Fluorescent covalent organic framework sensing array for detecting disease marker polypeptide

Abstract

The invention discloses a fluorescent covalent organic framework sensing array for detecting a disease marker polypeptide. Six fluorescent covalent organic frameworks with different topological structures are designed and synthesized to form a sensing unit, and the sensing unit has differential interaction with different polypeptides. By integrating fluorescence signal changes, the sensor array can detect a variety of polypeptides. The detected polypeptide comprises the disease marker, an efficient and sensitive disease marker polypeptide visual detection means can be provided, and an important basis is provided for early diagnosis, prognosis evaluation and treatment monitoring of diseases.

Description

Technical Field

[0001] The present invention belongs to the fields of fluorescence sensing technology and disease diagnosis, and relates to a fluorescence covalent organic framework sensing array for detecting disease biomarker polypeptides, which can detect and distinguish disease biomarker polypeptides in a simple and rapid visualization manner, providing an important basis for the early diagnosis, prognosis evaluation and treatment monitoring of diseases. Background Art

[0002] In modern biomedical research, the accurate detection of disease biomarkers is crucial for the early diagnosis and treatment of diseases. Disease biomarkers refer to biochemical indicators that can mark changes or possible changes in the structure or function of systems, organs, tissues, cells and subcellular structures, and can be classified into various types according to their characteristics and sources. Among them, endogenous polypeptides play important roles in various physiological processes such as metabolism and immune responses and stably exist in biological samples, and can be used as indicators of early pathophysiological changes. At present, there are various methods for detecting disease biomarker polypeptides. Immunoassay is relatively simple to operate and low in cost, but it is easily interfered by cross-reactions and has poor sensitivity for detecting low-abundance polypeptides; mass spectrometry can accurately identify and quantify and is high-throughput, such as MALDI-TOF MS and LC-MS / MS, but the equipment is expensive, the requirements for operators are high, and the pretreatment is complex; the biosensor method is fast and can be monitored in real time, but its stability and repeatability need to be improved. These deficiencies limit the more efficient and extensive application of disease biomarker polypeptide detection in clinical diagnosis and other aspects.

[0003] Covalent organic frameworks (abbreviated as COFs) are a class of emerging porous crystalline organic materials, and their advantages of tunability, porosity and crystallinity endow them with broad application potential. Among them, fluorescent COFs show unique advantages in sensing due to their unique photophysical properties, which can accommodate various types of guest analytes through specific or non-specific chemical bonds and non-bonding interactions. Nanoscale fluorescent COFs have the performance of polypeptide enrichment and can produce different fluorescence responses according to different polypeptide types. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a fluorescence covalent organic framework sensing array for detecting polypeptides. The present invention can simply and quickly distinguish and detect polypeptides.

[0005] The purpose of the present invention is achieved by the following technical solutions: A fluorescence covalent organic framework sensing array for detecting polypeptides is constructed by six fluorescent COFs with different topological structures as sensing units to form a sensing array. The structures of the six COFs are as follows:

[0006]

[0007] Furthermore, the fluorescence responses of the six selected different COFs to different types of polypeptides are different.

[0008] Furthermore, it is prepared according to the following steps:

[0009] Step 1. Preparation of fluorescent COFs:

[0010] (1) Preparation method of COF1: Put 1,3,6,8-tetrakis(4-formylphenyl)pyrene and trans-1,4-cyclohexanediamine in a molar ratio of 1:2 into a flame-sealed tube, then add n-butanol, 1,4-dioxane and 3M acetic acid with a volume ratio of 4:1:3, ultrasonicate for 5 - 10 min, and then perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame-sealing, react at a temperature of 100 - 120 °C for 3 - 5 days. Filter and collect the resulting precipitate, wash it with acetone multiple times, and then Soxhlet extract it with tetrahydrofuran at 130 - 140 °C for 1 - 2 days. Finally, vacuum dry it at 60 - 80 °C for 1 - 2 days to obtain COF1 with a yield of 80 - 84%;

