Magnetic fluorescent molecularly imprinted nanoparticles, and preparation method and application thereof

Abstract

This invention discloses a magnetic fluorescently imprinted nanoparticle, its preparation method, and its application. The nanoparticle comprises amino-functionalized magnetic nanoparticles and a fluorescently imprinted polymer layer coated on their surface, wherein the fluorescently imprinted polymer layer has an imprinted cavity; the imprinted molecule in the fluorescently imprinted polymer layer is a cTnI-specific C-terminal epitope peptide. Functional monomers are screened using computational chemistry, and nanomolecularly imprinted polymers (MIPs) are synthesized using epitope imprinting technology. Fluorescent monomers are introduced to impart autofluorescence response properties, forming a label-free detection system. These nanoparticles exhibit highly efficient adsorption and specific recognition of cTnI protein, with a detection sensitivity of 1 pg / mL (R²=0.98). They show extremely low non-specific adsorption to common interfering proteins and effectively resist interference from complex biological matrices, providing a new approach for rapid clinical cTnI detection.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and detection technology, specifically designing a magnetic fluorescent molecularly imprinted nanoparticle, its preparation method, and its application. Background Technology

[0002] Acute myocardial infarction (AMI) is one of the leading causes of cardiovascular disease mortality worldwide. The golden window for treatment is within 3-6 hours of onset; therefore, rapid and accurate diagnosis is crucial for the treatment of AMI patients. Cardiac troponin I (cTnI), as a specific biomarker for myocardial injury, typically possesses high sensitivity and specificity, and can be stably detected even in the early stages of the disease. It provides core evidence for the diagnosis and prognostic assessment of cardiovascular diseases and has become the "gold standard" for clinical diagnosis of AMI. However, existing commercial cTnI detection technologies still face many challenges. For example, enzyme-linked immunosorbent assay (ELISA) is cumbersome and time-consuming, typically requiring 2-4 hours, and is easily affected by matrix interference. While chemiluminescence immunoassay has high sensitivity, it relies on expensive instruments and specific antibodies; antibody preparation is time-consuming, unstable, and costly, making it difficult to meet the rapid testing needs of primary healthcare institutions. Furthermore, traditional detection methods often require complex sample pretreatment and washing steps, further limiting their application in emergency rapid diagnostic scenarios. Therefore, developing a rapid cTnI detection technology that is highly sensitive, highly specific, easy to operate, and cost-effective has become an important requirement for clinical laboratory AMI.

[0003] Molecular imprinting technology (MIT) is a biomimetic technique for preparing "artificial antibodies." Through the specific interaction between template molecules and functional monomers, recognition sites complementary to the spatial structure and functional groups of the template molecule are formed in the polymer, exhibiting affinity and specificity comparable to natural antibodies. Compared to natural antibodies, molecularly imprinted polymers (MIPs) offer significant advantages such as shorter preparation cycles, lower costs, higher stability, and ease of mass production, showing broad application prospects in biomarker detection, drug analysis, and environmental monitoring. Epitope imprinting technology, as an important branch of protein molecular imprinting, selects specific short peptide fragments of the target protein as templates, effectively solving problems such as the susceptibility of intact protein templates to denaturation, difficulty in forming imprinted sites, low elution efficiency, and high costs, significantly improving the recognition performance of MIPs for large protein molecules.

[0004] In recent years, the combination of magnetic nanomaterials and molecular imprinting technology has provided a new technical approach for rapid detection. Magnetic nanoparticles (MNPs) not only facilitate the rapid separation and enrichment of target analytes, but also serve as immobilization carriers for epitope templates. Simultaneously, by introducing fluorescent monomers into the polymerization system, molecularly imprinted polymers (MIPs) can be endowed with autofluorescence response properties, constructing a label-free "self-signal" detection platform, simplifying the detection process and improving detection efficiency. For example, Liu et al. prepared magnetic fluorescent microinjections (MIPs) targeting angiotensin-converting enzyme 2 (ACE2) using epitope imprinting technology, achieving rapid, wash-free detection with a detection limit as low as 0.081 pg / mL (Liu et al., Anal. Chem. 2024, 96, 7602−7608); Zhang et al. developed brain natriuretic peptide (BNP)-targeting magnetic MIPs, with a detection time of only 7 minutes and high consistency with clinical detection methods (Zhang et al., Adv. Healthcare Mater. 2023, 2300146). These studies demonstrate that magnetic fluorescent epitope imprinting technology has unique advantages in the rapid detection of protein biomarkers.

[0005] Despite the significant progress made in the field of biological detection by molecular imprinting technology, there are still areas for optimization in the epitope imprinting detection of cTnI. First, the screening of cTnI epitopes lacks a systematic approach and needs to be combined with bioinformatics tools to screen for epitope fragments with high specificity and stable structure. Second, the response speed and sensitivity of existing detection systems still need to be further improved to meet the rapid detection needs of clinical emergencies. Summary of the Invention

[0006] Purpose of the invention: To address the problems existing in the prior art, this invention provides magnetic fluorescent molecularly imprinted nanoparticles. By systematically screening cardiac troponin I (cTnI) specific epitopes, optimizing the combination of functional monomers, and improving the response speed and sensitivity of the detection system, this invention solves the technical problems of insufficient recognition accuracy and affinity, limited recognition specificity, and difficulty in meeting the rapid detection needs of clinical emergency departments in existing cTnI epitope imprinting detection technologies.

[0007] The present invention also provides a method for preparing the magnetic fluorescent molecularly imprinted nanoparticles and their applications.

[0008] Technical solution: The present invention discloses a magnetic fluorescent molecularly imprinted nanoparticle, comprising amino-functionalized magnetic nanoparticles and a fluorescent molecularly imprinted polymer layer coated on its surface, wherein the fluorescent molecularly imprinted polymer layer has an imprinted cavity; the imprinted molecule of the fluorescent molecularly imprinted polymer layer is a cTnI-specific C-terminal epitope peptide.

[0009] Furthermore, the amino acid sequence of the cTnI-specific C-terminal epitope peptide is ALSGMEGRKKKFES (SEQ ID NO.1).

[0010] The method for preparing magnetic fluorescent molecularly imprinted nanoparticles according to the present invention includes the following steps:

[0011] (1) Fe3O4 magnetic nanoparticles were synthesized by solvothermal method, and amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 were synthesized by sol-gel method;

[0012] (2) The amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 obtained in step (1) are dispersed in a buffer solution, and after reacting with a heterobifunctional crosslinking agent, cTnI-specific C-terminal epitope peptide is added for coupling to obtain a magnetic peptide template (magNP-P) with template peptide immobilized on the surface.

[0013] (3) Dissolve the thermosensitive monomer, hydrophilic monomer, cationic functionalized monomer, crosslinking agent and fluorescent monomer in water to obtain an aqueous solution. Dissolve the hydrophobic monomer in an organic solvent and mix it with the above aqueous solution to obtain a mixed solution. Disperse the product obtained in step (2) in water and add it to the mixed solution. Under the protection of inert gas, add an initiator and a catalyst to carry out a polymerization reaction. After treatment, obtain magnetic fluorescent molecularly imprinted nanoparticles (MIPs) for detecting cardiac troponin I (cTnI).

