Preparation method and application of universal renewable immunoaffinity magnetic beads

Abstract

The invention discloses a preparation method and application of a universal renewable immunoaffinity magnetic bead, and belongs to the technical field of chemical detection.The preparation method comprises the steps that firstly, an engineered bacteriophage H6G5-M13 is constructed, then a polypeptide module is coupled to the engineered bacteriophage H6G5-M13, the polypeptide module comprises a part A, a part B and a part C from the N end to the C end, the part A is a target object recognition sequence located at the N end, and the part C is a target object recognition sequence located at the C end; the part B is a selectively added connecting sequence, the part C is an LPXTG substrate sequence and is located at the C end, and X refers to any amino acid; finally, the engineered bacteriophage is directionally coupled to the Ni < 2 + >-IDA magnetic bead through the action of Ni-6His, and then the universal and novel immunoaffinity magnetic bead is formed. The universal and replaceable immunoaffinity magnetic bead prepared by the method can be used for preparing a chemical detection reagent, and can also be used for capturing, releasing and sensing detection of cells and extracellular vesicles.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and in particular relates to a preparation method and application of universal renewable immunoaffinity magnetic beads. Background Art

[0002] Immunoaffinity magnetic materials, such as immunomagnetic beads, are a type of biological separation medium based on the principle of magnetic separation, which has the advantages of high separation efficiency, strong selectivity, and simple operation. The principle is to use a certain recognition molecule (such as antibody, nucleic acid aptamer, peptide, etc.) modified on the surface of the magnetic material to enable the magnetic material to specifically bind to the target (such as cells, proteins, nucleic acids, etc.), thereby achieving the separation and enrichment of the target. At present, immunomagnetic beads are widely used in the fields of immunoassay, cell sorting, protein separation, nucleic acid extraction, etc. 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 immunoseparation, the efficiency of protein or nucleic acid extraction is effectively improved.

[0003] At present, immunoaffinity magnetic beads mainly rely on the binding of recognition molecules to targets, and the fixation methods of these recognition molecules on the surface of magnetic beads have significant deficiencies, which are mainly reflected in the following three aspects. First, the current immobilization method cannot achieve the orderly 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 between carboxyl and amino groups, nucleophilic addition reactions between maleimide and thiol groups, and Schiff base reactions between aldehydes (or ketones) and amino-containing compounds. 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 that is conducive to target binding. There may be functional groups of the recognition sites occupied by the coupling reaction, and the spatial orientation of the recognition sites is not conducive to target binding, which leads to a decrease in the capture efficiency of the recognition molecules. Second, the current immobilization method cannot achieve modular and reversible coupling of any recognition molecules on the surface of magnetic beads. At a time when the demand for precision medicine is increasing, end users (such as clinical laboratory departments) have an increasing demand for personalized customization of various targets. There is an urgent need to develop universal, modular, and flexible immunoaffinity magnetic beads that are convenient for non-professional end users to operate. Although there are some universal coupling reactions that can achieve this, such as the use of streptavidin-biotin coupling system, Spy-Tag / Spy-Catcher coupling system, etc., these coupling methods are usually irreversible, and immunoaffinity magnetic beads cannot be reused, which increases the cost of use. Third, the current immobilization method is difficult to meet the demand for controlled release of targets. For many application requirements of immunomagnetic beads, such as CTC separation and drug release, the targets that need to be captured can be released efficiently and non-destructively in a gentle way for downstream analysis. At present, related fields mostly release targets by enzymatic digestion. The magnetic beads treated in this way cannot easily regenerate the recognition molecules, which further increases the cost of using the magnetic beads. In summary, developing new preparation methods to construct immunomagnetic beads with flexible switching of recognition molecules, renewable and regenerable surfaces, and gentle release of targets has important practical value in clinical diagnosis and other application fields. However, there is currently no coupling method that can meet the above requirements at the same time. Summary of the invention

[0004] The primary purpose of the present invention is to provide a method for preparing universal renewable immunoaffinity magnetic beads, which can easily replace a variety of recognition ligands to meet diverse usage requirements. At the same time, by using modification technology based on transpeptidase A and Ni-6His, the directional and orderly modification, reversible disassembly or replacement of the recognition ligands can be achieved, thereby reducing the cost of use.

