Peptide probes for detecting influenza virus, sensing array and preparation method and application thereof

Abstract

This invention relates to the field of biodetection technology, and particularly to peptide probes, sensor arrays, preparation methods, and applications for detecting influenza viruses. A peptide probe for detecting influenza virus comprises polypeptide chains and fluorescent dyes, wherein the polypeptide chains are any one of a first, second, third, and fourth polypeptide chains. The peptide probe for detecting influenza virus and its preparation method enable the design of broad-spectrum fluorescent peptide probes that specifically recognize two influenza virus antigens, solving the technical problems of time-consuming and costly identification of influenza virus subtypes and the difficulty in peptide probe design. Using the above-mentioned sensor array for detecting influenza virus peptide probes and its preparation method, a sensor array based on the resonance transfer signal of dual-antigen activated fluorescence energy can be constructed, providing an effective strategy for achieving high-throughput, zero-background, and high-specificity detection of influenza viruses and subtype differentiation.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to peptide probes, sensor arrays, preparation methods, and applications for detecting influenza viruses. Background Technology

[0002] Influenza viruses are highly contagious and frequently spread across species, leading to continuous antigenic variation and genetic recombination. Influenza virus infection can cause severe respiratory symptoms, leading to complications such as pneumonia and acute respiratory distress syndrome (ARDS), and even inducing multiple organ failure, posing a serious threat to high-risk groups. Influenza viruses can be classified into different types and subtypes based on their surface antigens hemagglutinin (HA) and neuraminidase (NA). Currently, the main methods for identifying influenza virus subtypes in clinical practice are viral nucleic acid detection and viral isolation and culture. However, these methods are time-consuming and costly. Therefore, the development of precise antigenic epitope identification technology for virus typing is of significant value.

[0003] The high selectivity of peptide dyes for specific biological targets demonstrates unique application value in the field of molecular diagnostics. However, significant challenges remain in designing and developing peptide dyes for rapid typing of influenza A virus. This is because influenza A virus has 18 HA subtypes and 11 NA subtypes, which differ in structure and antigenicity. The existence of numerous subtypes means that the designed peptide probes must possess broad-spectrum targeting capabilities, effectively recognizing multiple HA or NA subtypes, which undoubtedly greatly increases the difficulty of peptide probe design. Therefore, screening and identifying broad-spectrum peptide sequences that can specifically bind to specific antigens of different subtypes has become one of the core challenges in peptide probe design. Summary of the Invention

[0004] In response to the problems raised in the background art, the purpose of this invention is to propose a peptide probe for detecting influenza virus and its preparation method, which can design a broad-spectrum peptide fluorescent probe that can specifically recognize influenza virus dual antigens (hemagglutinin and neuraminidase), thus solving the technical problems of long time consumption, high cost and difficulty in peptide probe design in existing influenza virus subtype identification.

[0005] Another objective of this invention is to propose a sensor array using the aforementioned peptide probes for detecting influenza viruses and its preparation method, thereby constructing a sensor array based on dual antigen activated fluorescence energy resonance transfer (FRET) signals, providing an effective strategy for achieving high-throughput, zero-background, and high-specificity detection of influenza viruses and subtype differentiation.

[0006] Another object of the present invention is to propose the application of the above-mentioned peptide probe sensing array in the preparation of products for high-throughput differentiation of influenza virus subtypes.

[0007] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0008] A peptide probe for detecting influenza virus, comprising polypeptide chains and fluorescent dyes, wherein the polypeptide chains are any one of a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain;

[0009] The amino acid sequence of the first polypeptide chain is shown in SEQ NO:1, and the sequence is: Ala-Arg-Asp-Arg-Gly-Cys-Asn-Ser-Asn-Ile-Cys-Tyr-Val-Val-Gly-Tyr-Tyr-Phe-Tyr-Gly-Met-Asp-Asp (ARDRGCNSNICYVVGYYFYGMDD). The peptide probe obtained from the first polypeptide chain is used for broad-spectrum recognition of influenza A virus hemagglutinin.

[0010] The amino acid sequence of the second polypeptide chain is shown in SEQ NO:2, and is: Cys-Ala-Arg-Val-Arg-Thr-Trp-Gly-Glu-Tyr-Thr-Thr-Arg-Glu-Glu-Pro-Ile-His-Ser-Trp-Tyr-Phe-Asp-Leu (CARVRTWGEYTTREEPIHSWYFDL). The peptide probe obtained from the second polypeptide chain is used for broad-spectrum recognition of influenza A virus neuraminidase.

[0011] The amino acid sequence of the third polypeptide chain is shown in SEQ NO:3, and is: Cys-Ala-Lys-Asp-Phe-Arg-Ser-Ser-Ser-Ser-Met-Asp-Tyr-Phe-Tyr-Tyr-Tyr-Gly-Met-Gly-Val-Trp (CAKDFRSSSSSMDYFYYYGMGVW). The peptide probe obtained from the third polypeptide chain is used for broad-spectrum recognition of influenza B virus hemagglutinin.

[0012] The amino acid sequence of the fourth polypeptide chain is shown in SEQ NO:4, and is: Cys-Ala-Arg-Gly-Asp-Tyr-Ser-Gly-Tyr-Asp-Arg-Asp-Val-Gln-Val-Glu-Leu-Met-Asp-Val-Trp (CARGDYSGYDRDVQVELMDVW). The peptide probe obtained from the fourth polypeptide chain is used for broad-spectrum recognition of influenza B virus neuraminidase.

[0013] To further clarify, the molar ratio of the peptide chain to the fluorescent dye is 1:3.

[0014] Furthermore, the fluorescent dye is selected from at least one of fluorescein, tetramethylrhodamine, anthocyanin 3, anthocyanin 5, anthocyanin 7, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, and Alexa Fluor 680.

[0015] A method for preparing a peptide probe for detecting influenza virus, the method comprising the following steps:

[0016] Step A1: Obtain the crystal structures of the broad-spectrum neutralizing antibody and influenza virus antigen from the PDB protein database, analyze the binding region in the antibody-antigen complex using protein visualization software, identify the key amino acid residues in the antibody binding domain, and extract the key polypeptide chain.

[0017] Step A2: Use AlphaFold3 to predict the structure of peptide-antigen complexes, use PRODIGY to evaluate the Gibbs free energy and dissociation constant of peptide-antigen complexes, and analyze and screen out the polypeptide chains with the strongest affinity for influenza virus antigens.

[0018] Step A3: Synthesize the polypeptide chain obtained in step A2, add fluorescent dye, stir the reaction, purify the reaction solution by reversed-phase high-performance liquid chromatography, and freeze-dry to obtain the peptide probe.

[0019] To further clarify, the broad-spectrum neutralizing antibody is selected from VH3-48 lineage monoclonal antibodies, VK1-12 lineage monoclonal antibodies, CR6261 antibody, CAV-F6 antibody, CAV-F34 antibody, NA-1G05 antibody, NA-2E01 antibody, 1E01 antibody, 1G01 antibody, 1G04 antibody, 1G05 antibody, FNI9 antibody, H1209 antibody, hVHH-69 antibody, and hVHH-75 antibody;

[0020] The VH3-48 lineage monoclonal antibody and the VK1-12 lineage monoclonal antibody were selected from 28-2, 28-4, 28-6 and 28-12, respectively.

[0021] To further explain, in step A3, after adding the fluorescent dye, the mixture is stirred and reacted at room temperature for 24 hours.

[0022] A sensor array of peptide probes, comprising two-dimensional nanosheets and a peptide probe combination, wherein the peptide probe combination is a combination of peptide probes obtained from a first polypeptide chain and peptide probes obtained from a second polypeptide chain;

[0023] Alternatively, the peptide probe combination may be a combination of the peptide probe obtained from the third polypeptide chain and the peptide probe obtained from the fourth polypeptide chain.

[0024] Furthermore, the two-dimensional nanosheets are selected from any one of graphene oxide, graphitic carbon nitride, transition metal disulfides, metal oxide sheets, metal hydroxide sheets, metal-organic framework nanosheets, and covalent organic framework nanosheets.