[0014] Further, the buffer solution mentioned in step (2) is a phosphate buffer with a pH of 7-8 and a concentration of 0.01-0.1M; the heteromorphic bifunctional crosslinking agent is selected from at least one of succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC), sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (Sulfo-SMCC), and sulfosuccinimide 6-((maleimide)hexamethylene)hexanoate (Sulfo-EMCS); The mass-to-volume ratio of the amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 to the buffer solution is (5-15):(0.5-2.0) (g / L); the mass ratio of the heteromorphic bifunctional crosslinking agent to the amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 is (0.5-1.5):(45-55); the mass ratio of the cTnI-specific C-terminal epitope peptide to the amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 is (1-2.0):(45-55).

[0015] Preferably, the buffer solution in step (2) is a phosphate buffer solution with a pH of 7.4 and a concentration of 0.01M; the heteromorphic bifunctional crosslinking agent is sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (Sulfo-SMCC); the mass-to-volume ratio of the amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 to the buffer solution is 10:1 (g / L); the mass ratio of the sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (Sulfo-SMCC) to the amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 is 1:50; and the mass ratio of the cTnI-specific C-terminal epitope peptide to the amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 is 1:50.

[0016] Further, the organic solvent mentioned in step (3) is at least one of ethanol and methanol.

[0017] Preferably, the organic solvent in step (3) is ethanol.

[0018] Further, in step (3), the thermosensitive monomer is selected from at least one of N-isopropylacrylamide (NIPAm), N-n-propylacrylamide (NNPAm), N,N'-diethylacrylamide (DEAm), and N-vinylcaprolactam (NVCL); the hydrophilic monomer is selected from at least one of acrylic acid (AAc), methacrylic acid (MAA), acrylamide (AM), and N-hydroxymethylacrylamide (NMA); the hydrophobic monomer is selected from at least one of N-isopropylacrylamide (TBAm), N-phenylacrylamide (NPAm), methyl methacrylate (MMA), and tert-butyl methacrylate (t-BMA); and the cationic functionalized monomer is N-(3-aminopropyl)methacrylamide hydrochloride (APM), [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC), and [3-(methacryloylamino)propyl]trimethylammonium chloride (MPTAC). The fluorescent monomer is selected from at least one of dimethylaminopropyl methacrylamide (DMAPMA); the fluorescent monomer is selected from at least one of fluorescein acrylamide (FITC-AAm), fluorescein methacrylate (Fl-MA), rhodamine B acrylamide (Rh-AAm), and coumarin acrylamide (Cou-AAm); the crosslinking agent is selected from at least one of N,N'-methylenebisacrylamide (BIS), N,N'-bis(acryloyl)cysteine ​​(BAC), polyethylene glycol diacrylate (PEGDA), and ethylene glycol dimethacrylate (EGDMA); the initiator is selected from at least one of ammonium persulfate (APS), potassium persulfate (KPS), azobisisobutyronitrile (AIBN), and benzoyl peroxide (BPO); the catalyst is selected from at least one of tetramethylethylenediamine (TEMED), N,N-dimethylaniline (DMA), N,N-diethylaniline (DEA), and sodium bisulfite (SBS).

[0019] Preferably, in step (3), the thermosensitive monomer is N-isopropylacrylamine (NIPAm), the hydrophilic monomer is acrylic acid (AAc), the hydrophobic monomer is N-isopropylacrylamide (TBAm), the cationic functionalized monomer is N-(3-aminopropyl)methacrylamide hydrochloride (APM), the fluorescent monomer is fluorescein acrylamide; the crosslinking agent is N,N'-methylenebisacrylamide (BIS); the initiator is ammonium persulfate (APS); and the catalyst is tetramethylethylenediamine (TEMED).

[0020] Further, in step (3), the molar ratio of the thermosensitive monomer, hydrophilic monomer, hydrophobic monomer, and cationic functionalized monomer is (5-8):(1-3):(1-5):(0-2), and the total concentration is 10mM.

[0021] Preferably, the molar ratio of the thermosensitive monomer, hydrophilic monomer, hydrophobic monomer, and cationic functionalized monomer in step (3) is 5.5:1.5:2.5:0.5.

[0022] Further, in step (3), the mass ratio of the product obtained in step (2) to the initiator is (0.5-2.0):(0.5-1.5); the mass-volume ratio of the initiator to the catalyst is (1000-2000):(0.5-2.0) (mg / mL); and the mass ratio of the crosslinking agent to the initiator is (3-5):(40-55).

[0023] Preferably, in step (3), the mass ratio of the product obtained in step (2) to the initiator is 1:1; the mass-volume ratio of the initiator to the catalyst is 1000:1 (mg / mL); and the mass ratio of the crosslinking agent to the initiator is (3-5):(40-55).

[0024] Furthermore, the polymerization reaction in step (3) is carried out at a temperature of 35-45°C and for a reaction time of 10-14 hours.

[0025] Preferably, the method for preparing nanoparticles according to the present invention includes the following steps:

[0026] (1) Preparation of fluorescein acrylamide: Weigh 5-aminofluorescein and dissolve it in acetone, stirring in an ice bath. Slowly add 50 μL of acryloyl chloride dissolved in 2 ml of acetone to the solution under a nitrogen atmosphere and incubate overnight at room temperature; wash with ether to obtain an orange solid, and dry the product under vacuum at 40 °C to obtain fluorescein acrylamide (FITC-AAm).

[0027] (2) Synthesis of magnetic nanoparticles: FeCl3·6H2O and sodium acetate were dissolved in ethylene glycol and hydrothermally reacted at 200℃ for 8 hours to obtain Fe3O4 magnetic nanoparticles magNP;

[0028] (3) Silica coating: The SiO2 layer was coated on the surface of magnetic nanoparticles in an ethanol / water mixed solution with ammonia as catalyst and tetraethyl orthosilicate (TEOS) as silicon source to obtain magNP@SiO2.

[0029] (4) Aminofunctionalization: magNP@SiO2 was dispersed in ethanol / water solution and reacted with APTMS under nitrogen protection to prepare aminofunctionalized magnetic nanoparticles Fe3O4@SiO2-NH2 (magNP@SiO2-NH2).

[0030] (5) Preparation of magnetic peptide template: Using sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (Sulfo-SMCC) as a crosslinking agent, the cysteine-modified epitope peptide ALSGMEGRKKKFES was covalently coupled to the surface of magNP@SiO2-NH2 to obtain magNP-P;

[0031] (6) Epitope imprinting polymerization: magNP-P was dispersed in a mixed solution containing NIPAm, AAc, TBaAm, BIS and FITC-AAm, and ammonium persulfate (APS) initiator and tetramethylethylenediamine (TEMED) catalyst were added. The polymerization reaction was carried out at 40°C under a nitrogen atmosphere for 12 hours. After the template was eluted, the nanomaterial was obtained.

[0032] The application of the magnetic fluorescently imprinted nanoparticles described in this invention in the preparation of a cardiac troponin I detection kit.

