A bionic dual-mode biosensing probe and a preparation method and application thereof

Abstract

This invention provides a biomimetic bimodal biosensing probe, its preparation method, and its application, relating to the technical field of biosensing probes. The biosensing probe comprises: a fluorescent covalent organic framework material, platinum nanoparticles connected to the surface of the fluorescent covalent organic framework material, and a biotinylated anti-human immunoglobulin antibody. The biotinylated anti-human immunoglobulin antibody is used to bind to an antinuclear antibody, the fluorescent covalent organic framework material is used to generate a fluorescent detection signal, and the platinum nanoparticles are used to generate a colorimetric detection signal. This biosensing probe solves the problem of low detection accuracy of current ANA (anti-nuclear antibody) assays.

Description

Technical Field

[0001] This application relates to the field of biosensing probe technology, and in particular to a biomimetic dual-modal biosensing probe, its preparation method and application. Background Technology

[0002] Antinuclear antibodies (ANA) are important serological markers for diagnosing various autoimmune diseases (AIDs), such as systemic lupus erythematosus and Sjögren's syndrome. However, current clinical ANA testing mostly uses methods such as indirect immunofluorescence (IIF), and the results are usually given in the form of positive / negative or fluorescence titer grading. This is a qualitative or semi-quantitative test, lacking precise quantitative data and failing to reflect the true concentration level of ANA in the patient's body. At the same time, the detection method represented by IIF relies on manual microscopic observation and subjective interpretation. The interpretation standards of different operators and different laboratories are different, resulting in poor repeatability and consistency of the test results, and it is easy to produce false positives or false negatives. The accuracy and sensitivity of the test are limited. Therefore, there is an urgent need for a detection method that can achieve precise quantitative detection of ANA, reduce human subjective interference, and improve detection accuracy and repeatability. Summary of the Invention

[0003] In view of this, the purpose of this application is to propose a biomimetic bimodal biosensing probe, its preparation method and application, in order to solve the problem of low detection accuracy of ANA.

[0004] To achieve the above objectives, this application provides a biomimetic dual-modal biosensing probe, comprising:

[0005] The invention comprises a fluorescent covalent organic framework material, platinum nanoparticles attached to the surface of the fluorescent covalent organic framework material, and a biotinylated anti-human immunoglobulin antibody, wherein the biotinylated anti-human immunoglobulin antibody is used to bind to an antinuclear antibody, the fluorescent covalent organic framework material is used to generate a fluorescent detection signal, and the platinum nanoparticles are used to generate a colorimetric detection signal.

[0006] Optionally, the biotinylated anti-human immunoglobulin antibody is linked to the surface of the fluorescent covalent organic framework material via glutaraldehyde and streptavidin.

[0007] Based on the same inventive concept, this application also discloses a method for preparing a biomimetic dual-modal biosensing probe, comprising the following steps: The polyamino aromatic monomer and the dialdehyde aromatic monomer were dissolved in a solvent, glacial acetic acid was added, and the mixture was reacted at room temperature. The precipitate was collected, washed, and dried to obtain a fluorescent covalent organic framework material. A fluorescent covalent organic framework material dispersion was mixed with a platinum nanoparticle dispersion, followed by sonication, centrifugation, and washing to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface. Biotinylated anti-human immunoglobulin antibodies were linked to the surface of a fluorescent covalent organic framework material loaded with platinum nanoparticles via glutaraldehyde and streptavidin to obtain a biosensing probe for the assessment of autoimmune diseases.

[0008] Optionally, the biotinylated anti-human immunoglobulin antibody is linked to the surface of the fluorescent covalent organic framework material loaded with platinum nanoparticles via glutaraldehyde and streptavidin to obtain a biosensing probe for assessing autoimmune diseases, comprising: The fluorescent covalent organic framework material dispersion and the platinum nanoparticle dispersion are mixed, sonicated, centrifuged, and washed to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface. The fluorescent covalent organic framework material loaded with platinum nanoparticles was dispersed in PBS buffer, activated with glutaraldehyde, and washed to remove excess glutaraldehyde, thus obtaining a glutaraldehyde-modified composite framework. The glutaraldehyde-modified composite backbone was added to a streptavidin solution and incubated. After incubation, glycine was added, centrifuged, and washed to obtain the streptavidin-modified composite backbone. The streptavidin-modified composite backbone and biotinylated anti-human immunoglobulin antibody were mixed and incubated to obtain an incubation product. The incubation product was washed to remove free antibodies, resulting in a biosensing probe for the assessment of autoimmune diseases.

[0009] Optionally, the preparation of the platinum nanoparticle dispersion includes: Chloroplatinic acid and polyvinylpyrrolidone were dissolved in deionized water and stirred until homogeneous to obtain a precursor solution. Sodium borohydride solution was added to the precursor solution and stirred to obtain the platinum nanoparticle dispersion.

[0010] Optionally, the step of mixing and incubating the streptavidin-modified composite backbone with the biotinylated anti-human immunoglobulin antibody to obtain an incubation product includes: The streptavidin-modified composite backbone and the biotinylated anti-human immunoglobulin antibody were mixed at a mass ratio of (8-12):1 and incubated at (3-5) °C for (10-15) h to obtain the incubation product.

[0011] Optionally, the polyamino aromatic monomer is 1,3,5-tris(4-aminophenyl)benzene, and the dialdehyde aromatic monomer is 2,5-dimethoxyterephthalaldehyde.

[0012] Optionally, the process of mixing the fluorescent covalent organic framework material dispersion with the platinum nanoparticle dispersion, followed by sonication, centrifugation, and washing to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface includes: Fluorescent covalent organic framework material dispersion and platinum nanoparticle dispersion were mixed at a volume ratio of 1:(1-2), sonicated at room temperature for 30-60 min, centrifuged, and washed with ultrapure water to obtain fluorescent covalent organic framework material with platinum nanoparticles loaded on the surface.

[0013] Optionally, the step of dispersing the fluorescent covalent organic framework material loaded with platinum nanoparticles in PBS buffer, activating it with glutaraldehyde, and washing to remove excess glutaraldehyde to obtain a glutaraldehyde-modified composite framework includes: The fluorescent covalent organic framework material loaded with platinum nanoparticles was dispersed in PBS buffer, and glutaraldehyde (2-3% by volume) was added. The mixture was activated at room temperature for 1-3 hours, and excess glutaraldehyde was removed by washing to obtain the glutaraldehyde-modified composite framework.

[0014] Based on the same inventive concept, this disclosure also provides an application of a biosensing probe in the detection of antinuclear antibodies.