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

[0006] The present invention provides a universal renewable immunoaffinity magnetic bead by constructing an engineered bacteriophage 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, which can meet the separation and analysis of multiple targets (such as cells, proteins, bacteria, etc.) in complex samples.

[0007] The preparation method of a universal renewable immunoaffinity magnetic bead described in the present invention comprises the following steps: firstly constructing an engineered bacteriophage H6G5-M13, and then coupling a polypeptide module to the engineered bacteriophage H6G5-M13. The polypeptide module described in the present invention comprises 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 a selectively added connection sequence, which mainly serves to meet potential application designs and improve the hydrophilicity of the polypeptide. Part C is a LPXTG substrate sequence located at the C-terminus, wherein X refers to any amino acid. Finally, the engineered bacteriophage is directionally coupled to Ni through the action of Ni-6His. 2+ -IDA magnetic beads (made by combining iminodiacetic acid (IDA) with nickel ions (Ni 2+ ) to form a universal and renewable immunoaffinity magnetic bead (SortPhace magnetic bead for short). SortPhace magnetic beads have the advantage that the recognition molecules can be quickly replaced and updated. The flexibility of SortPhace magnetic beads enables them to easily adapt to different application scenarios. After the target is captured by the SortPhace magnetic beads, it is released by non-endogenous transpeptidase A. Since transpeptidase A is a bacterial enzyme, the transpeptidase A cleavage sequence contained in part C is usually not present in mammalian blood. Therefore, SortPhace magnetic beads can stably exist in complex matrices such as human blood.

[0008] Further, the engineered phage H6G5-M13 was constructed, and the specific steps included: The wild-type M13KE phage vector was subjected to site-directed mutagenesis by site-directed mutagenesis, and the mutation sites included bases 1372, 1381, and 6246. Then, the exogenous gene fragment encoding 5Gly was inserted into the gpVIII region of the M13KE pVIII protein, and the exogenous gene fragment encoding 6His was inserted into the gpIII region of the M13KE pIII protein. 5Gly was located at the N-terminus of the M13 phage pVIII protein. The sequence accuracy was verified by sequencing to construct the H6G5-M13KE phage vector. Then, the genetically modified H6G5-M13KE phage vector was transformed into Escherichia coli XL1-Blue, and the H6G5-M13 phage particles that were completely consistent with the theoretical sequence were screened and sequenced by the double-layer plate method, and were screened and amplified in large quantities.

[0009] Furthermore, the polypeptide module is coupled to the engineered bacteriophage H6G5-M13, and the specific steps include: Under the action of transpeptidase A, the peptide module with the 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, thereby anchoring the peptide module on the surface of the engineered phage. Similarly, the generated connection product also has the LPXTG motif, which can be recognized by transpeptidase A and continue to be cut and connected, thereby realizing the renewal and replacement of the peptide module.

[0010] Furthermore, the engineered phage was directed to couple to Ni through Ni-6His. 2+ -IDA magnetic beads, the specific steps include: The minor capsid protein of engineered phage H6G5-M13 displays a 6His-tag, which is fixed on Ni 2+ -IDA magnetic beads provide specific sites. In a 500 μL reaction system (1×PBS, containing 3% BSA, pH 7.4), the anchoring products of the peptide modules on the surface of the engineered phages are bound to Ni 2+ -IDA-coated magnetic beads were incubated at room temperature for 3 h, the centrifuge tube was placed on a magnetic stand for magnetic separation, and the supernatant was removed to obtain universal renewable immunoaffinity magnetic beads.

[0011] Furthermore, the polypeptide module is composed of a polypeptide TBP (sequence shown in SEQ ID NO: 3) with a target sequence of lysosomal tetraspanin B (LAPTM4B) as part A, a specific cleavage sequence of cathepsin B as part B, and a polypeptide TBP with a LPETG sequence as part C. The anchor product TBP-H6G5-M13 of the polypeptide module on the surface of the engineered phage is bound to Ni 2+-IDA-coated magnetic beads were co-incubated at room temperature, magnetically separated, and the supernatant was removed to obtain the universal renewable immunoaffinity magnetic beads SortPhace-TBP.