[0025] A method for preparing a sensor array of a peptide probe, comprising the following steps:

[0026] Step B1: Prepare two-dimensional nanosheets with fluorescence quenching properties;

[0027] Step B2: Using two-dimensional nanosheets as fluorescence quenchers, add them to the peptide probe array for incubation to obtain a peptide probe sensing array.

[0028] Application of the sensor array of the peptide probe in the preparation of high-throughput products that distinguish influenza virus subtypes;

[0029] The influenza viruses are selected from influenza A and influenza B viruses, wherein the influenza A viruses are selected from H2N2, H4N2, H6N1, H6N2, H6N8, H7N1, H7N3, H7N7, H8N4, H10N3, H10N7, H10N8, H11N2, H11N9 and H12N5, and the influenza B viruses are selected from the Victoria lineage and the Yamagata lineage.

[0030] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0031] By using AlphaFold3 to assist in the design of broad-spectrum peptide fluorescent probes that specifically recognize two influenza virus antigens (hemagglutinin and neuraminidase), and constructing a sensing array based on the dual-antigen activation fluorescence energy resonance transfer (FRET) signal: A dual-antigen activation response system was initially constructed using the classic influenza virus antigens (hemagglutinin and neuraminidase); drawing on the fluorescence-activated probe response mechanism, two-dimensional nanosheets with fluorescence quenching properties were prepared. The peptide probes were then combined with the two-dimensional nanosheets through supramolecular self-assembly to construct a peptide probe sensing array. The fluorescence of the peptide probe sensing array transitioned from "dark → bright → FRET" using dual-antigen synergistic activation, improving detection accuracy. This provides an effective strategy for achieving high-throughput, zero-background, and highly specific detection of influenza virus and subtype differentiation.

[0032] The detection using a sensor array of dual-antigen activated peptide probes has the following advantages:

[0033] 1. High specificity: The response strategy of influenza virus dual epitope recognition activating fluorescence energy resonance transfer provides a novel paradigm for specific responses to influenza virus;

[0034] 2. Differentiating different influenza virus subtypes: Principal component analysis (PCA) is used to reduce the dimensionality of fluorescence resonance transfer (FRET) signals generated by different subtypes, effectively obtaining the "characteristic fingerprints" of different influenza virus subtypes. In the embodiments of this invention, the peptide probe sensing array exhibits excellent influenza virus subtype differentiation performance. This sensing array can achieve high-throughput differentiation of different types and subtypes of influenza viruses, and no virus lysis and nucleic acid separation operations are required during the detection process. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the fluorescence energy resonance transfer activated by the dual epitope recognition of the influenza virus.

[0036] Figure 2 This is a schematic diagram of step A1 in Example 3, where Figure a is a schematic diagram of analyzing the binding region in the antibody-antigen complex, Figure b is a schematic diagram of analyzing amino acid residues within a 5 Å radius of the binding interface, Figure c is a schematic diagram of identifying native contact residues on the antibody surface, and Figure d is a schematic diagram of truncation of key binding peptides from contact residues of antibody H1209.

[0037] Figure 3 This is a schematic diagram of step A2 in Example 3, where Figures a, b, and c are schematic diagrams of the complex models of the interaction between the PBH1, PBH2, and PBH3 peptide chains and the hemagglutinin of influenza B virus constructed using AlphaFold3, respectively. Figure d is a diagram for evaluating the Gibbs free energy (ΔG) and dissociation constant (K) of the peptide-antigen complex. d A schematic diagram of ( ).

[0038] Figure 4 This is the mass spectrum of PAH3-FITC from Example 1.

[0039] Figure 5 This is a high-performance liquid chromatogram of PAH3-FITC from Example 1.

[0040] Figure 6 This is the mass spectrum of PAN2-TMR from Example 2.

[0041] Figure 7 This is the high-performance liquid chromatogram of PAN2-TMR in Example 2.

[0042] Figure 8 This is the mass spectrum of PBH2-Cy3 from Example 3.

[0043] Figure 9 This is the high-performance liquid chromatogram of PBH2-Cy3 in Example 3.

[0044] Figure 10This is the mass spectrum of PBN3-Cy5 from Example 4.

[0045] Figure 11 This is the high-performance liquid chromatogram of PBN3-Cy5 from Example 4.

[0046] Figure 12 Figure 5 shows the spectral test results of the peptide probes. Figure A shows the fluorescence emission of PAH3-FITC, the UV absorption and fluorescence emission of PAN2-TMR, and Figure B shows the fluorescence emission of PBH2-Cy3, the UV absorption and fluorescence emission of PBN3-Cy5.

[0047] Figure 13 These are high-resolution transmission electron microscope (TEM) images of the four types of two-dimensional nanosheets prepared in step B1 of Example 6. Figures (A), (B), (C), and (D) are high-resolution TEM images of Cu-TCPP nanosheets, Zr-TCPP nanosheets, GO nanosheets, and MoS2 nanosheets, respectively.

[0048] Figure 14 This is a schematic diagram of step B2 of Example 6, which uses an orthogonal arrangement and combination strategy to construct the sensor array.

[0049] Figure 15 This is a schematic diagram of step B2 of embodiment 7, which uses an orthogonal arrangement and combination strategy to construct the sensor array.

[0050] Figure 16 The figures show fluorescence quenching test results of the sensor array of peptide probes. Figure (A1) is the fluorescence titration curve of PAH3-FITC and Cu-TCPP nanosheets, and Figure (A2) is a line graph converted from the fluorescence titration curve of Figure (A1). Figure (B1) is the fluorescence titration curve of PAN2-TMR and Cu-TCPP nanosheets, and Figure (B2) is a line graph converted from the fluorescence titration curve of Figure (B1). Figure (C1) is the fluorescence titration curve of PBH2-Cy3 and Cu-TCPP nanosheets, and Figure (C2) is a line graph converted from the fluorescence titration curve of Figure (C1). Figure (D1) is the fluorescence titration curve of PBN3-Cy5 and Cu-TCPP nanosheets, and Figure (D2) is a line graph converted from the fluorescence titration curve of Figure (D1).

[0051] Figure 17 This is a test diagram of the classification of influenza A virus using a sensor array (P1-P4) of peptide probes in Example 8. Figure A is a test diagram of the FRTE signal changes of P1-P4 to distinguish influenza A virus from other viruses. Figures B and C are two-dimensional scatter plots of the fluorescence values ​​of Figure A converted by principal component analysis. Figure D is a schematic diagram of the FRET response of the dual antigen activated peptide probe of influenza A virus.

[0052] Figure 18 This is a test diagram of the classification of influenza B virus using a sensor array (P5-P8) of peptide probes in Example 9. Figure A is a test diagram of the FRTE signal changes of P5-P8 to distinguish influenza B virus from other viruses. Figures B and C are two-dimensional scatter plots of the fluorescence values ​​of Figure A converted by principal component analysis. Figure D is a schematic diagram of the FRET response of the influenza B virus dual antigen activated peptide probe. Detailed Implementation

[0053] A peptide probe for detecting influenza virus, comprising polypeptide chains and fluorescent dyes, wherein the polypeptide chains are any one of a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain;

[0054] The amino acid sequence of the first polypeptide chain is shown in SEQ NO:1, and the sequence is: Ala-Arg-Asp-Arg-Gly-Cys-Asn-Ser-Asn-Ile-Cys-Tyr-Val-Val-Gly-Tyr-Tyr-Phe-Tyr-Gly-Met-Asp-Asp (ARDRGCNSNICYVVGYYFYGMDD). The peptide probe obtained from the first polypeptide chain is used for broad-spectrum recognition of influenza A virus hemagglutinin.

[0055] The amino acid sequence of the second polypeptide chain is shown in SEQ NO:2, and is: Cys-Ala-Arg-Val-Arg-Thr-Trp-Gly-Glu-Tyr-Thr-Thr-Arg-Glu-Glu-Pro-Ile-His-Ser-Trp-Tyr-Phe-Asp-Leu (CARVRTWGEYTTREEPIHSWYFDL). The peptide probe obtained from the second polypeptide chain is used for broad-spectrum recognition of influenza A virus neuraminidase.