[0033] This invention constructs a rapid label-free detection platform for cTnI based on magnetic fluorescent epitope imprinting technology. Specific C-terminal epitopes (ALSGMEGRKKKFES) of cTnI were screened from a database and immobilized on the surface of amino-functionalized magnetic nanoparticles to prepare magnetic peptide templates (magNP-P). The ratios of monomers such as N-isopropylacrylamide (NIPAm), acrylic acid (AAc), and N-tert-butylacrylamide (TBAm) were optimized. Fluorescein acrylamide (FITC-AAm) was introduced as a fluorescent monomer during polymerization to prepare MIPs with both specific recognition and autofluorescence response characteristics. Finally, a fluorescence sensing system based on a competitive substitution mechanism was constructed to achieve rapid and highly sensitive detection of cTnI. This study provides a novel technical solution for the rapid clinical diagnosis of cTnI and also offers a valuable approach for the imprinting detection of protein biomarkers.

[0034] Introducing fluorescein acrylamide (FITC-AAm) as a fluorescent monomer into the polymerization system imbues the MIP with an inherent fluorescent response, eliminating the need for additional labeled signal probes and simplifying the detection process. A wash-free sensing mechanism based on competitive substitution is employed through the selection of cTnI epitopes. This results in high sensitivity, short detection time, and low cost.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0036] (1) The magnetic fluorescent molecularly imprinted nanoparticles provided by the present invention are made by immobilizing the cTnI-specific C-terminal epitope peptide ALSGMEGRKKKFES as an epitope on the surface of amino-functionalized magnetic nanoparticles and coating them with a molecularly imprinted polymer layer, so that the nanoparticles have both fluorescence response and specific recognition functions, and realize high sensitivity detection of cTnI with a detection sensitivity of 1 pg / mL.

[0037] (2) The preparation method provided by the present invention uses thermosensitive monomers, hydrophilic monomers, hydrophobic monomers and cationic functionalized monomers to synergistically polymerize and introduce fluorescent monomers, so that the resulting molecularly imprinted polymer layer has an imprinted cavity that is complementary to the spatial structure and functional groups of cTnI epitope peptide, giving the nanoparticles self-fluorescent properties, constructing a detection system that does not require additional labeling, and simplifying the detection process.

[0038] (3) The magnetic fluorescent molecularly imprinted nanoparticles provided by the present invention are used in cTnI detection. They utilize magnetic separation and competitive displacement mechanisms to exhibit extremely low non-specific adsorption to common interfering proteins such as BSA and BHb. They can effectively resist interference from complex biological matrices and are suitable for early diagnosis of acute myocardial infarction. Attached Figure Description

[0039] Figure 1 (A) Schematic diagram of epitope immobilization on magnetic nanoparticles to prepare magNP-P, and the synthesis, collection and purification of molecularly imprinted polymers (MIPs). (B) Schematic diagram of protein-substituted magnetic peptide template (magNP-P) and MIPs conjugate.

[0040] Figure 2 TEM images of (A) magNP (B) magNP@SiO2 (C) magNP@SiO2-NH2 (D) magNP-P;

[0041] Figure 3 (A) Infrared spectra of magNP, magNP@SiO2, magNP@SiO2-NH2, and magNP-P; (B) Zate potential diagram of magNP, magNP@SiO2, magNP@SiO2-NH2, and magNP-P; (C) XPS analysis of magNP@SiO2-NH2 and magNP-P; (D) XRD of magNP, magNP@SiO2, magNP@SiO2-NH2, and magNP-P.

[0042] Figure 4 The NMR spectrum of the fluorescent monomer fluorescein acrylamide (FITC-AAm);

[0043] Figure 5Fluorescence emission spectra of MIP, magNP-P, peptides, cTnI, and different mixtures;

[0044] Figure 6 (A) is the TEM image of MIP, (B) is the TEM image of MIP combined with magNP-P, and (C) is the mapping image of MIP combined with magNP-P.

[0045] Figure 7 (A) Effect of functional monomers on fluorescence intensity of MIPs; (B) Effect of fluorescent monomer dosage on fluorescence intensity; (C) Normalized fluorescence intensity curve after optimization of molecularly imprinted polymer template adsorption time; (D) Optimization of protein replacement time.

[0046] Figure 8 Fluorescence intensity and calibration plots of MIP at different cTnI protein concentrations;

[0047] Figure 9 (A) Selectivity experiment of MIP for interfering proteins (B) Fluorescence response of MIP to cTnI at different storage periods (C) Reproducibility of cTnI fluorescence;

[0048] Figure 10 The combined isotherm of MIP;

[0049] Figure 11 (A) Fluorescence detection results of MIP (B) Results of MIP fluorescence detection and troponin I assay kit (chemiluminescence method) in detecting cTnI' in serum samples. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0051] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0052] The amino acid sequence SEQ ID NO.1 was purchased by Shanghai Sangon Biotech (Shanghai) Co., Ltd.

[0053] Target protein cTnI: Xibao Biotechnology, catalog number EDD0410A; Bovine serum albumin (BSA): Aladdin, batch number H2321488; Cysteine ​​(Cys): Anaiji, batch number GA190063; Aspartate (Asp): Maclean, batch number C14414622; Bovine hemoglobin (BHb): purchased from Maclean, batch number C18313424; Serum from patients with acute myocardial infarction was obtained from Subei People's Hospital of Jiangsu Province.

[0054] Example 1: Preparation of magnetic fluorescent molecularly imprinted nanoparticles

[0055] (1) Preparation of fluorescein acrylamide (FITC-AAm)

[0056] 100 mg of 5-aminofluorescein was weighed and dissolved in 10 mL of acetone, and stirred in an ice bath for 30 min. 50 μL of acryloyl chloride dissolved in 2 mL of acetone was slowly added to the solution under a nitrogen atmosphere, and the mixture was incubated overnight at room temperature. After washing with ether, an orange solid was obtained. The product was dried under vacuum at 40 °C to obtain fluorescein acrylamide. The properties and purity of the product were characterized by NMR, and the results are as follows: Figure 4 As shown.

[0057] (2) Synthesis of magnetic nanoparticles (magNP)

[0058] 2.7 g FeCl3·6H2O was added to 100 ml ethylene glycol, followed by 7.2 g sodium acetate. The mixture was stirred for 30 min, then transferred to a reaction vessel and heated at 200 °C for 8 h. The mixture was washed three times sequentially with deionized water and anhydrous ethanol, separated using an external magnet, and vacuum dried overnight at 60 °C. Finally, it was ground to obtain magNP magnetic nanoparticles.

[0059] (3) Silica coating: Preparation of magNP@SiO2

[0060] A SiO2 layer was modified on the surface of magNP using a sol-gel method. Specifically, 200 mg of magNP was added to a mixture of 100 mL anhydrous ethanol and 20 mL deionized water, and sonicated for 30 min. Then, 5.0 mL of ammonia and 2.0 mL of LTEOS were added sequentially. The mixture was stirred continuously at room temperature for 12 hours. The resulting product was separated by a magnetic field, washed three times sequentially with deionized water and ethanol, and then vacuum dried at 60°C. The product was then ground to obtain magNP@SiO2.

[0061] (4) Preparation of amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 (magNP@SiO2-NH2)

[0062] 200 mg of magNP@SiO2 was suspended in an ethanol / water (3:1, v / v) solution (180 mL) in a flask. Under nitrogen protection, 1.8 mmol LAPTMS was added, followed by mechanical stirring. The mixture was incubated overnight at 40°C. The final magNP@SiO2-NH2 product was separated using an external magnet, washed three times sequentially with deionized water and ethanol, and dried overnight in a vacuum oven at 60°C to obtain magNP@SiO2-NH2.

