Nucleic acid aptamer capable of specifically recognizing serum alpha fetoprotein and application of nucleic acid aptamer

Abstract

The invention belongs to the technical field of biomedical detection, and particularly discloses a nucleic acid aptamer for specifically recognizing serum alpha fetoprotein and application of the nucleic acid aptamer. The nucleotide sequence of the nucleic acid aptamer comprises a sequence as shown in SEQ ID No.1, or has more than 90% of homology with the sequence as shown in SEQ ID No.1, or one or more nucleotides are deleted or added in the sequence as shown in SEQ ID No.1. The nucleic acid aptamer can realize specific efficient recognition of AFP protein, also shows extremely high selectivity in a complex serum sample environment, eliminates non-specific adsorption interference of homologous protein and other serum interference protein, greatly reduces the false positive rate, and improves the accuracy and specificity of clinical detection. And the material constructed by directionally functionalizing the aptamer on the surface of the magnetic nano-carrier has rapid magnetic response performance, separation and enrichment of target protein can be realized within seconds, and the clinical diagnosis time is greatly shortened.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to nucleic acid aptamers that specifically recognize serum alpha-fetoprotein and their uses. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in clinical practice, characterized by insidious onset, rapid progression, and high malignancy, seriously threatening human health and life. However, due to the limitations of traditional antibody molecules themselves, especially the poor structural stability of antibody proteins, their sensitivity to heat, susceptibility to environmental factors leading to inactivation, and batch-to-batch differences in activity, there are uncertainties in the preparation and storage of antibody preparations. This results in significant deficiencies in the sensitivity, specificity, and batch-to-batch stability of antibody-based serum alpha-fetoprotein (AFP) detection methods, severely impacting the accuracy and reliability of AFP clinical testing.

[0003] Despite the wide variety of existing AFP detection methods, significant limitations restrict their effectiveness, particularly in early cancer detection. Standard enzyme-linked immunosorbent assays (ELISA), while reliable, involve multi-step procedures (binding, washing, signal visualization), are time-consuming (each assay typically takes several hours), and generally have limited linear ranges. Traditional ELISA kits may struggle to accurately quantify extremely low AFP levels because they often require the analyte to exceed a certain threshold to produce a measurable signal. More sensitive immunoassays based on fluorescence or radioimmunoassays can expand the detection range, but they introduce additional complexity and cost. Fluorescence detection requires laborious labeling steps and specialized readout equipment, while radioimmunoassays suffer from isotope decay and poor reproducibility. Similarly, a drawback of surface-enhanced Raman scattering immunoassays is their inherently weak signal, necessitating enhancement strategies for trace AFP analysis. Even highly sensitive and easily miniaturized electrochemical immunosensors face issues such as nonspecific binding signals and are difficult to multiplex for high-throughput applications. Generally, current AFP testing methods often require relatively large sample sizes and involve complex procedures, increasing turnaround time and cost per test. They also lack sensitivity for detecting extremely low concentrations of AFP; small increases in AFP associated with early-stage HCC often fall below the detection limits of standard methods. Therefore, routine AFP testing misses a significant portion of early-stage HCC cases. In fact, the clinical sensitivity of AFP for HCC is only around 40%-60%, meaning many cancer patients with small tumors or low AFP production go undetected. Therefore, there is an urgent need for a new AFP testing technology that can provide higher sensitivity, faster results, and lower cost at low concentrations, thereby enabling more effective early diagnosis of HCC. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of the prior art by providing a nucleic acid aptamer that specifically recognizes serum alpha-fetoprotein and its application. This nucleic acid aptamer overcomes the technical limitations of existing AFP immunoassay techniques in terms of stability, batch-to-batch consistency, and cross-reactivity, thereby comprehensively improving the specificity, sensitivity, and accuracy of AFP in clinical sample testing.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention is to provide a nucleic acid aptamer that specifically recognizes serum alpha-fetoprotein, said nucleic acid aptamer comprising at least one of A1)-A3): A1) The nucleotide sequence is the sequence shown in SEQ ID No. 1; The nucleotide sequences defined by A2) and A1) have more than 90% homology and specifically recognize serum alpha-fetoprotein single-stranded DNA. A3) Delete or add one or more nucleotides to the nucleotide sequence defined in A1) and specifically recognize serum alpha-fetoprotein single-stranded DNA.

[0006] Furthermore, the nucleic acid aptamer specifically recognizes serum alpha-fetoprotein by chemically modifying its 5′ or 3′ end, wherein the modification is selected from one or more of biotin labeling, fluorescent / luminescent group linkage, phosphorylation, methylation, amination, thiolation, sulfur substitution of oxygen, selenium substitution of oxygen, and isotopization; and / or, signal molecules and / or active molecules and / or functional groups are linked to the nucleotide sequence of the nucleic acid aptamer, and the linked nucleic acid aptamer specifically recognizes serum alpha-fetoprotein.

[0007] A second aspect of the present invention is to provide the use of the above-described nucleic acid aptamers in at least one of B1)-B5): B1) Use in the preparation of products for enriching serum alpha-fetoprotein; B2) Use in the preparation of products for the detection of serum alpha-fetoprotein; B3) Uses in the purification of serum alpha-fetoprotein; B4) Use in the preparation of products targeting serum alpha-fetoprotein; B5) Use in the preparation of screening, diagnostic or auxiliary diagnostic materials for diseases related to serum alpha-fetoprotein.

