Preparation method and application of a general updatable immune affinity magnetic bead

Abstract

The present invention discloses a preparation method and application of a general updateable immunomagnetic bead, belonging to the technical field of chemical detection, including: first constructing an engineered phage H6G5-M13, and then coupling a polypeptide module to the engineered phage H6G5-M13. The polypeptide module includes three parts, A, B, and C, from the N-terminus to the C-terminus. Part A is a target recognition sequence located at the N-terminus, part B is an optionally added linker sequence, and part C is an LPXTG substrate sequence located at the C-terminus, where X represents any amino acid. Finally, the engineered phage is directionally coupled to the Ni2+-IDA magnetic bead through the Ni-6His interaction, thereby forming a general updateable immunomagnetic bead. The general updateable immunomagnetic bead prepared by the method of the present invention can be used for preparing chemical detection reagents, and can also be used in the capture, release, and sensing detection of cells and extracellular vesicles.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical detection, and particularly relates to a preparation method and application of a general-purpose updatable immune affinity magnetic bead. Background Art

[0002] Immune affinity magnetic materials, such as immunomagnetic beads, are a kind of biological separation medium based on the principle of magnetic separation, with advantages of high separation efficiency, strong selectivity, and simple operation. Its principle is to use a certain recognition molecule (such as antibody, nucleic acid aptamer, polypeptide, etc.) modified on the surface of the magnetic material, so that the magnetic material can specifically bind to the target (such as cells, proteins, nucleic acids, etc.), thereby realizing the separation and enrichment of the target. Currently, immunomagnetic beads are widely used in fields such as immunoassay, cell sorting, protein separation, and nucleic acid extraction. For example, in tumor diagnosis, immunomagnetic beads can be used to specifically capture and enrich circulating tumor cells (CTC) or protein and nucleic acid tumor markers, greatly improving the sensitivity of early tumor diagnosis. In molecular biology research, immunomagnetic beads are often used for the purification and detection of protein or nucleic acid molecules. Through precise and convenient immune separation, the efficiency of protein or nucleic acid extraction is effectively improved.

[0003] Currently, immune affinity magnetic beads mainly rely on the binding of recognition molecules to target substances, but there are significant deficiencies in the immobilization methods of these recognition molecules on the surface of magnetic beads, which are mainly reflected in the following three aspects. First, the current immobilization methods cannot achieve the ordered arrangement of recognition molecules on the surface of magnetic beads. The coupling of recognition molecules to magnetic beads mostly relies on functional group-specific coupling reactions, such as amidation reactions based on carboxyl and amino groups, nucleophilic addition reactions based on maleimide and thiol groups, and Schiff base reactions based on aldehydes (or ketones) and compounds with amino groups. Fixing recognition molecules on the surface of magnetic beads through reactions between functional groups cannot ensure that the recognition molecules are in a spatial position favorable for target binding. There may be situations where the functional groups of the recognition sites are occupied by coupling reactions, and the spatial orientation of the recognition sites is not conducive to target binding, etc., resulting in a decrease in the capture efficiency of recognition molecules. Second, the current immobilization methods cannot achieve modular and reversible coupling of any recognition molecule on the surface of magnetic beads. In the current context of the increasing demand for precision medicine, end-users (such as clinical laboratories) have an increasing need for personalized customization of various targets. There is an urgent need to develop immune affinity magnetic beads that are convenient for non-professional end-users to operate, universal, modular, and with flexible target variability. Although there are currently some general coupling reactions that can achieve this, such as the streptavidin-biotin coupling system, Spy-Tag / Spy-Catcher coupling system, etc., these coupling methods are usually irreversible, and the immune affinity magnetic beads cannot be reused, increasing the usage cost. Third, the current immobilization methods are difficult to meet the demand for controllable release of targets. Many application requirements of immune magnetic beads, such as the separation of CTCs, drug release, etc., require the captured targets to be released efficiently and without damage through a mild method for downstream analysis. Currently, in the relevant fields, targets are mostly released through enzymatic digestion. Magnetic beads treated in this way cannot be conveniently updated and regenerated with recognition molecules, further increasing the usage cost of magnetic beads. In summary, developing a new preparation method to construct immune magnetic beads with flexible switching of recognition molecules, renewable surfaces, and mild release of targets has important practical value in application fields such as clinical diagnosis. However, there is currently no coupling method that can meet the above requirements simultaneously. Summary of the Invention

[0004] The primary objective of the present invention is to provide a preparation method for a universal renewable immune affinity magnetic bead, which can conveniently replace various recognition ligands to meet diverse usage requirements. At the same time, by using the modification technology based on transpeptidase A and Ni-6His, the directional and ordered modification, reversible disassembly, or replacement of recognition ligands is achieved, reducing the usage cost.

[0005] Another object of the present invention is to achieve gentle release of biomarkers such as circulating tumor cells and extracellular vesicles based on the general updateable immune affinity magnetic beads, avoid damage to such biomarkers, improve the capture and release efficiency and facilitate downstream analysis.

[0006] The present invention provides a general updateable immune affinity magnetic bead by constructing an engineered phage H6G5-M13 and combining the action characteristics of sortase A. It is designed as an efficient separation tool with modularity, high binding affinity, and self-renewal ability, and can meet the separation and analysis of multiple targets (such as cells, proteins, bacteria, etc.) in complex samples.