[0012] The polypeptide module of the present invention, its part A can also be an Anti-EpCAM sequence targeting epithelial cell adhesion factor (EpCAM) protein as shown in SEQ ID NO: 4, an Anti-Ncadherin sequence targeting neural cadherin (N-cadherin) as shown in SEQ ID NO: 5, an Anti-CD63 sequence targeting extracellular vesicle marker protein (CD63) as shown in SEQ ID NO: 6, and a peptide nucleic acid sequence as shown in SEQ ID NO: 7. Part B of the polypeptide module can also be a sequence that enhances the water solubility of the polypeptide (preferably KKK), a thrombin-specific cleavage sequence (preferably LVPRGS) or a combination thereof as shown in SEQ ID NO: 8.

[0013] In the method of the present invention, the corresponding affinity polypeptide can be replaced according to application needs, and more universal and renewable immunoaffinity magnetic beads can be obtained, such as SortPhace-Anti-EpCAM targeting epithelial cell adhesion factor (EpCAM), SortPhace-Anti-Ncad targeting neural cadherin (N-cadherin), SortPhace-Anti-CD63 targeting extracellular vesicle marker protein (CD63), and SortPhace-PNA targeting nucleic acid.

[0014] Specifically, in the SortPhace-Anti-EpCAM, part A of the polypeptide module is as shown in SEQ ID NO: 4, part B is the specific cleavage sequence of cathepsin B as shown in SEQ ID NO: 2, part C is the LPETG sequence, and the polypeptide module sequence is shown in SEQ ID NO: 9.

[0015] In the SortPhace-Anti-Ncad, part A of the polypeptide module is shown in the sequence of SEQ ID NO: 5, part B is shown in the sequence of SEQ ID NO: 8, part C is the LPETG sequence, and the polypeptide module sequence is shown in SEQ ID NO: 10.

[0016] In the SortPhace-Anti-CD63, the sequence of part A of the polypeptide module is shown in SEQ ID NO: 6, the sequence of part C is LPETG, and the sequence of the polypeptide module is shown in SEQ ID NO: 11.

[0017] In the SortPhace-PNA, the sequence of part A of the polypeptide module is shown in SEQ ID NO: 7, the sequence of part C is LPETG, and the sequence of the polypeptide module is shown in SEQ ID NO: 12.

[0018] The present invention also provides the use of the universal renewable immunoaffinity magnetic beads in the preparation of immunoaffinity materials.

[0019] The present invention also provides the use of the universal renewable immunoaffinity magnetic beads in the capture, release and sensing detection of cells and extracellular vesicles.

[0020] Capture and release of cells: Add SortPhace magnetic bead dispersion to the sample to be tested, incubate at 37°C for 20-30 minutes, place the centrifuge tube on a magnetic stand for magnetic separation, and remove the supernatant. After separation, add a release agent (transpeptidase A 20μM, 3Gly peptide 100μM, transpeptidase A reaction buffer) to the magnetic bead precipitate, incubate at 37°C for 20-30 minutes, place the centrifuge tube on a magnetic stand for magnetic separation, and the released cells are present in the supernatant. Collect the supernatant, use a cell solid immunostaining kit (Cytointelligen, IFH-001, China) to immunostain the separated cells, and identify and count them under a fluorescence microscope.

[0021] Renewal of magnetic beads: After the captured target cells are selectively released from the SortPhace magnetic beads, they are digested with 0.25% EDTA-trypsin for 3-5 minutes and washed 3-5 times with PBS to remove other cells remaining on the surface. The targeted peptides can then be reconnected for a new round of target capture and release.

[0022] Cell sensing detection: The aptamer probe Anti-EpCAM Apt (3'FAM modified) and the competitive probe C-Apt (5'BHQ1 modified) with specific recognition effect on EpCAM were designed. The 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 and SortPhace-PNA were reacted at 45°C for 10-15 minutes according to the molar ratio of aptamer to PNA of 1:1.2, and then slowly cooled to room temperature to complete the coupling. In order to detect CTCs, cells with different concentrations were incubated with SortPhace-PNA-DNA at 37°C for 20-30 minutes. 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.

[0023] Isolation and labeling of extracellular vesicles (EVs): SortPhace-PNA-DNA was constructed and co-incubated with extracellular vesicles secreted by cells. The extracellular vesicles were captured and the fluorescence of Anti-EpCAM Apt on SortPhace-PNA-DNA was restored. The surface of the microspheres was photographed using a fluorescence microscope and the fluorescence intensity was quantitatively analyzed.