[0008] Further, the serum alpha-fetoprotein-related diseases include one or more of the following: primary hepatocellular carcinoma, germ cell tumors, gastric cancer, pancreatic cancer, and cholangiocarcinoma.

[0009] A third aspect of the present invention is to provide a probe, wherein the probe is a substance obtained by labeling the above-mentioned nucleic acid aptamer with a label, the label being selected from signal molecules and / or functional groups.

[0010] A fourth aspect of the present invention is to provide a magnetic nanomaterial, wherein the magnetic nanomaterial is a material in which the above-mentioned nucleic acid aptamer is functionalized and immobilized on the surface of streptavidin magnetic nanoparticles through streptavidin-biotin high affinity interaction.

[0011] Furthermore, the magnetic nanomaterial is used to enrich or capture serum alpha-fetoprotein.

[0012] A fifth aspect of the present invention is to provide a product for enriching or detecting serum alpha-fetoprotein, the product comprising the above-described nucleic acid aptamers and / or the above-described magnetic nanomaterials.

[0013] Furthermore, the products include reagent kits, affinity purification reagents, sensors, and chips.

[0014] A sixth aspect of the present invention is to provide a method for detecting serum alpha-fetoprotein, the method comprising co-incubating a sample to be tested with the aforementioned nucleic acid aptamer or the aforementioned magnetic nanomaterial, and detecting serum alpha-fetoprotein by observing the change in signal before and after the interaction between the nucleic acid aptamer and serum alpha-fetoprotein in the sample to be tested.

[0015] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows: (1) The nucleic acid aptamer provided by the present invention can achieve specific and efficient recognition of AFP protein itself, and also exhibits extremely high selectivity in complex serum sample environments. It eliminates non-specific adsorption interference from homologous proteins and other serum interfering proteins, greatly reduces the false positive rate, and improves the accuracy and specificity of clinical testing.

[0016] (2) This invention screened a new nucleic acid aptamer, AP_Apt 47, using Magnetic beads-SELEX screening technology. Surface plasmon resonance (SPR) technology revealed that the binding affinity between AP_Apt 47 and AFP reached the nanomolar level (K0). D = 0.6 nM), exhibiting binding tightness and stability far exceeding those of traditional antibodies. Furthermore, Apt47 showed no significant binding signals to multiple AFP homologs and high-abundance serum proteins, including vitamin D-binding protease (VDB), human serum albumin (ALB), immunoglobulin G (IgG), bovine serum albumin (BSA), and fucosylated AFP subtype (AFP-L3), further validating its excellent specific recognition ability.

[0017] (3) The nucleic acid aptamer provided by this invention can replace traditional antibody molecules as AFP recognition elements. Compared with antibodies, nucleic acid aptamers have better thermal stability, chemical stability and environmental adaptability, which fundamentally solves the problems of batch variation, poor stability and easy inactivation of traditional antibodies, and significantly improves the stability and controllability of the detection system.

[0018] (4) Based on the non-covalent interaction mechanism with extremely high affinity between streptavidin and biotin, this invention functionalizes and immobilizes the screened AFP-specific single-stranded DNA aptamer (AP_Apt 47) onto the surface of streptavidin-modified carboxyl magnetic nanoparticles by biotin labeling at the 5′ end, thereby constructing a functionalized magnetic nanocomposite material (SMBs@AP_Apt 47) with AFP-targeting recognition capability. This construction process is stable and mild, approaching the effect of covalent binding, ensuring the conformational integrity and biological activity of the aptamer. The prepared SMBs@AP_Apt 47 composite material exhibits rapid response capability under the action of an external magnetic field, achieving magnetic enrichment and separation within seconds, effectively improving the capture efficiency of AFP in complex biological samples.

[0019] (5) The SMBs@AP_Apt 47 material provided by the present invention breaks through the bottlenecks of long incubation time and complicated operation of traditional antibody magnetic beads, and greatly improves the processing efficiency and detection speed of clinical samples. Attached Figure Description

[0020] Figure 1 The graph shows the binding-specific SPR response curves of AP_Apt 47 to AFP protein and other non-target proteins. Figure 2 SPR binding curves and affinity (Kb) of different concentrations of AP_Apt 47 aptamers to immobilized AFP protein chips. D (Fitting results graph;) Figure 3 The figures show the 3D structure and docking prediction results. In the figure, (a) is the 3D structural model of AP_Apt 47, (b) is the 3D docking model between AP_Apt 47 and AFP protein, and (c) is a magnified 3D interaction model observed from the two views. Figure 4 Images show the surface morphology of magnetic materials at different construction stages under focused ion beam scanning electron microscopy (FIB-FESEM). In the figure, (a) is a carboxyl-modified magnetic microsphere (MBs), (b) is a magnetic microsphere (SMBs) coupled with streptavidin, and (c) is a functionalized magnetic composite material (SMBs@AP_Apt 47) with biotinylated nucleic acid aptamer immobilized on the surface. Figure 5The figure shows the specific enrichment verification of AFP by SMBs@AP_Apt 47. In the figure, (a) is the SDS-PAGE staining map and (b) is the Western blot result. Figure 6 The stacked Venn diagram illustrates the differences and intersections of the captured proteins between the experimental and control groups; Figure 7 To further clarify the distribution relationships and classification of proteins in the Venn diagram; Figure 8 Protein interaction network analysis diagram constructed for Cytoscape; Figure 9 This is a volcano diagram showing the protein enrichment in the experimental group compared to the control group. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific test methods, instruments, or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0022] This invention utilizes an aptamer screening method to obtain a novel nucleic acid aptamer, AP_Apt 47, which possesses independent nucleotide sequence, spatial conformation, and biological functional characteristics. It differs fundamentally from the aptamer disclosed in CN117106784A in sequence, affinity, specificity, structural recognition site, and specific capture of AFP in complex matrix samples. AP_Apt 47 is significantly enriched in the SELEX library, exhibits a high sequence frequency, and molecular docking reveals a stable binding structure. Subsequent validation experiments demonstrated excellent recognition performance. The nucleotide sequence of this aptamer (SEQ ID No. 1) is as follows: attggcactccacgcataggCACAATGGAGGGAGGGTGGGTGGATTTGTGCATCGTGTTTTttcacggtagcacgcatagg.