[0007] The preparation method of a general updateable immune affinity magnetic bead described in the present invention includes the following steps: First, construct an engineered phage H6G5-M13, and then couple a polypeptide module to the engineered phage H6G5-M13. The polypeptide module involved in the present invention includes three parts, A, B, and C, from the N-terminus to the C-terminus. Part A is the target recognition sequence, located at the N-terminus. Part B is an optionally added linker sequence, whose main function is to meet potential application designs and improve the hydrophilicity of the polypeptide. Part C is the LPXTG substrate sequence, located at the C-terminus, where X refers to any amino acid. Finally, the engineered phage is directionally coupled to Ni 2+ -IDA magnetic beads (formed by a tricoordination structure of iminodiacetic acid (IDA) and nickel ions (Ni 2+ ), thereby forming a general updateable immune affinity magnetic bead (abbreviated as SortPhace magnetic bead). The SortPhace magnetic bead has the advantage that the recognition molecule can be quickly replaced and updated. The flexibility of the SortPhace magnetic bead enables it to easily adapt to different application scenarios. After the target is captured by the SortPhace magnetic bead, it is released by the mediation of non-endogenous sortase A. Since sortase A is a bacterial-derived enzyme, the sortase A cleavage sequence contained in part C usually does not exist in mammalian blood. Therefore, the SortPhace magnetic bead can stably exist in complex matrices such as human blood.

[0008] Further, the construction of the engineered phage H6G5-M13 specifically includes the following steps:

[0009] The wild-type M13KE phage vector was subjected to site-directed mutagenesis by the site-directed mutagenesis method. The mutation sites included the 1372nd base, the 1381st base, and the 6246th base. Then, the exogenous gene fragment encoding 5Gly was inserted into the gpVIII region encoding the pVIII protein of M13KE, and the exogenous gene fragment encoding 6His was inserted into the gpIII region encoding the pIII protein of M13KE. 5Gly was located at the N-terminus of the pVIII protein of M13 phage. After verifying the sequence accuracy by sequencing, the H6G5-M13KE phage vector was constructed. Then, the genetically modified H6G5-M13KE phage vector was transformed into Escherichia coli XL1-Blue, and through the double-layer plate method for screening and sequencing, the H6G5-M13 phage particles that were completely consistent with the theoretical sequence were screened out and amplified in large quantities.

[0010] Furthermore, a polypeptide module was conjugated to the engineered phage H6G5-M13. The specific steps included:

[0011] Under the action of sortase A, the polypeptide module with an LPXTG motif at the C-terminus bound to the 5Gly motif displayed at the N-terminus of the major capsid protein pVIII of the engineered phage H6G5-M13, realizing the anchoring of the polypeptide module on the surface of the engineered phage. Similarly, the generated ligation product also had an LPXTG motif, which could be recognized by sortase A and continue to be cleaved and ligated, thus realizing the update and replacement of the polypeptide module.

[0012] Furthermore, the engineered phage was directionally conjugated to Ni 2+ -IDA magnetic beads. The specific steps included:

[0013] The 6His-tag displayed by the minor capsid protein of the engineered phage H6G5-M13 provided a specific site for it to be immobilized on the surface of the Ni 2+ -IDA magnetic beads. In a 500 μL reaction system (1×PBS, containing 3% BSA, pH 7.4), the anchoring product of the polypeptide module on the surface of the engineered phage was co-incubated with the Ni 2+ -IDA-coated magnetic beads at room temperature for 3 h. The centrifuge tube was placed on a magnetic rack for magnetic separation, and the supernatant was removed to obtain a general updateable immunoaffinity magnetic bead.

[0014] Further, taking the sequence of the four-transmembrane protein B (LAPTM4B) targeting the lysosome of part A as shown in SEQ ID NO: 1, the specific cleavage sequence of cathepsin B of part B as shown in SEQ ID NO: 2, and the polypeptide TBP (sequence as shown in SEQ ID NO: 3) of part C as the LPETG sequence as the polypeptide module, the anchored product TBP-H6G5-M13 of the polypeptide module on the surface of the engineered phage is co-incubated with Ni 2+ -IDA-coated magnetic beads at room temperature, magnetically separated, and the supernatant was removed to obtain the general renewable immunomagnetic beads SortPhace-TBP.

[0015] For the polypeptide module of the present invention, the A part can also be the Anti-EpCAM sequence targeting the epithelial cell adhesion molecule (EpCAM) protein as shown in SEQ ID NO: 4, the Anti-Ncadherin sequence targeting N-cadherin as shown in SEQ ID NO: 5, the Anti-CD63 sequence targeting and binding to the extracellular vesicle marker protein (CD63) as shown in SEQ ID NO: 6, and the peptide nucleic acid sequence as shown in SEQ ID NO: 7. The B part of the polypeptide module can also be a sequence enhancing the water solubility of the polypeptide (preferably KKK), a thrombin-specific cleavage sequence (preferably LVPRGS), or a combined sequence as shown in SEQ ID NO: 8.

[0016] In the method of the present invention, corresponding affinity polypeptides can be replaced according to application needs, and more general renewable immunomagnetic beads can also be obtained, such as SortPhace-Anti-EpCAM targeting the epithelial cell adhesion molecule (EpCAM), SortPhace-Anti-Ncad targeting N-cadherin, SortPhace-Anti-CD63 targeting and binding to the extracellular vesicle marker protein (CD63), and SortPhace-PNA targeting nucleic acids.

[0017] Specifically, in the SortPhace-Anti-EpCAM, the A part of the polypeptide module is as shown in the sequence SEQ ID NO: 4, the B part is the specific cleavage sequence of cathepsin B as shown in SEQ ID NO: 2, the C part is the LPETG sequence, and the polypeptide module sequence is as shown in SEQ ID NO: 9.

[0018] In the SortPhace-Anti-Ncad, part A of the polypeptide module is shown as SEQ ID NO: 5, part B is shown as SEQ ID NO: 8, part C is the LPETG sequence, and the polypeptide module sequence is shown as SEQ ID NO: 10.

[0019] In the SortPhace-Anti-CD63, the sequence of part A of the polypeptide module is shown as SEQ ID NO: 6, part C is the LPETG sequence, and the polypeptide module sequence is shown as SEQ ID NO: 11.

[0020] In the SortPhace-PNA, the sequence of part A of the polypeptide module is shown as SEQ ID NO: 7, part C is the LPETG sequence, and the polypeptide module sequence is shown as SEQ ID NO: 12.

