Protein-functionalized paper substrates, their preparation methods, and their application in the detection of Texas red fluorophores.

Abstract

The application discloses a GST-TR512-6xHis protein functionalized modified paper base, a preparation method thereof and application of the paper base in Texas red fluorophore enrichment detection, and belongs to the paper base sensor field. The paper base is functionalized and modified with a fusion protein containing a TR512 polypeptide. The paper base preparation method is simple, detection time is fast, and detection cost is low.

Description

Technical Field

[0001] This application belongs to the field of paper-based sensors, specifically relating to a paper substrate functionalized with GST-TR512-6×His protein, its preparation method, and its application in the detection of Texas red fluorophores. Background Technology

[0002] The Whitesides team used paper as a microfluidic platform to simultaneously detect various analytes, subsequently defining it as a paper-based analytical device (PAD). Since then, paper-based sensors have attracted significant attention due to their portability and low cost. For example, Teengam et al. introduced a paper-based hepatitis C virus (HCV) DNA detection device that uses a chemical method to immobilize the acpcPNA probe, which specifically binds to HCV DNA for fluorescence detection. Cinti et al. designed an electroanalytical paper-based sensor with a recognition probe for detecting breast cancer mutations. Henry's group developed a distance-based paper-based sensor that uses fluorescence to quantitatively detect aluminum in environmental pollutants without requiring sample pretreatment or preconcentration. Although PAD technology can serve as a point-of-care detection platform due to its ideal portability, low cost, and biocompatibility, insufficient analyte enrichment leads to high detection limits and low sensitivity, significantly limiting the application of paper-based sensors.

[0003] TR512, an important fluorophore dye-binding peptide, binds to the Texas red fluorophore with non-covalent high affinity. In recent years, TR512 peptides have been widely used in fluorescence-based detection experiments in both in vitro and in vivo. However, most research on TR512 is limited to solutions, with limited studies on its immobilization on paper-based sensors. Summary of the Invention

[0004] Based on the above, this application proposes to immobilize the TR512 peptide onto a paper-based sensor while maintaining its high affinity for Texas red, thereby achieving the capture and enrichment of Texas red fluorophores and further solving the problem of insufficient analyte enrichment faced by paper-based sensors.

[0005] Therefore, this application provides a paper-based sensor with GST-TR512-6×His functionalization modification, and a method for enriching Texas red fluorophores based on the paper-based sensor. The paper-based sensor of this application has a simple preparation process, low cost, and can specifically capture and enrich Texas red fluorophores, achieving the filtering and enrichment effect of Texas red fluorophores.

[0006] According to one aspect of this application, a protein-modified paper substrate is provided, wherein a fusion protein containing a TR512 polypeptide is immobilized on the paper substrate.

[0007] Optionally, the fusion protein containing the TR512 polypeptide is a GST-TR512-6×His fusion protein.

[0008] Optionally, the fusion protein containing the TR512 peptide is immobilized on a paper substrate using a sol-gel; the fusion protein containing the TR512 peptide and the sol-gel are bound together by electrostatic interaction.

[0009] The sol-gel body carries a positive charge after hydrolysis.

[0010] Optionally, the loading of the fusion protein containing the TR512 polypeptide onto the paper substrate is 1 × 10⁻⁶. -10 mol~5×10 -10 mol;

[0011] The sol-gel is a 3-aminopropyltriethoxysilane sol-gel.

[0012] Optionally, the GST-TR512-6×His fusion protein in the paper base, wherein the TR512 polypeptide is the main functional group, can bind non-covalently and with high affinity to the Texas red fluorescent group; glutathione S-transferase (GST, PDB ID: IM99) is a highly soluble protein with high expression levels in E. coli and is widely used for the expression of various fusion proteins, therefore, it is used as a carrier protein in this application; the 6×His at the tail of the GST-TR512-6×His protein is mainly used for protein purification.

[0013] Optionally, the design, synthesis, and purification of the GST-TR512-6×His fusion protein in the paper base include:

[0014] (1) The gene sequence of GST-TR512-6×His fusion protein was designed (as shown in Sequence 2) and synthesized by GenScript. It was then subcloned into the pET-29b(+) vector, where the sequence of the TR512 polypeptide is shown in Sequence 1.

