Fluorescent immunoassay kit based on multi-epitope imprinting and application thereof in detection of liver cancer markers

Abstract

The application provides a multi-epitope imprinting-based fluorescent immunoassay kit and application thereof in detection of liver cancer markers, and the kit comprises: an N-terminal multi-epitope imprinting microplate, the surface of the microplate is simultaneously imprinted with N-terminal epitope peptide templates of alpha-fetoprotein (AFP), alpha-L-fucosidase (AFU) and alpha1-antitrypsin (AAT); and three kinds of C-terminal epitope imprinted fluorescent silica nanoparticles Cy3@CEIPs, Cy5@CEIPs and Cy7@CEIPs, which specifically recognize the three kinds of markers respectively. By constructing a sandwich structure of microplate@NMEIPs target protein Cy@CEIPs, high specificity is ensured by double molecular imprinting recognition, and multi-element synchronous detection is realized based on different fluorescent signals. The method does not need natural antibodies, can realize high-specificity, ultra-sensitivity and multi-element synchronous detection of multiple liver cancer markers in complex biological samples, and has important application value in early diagnosis of liver cancer.

Description

Technical Field

[0001] This invention relates to the fields of molecular imprinting technology and fluorescence immunoassay technology, and more specifically, to a fluorescence immunoassay kit based on multi-epitope imprinting and its application in the detection of liver cancer biomarkers. Background Technology

[0002] Disease biomarkers, as important biochemical indicators reflecting normal physiological and pathological processes and aiding in clinical diagnosis, play an irreplaceable role in disease diagnosis, classification, treatment guidance, disease progression monitoring, and prognostic assessment. However, relying solely on a single disease biomarker often has limited sensitivity and specificity, potentially leading to false positives and failing to comprehensively and accurately reflect the disease type and stage. Therefore, disease biomarker detection is gradually evolving from single-marker to combined and diversified approaches. By jointly detecting multiple disease biomarkers, the accuracy of disease diagnosis can be significantly improved, enabling the development of more scientific and rational individualized treatment plans for different patients.

[0003] Taking liver cancer as an example, it includes primary and secondary liver cancer. Primary liver cancer is currently the fourth most common malignant tumor and the second leading cause of cancer death in my country, seriously threatening people's lives and health. Currently, alpha-fetoprotein (AFP) is the most important serum biomarker for diagnosing liver cancer and monitoring treatment efficacy. However, AFP's specificity is not absolute; its levels can also rise in pregnancy, germ cell tumors of the gonads, and gastrointestinal tumors, easily leading to false-positive diagnoses. Alpha-L-fucosidase (AFU) is another important diagnostic biomarker for liver cancer, with abnormally elevated activity in the serum of liver cancer patients. Furthermore, acute-phase reactive proteins such as alpha-1-antitrypsin (AAT) also show abnormal increases in pathological conditions such as tumors. Clinical practice shows that combined detection of these three biomarkers—AFP, AFU, and AAT—can complement and validate each other, thereby greatly improving the accuracy and reliability of liver cancer diagnosis.

[0004] Currently, the most commonly used methods for detecting disease biomarkers are immunoassays, such as fluorescence immunoassay and enzyme-linked immunosorbent assay (ELISA). In these methods, antibodies play a crucial role in the specific recognition of disease biomarkers. However, the preparation of natural antibodies is complex, involves long screening cycles, and is extremely expensive. Furthermore, their storage conditions are demanding, they are unstable, and prone to inactivation; in some cases, it is even impossible to obtain high-quality antibodies targeting specific targets. More importantly, these traditional immunoassays typically only detect one disease biomarker at a time. When multiple biomarkers need to be detected together, multiple antibodies must be prepared and modified separately, requiring multiple independent experimental operations. This not only results in a huge waste of reagents, time, and manpower, leading to low detection efficiency, but also significantly increases sample consumption and overall detection costs, making it difficult to meet the clinical demand for rapid, efficient, and low-cost multivariate detection. Therefore, establishing a novel immunoassay method that does not require antibodies but still possesses high specificity and ultrasensitivity, and can achieve multivariate detection, has become an urgent technical need in this field.

[0005] Molecularly imprinted polymers (MIPs), hailed as "artificial antibodies," are chemically synthesized materials capable of mimicking the specific recognition interaction between antibodies and antigens. MIPs possess a pre-defined specific recognition capability for template molecules and boast significant advantages such as simple preparation, high physicochemical stability, tolerance to harsh environments (e.g., extreme pH, organic solvents, high temperatures), and low cost, offering a potential solution to overcome the limitations of antibodies themselves. As early as 1985, Glad et al. successfully applied MIPs to the imprinting of biomacromolecules, primarily using the whole-molecule imprinting method. However, this method faced challenges such as difficulty in obtaining low-abundance protein templates, irreversible conformational changes during imprinting, and difficulty in completely removing the protein template after imprinting, severely limiting its application in protein biomarker detection. To address these challenges, epitope imprinting was proposed in 2001. This strategy selects a short, characteristic peptide sequence (i.e., an epitope) from a protein as a template for imprinting, thereby achieving specific recognition of intact proteins. The epitope peptides used can be artificially obtained through solid-phase synthesis, making them easy to prepare and structurally stable. For example, Shao et al. successfully developed an electrochemical sensor using the C-terminal dodecapeptide of AFP as an epitope template. Nevertheless, traditional epitope imprinting methods still suffer from drawbacks such as disordered imprinting site orientation, low imprinting efficiency, and limited adaptability to substrate materials. To further optimize this method, Xing et al. proposed a boron affinity-anchored epitope controllable directional surface imprinting method in 2019. By glycosylating the epitope peptide and utilizing the reversible covalent bonding between boric acid groups and cis-dihydroxy groups, they achieved precise immobilization and convenient removal of the template on the substrate, thereby preparing imprinted materials with strong affinity and high imprinting efficiency.

[0006] With the advancement of research, multi-epitope imprinting has emerged, providing a new technical pathway for the simultaneous detection of multiple targets. For example, Yang et al. prepared multi-epitope imprinted polymers using a phase-inversion-based polyethersulfone self-assembly method, which were able to selectively extract multiple proteins from human plasma simultaneously. This demonstrates the significant advantages of multi-epitope imprinting strategies in simultaneously recognizing multiple proteins. The choice of signal output method is crucial when constructing MIPs sensors. Fluorescence detection is favored due to its high sensitivity and ease of operation. Silica nanoparticles, due to their excellent optical transparency and modifiability, are often used as carriers of fluorescent signals. By doping fluorescent dyes such as the Cy series into the interior of silica nanoparticles or modifying their surface, nanoprobes with stable and tunable fluorescence properties can be prepared. Combining such fluorescent nanoparticles with molecular imprinting technology can provide highly specific recognition using the imprinted cavity and achieve ultrasensitive detection using the fluorescence signal, demonstrating enormous application potential.