[0063] (5) Preparation of magnetic peptide template magNP-P

[0064] First, 50 mg of Fe3O4@SiO2-NH2 was dispersed in 5 mL of 0.01 M, pH 7.4 deoxyphosphate-buffered saline (PBS). Then, 1 mg of sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (Sulfo-SMCC) was added, and the reaction mixture was shaken at room temperature for 1 h. After the reaction, the product was washed three times with PBS (0.01 M, pH 7.4). Next, 1 mg of the cysteine-modified template peptide ALSGMEGRKKKFESC[cTnI] (the amino acid sequence of the specific C-terminal epitope peptide is ALSGMEGRKKKFES (SEQ ID NO.1)) was added. The addition of cysteine ​​was for coupling with magnetic beads, and the reaction mixture was shaken again for 2 h. After washing with PBS (0.01 M, pH 7.4), the magnetic peptide template (magNP-P) was obtained, quantified in PBS (0.01 M, pH 7.4), and stored at 4°C.

[0065] (6) Epitope Imprinting Polymerization: Synthesis of Magnetic Fluorescent Molecularly Imprinted Nanoparticles (MIPs)

[0066] Nano-sized MIPs were synthesized according to literature. A mild polymerization method was employed. Monomers were further determined based on the sequence of the template peptide. N-isopropylacrylamine (NIPAm), acrylic acid (AAc), N-isopropylacrylamide (TBAm), and N-(3-aminopropyl)methacrylate hydrochloride (APM) were used as functional monomers for polymerization. N,N'-methylenebisacrylamide (BIS) was used as a crosslinking agent, FITC-AAm as a fluorescent monomer, ammonium persulfate (APS) as an initiator, and tetramethylethylenediamine (TEMED) as a catalyst. The optimal ratio of NIPAM / TBAM / APM / AAc was determined by adjusting the content, with the total concentration controlled at 10 mM.

[0067] In a round-bottom flask, 5.5 mM NIPAm, 1.5 mM AAc, 0.5 mM APM, 3.6 mg BIS, and FITC-AAm were dissolved in 40 mL of water to obtain an aqueous solution. 2.5 mol TBAm was dissolved in 1 mL of ethanol and added to the aqueous solution. The mixture was sonicated for 10 min to obtain a mixed solution. 50 mg of MagNP-P prepared in step (4) was dispersed in 10 mL of water and added to the mixed solution. Under a nitrogen atmosphere, 50 mg of ammonium persulfate (APS) and 50 μL of tetramethylethylenediamine (TEMED) were added, and the mixture was mechanically stirred at 40°C for 12 hours. The final product was thoroughly washed with deionized water and collected by a magnet. Finally, the nano-MIPs were eluted by incubating the magnetic particles in deionized water at 4°C for 24 hours. The preparation process of non-imprinted nanogels (NIPs) followed the same method, the only difference being the use of unassembled peptides in magNP@SiO2-NH2.

[0068] Combination Figure 1 Analysis shows that this invention employs a stepwise modification strategy to construct magNP-P, and the preparation process is as follows: Figure 1 As shown in Figure A, nanoscale Fe3O4 magnetic particles (magNP) were first prepared via a hydrothermal method. Subsequently, a hydrophilic SiO2 shell was coated onto their surface using a sol-gel method to construct a Fe3O4@SiO2 core-shell composite material. The SiO2 shell was then functionalized with amino groups via APTMS, grafting primary amino functional groups onto the material surface to create active reaction sites for subsequent covalent coupling with biomolecules. To achieve directional and efficient coupling between the carrier and the target peptide, a bifunctional crosslinking agent, sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (Sulfo-SMCC), was introduced as a connecting bridge. The NHS ester group at one end undergoes an amide condensation reaction with the primary amino groups on the surface of the aminated magnetic particles to form a covalent bond. At the other end, the maleimide group undergoes a thiol-maleimide click chemistry reaction with the free thiol group of cysteine ​​in the peptide sequence, ultimately achieving directional coupling between the magnetic carrier and the target peptide, resulting in the magnetic template magNP-P.

[0069] The preparation of nano-MIPs involves three steps, such as... Figure 1 As shown in Figure B, the process includes polymerization, separation, and collection. In the MIP polymerization monomer, NIPam serves as the main monomer, giving the polymer thermal responsiveness; TBAM provides hydrophobicity; AAc and APM provide cationic and anionic binding sites, respectively; and BIS acts as a crosslinking agent. Furthermore, the fluorescent monomer fluorescein acrylamide (FITC-AAm) was successfully synthesized as a fluorescent signal probe, such as... Figure 4The figure shows the 1H NMR spectrum of FITC-AAm, confirming its synthesis. APS and TEMED were added as initiators for the polymerization of MIPs. After polymerization, the polymer-coated magnetic nanoparticles were collected by magnetic separation, and then MIPs with cTnI affinity were eluted from the nanoparticles.

[0070] To verify the successful implementation of each step of the magnetic template modification reaction and the structural stability, this study employed multiple characterization techniques, including TEM, FT-IR, Zeta potential, XPS, and XRD, to systematically analyze the material. The TEM characterization results are as follows: Figure 2 As shown, the original magNP exhibits good monodispersity. After SiO2 coating, a uniform amorphous silica layer is visible on the particle surface. Following amino functionalization and peptide coupling, the overall morphology of magNP-P did not change significantly, indicating that the modification process did not cause particle agglomeration or structural damage. FT-IR spectral characterization results are shown below. Figure 3 As shown in Figure A, the four materials are at 570cm. -1 Stable characteristic absorption peaks were observed at all locations, corresponding to the stretching vibrations of the Fe-O bonds in the Fe3O4 lattice, confirming that the Fe3O4 magnetic core maintained its structural integrity after multi-step modification reactions including SiO2 coating, amination, and peptide coupling. Compared to the original magNP, magNP@SiO2 showed a higher absorption peak at 1089 cm⁻¹. -1 The newly enhanced absorption peak is attributed to the asymmetric stretching vibration of the Si-O-Si bond in the SiO2 shell. The characteristic peak at 2929 cm⁻¹ in magNP@SiO2-NH2 corresponds to the stretching vibration of the -CH2- group in the aminopropyl group introduced by APTMS, indicating that primary amino functional groups have been successfully grafted onto the surface of the SiO2 shell. The final product magNP-P shows an absorption peak at 1641 cm⁻¹. -1 and 1550cm -1 Significant characteristic peaks appeared at the respective locations, corresponding to the amide I band (C=O stretching vibration) and the amide II band (superposition of NH bending vibration and CN stretching vibration). The appearance of these characteristic peaks proves that sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (Sulfo-SMCC) undergoes amide condensation with the amino group and further covalently couples with the peptide thiol group. Zeta potential characterization further corroborates the effectiveness of each modification step from the perspective of surface charge change. Figure 3B indicates that the original magNP's Zeta potential was +9.94 mV, exhibiting weak positive charge. After SiO2 coating, the potential of magNP@SiO2 plummeted to -28 mV, attributed to the dissociation of silanol groups on the SiO2 surface in aqueous solution, generating a large amount of negative charge. After APTMS amination modification, the potential of magNP@SiO2-NH2 rebounded to +13.5 mV, due to the protonation of the primary amino groups grafted onto the material surface. After peptide coupling, the potential of magNP-P further increased to +19.4 mV, correlated with the positive charge of the epitope peptide, directly proving that the peptide had been successfully coupled to the magnetic support surface.