[0023] In some embodiments, the aptamer may also be a single-stranded DNA that has more than 90% homology with the nucleotide sequence shown in SEQ ID No. 1 and specifically recognizes serum alpha-fetoprotein. In a preferred embodiment, the nucleotide sequence may have more than 90%, 93%, 95%, 97%, or 99% homology with SEQ ID No. 1.

[0024] In some embodiments, one or more nucleotides may be deleted or added to the nucleotide sequence shown in SEQ ID No. 1, and the single-stranded DNA specifically recognizes serum alpha-fetoprotein. In a preferred embodiment, the nucleotide sequence shown in SEQ ID No. 1 is obtained by substituting one or more (specifically, 1-10, 1-5, or 1-3) bases.

[0025] In some embodiments, the nucleic acid aptamer specifically recognizes serum alpha-fetoprotein by chemically modifying its 5′ or 3′ end, wherein the modification is selected from at least one of biotin labeling, fluorescent / luminescent group linkage, phosphorylation, methylation, amination, thiolation, sulfur substitution of oxygen, selenium substitution of oxygen, and isotopization; and / or, a signal molecule and / or an active molecule and / or a functional group are linked to the nucleotide sequence of the nucleic acid aptamer, and the linked nucleic acid aptamer specifically recognizes serum alpha-fetoprotein.

[0026] In some embodiments, the nucleotide sequence of the nucleic acid aptamer is linked to a signaling molecule and / or an active molecule and / or a functional group, and the linked nucleic acid aptamer specifically recognizes serum alpha-fetoprotein.

[0027] Based on the excellent specific recognition and / or enrichment of serum alpha-fetoprotein by the nucleic acid aptamers provided by the present invention, another aspect of the present invention protects the use of the nucleic acid aptamers in at least one of B1)-B5): B1) Use in the preparation of products for enriching serum alpha-fetoprotein; B2) Use in the preparation of products for the detection of serum alpha-fetoprotein; B3) Uses in the purification of serum alpha-fetoprotein; B4) Use in the preparation of products targeting serum alpha-fetoprotein; B5) Use in the preparation of screening, diagnostic or auxiliary diagnostic materials for diseases related to serum alpha-fetoprotein.

[0028] According to the technical solution of the present invention, the serum alpha-fetoprotein-related disease is hepatocellular carcinoma.

[0029] To better utilize newly discovered nucleic acid aptamers for AFP capture or detection, another aspect of the present invention protects a probe, which is a substance obtained by labeling the aforementioned nucleic acid aptamer with a marker. As a preferred embodiment, the marker is a signal molecule and / or a functional group. The marker refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive labels, such as 32P; conjugated coupling groups, such as biotin; haptens, such as digoxigenin (DIG); chemiluminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). Labels can provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetric analysis, quantum dots, electrochemistry, X-ray diffraction or absorption, magnetism, enzyme-linked immunosorbent assays (ELISA), filter paper-based immunoassays, affinity precipitation, affinity chromatography, enzyme activity, projection microscopy or scanning imaging, super-resolution imaging, cell tracing, in vivo nanoparticle tracking imaging in animals or humans, nanoflow cytometry, tunable resistance pulse sensing, fluorescence-correlated spectroscopy, surface plasmon resonance, fluorescence polarization, surface-enhanced Raman spectroscopy, electrochemical sensing, microfluidics or microfluidics, microarray analysis, proteomics, genomics, metabolomics, microbiome, and RNA (mRNA, lnRNA, snRNA), miRNA, etc. Labels can be charged portions (positive or negative) or selected as needed, and can be charge-neutral. Labels can include nucleic acid or protein sequences or combinations thereof, provided the sequence containing the label is detectable.

[0030] In some implementations, nucleic acids are detected directly without labels (e.g., direct sequence reading). The labels can also be used for targeted drug delivery.

[0031] In some embodiments, the label is a fluorophore, colorimetric label, quantum dot, biotin, and other tag molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cycloalkenes for click reactions, and initiating groups for polymer labeling). It can also be selected from peptide / protein molecules, LNA / PNA, non-natural amino acids and their analogs (such as peptides), non-natural nucleic acids and their analogs (nucleotides), and nanostructures (including inorganic nanoparticles, NV-centers, aggregation / assembly-induced luminescent molecules, rare earth ion ligand molecules, polyoxometalates, etc.).