[0056] The amino acid sequence of the third polypeptide chain is shown in SEQ NO:3, and is: Cys-Ala-Lys-Asp-Phe-Arg-Ser-Ser-Ser-Ser-Met-Asp-Tyr-Phe-Tyr-Tyr-Tyr-Gly-Met-Gly-Val-Trp (CAKDFRSSSSSMDYFYYYGMGVW). The peptide probe obtained from the third polypeptide chain is used for broad-spectrum recognition of influenza B virus hemagglutinin.

[0057] The amino acid sequence of the fourth polypeptide chain is shown in SEQ NO:4, and is: Cys-Ala-Arg-Gly-Asp-Tyr-Ser-Gly-Tyr-Asp-Arg-Asp-Val-Gln-Val-Glu-Leu-Met-Asp-Val-Trp (CARGDYSGYDRDVQVELMDVW). The peptide probe obtained from the fourth polypeptide chain is used for broad-spectrum recognition of influenza B virus neuraminidase.

[0058] The peptide probe for detecting influenza virus of the present invention is a broad-spectrum peptide fluorescent probe that can specifically recognize the two antigens of influenza virus (hemagglutinin and neuraminidase), providing a highly specific peptide probe for distinguishing influenza virus subtypes and solving the technical problems of long time and high cost in the existing identification of influenza virus subtypes.

[0059] To further clarify, the molar ratio of the peptide chain to the fluorescent dye is 1:3.

[0060] Furthermore, the fluorescent dye is selected from at least one of fluorescein, tetramethylrhodamine, anthocyanin 3 (Cy3), anthocyanin 5 (Cy5), anthocyanin 7 (Cy7), Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, and Alexa Fluor 680.

[0061] A method for preparing a peptide probe for detecting influenza virus, the method comprising the following steps:

[0062] Step A1: Obtain the crystal structures of the broad-spectrum neutralizing antibody and influenza virus antigen from the PDB protein database, analyze the binding region in the antibody-antigen complex using protein visualization software, determine the key amino acid residues in the antibody binding domain, and extract the key polypeptide chain to simulate the antibody recognition epitope.

[0063] Step A2: Use AlphaFold3 to predict the structure of the peptide-antigen complex, and use the PRODIGY web server to evaluate the Gibbs free energy (ΔG) and dissociation constant (K) of the peptide-antigen complex. d ), and analyzed and screened out the polypeptide chains with the strongest affinity for influenza virus antigens;

[0064] Step A3: The polypeptide chain screened in step A2 is synthesized by solid-phase method, fluorescent dye is added, the reaction is stirred, the reaction solution is purified by reversed-phase high-performance liquid chromatography (C18 column), and the peptide probe is obtained by freeze drying.

[0065] This invention utilizes AlphaFold3 to assist in the design and synthesis of broad-spectrum peptide probes. AlphaFold3 can predict almost all types of biological complexes with extremely high accuracy and accurately predict the interfacial residue interaction network between peptides and target proteins, which can be used to guide the design of high-affinity peptides. This invention provides a universal targeted peptide design strategy, using AlphaFold3 to assist in the design of peptide sequences to achieve specific recognition of target proteins. By using AlphaFold3 to assist in the design of broad-spectrum peptide fluorescent probes that specifically recognize influenza virus dual antigens (hemagglutinin and neuraminidase), the technical problem of difficult peptide probe design in existing systems is solved, and a sensing array based on dual-antigen activated fluorescence energy resonance transfer (FRET) signals (such as...) is constructed. Figure 1 As shown in the figure, this provides an effective strategy for achieving high-throughput, zero-background, and high-specificity detection of influenza viruses and subtype differentiation.

[0066] Furthermore, the more negative the value of the Gibbs free energy (ΔG), the easier the binding reaction occurs, the stronger the binding affinity, and the higher the dissociation constant (K). d The smaller the value, the stronger the binding affinity.

[0067] To further clarify, the broad-spectrum neutralizing antibody is selected from VH3-48 lineage monoclonal antibodies, VK1-12 lineage monoclonal antibodies, CR6261 antibody, CAV-F6 antibody, CAV-F34 antibody, NA-1G05 antibody, NA-2E01 antibody, 1E01 antibody, 1G01 antibody, 1G04 antibody, 1G05 antibody, FNI9 antibody, H1209 antibody, hVHH-69 antibody, and hVHH-75 antibody;

[0068] The VH3-48 lineage monoclonal antibody and the VK1-12 lineage monoclonal antibody were selected from 28-2, 28-4, 28-6 and 28-12, respectively.

[0069] It should be noted that antibodies 28-2, 28-4, 28-6, and 28-12 together constitute a rare antibody lineage. Their heavy chains use the VH3-48 / DH2-2 / JH6 germline gene fragment, and their light chains use the VK1-12 / JK5 germline gene fragment. Therefore, they belong to both the VH3-48 and VK1-12 lineages.

[0070] To further explain, in step A3, after adding the fluorescent dye, the mixture is stirred and reacted at room temperature for 24 hours.

[0071] A sensor array of peptide probes, comprising two-dimensional nanosheets and a peptide probe combination, wherein the peptide probe combination is a combination of peptide probes obtained from a first polypeptide chain and peptide probes obtained from a second polypeptide chain;

[0072] Alternatively, the peptide probe combination may be a combination of the peptide probe obtained from the third polypeptide chain and the peptide probe obtained from the fourth polypeptide chain.

[0073] The sensor array of the peptide probe is a "dual antigen activation" type peptide probe sensor array.

[0074] Dye molecules that "recognize single epitopes" are not very effective at recognizing influenza viruses that are prone to antigenic drift. This invention uses peptide probes designed with the assistance of AlphaFold3 to specifically recognize the dual antigens (hemagglutinin and neuraminidase) of the influenza virus. The sensor array obtained by combining peptide probes can more effectively identify and distinguish influenza virus subtypes.

[0075] This invention preliminarily constructs a dual-antigen activation response system using classic influenza virus antigens (hemagglutinin and neuraminidase). Drawing on the fluorescence-activated probe response mechanism, two-dimensional nanosheets with fluorescence quenching properties are prepared. Peptide probes can be composited with these two-dimensional nanosheets through supramolecular self-assembly to construct a peptide probe sensing array. The synergistic activation of the dual antigens enables the fluorescence of the peptide probe sensing array to transition from "dark → bright → FRET," thereby improving detection accuracy.

[0076] The detection using a sensor array of dual-antigen activated peptide probes has the following advantages:

[0077] 1. High specificity: The response strategy of influenza virus dual epitope recognition activating fluorescence energy resonance transfer provides a novel paradigm for specific responses to influenza virus;

[0078] 2. Differentiating different influenza virus subtypes: Principal component analysis (PCA) is used to reduce the dimensionality of fluorescence resonance transfer (FRET) signals generated by different subtypes, effectively obtaining the "characteristic fingerprints" of different influenza virus subtypes. In the embodiments of this invention, the peptide probe sensing array exhibits excellent influenza virus subtype differentiation performance. This sensing array can achieve high-throughput differentiation of different types and subtypes of influenza viruses, and no virus lysis and nucleic acid separation operations are required during the detection process.

[0079] Furthermore, the two-dimensional nanosheets are selected from any one of graphene oxide, graphitic carbon nitride, transition metal disulfides, metal oxide sheets, metal hydroxide sheets, metal-organic framework nanosheets, and covalent organic framework nanosheets.

[0080] A method for preparing a sensor array of a peptide probe, comprising the following steps:

[0081] Step B1: Prepare two-dimensional nanosheets with fluorescence quenching properties;

[0082] Step B2: Using two-dimensional nanosheets as fluorescence quenchers, add them to the peptide probe array for incubation to obtain a peptide probe sensing array.

[0083] Specifically, in step B1, two-dimensional nanosheets with fluorescence quenching properties can be prepared by ultrasonic-assisted liquid phase exfoliation or solvothermal synthesis.

[0084] Preferably, in step B2, the incubation is carried out at 37°C for 10 minutes.