[0015] As described above, this application provides a biomimetic dual-modal biosensing probe comprising: a fluorescent covalent organic framework material, platinum nanoparticles connected to the surface of the fluorescent covalent organic framework material, and a biotinylated anti-human immunoglobulin antibody. The biotinylated anti-human immunoglobulin antibody binds to an antinuclear antibody, the fluorescent covalent organic framework material generates a fluorescent detection signal, and the platinum nanoparticles generate a colorimetric detection signal. The fluorescent covalent organic framework material possesses a self-luminescent structure, eliminating the need for external fluorescent dye labeling and avoiding background interference caused by the easy quenching and non-specific adsorption of exogenous fluorescent molecules. It stably outputs a fluorescent detection signal for quantifying ANA. The surface-modified platinum nanoparticles possess peroxidase-like catalytic activity, catalyzing the oxidation and colorimetric reaction of TMB substrates to output a colorimetric detection signal. The terminally coupled biotinylated anti-human immunoglobulin antibody specifically recognizes and captures ANA in the sample. Once ANA is captured, the two different detection signals displayed by the biosensing probe (fluorescent detection signal and colorimetric detection signal) can quantitatively analyze the ANA concentration. The two signal channels are independent of each other and do not interfere with each other. They can verify each other and achieve cross-validation of dual data for the same sample, which greatly reduces the probability of false positives and false negatives. At the same time, it eliminates the subjective error of traditional detection relying on manual interpretation and solves the defect of inaccurate single signal output results of existing biosensors. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in this application or related technologies, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A is a scanning electron microscope (SEM) image of a fluorescent covalent organic framework (COF) material; Figure 1 B is a scanning electron microscope (SEM) image of the COF@PtNPs composite framework; Figure 1 C is a scanning electron microscope (SEM) image of the MoB biosensing probe. Figure 1 D is a transmission electron microscope (TEM) image of the fluorescent covalent organic framework material COF; Figure 1 E is a transmission electron microscope (TEM) image of the COF@PtNPs composite framework; Figure 1 F is a transmission electron microscope (TEM) image of the MoB probe; Figure 1 G is the EDS element distribution mapping diagram of the MoB probe; Figure 2 A is a comparison of 560nm fluorescence emission spectra with and without ANA; Figure 2 B is a comparison of the 450nm UV-Vis absorption spectra with and without ANA; Figure 3 A shows the fluorescence and colorimetric dual signal change curves corresponding to different MoB probe concentrations; Figure 3 B represents the fluorescence and colorimetric dual signal change curves corresponding to different probe incubation times; Figure 3 C represents the fluorescence and colorimetric dual signal change curves corresponding to different probe incubation temperatures; Figure 4 A shows the fluorescence intensity variation at 560 nm under different gradient ANA concentrations; Figure 4 B is the standard curve for the linear fit between the logarithm of the ANA concentration in the fluorescence channel and the difference in fluorescence. Figure 4 C is the absorption spectrum of absorbance changes at 450 nm under different gradient ANA concentrations; Figure 4 D is the linear fitting standard curve of the difference between the logarithm of the ANA concentration in the colorimetric channel and the absorbance; Figure 5A represents the signal bubble distribution diagram of the healthy control group and AID patient samples under fluorescence detection mode; Figure 5 B is a bubble distribution diagram of signals in the healthy control group and AID patient samples under colorimetric detection mode; Figure 5 C represents the bubble distribution of signals in samples from healthy controls and AID patients detected by a commercial ELISA kit. Figure 5 D is a Bland-Altman consistency analysis chart of fluorescence channel detection results and ELISA detection results; Figure 5 E is a Bland-Altman consistency analysis graph between the colorimetric channel detection results and the ELISA detection results; Figure 5 F is the Bland-Altman consistency analysis graph between the fluorescence and colorimetric correction results and the ELISA detection results; Figure 6 A is a comparison chart of the distribution of ALT, AST, IgG, and ANA levels between healthy individuals and AIH patients; Figure 6 B is a comparison chart of ROC curves for a single biomarker and a combined diagnostic model of four indicators. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0019] It should be noted that, unless otherwise defined, the technical or scientific terms used in the embodiments of this application should have the ordinary meaning understood by one of ordinary skill in the art to which this application pertains. The terms "first," "second," and similar terms used in the embodiments of this application do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are only used to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0020] As described in the background section, autoimmune diseases (AIDs) are a group of chronic diseases in which the immune system abnormally attacks its own tissues, affecting approximately 5-8% of the global population, and their incidence continues to rise. AIDs can affect multiple organ systems, and if not diagnosed and intervened in a timely manner, they can lead to irreversible damage such as kidney failure, pulmonary fibrosis, and cardiovascular complications, hence the term "silent destroyer." AIDs typically have an insidious onset, with atypical early symptoms, and diagnosis has traditionally relied on a combination of clinical manifestations and laboratory tests. However, in many cases, irreversible organ damage has already occurred by the time of diagnosis. Therefore, early and accurate diagnosis of AIDs through monitoring specific biochemical markers in serum is of great significance for timely intervention and improving patient prognosis. Antinuclear antibodies (ANA) are the most important serological marker in the diagnosis of AIDs, and have significant diagnostic value in various AIDs, including systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, and autoimmune hepatitis.

[0021] Currently, the main methods for ANA detection in clinical practice include indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and Western blotting. Indirect immunofluorescence is the currently accepted standard. It involves incubating patient serum with HEp-2 cell matrix, staining with fluorescently labeled secondary antibody, and interpreting the fluorescence pattern and intensity under a fluorescence microscope by a professional, reporting the results qualitatively or semi-quantitatively. ELISA involves coating ANA target antigens onto microplates, incubating the sample, binding with enzyme-labeled secondary antibody, and developing the substrate color, then reading the absorbance value at 450 nm for semi-quantitative detection. However, existing ANA detection methods have significant limitations. The interpretation of results from indirect immunofluorescence relies on the operator's subjective experience, resulting in poor reproducibility between different laboratories. Especially in samples with weakly positive gray areas, subjective interpretation can easily lead to misclassification, producing false positive or false negative results.

[0022] More importantly, the diagnostic accuracy of a single qualitative ANA test is limited, making it difficult to meet the precise diagnostic needs of specific diseases such as autoimmune hepatitis. Existing biosensors mostly employ a single signal output mode, making them susceptible to environmental interference and non-specific adsorption, thus requiring improvement in detection reliability. Therefore, developing a sensitive, objective, and quantifiable dual-modal ANA detection platform, and combining its quantitative results with multiple clinical indicators for the precise diagnosis of autoimmune diseases, has significant clinical translational value.

[0023] The following is in conjunction with the appendix Figure 1-6 The embodiments of this application will be described in detail below.

[0024] In some embodiments, a biomimetic bimodal biosensing probe includes: The invention comprises a fluorescent covalent organic framework material, platinum nanoparticles attached to the surface of the fluorescent covalent organic framework material, and a biotinylated anti-human immunoglobulin antibody, wherein the biotinylated anti-human immunoglobulin antibody is used to bind to an antinuclear antibody, the fluorescent covalent organic framework material is used to generate a fluorescent detection signal, and the platinum nanoparticles are used to generate a colorimetric detection signal.