[0071] XPS elemental analysis provided direct elemental evidence for the successful modification of the peptide. The XPS elemental analysis results of the material are shown in Table 1.

[0072] Table 1 XPS elemental analysis of materials

[0073]

[0074] See Figure 3 According to Table 1, the elemental composition of magNP@SiO2-NH2 is Fe 0.51%, C 13.57%, O 53.84%, N 1.54%, and Si 24.11%. After peptide coupling, the C and N contents in magNP-P increased to 16.47% and 5.05%, respectively, while the O and Si contents decreased to 46.64% and 21.58%. The enrichment of C and N elements is directly related to the introduction of peptide molecules, while the decrease in O and Si contents is due to the coverage of the SiO2 shell surface by the organic modification layer. XRD characterization verified the crystal stability of the material, such as... Figure 3 D. The original magNP showed characteristic diffraction peaks at 2θ=30.02°, 35.42°, 43.02°, 53.44°, 56.93°, and 62.51°, corresponding to the (220), (311), (400), (422), (511), and (440) crystal planes of spinel-type Fe3O4, respectively, which are highly consistent with the standard PDF card. After SiO2 coating, amination, and peptide coupling, the above-mentioned characteristic diffraction peaks of Fe3O4 still exist stably, and no impurity phase diffraction peaks appear, confirming that the crystal form of the Fe3O4 magnetic core was not destroyed during the multi-step modification process. At the same time, magNP@SiO2, magNP@SiO2-NH2, and magNP-P all showed broadened diffuse peaks in the 2θ=20~30° region, corresponding to the characteristic diffraction peaks of amorphous SiO2, further confirming the successful coating of the SiO2 shell.

[0075] Example 2: Optimization of NIPAM / TBAM / APM / AAc content

[0076] Example 2 The synthesis steps of magnetic fluorescent molecularly imprinted nanoparticles (MIPs) are the same as those in Example 1, except that the content ratio of NIPAM / TBAM / APM / AAc is different, as shown in Table 2.

[0077] Table 2 Optimization of NIPAM / TBAM / APM / AAc content ratio

[0078]

[0079] Combine Table 2 and Figure 7 Analysis A revealed significant differences in fluorescence intensity among the eight groups of MIPs, ranked as follows: MIP8 > MIP4 > MIP5 > MIP1 / MIP2 / MIP3 / MIP6 / MIP7. This result indicates that the type and ratio of functional monomers are key factors affecting the fluorescence performance of magnetic fluorescently imprinted nanoparticles.

[0080] Example 3 Epitope Screening and Prediction

[0081] 1.1 Materials and Databases

[0082] The complete amino acid sequence of human cardiac troponin I (cTnI) was obtained from the UniProt database. The online tools used for epitope prediction, antigenicity verification, physicochemical property analysis, and homology comparison were all open-source public platforms, specifically the BepiPred-2.0 tool for the IEDB database, the Vaxijen 2.0 server, ExPASy-ProtParam, and the NCBIProtein BLAST platform.

[0083] 1.2 Linear B-cell epitope prediction and screening

[0084] Free online access to the IEBDB (Immunotope Database and Analysis Resources) server was used for predicting B cell epitopes. Log in to the IEBDB database, select the BepiPred-2.0 linear epitope prediction tool, input the complete amino acid sequence of cTnI, and use a scoring threshold of 0.5 to filter for highly immunogenic epitope regions. Finally, the primary candidate epitope and four alternative epitopes were determined for subsequent validation experiments.

[0085] 1.3 Epitope antigenicity verification

[0086] The Vaxijen 2.0 server is used to study the antigenicity of selected epitopes. Candidate epitope sequences are submitted to the Vaxijen 2.0 online server, a tumor model is selected for antigenicity assessment, an antigenicity threshold of 0.4 is set, and the antigenicity score of the epitope is output. A score higher than the threshold is considered to have strong antigenicity.

[0087] 1.4 Analysis of the physicochemical properties of the episite

[0088] The ExPASy-ProtParam online tool is used to analyze key physicochemical properties of candidate epitopes. These include theoretical isoelectric point (pI), hydrophilicity (GRAVY value), and instability index, with default values ​​for the analytical parameters. A negative GRAVY value indicates hydrophilicity of the epitope; an instability index < 40 indicates protein stability, 40–50 indicates moderate stability, and > 50 indicates instability.

[0089] 1.5 Verification of Epitope Homology and Specificity

[0090] The NCBI Protein BLAST platform was used to validate the homology and specificity of epitopes. Homology alignment was performed using the human Swiss-Prot protein database, with alignment parameters set to default values. Epitope specificity was assessed through sequence consistency and coverage, with a focus on comparing the match between troponin family members (fast skeletal muscle troponin I, slow skeletal muscle troponin I, cardiac troponin T, and troponin C) and the target epitope to evaluate potential cross-reactivity risks.

[0091] 1.6 Predictive Epitope Analysis

[0092] Table 3. Epitope scores predicted by IEDB database analysis, antigenicity scores predicted by Vaxijen 2.0 server, and physicochemical properties obtained from the ExPASy-ProtParam online tool.

[0093]

[0094] Table 3 shows that the BepiPred epitope scores of the six candidate epitopes range from 0.498 to 0.600, with five epitopes scoring ≥0.5, meeting the screening criteria for epitopes with high immunogenicity potential. Epitopes 141-153 have the highest score, reaching 0.600, indicating optimal performance in immunogenicity prediction. Epitopes 197-210 have a score of 0.498, slightly below the 0.5 threshold, placing them at a relative disadvantage in the immunogenicity potential prediction dimension. However, considering the combined performance of antigenicity and physicochemical properties, the ALSGMEGRKKKFES (SEQ ID NO.1) polypeptide sequence demonstrates a significant advantage over other candidate epitopes. Firstly, in terms of antigenicity, this epitope has an antigenicity score of 1.1082, only slightly lower than epitopes 65-79, significantly higher than the other four epitope sequences, and far exceeding the 0.4 antigenicity threshold, indicating extremely strong antigen recognition potential. In terms of stability, the ALSGMEGRKKKFES (SEQ ID NO.1) peptide has a greater advantage, with an instability index of only 23.04, classifying it as a strongly stable epitope. This means that under physiological conditions and in vitro molecular imprinting experiments, this epitope is not prone to conformational deformation or degradation and can maintain a stable spatial structure over a long period. Compared to ALSGMEGRKKKFES (SEQ ID NO.1), the other five candidate epitopes exhibit weaker stability and cannot meet the template stability requirements of molecular imprinting experiments. Regarding hydrophilicity, the ALSGMEGRKKKFES (SEQ ID NO.1) peptide has a GRAVY value of -1.093, which is negative and has a large absolute value, indicating strong hydrophilicity. Strongly hydrophilic epitopes are more easily exposed on the surface of cTnI protein molecules. On the one hand, they can form efficient interactions with functional monomers such as hydrogen bonds and electrostatic interactions, improving the binding specificity and affinity of the imprinted cavity; on the other hand, they are better suited for detection scenarios of body fluid samples such as serum and plasma, reducing non-specific adsorption and improving the detection sensitivity of biosensors. Although the hydrophilicity of this epitope is slightly weaker than that of epitopes at positions 65–79 (-1.82) and 186–196 (-1.773), it is far superior to the weakly hydrophilic epitope at position 116–125 (-0.12). Furthermore, considering its stability, its overall performance is significantly better than the two extremely hydrophilic but unstable epitopes mentioned above. In addition, the theoretical isoelectric point of ALSGMEGRKKKFES (SEQ ID NO.1) is 9.70, which falls within the physiologically compatible pH range. It will not undergo conformational changes due to charge abnormalities, nor will it interfere with the specific binding of the imprinted cavity.