[0021] The present invention also provides the application of the described general updatable immune affinity magnetic beads in the preparation of immune affinity materials.

[0022] The present invention also provides the application of the described general updatable immune affinity magnetic beads in the capture, release, and sensing detection of cells and extracellular vesicles.

[0023] Capture and release of cells: Add the SortPhace magnetic bead dispersion to the sample to be tested, incubate at 37 °C for 20 min - 30 min, place the centrifuge tube on a magnetic rack for magnetic separation, and remove the supernatant. After separation, add a releasing agent (transpeptidase A 20 μM, 3Gly polypeptide 100 μM, transpeptidase A reaction buffer) to the magnetic bead precipitate, incubate at 37 °C for 20 min - 30 min, place the centrifuge tube on a magnetic rack for magnetic separation. At this time, the released cells are present in the supernatant. Collect the supernatant, use a cell solid-phase immunostaining kit (Cytointelligen, IFH - 001, China) to immunostain the separated cells, and identify and count them under a fluorescence microscope.

[0024] Update of magnetic beads: After the captured target cells are selectively released from the SortPhace magnetic beads, digest them with 0.25% EDTA-trypsin for 3 min - 5 min, wash with PBS 3 - 5 times to remove other residual cells on the surface, and then the magnetic beads can be reconnected with the targeting polypeptide for a new round of capture and release of the target.

[0025] Cellular sensing assay: EpCAM-specific aptamer probes Anti-EpCAM Apt (3'FAM modified) and competitive probes C-Apt (5'BHQ1 modified) were designed. Their sequences are shown in SEQ ID NO: 13 and SEQ ID NO: 14. After annealing and hybridization at 95°C for 5 minutes, the fluorescent aptamer probe Anti-EpCAM Apt was reacted with SortPhace-PNA at a molar ratio of aptamer to PNA of 1:1.2 at 45°C for 10-15 minutes, and then the reaction mixture was slowly cooled to room temperature to complete coupling. To detect CTCs, cells at different concentrations were incubated with SortPhace-PNA-DNA at 37°C for 20-30 minutes. The C-Apt in the SortPhace-PNA-DNA complex was competed off by EpCAM on the cell surface, and the fluorescence of Anti-EpCAM Apt was restored. The restored fluorescence intensity was measured at Ex / Em=484nm / 530nm, and a standard curve was drawn for quantitative analysis of the cells.

[0026] Isolation and labeling of extracellular vesicles (EVs): SortPhace-PNA-DNA was constructed and co-incubated with cell-secreted EVs. The EVs were captured and the fluorescence of the Anti-EpCAM Apt on the SortPhace-PNA-DNA was restored. The microsphere surface was imaged using a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed.

[0027] Key points of the present invention:

[0028] An engineered phage H6G5-M13 was constructed. The major capsid protein pVIII and the minor capsid protein pIII of this engineered phage displayed 5Gly and 6His peptide segments, respectively, providing more active sites for displaying polypeptide modules on the phage surface and immobilizing them on a solid phase carrier.

[0029] Through transpeptidase A-mediated peptide ligation, a modular and universally renewable immunoaffinity magnetic bead preparation method and application have been developed. Functional peptide modules can be multivalently displayed and reversibly loaded and unloaded on the SortPhace beads, enabling efficient capture and gentle release of targets. The SortPhace beads can also be renewed for multiple applications.

[0030] Beneficial effects of the present invention:

[0031] The present invention constructs an engineered phage H6G5-M13, whose major capsid protein pVIII and minor capsid protein pIII display 5Gly (2700 copies) and 6His peptide segments (5 copies) respectively, and has bioorthogonal functions. By utilizing the specificity of sortase A for polypeptide substrate recognition and the bifunctional characteristics of the engineered phage H6G5-M13, the present invention provides a versatile tool. Benefiting from the simplicity of the sortase A reaction, the polypeptide modules of the SortPhace magnetic beads can be easily switched, including but not limited to the polypeptide module TBP in Example 1. The engineered phage H6G5-M13 provides an easy-to-operate scaffold for the directional multivalent display of peptide modules or their analog modules (such as peptide nucleic acid PNA); moreover, the reaction mediated by sortase A provides a method for non-destructively releasing biological targets and ensures the self-renewal ability of the SortPhace magnetic beads.

[0032] The SortPhace magnetic beads disclosed in the present invention are characterized by efficient capture and release of CTCs for subsequent analysis and research; they can bind to fluorescent aptamer probes to achieve specific detection and quantitative analysis of CTCs; they can isolate and label tumor-derived extracellular vesicles (tEVs) for studying the tumor microenvironment and liquid biopsy; the reusable magnetic bead system reduces experimental costs; they can be widely applied in biomedical research and clinical diagnosis, especially in the field of cancer. Brief Description of the Drawings

[0033] Figure 1 It is the construction process and amino acid sequence of the engineered phage H6G5-M13 in the present invention; wherein (a) is the gene map of the M13KE plasmid and the schematic diagram of genetic engineering operation sites; (b) is the gene map of the H6G5-M13KE plasmid; (c) is the DNA fragment of the key region in the H6G5-M13KE plasmid and the corresponding amino acid sequence.

[0034] Figure 2 In Example 1 of the present invention, the modification efficiency of the polypeptide TBP on the phage surface is 36.1%.

[0035] Figure 3 In Example 1 of the present invention, the immobilization efficiency of the phage on the magnetic bead surface >90%.

[0036] Figure 4 It is the schematic diagram and fluorescence image (scale bar = 25 μm) of different SortPhace magnetic beads used for the isolation of circulating tumor cells or extracellular vesicles in Example 1 of the present invention.

[0037] Figure 5Comparison of the affinity (Kd value) of free TBP polypeptide, TBP-H6G5-M13, and SortPhace-TBP for circulating tumor cells in Example 1 of the present invention.