[0025] In this embodiment, the fluorescent covalent organic framework material possesses its own luminescent structure, eliminating the need for external fluorescent dye labeling and avoiding background interference caused by the easy quenching and non-specific adsorption of exogenous fluorescent molecules. It stably outputs a 560nm characteristic fluorescence signal for quantitative ANA. The surface-modified platinum nanoparticles possess peroxidase-like catalytic activity, catalyzing the oxidation and colorimetric reaction of TMB substrates, outputting a 450nm characteristic colorimetric absorption signal. The terminally coupled biotinylated anti-human immunoglobulin antibody specifically recognizes and captures ANA targets in the sample, establishing a bridge between immune recognition and dual optical signals. The fluorescence detection signal and the colorimetric detection signal have different detection wavelengths; the two signal channels are independent and do not interfere with each other, mutually verifying each other. This enables cross-validation of dual data for the same sample, significantly reducing the probability of false positives and false negatives. Simultaneously, it eliminates the subjective errors of traditional detection relying on manual visual interpretation, overcoming the inaccuracy of single-signal output results in existing biosensors.

[0026] In some embodiments, the biotinylated anti-human immunoglobulin antibody is linked to the surface of the fluorescent covalent organic framework material via glutaraldehyde and streptavidin.

[0027] In this embodiment, glutaraldehyde, as a bifunctional crosslinking agent, covalently binds to the surface-active groups of the composite nanoframework at one end and to streptavidin at the other, achieving stable immobilization of streptavidin. The highly specific and strong-affinity biological interaction between streptavidin and biotin enables targeted anchoring of biotinylated antibodies, allowing the antibody-antigen recognition ends to be arranged outwards in an orderly manner, maximizing the exposure of active binding sites for recognition and binding to antinuclear antibodies. Compared to traditional processes that directly physically adsorb antibodies, this method captures ANA more efficiently, thereby improving the accuracy of detection.

[0028] In some embodiments, a method for preparing a biomimetic bimodal biosensing probe includes the following steps: The polyamino aromatic monomer and the dialdehyde aromatic monomer were dissolved in a solvent, glacial acetic acid was added, and the mixture was reacted at room temperature. The precipitate was collected, washed, and dried to obtain a fluorescent covalent organic framework material. A fluorescent covalent organic framework material dispersion was mixed with a platinum nanoparticle dispersion, followed by sonication, centrifugation, and washing to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface. Biotinylated anti-human immunoglobulin antibodies were linked to the surface of a fluorescent covalent organic framework material loaded with platinum nanoparticles via glutaraldehyde and streptavidin to obtain a biosensing probe for the assessment of autoimmune diseases.

[0029] In this embodiment, a stepwise probe preparation process is used to connect a fluorescent covalent organic framework material with platinum nanoparticles and a biotinylated anti-human immunoglobulin antibody. The first step synthesizes a spiky covalent organic framework skeleton, which relies on monomer condensation to form a protruding spiky structure, providing numerous binding sites and simultaneously imparting stable endogenous fluorescence. The second step loads platinum nanoparticles, uniformly dispersing 5nm platinum particles on the surface of the spiky skeleton to ensure sufficient catalytic active sites. The third step connects the biotinylated anti-human immunoglobulin antibody to the surface of the platinum-loaded fluorescent covalent organic framework material using glutaraldehyde and streptavidin to form an immunorecognition unit for the antinuclear antibody. The entire preparation process is mild and reproducible, requiring no complex high-temperature and high-pressure equipment. During large-scale preparation, the probe morphology, linear detection range, and detection limit show minimal fluctuations, solving the problems of poor reproducibility, uneven functional component loading, and large batch-to-batch variations in detection performance in existing nanosensor probes, thus meeting the needs of clinical batch sample testing.

[0030] In some embodiments, the biotinylated anti-human immunoglobulin antibody is linked to the surface of the fluorescent covalent organic framework material loaded with platinum nanoparticles via glutaraldehyde and streptavidin to obtain a biosensing probe for assessing autoimmune diseases, comprising: The fluorescent covalent organic framework material dispersion and the platinum nanoparticle dispersion are mixed, sonicated, centrifuged, and washed to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface. The fluorescent covalent organic framework material loaded with platinum nanoparticles was dispersed in PBS buffer, activated with glutaraldehyde, and washed to remove excess glutaraldehyde, thus obtaining a glutaraldehyde-modified composite framework. The glutaraldehyde-modified composite backbone was added to a streptavidin solution and incubated. After incubation, glycine was added, centrifuged, and washed to obtain the streptavidin-modified composite backbone. The streptavidin-modified composite backbone and biotinylated anti-human immunoglobulin antibody were mixed and incubated to obtain an incubation product. The incubation product was washed to remove free antibodies, resulting in a biosensing probe for the assessment of autoimmune diseases.

[0031] In this embodiment, the first step involves activating the composite backbone with glutaraldehyde and thoroughly washing away free glutaraldehyde. This introduces sufficient active aldehyde groups onto the surface of the nanomaterial, providing covalent binding sites for streptavidin, while preventing residual glutaraldehyde from damaging subsequent protein activity. The second step involves incubating streptavidin followed by adding glycine to block unreacted aldehyde groups, eliminating the problem of exposed active groups on the carrier adsorbing serum contaminants, reducing background signal, and improving the signal-to-noise ratio of both fluorescence and colorimetric channels. The third step relies on the highly specific interaction between streptavidin and biotin to directionally bind biotinylated antibodies, ensuring that the antigen recognition ends of the antibodies are arranged outwards in an orderly manner, fully exposing active binding sites and improving ANA capture efficiency. Finally, washing removes unbound free antibodies, preventing excess free antibodies from raising the detection baseline. This layered modification process is conducted under mild conditions throughout, without damaging the intrinsic fluorescence properties of the fluorescent covalent organic framework material or causing the detachment and aggregation of surface platinum nanoparticles. The resulting probe retains a stable fluorescence signal, excellent enzyme-like catalytic ability, and efficient immune recognition capability. It has higher dual-modal detection sensitivity and can accurately quantify ANA in complex clinical serum matrices, effectively reducing false positives and false negatives, and is suitable for batch and accurate screening of autoimmune disease samples.

[0032] In some embodiments, the preparation of the platinum nanoparticle dispersion includes: Chloroplatinic acid and polyvinylpyrrolidone were dissolved in deionized water and stirred until homogeneous to obtain a precursor solution. Sodium borohydride solution was added to the precursor solution and stirred to obtain the platinum nanoparticle dispersion.