[0011] (2) Preparation method of COF2: Put 1,3,6,8-tetrakis(4-formylphenyl)pyrene and p-phenylenediacetonitrile in a molar ratio of 1:2 into a flame-sealed tube, then add 1,4-dioxane and 4M potassium hydroxide with a volume ratio of 5:1, ultrasonicate for 10 - 20 min, and then perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame-sealing, react at a temperature of 100 - 120 °C for 3 - 4 days. Filter and collect the resulting precipitate, wash it with deionized water, dichloromethane and methanol multiple times, and then Soxhlet extract it with tetrahydrofuran at 130 - 140 °C for 1 - 2 days. Finally, vacuum dry it at 90 - 100 °C for 1 - 2 days to obtain COF2 with a yield of 70 - 72%;

[0012] (3) Preparation method of COF3: Put terephthalaldehyde and 2,2'-bipyridine-5,5'-diamine in a molar ratio of 2:3 into a flame-sealed tube, then add 1,4-dioxane and mesitylene with a volume ratio of 2:1, ultrasonicate for 10 - 15 min, and then perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame-sealing, react at a temperature of 110 - 120 °C for 5 - 7 days. Filter and collect the resulting precipitate, wash it with methanol multiple times, and then Soxhlet extract it with tetrahydrofuran at 130 - 140 °C for 1 - 2 days. Finally, vacuum dry it at 100 - 120 °C for 12 - 24 h to obtain COF3 with a yield of 55 - 59%;

[0013] (4) Preparation method of COF4: Put 2,2'-bipyridine-5,5'-dicarboxaldehyde and melamine into a flame-sealed tube according to a molar ratio of 3:2, then add mesitylene, 1,4-dioxane and acetic acid with a volume ratio of 25:5:1. After ultrasonic treatment for 5 - 10 min, perform three freeze-vacuum-thaw cycles under a liquid nitrogen environment. After flame sealing, react at a temperature of 120 - 125 °C for 3 - 4 days. Filter and collect the generated precipitate. After washing it with acetone and absolute ethanol for multiple times, perform Soxhlet extraction with tetrahydrofuran at 130 - 140 °C for 24 - 36 h. Finally, perform vacuum drying at 60 - 70 °C for 24 - 36 h to obtain COF4 with a yield of 60 - 65%;

[0014] (5) Preparation method of COF5: Put 2,5-bis(2-methoxyethoxy)terephthalohydrazide and 4,4',4”-(1,3,5-triazine-2,4,6-triyl)tribenzaldehyde into a flame-sealed tube according to a molar ratio of 3:2, then add acetonitrile and 6M acetic acid with a volume ratio of 4:1. After ultrasonic treatment for 15 - 20 min, perform three freeze-vacuum-thaw cycles under a liquid nitrogen environment. After flame sealing, react at room temperature for 3 - 4 days. Filter and collect the generated precipitate. After washing it with absolute ethanol for multiple times, perform Soxhlet extraction with tetrahydrofuran at 130 - 140 °C for 24 - 30 h. Finally, perform vacuum drying at 50 - 60 °C for 12 - 24 h to obtain COF5 with a yield of 77 - 80%;

[0015] (6) Preparation method of COF6: Put tetrakis(4-aminophenyl)ethylene and naphthalene-2,6-dicarboxaldehyde into a flame-sealed tube according to a molar ratio of 1:2, then add o-dichlorobenzene, 1,4-dioxane and 6M acetic acid with a volume ratio of 10:10:1. After ultrasonic treatment for 5 - 10 min, perform three freeze-vacuum-thaw cycles under a liquid nitrogen environment. After flame sealing, react at a temperature of 120 - 125 °C for 3 - 4 days. Filter and collect the generated precipitate. After washing it with dimethylformamide, acetone and methanol for multiple times, perform Soxhlet extraction with tetrahydrofuran at 120 - 130 °C for 24 - 48 h. Finally, perform vacuum drying at 80 - 90 °C for 12 - 24 h to obtain COF6 with a yield of 58 - 62%;

[0016] Step 2. Preparation of the COFs nanosheet solution for the sensor array unit to be measured: Mix the six COFs with ultrapure water respectively to form a suspension of 1 mg / mL, and ultrasonically treat it into a nanosheet structure through sufficient ultrasonic treatment. The ultrasonic time ranges from 20 to 60 min.