[0007] In summary, although molecular imprinting technology, especially epitope imprinting, has shown great potential as an antibody alternative, current technologies lack a complete solution that integrates multiplex capture, dual-specificity recognition, and multivariate fluorescence signal detection to achieve rapid, low-cost, highly sensitive, and highly specific simultaneous detection of biomarkers for various diseases such as liver cancer. Therefore, there is an urgent need in this field for an innovative, integrated technological solution to overcome these multiple technical bottlenecks. Summary of the Invention

[0008] In view of this, the present invention proposes a fluorescence immunoassay kit based on multi-epitope imprinting and its application in the detection of liver cancer biomarkers, in order to solve the problems existing in the prior art.

[0009] To achieve the above objectives, this invention proposes a fluorescence immunoassay kit based on multi-epitope blotting, comprising: An N-terminal multi-epitope imprinted microplate, the surface of which is simultaneously imprinted with N-terminal epitope peptide templates of AFP, AFU and AAT, forming a multi-specific recognition cavity; Three types of C-terminal epitope-imprinted fluorescent silica nanoparticles, namely Cy3@C-EIPs, Cy5@C-EIPs and Cy7@C-EIPs, specifically recognize and label the C-terminal epitopes of AFP, AFU and AAT, respectively. The N-terminal multi-epitope imprinted microplate and the three C-terminal imprinted fluorescent silica nanoparticles together constitute a sandwich structure detection system for detecting AFP, AFU and AAT.

[0010] Furthermore, the N-terminal multi-epitope imprinted microplate is prepared using a multi-epitope directional precise imprinting strategy. Its imprinted layer is formed by polymerization of a mixed silanizing agent around the N-terminal epitope peptide template. The mixed silanizing agent includes aminopropyltriethoxysilane (APTES), 3-ureapropyltriethoxysilane (UPTES), benzyltriethoxysilane (BnTES), isobutyltriethoxysilane (IBTES), and tetraethyl orthosilicate (TEOS).

[0011] Furthermore, in the mixed silanizing reagent, the molar ratio of APTES, UPTES, BnTES, IBTES and TEOS is (10-30):(10-30):10:(10-30):(10-60), preferably 20:20:10:20:30.

[0012] Furthermore, the Cy3@C-EIPs, Cy5@C-EIPs and Cy7@C-EIPs are prepared by a boron affinity anchored epitope controllable directional surface imprinting strategy, and their imprinted layers are formed by the polymerization of mixed silanizing reagents around the corresponding C-terminal epitope peptide template.

[0013] Furthermore, in the mixed silanizing reagents used in the preparation of Cy3@C-EIPs, the molar ratio of APTES, UPTES, BnTES, IBTES and TEOS is (10-20):(10-20):0:(10-20):(40-70), preferably 20:10:0:20:50; In the preparation of Cy5@C-EIPs, the molar ratio of APTES, UPTES, BnTES, IBTES and TEOS in the mixed silanizing reagents is 10:10:(10-20):(10-30):(30-60), preferably 10:10:10:30:40. In the preparation of Cy7@C-EIPs, the molar ratio of APTES, UPTES, BnTES, IBTES and TEOS in the mixed silanizing reagents is (10-20):(10-20):0:(10-20):(40-70), preferably 15:15:0:20:50.

[0014] Furthermore, the fluorescent dyes in Cy3@C-EIPs and Cy5@C-EIPs are doped in an outer layer, while the fluorescent dyes in Cy7@C-EIPs are doped in both inner and outer layers.

[0015] The present invention also provides a detection system for the simultaneous detection of AFP, AFU and AAT, comprising the multi-epitope-based fluorescent immunoassay kit as described above.

[0016] The present invention also provides the use of the kit or detection system described above in the preparation of products for the in vitro quantitative detection of AFP, AFU and AAT concentrations in biological samples.

[0017] The present invention also provides the use of the kit or detection system described above in the preparation of in vitro diagnostic products for auxiliary diagnosis, efficacy monitoring or prognostic assessment of liver cancer.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention targets liver cancer biomarkers alpha-fetoprotein (AFP), α-L-fucosidase (AFU), and α1-antitrypsin (AAT) as proteins. A multi-epitope imprinting strategy is employed to prepare N-terminal multi-epitope imprinted microplates (microplate@N-MEIPs) as extraction arrays. Three types of C-terminal epitope imprinted Cy-series fluorescent dye-doped silica nanoparticles (Cy@C-EIPs) are prepared as labeling particles using the same epitope imprinting strategy, forming a sandwich structure of "microplate@N-MEIPs-target protein-Cy@C-EIPs". This establishes a fluorescence immunoassay based on multi-epitope imprinting. The established immunoassay method is antibody-free and offers advantages such as simple operation, fast detection speed, low cost, and good stability. It can perform highly specific, ultrasensitive, and multivariate detection of multiple disease biomarkers in complex biological samples, playing a significant role in the biomedical field. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the construction of a fluorescence immunoassay based on multi-epitope imprinting and its principle of high specificity, ultrasensitivity, and multivariate detection of three liver cancer biomarkers.

[0020] Figure 2 The images are SEM characterization images of the microplates; where (a) is an untreated microplate, (b) is a microplate @N-MEIPs, and (c) is a microplate @N-MNIPs.

[0021] Figure 3 The images show the fluorescence emission spectra of different Cy series fluorescent dyes with different doping methods; (a) is a comparison of the doping methods of Cy3, (b) is a comparison of the doping methods of Cy5, and (c) is a comparison of the doping methods of Cy7.

[0022] Figure 4 TEM characterization and particle size distribution of Cy series silica nanoparticles at different stages are shown; (a) Cy3@SiO2, (b) Cy5@SiO2, (c) Cy7@SiO2, (d) Cy3@BA, (e) Cy5@BA, (f) Cy7@BA, (g) Cy3@C-EIPs, (h) Cy5@C-EIPs, (i) Cy7@C-EIPs, (j) Cy3@C-NIPs, (k) Cy5@C-NIPs, and (l) Cy7@C-NIPs.

[0023] Figure 5SEM images of Cy series silica nanoparticles at different stages are shown; (a) Cy3@SiO2, (b) Cy5@SiO2, (c) Cy7@SiO2, (d) Cy3@BA, (e) Cy5@BA, (f) Cy7@BA, (g) Cy3@C-EIPs, (h) Cy5@C-EIPs, (i) Cy7@C-EIPs, (j) Cy3@C-NIPs, (k) Cy5@C-NIPs, and (l) Cy7@C-NIPs.

[0024] Figure 6 The images show the fluorescence spectra during the synthesis of Cy@C-EIPs; where (a) represents Cy3@C-EIPs, (b) represents Cy5@C-EIPs, and (c) represents Cy7@C-EIPs.

[0025] Figure 7 The fluorescence intensity diagrams of microplates @BA and Cy@BA for different test compounds are shown; (a) shows the response of microplate @BA to puerarin and psoralen, (b) shows the response of Cy3@BA to HRP and BSA, (c) shows the response of Cy5@BA to HRP and BSA, and (d) shows the response of Cy7@BA to HRP and BSA.