[0085] Furthermore, the optimal ratio of two-dimensional nanosheets to peptide probes can be screened by systematically evaluating fluorescence quenching efficiency (the concentration of two-dimensional nanosheets is selected based on the degree of fluorescence quenching). Sensing arrays can then be constructed by arranging and combining various types of two-dimensional nanosheets and peptide probes.

[0086] Application of the sensor array of the peptide probe in the preparation of high-throughput products that distinguish influenza virus subtypes;

[0087] The influenza viruses are selected from influenza A and influenza B viruses, wherein the influenza A viruses are selected from H2N2, H4N2, H6N1, H6N2, H6N8, H7N1, H7N3, H7N7, H8N4, H10N3, H10N7, H10N8, H11N2, H11N9 and H12N5, and the influenza B viruses are selected from the Victoria lineage and the Yamagata lineage.

[0088] The peptide probe sensing array can be used in high-throughput products for differentiating influenza virus subtypes, such as detection kits and influenza virus immunoassay chips. Because the peptide probes specifically target and bind to the characteristic antigens of the influenza virus, the two peptide probes in the sensor array's peptide probe combination target and bind to influenza virus hemagglutinin and neuraminidase, respectively, leading to fluorescence energy resonance transfer. Therefore, the "dual-antigen activation" peptide probe sensing array of this invention is suitable for high-throughput differentiation of influenza virus subtypes.

[0089] The sensor array of the "dual antigen activation" peptide probe of this invention is mainly targeted at influenza A viruses (including H2N2, H4N2, H6N1, H6N2, H6N8, H7N1, H7N3, H7N7, H8N4, H10N3, H10N7, H10N8, H11N2, H11N9, H12N5) and influenza B viruses (Victoria lineage and Yamagata lineage).

[0090] To facilitate understanding of the present invention, a more complete description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0091] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0092] Example 1

[0093] A method for preparing a peptide probe for detecting influenza virus, using AlphaFold3-assisted design of a peptide probe that broadly recognizes hemagglutinin of influenza A virus, includes the following steps:

[0094] Step A1: Obtain the crystal structures of the broad-spectrum neutralizing antibody and influenza virus antigen from the PDB protein database. The obtained crystal structure is that of neutralizing antibody 28-12 (which targets hemagglutinin H1 from A / Washington / 05 / 2011, PDB code: 7X6O). The binding region in the antibody-antigen complex was analyzed using protein visualization software (Pymol visualization software). Several key residues that interact with influenza A virus hemagglutinin were identified in antibody 28-12, and the key binding peptide in antibody 28-12 was extracted.

[0095] The three candidate key binding peptides are:

[0096] PAH1, whose amino acid sequence is shown in SEQ NO:5, is as follows:

[0097] Ala-Arg-Asp-Arg-Gly-Cys-Ser-Ser-Thr-Asn-Cys-Tyr-Val-Val-Gly-Tyr-Tyr-Phe-Tyr-Gly-Met-Asp-Val (ARDRGCSSTNCYVVGYYFYGMDV);

[0098] PAH2, whose amino acid sequence is shown in SEQ NO:6, is as follows:

[0099] Glu-Trp-Leu-Ser-Tyr-Ile-Ser-Thr-Ser-Ser-Asn-Thr-Ile-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Ile (EWLSYISTSSNTIYYADSVKGRFTI);

[0100] PAH3, whose amino acid sequence is shown in SEQ NO:1, is as follows:

[0101] Ala-Arg-Asp-Arg-Gly-Cys-Asn-Ser-Asn-Ile-Cys-Tyr-Val-Val-Gly-Tyr-Tyr-Phe-Tyr-Gly-Met-Asp-Asp (ARDRGCNSNICYVVGYYFYGMDD);

[0102] Step A2: Use AlphaFold3 to predict the structure of the peptide-antigen complex, construct complex models of the interaction between three candidate key binding peptides and influenza A virus hemagglutinin, and use the PRODIGY web server to evaluate the Gibbs free energy (ΔG) and dissociation constant (K) of the peptide-antigen complex. d The peptide chain PAH3 with the strongest affinity for binding to the hemagglutinin of influenza A virus was identified through PRODIGY analysis and screening.

[0103] Gibbs free energy (ΔG) and dissociation constant (K) of three candidate key peptides d As shown in the table below:

[0104] Table 1. Gibbs free energy (ΔG) and dissociation constant (K) of the three candidate key peptides in Example 1. d )

[0105]

[0106] Step A3: The polypeptide chain PAH3 obtained in step A2 was synthesized using a solid-phase method. The specific operation steps are as follows: Using Wang resin with a loading of 0.6 mmol / g as the solid-phase support, the target polypeptide PAH3 was synthesized at a scale of 0.1 mmol. 25 amino acids were sequentially linked from the C-terminus to the N-terminus. Each cycle included: two deprotection reactions using 20% ​​piperidine / DMF solution (3 min + 7 min), followed by coupling reaction (30-45 min / cycle) using 0.1 mmol as the reference, activated in DMF with 4 equivalents of Fmoc-amino acids, 3.9 equivalents of HATU (peptide condensation reagent), 0.4 equivalents of HOAt, and 8 equivalents of DIEA (N,N-diisopropylethylamine). The Fmoc protecting group was then removed twice (3 min and 10 min) with 20% piperidine / DMF solution. The cysteine ​​(C) side chain in the sequence was protected with Trt (triphenylmethyl). After all amino acid coupling was completed, the resin was dried, and the peptide was cleaved from the resin and all side-chain protecting groups were removed simultaneously using a pre-prepared cleavage reagent (TFA / water / TIS / EDT = 94 / 2.5 / 2.5 / 2.5, v / v) at room temperature for 3 hours. The crude PAH3 product was precipitated with cold diethyl ether, washed, and dried. The PAH3 peptide chain was dissolved in dimethylformamide, and fluorescein isothiocyanate (FITC) and an appropriate amount of diisopropylethylamine were added (to adjust the pH to 8-9). The molar ratio of PAH3 to FITC was 1:3, and the reaction was stirred at room temperature for 24 hours. The reaction solution was purified by reversed-phase high-performance liquid chromatography (C18 column) to remove unreacted FITC and byproducts, and then freeze-dried to obtain the peptide probe (abbreviated as PAH3-FITC).

[0107] The structural formula of the peptide probe PAH3-FITC is as follows:

[0108] .

[0109] Characterization of the peptide probe PAH3-FITC: The structure and purity of the peptide probe were determined by mass spectrometry and high-performance liquid chromatography, respectively. Figure 4 and 5 As shown. The molecular weight C of PAH3-FITC 137 H 177 N 32 O 42 S4 3+ [M+3H] 3+ The value is 1062.5, measured by time-of-flight mass spectrometry [M+3H]. 3+ The value was 1062.4. High performance liquid chromatography conditions: flow rate 1 mL / min. -1The mobile phase was 45% solvent A (acetonitrile solution of 0.1% trifluoroacetic acid) / 55% solvent B (aqueous solution of 0.1% trifluoroacetic acid), tR = 13.356 min, 30 min, purity 96.48%.

[0110] Example 2

[0111] A method for preparing a peptide probe for detecting influenza virus, using AlphaFold3-assisted design to create a peptide probe that broadly recognizes the neuraminidase of influenza A virus, includes the following steps:

[0112] Step A1: Obtain the crystal structures of the broad-spectrum neutralizing antibody and influenza virus antigen from the PDB protein database. Among them, the obtained crystal structure is that of antibody 1G04 (which targets neuraminidase N9 from A / Hunan / 02650 / 2016, PDB code: 6Q1Z). The binding region in the antibody-antigen complex is analyzed using protein visualization software (Pymol visualization software). Several key residues that interact with influenza A virus neuraminidase are identified in antibody 1G04. The key binding peptide in antibody 1G04 is extracted.