[0017] Furthermore, the molar ratio of 1,3,6,8-tetrakis(4-formylphenyl)pyrene and trans-1,4-cyclohexanediamine is 1:2; the volume ratio of n-butanol, 1,4-dioxane and 3M acetic acid is 4:1:3;

[0018] The molar ratio of 1,3,6,8 - tetrakis(4 - formylphenyl)pyrene to p - xylylenedicyanide is 1:2; the volume ratio of 1,4 - dioxane to 4M potassium hydroxide is 5:1;

[0019] The molar ratio of phloroglucinol trialdehyde to 2,2'-bipyridine - 5,5'-diamine is 2:3; the volume ratio of 1,4 - dioxane to mesitylene is 2:1.

[0020] Furthermore, the polypeptide includes peptide segments from Alzheimer's disease Aβ, cardiovascular disease marker C - reactive protein, calcitonin gene - related peptide, incretin, atrial natriuretic peptide fragment or glucagon - like peptide hormones.

[0021] A fluorescence covalent organic framework sensing array for detecting polypeptides according to the present invention includes six COFs nanosheet solutions with different topological structures; the test solution includes six common disease marker polypeptides Pep - 1 to Pep - 6, which are key peptide segments from Alzheimer's disease Aβ, cardiovascular disease marker C - reactive protein, calcitonin gene - related peptide, incretin, atrial natriuretic peptide fragment, glucagon - like peptide hormones respectively. Compared with a single COF, selecting six COFs can increase the recognition sites, synergistically reduce the detection limit, improve the detection accuracy and reliability; the respective specificities can be utilized to broaden the detection range, improve the discrimination ability, and achieve the detection of multiple polypeptides; the different COFs have complementary properties and fault - tolerance ability, enhancing the stability and adaptability of the array to meet the actual application requirements. Specifically, six COFs with different topological structures are synthesized by reasonably selecting building units, and their nanosheet suspensions are prepared as the units of the sensing array to detect polypeptides. The different hydrophilic - hydrophobic properties and charges of the polypeptides will produce different interactions with the COFs, thereby affecting their fluorescence intensities, so as to achieve the detection of the six disease marker polypeptides mentioned above.

[0022] The beneficial effects of the present invention are as follows: The present invention provides a fluorescence covalent organic framework sensing array for detecting polypeptides. By designing and synthesizing six kinds of fluorescence COFs with different topological structures and using them as sensing units to construct a sensing array, it is experimentally determined that the fluorescence responses of this sensing array to different polypeptides are different, thereby realizing the specific detection of the six selected polypeptides. Since the fluorescence response between polypeptides and COFs is a non-specific response, compared with using a single COF, using six COFs can increase the recognition sites, synergistically reduce the detection limit, improve the detection accuracy and reliability; the respective specificities can be utilized to broaden the detection range and improve the discrimination ability to achieve the detection of multiple polypeptides; the performances of different COFs are complementary and have fault tolerance ability, enhancing the stability and adaptability of the array to meet the actual application requirements. Considering multiple factors comprehensively, six COFs are the optimal choice. This innovative method not only improves the efficiency and sensitivity of biosensors but also brings an efficient and sensitive detection means to the field of biosensing.

[0023] The present invention uniformly adopts the vacuum flame sealing method for preparation, which improves the synthesis yield of COFs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings of the present invention are intended to provide an in-depth understanding of the present invention. The embodiments shown in these drawings and their descriptions are intended to illustrate various aspects of the present invention but do not impose any undue limitation on the scope of the present invention.