[0032] In some embodiments, the fluorophore may be selected from fluorescein dyes, rhodamine dyes, and cyanine dyes. Preferably, the fluorescein dyes include standard fluorophores and their derivatives, such as fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), Cy5, Cy3, Quasar 670, Alexa Fluor 488 / 555 / 647 / 750, etc. Preferably, the rhodamine dyes include R101, tetraethylrhodamine (RB200), and carboxytetramethylrhodamine (TAMRA), etc. Preferably, the cyanine dyes are mainly selected from two categories: one is the thiazole orange (TO) and oxazole orange (YO) series and their dimer dyes, and the other is the polymethyl cyanine dye series.

[0033] In some embodiments, the fluorophore may also be selected from dyes such as stilbene, naphthalimide, coumarins, acridines, and pyrene. The fluorophore is usually labeled at the 5' end of the probe sequence, but it can also be placed at the 3' end by changing the modifying bond (e.g., -OH or -NH bond).

[0034] In one embodiment of the present invention, the 5' end of the single-stranded DNA (ssDNA) aptamer described in SEQ ID No. 1 is labeled with FITC / FAM / Alexa Fluor 488 / Cy3. The probe is then injected into the blood or incubated in vitro with relevant tissues or cells. The tissues or cells expressing serum alpha-fetoprotein can then be identified and imaged using a fluorescence recognition instrument.

[0035] To improve the specific capture ability of AFP protein, the present invention also provides a magnetic nanomaterial SMBs@AP_Apt 47 obtained by functionalizing and immobilizing the above-mentioned nucleic acid aptamer on the surface of streptavidin magnetic nanoparticles through streptavidin-biotin high affinity interaction, which can achieve rapid and specific capture and enrichment of AFP.

[0036] Furthermore, another aspect of the present invention protects a product for enriching or detecting serum alpha-fetoprotein, said product comprising the nucleic acid aptamers as described above.

[0037] According to the technical solution of the present invention, the product is selected from kits, affinity purification reagents, sensors, and chips. Preferably, the kit further includes one or more of Taq DNA polymerase, dNTPs, PCR buffer, and metal ions required for PCR amplification. Preferably, the various reagent components of the kit can be present in separate containers, or can be pre-assembled into a reagent mixture, either wholly or partially.

[0038] According to the technical solution of the present invention, a method for detecting serum alpha-fetoprotein is also provided. The method includes co-incubating the sample to be tested with the above-mentioned nucleic acid aptamer or the above-mentioned magnetic nanomaterial, and realizing the detection of serum alpha-fetoprotein by observing the change in signal before and after the interaction between the nucleic acid aptamer and serum alpha-fetoprotein in the sample.

[0039] The embodiments of the present invention have achieved some positive results during the research and development or use process, and have indeed great advantages compared with the prior art. The following content describes them in conjunction with the data, charts and other information of the experimental process.

[0040] Example 1 Screening and preparation of nucleic acid aptamers.

[0041] In this embodiment, nucleic acid aptamers that specifically recognize serum alpha-fetoprotein were screened using Magneticbeads-SELEX screening and sequencing technologies. For the specific screening process, please refer to the method disclosed in patent CN117106784A. The sequence of the nucleic acid aptamer (AP_Apt 47) is as follows: attggcactccacgcataggCACAATGGAGGGAGGGTGGGTGGATTTGTGCATCGTGTTTTttcacggtagcacgcatagg.

[0042] Example 2 Specificity studies of nucleic acid aptamers.

[0043] To further confirm the specificity and binding strength of the nucleic acid aptamer AP_Apt47 provided in this invention for AFP recognition, the binding kinetics of AP_Apt47 were systematically evaluated using surface plasmon resonance (SPR) technology on a Biacore 8K platform (Cytiva, Sweden). All buffers (DPBS mobile phase and 1 M NaCl regeneration solution) were filtered through a 0.22 μm filter and degassed before use. All experiments were performed at a constant temperature of 25°C. Data analysis was performed using Biacore Insight Evaluation 5.0 software, and all response data represent the difference between the detection channel and the reference channel.

[0044] AFP and various structure-related or common serum interfering proteins were surface-coupled and immobilized, and then injected into the AP_Apt 47 aptamer under uniform conditions. The specific steps are as follows: (1) AFP protein was immobilized on the detection channel surface of the CM5 chip using the standard amine coupling method (EDC / NHS). Simultaneously, non-target proteins that might cause interference were immobilized in other channels of the chip, including human serum albumin (ALB), AFP glycosylated subtype (AFP-L3), GPC3, the C-chain subunit of the C1q complex in the complement pathway (C1QC), vitamin D-binding protein (VDB), anti-AFP antibody (Anti-AFP), immunoglobulin G (IgG), immunoglobulin M (IgM), bovine serum albumin (BSA), and streptavidin (SA). The immobilized concentration of each protein was 50 μg / mL. Ethanolamine was used to block any uncoupled residual active sites on the chip.

[0045] (2) Prepare an AP_Apt47 aptamer solution with a concentration of 1000 nM and inject it into all the above chip channels at a flow rate of 30 µL / min. The binding time between the aptamer and the protein is set to 240 s and the dissociation time is set to 120 s. During the experiment, the real-time SPR response curve (Sensorgram) of each channel is recorded.

[0046] (3) By comparing the difference in response signals between the AFP channel and the non-AFP channel, the specific recognition ability of aptamer AP_Apt47 and AFP is evaluated.