[0033] In this embodiment, the entire preparation process of the platinum nanoparticle dispersion is defined. First, a precursor aqueous solution is prepared using chloroplatinic acid as the platinum source and polyvinylpyrrolidone (PVP) as a stabilizer. Then, platinum nanoparticles are prepared by dropwise addition of sodium borohydride as a reducing agent, resulting in uniformly dispersed platinum nanoparticles that are not prone to agglomeration. PVP can coat the surface of the newly formed platinum particles to form a protective layer, preventing agglomeration during synthesis and ensuring uniform particle size. Sodium borohydride has strong reducing power; its slow, dropwise addition ensures stable and complete reduction of platinum ions, allowing the catalytic active sites on the particle surface to remain intact. If PVP is lacking in the preparation process, the platinum particles are prone to agglomeration, leading to localized particle accumulation when subsequently loaded onto the fluorescent framework, resulting in a significant decrease in catalytic colorimetric efficiency, weak absorbance signal, and a narrowed detection linear range. If sodium borohydride is added rapidly all at once, a large number of particles will precipitate instantly, resulting in inconsistent particle sizes and significant differences in catalytic performance between different batches of platinum particles. The platinum nanoparticle dispersion prepared by this process has good stability. After being loaded onto a fluorescent covalent organic framework material, it can be uniformly distributed on the surface of the barbed structure. It can stably catalyze the TMB colorimetric system to output colorimetric signals, ensuring the stability of the colorimetric channel signals and the clarity of the quantitative gradient of the entire dual-modal sensing probe, thereby improving the repeatability and accuracy of the detection results.

[0034] In some embodiments, the step of mixing and incubating the streptavidin-modified composite backbone with the biotinylated anti-human immunoglobulin antibody to obtain an incubation product includes: The streptavidin-modified composite backbone and the biotinylated anti-human immunoglobulin antibody were mixed at a mass ratio of (8-12):1 and incubated at (3-5) °C for (10-15) h to obtain the incubation product.

[0035] In this embodiment, the streptavidin-modified composite backbone and biotinylated anti-human immunoglobulin antibody are controlled at a mass ratio of (8-12):1. This ensures sufficient recognition antibody binds to the surface of the composite backbone, guaranteeing subsequent ANA capture capability, while avoiding excessive consumption of antibody raw materials and reducing background interference caused by unbound free antibody residues. A low-temperature environment of 3-5°C combined with 10-15 hours of incubation is used. The gentle low temperature avoids high temperature damage to the spatial conformation of the antibody protein, causing inactivation of immunological activity. Continuous incubation allows for sufficient contact between the solid and liquid phases, ensuring uniform antibody binding on the carrier surface. If the ratio of the streptavidin-modified composite backbone to the biotinylated anti-human immunoglobulin antibody is too low, insufficient antibody will be loaded on the carrier, resulting in decreased ANA capture efficiency in the sample and overall low fluorescence and colorimetric signals. If the ratio is too high, excess antibody will be difficult to wash away, raising the baseline signal and making it difficult to distinguish weak positive samples. High temperature and insufficient incubation time will lead to insufficient antibody binding and impaired activity, easily resulting in false negative results. The probes prepared using this range of parameters have stable specific recognition capabilities, and when used in clinical serum testing, the test results show small deviations and better overall repeatability.

[0036] In some embodiments, the polyamino aromatic monomer is 1,3,5-tris(4-aminophenyl)benzene, and the dialdehyde aromatic monomer is 2,5-dimethoxyterephthalaldehyde.

[0037] In this embodiment, two specific monomers are used to synthesize a fluorescent covalent organic framework material. These monomers undergo an imine condensation reaction to generate a covalent organic framework material with a typical spiky spherical structure and stable intrinsic fluorescence. 1,3,5-Tris(4-aminophenyl)benzene is a polyamino monomer, and 2,5-dimethoxyterephthalaldehyde is a dialdehyde monomer. The combination of these two monomers constructs a large π-conjugated molecular framework, generating a stable fluorescence signal without the need for additional fluorescent dyes, providing an independent fluorescence detection channel for the probe. Simultaneously, this monomer combination allows for precise control of the nanomaterial morphology, forming spherical particles with protruding spiky surfaces, significantly increasing the number of binding sites on the carrier surface. This facilitates subsequent loading of platinum nanoparticles and conjugated antibodies, improving ANA capture efficiency.

[0038] In some embodiments, the mixing of the fluorescent covalent organic framework material dispersion and the platinum nanoparticle dispersion, followed by sonication, centrifugation, and washing to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface includes: Fluorescent covalent organic framework material dispersion and platinum nanoparticle dispersion were mixed at a volume ratio of 1:(1-2), sonicated at room temperature for 30-60 min, centrifuged, and washed with ultrapure water to obtain fluorescent covalent organic framework material with platinum nanoparticles loaded on the surface.

[0039] In this embodiment, the volume ratio of the fluorescent covalent organic framework material dispersion to the platinum nanoparticle dispersion is 1:(1-2). This ensures that a sufficient amount of platinum particles are adsorbed onto the framework surface, providing ample catalytic sites for subsequent colorimetric detection, while preventing excessive free platinum particles from increasing the blank absorbance. Sonication at room temperature for 30-60 minutes breaks up platinum particle agglomerates, allowing fine platinum particles to be uniformly embedded in the framework's uneven, spiky structure, preventing localized accumulation or absence of particles. The composite framework prepared using these parameters exhibits fluorescence signals unaffected by the loading process, stable catalytic performance of the colorimetric channel, and clear signal gradients corresponding to different ANA concentrations, effectively improving detection repeatability and quantitative accuracy.

[0040] In some embodiments, the step of dispersing the fluorescent covalent organic framework material loaded with platinum nanoparticles in PBS buffer, activating it with glutaraldehyde, and washing to remove excess glutaraldehyde to obtain a glutaraldehyde-modified composite framework includes: The fluorescent covalent organic framework material loaded with platinum nanoparticles was dispersed in PBS buffer, and glutaraldehyde (2-3% by volume) was added. The mixture was activated at room temperature for 1-3 hours, and excess glutaraldehyde was removed by washing to obtain the glutaraldehyde-modified composite framework.

[0041] In this embodiment, 2%~3% volume fraction of glutaraldehyde is used to activate the fluorescent covalent organic framework material loaded with platinum nanoparticles at room temperature for 1~3 hours. This can generate a sufficient amount of active aldehyde groups on the surface of the fluorescent covalent organic framework material loaded with platinum nanoparticles, laying the foundation for subsequent stable binding of streptavidin. The entire activation process is carried out under mild room temperature conditions, which will not destroy the conjugated fluorescent structure of the fluorescent covalent organic framework material, nor will it cause the platinum nanoparticles on the surface to detach and aggregate.

[0042] In some embodiments, the step of adding the glutaraldehyde-modified composite backbone to a streptavidin solution for incubation, followed by the addition of glycine, centrifugation, and washing to obtain the streptavidin-modified composite backbone includes: The glutaraldehyde-modified composite backbone was added to a streptavidin solution and incubated for 3-5 hours. Glycine was added to make the glycine concentration in the solution reach 90-110 mM. After centrifugation and washing, the streptavidin-modified composite backbone was obtained.