[0025] Figure 1 It is a schematic diagram of the principle for the present invention to detect disease biomarker polypeptides;

[0026] Figure 2 It is the SEM characterization diagram and morphology diagram of the six COFs prepared in the embodiments of the present invention, where (a) - (f) respectively correspond to the SEM characterization diagrams of COF1 - COF6, and the insets are the corresponding morphology diagrams;

[0027] Figure 3 It is the XRD pattern of the six COFs prepared in the embodiments of the present invention;

[0028] Figure 4 It is the infrared spectrum diagram of the six COFs prepared in the embodiments of the present invention;

[0029] Figure 5 It is the fluorescence response spectrum diagram of the six COFs prepared in the embodiments of the present invention to 6 disease biomarker polypeptides pep1 - pep6. (A) - (F) respectively correspond to COF1 - COF6 for detecting polypeptides;

[0030] Figure 6Thermal map of fluorescence intensity quenched by six disease biomarker polypeptides for the six COFs prepared in the embodiments of the present invention. (A)-(E) correspond to pep1-pep6 with concentrations of 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, and 2000 ng / mL, respectively. Detailed implementation mode

[0031] Here, the exemplary embodiments will be described in detail, and the examples are shown in the drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementation modes described in the following exemplary embodiments do not represent all implementation modes consistent with the present invention. On the contrary, they are only examples of devices and methods consistent with some aspects of the present invention as detailed in the appended claims.

[0032] The present invention will be described in detail below with reference to the drawings. Without conflict, the features in the following embodiments and implementation modes can be combined with each other.

[0033] A polypeptide detection sensing array based on fluorescent covalent organic frameworks of the present invention, and its detection implementation approach is as Figure 1 shown. Different COFs will produce special fluorescence responses to different polypeptides, and these non-specific fluorescence responses are integrated to achieve the detection of polypeptides. It is obtained through the following preparation method:

[0034] Step 1. Preparation of fluorescent COFs:

[0035] (1) Preparation method of COF1: Weigh 27.1 mg of 1,3,6,8-tetra(4-formylphenyl)pyrene and 10 mg of trans-1,4-cyclohexanediamine and put them into a flame-sealed tube. Then add 1.2 mL of n-butanol, 0.3 mL of 1,4-dioxane, and 0.9 mL of 3M acetic acid. After ultrasonic treatment for 5-10 min, perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame sealing, react at a temperature of 100-120 °C for 3-5 days. Filter and collect the generated precipitate. After washing with acetone multiple times, perform Soxhlet extraction with tetrahydrofuran at 130-140 °C for 1-2 days. Finally, dry under vacuum at 60-80 °C for 1-2 days to obtain COF1, and the yield is 80-84%;

[0036]

[0037] (2) Preparation method of COF2:: Weigh 15 m of 1,3,6,8-tetrakis(4-formylphenyl)pyrene and 10 mg of p-phthalonitrile separately and put them into a flame-sealed tube. Then add 1 mL of 1,4-dioxane and 0.2 mL of 4 M potassium hydroxide. After ultrasonic treatment for 10 - 20 min, perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame-sealing, react at a temperature of 100 - 120 °C for 3 - 4 days. Filter and collect the generated precipitate. After washing it with deionized water, dichloromethane and methanol for multiple times, perform Soxhlet extraction with tetrahydrofuran at 130 - 140 °C for 1 - 2 days. Finally, dry it under vacuum at 90 - 100 °C for 1 - 2 days to obtain COF2 with a yield of 70 - 72%;

[0038]