[0047] The results are as follows Figure 1 As shown, AP_Apt 47 exhibits a strong SPR response signal when interacting solely with AFP, with a maximum response value of 320 RU. The rapid binding and low dissociation rates indicate a stable and specific binding with AFP. In contrast, other proteins show response curves that largely coincide with the baseline, with maximum response values ​​not exceeding 20 RU and minimal variation. Some even spontaneously dissociate in the dissociation phase, returning to baseline, indicating almost no detectable binding. Notably, AFP-L3, a glycan-modified isoform of AFP, did not show a significant response to this aptamer. This suggests that the recognition site of AP_Apt 47 is primarily located in the AFP protein backbone rather than the glycan region. This recognition characteristic will significantly enhance the applicability of AP_Apt 47 in clinically polymorphic AFP samples, avoiding recognition bias caused by conformational changes in AFP due to glycan modification. Furthermore, GPC3 and C1QC, as control proteins for liver cancer tumor markers, also showed no significant response, further demonstrating that the aptamer of this invention has extremely high target specificity and does not exhibit cross-binding with homologous or non-specific proteins, thus possessing significant structural recognition selectivity.

[0048] Example 3 Affinity study of nucleic acid aptamers.

[0049] To determine the exact binding dissociation constant (K) between AP_Apt 47 and AFP.D This invention further conducts affinity determination experiments, with the specific procedures and parameter settings as follows: (1) AFP protein (50 μg / mL) was immobilized on the surface of the detection channel of the CM5 chip by standard amide coupling method, and unbound active sites were blocked with ethanolamine; (2) AP_Apt 47 was injected into the chip detection channel in a series of concentration gradients (0, 15.7, 31.3, 62.5, 125, 250, 500, 750, 1000 nM) at a flow rate of 30 µL / min. The binding time was 240 s and the dissociation time was 120 s under each concentration condition. After each binding and dissociation experiment, the chip surface was regenerated with 1 M NaCl until the baseline was stable. (3) Data was acquired using multi-cycle kinetics on the Biacore 8K platform to obtain a series of dose-dependent binding curves; (4) The binding-dissociation curves were recorded using a multi-cycle kinetics model, and the K-value between AP_Apt 47 and AFP was calculated using the Langmuir 1:1 model. D .

[0050] The results are as follows Figure 2 As shown, the RU value increases linearly with increasing concentration, and the response curves at each concentration exhibit typical biphasic curves (increase + dissociation), indicating stable and well-defined binding kinetics. The accurate dissociation constant (K0) is obtained through full-curve nonlinear fitting (1:1 Langmuir model). D The affinity is 0.6 nM. This nanomolar affinity indicates that AP_Apt 47 has a very strong binding capacity for AFP, achieving stable recognition even at low concentrations, thus meeting the requirements for constructing biosensors and biorecognition probes. This lays a solid molecular recognition foundation for constructing functionalized magnetic trapping materials (SMBs@AP_Apt 47) and provides key molecular components to support subsequent expansion into pull-down experiments, mass spectrometry analysis, and the enrichment and recognition of clinical biomarkers.

[0051] Example 4 Research on the mechanism of action between nucleic acid aptamers and serum alpha-fetoprotein.

[0052] The 3D molecular docking model was used, and the specific steps are as follows: (1) Structural preparation of serum alpha-fetoprotein The amino acid sequence information of serum alpha-fetoprotein was obtained from the UniProt database (https: / / www.uniprot.org / , UniProt ID: P02771), and its high-confidence predicted three-dimensional structure model was obtained from the AlphaFold Protein Structure Database (https: / / alphafold.ebi.ac.uk / ) for subsequent identification of aptamer binding sites.

[0053] (2) Three-dimensional modeling of AP_Apt 47 nucleic acid aptamers The nucleic acid sequence of AP_Apt 47 was first used to predict its secondary structure using the Mfold online tool to determine its potential stem-loop structure and loop conformation. The nucleic acid sequence was then input into the AlphaFold 3 platform (https: / / alphafoldserver.com / welcome) for structure construction, generating a 3D model. The resulting aptamer structure model was further optimized for conformation and energy minimization in PyMOL (Version 3.0, Schrödinger, LLC) to obtain a stable spatial folding conformation.

[0054] (3) Molecular docking and complex construction The optimized AFP protein structure and the three-dimensional structure of AP_Apt47 nucleic acid were imported into the PyMOL platform for docking analysis based on the protein-nucleic acid flexible docking mechanism. The positively charged hydrophobic groove region on the AFP protein surface was used as the active constraint site to guide the aptamer to approach and establish a potential interaction. During docking, the binding energy score (PyMOL score), interface, number of hydrogen bonds, and binding stability of all conformations were systematically evaluated. Based on the comprehensive score ranking, the docking model with the lowest energy and the most reasonable conformation was selected as the representative binding mode of the AFP–AP_Apt47 complex.