[0015] (2) GST-TR512-6×His fusion protein particle was transformed into BL21 DE3 E. coli cells. When the OD600 value of the cells was in the range of 0.6 to 0.8, they were induced with IPTG (0.5 mM) at 30°C for 4 hours. The cells were collected and resuspended in resuspension buffer (50 mM Tris·HCl, 100 mM NaCl, pH=8.0). The cells were then sonicated at 4°C (12000 rpm, 30 min).

[0016] (3) The supernatant was then collected onto a 10 mL Ni-NTA column and washed with buffer (50 mM Tris·HCl, 100 mM NaCl, pH = 8.0). The protein was then eluted using a gradient of imidazole buffer (50 mM Tris·HCl, 100 mM NaCl, 500 mM imidazole, pH = 8). Purity was then determined by SDS-PAGE gel chromatography. The protein was then purified again using an SEC column with a buffer solution of (10 mM sodium phosphate, 100 mM potassium chloride, 1 mM EDTA, acidified to pH = 7.40 with hydrochloric acid). Subsequent SDS-PAGE gel chromatography analysis revealed no significant impurities, and the purity was 98%, meeting the purity requirements.

[0017] According to one aspect of this application, a method for preparing the above-mentioned paper base is provided, wherein a solution I containing a sol-gel is dropped onto the detection area of ​​the paper base, dried, and then a solution II containing a fusion protein is added to the detection area of ​​the paper base, dried, to obtain the paper base modified with protein; wherein the fusion protein is a fusion protein containing a TR512 polypeptide.

[0018] Optionally, the volume ratio of solution I to solution II is 1:100 to 1:500;

[0019] In solution I, the concentration of the sol-gel is 0.61 mM to 5 mM;

[0020] In solution II, the concentration of the fusion protein is 1 μM to 20 μM.

[0021] According to one aspect of this application, a paper-based sensor is provided, the paper-based sensor comprising one of the paper-based materials described above or the paper-based materials prepared by the above preparation method.

[0022] According to one aspect of this application, a method for detecting Texas red fluorophores is provided, wherein the detection method uses the aforementioned paper-based sensor to capture and enrich Texas red fluorophores.

[0023] Optionally, the paper-based sensor is placed in a detection device to capture and enrich Texas red fluorophores, acquire images, and obtain the red channel value R in the RGB values ​​of the image.

[0024] As one embodiment of this application, a method for preparing a paper substrate functionalized with GST-TR512-6×His fusion protein is provided, comprising the following steps:

[0025] (1) Obtain a paper base with a circular detection area with a diameter of 5 mm;

[0026] (2) Obtain APTES sol-gel solution;

[0027] (3) Add 1 μL of APTES sol-gel to the detection area of ​​the paper-based sensor, let it stand and dry naturally, and store it in a sealed container away from light;

[0028] (4) Add GST-TR512-6×His fusion protein (1μM, 100μL) to the detection area of ​​the paper-based sensor and let it air dry for about 10 minutes before use for detection.

[0029] Optionally, the paper base with a 5mm diameter circular detection area described in step (1) is prepared by: a pattern consisting of a square hydrophobic area (13.5mm × 13.5mm) and a circular hydrophilic sensing area (5.0mm in diameter) designed using Adobe Photoshop software, which is then printed onto Whatman no. 41 quantitative filter paper using a wax printing machine. The printed paper is then 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.

[0030] Optionally, the APTES sol-gel solution described in step (2) is prepared by reacting 200 μL of APTES with 200 μL of tetramethylammonium hydroxide (25% aqueous solution) and 1 mL of ethanol in an ice-water bath for 1 hour.

[0031] As one embodiment of this application, a method for capturing and enriching Texas red fluorophores is provided. The method uses a paper-based sensor functionalized with the prepared GST-TR512-6×His fusion protein to capture and enrich Texas red fluorophores, which are then detected by a detection device.