[0026] Figure 8 Figures show the optimization results of microplates @N-MEIPs and Cy@C-EIPs under different monomer ratios and blotting times; where (a) to (c) are the fluorescence intensities and IF values ​​of microplates @N-MEIPs / N-MNIPs for AFP, AFU, and AAT under different monomer ratios, and (d) to (f) are the fluorescence intensities of Cy3@C-EIPs / C-NIPs for AFP, Cy5@C-EIPs / C-NIPs for AFU, and Cy7@C-EIPs for AAT under different monomer ratios. The fluorescence intensity and IF value of Ps / C-NIPs for AAT are shown in (g) to (i), which represent the fluorescence intensity and IF value of microplates @N-MEIPs / N-MNIPs for AFP, AFU, and AAT at different blotting times. The fluorescence intensity and IF value of Cy3@C-EIPs / C-NIPs for AFP, Cy5@C-EIPs / C-NIPs for AFU, and Cy7@C-EIPs / C-NIPs for AAT at different blotting times are shown in (j) to (l).

[0027] Figure 9Figure 1 shows the optimization results of extraction time and labeling time. Among them, (a) to (c) are the fluorescence intensities of microplates @N-MEIPs / N-MNIPs for AFP, AFU, and AAT at different extraction times, and (d) to (f) are the fluorescence intensities of Cy3@C-EIPs / C-NIPs for AFP, Cy5@C-EIPs / C-NIPs for AFU, and Cy7@C-EIPs / C-NIPs for AAT at different labeling times.

[0028] Figure 10 The figure shows the results of the specificity assessment of different proteins by fluorescence immunoassay based on multi-epitope blotting; where (a) is the response to AFP and its competing proteins, (b) is the response to AFU and its competing proteins, and (c) is the response to AAT and its competing proteins.

[0029] Figure 11 The figure shows the stability results of the fluorescence immunoassay based on multi-epitope blotting; where (a) is the fluorescence intensity and percentage of AFP within 6 months, (b) is the fluorescence intensity and percentage of AFU within 6 months, and (c) is the fluorescence intensity and percentage of AAT within 6 months.

[0030] Figure 12 The graphs show the concentration response curves of three liver cancer biomarkers based on multi-epitope immunoassay; where (a) is the standard curve for AFP, (b) is the standard curve for AFU, and (c) is the standard curve for AAT. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0033] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0034] Example 1 1. Experimental Section 1.1 Reagents and Materials Alpha-fetoprotein (AFP), α-L-fucosidase (AFU), human serum albumin (HSA), transferrin (TRF), horseradish peroxidase (HRP), and bovine serum albumin (BSA) were purchased from Shanghai Guchen Biotechnology Co., Ltd. (Shanghai, China); AAT was purchased from Abcam (Shanghai, China); the glycated N-terminal epitope templates Fru-KRTLHRNEYG, Fru-KHSATRFDPT, Fru-KEDPQGDAAQ and the glycated C-terminal epitope templates SKTRAALGVK-Fru, WALALTNVIK-Fru, and GKVVNPTQKK-Fru of AFP, AFU, and AAT were synthesized from Nanjing Peptide Valley Biotechnology Co., Ltd., with a purity ≥98% (Nanjing, China); puerarin and psoralen were purchased from Chengdu Manster Biotechnology Co., Ltd. (Chengdu, China); the human AFP, AFU, and AAT ELISA kits were purchased from Wuhan Kelu Biotechnology Co., Ltd. (Wuhan, China); Sulfo-Cy3 NHS Sulfo-Cy5NHS ester and Sulfo-Cy7 NHS ester were purchased from Beijing Oukenas Biochemical Technology Co., Ltd. (Beijing, China); concentrated sulfuric acid (H2SO4) was purchased from Beijing Beihua Fine Chemicals Co., Ltd. (Beijing, China); concentrated nitric acid (HNO3) was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd. (Tianjin, China); dimethyl sulfoxide (DMSO), 50% glutaraldehyde, anhydrous ethanol, methanol, ammonia (NH3·H2O, 25%), acetonitrile, and glacial acetic acid were purchased from Tianjin Kemio Chemical Reagent Co., Ltd. (Tianjin, China); aminopropyltriethoxysilane (APTES) 3-Uretopropyltriethoxysilane (UPTES), benzyltriethoxysilane (BnTES), isobutyltriethoxysilane (IBTES), tetraethyl orthosilicate (TEOS), 4-formylphenylboronic acid (FPBA), and sodium cyanoborohydride (NaBH3CN) were purchased from Shanghai Maclean Biotechnology Co., Ltd. (Shanghai, China); black microplates were purchased from White Shark Biotechnology Co., Ltd. (Hefei, China); the concentration of phosphate buffered saline (PBS) used in the experiments was 10 mM, and the pH was 7.4; the water used in the experiments was double-distilled water.

[0035] 1.2 Instruments Scanning electron microscopy (SEM) was performed using a Gemini Sigma 300 (Carl Zeiss AG, Oberkochen, Germany); transmission electron microscopy (TEM) was performed using a JEM-F200 (JEOL Ltd., Tokyo, Japan); a nanolaser particle size analyzer was performed using a ZEN3690 (Malvern Panalytical, Malvern, UK); and an enzyme-linked immunosorbent assay (ELISA) reader was performed using a SpectraMax iD5 (Molecular Devices, LLC, California, USA). The excitation / emission wavelengths for fluorescence detection were Cy3: 525 / 565 nm, Cy5: 625 / 665 nm, and Cy7: 720 / 765 nm.

[0036] 1.3 Preparation of microplates @N-MEIPs This embodiment details the preparation process of a multi-epitope imprinted microplate for simultaneously capturing three liver cancer biomarkers (AFP, AFU, and AAT). A schematic diagram of the principle can be found in the appendix. Figure 1 .

[0037] 1.3.1 Preparation of microplates @NH2. Microplates were immersed in a H2SO4 / HNO3 (v / v, 3:1) mixed solution for 30 min. The activated microplates were then washed with plenty of water to neutralize the pH. 250 μL of a 5% (v / v) APTES aqueous solution (pH 6.9) was added to the activated microplates and shaken for 2 h. The microplates were then cured in an oven at 62 °C for 2 h. The resulting @NH2 microplates were washed three times each with water and anhydrous ethanol, and then dried in an oven at 50 °C.

[0038] 1.3.2 Preparation of microplates @CHO. 250 μL of 0.5% (v / v) glutaraldehyde aqueous solution was added to microplates @NH2, shaken for 2 h, washed six times with water, and dried in an oven at 50°C. The prepared microplates @CHO were stored at 4°C for later use.

[0039] 1.3.3 Preparation of microplates @BA. Add 150 μL each of 10.0 mg / mL FPBA and 10.0 mg / mL NaBH3CN methanol solutions to microplates @NH2, seal and shake for 24 h, then wash three times each with water and anhydrous ethanol. Air dry at room temperature, and store the prepared microplates @BA at 4℃ for later use.

[0040] 1.3.4 Preparation of microplates @N-MEIPs. The preparation process of microplates @N-MEIPs mainly includes the following three steps: 1) Immobilization of multiple glycation epitopes. The N-terminal glycation epitopes of three proteins, AFP, AFU, and AAT, were prepared into a 0.1 mg / mL solution using PBS. 200 μL of the solution was added to a microplate @BA and shaken for 2 h. The microplate with the three glycation epitopes immobilized was then washed three times with PBS.