[0113] The three candidate key binding peptides are:

[0114] PAN1, whose amino acid sequence is shown in SEQ NO:7, has the following sequence:

[0115] Ile-Ser-Ser-Phe-Leu-Ala-Trp-Tyr-Gln-Gln-Lys-Pro-Gly-Asn-Ala-Pro-Lys-Val-Leu-Ile-Tyr-Ala-Ala-Ser (ISSFLAWYQQKPGNAPKVLIYAAS);

[0116] PAN2, whose amino acid sequence is shown in SEQ NO:2, has the following sequence:

[0117] Cys-Ala-Arg-Val-Arg-Thr-Trp-Gly-Glu-Tyr-Thr-Thr-Arg-Glu-Glu-Pro-Ile-His-Ser-Trp-Tyr-Phe-Asp-Leu (CARVRTWGEYTTREEPIHSWYFDL);

[0118] PAN3, its amino acid sequence is shown in SEQ NO:8, and the sequence is as follows:

[0119] Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Phe-Val-Ser-Gly-Leu-Asn-Trp-Asn-Gly-Asp-Ile-Thr-Ala-Phe-Thr (RQAPGKGLEFVSGLNWNGDITAFT);

[0120] Step A2: Use AlphaFold3 to predict the structure of the peptide-antigen complex, construct complex models of the interaction between three candidate key binding peptides and influenza A virus neuraminidase, and use the PRODIGY web server to evaluate the Gibbs free energy (ΔG) and dissociation constant (K) of the peptide-antigen complex. d ), through PRODIGY analysis and screening, the peptide chain PAN2 with the strongest affinity for binding to influenza A taurine enzyme was identified;

[0121] Gibbs free energy (ΔG) and dissociation constant (K) of three candidate key peptides d As shown in the table below:

[0122] Table 2. Gibbs free energy (ΔG) and dissociation constant (K) of the three candidate key peptides in Example 2. d )

[0123]

[0124] Step A3: The polypeptide chain PAN2 obtained in step A2 was synthesized using a solid-phase method. The specific operation steps are as follows: Using Wang resin with a loading of 0.6 mmol / g as a solid-phase support, the target polypeptide PAN2 was synthesized at a scale of 0.1 mmol. 24 amino acids were sequentially linked from the C-terminus to the N-terminus. Each cycle included: two deprotection treatments using 20% ​​piperidine / DMF solution (3 min + 7 min), followed by activation in DMF with 4 equivalents of Fmoc-amino acid, 3.9 equivalents of HATU (peptide condensation reagent), 0.4 equivalents of HOAt, and 8 equivalents of DIEA (N,N-diisopropylethylamine) as the reference, followed by coupling reaction (30-45 min / cycle). For difficult sequence sites (such as consecutive Ser / Tyr), a double coupling strategy was adopted (the coupling reaction of the same amino acid was performed twice consecutively for difficult sequence sites) to ensure efficiency. All amino acid side chains were protected with standard acid-sensitive groups (e.g., acetaminomethyl Acm was used to protect the thiol group on the cysteine ​​(Cys) side chain, and tert-butyloxycarbonyl Boc was used to protect the nitrogen atom on the indole ring of the tryptophan (Trp) side chain). After all amino acids were coupled, the resin was dried, and the peptide was cleaved from the resin and all side chain protecting groups were removed simultaneously using a pre-prepared cleavage reagent (TFA / water / TIS / EDT = 94 / 2.5 / 2.5 / 2.5, v / v) at room temperature for 3 hours. The crude PAN2 product was precipitated with cold diethyl ether, washed, and dried. The peptide chain PAN2 was dissolved in dimethylformamide, and tetramethylrhodamine (TMR) and an appropriate amount of diisopropylethylamine were added (to adjust the pH to 8-9). The molar ratio of PAN2 to TMR was 1:3, and the reaction was stirred at room temperature for 24 hours. The reaction solution was purified by reversed-phase high-performance liquid chromatography (C18 column) to remove unreacted TMR and byproducts, and then freeze-dried to obtain the peptide probe (abbreviated as PAN2-TMR).

[0125] The structural formula of the peptide probe PAN2-TMR is as follows:

[0126] .

[0127] Characterization of the peptide probe PAN2-TMR: The structure and purity of the peptide probe were determined by mass spectrometry and high-performance liquid chromatography, respectively. Figure 6 and 7 As shown. The molecular weight C of PAN2-TMR 168 H 224 N 42 O 44 S + [M+5H] 5+ The value was 714.2, measured by time-of-flight mass spectrometry [M+5H]. 5+ The value was 714.2. High performance liquid chromatography conditions: flow rate 1 mL / min.-1 The mobile phase consisted of 43% solvent A (acetonitrile solution of 0.1% trifluoroacetic acid) / 57% solvent B (aqueous solution of 0.1% trifluoroacetic acid), with a tR of 12.739 min for 30 min and a purity of 95.37%.

[0128] Example 3

[0129] A method for preparing a peptide probe for detecting influenza virus, using AlphaFold3-assisted design of a peptide probe that broadly recognizes hemagglutinin of influenza B virus, includes the following steps:

[0130] Step A1: Obtain the crystal structures of the broad-spectrum neutralizing antibody and influenza virus antigen from the PDB protein database. The obtained structure is that of neutralizing antibody H1209 (which targets the hemagglutinin crystal structure derived from B / Hong Kong / 8 / 1973, PDB code: 7KQI). Figure 2 As shown, the binding region in the antibody-antigen complex was analyzed using protein visualization software (Pymol visualization software). Several key residues interacting with the hemagglutinin of influenza B virus were identified in antibody H1209. Key binding peptides in antibody H1209 were truncated. Specifically, by analyzing amino acid residues within a 5 Å radius of the binding interface, the native contact residues on the antibody surface were identified, and then the key binding peptides in the recognition domain of antibody H1209 were truncated based on these contact residues.

[0131] The three candidate key binding peptides are:

[0132] PBH1, whose amino acid sequence is shown in SEQ NO:9, has the following sequence:

[0133] Gly-Leu-Glu-Trp-Ile-Ser-Ser-Ile-Asn-Trp-Lys-Gly-Asn-Ala-Leu-Asp-Tyr-Ala-Gly-Ser-Val-Arg-Gly (GLEWISSINWKGNALDYAGSVRG);

[0134] PBH2, whose amino acid sequence is shown in SEQ NO:3, is as follows:

[0135] Cys-Ala-Lys-Asp-Phe-Arg-Ser-Ser-Ser-Ser-Met-Asp-Tyr-Phe-Tyr-Tyr-Tyr-Gly-Met-Gly-Val-Trp (CAKDFRSSSSMDYFYYYGMGVW);

[0136] PBH3, whose amino acid sequence is shown in SEQ NO:10, has the following sequence:

[0137] Cys-Gln-Ala-Ser-Gln-Asp-Ile-Arg-Gln-Cys-Leu-Asn-Trp-Tyr-Gln-Gln-Lys-Pro-Gly-Lys-Pro-Pro-Lys (CQASQDIRQCLNWYQQKPGKPPK);

[0138] Step A2, as follows Figure 3 As shown, AlphaFold3 was used to predict the structure of peptide-antigen complexes, and complex models of the interaction between three candidate key binding peptides and influenza B virus hemagglutinin were constructed. The Gibbs free energy (ΔG) and dissociation constant (K) of the peptide-antigen complexes were evaluated using the PRODIGY web server. d ), and through PRODIGY analysis and screening, the peptide chain PBH2 with the strongest affinity for binding to the hemagglutinin of influenza B virus was identified;

[0139] Gibbs free energy (ΔG) and dissociation constant (K) of three candidate key peptides d As shown in the table below:

[0140] Table 3. Gibbs free energy (ΔG) and dissociation constant (K) of the three candidate key peptides in Example 3. d )

[0141]