[0032] Optionally, the method includes:

[0033] S1: As the volume of a certain concentration of Texas red solution added to the prepared paper substrate increases, the fluorescence intensity signal gradually accumulates and increases, exhibiting an "S" shape. This clearly demonstrates the capture and enrichment effect of the paper substrate on Texas red fluorophores.

[0034] S2: As the concentration of a certain volume of Texas red solution added to the prepared paper substrate increases, the fluorescence intensity signal gradually accumulates and increases, exhibiting an "S" shape. This clearly demonstrates the capture and enrichment effect of the paper substrate on Texas red fluorophores.

[0035] S3: The detection device is inexpensive and operates in a darkroom. The key components consist of an excitation beam, two filters, and a camera. Subsequent data processing is performed by Adobe Photoshop using the red channel value (R value) in RGB.

[0036] Specifically, the application of the protein-modified paper-based sensor in the detection of Texas red fluorophores includes:

[0037] (1) Design and preparation of the paper base: The pattern consists of a square hydrophobic area (13.5mm × 13.5mm) and a circular hydrophilic detection area (5.0mm in diameter) designed using Adobe Photoshop software, and is printed on Whatman no. 41 quantitative filter paper using a wax printing machine. The printed paper is heated in an oven at 200℃ for 4 minutes to allow the wax to penetrate the paper. Finally, the test paper is cooled and kept at room temperature.

[0038] (2) Design, synthesis, and purification of the fusion protein: The gene sequence of the GST-TR512-6×His fusion protein was designed and synthesized using GenScript, then subcloned into the pET-29b(+) vector. The GST-TR512-6×His fusion protein particle was transformed into BL21 DE3 E. coli cells, and when the cell OD... 600 When the pH value is in the range of 0.6–0.8, induce with IPTG (0.5 mM) at 30 °C for 4 hours. Collect cells and resuspend in resuspension buffer (50 mM Tris·HCl, 100 mM NaCl, pH = 8.0). Then, sonicate the cells at 4 °C (12000 rpm, 30 min), collect the supernatant onto a 10 mL Ni-NTA column, wash with buffer (50 mM Tris·HCl, 100 mM NaCl, pH = 8.0), and then increase the elution protein with a gradient of imidazole buffer (50 mM Tris·HCl, 100 mM NaCl, 500 mM imidazole, pH = 8). The purity was then determined by SDS-PAGE gel analysis. The protein was then purified again by SEC column with a buffer solution of (10 mM sodium phosphate, 100 mM potassium chloride, 1 mM EDTA, acidified to pH 7.40 with hydrochloric acid). Subsequent SDS-PAGE gel analysis showed no obvious impurities, and the purity was 98%, which met the purity requirements.

[0039] (3) Preparation of APTES sol-gel solution: 200 μL of APTES was reacted with 200 μL of tetramethylammonium hydroxide (25% aqueous solution) and 1 mL of ethanol in an ice-water bath for 1 hour.

[0040] (4) Add 1 μL of APTES sol-gel to the detection area of ​​the paper-based sensor, let it stand and dry naturally, and store it in a sealed container away from light.

[0041] (5) Add GST-TR512-6×His fusion protein (1μM, 100μL) to the detection area of ​​the paper-based sensor and let it air dry for about 10 minutes before use for detection.

[0042] (6) Texas red solutions of the same concentration but different volumes were dropped onto the prepared paper-based sensor, and the R value in its RGB was detected by a detection device. It has a double reciprocal relationship I.

[0043] (7) Texas red solutions of different concentrations and the same volume were dropped onto the prepared paper-based sensor, and the R value in its RGB was detected by the detection device. It has a double reciprocal relationship II.

[0044] The paper-based sensor can detect Texas red solution volumes ranging from 0 to 300 μL (0.25 μM); and can detect Texas red solution concentrations ranging from 2.5 to 65 nM (100 μL).

[0045] The double reciprocal relation I is ΔR -1 =1.7030V -1 -0.0002, ΔR is the R value of the paper-based sensor (the background value R0 has been subtracted, and R0 is the R value of the paper-based sensor modified with protein), and V is the volume of the Texas red solution added.