[0041] 2) Directional Imprinting. 160 μL of anhydrous ethanol solution containing 4.5 μL NH3·H2O and 10 μL water was added to a microplate with three fixed glycosylation epitopes. Then, 40 μL of anhydrous ethanol solution containing 0.5% silanizing reagent (molar ratio of APTES, UPTES, BnTES, IBTES, and TEOS of 20:20:10:20:30) was added. After shaking for 1 h, the plate was washed three times with anhydrous ethanol.

[0042] 3) Removal of multiple glycosylated epitopes. Add 200 μL of acetonitrile / water / glacial acetic acid (50:49:1, v / v / v) to the microplate, shake for 20 min, and repeat the template removal process three times. Finally, wash three times each with water and anhydrous ethanol. Air-dry the prepared microplates @N-MEIPs at room temperature for later use. The corresponding microplates @N-MNIPs follow the same steps except that the three glycosylated N-terminal epitope templates are not added.

[0043] 1.4 Preparation of Cy@C-EIPs This embodiment details the preparation of three types of C-terminal epitope-imprinted fluorescently labeled particles targeting AFP, AFU, and AAT, respectively. A schematic diagram illustrating the underlying principles can be found in the appendix. Figure 1 In this embodiment, three Cy@C-EIPs were synthesized using Cy3, Cy5, and Cy7 fluorescent dyes. Cy3@C-EIPs and Cy5@C-EIPs were synthesized using an outer-layer doping method with fluorescent dyes, while Cy7@C-EIPs were synthesized using a two-layer doping method with fluorescent dyes on both the inner and outer layers. For the detection wavelengths, Cy3 was 525 nm / 565 nm, Cy5 was 625 nm / 665 nm, and Cy7 was 720 nm / 765 nm.

[0044] This embodiment details the preparation of Cy7@C-EIPs (for AAT) as an example. The preparation processes of Cy3@C-EIPs (for AFP) and Cy5@C-EIPs (for AFU) are similar, differing only in the fluorescent dye, the corresponding template peptide sequence, and the optimal monomer ratio.

[0045] 1.4.1 Preparation of Cy7-APTES precursor solution. A 5 mg / mL solution of Cy7 fluorescent dye was prepared using DMSO. 80 μL of this solution was then added to 300 μL of anhydrous ethanol, followed by 20 μL of APTES. The solution was sealed and shaken in the dark for 24 h.

[0046] 1.4.2 Preparation of Cy7@SiO2@NH2. 16 mL of anhydrous ethanol, 8 mL of water, and 2 mL of NH3·H2O were sequentially added to a 100 mL brown conical flask. The mixture was magnetically stirred at 1,000 rpm. 2 mL of TEOS and 16 mL of anhydrous ethanol were thoroughly mixed and quickly added to the conical flask. Then, 300 μL of Cy7-APTES precursor solution was added. After 1 min, the stirring speed was reduced to 600 rpm, and the reaction was carried out in the dark for 2 h. The mixture was centrifuged at 10,000 rpm for 10 min, and Cy7@SiO2 was collected. The mixture was washed twice with water and twice with anhydrous ethanol, and finally dispersed in 10 mL of anhydrous ethanol. 50 μL of APTES was added to the reaction product, and the mixture was shaken in the dark for 2 h. The mixture was centrifuged at 10,000 rpm for 10 min, and Cy7@SiO2@NH2 was collected. The mixture was washed twice with water and twice with anhydrous ethanol, and finally dispersed in 10 mL of anhydrous ethanol.

[0047] 1.4.3 Preparation of Cy7@SiO2@NH2@Cy7@SiO2@NH2. 50 μL of 5 mg / mL Cy7 fluorescent dye was added to Cy7@SiO2@NH2, and the mixture was shaken in the dark for 12 h to obtain Cy7@SiO2@NH2@Cy7. 16 mL of anhydrous ethanol, 8 mL of water, and 2 mL of NH3·H2O were sequentially added to a 100 mL brown conical flask, and the mixture was magnetically stirred at 1,000 rpm. 2 mL of TEOS and 16 mL of anhydrous ethanol were thoroughly mixed and then rapidly added to the conical flask. After 1 min, the stirring speed was reduced to 600 rpm, and prepolymerization was carried out for 5 min. Subsequently, Cy7@SiO2@NH2@Cy7 was added to the conical flask, and the reaction was carried out in the dark for 2 h. Centrifuge at 10,000 rpm for 10 min to collect Cy7@SiO2@NH2@Cy7@SiO2, wash twice with water and twice with anhydrous ethanol, and finally disperse in 15 mL of anhydrous ethanol. Add 75 μL of APTES to the above reaction product and shake in the dark for 2 h. Centrifuge at 10,000 rpm for 10 min to collect Cy7@SiO2@NH2@Cy7@SiO2@NH2, wash twice with water and twice with anhydrous ethanol, and finally disperse in 15 mL of methanol.

[0048] 1.4.4 Preparation of Cy7@BA. 5 mg / mL of FPBA and 5 mg / mL of NaBH3CN were added to 15 mL of a methanol solution of Cy7@SiO2@NH2@Cy7@SiO2@NH2, and the solution was sealed and shaken for 24 h in the dark. Cy7@BA was obtained by centrifugation at 10,000 rpm for 10 min, washed twice with water and twice with anhydrous ethanol, and finally dispersed in PBS and stored at 4 °C in the dark.

[0049] 1.4.5 Preparation of Cy7@C-EIPs. The preparation process of Cy7@C-EIPs mainly includes the following three steps: 1) Immobilization of glycated epitopes. 0.1 mg / mL of the glycated C-terminal epitope of AAT was added to Cy7@BA and shaken in the dark for 2 hours. The mixture was then centrifuged at 10,000 rpm for 10 minutes, and the Cy7@BA with the glycated C-terminal epitope immobilized was collected and washed three times with PBS.

[0050] 2) Directional Imprinting. Cy7@BA with glycosylated C-terminal epitopes fixed were uniformly dispersed in 16 mL of anhydrous ethanol solution containing 450 μL NH3·H2O and 1 mL water and shaken in the dark for 5 min. Then, 4 mL of anhydrous ethanol solution containing 0.5% silanizing reagent (molar ratio of APTES, UPTES, BnTES, IBTES, and TEOS of 15:15:0:20:50) was added, and the mixture was shaken in the dark for 1 h. After centrifugation at 10,000 rpm for 10 min, Cy7@C-EIPs were collected and washed three times with anhydrous ethanol.

[0051] 3) Remove glycosylated epitopes. Disperse Cy7@C-EIPs evenly in 15 mL of acetonitrile / water / glacial acetic acid (…). v / v / v In a solution of 50:49:1, the sample was shaken in the dark for 20 min. The template removal process was repeated three times, followed by washing twice with water and twice with anhydrous ethanol. Finally, the Cy7@C-EIPs were redispersed in PBS and stored at 4°C in the dark for later use.