[0043] In this embodiment, the incubation time of streptavidin and the blocking concentration of glycine were limited to ensure that streptavidin could fully bind to the surface of the composite matrix while completely blocking unreacted aldehyde groups, thus reducing non-specific adsorption during detection. An incubation time of 3-5 hours allows streptavidin to fully covalently bind to the aldehyde groups on the matrix surface, maximizing the sites on the matrix surface that can bind biotinylate antibodies. After incubation, glycine was added to 90-110 mM. The glycine reacts with the remaining free aldehyde groups, eliminating excess active groups on the matrix and preventing the adsorption of contaminating proteins during subsequent detection of serum samples, thus reducing interference from fluorescence and absorbance in the blank baseline.

[0044] In some embodiments, the application of a biomimetic bimodal biosensing probe in the assessment of autoimmune diseases includes: Add the antigen mixture corresponding to the antinuclear antibody to the solid-phase adsorption carrier, wash after incubation, add blocking solution to block, wash again to obtain the antigen-modified solid-phase carrier; An antinuclear antibody solution was added to the antigen-modified solid-phase support, and the mixture was incubated to allow the antinuclear antibody to specifically bind to the antigen. After washing, a solid-phase support loaded with antinuclear antibody was obtained. The biosensing probe is added to the solid-phase carrier loaded with antinuclear antibody, and incubated to allow the biosensing probe to specifically bind to the antinuclear antibody. The free probe is then washed away to obtain the solid-phase carrier to be detected. The solid-phase carrier to be tested is subjected to fluorescence detection and colorimetric detection to obtain the concentration of antinuclear antibodies for the assessment of autoimmune diseases.

[0045] In this embodiment, a multi-type ANA antigen mixture is first used to coat a solid-phase microporous carrier, which can broadly capture various subtypes of antinuclear antibodies in serum and effectively avoid the problem of partial ANA missed detection caused by coating with a single antigen. After coating, incubation, and washing, a blocking solution is introduced to block the exposed adsorption sites of the carrier, reducing the background signal rise caused by non-specific adsorption of contaminating proteins in serum from the source and improving the detection signal-to-noise ratio. Then, the serum sample to be tested is added for incubation, so that the ANA in the sample can specifically bind to the solid-phase antigen (antigen on the solid-phase carrier). After washing, unbound serum impurities are thoroughly removed, eliminating the interference of free contaminating proteins on subsequent signals. Then, the specially designed biosensor probe of this invention is added for incubation. The biotinylated anti-human immunoglobulin antibody on the probe can accurately anchor the ANA already bound to the solid phase. Washing again removes unbound free probes, minimizing baseline interference caused by irrelevant probes. Finally, fluorescence and colorimetric detection were performed sequentially on the same solid-phase carrier. ANA concentration was calculated using two independent optical signals, achieving dual-signal cross-validation. This overcomes the shortcomings of traditional indirect immunofluorescence methods, which rely on subjective interpretation by laboratory personnel and suffer from poor reproducibility between different laboratories. The entire operation process is clear and the conditions are mild. It can be directly adapted to general laboratory ELISA readers for automated readings, outputting continuous and comparable precise quantitative ANA values. This not only allows for direct differentiation of serum ANA levels between healthy individuals and patients with autoimmune diseases, enabling early disease screening, but also allows for the combination of quantitative ANA values ​​with routine liver function and immune indicators such as ALT, AST, and IgG to build multi-parameter joint diagnostic models. This significantly improves the diagnostic efficacy for specific autoimmune diseases such as autoimmune hepatitis, providing objective and quantitative data support for clinical disease staging, dynamic monitoring of treatment effects, and prognostic assessment. It possesses strong clinical translational and practical application value.

[0046] In some embodiments, the step of performing fluorescence detection and colorimetric detection on the solid-phase support to be detected to obtain the antinuclear antibody concentration includes: An aqueous solution was added to the solid support to be tested, and a fluorescence signal was collected to obtain the concentration of the first antinuclear antibody. A chromogenic substrate system was added to the solid support to be tested to carry out a chromogenic reaction. After the reaction was terminated by adding a stop solution, the absorbance signal was collected to obtain the concentration of the second antinuclear antibody. The concentration of the first antinuclear antibody is corrected by the concentration of the second antinuclear antibody to obtain the concentration of the antinuclear antibody.

[0047] In this embodiment, pure water is first added to the solid-phase support to directly read the intrinsic fluorescence signal generated by the fluorescent covalent organic framework material, quickly obtaining the first set of ANA concentration data. The detection process requires no additional reaction reagents, making the operation simple and rapid. Subsequently, TMB chromogenic substrate is added to the same well, relying on the enzyme-like activity of platinum nanoparticles on the probe surface to catalyze the colorimetric reaction. After adding a stop solution to stabilize the chromogenic product, the absorbance at 450 nm is read, obtaining the second set of independent quantitative values. The fluorescence channel is susceptible to interference from endogenous fluorescent impurities in serum, while the colorimetric channel is susceptible to the influence of reducing substances in the sample. These two sources of interference are completely different. Correcting the fluorescence data with colorimetric data can mutually cancel out the errors caused by both types of interference, filtering abnormal fluctuation signals and avoiding false positives and false negatives in single-channel detection. This correction method requires no additional test samples, does not increase consumables or operation time. Validated with clinical serum samples, the corrected final ANA quantitative results show higher consistency with commercial ELISA kits, significantly improving the resolution of weakly positive borderline samples, making the detection results of low-concentration early ANA samples more accurate and reliable, and providing objective and stable quantitative data for early screening and dynamic monitoring of autoimmune diseases.

[0048] In some embodiments, adding the biosensing probe to the solid-phase support loaded with the antinuclear antibody and incubating it to allow the biosensing probe to specifically bind to the antinuclear antibody includes: Add 2.0 mg / mL of the biosensing probe to the solid-phase carrier loaded with the antinuclear antibody, and incubate at 37°C for 60 min to allow the biosensing probe to specifically bind to the antinuclear antibody.

[0049] In this embodiment, the probe concentration was fixed at 2.0 mg / mL. At this concentration, the ANA binding sites on the solid-phase support surface reached saturation. Further increasing the probe concentration would not further enhance the signal but would only waste probe reagents and increase the amount of unbound free probe residue, raising the blank detection baseline. Incubation at 37°C for 60 min ensured sufficient binding of the probe to ANA, resulting in high detection accuracy. Using these parameters for the incubation reaction, the sensor could stably maintain a wide linear detection range of 0.1–1000 ng / mL, with a lower detection limit and small repeated detection error for the same batch of samples. It could form a clear and distinguishable signal gradient for both low-ANA serum from healthy individuals and high-ANA serum from patients, effectively improving the accuracy and repeatability of autoimmune disease sample screening.

[0050] The following describes specific embodiments and appendices. Figure 1-6 The above embodiments are explained.