[0055] (4) Structural integration feature analysis and functional interpretation like Figure 3 The figure shown is a 3D structure and docking prediction result diagram. Figure 3 (a) shows the overall 3D structure of AP_Apt47, which is a left-handed helical structure. It inserts from the central region into the surface binding groove region of the AFP protein, as shown in Figure 1. Figure 3 As shown in (b), a tight protein-nucleic acid complex structure is formed, with its binding region located near the α-helix surface of the C-terminal region of the AFP protein, forming a stable docking interface. This suggests that this region is the main binding site for aptamer recognition. A magnified view of the local key sites is shown below. Figure 3As shown in (c), several key bases of AP_Apt47 form stable hydrogen bonds and hydrophobic interactions with amino acids on the AFP surface. Specific binding sites are as follows: Key amino acid residues forming hydrogen bonds / π–π stacking: TYR-425, TYR-426, LEU-432, ASN-429, GLN-546, VAL-433, ALA-434. Corresponding key recognition bases: dT-58, dT-59, dT-60, dT-61, dC-63, dG-57. Amino acids contributing to boundary stability: LYS-421, SER-415, TYR-435, and GLN-546. The interaction distances between these amino acids and the nucleic acid are between 2.8 and 3.4 Å, which is typical for hydrogen bonding, indicating a tight and stable binding interface. Simultaneously, AP_Apt47 forms a cyclic conformation that embeds itself in the grooves on the AFP protein surface, enhancing spatial matching. In summary, this invention clearly elucidates the aptamer-protein recognition mechanism through molecular docking, possessing advantages such as well-defined structure, clear target sites, and stable spatial conformation. It can be further applied to aptamer structure optimization, derivatized probe construction, and the development of multimodal recognition platforms. This provides mechanistic support for the development of AFP molecular recognition probes and aptamers with higher specificity and affinity in this field.

[0056] Example 5 This embodiment provides a magnetic nanomaterial (SMBs@AP_Apt 47), which can be constructed by referring to the method disclosed in patent CN118393140A.

[0057] Compared with the one disclosed in CN118393140A, the nucleic acid aptamer sequence, target protein, and application provided in this embodiment are different. It has significant advantages in the high-specificity capture and clinical detection of AFP, a biomarker related to hepatocellular carcinoma (HCC).

[0058] (1) Microscopic morphology characterization and analysis The hierarchical structure of the magnetic bead surface was characterized using a Thermo Fisher Helios 5 UC focused ion beam field emission scanning electron microscope (FIB-FESEM). Details are as follows: (1.1) Sample preparation The magnetic nanoparticles SMBs@AP_Apt 47 were ultrasonically treated (frequency 40 kHz, power 50 W) for 10 min to ensure that the particles were fully and uniformly dispersed.

[0059] The ultrasonically dispersed sample was dried using a vacuum drying method (pressure less than 10 Pa) at room temperature for about 30 minutes until it was completely dry.

[0060] After drying, the magnetic bead samples are spotted onto the surface of the conductive adhesive glass slide (the glass slide is cleaned with ethanol before use). The sample amount should be enough to cover the central area of ​​the conductive adhesive slide, and the sample should be evenly dispersed.

[0061] After spotting, the glass slides are treated with gold sputtering using a magnetron sputtering instrument (gold plating thickness of about 5–10 nm) to improve the conductivity of the sample, reduce the charging effect generated during scanning electron microscopy, and improve the imaging quality.

[0062] (1.2) FIB-FESEM detection method The prepared gold-sprayed sample was fixed inside the cavity of a Thermo Fisher Helios 5 UC focused ion beam field emission scanning electron microscope.

[0063] Set the specific parameters for FIB-FESEM detection: Accelerating voltage (HV): 5.00 kV; Probe current (curr): 0.10 nA; Detector type (det): Lens detector (TLD); Imaging mode (mode): SE (Secondary Electron Imaging); Working distance (WD): 4.6 mm.

[0064] The samples were imaged and scanned under an electron microscope at the following three magnifications: low magnification: 5,000×, for overall observation of macroscopic morphology; medium magnification: 15,000×, for observation of particle morphology, particle size distribution and agglomeration; and high magnification: 200,000×, for observation of fine surface structure of particles, aptamer modification layer and particle-aptamer interface details.

[0065] like Figure 4 The images show the surface morphology of magnetic materials at different construction stages under a focused ion beam scanning electron microscope. In image 4(a), the MBs exhibit typical characteristics of uniform particle size, regular spherical shape, and smooth surface, with no obvious aggregation or depressions. Low, medium, and high magnification images respectively reflect a compact overall arrangement and clear individual particle outlines, with an average particle size of approximately 1000 nm, indicating excellent synthesis quality and a good support structure. Figure 4 As shown in (b), after streptavidin coupling treatment, the surface morphology of the particles underwent slight changes, with obvious particle coating and edge roughening, presumably due to surface structure variations caused by SA molecule coverage. The particles were relatively... Figure 4(a) Slight aggregation suggests that protein coupling may introduce some changes in surface charge, but it still maintains basic dispersion and morphological stability, indicating that the coupling process is structurally controllable; Figure 4 (c) The final functionalized nucleic acid aptamer magnetic bead composite material SMBs@AP_Apt 47. The image shows the overall morphology of the particles and... Figure 7 While maintaining consistency, the surface further exhibits slight thickening and a finely uneven "membrane-like coating," possibly originating from a high-density aptamer layer attachment. In the 200,000× image, some particle surfaces show "chain-like distribution" or dotted denser areas, representing physical signals generated by immobilized nucleic acids, verifying the binding process of Bio-AP_Apt 47 on the magnetic bead surface.