[0142] Step A3: The polypeptide chain PBH2 obtained in step A2 was synthesized using a solid-phase method. The specific operation steps are as follows: Wang resin with a loading of 0.6 mmol / g was used as the solid-phase support, and the target polypeptide PBH2 was synthesized at a scale of 0.1 mmol. 22 amino acids were sequentially linked from the C-terminus to the N-terminus. Each cycle included: two deprotection treatments using 20% ​​piperidine / DMF solution (3 min + 7 min), followed by coupling reaction (30-45 min / cycle) after activation in DMF with 0.1 mmol as the reference, using 4 equivalents of Fmoc-amino acid, 3.9 equivalents of HATU (peptide condensation reagent), 0.4 equivalents of HOAt, and 8 equivalents of DIEA (N,N-diisopropylethylamine). For difficult sequence sites (such as consecutive Ser / Tyr sequences), a double coupling strategy was adopted (the coupling reaction of the same amino acid was performed twice consecutively for difficult sequence sites) to ensure efficiency. All amino acid side chains were protected with standard acid-sensitive groups (e.g., acetaminomethyl Acm was used to protect the thiol group on the cysteine ​​(Cys) side chain, and tert-butyloxycarbonyl Boc was used to protect the nitrogen atom on the indole ring of the tryptophan (Trp) side chain). After all amino acids were coupled, the resin was dried, and the peptide was cleaved from the resin and all side chain protecting groups were removed simultaneously using a pre-prepared cleavage reagent (TFA / water / TIS / EDT = 94 / 2.5 / 2.5 / 2.5, v / v) at room temperature for 3 hours. The crude PBH2 product was precipitated with cold diethyl ether, washed, and dried. The peptide chain PBH2 was dissolved in dimethylformamide, anthocyanin 3 (Cy3) and an appropriate amount of diisopropylethylamine were added (to adjust the pH to 8-9), the molar ratio of PBH2 to Cy3 was 1:3, and the reaction was stirred at room temperature for 24 hours. The reaction solution was purified by reversed-phase high-performance liquid chromatography (C18 column) to remove unreacted Cy3 and byproducts, and then freeze-dried to obtain the peptide probe (abbreviated as PBH2-Cy3).

[0143] The structural formula of the peptide probe PBH2-Cy3 is as follows:

[0144] .

[0145] Characterization of the peptide probe PBH2-Cy3: The structure and purity of the peptide probe were determined using mass spectrometry and high-performance liquid chromatography, respectively. Figure 8 and 9 As shown. The molecular weight C of PBH2-Cy3. 153 H 201 N 29 O 42 S5 + [M+2H] 2+ The value was 1636.3, measured by time-of-flight mass spectrometry [M+2H]. 2+ The value was 1636.6. High performance liquid chromatography conditions: flow rate 1 mL / min.-1 The mobile phase consisted of 43% solvent A (acetonitrile solution of 0.1% trifluoroacetic acid) / 57% solvent B (aqueous solution of 0.1% trifluoroacetic acid), with a tR of 12.657 min for 30 min and a purity of 95.71%.

[0146] Example 4

[0147] A method for preparing a peptide probe for detecting influenza virus, using AlphaFold3-assisted design to create a peptide probe that broadly recognizes the neuraminidase of influenza B virus, includes the following steps:

[0148] Step A1: Obtain the crystal structures of the broad-spectrum neutralizing antibody and influenza virus antigen from the PDB protein database. The obtained crystal structure is that of neutralizing antibody 1G05 (which targets the neuraminidase crystal structure derived from B / Phuket / 3073 / 2013, PDB code: 6V4N). The binding region in the antibody-antigen complex is analyzed using protein visualization software (Pymol visualization software). Several key residues that interact with influenza B virus neuraminidase are identified in antibody 1G05. The key binding peptide in antibody 1G05 is extracted.

[0149] The three candidate key binding peptides are:

[0150] PBN1, whose amino acid sequence is shown in SEQ NO:11, has the following sequence:

[0151] Ser-Leu-Thr-Cys-Thr-Val-Ser-Gly-Asp-Ser-Ile-Gly-Gly-Ser-Tyr-Trp-Asn-Trp-Ile-Arg-Gln-Pro-Pro (SLTCTVSGDSIGGSYWNWIRQPP);

[0152] PBN2, its amino acid sequence is shown in SEQ NO:12, the sequence is as follows:

[0153] Gln-Trp-Ile-Gly-Tyr-Ile-Tyr-Tyr-Thr-Gly-Ile-Thr-Asn-Tyr-Asn-Pro-Ser-Leu-Lys-Ser-Arg-Val-Thr-Met-Ser (QWIGYIYYTGITNYNPSLKSRVTMS);

[0154] PBN3, its amino acid sequence is shown in SEQ NO:4, the sequence is as follows:

[0155] Cys-Ala-Arg-Gly-Asp-Tyr-Ser-Gly-Tyr-Asp-Arg-Asp-Val-Gln-Val-Glu-Leu-Met-Asp-Val-Trp (CARGDYSGYDRDVQVELMDVW);

[0156] Step A2: Use AlphaFold3 to predict the structure of the peptide-antigen complex, construct complex models of the interaction between three candidate key binding peptides and influenza B virus neuraminidase, and use the PRODIGY web server to evaluate the Gibbs free energy (ΔG) and dissociation constant (K) of the peptide-antigen complex. d ), through PRODIGY analysis and screening, the peptide chain PBN3 with the strongest affinity for binding to influenza B virus neuraminidase was identified;

[0157] Gibbs free energy (ΔG) and dissociation constant (K) of three candidate key peptides d As shown in the table below:

[0158] Table 4. Gibbs free energy (ΔG) and dissociation constant (K) of the three candidate key peptides in Example 4. d )

[0159]

[0160] Step A3: The polypeptide chain PBN3 obtained in step A2 was synthesized using a solid-phase method. The specific operation steps are as follows: Wang resin with a loading of 0.6 mmol / g was used as the solid-phase support, and the target polypeptide PBN3 was synthesized at a scale of 0.1 mmol. 21 amino acids were sequentially linked from the C-terminus to the N-terminus. Each cycle included: two deprotection treatments (3 min + 7 min) using 20% ​​piperidine / DMF solution, followed by coupling reaction (30-45 min / cycle) after activation in DMF using 0.1 mmol as the reference, with 4 equivalents of Fmoc-amino acid, 3.9 equivalents of HATU (peptide condensation reagent), 0.4 equivalents of HOAt, and 8 equivalents of DIEA (N,N-diisopropylethylamine). Subsequently, the Fmoc protecting group was removed twice (3 min and 10 min) with 20% piperidine / DMF solution. After all amino acid coupling was completed, the resin was dried, and the peptide was cleaved from the resin and all side-chain protecting groups were removed simultaneously using a pre-prepared cleavage reagent (TFA / water / TIS / EDT = 94 / 2.5 / 2.5 / 2.5, v / v) at room temperature for 3 hours. The crude PBN3 product was precipitated with cold diethyl ether, washed, and dried. The PBN3 peptide chain was dissolved in dimethylformamide, and anthocyanin 5 (Cy5) and an appropriate amount of diisopropylethylamine were added (to adjust the pH to 8-9). The molar ratio of PBN3 to Cy5 was 1:3, and the reaction was stirred at room temperature for 24 hours. The reaction solution was purified by reversed-phase high-performance liquid chromatography (C18 column) to remove unreacted Cy5 and byproducts, and then freeze-dried to obtain the peptide probe (abbreviated as PBN3-Cy5).

[0161] The structural formula of the peptide probe PBN3-Cy5 is as follows:

[0162] .

[0163] Characterization of the peptide probe PBN3-Cy5: The structure and purity of the peptide probe were determined by mass spectrometry and high-performance liquid chromatography, respectively. Figure 10 and 11 As shown. The molecular weight C of PBN3-Cy5 139 H 196 N 31 O 43 S4 + [M+2H] 2+ The value was 1556.2, measured by time-of-flight mass spectrometry [M+2H]. 2+ The value was 1556.9. High-performance liquid chromatography (HPLC) conditions: flow rate 1 mL / min. -1 The mobile phase was 32% solvent A (acetonitrile solution of 0.1% trifluoroacetic acid) / 68% solvent B (aqueous solution of 0.1% trifluoroacetic acid), tR = 12.306 min, 30 min, purity 96.03%.

[0164] Example 5

[0165] Spectroscopic testing of peptide probes:

[0166] A 1 mM stock solution of peptide probes was prepared (using biological-grade dimethyl sulfoxide, DMSO) for the following peptide probe types: PAH3-FITC, PAN2-TMR, PBH2-Cy3, and PBN3-Cy5. The UV-Vis absorption and fluorescence emission spectra of the peptide probes in phosphate-buffered saline (PBS, pH 7.4) were measured using UV-Vis absorption and fluorescence spectroscopy at a concentration of 2 μM and a temperature of 25 °C.