[0039] (3) Preparation method of COF3: Weigh 89.1 mg of trimesic aldehyde and 160.93 mg of 2,2'-bipyridine-5,5'-diamine separately and put them into a flame-sealed tube. Then add 2 mL of 1,4-dioxane and 1 mL of mesitylene. After ultrasonic treatment for 10 - 15 min, perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame-sealing, react at a temperature of 110 - 120 °C for 5 - 7 days. Filter and collect the generated precipitate. After washing it with methanol for multiple times, perform Soxhlet extraction with tetrahydrofuran at 130 - 140 °C for 1 - 2 days. Finally, dry it under vacuum at 100 - 120 °C for 12 - 24 h to obtain COF3 with a yield of 55 - 59%;

[0040]

[0041] (4) Preparation method of COF4: Weigh 82.7 mg of 2,2'-bipyridine-5,5'-dialdehyde and 32.8 mg of melamine separately and put them into a flame-sealed tube. Then add 5.1 mL of mesitylene, 0.9 mL of 1,4-dioxane and 0.2 mL of acetic acid. After ultrasonic treatment for 5 - 10 min, perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame-sealing, react at a temperature of 120 - 125 °C for 3 - 4 days. Filter and collect the generated precipitate. After washing it with acetone and absolute ethanol for multiple times, perform Soxhlet extraction with tetrahydrofuran at 130 - 140 °C for 24 - 36 h. Finally, dry it under vacuum at 60 - 70 °C for 24 - 36 h to obtain COF4 with a yield of 60 - 65%;

[0042]

[0043] (5) Preparation method of COF5: Weigh 12.3 mg of 2,5-bis(2-methoxyethoxy)terephthalohydrazide and 10.5 mg of 4,4',4''-(1,3,5-triazine-2,4,6-triyl)tribenzaldehyde separately and put them into a flame-sealed tube. Then add 2 mL of acetonitrile and 0.6 mL of 6 M acetic acid. After ultrasonic treatment for 15 - 20 min, perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame sealing, react at room temperature for 3 - 4 days. Filter to collect the generated precipitate. After washing it with absolute ethanol for multiple times, perform Soxhlet extraction with tetrahydrofuran at 130 - 140 °C for 24 - 30 h. Finally, dry it under vacuum at 50 - 60 °C for 12 - 24 h to obtain COF5, and the yield is 77 - 80%;

[0044]

[0045] (6) Preparation method of COF6: Weigh 15.7 mg of tetrakis(4-aminophenyl)ethylene and 14.7 mg of naphthalene-2,6-dicarbaldehyde separately and put them into a flame-sealed tube. Then add 1 mL of o-dichlorobenzene, 1 mL of 1,4-dioxane and 0.1 mL of 6 M acetic acid. After ultrasonic treatment for 5 - 10 min, perform three freeze-vacuum-thaw cycles under liquid nitrogen environment. After flame sealing, react at 120 - 125 °C for 3 - 4 days. Filter to collect the generated precipitate. After washing it with dimethylformamide, acetone and methanol for multiple times, perform Soxhlet extraction with tetrahydrofuran at 120 - 130 °C for 24 - 48 h. Finally, dry it under vacuum at 80 - 90 °C for 12 - 24 h to obtain COF6, and the yield is 58 - 62%;

[0046]

[0047] Step 2. Preparation of the COFs solution of the sensing array unit to be measured: Mix COFs with ultrapure water to form a suspension of 1 mg / mL, and ultrasonically treat it into a nanosheet structure. The ultrasonic time ranges from 20 to 60 min (depending on the dispersion of COFs).

[0048] Example 1: Detection of six disease biomarker polypeptides

[0049] 1. Prepare the reaction solution: First, dissolve the six polypeptide solutions separately in pure water to form solutions of 1 mg / mL, and then dilute them separately to five concentration gradients of 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, and 2000 ng / mL, and perform fluorescence detection using the six pre-prepared COFs nanosheet solutions respectively.