[0051] Example 1 The fabrication of a biosensor includes: S1: Synthesis of Fluorescent Covalent Organic Framework (COF): 10.5 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 8.74 mg of 2,5-dimethoxyterephthalaldehyde (DMTP) were completely dissolved in 4.5 mL of acetonitrile, and the dissolution was promoted by sonication. Then, 0.4 mL of glacial acetic acid was added as a catalyst, and the mixture was thoroughly mixed and allowed to stand at room temperature for 72 h. After the reaction was completed, the precipitate was collected by centrifugation, washed three times each with tetrahydrofuran and anhydrous ethanol, and finally dried under vacuum. The resulting precipitate was the fluorescent covalent organic framework material, which was collected for later use.

[0052] S2: The preparation of the platinum nanoparticle dispersion (PtNPs solution) includes: dissolving 39 μL of chloroplatinic acid (H2PtCl6, 1M) and 51.4 mg of polyvinylpyrrolidone (PVP, MW=10,000) in 50 mL of deionized water and stirring until homogeneous to obtain a precursor solution. Separately, dissolve 17.6 mg of sodium borohydride (NaBH4) in 30 mL of deionized water. Under vigorous stirring, slowly add the NaBH4 solution dropwise to the above precursor solution until the reaction system turns dark brown and bubbles are generated. Continue stirring for 30 min to ensure complete reduction, obtaining the PtNPs solution, which is then stored in the dark for later use.

[0053] S3: Preparation of fluorescent covalent organic framework material (COF@PtNPs) loaded with platinum nanoparticles: 500 μL of COF dispersion (300 μg / mL) was mixed with 150 μL of PtNPs solution (volume ratio 1:0.5), and ultrasonicated at room temperature for 30 min to uniformly load PtNPs onto the COF surface. The precipitate was collected by centrifugation to obtain COF@PtNPs, which were washed three times with ultrapure water and resuspended for later use.

[0054] S4: Preparation of the streptavidin-modified composite backbone (COF@PtNPs@SA): 1 mg COF@PtNPs was dispersed in 2 mL PBS (phosphate-buffered saline, 0.01 M, pH 7.4), and glutaraldehyde (GA) was added to a final concentration of 2.5% (v / v). Activation was performed at room temperature for 2 h, followed by repeated centrifugation and washing to remove free GA. The resulting solid was redispersed and 100 μL of streptavidin (SA, 1 mg / mL) was added, followed by sonication for 4 h. Glycine was then added to a final concentration of 100 mM, and incubation was performed for 30 min to block unreacted aldehyde groups. The mixture was then centrifuged, washed, and resuspended for later use.

[0055] S5: Preparation of biosensing probes (MoB probes, COF@PtNPs@SA@Bio-Ab): The COF@PtNPs@SA complex (0.5 mg / mL) was mixed with biotinylated rabbit anti-human IgG antibody (Bio-Ab) at a mass ratio of 10:1 and incubated gently at 4°C for 12 h. After incubation, the free antibody was removed by centrifugation at 4°C, and the probes were washed twice with PBS containing 0.1% BSA. The resulting MoB probes were resuspended in PBS and stored at 4°C for later use.

[0056] Example 2 Application methods of biosensing probes include: 1. Solid-phase antigen coating procedure Reagent abbreviation notes: PBST: Phosphate buffer (PBS, pH=7.4) containing 0.05% Tween-20. Tween-20: Tween 20 is a nonionic surfactant used to wash away non-specifically adsorbed proteins in microplates.

[0057] Detailed operating procedures: (1) Preparation of ANA target antigen mixture: Mix ten antigens, namely dsDNA (double-stranded DNA), histone, ribosomal P protein, nRNP / Sm (ribonucleoprotein / Smith antigen), Sm (Smith antigen), SS-A (Sjögren's syndrome antigen A), SS-B (Sjögren's syndrome antigen B), Scl-70 (scleroderma 70 antigen), Jo-1 (polymyositis antigen 1) and centromere protein in equal mass ratio, dilute with PBS buffer, and prepare an antigen mixture working solution with a concentration of 5 μg / mL.

[0058] (2) Antigen adsorption coating: Take a 96-well polystyrene microplate with high binding force, add 100 μL of the above-prepared ANA target antigen mixed working solution to each microplate, seal the microplate and place it in a low temperature environment of 4℃ for 12h. Relying on physical adsorption, multiple antigens are uniformly fixed on the solid surface of the inner wall of the microplate.

[0059] (3) Washing to remove unbound free antigens: After incubation, discard the liquid in the wells, add sufficient PBST washing solution to each well, soak and wash 3 times, and thoroughly shake off the residual liquid in the wells after each wash to remove free antigens that have not been adsorbed on the plate wall, so as to avoid free antigens interfering with subsequent immune binding reactions.

[0060] (4) Microplate blocking treatment: Add 200 μL of 1% casein-PBS blocking solution to each well and incubate the microplate at 37°C for 2 hours. Casein can fill the blank adsorption sites on the inner wall of the microwell that are not occupied by antigens, block the non-specific adsorption of impurities in serum, and reduce the background signal of detection.

[0061] (5) Secondary washing and storage: After sealing, wash the microwells three times with PBST washing solution to remove excess blocking solution; the processed antigen-coated microplates can be directly used for subsequent testing, or sealed and stored at 4°C for short-term use.

[0062] 2. Complete ANA sample detection steps Notes on reagent / material abbreviations: TMB: 3,3',5,5'-Tetramethylbenzidine, a chromogenic substrate, undergoes oxidation and color development catalyzed by platinum nanoparticle peroxidase.

[0063] Detailed step-by-step instructions: (1) Sample loading and target capture: The serum sample to be tested was diluted 5-fold with PBS buffer beforehand; 100 μL of diluted serum sample or an equal volume of ANA standard solutions of different concentration gradients was added to the wells after antigen coating, and the microplate was incubated at 37°C for 60 min. In this step, the various antigens fixed on the walls of the microwells will specifically capture the antinuclear antibodies (ANA) present in the sample, forming a solid-phase antigen-ANA immune complex. After incubation, the microwells were washed 3 times with PBST washing solution to thoroughly remove unbound proteins and free impurities from the serum.

[0064] (2) Biomimetic probe specific binding incubation: 100 μL of 2.0 mg / mL MoB probe working solution (from Example 1) was added to the washed microwells and incubated at 37°C in the dark for 60 min. The biotinylated anti-human IgG antibody conjugated to the probe surface can specifically recognize and bind to ANA that has been immobilized on the plate wall, forming a solid-phase antigen-ANA-MoB probe ternary stable immune complex. After incubation, the microwells were washed three times with PBST to completely wash away the free MoB probes that were not bound to ANA, thus eliminating baseline interference caused by the free probes.

[0065] (3) Acquisition and quantitative calculation of fluorescence mode signal: Add 100 μL of ultrapure water to the cleaned microwells, use a multi-functional microplate reader, set the excitation wavelength to 490 nm, and collect the fluorescence intensity values ​​of each well at the emission wavelength of 560 nm, which are recorded as F; simultaneously set blank control wells (without ANA target) and read the blank fluorescence intensity F0; use the difference F-F0 as the effective signal of the fluorescence channel, and convert it with the pre-established fluorescence standard curve to obtain the corresponding ANA quantitative concentration.