[0052] The preparation of Cy7@C-NIPs was identical except for the absence of a glycosylated C-terminal epitope template. The preparation of Cy3@C-EIPs and Cy5@C-EIPs followed the experimental steps in 1.4.2 to 1.4.5, except that the precursor solution was not added, Cy7 was replaced with Cy3 or Cy5, the corresponding template was replaced with a glycosylated C-terminal epitope of AFP or AFU, and the molar ratio of APTES, UPTES, BnTES, IBTES, and TEOS was changed to 20:10:0:20:50 or 10:10:10:30:40, respectively.

[0053] 1.5 Boron affinity selectivity To verify whether the boric acid groups on the surface of microplates @BA and Cy@BA were successfully modified and possessed specific binding ability, the following experiments were conducted.

[0054] 1.5.1 Boron affinity selectivity of microplates @BA. 200 μL of 10 ng / mL PBS solutions of puerarin and psoralen were added to microplates @BA, respectively. After shaking for 30 min, the plates were washed three times with PBS and then subjected to fluorescence detection using a microplate reader. The excitation / emission wavelengths of puerarin and psoralen were 270 nm / 480 nm and 290 nm / 460 nm, respectively. The experiment was performed in triplicate. For the control experiment, the experimental procedures were the same as described above, except that the analyte was not added to the extraction solution.

[0055] 1.5.2 Boron affinity selectivity of Cy@BA. 200 μL of PBS solutions containing 0.1 mg / mL HRP and 0.1 mg / mL BSA were added to each well of a CHO microplate, and the plates were shaken for 2 h, followed by three washes with PBS. Then, 200 μL of Cy3@BA, Cy5@BA, and Cy7@BA were added, and the plates were shaken for 1 h, followed by three washes with PBS. Fluorescence detection was performed using a microplate reader. The experiment was performed in triplicate. For the control experiment, the experimental procedures were the same as described above, except that the target protein was not added to the extraction solution.

[0056] 1.6 Optimization of Imprint Conditions To obtain optimal recognition performance, the preparation conditions (monomer ratio and imprinting time) of microplates @N-MEIPs and Cy@C-EIPs were systematically optimized, with the imprinting factor (IF = Q MIPs / Q NIPs () as an evaluation indicator.

[0057] 1.6.1 Optimization of Cy@C-EIPs monomer ratios. 200 μL of PBS solution containing 0.1 μg / mL AFP, 0.25 ng / mL AFU, and 0.1 μg / mL AAT were added to each well of the @CHO microplate. After shaking for 2 h, the plate was washed three times with PBS. Then, 200 μL of PBS solution containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs prepared with different molar ratios of silanizing reagent monomers (APTES, UPTES, BnTES, IBTES, and TEOS) were added to each well of the @CHO microplate. After shaking for 40 min, the plate was washed three times with PBS. Finally, fluorescence detection was performed using a microplate reader. The experimental procedures for the corresponding Cy3@C-NIPs, Cy5@C-NIPs, and Cy7@C-NIPs were the same as described above. The experiments were performed in triplicate. For the control experiment, the experimental procedures were the same as described above, except that the three target proteins were not added to the extraction solution.

[0058] 1.6.2 Optimization of Cy@C-EIPs Blotting Time. 200 μL of PBS solution containing 0.1 μg / mL AFP, 0.25 ng / mL AFU, and 0.1 μg / mL AAT were added to each well of the @CHO microplate. After shaking for 2 h, the plates were washed three times with PBS. Then, 200 μL of PBS solution containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs prepared at different blotting times (40 min, 50 min, 60 min, 70 min, and 80 min) were added to each well of the @CHO microplate. After shaking for 40 min, the plates were washed three times with PBS. Finally, fluorescence detection was performed using a microplate reader. The experimental procedures for the corresponding Cy3@C-NIPs, Cy5@C-NIPs, and Cy7@C-NIPs were the same as described above. The experiments were performed in triplicate. For the control experiment, the experimental procedures were the same as described above, except that the three target proteins were not added to the extraction solution.

[0059] 1.6.3 Optimization of monomer ratios for @N-MEIPs microplates. 200 μL of a PBS mixture containing 0.1 μg / mL AFP, 0.25 ng / mL AFU, and 0.1 μg / mL AAT was added to @N-MEIPs microplates prepared with different molar ratios of silanizing reagent monomers (APTES, UPTES, BnTES, IBTES, and TEOS). After shaking for 40 min, the plates were washed three times with PBS. Then, 200 μL of a PBS mixture containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs was added to the same @N-MEIPs microplates. After shaking for 40 min, the plates were washed three times with PBS. Finally, fluorescence detection was performed using a microplate reader. The experimental procedures for the corresponding @N-MNIPs microplates were the same as described above. Experiments were performed in triplicate. For the control experiment, the experimental procedures were the same as described above, except that the three target proteins were not added to the extraction solution.

[0060] 1.6.4 Optimization of Blotting Time for @N-MEIPs Microplates. 200 μL of a PBS mixture containing 0.1 μg / mL AFP, 0.25 ng / mL AFU, and 0.1 μg / mL AAT was added to @N-MEIPs microplates prepared at different blotting times (40 min, 50 min, 60 min, 70 min, and 80 min). After shaking for 40 min, the plates were washed three times with PBS. Then, 200 μL of a PBS mixture containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs was added to the same @N-MEIPs microplates. After shaking for 40 min, the plates were washed three times with PBS. Finally, fluorescence detection was performed using a microplate reader. The experimental procedures for the corresponding @N-MNIPs microplates were the same as described above. Experiments were performed in triplicate. For the control experiment, the experimental procedures were the same as described above, except that the three target proteins were not added to the extraction solution.

[0061] 1.7 Investigation of extraction time for microplates@N-MEIPs Add 200 μL of a PBS mixture containing 0.1 μg / mL AFP, 0.25 ng / mL AFU, and 0.1 μg / mL AAT to the @N-MEIPs microplates. Shake for 20 min, 30 min, 40 min, 50 min, and 60 min, respectively, and wash three times with PBS. Then, add 200 μL of a PBS mixture containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs to the same @N-MEIPs microplates. Shake for 40 min and wash three times with PBS. Finally, perform fluorescence detection using a microplate reader. The experimental procedures for the corresponding @N-MNIPs microplates are the same as above. Experiments are performed in triplicate. For the control experiment, the experimental procedures are the same as above, except that the three target proteins are not added to the extraction solution.

[0062] 1.8 Examination of the labeling time of Cy@C-EIPs Add 200 μL of a PBS mixture containing 0.1 μg / mL AFP, 0.25 ng / mL AFU, and 0.1 μg / mL AAT to the @N-MEIPs microplates, shake for 40 min, and wash three times with PBS. Then, add 200 μL of a PBS mixture containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs to the same @N-MEIPs microplates, shake for 20 min, 30 min, 40 min, 50 min, and 60 min, and wash three times with PBS. Finally, perform fluorescence detection using a microplate reader. The experimental procedures for Cy@C-NIPs are the same as above. Experiments are performed in triplicate. For the control experiment, the experimental procedures are the same as above, except that the three target proteins are not added to the extraction solution.