[0050] 2. Detection: Mix 80 μL of a 1 mg / mL concentrated suspension of a certain COF with 20 μL of a concentration solution of a disease biomarker polypeptide. Aspirate repeatedly with a pipette for more than 10 times to ensure sufficient reaction between the two, and then detect with a fluorescence spectrometer. Repeat the above detection steps until each COF solution has reacted with each concentration of each polypeptide solution.

[0051] Characterization of Six COFs

[0052] Structurally stable COF particles were prepared through an aldehyde-amine condensation reaction. In this paper, a variety of characterization methods were used to confirm the accurate synthesis and preparation of COFs. To verify that the COFs we synthesized have good morphology, we used scanning electron microscopy to characterize the morphology images of COFs, as Figure 2 shown. The 6 COFs all have unique microscopic features in terms of SEM images and morphology. (a) COF1 has a fibrous structure, with obvious gaps between the fibers, which are relatively evenly distributed, forming a porous microstructure. (b) COF2 has a rod-like structure, with the columnar structures arranged relatively neatly, having regular spacing, and the surface structure is relatively smooth. (c) COF3 is composed of plate-like structures, and the surface of the structure is relatively flat. (d) COF4 presents multiple slender fibrous structures, showing obvious bending and interlacing characteristics. (e) COF5 is a complex flower-like microstructure, composed of multiple petal-like structures, presenting a three-dimensional, highly ordered arrangement, and these flower-like structures are interconnected to form a network-like microscopic morphology. (f) COF6 presents a complex layered structure, and these layered structures are superimposed on each other to form a three-dimensional, petal-like microscopic morphology. The edge of each layer structure is relatively clear, and the spacing between layers is relatively uniform. The inset shows the corresponding powder appearance morphology, which matches its macroscopic performance. It was observed that the six COF samples presented powdery appearances of different colors, with relatively uniform textures, and there was a certain electrostatic phenomenon when collecting the samples. And refer to the experimental results obtained from the XRD patterns ( Figure 3 (A) - (F) in Figure 4 and the Fourier transform infrared spectra (

[0053] Fluorescence Spectrometer Detection of Six Disease Biomarker Polypeptides

[0054] Figure 5 are the fluorescence response spectra of six COFs to six disease biomarker polypeptides pep1 - pep6. Figure 5(A)-(F) therein respectively correspond to COF1-COF6 for detecting polypeptides. Among them, pep1 and pep2 showed fluorescence quenching with increasing concentration for all six COFs. The reason is also due to the hydrophilic-hydrophobic interaction between insoluble COFs and hydrophobic peptides, which leads to a decrease in fluorescence intensity. However, the hydrophobic disease biomarker pep3 has different fluorescence responses to different COFs, which may be because its average hydrophilicity index is lower than that of pep1 and pep2. Therefore, when pep3 interacts with different COFs, this hydrophilic-hydrophobic interaction is not the dominant one, and other interactions between different COFs and pep3 need to be considered. For the two hydrophilic disease biomarker polypeptides pep4 and pep6, there is no obvious quenching pattern in the fluorescence responses of pep4 and pep6 to different COFs. This may be because the fluorescence response of COFs to polypeptides is mainly due to hydrophilic-hydrophobic interaction. When hydrophilic polypeptides bind to COFs, there is no such interaction force, so there is no obvious fluorescence response pattern, further verifying the fluorescence response mechanism between COFs and polypeptides. For the disease biomarker pep5, the isoelectric point of pep5 is 11.70, which is positively charged in a neutral aqueous solution and can undergo electrostatic interaction with some negatively charged groups in COFs, thereby causing pep5 to adsorb on the pore size or surface of COFs, resulting in fluorescence quenching of COFs.

