL of the APTES sol-gel to the detection zone of the functionalized modification paper-based material, and allow it to dry naturally. Seal and store in the dark;

[0065] (4) Add GST-TR512-6xHis protein (1 μM, 100 μL) to the detection zone of the paper-based material, and allow it to dry naturally for about 10 minutes, and then use it for detection;

[0066] Specifically, the paper-based material with a 5 mm diameter circular detection zone in step (1) is prepared as follows: a square hydrophobic region (13.5 mm x 13.5 mm) and a circular hydrophilic sensing region (5.0 mm in diameter) are designed by Adobe Photoshop software, and are printed on Whatman no. 41 quantitative filter paper using a wax printing machine. The printed paper is heated in an oven at 200°C for 4 minutes to allow the wax to penetrate the paper. Finally, the test paper is cooled and kept at room temperature.

[0067] Specifically, the APTES sol-gel solution in step (2) is prepared as follows: 200 μL of APTES is reacted with 200 μL of tetramethylammonium hydroxide (25% aqueous solution) and 1 mL of ethanol in an ice water bath for 1 hour.

[0068] Example 3

[0069] This example illustrates the use process of the low-cost HBV virus nucleic acid rapid detection device based on the functionalized modification paper-based material of the present application as follows:Figure 5 As shown in the figure, (1): the virus-free nucleic acid solution and the extracted DNA solution are respectively placed in the left and right amplification pools of the heating device, and pre-amplification is performed by RAA method, the pre-amplification time is 5 minutes, 39°C; (2): the functionalized modified paper base is placed in the mold, and the virus-free nucleic acid pre-amplification solution and the extracted DNA pre-amplification solution (300 μL) are respectively dropped into the left and right sample pools. The detection points in the solid frame in the upper right corner of the picture are used for detection, and the fusion protein is functionalized in advance; other detection points are used for device positioning; (3): the paper is placed in the card slot, and then placed in the handheld fluorescence detector; (4)-(6): select "enter test", and then display "in test". Finally, the ΔR is 71 (ΔR = R value obtained by test-R0, R0 is the R value of the blank functionalized modified paper base), and the virus detection result is "positive"; the upper right corner is an enlarged view of the screen.

[0070] The extracted DNA solution is extracted according to the DNA extraction step in the hepatitis B virus (HBV) nucleic acid (DNA) detection kit (fluorescent probe method) (manufacturer: Northeast Pharmaceutical Group Co., Ltd.). According to the operation in the kit: add 400 μL of lysis solution, 200 μL of serum, and 10 μL of magnetic bead suspension into the centrifugal tube, shake and mix for 10 s, then stand at room temperature for 10 mins, and centrifuge instantly; place the centrifugal tube on the magnetic stand and adsorb for 2 mins, discard the supernatant, and stand for 30 s before absorbing the residual liquid; add 600 μL of rinse solution, and repeatedly suck and beat 10 times with a pipette, then centrifuge instantly. Again, adsorb for about 2 mins on the magnetic stand, absorb the residual liquid, and stand for 2 mins; add 10 μL of eluent, shake and mix for 10 s, then stand for 2 mins; place the centrifugal tube on the magnetic stand and adsorb for 2 mins, and the supernatant is the extracted DNA solution for use.

[0071] Example 4

[0072] This example shows the detection method of the GST-TR512-6xHis protein functionalized paper base for HBV virus, specifically, the time of traditional HBV PCR is shortened to 25 cycles in the heating device (at this time the real-time fluorescence and the fluorescence spectrum after amplification are difficult to distinguish between positive and negative control groups), then the amplified negative control and positive solution are dropped on the prepared GST-TR512-6xHis protein functionalized paper base for Texas red labeled nucleic acid capture and enrichment, and then detected by the self-made handheld fluorescence detector. It can be seen that the distinction between negative and positive signals is more and more obvious.

[0073] The results are as follows Figure 6As shown, (a) illustrates the principle of PCR amplification: when target DNA is present, PCR amplification causes probe breakage, producing Texas Red-labeled nucleic acid; (b) shows real-time fluorescence readings: the HBV PCR time was shortened from 60 cycles to 25 cycles; (c) shows fluorescence spectral readings after amplification: a comparison of fluorescence intensity at 60 cycles, 25 cycles, and the negative control solution, where the fluorescence intensity at 25 cycles was 1.9 times that of the negative control, making it difficult to distinguish; (d) 50-300 μL of the pre-amplified 25-cycle solution and the negative control solution were gradually added to a functionalized paper substrate, and the ΔR value was read using a self-made handheld fluorescence detector after 5 minutes. When the added volume was 300 μL, the fluorescence signal difference between the 25-cycle solution and the negative control was 2.8 times. This achieved differentiation between the positive and negative control groups.

[0074] Example 5

[0075] This embodiment demonstrates a method for detecting HBV virus using a paper substrate functionalized with GST-TR512-6×His protein. Specifically, the traditional HBV RAA amplification time is shortened from 30 minutes to 5 minutes in a heating device. Subsequently, the amplified negative and positive solutions are added to the prepared GST-TR512-6×His protein functionalized paper substrate for Texasred-labeled nucleic acid capture and enrichment, followed by detection using a self-made handheld fluorescence detector. The distinction between negative and positive signals becomes increasingly clear.