[0063] 1.9 Specificity assessment Add 200 μL of PBS solution containing 0.1 μg / mL AFP, 0.25 ng / mL AFU, 0.1 μg / mL AAT, 0.1 μg / mL BSA, 0.1 μg / mL HSA, 0.1 μg / mL HRP, and 0.1 μg / mL TRF to each well of the @N-MEIPs microplate. Shake for 40 min and wash three times with PBS. Then, add 200 μL of a PBS mixture containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs to each well of the @N-MEIPs microplate. Shake for 40 min and wash three times with PBS. Finally, perform fluorescence detection using a microplate reader. The experimental procedures for the corresponding @N-MNIPs and Cy@C-NIPs microplates are the same as above. Experiments are performed in triplicate. For the control experiment, the experimental procedures are the same as above, except that the target protein is not added to the extraction solution.

[0064] 1.10 Stability Test Add 200 μL of a PBS mixture containing 0.1 μg / mL AFP, 0.25 ng / mL AFU, and 0.1 μg / mL AAT to the @N-MEIPs microplates, shake for 40 min, and wash three times with PBS. Then, add 200 μL of a PBS mixture containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs to the same @N-MEIPs microplates, shake for 40 min, and wash three times with PBS. Finally, perform fluorescence detection using a microplate reader. The above experiments were performed for six consecutive months at one-month intervals using @N-MEIPs and Cy@C-EIPs prepared from the same batch. The experiments were performed in triplicate. For the control experiment, the experimental procedures were the same as above, except that the three target proteins were not added to the extraction solution.

[0065] 1.11 Concentration Response Curve AFP was prepared in PBS at concentrations of 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, and 10 μg / mL. AFU was prepared in PBS at concentrations of 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL. AAT was prepared in PBS at concentrations of 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL. Mixed protein solutions were prepared at these concentrations, from lowest to highest. 200 μL of each of these mixed protein solutions was added to a microplate @N-MEIPs, and the plate was shaken for 40 min, followed by washing three times with PBS. Then, 200 μL of a PBS mixture containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs was added to the above-mentioned @N-MEIPs microplates. After shaking for 40 min, the plates were washed three times with PBS. Finally, fluorescence detection was performed using a microplate reader. The experiment was performed in triplicate. For the control experiment, the experimental procedures were the same as above, except that the three target proteins were not added to the extraction solution.

[0066] 1.12 Application of actual samples After centrifuging blood samples from healthy individuals and liver cancer patients, 50 μL of the supernatant serum was collected and diluted 100-fold with PBS. 200 μL of this diluted serum solution was added to a microplate @N-MEIPs, shaken for 40 min, and then washed three times with PBS. Next, 200 μL of a PBS mixture containing Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs was added to the same microplate @N-MEIPs, shaken for 40 min, and then washed three times with PBS. Finally, fluorescence detection was performed using a microplate reader. The experiment was performed in triplicate. For the control experiment, the experimental procedures were the same as above, except that PBS was used instead of the serum sample.

[0067] 2. Results and Discussion 2.1 Characterization of microplates@N-MEIPs The microplates @N-MEIPs were characterized by SEM, and the results are as follows: Figure 2 As shown. The surface of the microplate is untreated, therefore its surface is very smooth. Figure 2 a). The surface of the microplates @N-MEIPs underwent a series of chemical modifications and imprinting processes, resulting in a rough and uneven surface. Figure 2 b); However, since no template molecules are added during the imprinting process of microplates @N-MNIPs, imprint cavities cannot be formed, so their surface is smoother and more uniform than that of microplates @N-MEIPs. Figure 2 c) indicates that an imprinted cavity has been successfully formed on the surface of the microplate @N-MEIPs.

[0068] 2.2 Characterization of Cy@C-EIPs 2.2.1 Selection of Cy fluorescent dye doping mode. The doping mode of Cy fluorescent dye is mainly selected based on the intensity of the fluorescence emission spectrum signal. Three doping modes were investigated: inner layer doping, outer layer doping, and both inner and outer layer doping. The results are as follows: Figure 3 As shown. Although the fluorescence intensity of Cy3 with both inner and outer doping (Cy3@SiO2@Cy3) ​​is higher than that of Cy3 with only inner doping (Cy3@SiO2) and only outer doping (SiO2@Cy3), the fluorescence intensity of Cy3 with only outer doping is sufficient for subsequent analysis. Therefore, outer doping was chosen for Cy3. Figure 3 a); Cy5 outer-layer doping (SiO2@Cy5) has higher fluorescence intensity than inner-layer doping (Cy5@SiO2) and both inner and outer-layer doping (Cy5@SiO2@Cy5), therefore, outer-layer doping is preferred for Cy5. Figure 3 b); Cy7 with two layers of doping (Cy7@SiO2@Cy7) has higher fluorescence intensity than Cy7 with only one layer of doping (Cy7@SiO2) and only one layer of doping (SiO2@Cy7). Therefore, Cy7 is selected for two-layer doping (Cy7@SiO2@Cy7). Figure 3 c).

[0069] 2.2.2 Particle size analysis. The particle size of the three Cy@C-EIPs was characterized using TEM and a particle size analyzer. The results are as follows: Figure 4 As shown. All three types of Cy@SiO2 particles prepared were spherical with uniform size, and a particle size of approximately 200 nm. Figure 4 ac); its morphology was not changed after modification with boric acid groups (ac); Figure 4 df); after further imprinting, the three types of Cy@C-EIPs and Cy@C-NIPs prepared separately showed a thin, rough imprint layer on the surface ( Figure 4 (gl), among which the surface of Cy@C-EIPs is rougher than that of Cy@C-NIPs due to the presence of imprinted cavities. These results indicate that all three types of Cy@C-EIPs have been successfully prepared.

[0070] 2.2.3 Morphology Analysis. The morphology of the three Cy@C-EIPs was characterized by SEM, and the results are as follows: Figure 5 As shown. The prepared Cy@SiO2 ( Figure 5 ac), Cy@BA ( Figure 5 df), Cy@C-EIPs ( Figure 5 gi) and Cy@C-NIPs Figure 5 The jl) are all spherical, uniform in size, and well dispersed.

[0071] 2.2.4 Fluorescence spectrum of labeled particles. Figure 6 The images show the fluorescence spectra of the three Cy@C-EIPs during their synthesis. Although the fluorescence intensity of the fluorescent dyes Cy3, Cy5, and Cy7 decreased after a series of modifications and imprinting, the final Cy@C-EIPs still exhibited high fluorescence intensity, fully meeting the requirements for subsequent analysis. The synthesis process of Cy@C-NIPs was identical to that of Cy@C-EIPs, except for the absence of a template solution; therefore, their spectra were almost identical.

[0072] 2.3 Boron affinity selectivity of microplates @BA and Cy@BA Boric acid can reversibly bind to compounds containing a cis-dihydroxy structure by adjusting the pH. Therefore, the success of boric acid modification is crucial for subsequent fixation and directional imprinting of glycosylated epitopes in this experiment. Thus, puerarin containing a cis-dihydroxy structure and psoralen without a cis-dihydroxy structure were selected as test compounds to investigate the boron affinity selectivity of the microplate @BA. Glycoprotein HRP and non-glycoprotein BSA were selected as test compounds to investigate the boron affinity selectivity of Cy@BA. The results are as follows: Figure 7 As shown. The microplate @BA exhibits good selectivity for puerarin, but no boron affinity for psoralen. Figure 7 a). Similarly, all three Cy@BA species exhibited good selectivity for HRP, but no boron affinity for BSA. Figure 7 (bd). The above results indicate that the surfaces of both microplates @BA and Cy@BA have been successfully modified with boric acid groups, which can be used for subsequent fixation of saccharification epitopes.