[0066] (4) Colorimetric signal acquisition and quantitative calculation: No system replacement is required for the same microwell. 100 μL of commercially available single-component TMB chromogenic substrate solution is added directly, and the mixture is allowed to stand at 37°C in the dark for 15 min. The platinum nanoparticles loaded on the probe surface possess peroxidase-like catalytic activity, catalyzing the oxidation of TMB to produce a blue chromogenic product. After the chromogenic time is reached, 100 μL of 2 mol / L sulfuric acid is added to each well to terminate the chromogenic reaction, and the solution changes from blue to a stable yellow. The absorbance value of each well is then read at a detection wavelength of 450 nm using a microplate reader and recorded as A. Simultaneously, the absorbance A0 of the blank control well is read. The difference A-A0 is used as the effective signal of the colorimetric channel, and the ANA concentration is calculated by referring to the colorimetric standard curve.

[0067] (5) Data correction: The ANA quantitative value obtained from the colorimetric channel is used as the correction benchmark to correct the quantitative result of the fluorescence channel, so as to obtain the final accurate ANA detection concentration for subsequent autoimmune disease assessment and analysis.

[0068] Example 3 Performance verification experiments and results analysis of biosensor probes: The biosensing probe prepared in Example 1 was tested, and the detection process and results are as follows: The morphology of the synthesized COF, COF@PtNPs and MoB probes was observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 1 A and Figure 1 D represents the SEM and TEM images of COF, respectively. From these images, it can be observed that COF has a uniform morphology, exhibiting a typical spiky spherical structure, with a particle size of approximately 357.91 ± 20.37 nm. Compared to COF, the SEM images of COF@PtNPs show... Figure 1 B) and TEM Figure 1 In the E image, a large number of PtNPs are clearly identifiable on the COF surface. The PtNPs have a particle size of approximately 5 nm and are uniformly dispersed on the COF surface, which has a rougher surface texture than the COF itself. In the MoB probe SEM (…),… Figure 1 C) and TEM Figure 1 Image F shows that it retains its characteristic spiky morphology, with a denser and rougher surface, consistent with the introduction of SA and Bio-Ab protein layers. Energy dispersive spectroscopy (EDS) elemental mapping image ( Figure 1 G) shows that C, N, O, S, and Pt elements are uniformly distributed in the MoB probe nanostructure, and the presence of S element further confirms the successful introduction of SA and Bio-Ab proteins. The above TEM, SEM, and EDS characterization results indicate that the preparation of the MoB probe was successful.

[0069] like Figure 2As shown in Figure A, fluorescence detection indicates that the characteristic fluorescence peak at approximately 560 nm is significantly stronger in the presence of ANA than in the absence of ANA, demonstrating the feasibility of the fluorescence detection channel in this sensor. Similarly, as... Figure 2 As shown in Figure B, a strong characteristic absorption peak can be detected at approximately 450 nm when ANA is present, while only a weak background absorption peak is detected when ANA is absent, proving the feasibility of the colorimetric detection channel of this sensor.

[0070] Example 4 Experiment on optimization of reaction conditions for ANA detection To achieve higher sensing performance, the reaction conditions for ANA detection were optimized. Considering that the MoB probe concentration is the main factor controlling reaction efficiency, it was optimized first. For example... Figure 3 As shown in Figure A, when detecting 1 ng / mL ANA, the dual-modal signal gradually increased as the MoB probe concentration increased from 0 to 2.0 mg / mL, reaching a plateau at 2.0 mg / mL. Further increases in concentration did not result in significant gain. This may be because the binding sites of solid-phase ANA have reached saturation, and excessively high probe concentrations cannot further enhance the signal. Therefore, 2.0 mg / mL is the optimal concentration of the MoB probe.

[0071] Subsequently, the incubation time of the MoB probe and solid-phase trapped ANA was optimized by comparing the intensity of the dual-modal signal generated at different incubation times. Figure 3 As shown in Figure B, when detecting 1 ng / mL ANA, increasing the incubation time significantly improved the dual-modal signal intensity, reaching its peak at 60 min. After 60 min, the signal intensity no longer increased with further incubation, indicating that the antigen-ANA-MoB probe ternary immune complex had reached binding equilibrium. Therefore, 60 min was selected as the optimal incubation time.

[0072] In addition, the effect of incubation temperature on sensor performance was investigated. Figure 3 As shown in Figure C, the bimodal signal increases with increasing temperature, reaching a maximum at 37°C. Above 37°C, the signal decreases significantly, likely due to partial denaturation of Bio-Ab and dissociation of the immune complex caused by high temperatures. Therefore, 37°C was chosen as the optimal incubation temperature.

[0073] To evaluate the analytical performance of the proposed biosensor under optimal experimental conditions, a range of ANA standard solutions at various concentrations were detected using the prepared biosensor. For fluorescence-based detection, such as... Figure 4 As shown in Figure A, with increasing ANA concentration, the amount of MoB probe enriched on the solid phase increased, and the fluorescence intensity at approximately 560 nm gradually increased. We also observed a significant linear relationship between F-F0 and the logarithm of ANA concentration (0.1-1000 ng / mL). Figure 4The linear regression equation for F-F0 in B is y = 223.878lgC + 503.786 (R²) 2 =0.9903). For colorimetric mode detection, such as Figure 4 As shown in C and D, the absorbance at approximately 450 nm gradually increases with increasing ANA concentration. The linear regression equation for the logarithm of ANA concentrations in the range of A-A0 and 0.1-1000 ng / mL is y = 0.14056lgC + 0.153 (R²). 2 =0.99654). The LODs calculated using the 3SD / m criterion were as low as 0.271 ng / mL and 0.516 ng / mL in fluorescence and colorimetric modes, respectively (SD is the standard deviation of 20 blank measurements, and m is the slope of the calibration curve). Both detection modes exhibited good linearity in the range of 0.1–1000 ng / mL, cross-validated each other, effectively eliminating matrix interference and achieving reliable quantification. This indicates that the constructed dual-modal biosensor has advantages such as high sensitivity, wide linear range, and accurate quantification.

[0074] To explore the applicability of the constructed biosensor, 160 clinical serum samples (80 healthy controls and 80 AID patients) were tested, and ANA levels were simultaneously detected using both a dual-modal biosensor and a commercial ELISA (enzyme-linked immunosorbent assay). Figure 5 A and B represent the bubble diagram signal results for the fluorescence mode and colorimetric mode of the dual-modal sensor, respectively. Figure 5 C represents the ELISA results. It can be seen that in both fluorescence and colorimetric modes, the signal intensity in AID patient samples was significantly higher than that in healthy controls. The two signal types showed significant differences between the two groups, indicating that both biosensing and ELISA methods are sensitive to ANA and can be used to distinguish AID patients from healthy individuals. Furthermore, we explored the methodological consistency between the dual-modal sensor and ELISA detection. Figure 5 As shown in D, E, and F, the Bland-Altman analysis results indicate that the sensor's overall output, fluorescence mode, and colorimetric mode are highly consistent with ELISA. The mean deviation of the sensor's overall output is -0.3819%, and the 95% agreement limit ranges from -3.624% to 2.860%, further demonstrating the reliability of the dual-modal cross-validation strategy and the feasibility of the constructed biosensor for the quantitative detection of ANA in complex serum samples.