[0095] In summary, although the epitope score of the ALSGMEGRKKKFES (SEQ ID NO.1) peptide is slightly below the screening threshold, its comprehensive physicochemical properties—strong antigenicity, high stability, and good hydrophilicity—make it the best-performing target epitope among the six candidate sequences and the most suitable for use as a molecular imprinting template. Therefore, ALSGMEGRKKKFES is selected as a candidate epitope for MIP synthesis.

[0096] 1.7 Results of verification of homology and specificity of candidate epitopes

[0097] Table 4. Validation results of candidate epitopes homology and specificity based on the NCBI Protein BLAST platform.

[0098]

[0099] Table 4 shows that the candidate epitope has 100.00% sequence identity with the target protein (UniProt ID: P19429), with 100% coverage and an E-value of 1.00E-08. This result confirms that the epitope is a specific fragment of cTnI, with extremely high statistical significance, making it highly unlikely to be caused by random factors. The sequence identity with fast skeletal muscle troponin I (P48788.2) (P48788.2) is 91.67%, with 86% coverage and an E-value of 3.00E-05, and with slow skeletal muscle troponin I (P19237.3) is 76.92%, with 93% coverage and an E-value of 2.00E-04, indicating some homology, but the risk of cross-reactivity is low. The sequence identity with cardiac troponin T (P45379.3) was 83.33%, but the coverage was only 43%, with an E value of 5.00E-03, indicating that the match was only a local short fragment. The sequence identity with cardiac troponin C (P02585) was 100%, but the coverage was only 14%, with an E value of 3.7, indicating that the match was a random short fragment homology with no significant biological significance.

[0100] In summary, the candidate epitope ALSGMEGRKKKFES only shows a full-sequence, highly significant match with the target protein cTnI. Its matches with other homologous proteins are mostly local fragments or random homologs, demonstrating high cTnI specificity and effectively avoiding cross-reactions in subsequent molecular imprinting experiments.

[0101] Therefore, the amino acid sequence of the cTnI-specific C-terminal epitope peptide, ALSGMEGRKKKFES (SEQ ID NO.1), was selected as the imprinting molecule for the synthesis of magnetic fluorescent molecularly imprinted nanoparticles (MIPs).

[0102] Example 4: Competitive fluorescence determination of cTnI

[0103] Since the MIP synthesized in this study has a fluorescent signal, it was used as an optical probe to construct a fluorescence-responsive competitive substitution experiment, which aimed to verify the competitive substitution ability of the target protein on MIP bound to the magNP-P surface.

[0104] 1 mL of 1 mg / mL nano-MIPs dispersion was mixed with 5 mL of 2 mg / mL magNP-P dispersion (both prepared with 0.01 M PBS, pH 7.4) and incubated at 37°C for 1 h in a shaker. The mixture was then collected using an external magnet, washed three times with PBS (0.01 M, pH 7.4), and redispersed in an equal volume of PBS (0.01 M, pH 7.4). 1 mL of the mixture and 200 μL of cTnI standard solution (PBS, 0.01 M, pH 7.4) were added to a 5 mL centrifuge tube. After incubation for 30 minutes, magNP-P was removed magnetically. The fluorescence response of the supernatant was read using a fluorescence spectrometer with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

[0105] As shown in Figure 5, at an excitation wavelength of 485 nm, neither the magnetic template magNP-P, the peptide, nor cTnI itself produced significant fluorescence emission signals. When MIP specifically bound to magNP-P to form a complex, the fluorescence intensity of the system decreased significantly compared to the free MIP. This phenomenon stems from a conformational change or fluorescence quenching effect after MIP binds to magNP-P. Adding the peptide or cTnI to the complex system resulted in a significant rebound in fluorescence intensity, which was significantly higher than that of the MIP-magNP-P complex system. This indicates that the peptide or cTnI successfully displaced the MIP bound to the magNP-P surface by competitively binding to the specific recognition site of MIP, restoring the MIP to its free state and reproducing some fluorescence. This dynamic change in fluorescence response directly confirms the feasibility of the target protein's competitive replacement of MIP.

[0106] To further verify the binding properties of MIP and magNP-P at the microstructural level, we analyzed the morphology and elemental distribution of the complex using transmission electron microscopy and elemental mapping. As shown in Figure 6A, free MIP exhibits an amorphous nanoaggregate with a loose porous structure between particles. When MIP binds to magNP-P, as shown in Figure 6A... Figure 7B. TEM bright-field imaging reveals a distinct core-shell structure in the complex: the dark, dense region is the core of the magNP-P magnetic template, composed of Fe3O4@SiO2@peptide, surrounded by a uniformly thick, light-colored MIP layer. No obvious particle aggregation or interface separation is observed, indicating that the MIP and magNP-P achieve tight binding through molecular recognition. Elemental mapping further validates the elemental composition and spatial distribution of the complex (Figure 7C). The figure shows that the Fe element signal is concentrated in the core region of the complex, corresponding to the Fe3O4 magnetic core of magNP-P, indicating that the magnetic template did not undergo structural damage or leakage during binding. The Si element is distributed in a continuous shell-like pattern around the Fe element, corresponding to the SiO2 intermediate layer of magNP-P. C, O, and N elements are distributed throughout the entire complex region, which is due to the fact that the MIP is an organic polymer and the peptide on the template surface. The S element is uniformly distributed in the complex region, and its signal originates from the cysteine ​​thiol group in the template peptide. The uniform distribution indicates that the peptide coupling structure on the template surface remains stable after MIP binding, and there are no obvious gaps at the binding interface between MIP and template.