[0073] 2.4 Optimization of Imprinting Conditions for Microplates@N-MEIPs and Cy@C-EIPs According to the imprinting factor (IF= Q MIPs / Q NIPs The monomer ratio and imprinting time of microplates @N-MEIPs and Cy@C-EIPs were optimized, and the results are as follows: Figure 8 As shown. For monomer ratios, an appropriate monomer ratio allows microplate @N-MEIPs and Cy@C-EIPs to achieve optimal recognition performance with the epitope sequences of the target protein. In this experiment, APTES, UPTES, BnTES, and IBTES were selected as functional monomers, which can form hydrogen bonds, electrostatic attraction, π-π stacking, and hydrophobic interactions with different types of amino acids in the epitope sequence. TEOS was selected as a cross-linking agent to connect the functional monomers and form a hydrophilic silica backbone. For example... Figure 8As shown in Figure 4, with the increase of the functional monomer ratio, the adsorption capacity of microplates @N-MEIPs and Cy@C-EIPs first increases rapidly and then decreases slowly. This is mainly because more functional monomers can form more binding sites in the imprint cavity. When the functional monomer ratio increases further, the lack of TEOS to form a silica framework prevents the formation of an effective imprint cavity. Conversely, the adsorption capacity of microplates @N-MNIPs and Cy@C-NIPs increases with the increase of the functional monomer ratio, especially at higher functional monomer ratios. This is mainly because more functional monomers significantly increase the number of non-specific adsorption sites on their surfaces. Therefore, the IF of microplates @N-MEIPs and Cy@C-EIPs shows a trend of first increasing and then decreasing. When the monomer ratio of microplates @N-MEIPs is 20:20:10:20:30, the optimal IF values ​​are obtained for AFP, AFU, and AAT. Figure 8 When the monomer ratios of Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs are 20:10:0:20:50, 10:10:10:30:40, and 15:15:0:20:50, respectively, the optimal IF values ​​for AFP, AFU, and AAT are obtained. Figure 8 df).

[0074] Regarding imprinting time, the duration of imprinting is related to the thickness of the imprint layer, and the results are as follows: Figure 8 As shown in gl, the adsorption capacity of the imprinted cavities of microplates @N-MEIPs and Cy@C-EIPs showed a trend of first increasing and then decreasing with increasing imprinting time. This is mainly because when the imprinting time is short, the imprint layer is thin, resulting in incomplete imprinted cavities and fewer recognition sites; when the imprinting time is long, the imprint layer is thick, resulting in the saccharified epitope template being buried and unable to form an effective imprinted cavity. For microplates @N-MNIPs and Cy@C-NIPs, under a fixed monomer ratio, since they do not contain imprinted cavities, the imprinting time has no effect on their adsorption capacity. Therefore, the IF of the imprinted cavities of microplates @N-MEIPs and Cy@C-EIPs also showed a trend of first increasing and then decreasing. When the imprinting time of microplates @N-MEIPs was 60 min, the optimal IF values ​​for AFP, AFU, and AAT were obtained, which were 5.4, 5.2, and 5.5, respectively. Figure 8 When the blotting time of Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs is 60 min, the optimal IF values ​​for AFP, AFU, and AAT are 6.3, 5.9, and 6.1, respectively. Figure 8 jl).

[0075] 2.5 Optimization of extraction time and labeling time To obtain the strongest fluorescence intensity in the shortest operation time, the extraction and labeling times were optimized, and the results are as follows: Figure 9 As shown, the fluorescence intensity increased with the extension of extraction time for AFP, AFU, and AAT by microplates@N-MEIPs, while the fluorescence intensity remained basically unchanged after the extraction time exceeded 40 min, indicating that the optimal extraction time for AFP, AFU, and AAT by microplates@N-MEIPs was 40 min. Figure 9 Similarly, the labeling time followed the same trend as the extraction time. The fluorescence intensity increased with the extension of the labeling time of Cy3@C-EIPs, Cy5@C-EIPs, and Cy7@C-EIPs for AFP, AFU, and AAT, respectively. When the labeling time exceeded 40 min, the fluorescence intensity remained basically unchanged, indicating that the optimal labeling time for the three Cy@C-EIPs for AFP, AFU, and AAT was 40 min. Figure 9 df). Correspondingly, microplates @N-MNIPs and Cy@C-NIPs, lacking a blot cavity, exhibit weaker and almost constant fluorescence intensity. Figure 9 af).

[0076] 2.6 Specificity assessment By selecting AFP, AFU, and AAT as target proteins and HRP, BSA, HSA, and TRF as competing proteins, the specificity of the established multi-epitope-based fluorescence immunoassay method was investigated, and optimization was performed based on cross-reactivity, where cross-reactivity is the ratio of fluorescence intensity of the established immunoassay method to that of the competing proteins and the target proteins, respectively. Results are as follows: Figure 10 As shown, the established immunoassay method exhibits good specificity, with maximum cross-reactivity of only 3.1%, 3.7%, and 5.0% for AFP, AFU, and AAT, respectively. This is mainly attributed to the ability of the prepared microplates @N-MEIPs and Cy@C-EIPs to form dual MIP recognition for the target proteins, thereby ensuring the high specificity of the established immunoassay method.

[0077] 2.7 Stability Test Stability is crucial for the accuracy of test results and the lifespan of materials. The stability of the established immunoassay method was investigated by measuring AFP, AFU, and AAT every month for six months. The results are as follows: Figure 11 As shown, after 6 months of storage, the fluorescence intensity for AFP, AFU, and AAT still remained above 87.6%, indicating that the fluorescence immunoassay based on multi-epitope blotting has good stability.

[0078] 2.8 Concentration Response Curve To validate the analytical performance of the multi-epitope-based fluorescence immunoassay, linearity, linear range, LOD, and LOQ were investigated. A standard curve was plotted with the logarithm of protein concentration on the x-axis and the corresponding fluorescence intensity on the y-axis. The LOD was defined as the concentration of the target analyte at a signal-to-noise ratio (S / N) of 3, and the LOQ was defined as the concentration of the target analyte at a S / N of 10. The results are as follows: Figure 12 As shown, the linear equation for AFP based on multi-epitope blot fluorescence immunoassay is: y = 19519 x + 79130 ( r = 0.9914), the linear range was 10 pg / mL to 10 μg / mL, and the LOD and LOQ were 0.5 pg / mL and 1 pg / mL, respectively; the linear equation for AFU was... y = 16665 x + 52774 ( r = 0.9903), the linear range was 10 pg / mL to 1 μg / mL, and the LOD and LOQ were 1 pg / mL and 3 pg / mL, respectively; the linear equation for AAT was... y = 1210.2 x + 14243 ( r = 0.9932), with a linear range of 10 ng / mL to 100 μg / mL, and LOD and LOQ of 0.5 ng / mL and 1 ng / mL, respectively.