[0075] To validate the clinical value of quantitative ANA testing in precision diagnosis, the quantitative ANA results from the sensor were combined with ALT (alanine aminotransferase), AST (alanine aminotransferase), and IgG (immunoglobulin G) to construct a multi-indicator combined diagnostic model for AIH (autoimmune liver disease). Fifteen AIH patients and 18 healthy controls were selected for analysis. Figure 6As shown in Figure A, the distribution of the four biomarkers differed significantly between AIH patients and healthy controls, with all indicators being higher in AIH patients than in healthy controls. ROC curve analysis was performed on each individual indicator and the combined model, and the results are as follows. Figure 6 As shown in Figure B, the diagnostic capabilities of each individual indicator are limited. IgG had the highest AUC (diagnostic efficacy evaluation index) (0.785), followed by ANA (0.685), AST (0.563), and ALT (0.424). The combined AUC of the four indicators reached 0.967, significantly better than any single indicator. These results indicate that the constructed dual-modal biosensor has good clinical applicability in quantitative ANA detection, and combining quantitative ANA with multiple clinical indicators can significantly improve the accuracy of AIH diagnosis, demonstrating the important clinical translational value of quantitative ANA detection.

[0076] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of this application is limited to these examples; under the concept of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of the embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0077] Although this application has been described in conjunction with specific embodiments thereof, many substitutions, modifications, and variations of these embodiments will be apparent to those skilled in the art from the foregoing description. For example, other memory architectures (e.g., dynamic RAM (DRAM)) may be used with the embodiments discussed.

[0078] The embodiments of this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the claims of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the embodiments of this application should be included within the protection scope of this application.

Claims

1. A biomimetic dual-modal biosensing probe, characterized in that, include: The invention comprises a fluorescent covalent organic framework material, platinum nanoparticles attached to the surface of the fluorescent covalent organic framework material, and a biotinylated anti-human immunoglobulin antibody, wherein the biotinylated anti-human immunoglobulin antibody is used to bind to an antinuclear antibody, the fluorescent covalent organic framework material is used to generate a fluorescent detection signal, and the platinum nanoparticles are used to generate a colorimetric detection signal.

2. The biosensing probe according to claim 1, characterized in that, The biotinylated anti-human immunoglobulin antibody is linked to the surface of the fluorescent covalent organic framework material via glutaraldehyde and streptavidin.

3. A method for preparing a biomimetic dual-modal biosensing probe, characterized in that, Includes the following steps: The polyamino aromatic monomer and the dialdehyde aromatic monomer were dissolved in a solvent, glacial acetic acid was added, and the mixture was reacted at room temperature. The precipitate was collected, washed, and dried to obtain a fluorescent covalent organic framework material. A fluorescent covalent organic framework material dispersion was mixed with a platinum nanoparticle dispersion, followed by sonication, centrifugation, and washing to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface. Biotinylated anti-human immunoglobulin antibodies were linked to the surface of a fluorescent covalent organic framework material loaded with platinum nanoparticles via glutaraldehyde and streptavidin to obtain a biosensing probe for the assessment of autoimmune diseases.

4. The preparation method according to claim 3, characterized in that, The biotinylated anti-human immunoglobulin antibody is linked to the surface of the fluorescent covalent organic framework material loaded with platinum nanoparticles via glutaraldehyde and streptavidin to obtain a biosensing probe for assessing autoimmune diseases, comprising: The fluorescent covalent organic framework material dispersion and the platinum nanoparticle dispersion are mixed, sonicated, centrifuged, and washed to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface. The fluorescent covalent organic framework material loaded with platinum nanoparticles was dispersed in PBS buffer, activated with glutaraldehyde, and washed to remove excess glutaraldehyde, thus obtaining a glutaraldehyde-modified composite framework. The glutaraldehyde-modified composite backbone was added to a streptavidin solution and incubated. After incubation, glycine was added, centrifuged, and washed to obtain the streptavidin-modified composite backbone. The streptavidin-modified composite backbone and biotinylated anti-human immunoglobulin antibody were mixed and incubated to obtain an incubation product. The incubation product was washed to remove free antibodies, resulting in a biosensing probe for the assessment of autoimmune diseases.

5. The preparation method according to claim 3, characterized in that, The preparation of the platinum nanoparticle dispersion includes: Chloroplatinic acid and polyvinylpyrrolidone were dissolved in deionized water and stirred until homogeneous to obtain a precursor solution. Sodium borohydride solution was added to the precursor solution and stirred to obtain the platinum nanoparticle dispersion.

6. The preparation method according to claim 4, characterized in that, The process involves mixing and incubating the streptavidin-modified composite backbone with a biotinylated anti-human immunoglobulin antibody to obtain an incubation product comprising: The streptavidin-modified composite backbone and the biotinylated anti-human immunoglobulin antibody were mixed at a mass ratio of (8-12):1 and incubated at (3-5) °C for (10-15) h to obtain the incubation product.

7. The preparation method according to claim 3, characterized in that, The polyamino aromatic monomer is 1,3,5-tris(4-aminophenyl)benzene, and the dialdehyde aromatic monomer is 2,5-dimethoxy-terephthalaldehyde.

8. The preparation method according to claim 4, characterized in that, The process involves mixing a fluorescent covalent organic framework material dispersion with a platinum nanoparticle dispersion, followed by sonication, centrifugation, and washing to obtain a fluorescent covalent organic framework material with platinum nanoparticles loaded on its surface. This includes: Fluorescent covalent organic framework material dispersion and platinum nanoparticle dispersion were mixed at a volume ratio of 1:(1-2), sonicated at room temperature for 30-60 min, centrifuged, and washed with ultrapure water to obtain fluorescent covalent organic framework material with platinum nanoparticles loaded on the surface.

9. The preparation method according to claim 4, characterized in that, The process involves dispersing the fluorescent covalent organic framework material loaded with platinum nanoparticles in PBS buffer, activating it with glutaraldehyde, and washing to remove excess glutaraldehyde to obtain a glutaraldehyde-modified composite framework, comprising: The fluorescent covalent organic framework material loaded with platinum nanoparticles was dispersed in PBS buffer, and glutaraldehyde (2-3% by volume) was added. The mixture was activated at room temperature for 1-3 hours, and excess glutaraldehyde was removed by washing to obtain the glutaraldehyde-modified composite framework.

10. The application of the biosensing probe of claim 1 in the detection of antinuclear antibodies.

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