[0038] Figure 6 Performance of SortPhace magnetic beads in capturing and releasing circulating tumor cells in Example 1 of the present invention, including its stability, efficiency, and cell viability; where (a) shows that SortPhace loaded with different polypeptides can achieve the capture and release of various circulating tumor cells; (b) shows the cell viability of circulating tumor cells after separation by SortPhace magnetic beads; (c) shows the renewable stability of SortPhace magnetic beads.

[0039] Figure 7 Schematic diagram of the detection and analysis process of SortPhace magnetic beads for circulating tumor cells (CTC) in the peripheral blood of colorectal cancer patients in Example 1 of the present invention.

[0040] Figure 8 CTC count analysis in the peripheral blood of clinical patients and healthy volunteers in Example 1 of the present invention.

[0041] Figure 9 Immunofluorescence staining images of CTC and white blood cells (WBC) in Example 1 of the present invention (scale bar = 10 μm).

[0042] Figure 10 Receiver operating characteristic curve (ROC) analysis of SortPhace magnetic beads for colorectal cancer clinical diagnosis in Example 1 of the present invention.

[0043] Figure 11 Consistency comparison between the results of gene mutation analysis of CTC isolated from the peripheral blood of 11 patients in Example 1 of the present invention and the clinical reports.

[0044] Figure 12 Change in the number of CTC before and after different treatment methods in Example 1 of the present invention.

[0045] Figure 13 Schematic diagram and fluorescence image of SortPhace-PNA for sensing and detecting circulating tumor cells or extracellular vesicles in Example 2 of the present invention.

[0046] Figure 14 Standard curve of SortPhace-PNA for sensing and detecting MCF-7 cells in Example 2 of the present invention. Detailed implementation method

[0047] The present invention relates to the collection of venous blood samples from patients and volunteers for the isolation and analysis of circulating tumor cells (CTCs). Informed consent forms were signed by all samples with the full knowledge of patients and volunteers, consenting to the use of their blood samples for this study. The research protocol has passed the ethical review of the [Bioethics Committee of Northeastern University], with the approval number [NEU-EC-2024B035S] and the approval date [May 30, 2024]. During the research process, relevant regulations on patient privacy protection and data anonymization were strictly adhered to, and the samples were only used for the sample detection of the present invention and not for any commercial purposes.

[0048] The raw materials and reagents used in the present invention are all commercially available products and can be obtained from conventional biochemical reagent suppliers.

[0049] Example 1

[0050] A preparation method and application of a general-purpose updatable immune affinity magnetic bead SortPhace-TBP are as follows:

[0051] 1. Preparation of genetically engineered phage: First, site-directed mutagenesis was performed on the wild-type M13KE phage vector (the sequence is shown in SEQ ID NO: 15) by site-directed mutagenesis method. Specifically, using primers SDM1-F and SDM1-R (the sequences are shown in SEQ ID NO: 16 and SEQ ID NO: 17), the 1372nd base A was mutated to T, and the 1381st base G was mutated to C. Using primers SDM2-F and SDM2-R (the sequences are shown in SEQ ID NO: 18 and SEQ ID NO: 19), the 6246th base T was mutated to A. Then, through homologous recombination, the exogenous gene fragment encoding 5Gly was inserted into the gpVIII region encoding pVIII of M13KE using primers G5-F and G5-R (the sequences are shown in SEQ ID NO: 20 and SEQ ID NO: 21). Similarly, the exogenous gene fragment encoding 6His was inserted into the gpIII region encoding pIII of M13KE using primers H6-F and H6-R (the sequences are shown in SEQ ID NO: 22 and SEQ ID NO: 23). Finally, site-directed mutagenesis was performed on the 1371st base, changing from C to G, using primers SDM3-F and SDM3-R (the sequences are shown in SEQ ID NO: 24 and SEQ ID NO: 25), to ensure that 5Gly is located at the N-terminus of the pVIII protein of M13 phage. After verifying the sequence accuracy by sequencing, the H6G5-M13 phage vector was constructed. Then, the genetically modified H6G5-M13 phage vector was transformed into Escherichia coli XL1-Blue, and screened and sequenced by the double-layer plate method. The H6G5-M13 phage particles that were completely consistent with the theoretical sequence were screened out and amplified in large quantities. The construction process and amino acid sequence of the above engineered phage H6G5-M13 are as Figure 1 shown.

[0052] 2. Transpeptidase A-mediated polypeptide ligation reaction: Transpeptidase A first specifically recognizes and cleaves the peptide bond between T and G in the C-terminal LPXTG motif of the polypeptide module to form a thioester intermediate. Then, the 5Gly motif displayed at the N-terminus of the major capsid protein pVIII of the engineered phage H6G5-M13 nucleophilically attacks the thioester intermediate, realizing the anchoring of the polypeptide module on the surface of the engineered phage. Similarly, the generated ligation product also has an LPXTG motif, which can be recognized by transpeptidase A and continue to be cleaved and ligated, thus realizing the update and replacement of the polypeptide module.

[0053] Specifically, in this Example 1, a polypeptide module TBP was designed with part A being the sequence targeting the lysosomal tetraspanin B (LAPTM4B) of cells, as shown in SEQ ID NO: 1; part B being the specific cleavage sequence of cathepsin B, as shown in SEQ ID NO: 2; and part C being the LPETG sequence, with the sequence shown in SEQ ID NO: 3. In 500 μL of transpeptidase A reaction buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.5), the engineered phage H6G5-M13 and the polypeptide TBP were added to the reaction at an optimized molar ratio (pVIII:TBP = 1:10). Under the action of transpeptidase A (20 μM), the reaction was carried out at 37 °C for 1 h. After the reaction, excess components were removed by PEG / NaCl precipitation or ultrafiltration centrifugation to obtain TBP-H6G5-M13.