[0079] 2.9 Application of actual samples To evaluate the practical application performance of the established immunoassay method, serum samples from healthy individuals and liver cancer patients were analyzed, and the results were compared with those obtained from commercial ELISA kits. The results are shown in Table 1. Based on multi-epitope blotting, the levels of AFP, AFU, and AAT in the serum of healthy individuals were 11.9 ng / mL, 2.56 μg / mL, and 1.39 mg / mL, respectively. In liver cancer patient 1, the levels were 597.3 ng / mL, 4.49 μg / mL, and 2.56 mg / mL, respectively. In liver cancer patient 2, the levels were 857.1 ng / mL, 6.25 μg / mL, and 4.31 mg / mL, respectively. Compared with the results obtained from commercial ELISA kits, the errors for AFP were ≤2.0%, AFU ≤3.7%, and AAT ≤2.1%, indicating that the established immunoassay method is accurate and reliable and can be used for the analysis of actual samples.

[0080] Table 1. Determination of AFP, AFU, and AAT levels in serum of healthy individuals and liver cancer patients using a multi-epitope-based fluorescence immunoassay and commercially available ELISA kits.

[0081] 2.10 Performance Comparison with Commercially Available ELISA Kits The performance of the multi-epitope-blotting-based fluorescence immunoassay was compared with that of a commercial ELISA kit, and the results are shown in Table 2. Compared with commercial ELISA kits, the established immunoassay method not only has significant advantages in terms of linear range, detection limit, operation time, and cost, but also enables the simultaneous detection of multiple disease biomarkers.

[0082] Table 2. Performance comparison between multi-epitope-blotting-based fluorescence immunoassay and commercially available ELISA kits.

[0083] 4. Conclusion In summary, this invention addresses the technical limitations of traditional immunoassays, which rely on natural antibodies and struggle to achieve multiplex simultaneous detection. Using the core liver cancer biomarkers AFP, AFU, and AAT as detection targets, a multi-epitope targeted blotting strategy was employed to prepare microplate @N-MEIPs, and a boron affinity-anchored epitope controllable targeted surface blotting strategy was used to prepare three types of Cy@C-EIPs, which respectively replace traditional capture and labeled antibodies. This successfully constructed a sandwich-type fluorescent immunoassay system that does not require natural antibodies. This system achieves high specificity detection through a dual molecular blotting recognition mechanism of N-terminal epitope capture and C-terminal epitope labeling. The maximum cross-reactivity for the three biomarkers is less than 5.0%, and the detection sensitivity is approximately 500-fold, 115-fold, and 5.66-fold higher than commercial ELISA kits, respectively. The LODs are as low as 0.5 pg / mL, 1 pg / mL, and 0.5 ng / mL, respectively, with a linear range covering 4-6 orders of magnitude. A single detection takes only 1.5 hours, and the detection efficiency is approximately 57% higher than commercial ELISA kits. This kit can be stably stored at room temperature for more than 6 months, and the signal retention rate for the three biomarkers is over 87.6%. The deviation between the actual serum sample test results and those of commercial ELISA kits is less than 4%. The multi-epitope-based fluorescent immunoassay kit provided by this invention integrates three core technologies: multiple target capture, dual specificity recognition, and multivariate fluorescence signal detection. It has advantages such as simple operation, rapid detection, high sensitivity, strong specificity, good stability, low cost, and multivariate simultaneous detection. It not only has important clinical application value in the early auxiliary diagnosis, efficacy monitoring, and prognostic assessment of liver cancer, but also has good versatility of core technologies. By changing the corresponding epitope peptide template, it can be quickly extended to the detection of other disease biomarkers, providing a new, efficient, and low-cost technical solution for the accurate diagnosis of various diseases.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fluorescence immunoassay kit based on multi-epitope imprinting, characterized in that, include: An N-terminal multi-epitope imprinted microplate, the surface of which is simultaneously imprinted with N-terminal epitope peptide templates of AFP, AFU and AAT, forming a multi-specific recognition cavity; Three types of C-terminal epitope-imprinted fluorescent silica nanoparticles, namely Cy3@C-EIPs, Cy5@C-EIPs and Cy7@C-EIPs, specifically recognize and label the C-terminal epitopes of AFP, AFU and AAT, respectively. The N-terminal multi-epitope imprinted microplate and the three C-terminal imprinted fluorescent silica nanoparticles together constitute a sandwich structure detection system for detecting AFP, AFU and AAT.

2. The reagent kit according to claim 1, characterized in that, The N-terminal multi-epitope imprinted microplate is prepared using a multi-epitope directional precise imprinting strategy. Its imprinted layer is formed by polymerization of a mixed silanizing agent around the N-terminal epitope peptide template. The mixed silanizing agent includes aminopropyltriethoxysilane (APTES), 3-ureapropyltriethoxysilane (UPTES), benzyltriethoxysilane (BnTES), isobutyltriethoxysilane (IBTES), and tetraethyl orthosilicate (TEOS).

3. The reagent kit according to claim 2, characterized in that, In the mixed silanizing reagent, the molar ratio of APTES, UPTES, BnTES, IBTES and TEOS is (10-30):(10-30):10:(10-30):(10-60), preferably 20:20:10:20:

30.

4. The reagent kit according to claim 1, characterized in that, The Cy3@C-EIPs, Cy5@C-EIPs and Cy7@C-EIPs were prepared by a boron affinity anchored epitope controllable directional surface imprinting strategy, and their imprinted layers were formed by the polymerization of mixed silanizing reagents around the corresponding C-terminal epitope peptide template.

5. The reagent kit according to claim 4, characterized in that: In the preparation of Cy3@C-EIPs, the molar ratio of APTES, UPTES, BnTES, IBTES and TEOS in the mixed silanizing reagents is (10-20):(10-20):0:(10-20):(40-70), preferably 20:10:0:20:

50. In the preparation of Cy5@C-EIPs, the molar ratio of APTES, UPTES, BnTES, IBTES and TEOS in the mixed silanizing reagents is 10:10:(10-20):(10-30):(30-60), preferably 10:10:10:30:

40. In the preparation of Cy7@C-EIPs, the molar ratio of APTES, UPTES, BnTES, IBTES and TEOS in the mixed silanizing reagents is (10-20):(10-20):0:(10-20):(40-70), preferably 15:15:0:20:

50.

6. The reagent kit according to claim 1, characterized in that, The fluorescent dyes in Cy3@C-EIPs and Cy5@C-EIPs are doped in the outer layer, while the fluorescent dyes in Cy7@C-EIPs are doped in both the inner and outer layers.

7. A detection system for simultaneously detecting AFP, AFU, and AAT, characterized in that, A fluorescent immunoassay kit based on multi-epitope blotting as described in any one of claims 1-6.

8. The kit according to any one of claims 1-6 or the detection system according to claim 7 in the preparation of products for the in vitro quantitative detection of AFP, AFU and AAT content in biological samples.

9. The kit according to any one of claims 1-6 or the detection system according to claim 7 in the preparation of in vitro diagnostic products for auxiliary diagnosis, efficacy monitoring or prognostic assessment of liver cancer.

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