[0046] Optionally, V is any one of 25 μL, 50 μL, 75 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, or a range determined by any two values.

[0047] The double reciprocal relation II is ΔR -1 =0.53183C -1 -0.00201, ΔR is the R value of the paper-based sensor (after subtracting the background value R0), and C is the concentration of the added Texas red solution.

[0048] Optionally, C is any one of 2.5nM, 5nM, 15nM, 25nM, 35nM, 45nM, 55nM, 65nM, or a range of values ​​determined by any two values.

[0049] The beneficial effects of this application include:

[0050] 1) A paper-based sensor with functionalized GST-TR512-6×His fusion protein was constructed, which fully preserved the protein's activity.

[0051] 2) The high affinity between the TR512 peptide and the Texas red fluorophore enabled the capture and enrichment of the Texas red fluorophore on a paper substrate.

[0052] 3) This application is the first to propose using the APTES sol-gel system to immobilize proteins on paper substrates, which increases the immobilization efficiency while maintaining their activity.

[0053] 4) The GST-TR512-6×His fusion protein was synthesized, which is innovative in protein construction.

[0054] 5) The constructed paper-based sensor is used to capture and enrich Texas red fluorophores. Detection can be performed on a simple detection device, which is easy to operate, fast, and inexpensive.

[0055] 6) Based on the capture and enrichment of Texas red fluorophores, the paper-based sensor provided in this application is expected to be used in other areas such as virus detection of Texas red labeled molecules. Attached Figure Description

[0056] Figure 1 A schematic diagram illustrating the fabrication and detection of a paper-based sensor is shown.

[0057] Figure 2 The MALDI-TOF analysis results of the GST-TR512-6×His fusion protein are shown, indicating that the fusion protein was successfully expressed.

[0058] Figure 3 The results of fluorescence microscopy observation of the fusion protein in Texas red solution are shown. Microscopic analysis shows that the fusion protein has binding activity with the Texas red fluorophore in solution.

[0059] Figure 4 A schematic diagram illustrating the binding mechanism of the fusion protein to Texas Red solution is shown.

[0060] Figure 5 The surface charge of the fusion protein is shown, with the darker areas indicating that the Asp and Glu residues in the fusion protein are negatively charged.

[0061] Figure 6 The detection device used in this application is shown.

[0062] Figure 7 The diagram illustrates how the fluorescence color of the paper-based sensor in Embodiment 6 of this application changes with the volume of Texas red solution added.

[0063] Figure 8 The relationship between ΔR of the paper-based sensor and the volume of the added Texas red solution is shown in Embodiment 6 of this application.

[0064] Figure 9 The ΔR of the paper-based sensor in Embodiment 6 of this application is shown. -1 The volume V of the added Texas red solution -1 The relationship between the two reciprocals of the function.

[0065] Figure 10 The diagram illustrates how the fluorescence color of the paper-based sensor in Embodiment 7 of this application changes with the concentration of Texas red solution added.

[0066] Figure 11 The relationship between ΔR of the paper-based sensor and the concentration of the added Texas red solution is shown in Embodiment 7 of this application.

[0067] Figure 12 The ΔR of the paper-based sensor in Embodiment 7 of this application is shown. -1 The concentration C of the added Texas red solution -1 The relationship between the two reciprocals of the function. Detailed Implementation

[0068] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0069] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, but should be understood to include those approximations of such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0070] Unless otherwise specified, all raw materials used in the embodiments of this application were purchased through commercial channels.

[0071] In this embodiment, the instrument used for MALDI mass spectrometry detection is a Bruker Ultra Flex III MALDI-TOF-TOF.

[0072] The fluorescence microscopy analysis results in this embodiment were obtained using an Olympus FV1000 FluoView instrument. TM confocal microscope.