[0167] Figure 12 The UV absorption and fluorescence emission spectra of different peptide probes (PAH3-FITC, PAN2-TMR, PBH2-Cy3, and PBN3-Cy5) in Examples 1-4 are shown. The spectroscopic results indicate that fluorescence energy resonance transfer (FERT) occurs between PAH3-FITC and PAN2-TMR, and also between PBH2-Cy3 and PBN3-Cy5.

[0168] Example 6

[0169] A method for preparing a peptide probe sensing array, comprising a peptide probe sensing array responsive to dual antigen activation of influenza A virus, including peptide probes targeting dual antigens and two-dimensional nanosheets, including the following steps:

[0170] Step B1: Prepare two-dimensional nanosheets with fluorescence quenching properties, including Cu-TCPP nanosheets (Cu-MOF nanosheets), Zr-TCPP nanosheets (Zr-MOF nanosheets), graphene nanosheets (GO nanosheets), and molybdenum disulfide nanosheets (MoS2 nanosheets). The preparation methods are as follows:

[0171] Preparation of Cu-TCPP nanosheets (Cu-MOF nanosheets): Cu(NO3)2·3H2O (3.0 mg, 0.01 mmol), pyridine (0.8 mg, 0.01 mmol), and polyvinylpyrrolidone (20.0 mg) were dissolved in 12 mL of a mixed solvent of dimethylformamide and ethanol (V:V = 3:1). Subsequently, under vigorous stirring, 4 mL of a mixed solvent of dimethylformamide and ethanol containing tetrakis(4-carboxyphenyl)porphyrin (TCPP, 0.005 mmol) (V:V = 3:1) was added dropwise. The reaction solution was sonicated for 10 minutes, heated to 80 °C and maintained for 24 hours. After the mixture cooled to room temperature, it was centrifuged at 8000 rpm for 10 minutes and washed three times with ethanol. Finally, Cu-TCPP was redispersed in PBS solvent and stored at 4 °C for later use.

[0172] Preparation of Zr-TCPP nanosheets (Zr-MOF nanosheets): 20 mg of ZrCl4 (0.08 mmol), 100 μL of water and 480 μL of acetic acid were added to 4 mL of dimethylformamide; then, 18 mg of TCPP (0.02 mmol) was added to the above solution and the mixture was stirred for 10 minutes; the resulting mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 120 °C for 24 hours; after the mixture cooled to room temperature, it was centrifuged at 8000 rpm for 10 minutes and washed three times with ethanol; finally, Zr-TCPP was redispersed in PBS solvent and stored at 4 °C for later use.

[0173] Preparation of graphene nanosheets (GO): 200 mg of powdered graphite was added to 80 mL of a 1:1 mixture of 100 mL of 1 ...

[0174] Preparation of molybdenum disulfide nanosheets (MoS2): 200 mg of blocky molybdenum disulfide was placed in 100 mL of ethanol / water (1:1) mixture, deoxygenated by nitrogen gas, and then sonicated at 600 W for 8 hours in an ice-water bath; the resulting dispersion was centrifuged at 4000 rpm for 30 min, the supernatant was collected, and then centrifuged at 10000 rpm for 20 min, and the precipitate was discarded; the mixture was filtered through a 0.22 μm filter membrane and dried under vacuum at 60 ℃ for 12 hours to obtain black molybdenum disulfide nanosheets.

[0175] High-resolution transmission electron microscopy (TEM) images of the prepared Cu-TCPP nanosheets, Zr-TCPP nanosheets, graphene nanosheets, and molybdenum disulfide nanosheets are shown below. Figure 13 As shown in the figure, the typical morphology of these four two-dimensional materials is observed, all of which exhibit a thin two-dimensional layered structure.

[0176] Step B2: Using the two-dimensional nanosheets prepared in Step B1 as fluorescence quenchers, including Cu-TCPP nanosheets, Zr-TCPP nanosheets, graphene nanosheets, and molybdenum disulfide nanosheets, add the two-dimensional nanosheets to a cuvette, followed by 10 μL of a peptide probe combination (the four nanosheets and peptide probe combination PAH3-FITC+PAN2-TMR are arranged in an orthogonal combination strategy to construct a sensing array, such as...). Figure 14As shown, a total of 4 sensing arrays were constructed (referred to as P1-P4). After incubation at 37℃ for 10 minutes, the sensing arrays of peptide probes were obtained (the prepared peptide probe composite Cu-MOF nanosheets - referred to as P1, peptide probe composite Zr-MOF nanosheets - referred to as P2, peptide probe composite graphene nanosheets - referred to as P3, peptide probe composite molybdenum disulfide nanosheets - referred to as P4).

[0177] In step B2, the optimal combination of two-dimensional nanosheets and peptide probes was screened by systematically evaluating the fluorescence quenching efficiency: different concentrations of two-dimensional quenching nanosheets Zr-TCPP (0-15 μg·mL⁻¹) were used. -1 Cu-TCPP (0-15 μg·mL) -1 ), GO (0-18μg·mL) -1 ) and MoS2 (0-24 μg·mL -1 The peptide probe was incubated in PBS (0.01 M, pH 7.4) for 3 min, and then fluorescence was measured on a Varian Cary Eclipse fluorescence spectrophotometer. The concentration of the peptide probe in the sample was PAH3-FITC (2 × 10⁻⁶). -6 M), PAN2-TMR (3×10 -6 Based on the degree of fluorescence quenching, the concentration of nanosheets in the sample was selected as Zr-TCPP (15 μg·mL⁻¹). -1 Cu-TCPP (15 μg·mL) -1 ), GO (18 μg·mL) -1 ) and MoS2 (24 μg·mL -1 ).

[0178] Example 7

[0179] A method for preparing a peptide probe sensing array, comprising a peptide probe sensing array responsive to influenza B virus dual antigen activation, including peptide probes targeting dual antigens and two-dimensional nanosheets, including the following steps:

[0180] Step B1: Two-dimensional nanosheets with fluorescence quenching properties are prepared using the method in Step B1 of Example 6, resulting in Cu-TCPP nanosheets, Zr-TCPP nanosheets, graphene nanosheets, and molybdenum disulfide nanosheets.

[0181] Step B2: Using the two-dimensional nanosheets prepared in Step B1 as fluorescence quenchers, including Cu-TCPP nanosheets, Zr-TCPP nanosheets, graphene nanosheets, and molybdenum disulfide nanosheets, add the two-dimensional nanosheets to a cuvette, followed by 10 μL of each peptide probe combination (the four nanosheets and the peptide probe combination PBN3-Cy5+PBH2-Cy3 are arranged in an orthogonal combination strategy to construct a sensing array, such as...). Figure 15As shown, four sensor arrays were constructed (referred to as P5-P8). After incubation at 37°C for 10 minutes, sensor arrays of peptide probes were obtained (peptide probe composite Cu-MOF nanosheets - referred to as P5, peptide probe composite Zr-MOF nanosheets - referred to as P6, peptide probe composite graphene nanosheets - referred to as P7, peptide probe composite molybdenum disulfide nanosheets - referred to as P8).

[0182] In step B2, the optimal combination of two-dimensional nanosheets and peptide probes was screened by systematically evaluating the fluorescence quenching efficiency: different concentrations of two-dimensional quenching nanosheets Zr-TCPP (0-15 μg·mL⁻¹) were used. -1 Cu-TCPP (0-15 μg·mL) -1 ), GO (0-18μg·mL) -1 ) and MoS2 (0-24 μg·mL -1 The peptide probe was incubated in PBS (0.01 M, pH 7.4) for 3 min, and then fluorescence was measured on a Varian Cary Eclipse fluorescence spectrophotometer. The concentration of the peptide probe in the sample was PBH2-Cy3 (3 × 10⁻⁶). -6 M), PBN3-Cy5 (4×10 -6 Based on the degree of fluorescence quenching, the concentration of nanosheets in the sample was selected as Zr-TCPP (15 μg·mL⁻¹). -1 Cu-TCPP (15 μg·mL) -1 ), GO (18 μg·mL) -1 ) and MoS2 (24 μg·mL -1 ).