[0054] In addition to the above sequences, part A of the polypeptide module can also be Anti-EpCAM targeting the epithelial cell adhesion molecule (EpCAM) protein, with the sequence shown in SEQ ID NO: 4; Anti-Ncadherin targeting the neural cadherin (N-cadherin), with the sequence shown in SEQ ID NO: 5; and Anti-CD63 targeting and binding to the extracellular vesicle marker protein (CD63), with the sequence shown in SEQ ID NO: 6. Part B of the polypeptide module can also be a sequence enhancing the water solubility of the polypeptide (preferably KKK), a thrombin-specific cleavage sequence (preferably LVPRGS), or a combined sequence thereof, as shown in SEQ ID NO: 8.

[0055] 3. Construction of magnetic beads: The 6His-tag displayed on the minor capsid protein of the engineered phage H6G5-M13 provides a specific site for it to be immobilized on the surface of Ni-IDA magnetic beads. In a 500 μL reaction system (1×PBS, containing 3% BSA, pH 7.4), TBP-H6G5-M13 and Ni-IDA-coated magnetic beads were co-incubated at room temperature for 3 h. The centrifuge tube was placed on a magnetic rack for magnetic separation, and the supernatant was removed to obtain SortPhace-TBP. 2+ -IDA magnetic beads in a 500 μL reaction system (1×PBS, containing 3% BSA, pH 7.4), TBP-H6G5-M13 and Ni 2+ -IDA-coated magnetic beads were co-incubated at room temperature for 3 h. The centrifuge tube was placed on a magnetic rack for magnetic separation, and the supernatant was removed to obtain SortPhace-TBP.

[0056] Through the reaction mediated by transpeptidase A, the polypeptide TBP with a FITC fluorescent label was linked to the major capsid protein of the engineered phage H6G5-M13. Record the fluorescence intensity of the engineered phage H6G5-M13 at 520 nm, and substitute it into the standard curve to calculate the content of the polypeptide TBP on the surface of the engineered phage H6G5-M13, and further obtain the modification efficiency of the polypeptide TBP.

[0057] The engineered phage H6G5-M13 that has successfully linked the polypeptide TBP was co-incubated with Ni 2+ -IDA-coated magnetic beads at room temperature for 3 h. The centrifuge tube was placed on a magnetic rack for magnetic separation, and the supernatant and magnetic beads were collected separately. The phages not immobilized on the magnetic beads in the supernatant were quantified by plating to measure the titer, and thus the immobilization efficiency of the phages on the magnetic bead surface was obtained.

[0058] In Example 1, through the reaction mediated by sortase A, the modification efficiency of the polypeptide TBP on the surface of the engineered phage H6G5-M13 was 36.1%. Through the action of Ni-6His, the immobilization efficiency of the phage on the magnetic bead surface > 90% ( Figures 2 - 3 ).

[0059] According to application requirements, more SortPhace magnetic beads can be obtained by replacing the corresponding affinity polypeptides. For example, SortPhace-Anti-EpCAM targeting epithelial cell adhesion molecule (EpCAM), SortPhace-Anti-CD63 targeting the extracellular vesicle marker protein (CD63), and SortPhace-Anti-Ncad targeting N-cadherin are used for the sensing detection or separation and enrichment of circulating tumor cells (CTC) with high EpCAM expression, tumor extracellular vesicles (tEV), or cells with high N-Cadherin expression, respectively. The preparation method is the same as that of SortPhace-TBP magnetic beads.

[0060] In SortPhace-Anti-EpCAM, the sequence of part A of the polypeptide module is shown in SEQ ID NO: 4, part B is the specific cleavage sequence of cathepsin B shown in SEQ ID NO: 2, part C is the LPETG sequence, and the polypeptide module sequence is shown in SEQ ID NO: 9. In SortPhace-Anti-Ncad, the sequence of part A of the polypeptide module is shown in SEQ ID NO: 5, the sequence of part B is shown in SEQ ID NO: 8, part C is the LPETG sequence, and the polypeptide module sequence is shown in SEQ ID NO: 10. In SortPhace-Anti-CD63, the sequence of part A of the polypeptide module is shown in SEQ ID NO: 6, part C is the LPETG sequence, and the polypeptide module sequence is shown in SEQ ID NO: 11.

[0061] The present invention also provides the applications of different SortPhace magnetic beads in cell capture, release, and detection.

[0062] (1)Cell capture and release: Add SortPhace magnetic bead dispersion (500 μL PBS or 500 μL PBS containing 3% BSA solution) to 1 mL of the sample to be tested, incubate at 37 °C for 30 min, place the centrifuge tube on a magnetic stand for magnetic separation for 5 min, collect the supernatant, immunostain the separated cells using a circulating tumor cell solid-phase immunostaining kit (Cytointelligen, IFH - 001, China), count the cells under a microscope, and calculate the cell capture efficiency using the following formula:

[0063] .

[0064] After separation, add 1 mL of release agent (transpeptidase A 20 μM, 3Gly polypeptide 100 μM, transpeptidase A reaction buffer) to the magnetic bead precipitate, incubate at 37 °C for 30 min, place the centrifuge tube on a magnetic stand for magnetic separation for 5 min. At this time, the released circulating tumor cells are present in the supernatant. Collect the supernatant, identify and count them under a fluorescence microscope, and calculate the cell capture efficiency and release efficiency using the following formula:

[0065] .

[0066] Perform a live / dead cell staining experiment on the released cells, and calculate the cell viability using the following formula:

[0067] .

[0068] As Figure 4 shows, the A549 cell line expresses Ncadherin and TBPR; the MCF-7 cell line expresses EpCAM and TBPR; the MDA-MB-231 cell line expresses Ncadherin and TBPR; uEV (exosomes derived from healthy human urine) expresses CD63; the 16 HBE cell line is used as a control group and does not express the above markers. SortPhace-TBP can capture cells expressing TBPR (A549, MCF-7, MDA-MB-231), but has no capture effect on the 16 HBE cell line that does not express TBPR. SortPhace-Anti-EpCAM can effectively capture the MCF-7 cell line expressing EpCAM, but has no capture effect on other cell lines. SortPhace-Anti-Ncad can capture the A549 and MDA-MB-231 cell lines expressing Ncadherin, but has no capture effect on the MCF-7 and 16HBE cell lines that do not express Ncadherin. SortPhace-Anti-CD63 can effectively capture uEV expressing CD63, but has no capture effect on other cell lines.