[0073] The detection device used in this application is such as Figure 6 As shown, it includes: a dark box, a 570nm excitation light, a 570nm bandpass filter under the light, a 610nm longpass filter at the top of the dark box, and a camera;

[0074] The biosensor is located in the dark chamber 4. Under the excitation of the excitation light 1, the excitation light passes through the light filter 2 under the lamp, the paper-based sensor, and the filter 5 at the top of the dark chamber in sequence. The image is acquired by the camera 3 to obtain the R value in the RGB values.

[0075] The R values ​​in the embodiments of this application were all processed by Adobe Photoshop.

[0076] Example 1

[0077] This embodiment illustrates the design (sequence shown in Table 1), synthesis, and purification methods of the GST-TR512-6×His fusion protein, which includes the following steps:

[0078] (1) The gene sequence of the GST-TR512-6×His fusion protein was designed and synthesized using GenScript, and then subcloned into the pET-29b(+) vector.

[0079] (2) GST-TR512-6×His fusion protein particle was transformed into BL21 DE3 E. coli cells. When the OD600 value of the cells was in the range of 0.6-0.8, they were induced with IPTG (0.5 mM) at 30°C for 4 hours. The cells were collected and resuspended in resuspension buffer (50 mM Tris·HCl, 100 mM NaCl, pH=8.0); then the cells were sonicated at 4°C (12000 rpm, 30 min).

[0080] (3) The supernatant was then collected onto a 10 mL Ni-NTA column and washed with buffer (50 mM Tris·HCl, 100 mM NaCl, pH = 8.0). The protein was then eluted using a gradient of imidazole buffer (50 mM Tris·HCl, 100 mM NaCl, 500 mM imidazole, pH = 8). Purity was then determined by SDS-PAGE gel chromatography. The protein was then purified again using an SEC column with a buffer solution of (10 mM sodium phosphate, 100 mM potassium chloride, 1 mM EDTA, acidified to pH = 7.40 with hydrochloric acid). Subsequent SDS-PAGE gel chromatography analysis revealed no significant impurities, and the purity was 98%, meeting the purity requirements.

[0081] Table 1. Sequence of GST-TR512-6×His fusion protein

[0082]

[0083] Example 2

[0084] This example illustrates the MALDI-TOF detection process for the prepared GST-TR512-6×His fusion protein.

[0085] Specifically, the fusion protein solution was diluted to 1 g / L, and ionization was assisted by sinapic acid (SA). Then, 1 μL of the protein solution and 1 μL of the SA solution were simultaneously coated onto a steel plate, allowed to dry, and then detected. See [link to results] for details. Figure 2 The actual measured values ​​were not significantly different from the calculated values, indicating the successful preparation of the fusion protein.

[0086] Example 3

[0087] This example illustrates the fluorescence microscopy detection process of the prepared GST-TR512-6×His fusion protein.

[0088] Specifically, the TR512 peptide of the fusion protein binds to TALON metal affinity resin (Clontech) in TBS buffer via a His6 tag. After washing three times with 1 mL Tris buffer to remove unbound protein, the protein is incubated for 30 minutes in 40 μL Tris buffer with 2 μM Texasred solution. Finally, it is washed three times with 1 mL Tris buffer to remove unbound dye. The protein is then used in an Olympus FV1000 FluoView. TM Fluorescence images were captured using a confocal microscope. The results are as follows: Figure 3 As shown in the figure, the prepared protein solution maintains good binding activity for the Texas red fluorophore.

[0089] Example 4

[0090] This embodiment illustrates the fabrication process of a paper-based sensor functionalized with the GST-TR512-6×His fusion protein, as follows: Figure 1 As shown, it includes the following steps:

[0091] (1) Obtain a paper base with a circular detection area with a diameter of 5 mm;

[0092] (2) Obtain APTES sol-gel solution;

[0093] (3) Add 1 μL of APTES sol-gel to the detection area of ​​the paper-based sensor, let it stand and dry naturally, and store it in a sealed container away from light;

[0094] (4) Add GST-TR512-6×His fusion protein (1μM, 100μL) to the detection area of ​​the paper-based sensor and let it air dry for about 10 minutes before use for detection;

[0095] Specifically, the paper base with a 5mm diameter circular detection area described in step (1) is prepared as follows: the pattern consists of a square hydrophobic area (13.5mm × 13.5mm) and a circular hydrophilic sensing area (5.0mm in diameter) designed using Adobe Photoshop software, and is 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.