[0076] The results are as follows Figure 7 As shown, (a) illustrates the RAA amplification principle: when target DNA is present, RAA amplification causes probe breakage, producing Texas Red-labeled nucleic acid; (b) shows the real-time fluorescence reading: the HBV RAA time was shortened from 30 minutes to 5 minutes; (c) shows the fluorescence spectrum reading after amplification: a comparison of fluorescence intensity at 30 minutes, 5 minutes, and the negative control solution, where the fluorescence intensity at 5 minutes was 2.4 times that of the negative control group, making it difficult to distinguish; (d) 100-600 μL of the pre-amplified 5-minute solution and the negative control solution were gradually added to a functionalized paper substrate, and the ΔR value was read using a self-made handheld fluorescence detector after 5 minutes. When the added volume was 600 μL, the fluorescence signal difference between the 5-minute amplification and the negative control group was 3.7 times. This achieved the distinction between the positive and negative control groups.

[0077] Example 6

[0078] The embodiment shows a detection method of the GST-TR512-6xHis protein functionalized modified paper base for HBV virus, specifically, the traditional HBV CRISPR amplification time is shortened from 6 hours to 30 minutes in the heating device, then the amplified positive and negative solutions are added dropwise on the prepared GST-TR512-6xHis protein functionalized modified paper base for capture and enrichment of Texas red labeled nucleic acid, and then detected by a handheld fluorescence detector. It can be seen that the distinction between positive and negative signals is more and more obvious.

[0079] The results are shown in Figure 8 (a) is the principle of CRISPR amplification, when the target DNA exists, the activity of cas12a is activated, the ssDNA reporter group is broken, and Texas red labeled nucleic acid is produced; (b) is real-time fluorescence reading: the HBV CRISPR time is shortened from 6 hours to 30 minutes; (c) is the fluorescence spectrum reading after amplification: the fluorescence intensity comparison of 6 hours, 30 minutes and negative control solution, wherein the fluorescence intensity of 30 minutes is 2.9 times that of the negative control group; (d) 50-300 μL of pre-amplified 30-minute solution and negative control solution is added dropwise on the functionalized modified paper base, and ΔR value is read by a handheld fluorescence detector after 5 minutes. When the dropwise amount is 300 μL, the fluorescence signal difference between 30-minute amplification and negative control group is 3.8 times. The distinction between positive and negative control groups is realized.

[0080] The above is only a few embodiments of the present application, and does not limit the present application in any form. Although the preferred embodiments are disclosed as above, they are not intended to limit the present application. Any skilled person in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, which are equivalent to equivalent embodiments, and belong to the scope of the technical solution. SEQUENCE LISTING <110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences <120> A low-cost HBV virus nucleic acid rapid detection method based on functionalized modified paper base <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 38 <212> PRT <213> Artificial Sequence <400> 1 Gly Gly Gly Ser Lys Val Ile Leu Phe Glu Gly Pro Ala Gly Arg Trp 1 5 10 15 Thr Trp Pro Glu Ile Ser Glu Gly Ala Pro Gly Ser Lys Val Ile Leu 20 25 30 Phe Glu Gly Gly Pro Gly 35 <210> 2 <211> 786 <212> DNA <213> Artificial Sequence <400> 2 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaaggtggc 660 ggttccaaag ttattctgtt tgaaggtcct gcaggtcgtt ggacctggcc tgaaatctct 720 gaaggtgcgc cgggttctaa ggtaattctg ttcgaaggtg gccctggcca tcatcaccat 780 caccac 786

Claims

1. A method for detecting HBV viral nucleic acid based on functionally modified paper base for non-diagnostic purposes, characterized by, The method comprises the following steps: (1) pre-amplifying HBV viral nucleic acid by one of polymerase chain reaction (PCR), recombinase aided amplification (RAA) or clustered regularly interspaced short palindromic repeats (CRISPR), to obtain Texas red-labeled nucleic acid; (2) capturing and enriching the Texas red-labeled nucleic acid by using a functionalized paper substrate loaded with GST-TR512-6xHis fusion protein through 3-aminopropyl triethoxysilane sol-gel; the fusion protein containing TR512 polypeptide is loaded on the paper substrate through sol-gel; the fusion protein containing TR512 polypeptide and the sol-gel are combined with each other through electrostatic interaction; the sol-gel has positive charge after hydrolysis; the gene sequence of the GST-TR512-6xHis fusion protein is shown in SEQ ID NO. 2; (3) signal detection; The functionalized paper substrate has immobilized thereon a fusion protein comprising a TR512 polypeptide in an amount of 1 x 10 -10 mol to 5 x 10 -10 mol.

2. The detection method according to claim 1, wherein the preparation method of the functionalized paper substrate comprises: adding solution I containing sol-gel to the detection area of the paper substrate, and then adding solution II containing fusion protein to the detection area of the paper substrate after drying, and drying to obtain the functionalized paper substrate.

3. The detection method according to claim 2, wherein the volume ratio of solution I to solution II is 1:100-1:500; the concentration of sol-gel in solution I is 0.61 mM-5 mM; the concentration of fusion protein in solution II is 1 μM-20 μM.

4. The method of claim 1, wherein In step (1), a heating device is used for pre-amplification.

5. The method of claim 1, wherein In step (3), a handheld fluorescence detector is used for signal detection; the handheld fluorescence detector comprises LED excitation light, 570 nm filter I, 610 nm filter II and a CCD camera.

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