[0183] The sensor arrays of the peptide probes prepared in steps B2 of Example 6 and B2 of Example 7 were subjected to fluorescence quenching tests. Fluorescence titration experiments were used to determine the optimal quenching effect in 0.01M phosphate buffered saline solution at pH 7.4. Figure 16 As shown, two-dimensional nanosheets cause concentration-dependent fluorescence quenching of peptide probes (including PAH3-FITC, PAN2-TMR, PBH2-Cy3 and PBN3-Cy5), which indirectly indicates the successful assembly of peptide probes with two-dimensional nanosheets.

[0184] Example 8

[0185] High-throughput assay for distinguishing influenza A virus subtypes using a peptide probe sensing array (P1-P4):

[0186] Two-dimensional quenching nanosheets, including Cu-TCPP nanosheets (10 μL, 45 μg·mL), were added to black 384-well plates. -1 Zr-TCPP nanosheets (10 μL, 45 μg·mL)-1 ), graphene oxide nanosheets (10 μL, 54 μg·mL) -1 ) and molybdenum disulfide nanosheets (10 uL, 72 μg·mL) -1 Then, 10 μL of the peptide probe combination (PAH3-FITC / PAN2-TMR (6 μM / 9 μM)) was added and incubated with the two-dimensional nanosheets for 5 minutes. Next, different subtypes of influenza virus (10 μL, 2 × 10⁻⁶ mcg) were added to the peptide probe composite two-dimensional nanosheet solution. 3 TCID50mL -1 The reaction was carried out at 37°C with shaking for 15 minutes. FRET signals (λex=490nm, λem=590nm) were collected using a microplate reader, and six parallel experiments were performed for each sample to ensure data reliability.

[0187] The results are as follows Figure 17 As shown, Figure 17 This diagram illustrates the high-throughput differentiation results of influenza A virus subtypes and other virus types using a peptide probe sensing array (P1-P4). The fluorescence energy resonance transfer intensity in the figure indicates that the presence of influenza A virus activates a stronger fluorescence energy resonance transfer signal in the peptide probe sensing array (P1-P4) compared to other control viruses. This is because PAH3-FITC and PAN2-TMR specifically target and bind to the hemagglutinin and neuraminidase of influenza A virus, respectively. Principal component analysis (PCA) was used to convert the fluorescence response matrix into a two-dimensional scatter plot, obtaining the characteristic "fingerprints" of different subtypes, thus achieving high-throughput differentiation of different influenza A virus subtypes.

[0188] Example 9

[0189] High-throughput assay for distinguishing influenza B virus subtypes using peptide probe sensing arrays (P5-P8):

[0190] Two-dimensional quenching nanosheets, including Cu-TCPP nanosheets (10 μL, 45 μg·mL⁻¹), were added to black 384-well plates. -1 Zr-TCPP nanosheets (10 μL, 45 μg·mL) -1 ), graphene oxide nanosheets (10 μL, 54 μg·mL) -1 ) and molybdenum disulfide nanosheets (10 uL, 72 μg·mL) -1 Then, 10 μL of peptide probe (PBH2-Cy3 / PBN3-Cy5 (9 μM / 12 μM)) was added and incubated with the two-dimensional nanosheets for 5 minutes. Next, different subtypes of influenza virus (10 μL, 2 × 10⁻⁶) were added to the peptide probe composite two-dimensional nanosheet solution. 3 TCID50mL -1The reaction was carried out at 37°C with shaking for 15 minutes. FRET signals were collected using a microplate reader (λex=490nm, λem=590nm), and six parallel experiments were performed for each sample to ensure data reliability.

[0191] The results are as follows Figure 18 As shown, Figure 18 This diagram illustrates the high-throughput differentiation results of the peptide probe sensing array (P5-P8) for influenza B virus subtypes and other virus types. The fluorescence energy resonance transfer intensity in the figure indicates that the presence of influenza B virus activates a stronger fluorescence energy resonance transfer signal in the peptide probe sensing array (P5-P8) compared to other control viruses. This is because PBH2-Cy3 and PBN3-Cy5 specifically target and bind to the hemagglutinin and neuraminidase of influenza B virus, respectively. Principal component analysis (PCA) was used to convert the fluorescence response matrix into a two-dimensional scatter plot, obtaining the characteristic "fingerprints" of different subtypes, thus achieving high-throughput differentiation of the two influenza B virus subtypes.

[0192] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A sensor array for a peptide probe, characterized in that, The raw materials include two-dimensional nanosheets and a peptide probe combination, wherein the peptide probe combination is a combination of peptide probes obtained from a first polypeptide chain and peptide probes obtained from a second polypeptide chain. Alternatively, the peptide probe combination may be a combination of a peptide probe obtained from a third polypeptide chain and a peptide probe obtained from a fourth polypeptide chain. The raw materials for the peptide probe obtained from the first polypeptide chain include the first polypeptide chain and a fluorescent dye. The amino acid sequence of the first polypeptide chain is shown in SEQ NO:

1. The peptide probe obtained from the first polypeptide chain is used for broad-spectrum recognition of hemagglutinin of influenza A virus. The raw materials for the peptide probe obtained from the second polypeptide chain include the second polypeptide chain and a fluorescent dye. The amino acid sequence of the second polypeptide chain is shown in SEQ NO:

2. The peptide probe obtained from the second polypeptide chain is used for broad-spectrum recognition of influenza A virus neuraminidase. The raw materials for the peptide probe obtained from the third polypeptide chain include the third polypeptide chain and a fluorescent dye. The amino acid sequence of the third polypeptide chain is shown in SEQ NO:

3. The peptide probe obtained from the third polypeptide chain is used for broad-spectrum recognition of hemagglutinin of influenza B virus. The raw materials for the peptide probe obtained from the fourth polypeptide chain include the fourth polypeptide chain and a fluorescent dye. The amino acid sequence of the fourth polypeptide chain is shown in SEQ NO:

4. The peptide probe obtained from the fourth polypeptide chain is used for broad-spectrum recognition of influenza B virus neuraminidase. The two peptide probes in the peptide probe combination of the sensor array target and bind to influenza virus hemagglutinin and neuraminidase, respectively, thereby causing fluorescence energy resonance transfer.

2. The sensor array for the peptide probe according to claim 1, characterized in that, The molar ratio of the first polypeptide chain to the fluorescent dye is 1:3; The molar ratio of the second polypeptide chain to the fluorescent dye is 1:3; The molar ratio of the third polypeptide chain to the fluorescent dye is 1:3; The molar ratio of the fourth polypeptide chain to the fluorescent dye is 1:

3.

3. The sensor array for the peptide probe according to claim 1, characterized in that, The fluorescent dye is selected from at least one of fluorescein, tetramethylrhodamine, anthocyanin 3, anthocyanin 5, anthocyanin 7, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647 and Alexa Fluor 680.

4. The sensor array of the peptide probe according to claim 1, characterized in that, The two-dimensional nanosheets are selected from any one of graphene oxide, graphitic carbon nitride, transition metal disulfide, metal oxide sheets, metal hydroxide sheets, metal-organic framework nanosheets, and covalent organic framework nanosheets.

5. A method for preparing a sensor array for a peptide probe, characterized in that, The sensing array for preparing the peptide probe as described in claim 1 comprises the following steps: Step B1: Prepare two-dimensional nanosheets with fluorescence quenching properties; Step B2: Using two-dimensional nanosheets as fluorescence quenchers, add them to the peptide probe array for incubation to obtain a peptide probe sensing array.

6. The application of the peptide probe sensing array as described in claim 1 in the preparation of high-throughput products for distinguishing influenza virus subtypes; The influenza viruses are selected from influenza A and influenza B viruses, wherein the influenza A viruses are selected from H1N1, H3N2, H5N1 and H7N9, and the influenza B viruses are selected from the Victoria lineage and the Yamagata lineage.

Images