[0069] The observation results under the fluorescence microscope showed that after capture with SortPhace magnetic beads, fluorescence labels of different colors were observed under the fluorescence microscope, indicating the capture effects of different cell lines. For example, as Figure 4 shown, after the A549 cell line pre-stained with DAPI (blue under the fluorescence microscope) was captured with SortPhace-TBP (red under the fluorescence microscope), red and blue fluorescence labels could be seen; similarly, after capture with SortPhace-Anti-Ncad, red and blue fluorescence labels could be seen. Figure 4 The specific capture effects of different SortPhace magnetic beads on different cell lines were demonstrated, verifying the high efficiency and specificity of these magnetic beads in cell separation and capture.

[0070] After releasing the cells, the SortPhace magnetic beads were digested with 0.25% EDTA-trypsin for 3 - 5 minutes, washed 3 - 5 times with PBS to remove other residual cells on the surface, and could be reconnected with the targeting polypeptide for a new round of capture and release of the target.

[0071] (2) High efficiency and renewable stability of the magnetic beads: As Figure 5 shown, according to the test results of the dynamic equilibrium determination method (ELISA saturation concentration method), the affinity of SortPhace-TBP for the target cells (Kd = 1.93 pM) was increased by four orders of magnitude compared with the free polypeptide TBP (Kd = 390.33 nM) (4.54×10 4 ), which means that SortPhace-TBP can bind to the target cells more efficiently. This significantly improved affinity was verified in practical applications: As Figure 6 shown in (a), the capture efficiency of SortPhace-TBP for circulating tumor cells (CTCs) reached 85.5% - 91.6%, and the release efficiency reached 79.1% - 85.6%. This indicates that SortPhace-TBP not only has a significant improvement in affinity, but also shows high efficiency in the capture and release processes of CTCs, which is of great significance for the detection and analysis of CTCs. In addition, Figure 6 other types of SortPhace magnetic beads mentioned in (a) (such as SortPhace-Anti-EpCAM, SortPhace-Anti-Ncad) also showed high capture and release efficiencies. As Figure 6 shown in (b), the viability of the cells separated by SortPhace magnetic beads reached 96.7%, further proving the high efficiency and low damage of this technology in cell separation and processing. As Figure 6As shown in (c), the SortPhace-TBP in Example 1 was recycled 14 times with stable performance, indicating that it can still maintain a high capture and release efficiency after multiple cycles.

[0072] (3)SortPhace-TBP is used for clinical auxiliary evaluation of the effectiveness of treatment plans;

[0073] Collect 1 mL of venous blood samples from patients, and perform CTC separation through SortPhace-TBP within 24 hours, and complete CTC immunostaining identification and downstream analysis. The research subjects are patients aged >18 years old, with pathological diagnosis of colorectal cancer or benign digestive tract diseases, and meeting the inclusion criteria of this research project. Analyze 81 clinical samples (53 cases of colorectal cancer, 18 cases of benign digestive tract polyps, 10 cases of healthy volunteers). The number of CTCs in 1 mL of venous blood is correlated with the occurrence and development of malignant tumors (see Figures 7 - 9 , significant differences *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.). The AUC of this method is 0.981 (Sensitivity = 90.57%, Specificity = 100%) ( Figure 10 ).

[0074] Figure 7 Shows the process of capturing and releasing circulating tumor cells (CTCs) from whole blood using SortPhace magnetic bead technology, and further identification through immunofluorescence and gene mutation analysis.

[0075] Figure 8 Shows the number of CTCs in the peripheral blood of clinical patients with different TNM stages (tumor staging system) and healthy volunteers. It can be observed that: with the increase of TNM stage (from stage I / II to stage IV), the number of CTCs increases significantly. The number of CTCs in healthy / benign individuals is very low, with significant differences compared with cancer patients. These results indicate that CTC counting can be used as an indicator of cancer progression, helping to evaluate the severity of the disease and monitor the treatment effect.

[0076] Figure 9 Shows the immunofluorescence staining results of CTCs and white blood cells (WBCs), using different markers DAPI, EpCAM, CK, CD45 to distinguish CTCs and WBCs: DAPI is used to stain cell nuclei, showing all cells; EpCAM epithelial cell adhesion molecule, usually expressed on the surface of cancer cells; CK (cytokeratin) is a marker of epithelial cells, and CTCs usually express CK; CD45 is a marker of white blood cells, used to distinguish WBCs and CTCs. Figure 9As can be seen, CTC shows positive expression of EpCAM and CK, while CD45 is negative, which is consistent with the characteristics of cancer cells. WBC mainly shows positive CD45, while EpCAM and CK are negative, which is consistent with the characteristics of white blood cells. These results further verify the effectiveness of the SortPhace technology in accurately isolating and identifying CTC.

[0077] Figure 10 The receiver operating characteristic curve (ROC curve) of SortPhace magnetic beads in the clinical diagnosis of colorectal cancer is shown. The ROC curve shows the performance of SortPhace magnetic beads in distinguishing colorectal cancer patients from non-patients, with an AUC = 0.981, close to 1, indicating that this method has high sensitivity and specificity. The isolated CTCs were recultured and gene amplified, and whether mutations occurred in colorectal cancer-related genes (TP53, KRAS, APC) was analyzed by gene sequencing. The results showed that SortPhace-TBP isolated CTCs in clinical samples and performed whole-genome amplification, detecting common mutation sites in colorectal cancer (such as KRASG12C). Compared with the clinical gene diagnosis report, the accuracy rate reached 93.94% (31 / 33) ( Figure 11 ), and × indicates inconsistency with the clinical gene diagnosis.