[0107] After realizing the fluorescence-responsive competitive substitution experiment, the synthesis of MIP and the competitive substitution experiment were optimized. Figure 7 The results of optimizing key preparation conditions in the MIP synthesis process are presented, and the recognition performance of MIP under different conditions is characterized by normalized fluorescence intensity. Figure 7 A compares the normalized fluorescence intensity of MIPs synthesized with different functional monomer ratios. The results show that the normalized fluorescence intensity of MIPs with monomer ratio 8 (NIPAM 55%, TBAM 25%, AAC 15%, APM 5%) is significantly higher than that of other groups. This indicates that this monomer combination can form recognition sites that are highly complementary to the spatial structure and functional groups of the template peptide through synergistic effects, thereby improving the binding specificity and affinity of MIPs to the template. The other ratios may have resulted in a decrease in the matching degree of the imprinted cavity and a decline in recognition performance due to the imbalance of hydrophilic and hydrophobic interactions and insufficient ion binding sites. Figure 7 B represents the optimized dosage of the fluorescent monomer FITC-AAm. With increasing monomer dosage, the normalized fluorescence intensity initially increases and then stabilizes. When the dosage reaches a certain threshold, the fluorescence signal no longer significantly increases, indicating that at this dosage, the fluorescent monomer is uniformly distributed in the polymer, ensuring sufficient fluorescence response signal without causing non-specific polymerization due to excessive monomer, thus affecting the recognition performance of MIP. To investigate the effect of the adsorption-binding time of MIP and magNP-P on the subsequent competitive substitution fluorescence response, the substitution fluorescence response characteristics of MIP under different adsorption-binding times were examined. The results are as follows: Figure 7As shown in Figure C, with the extension of the adsorption and binding time of MIP and magNP-P, the normalized fluorescence intensity of the system exhibits a pattern of first rapidly increasing and then stabilizing. In the initial stage of adsorption and binding, the fluorescence intensity increases significantly with increasing incubation time. At this stage, the specific binding of MIP and magNP-P has not yet reached saturation. Extending the binding time promotes more MIP recognition of magNP-P, thereby increasing the amount of MIP that can be released during subsequent peptide competitive replacement, resulting in a significant upward trend in fluorescence intensity. When the adsorption and binding time reaches 60 min, further extending the incubation time no longer increases the normalized fluorescence intensity, indicating that the specific adsorption and binding of MIP and magNP-P has reached saturation, and the amount of MIP replaced by the subsequent peptide reaches its maximum. If the adsorption and binding time is further extended, the fluorescence intensity of the system shows a downward trend. This phenomenon may be due to the combined effect of enhanced non-specific adsorption caused by excessively long incubation time and the aggregation of the MIP-magNP-P complex. On the one hand, once specific binding reaches saturation, prolonged incubation time allows unspecifically bound MIPs to adsorb onto the magNP-P surface or the outer layer of the existing MIP-magNP-P complex through non-specific interactions such as hydrophobic interactions and van der Waals forces. This forms adsorption structures without specific recognition function. Such non-specifically bound MIPs cannot be effectively competitively replaced by subsequent peptides, and steric hindrance hinders site competition between peptides and specifically bound MIPs, leading to a decrease in the amount of effectively replaceable MIPs. On the other hand, excessively long adsorption incubation time causes slight aggregation of the MIP-magNP-P complex in the system due to intermolecular interactions, reducing its effective contact sites with free peptides and exacerbating local fluorescence quenching, further attenuating the fluorescence response signal. In summary, the decrease in fluorescence intensity after the adsorption binding time exceeds the saturation threshold is essentially due to changes in material structure and molecular binding efficiency dominated by non-specific interactions after specific binding saturation. This result further confirms the necessity of using the shortest adsorption-binding time to reach the steady-state phase of the adsorption curve as the optimal condition for this experiment. It can effectively avoid interference from non-specific effects on the detection signal and ensure the stability of the competitive displacement fluorescence response and the accuracy of the detection results.

[0108] Figure 7D represents the optimized curve of peptide replacement time. This study used normalized fluorescence intensity as the evaluation index to investigate the competitive binding effect of the target peptide and MIP at different replacement times, in order to determine the optimal reaction time for MIP to replace magNP-P. The experiment used a complex of MIP and magNP-P saturated binding as the reaction substrate. After the addition of the target peptide, the peptide competitively replaced the bound MIP into a free state through specific sites. The free MIP then recovered fluorescence after escaping the quenching environment. The results showed that the normalized fluorescence intensity of the system rapidly increased with time after the addition of the peptide, and tended to stabilize at 30 min, indicating that the competitive binding reaction between the peptide and MIP reached equilibrium at this point, and the content of free MIP in the system reached its maximum value. The results confirm that the specific competitive binding of the target peptide to MIP and the efficient replacement of MIP can be completed within 30 minutes. This study also determined 30 minutes as the optimal reaction time for peptide replacement. This parameter provides key experimental basis for the subsequent establishment of a rapid cTnI detection system, effectively shortens the detection time, meets the core requirements of rapid and efficient cTnI detection in clinical emergency departments, and further highlights the technical advantages of the magnetic fluorescent molecular imprinting sensing platform constructed in this study in rapid detection applications.

[0109] Figure 8 The fluorescence response correction results of MIP to different concentrations of cTnI protein reflect the detection sensitivity and linearity of this sensing platform. Figure 9 A represents the fluorescence displacement emission spectra at different cTnI concentrations. As the cTnI concentration gradually increases from 1 pg / mL, the fluorescence intensity of the system increases significantly, indicating that the system can characterize the concentration of cTnI through quantitative changes in fluorescence signals. Figure 9 B is the calibration curve plotted with logC (logarithmic value of cTnI concentration) on the x-axis and fluorescence intensity on the y-axis. The results show that within the concentration range of 1 pg / mL to 100 ng / mL, fluorescence intensity and the logarithmic value of cTnI concentration have a good linear relationship, and the fitting equation is: Correlation coefficient This indicates that the detection system is significantly superior to the traditional antibody method for detecting cTnI in terms of sensitivity, and also has a wide linear detection range. The binding isotherm of MIP is as follows... Figure 10 As shown, the Kd value of MIPs is 1.09 × 10⁻⁶. -11 mol / L (R 2 =0.98).

[0110] Figure 9 The specificity, storage stability, and detection repeatability of the MIP are key indicators for evaluating the practical application value of this sensing platform. Figure 9A represents the selectivity experiment of MIP for interfering proteins. Commonly used clinical interfering proteins such as bovine serum albumin (BSA), cysteine ​​(Cys), aspartic acid (Asp), and bovine hemoglobin (BHb) were selected and their fluorescence responses were compared with cTnI at a concentration of 10:1. The results showed that the fluorescence response intensity of MIP to cTnI was significantly higher than that of other interfering proteins, while the fluorescence response to interfering proteins such as BSA and Cys showed almost no significant change. This indicates that MIP can effectively avoid interference from other proteins in biological samples on the detection results. Figure 9 B is a repeatability experiment for cTnI detection. Multiple parallel detections were performed on cTnI samples of the same concentration. The results showed that the fluorescence response intensity deviation of the multiple detections was small, with a relative standard deviation (RSD) of 5.17%, close to 5%, indicating that the detection system has good operational repeatability. Figure 9 C represents the change in fluorescence response of MIP to cTnI at different storage periods. The prepared MIP was stored at 4℃, and the fluorescence response of the same concentration of cTnI was detected at 0, 7, 14, and 21 days. The results showed that the fluorescence response intensity of MIP did not decrease significantly within 21 days, remaining above 90% of the initial intensity, with an RSD of 4.38%, indicating that the spatial structure of MIP was stable, the imprinted cavity did not undergo significant deformation or degradation, and it has good storage stability.

[0111] Experimental Example 1

[0112] To verify the effectiveness of this method, the concentration of cTnI in the serum of patients was determined using a troponin I assay kit (chemiluminescence method). The specific steps were as follows: 20 μL of serum was diluted to 1000 μL with PBS (0.01 M, pH 7.4) buffer; MIP was incubated with MagNP-P for 1 h; then the diluted solution was added; and after 30 min, the fluorescence intensity at 520 nm was measured.