[0066] (1.3) Preliminary evaluation of structural stability and application performance The FIB-FESEM stepwise scanning imaging results show that the SMBs@AP_Apt 47 material constructed in this invention did not exhibit severe aggregation, collapse, or interface damage at any of the modification stages. The streptavidin protein and nucleic acid aptamer modification layers introduced stepwise during the construction process all showed morphological changes in low, medium, and high magnification images, verifying the effectiveness of the immobilization process. The constructed SMBs@AP_Apt 47 material has good spherical structure retention, dispersibility, and functional layer adhesion characteristics, providing a physical basis for subsequent target protein enrichment, magnetic separation, and biorecognition applications.

[0067] Example 6 SMBs@AP_Apt 47 were used for AFP-specific enrichment and proteomic analysis.

[0068] To verify the specific capture ability of the SMBs@AP_Apt 47 magnetic nanomaterial constructed in this invention for AFP protein under complex clinical serum conditions, this embodiment uses SMBs@AP_Apt 47 to enrich AFP from the serum of hepatocellular carcinoma (HCC) patients, and compares it with bare magnetic beads without aptamers as a negative control. The entire process includes pull-down enrichment experiments, SDS-PAGE electrophoresis and Western blot verification, mass spectrometry identification analysis, and bioinformatics analysis to evaluate its capture performance for AFP and its potential interacting proteins, and to explore its application potential in the detection of liver cancer biomarkers and protein interaction research. Serum samples from hepatocellular carcinoma (HCC) patients were obtained from Zhongshan Hospital Affiliated to Xiamen University.

[0069] Specifically as follows: (1) Pull-down experiment The SMBs@AP_Apt 47 functionalized magnetic beads and SMBs prepared above were added to the serum of hepatocellular carcinoma patients (labeled C, concentration 2 µg / mL) and normal healthy human serum (labeled N, concentration 2 µg / mL), respectively. After incubation at room temperature for about 30 minutes, they were magnetically separated by an external magnetic field and thoroughly washed with phosphate-buffered saline (PBST) to remove non-specific adsorbed proteins.

[0070] (2) SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining After denaturing the proteins captured by the magnetic beads at 95°C using sample buffer, an appropriate amount of protein sample was loaded onto a 10% polyacrylamide gel for electrophoresis separation, and the gel was stained with Coomassie Brilliant Blue to observe the distribution characteristics of the protein bands.

[0071] (3) Western blot verification The proteins separated by electrophoresis were transferred onto a polyvinylidene fluoride (PVDF) membrane. Chemiluminescence detection was performed using AFP monoclonal antibody (AFP Mou mAb, Abclonal) as the primary antibody and horseradish peroxidase-labeled goat anti-mouse secondary antibody (Cell signaling technology) to specifically verify the capture of AFP protein.

[0072] (4) Protein strip mass spectrometry identification Protein bands obtained by SDS-PAGE gel electrophoresis, with a specific band of approximately 70 kDa, were recovered by gel cleavage. The recovered proteins were then digested with trypsin, and the products were identified by proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The mass spectrometry data were processed using Proteome Discoverer software and compared with the UniProt database to obtain a detailed list of the proteins.

[0073] (5) Bioinformatics statistics Mass spectrometry identification results were first standardized and differentially expressed proteins were screened in RStudio. R packages such as tidyverse, limma, and VennDiagram were used to perform differential analysis and visualization of proteomics data identified in the experimental group (SMBs@AP_Apt 47 + HCC serum) and the control group (SMBs + HCC serum). Venn diagrams were used to clearly compare the intersection and specific distribution of proteins between the two groups. Furthermore, igraph and ggraph were used to construct Venn network diagrams to visually represent the classification and proportion of various proteins between groups. Based on the list of differentially expressed proteins obtained through screening, combined with logarithmic analysis... 10The significance criteria for fold change and P-value were established. A volcano plot was generated using ggplot2 to highlight the significantly enriched target protein AFP and to indicate its significant upregulation trend in the experimental group (P < 0.05, log2FC > 1.3). Furthermore, the list of differentially expressed proteins from the experimental group was imported into Cytoscape software to construct a protein-protein interaction network (PPI network) to further identify key protein nodes that may synergize with AFP.

[0074] (6) Pull-down fishing results and analysis like Figure 5 The results show the experimental results of SMBs@AP_Apt 47 specifically capturing AFP protein. Figure 5 (a) SDS-PAGE gel staining results showed that in the experimental group (AP_Apt 47) incubated with serum from liver cancer patients (Group C), a clearly visible 70 kDa protein band was observed. The band brightness and clarity were significantly higher than those of serum samples incubated with other aptamers (such as Apt 14, Apt 31, Apt 32, Apt 33, Apt 46, Apt 48, Apt 49) and the negative control bare magnetic beads (SMBs), indicating that AP_Apt 47 has a significantly higher specific capture capacity for AFP. In normal human serum (Group N), no obvious 70 kDa band was observed with SMBs@AP_Apt 47, further suggesting that AP_Apt 47 has high disease specificity in capturing AFP. In addition, no obvious 70 kDa band was observed with the negative control magnetic beads (SMBs) in either serum, confirming that the magnetic beads themselves have extremely low non-specific adsorption capacity for AFP. Figure 5 (b) Further validation of Western blot results: Western blot specifically detected AFP protein. The results showed that only samples from hepatocellular carcinoma serum incubated with AP_Apt 47 functionalized magnetic beads (Group C) showed a specific signal band of AFP protein at approximately 70 kDa, while no AFP signal was detected in normal human serum (Group N) or in corresponding samples using other aptamers or negative control magnetic beads. These results further clarify that SMBs@AP_Apt 47 magnetic nanomaterials can specifically and efficiently capture AFP protein in complex clinical serum environments, and that this capture process is highly specific, stable, and reliable, making it suitable for clinical AFP detection and further molecular diagnostic research.