[0078] Patients receiving different treatment regimens were followed up. Three patients received stage 2 chemotherapy, chemotherapy + targeted therapy, and rectal Hartmann surgery respectively. The changes in the number of CTCs before and after treatment were compared, and they decreased by 52%, 70%, and 74% respectively ( Figure 12 ), proving the effectiveness of SortPhace-TBP in clinically assisting the evaluation of treatment regimens.

[0079] Example 2

[0080] The application of the general updatable row immunoaffinity SortPhace-PNA in cell sensing detection is as follows:

[0081] SortPhace-PNA was prepared in the same way as SortPhace-TBP in Example 1. First, the engineered phage H6G5-M13 was constructed, then PNA (polypeptide module, the A part sequence is as shown in SEQ ID NO: 7, the C part is the LPETG sequence, and the polypeptide module sequence is as shown in SEQ ID NO: 12) was conjugated to the engineered phage H6G5-M13, and finally the engineered phage was directionally conjugated to Ni 2+ -IDA magnetic beads to obtain SortPhace-PNA.

[0082] As Figure 13As shown, an aptamer probe Anti-EpCAM Apt (modified with 3’FAM) and a competitive probe C-Apt (modified with 5’BHQ1) that specifically recognize EpCAM were designed, and the sequences are shown in SEQ ID NO: 13 and SEQ ID NO: 14. After annealing and hybridization at 95°C for 5 min, according to the molar ratio of aptamer to PNA of 1:1.2, the fluorescent aptamer probe Anti-EpCAM Apt and SortPhace-PNA were reacted at 45°C for 15 min, and then slowly cooled to room temperature to complete the coupling to obtain the SortPhace-PNA-DNA complex. In this complex, Anti-EpCAM Apt and C-Apt are respectively labeled with the fluorescent group FAM and the quenching group BHQ1. When the two hybridize, due to the close distance between the two groups, the FAM group on Anti-EpCAM Apt is quenched by the BHQ1 group; when the complex binds to CTCs in the system, C-Apt is competitively displaced, and the fluorescence of the FAM group on Anti-EpCAM Apt is restored. Based on this principle, the off-on nucleic acid probe SortPhace-PNA was constructed in Example 2. To achieve sensitive detection of CTCs, different numbers of MCF-7 cells were incubated with SortPhace-PNA-DNA (10 μM) at 37°C for 30 min. C-Apt in the SortPhace-PNA-DNA complex was competitively displaced by EpCAM on the cell surface, and the fluorescence of Anti-EpCAM Apt was restored. The restored fluorescence intensity was measured at Ex / Em = 484 nm / 530 nm, and a standard curve was plotted for quantitative analysis of MCF-7 cells. As Figure 14 shown, the linear range for the detection of MCF-7 cells is 50 - 10,000 cells / mL, and the calculated linear regression equation is: F - F 0 = 1.048x - 21.23, R 2 = 0.9965, and the detection limit is 30 cells / mL (3σ / s, n = 11). Figure 14 This provides strong evidence for the application of immunosorbent SortPhace-PNA in cell sensing detection, demonstrating its advantages in sensitivity, accuracy, specificity, and rapid response, and laying a foundation for future research and clinical applications.

[0083] Example 3

[0084] Isolation and labeling of tEVs were carried out as follows:

[0085] Construct SortPhace-PNA-DNA by the method of Example 2 and co-incubate it with tumor-derived extracellular vesicles (tEVs) secreted by MCF-7 cells. At this time, the tEVs are captured, and at the same time, the fluorescence of Anti-EpCAM Apt on SortPhace-PNA is restored. The surface of the microspheres is photographed by a fluorescence microscope, and the fluorescence intensity is quantitatively analyzed. Capturing and labeling tEVs with the SortPhace-PNA-DNA complex can further study the role of tEVs in tumor development, metastasis, and treatment resistance, which not only helps to improve the sensitivity and specificity of tEVs detection but also provides new possibilities for future research and clinical applications.

[0086] Table 1 Primers and sequences used in the present invention:

[0087]

[0088] Those skilled in the art should understand that the above embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

Claims

1. A preparation method of a general updateable immune affinity magnetic bead, characterized in that, It includes the following steps: First, construct the engineered phage H6G5-M13, and then couple a polypeptide module to the engineered phage H6G5-M13. The polypeptide module contains three parts, A, B, and C, from the N-terminus to the C-terminus. Part A is the target recognition sequence located at the N-terminus, part B is an optionally added linker sequence, and part C is the LPXTG substrate sequence located at the C-terminus, where X represents any amino acid. Finally, the engineered phage is directionally coupled to Ni 2+ -IDA magnetic beads through the action of Ni Among them, the construction of the engineered phage H6G5-M13 specifically includes the following steps: site-directed mutagenesis is performed on the wild-type M13KE phage vector by the site-directed mutagenesis method, and the mutation sites include the 1372nd base, the 1381st base, and the 6246th base. Then, the exogenous gene fragment encoding 5Gly is inserted into the gpVIII region encoding the pVIII protein of M13KE, and the exogenous gene fragment encoding 6His is inserted into the gpIII region encoding the pIII protein of M13KE. 5Gly is located at the N-terminus of the pVIII protein of the M13 phage. After verifying the sequence accuracy by sequencing, the H6G5-M13KE phage vector is constructed. Then, the genetically modified H6G5-M13KE phage vector is transformed into Escherichia coli XL1-Blue, and through the double-layer plate method for screening and sequencing, the H6G5-M13 phage particles that are completely consistent with the theoretical sequence are screened out.

2. The preparation method of a general updatable immune affinity magnetic bead according to claim 1, characterized in that, Coupling the polypeptide module to the engineered phage H6G5-M13 specifically includes the following steps: under the action of sortase A, the polypeptide module with an LPXTG motif at the C-terminus binds to the 5Gly motif displayed at the N-terminus of the major capsid protein pVIII of the engineered phage H6G5-M13.