[0055] Figure 6 It is a heat map of the fluorescence intensity quenched by six disease biomarker polypeptides for six COFs. Figure 6 (A)-(E) therein are respectively heat maps of the fluorescence intensity quenched by disease biomarker polypeptides pep1-pep6 at different concentrations (100-2000 ng / mL) for COFs. The heat map can more intuitively show the fluorescence changes of COFs. The closer the color is to blue, the greater the degree of fluorescence reduction of COFs. As the concentration of pep1 and pep2 increases, their heat maps tend to be positively blue. As the concentration of pep3 increases, its heat map approaches light blue. In the heat map, since pep1-pep6 have unique color characteristics corresponding to different COFs at different concentrations, COFs can be developed into an array for fluorescently detecting disease biomarker polypeptides. Using different concentrations and different types of COFs as the basic units of the array, different fluorescence response signals are formed through the interaction between disease biomarker polypeptides and them, and a series of signals are used as the identity codes of the polypeptides to achieve the specific detection of disease biomarker polypeptides by non-specific interaction.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the present invention.

[0057] The above embodiments are only used to illustrate the design concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made according to the principles and design concepts disclosed by the present invention are within the protection scope of the present invention.

Claims

1. A fluorescent covalent organic framework sensing array for detecting disease biomarker polypeptides, characterized in that, A sensing array is jointly constructed by six fluorescent COFs with different topological structures as sensing units. The structures of the six COFs are as follows:

2. The fluorescent covalent organic framework sensing array for detecting disease biomarker polypeptides according to claim 1, wherein The six different COFs selected have different fluorescence responses to different types of polypeptides.

3. The fluorescence covalent organic framework sensing array for detecting disease biomarker polypeptides according to claim 2, wherein It is prepared according to the following steps: Step 1. Preparation of fluorescent COFs: (1) Preparation method of COF1: Put 1,3,6,8-tetrakis(4-formylphenyl)pyrene and trans-1,4-cyclohexanediamine in a flame-sealed tube according to a molar ratio of 1:2, and then add n-butanol, 1,4-dioxane and 3M acetic acid with a volume ratio of 4:1:

3. After ultrasonic treatment for 5-10 min, perform three freeze-vacuum-thaw cycles in a liquid nitrogen environment. After flame sealing, react at a temperature of 100-120 °C for 3-5 days. Filter and collect the generated precipitate. After washing with acetone multiple times, perform Soxhlet extraction with tetrahydrofuran at 130-140 °C for 1-2 days; finally, vacuum dry at 60-80 °C for 1-2 days to obtain COF1 with a yield of 80-84%; (2) Preparation method of COF2: Put 1,3,6,8-tetrakis(4-formylphenyl)pyrene and p-phenylenediacetonitrile in a flame-sealed tube according to a molar ratio of 1:2, and then add 1,4-dioxane and 4M potassium hydroxide with a volume ratio of 5:

1. After ultrasonic treatment for 10-20 min, perform three freeze-vacuum-thaw cycles in a liquid nitrogen environment. After flame sealing, react at a temperature of 100-120 °C for 3-4 days. Filter and collect the generated precipitate. After washing with deionized water, dichloromethane and methanol multiple times, perform Soxhlet extraction with tetrahydrofuran at 130-140 °C for 1-2 days; finally, vacuum dry at 90-100 °C for 1-2 days to obtain COF2 with a yield of 70-72%; (3) Preparation method of COF3: Put benzene-1,3,5-tricarbaldehyde and 2,2'-bipyridine-5,5'-diamine in a flame-sealed tube according to a molar ratio of 2:3, and then add 1,4-dioxane and mesitylene with a volume ratio of 2:

1. After ultrasonic treatment for 10-15 min, perform three freeze-vacuum-thaw cycles in a liquid nitrogen environment. After flame sealing, react at a temperature of 110-120 °C for 5-7 days. Filter and collect the generated precipitate. After washing with methanol multiple times, perform Soxhlet extraction with tetrahydrofuran at 130-140 °C for 1-2 days; finally, vacuum dry at 100-120 °C for 12-24 h to obtain COF3 with a yield of 55-59%; (4) Preparation method of COF4: 2,2'-Bipyridine-5,5'-dicarbaldehyde and melamine are put into a flame-sealed tube according to a molar ratio of 3:2, and then mesitylene, 1,4-dioxane and acetic acid with a volume ratio of 25:5:1 are added. After ultrasonic treatment for 5-10 min, three freeze-vacuum-thaw cycles are carried out under liquid nitrogen environment. After flame sealing, the reaction is carried out at a temperature of 120-125 °C for 3-4 days. The resulting precipitate is collected by filtration, washed repeatedly with acetone and absolute ethanol, and then Soxhlet extracted with tetrahydrofuran at 130-140 °C for 24-36 h; finally, vacuum dried at 60-70 °C for 24-36 h to obtain COF4 with a yield of 60-65%; (5) Preparation method of COF5: 2,5-Bis(2-methoxyethoxy)terephthalohydrazide and 4,4',4''-(1,3,5-triazine-2,4,6-triyl)tribenzaldehyde are put into a flame-sealed tube according to a molar ratio of 3:2, and then acetonitrile and 6M acetic acid with a volume ratio of 4:1 are added. After ultrasonic treatment for 15-20 min, three freeze-vacuum-thaw cycles are carried out under liquid nitrogen environment. After flame sealing, the reaction is carried out at room temperature for 3-4 days. The resulting precipitate is collected by filtration, washed repeatedly with absolute ethanol, and then Soxhlet extracted with tetrahydrofuran at 130-140 °C for 24-30 h; finally, vacuum dried at 50-60 °C for 12-24 h to obtain COF5 with a yield of 77-80%; (6) Preparation method of COF6: Tetrakis(4-aminophenyl)ethylene and naphthalene-2,6-dicarbaldehyde are put into a flame-sealed tube according to a molar ratio of 1:2, and then o-dichlorobenzene, 1,4-dioxane and 6M acetic acid with a volume ratio of 10:10:1 are added. After ultrasonic treatment for 5-10 min, three freeze-vacuum-thaw cycles are carried out under liquid nitrogen environment. After flame sealing, the reaction is carried out at a temperature of 120-125 °C for 3-4 days. The resulting precipitate is collected by filtration, washed repeatedly with dimethylformamide, acetone and methanol, and then Soxhlet extracted with tetrahydrofuran at 120-130 °C for 24-48 h; finally, vacuum dried at 80-90 °C for 12-24 h to obtain COF6 with a yield of 58-62%; Step 2. Preparation of the COFs nanosheet solution of the sensing array unit to be measured: Six COFs are respectively mixed with ultrapure water to form a suspension of 1 mg / mL, and ultrasonicated sufficiently to form a nanosheet structure.

4. The fluorescent covalent organic framework sensing array for detecting disease marker polypeptides according to claim 3, wherein, The ultrasonic time in Step 2 ranges between 20-60 min.

5. The fluorescent covalent organic framework sensing array for detecting disease marker polypeptides according to claim 3, characterized in that, The molar ratio of 1,3,6,8-tetrakis(4-formylphenyl)pyrene and trans-1,4-cyclohexanediamine is 1:2; the volume ratio of n-butanol, 1,4-dioxane and 3M acetic acid is 4:1:3; The molar ratio of 1,3,6,8-tetrakis(4-formylphenyl)pyrene and p-phenylenediacetonitrile is 1:2; the volume ratio of 1,4-dioxane and 4M potassium hydroxide is 5:1; The molar ratio of benzene-1,3,5-tricarbaldehyde and 2,2'-bipyridine-5,5'-diamine is 2:3; the volume ratio of 1,4-dioxane and mesitylene is 2:

1.

6. The fluorescent covalent organic framework sensing array for detecting disease biomarker polypeptides according to claim 1, wherein, The polypeptide includes peptide segments from Alzheimer's disease Aβ, cardiovascular disease marker C-reactive protein, calcitonin gene-related peptide, incretin, atrial natriuretic peptide fragment or glucagon-like peptide hormones.

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