[0075] (7) Proteomics identification results and analysis Figure 6As shown in the stacked Venn diagram, after incubating HCC serum with SMBs@AP_Apt 47, a total of 26 proteins were identified. Of these, 16 were specifically enriched in the experimental group, while the control group (naked SMBs magnetic beads) captured 18 proteins, including 8 specific proteins. A total of 10 proteins were identified across both groups. Notably, AFP protein was specifically identified only in the experimental group and was not found in the control group. Figure 7 The proteins are further visualized as a network, with purple nodes representing proteins common to both groups, and red and blue nodes representing proteins specific to the experimental and control groups, respectively. In the protein interaction network constructed using Cytoscape ( Figure 8 AFP is located at the core of the network, with the highest Degree value and the largest node, indicating that it is the most core functional node in the network. Other proteins are enriched together by interacting directly or indirectly with AFP, and are not directly recognized by the aptamer itself. Figure 9 The volcano plot further shows that the data point corresponding to AFP is much higher than the statistical significance threshold (P<0.05) and located in the upper right corner, indicating that AFP has the highest enrichment fold in the experimental group compared to the control group and has a statistically significant difference. Conversely, the data points of most other proteins are close to zero on the horizontal axis and below the significance threshold, indicating that they have no significant enrichment difference with or without aptamers. In summary, the main difference between the experimental and control groups comes from the specific recognition and enrichment of AFP by aptamer AP_Apt 47. Aptamer magnetic bead enrichment significantly increased the enrichment of AFP in the sample, making AFP the only protein that was statistically significantly upregulated, while other proteins did not show significant changes. This result fully demonstrates the high selectivity and effectiveness of the method of this invention for enriching the target protein AFP, and verifies the feasibility and effectiveness of the SMBs@AP_Apt 47 system in the detection of AFP in HCC serum.

[0076] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nucleic acid aptamer that specifically recognizes serum alpha-fetoprotein, characterized in that, The nucleic acid aptamer includes at least one of A1)-A3): A1) The nucleotide sequence is as shown in SEQ ID No. 1; The nucleotide sequences defined by A2) and A1) have more than 90% homology and specifically recognize serum alpha-fetoprotein single-stranded DNA. A3) Delete or add one or more nucleotides to the nucleotide sequence defined in A1) and specifically recognize serum alpha-fetoprotein single-stranded DNA.

2. The nucleic acid aptamer according to claim 1, characterized in that, The nucleic acid aptamer specifically recognizes serum alpha-fetoprotein by chemically modifying its 5′ or 3′ end, wherein the modification is selected from one or more of biotin labeling, fluorescent / luminescent group linkage, phosphorylation, methylation, amination, thiolation, sulfur substitution of oxygen, selenium substitution of oxygen, and isotopization; and / or, a signal molecule and / or an active molecule and / or a functional group are linked to the nucleotide sequence of the nucleic acid aptamer, and the linked nucleic acid aptamer specifically recognizes serum alpha-fetoprotein.

3. Use of the nucleic acid aptamer as described in claim 1 or 2 in at least one of B1)-B5): B1) Use in the preparation of products for enriching serum alpha-fetoprotein; B2) Use in the preparation of products for the detection of serum alpha-fetoprotein; B3) Uses in the purification of serum alpha-fetoprotein; B4) Use in the preparation of products targeting serum alpha-fetoprotein; B5) Use in the preparation of screening, diagnostic or auxiliary diagnostic materials for diseases related to serum alpha-fetoprotein.

4. The use according to claim 3, characterized in that, The serum alpha-fetoprotein-related diseases include one or more of hepatocellular carcinoma, germ cell tumors, gastric cancer, pancreatic cancer, and cholangiocarcinoma.

5. A probe, characterized in that, The probe is a substance obtained by labeling a nucleic acid aptamer as described in claim 1 or 2, wherein the label is selected from signal molecules and / or functional groups.

6. A magnetic nanomaterial, characterized in that, The magnetic nanomaterial is a material in which the nucleic acid aptamer described in claim 1 or 2 is functionalized and immobilized on the surface of streptavidin magnetic nanoparticles through streptavidin-biotin high affinity interaction.

7. The magnetic nanomaterial according to claim 6, characterized in that, The magnetic nanomaterials are used to enrich or capture serum alpha-fetoprotein.

8. A product for enriching or detecting serum alpha-fetoprotein, characterized in that, The product includes the nucleic acid aptamer as described in claim 1 or 2, and / or the magnetic nanomaterial as described in claim 6.

9. The product according to claim 8, characterized in that, The products include reagent kits, affinity purification reagents, sensors, and chips.

10. A method for detecting serum alpha-fetoprotein, the method comprising co-incubating a sample to be tested with a nucleic acid aptamer as described in claim 1 or 2, or a magnetic nanomaterial as described in claim 6, and detecting serum alpha-fetoprotein by observing the change in signal before and after the interaction between the nucleic acid aptamer and serum alpha-fetoprotein in the sample.

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