[0096] Specifically, the APTES sol-gel solution described in step (2) is prepared by reacting 200 μL of APTES with 200 μL of tetramethylammonium hydroxide (25% aqueous solution) and 1 mL of ethanol in an ice-water bath for 1 hour.

[0097] Example 5

[0098] The binding mechanism of the fusion protein to the Texas Red solution is as follows: Figure 4 As shown.

[0099] Fluorescence microscopy observations of the fusion protein and Texas red solution are as follows: Figure 3 As shown, microscopic analysis revealed that the fusion protein exhibits binding activity to the Texas red fluorophore in solution.

[0100] The surface charge of the fusion protein is as follows: Figure 5 As shown, the darker areas reveal the negatively charged Asp and Glu residues on the protein surface. This figure demonstrates that the protein is immobilized by the APTES sol-gel system through electrostatic interactions.

[0101] Example 6

[0102] This embodiment demonstrates the capture and enrichment of Texas red fluorophores by a paper-based sensor.

[0103] Specifically, 25 μL, 50 μL, 75 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL of Lexaas Red solution (0.25 μM) were dropped onto a paper-based sensor; images were taken after 5 minutes. Fluorescence images are shown below. Figure 7 As shown, and Figure 8 , Figure 9 The obtained double reciprocal relationship is shown: ΔR -1 =1.7030V -1 -0.0002, ΔR is the R value of the paper-based sensor (excluding the background value R0), and V is the volume of the added solution.

[0104] Example 7

[0105] This embodiment illustrates the lowest detection limit of a paper-based sensor for Texas red.

[0106] Specifically, 100 μL of 2.5 nM, 5 nM, 15 nM, 25 nM, 35 nM, 45 nM, 55 nM, and 65 nM Texas Red solutions were dropped onto a paper-based sensor; images were taken after 5 minutes. Fluorescence images are shown below. Figure 10 As shown, and Figure 11 , Figure 12 The obtained double reciprocal relationship is shown: ΔR-1 =0.53183C -1 -0.00201, ΔR is the R value of the paper-based sensor (subtracting the background value R0), and C is the concentration of the added solution. The limit of detection is 1.4 nM (3σ / k).

[0107] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution. sequence list <110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences <120> Protein-functionalized paper substrates, their preparation methods, and their application in the detection of Texas red fluorophores. <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 tggccttgc 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 paper base modified with protein, characterized in that, The paper substrate contains a fusion protein containing the TR512 polypeptide. The fusion protein containing the TR512 polypeptide is a GST-TR512-6×His fusion protein; The fusion protein containing the TR512 polypeptide is immobilized on a paper substrate via a sol-gel; the fusion protein containing the TR512 polypeptide and the sol-gel are bound together by electrostatic interaction. The sol-gel body carries a positive charge after hydrolysis; The fusion protein containing the TR512 polypeptide has a loading of 1×10 on the paper substrate. -10 mol~5×10 -10 mol; The sol-gel is a 3-aminopropyltriethoxysilane sol-gel.

2. A method for preparing the paper base according to claim 1, characterized in that, Solution I containing sol-gel was dropped onto the detection area of ​​the paper base and dried. Solution II containing fusion protein was then added to the detection area of ​​the paper base and dried to obtain the paper base modified with protein. The fusion protein is a fusion protein containing TR512 polypeptide.

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

4. A paper-based sensor, characterized in that, The paper-based sensor includes one of the paper-based materials described in claim 1 or the paper-based materials prepared by the preparation method described in claim 2 or 3.

5. A method for detecting Texas red fluorophores, characterized in that, The detection method uses the paper-based sensor described in claim 4 to capture and enrich Texas red fluorophores.

6. The detection method according to claim 5, characterized in that, The paper-based sensor is placed in the detection device to capture and enrich the Texas red fluorophore, acquire an image, and obtain the red channel value R in the RGB values ​​of the image.

Images