[0113] like Figure 11 Figure A shows the fluorescence intensity graphs of 5 patients detected by MIP. After substituting them into the standard curve, as shown... Figure 11 Figure B shows a comparison of the results of MIP fluorescence detection and troponin I assay kits. The results show that the two methods are highly consistent in the quantitative detection of cTnI in the serum of 5 patients with acute myocardial infarction, and there is no significant difference between the two results (P>0.05). This indicates that the magnetic fluorescent molecular imprinting sensing platform constructed in this study can still maintain good detection accuracy and reliability in complex biological matrices.

[0114] This study successfully constructed a rapid, label-free cTnI magnetic fluorescent molecular imprinting (MIP) sensing and detection platform using the cTnI-specific C-terminal epitope ALSGMEGRKKKFES as a template, combined with magnetic nanomaterials and fluorescent molecular imprinting technology. Optimal reaction parameters were determined through systematic optimization of key conditions for MIP synthesis and competitive substitution detection. The prepared MIP was confirmed to be structurally stable and possesses high affinity for cTnI through various characterization processes. The detection platform exhibits a linear detection range of 1 pg / mL to 100 ng / mL for cTnI, demonstrating excellent specificity, storage stability, and reproducibility, and effectively resisting interference from other proteins in biological samples. Clinical serum sample analysis showed that the quantitative results of cTnI in the serum of patients with acute myocardial infarction obtained by this platform were highly consistent with the clinical gold standard chemiluminescent immunoassay, with no statistically significant difference between the two methods (P>0.05). Compared with traditional detection methods, this platform does not require expensive instruments and specific antibodies, is easy to operate, and provides rapid detection, meeting the needs of rapid detection in clinical emergency departments. It provides a new and reliable technical means for the early diagnosis of acute myocardial infarction and also expands new pathways for the application of molecular imprinting technology in the clinical detection of protein biomarkers.

Claims

1. A magneto-fluorescent molecularly imprinted nanoparticle, characterized in that, It includes amino-functionalized magnetic nanoparticles and a fluorescently imprinted polymer layer coated on their surface, wherein the fluorescently imprinted polymer layer has an imprinted cavity; the imprinted molecule of the fluorescently imprinted polymer layer is a cTnI-specific C-terminal epitope peptide.

2. The magnetic fluorescent molecularly imprinted nanoparticle of claim 1, wherein, The amino acid sequence of the cTnI-specific C-terminal epitope peptide is ALSGMEGRKKKFES (SEQ ID NO.1).

3. A method for preparing the magnetic fluorescent molecularly imprinted nanoparticles of claim 1, characterized in that, Includes the following steps: (1) Fe3O4 magnetic nanoparticles were synthesized by solvothermal method, and amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 were synthesized by sol-gel method; (2) The amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 obtained in step (1) are dispersed in a buffer solution, and after reacting with a heterobifunctional crosslinking agent, cTnI-specific C-terminal epitope peptide is added for coupling to obtain a magnetic peptide template (magNP-P) with template peptide immobilized on the surface. (3) Dissolve the thermosensitive monomer, hydrophilic monomer, cationic functionalized monomer, crosslinking agent and fluorescent monomer in water to obtain an aqueous solution. Dissolve the hydrophobic monomer in an organic solvent and mix it with the above aqueous solution to obtain a mixed solution. Disperse the product obtained in step (2) in water and add it to the mixed solution. Under the protection of inert gas, add an initiator and a catalyst to carry out a polymerization reaction. After treatment, obtain magnetic fluorescent molecularly imprinted nanoparticles (MIPs) for detecting cardiac troponin I (cTnI).

4. The preparation method according to claim 3, characterized in that, The buffer solution in step (2) is a phosphate buffer with a pH of 7-8 and a concentration of 0.01-0.1M; the heteromorphic bifunctional crosslinking agent is selected from at least one of succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC), sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (Sulfo-SMCC), and sulfosuccinimide 6-((maleimide)hexamethylene)hexanoate (Sulfo-EMCS); the ammonia The mass-to-volume ratio of the functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 to the buffer solution is (5-15):(0.5-2.0) (g / L); the mass ratio of the heteromorphic bifunctional crosslinking agent to the amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 is (0.5-1.5):(45-55); the mass ratio of the cTnI-specific C-terminal epitope peptide to the amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2 is (1-2.0):(45-55).

5. The preparation method according to claim 3, characterized in that, The organic solvent mentioned in step (3) is at least one of ethanol and methanol.

6. The preparation method according to claim 3, characterized in that, The thermosensitive monomer in step (3) is selected from at least one of N-isopropylacrylamide (NIPAm), N-n-propylacrylamide (NNPAm), N,N'-diethylacrylamide (DEAm), and N-vinylcaprolactam (NVCL); the hydrophilic monomer is selected from at least one of acrylic acid (AAc), methacrylic acid (MAA), acrylamide (AM), and N-hydroxymethylacrylamide (NMA); the hydrophobic monomer is selected from at least one of N-isopropylacrylamide (TBAm), N-phenylacrylamide (NPAm), methyl methacrylate (MMA), and tert-butyl methacrylate (t-BMA); the cationic functionalized monomer is N-(3-aminopropyl)methacrylamide hydrochloride (APM), [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC), [3-(methacryloylamino)propyl]trimethylammonium chloride (MPTAC), and dimethyl... The fluorescent monomer is selected from at least one of aminopropyl methacrylamide (DMAPMA); the fluorescent monomer is selected from at least one of fluorescein acrylamide (FITC-AAm), fluorescein methacrylate (Fl-MA), rhodamine B acrylamide (Rh-AAm), and coumarin acrylamide (Cou-AAm); the crosslinking agent is selected from at least one of N,N'-methylenebisacrylamide (BIS), N,N'-bis(acryloyl)cysteine ​​(BAC), polyethylene glycol diacrylate (PEGDA), and ethylene glycol dimethacrylate (EGDMA); the initiator is selected from at least one of ammonium persulfate (APS), potassium persulfate (KPS), azobisisobutyronitrile (AIBN), and benzoyl peroxide (BPO); the catalyst is selected from at least one of tetramethylethylenediamine (TEMED), N,N-dimethylaniline (DMA), N,N-diethylaniline (DEA), and sodium bisulfite (SBS).

7. The preparation method according to claim 3, characterized in that, The molar ratio of the thermosensitive monomer, hydrophilic monomer, hydrophobic monomer, and cationic functionalized monomer mentioned in step (3) is (5-8):(1-3):(1-5):(0-1), and the total concentration is 10mM.

8. The preparation method according to claim 3, characterized in that, In step (3), the mass ratio of the product obtained in step (2) to the initiator is (0.5-2.0):(0.5-1.5); the mass-volume ratio of the initiator to the catalyst is (1000-2000):(0.5-2.0) (mg / mL); and the mass ratio of the crosslinking agent to the initiator is (3-5):(40-55).

9. The preparation method according to claim 3, characterized in that, The polymerization reaction in step (3) is carried out at a temperature of 35-45°C and for a reaction time of 10-14 hours.

10. The use of the magnetic fluorescent molecularly imprinted nanoparticles according to claim 1 in the preparation of a cardiac troponin I detection kit.

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