3. The preparation method of a general updatable immune affinity magnetic bead according to claim 1, characterized in that, Engineered phages are directionally coupled to Ni-IDA magnetic beads through the action of Ni-6His, and the specific steps include: in the reaction system: 1×PBS, containing 3% BSA, pH 7.4, the anchored product of the polypeptide module on the surface of the engineered phage and the Ni-IDA-coated magnetic beads are co-incubated at room temperature, and then magnetic separation is carried out to remove the supernatant to obtain a general renewable immunoaffinity magnetic bead. 2+ -IDA magnetic beads, and the specific steps include: in the reaction system: 1×PBS, containing 3% BSA, pH 7.4, the anchored product of the polypeptide module on the surface of the engineered phage and Ni 2+ -IDA-coated magnetic beads are co-incubated at room temperature, and then magnetic separation is carried out to remove the supernatant to obtain a general renewable immunoaffinity magnetic bead.

4. The preparation method of a general updatable immune affinity magnetic bead according to claim 1, characterized in that, The A part of the polypeptide module includes: the sequence targeting the tetraspanin B of the cell lysosome as shown in SEQ ID NO: 1, the Anti-EpCAM sequence targeting the epithelial cell adhesion factor protein as shown in SEQ ID NO: 4, the Anti-Ncadherin sequence targeting N-cadherin as shown in SEQ ID NO: 5, the Anti-CD63 sequence targeting and binding to the extracellular vesicle marker protein as shown in SEQ ID NO: 6, and the peptide nucleic acid sequence as shown in SEQ ID NO: 7; the B part of the polypeptide module includes: the specific cleavage sequence of cathepsin B as shown in SEQ ID NO: 2, the sequence enhancing the water solubility of the polypeptide, the specific cleavage sequence of thrombin, or a combined sequence thereof as shown in SEQ ID NO:

8.

5. The preparation method of a general updatable immune affinity magnetic bead according to claim 4, wherein, The general updatable immune affinity magnetic beads are: SortPhace-TBP targeting the tetraspanin B of the cell lysosome, SortPhace-Anti-EpCAM targeting the epithelial cell adhesion factor, SortPhace-Anti-Ncad targeting N-cadherin, SortPhace-Anti-CD63 targeting and binding to the extracellular vesicle marker protein, SortPhace-PNA targeting nucleic acid; In SortPhace-TBP, the A part of the polypeptide module is the sequence targeting the tetraspanin B of the cell lysosome as shown in SEQ ID NO: 1, the B part is the specific cleavage sequence of cathepsin B as shown in SEQ ID NO: 2, the C part is the LPETG sequence, and the polypeptide module sequence is as shown in SEQ ID NO: 3; In SortPhace-Anti-EpCAM, the sequence of part A of the polypeptide module is shown as SEQ ID NO: 4, part B is the specific cleavage sequence of cathepsin B shown as SEQ ID NO: 2, part C is the LPETG sequence, and the polypeptide module sequence is shown as SEQ ID NO: 9; In SortPhace-Anti-Ncad, the sequence of part A of the polypeptide module is shown as SEQ ID NO: 5, the sequence of part B is shown as SEQ ID NO: 8, part C is the LPETG sequence, and the polypeptide module sequence is shown as SEQ ID NO: 10; In SortPhace-Anti-CD63, the sequence of part A of the polypeptide module is shown as SEQ ID NO: 6, part C is the LPETG sequence, and the polypeptide module sequence is shown as SEQ ID NO: 11; In SortPhace-PNA, the sequence of part A of the polypeptide module is shown as SEQ ID NO: 7, part C is the LPETG sequence, and the polypeptide module sequence is shown as SEQ ID NO:

12.

6. Use of a general updateable immunoaffinity magnetic bead prepared by the preparation method of a general updateable immunoaffinity magnetic bead according to any one of claims 1-5 in the preparation of an immunoaffinity material.

7. Use of a general updateable immunoaffinity magnetic bead prepared by the preparation method of a general updateable immunoaffinity magnetic bead according to any one of claims 1-5 in the capture, release and sensing detection of cells and extracellular vesicles.

8. Use of a general updatable immunomagnetic bead according to claim 7 in capture, release, and sensing detection of cells and extracellular vesicles, characterized in that, The process of cell capture and release is as follows: Add the general updateable immunoaffinity magnetic bead dispersion to the sample to be tested, incubate at 37 °C, perform magnetic separation, remove the supernatant. After separation, add the release agent to the magnetic bead precipitate, incubate at 37 °C, perform magnetic separation. At this time, the released cells are present in the supernatant. Collect the supernatant, immunostain the separated cells using a cell solid-phase immunostaining kit, and identify and count them under a fluorescence microscope.

9. Use of a general updatable immunomagnetic bead according to claim 7 in capture, release and sensing detection of cells and extracellular vesicles, characterized in that, The process of cell sensing detection is as follows: Design fluorescent aptamer probes Anti-EpCAM Apt and competitive probe C-Apt that specifically recognize EpCAM. The sequences are shown in SEQ ID NO: 13 and SEQ ID NO:

14. After annealing and hybridization at 95°C for 5 minutes, according to the molar ratio of aptamer to PNA of 1:1.2, react the fluorescent aptamer probe Anti-EpCAM Apt with SortPhace-PNA described in claim 5 at 45°C for 10 - 15 minutes, and then slowly cool down to room temperature to complete the coupling to obtain SortPhace-PNA-DNA; incubate cells with different concentrations with SortPhace-PNA-DNA at 37°C. The C-Apt in SortPhace-PNA-DNA is competitively displaced by EpCAM on the cell surface, and the fluorescence of Anti-EpCAM Apt is restored. Measure the restored fluorescence intensity at Ex / Em = 484nm / 530nm, and draw a standard curve for quantitative analysis of cells.

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