[0024] The key points of the present invention are: An engineered phage H6G5-M13 was constructed, in which the major capsid protein pVIII and the minor capsid protein pIII of the engineered phage displayed 5Gly and 6His peptides, respectively, providing more active sites for displaying polypeptide modules on the phage surface and immobilizing them on a solid phase carrier.

[0025] Through peptide ligation mediated by transpeptidase A, a preparation method and application of modular and universal renewable immunoaffinity magnetic beads (SortPhace magnetic beads) were developed. The multivalent display and reversible loading and unloading of functional peptide modules on SortPhace magnetic beads were achieved, thereby achieving efficient capture and gentle release of targets, and the renewal of SortPhace magnetic beads was achieved, which was convenient for multiple applications.

[0026] Beneficial effects of the present invention: 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 (5 copies), respectively, and has bioorthogonal functions. The present invention provides a universal tool by utilizing the specificity of transpeptidase A in recognizing polypeptide substrates and the bifunctional characteristics of the engineered phage H6G5-M13. Thanks to the simplicity of the transpeptidase 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); and the transpeptidase A-mediated reaction provides a method for non-destructively releasing biological targets and ensures the self-renewal ability of the SortPhace magnetic beads.

[0027] The SortPhace magnetic beads disclosed in the present invention have the characteristics of efficiently capturing and releasing CTCs for subsequent analysis and research; can be combined with fluorescent aptamer probes to achieve specific detection and quantitative analysis of CTCs; can separate and label tumor-derived extracellular vesicles (tEVs) for studying tumor microenvironment and liquid biopsy; the reusable magnetic bead system reduces experimental costs; and can be widely used in biomedical research and clinical diagnosis, especially in the field of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention shows the construction process and amino acid sequence of the engineered phage H6G5-M13; (a) is the gene map of the M13KE plasmid and a schematic diagram of the genetic engineering operation sites; (b) is the gene map of the H6G5-M13KE plasmid; and (c) is the DNA fragment of the key region in the H6G5-M13KE plasmid and the corresponding amino acid sequence.

[0029] Figure 2 The modification efficiency of the polypeptide TBP on the phage surface in Example 1 of the present invention is 36.1%.

[0030] Figure 3 The fixation efficiency of the bacteriophage on the magnetic bead surface in Example 1 of the present invention is >90%.

[0031] Figure 4 Schematic diagram and fluorescence image (scale bar = 25 μm) of different SortPhace magnetic beads used for separation of circulating tumor cells or extracellular vesicles in Example 1 of the present invention.

[0032] Figure 5The figure shows the comparison of the affinity (Kd value) of free TBP polypeptide, TBP-H6G5-M13 and SortPhace-TBP to circulating tumor cells in Example 1 of the present invention.

[0033] Figure 6 The performance of the SortPhace magnetic beads in Example 1 of the present invention in capturing and releasing circulating tumor cells, including their stability, efficiency and cell viability; wherein (a) SortPhace carrying different polypeptides can achieve the capture and release of various circulating tumor cells; (b) The cell viability of circulating tumor cells after separation by SortPhace magnetic beads; (c) The renewable stability of SortPhace magnetic beads.

[0034] Figure 7 Schematic diagram of the detection and analysis process of SortPhace magnetic beads for circulating tumor cells (CTCs) in the peripheral blood of colorectal cancer patients in Example 1 of the present invention.

[0035] Figure 8 This is the CTC count analysis in the peripheral blood of clinical patients and healthy volunteers in Example 1 of the present invention.

[0036] Fig. 9 This is an immunofluorescence staining image of CTC and white blood cells (WBC) in Example 1 of the present invention (scale bar = 10 μm).

[0037] Fig.10 This is a receiver operating characteristic curve (ROC) analysis of the SortPhace magnetic beads in Example 1 of the present invention for clinical diagnosis of colorectal cancer.

[0038] Fig.11 The results of gene mutation analysis of CTCs isolated from the peripheral blood of 11 patients in Example 1 of the present invention were compared with the consistency of clinical reports.

[0039] Fig.12 The figure shows the change in the number of CTCs before and after different treatments in Example 1 of the present invention.

[0040] Fig.13 Schematic diagram and fluorescence image of SortPhace-PNA used for sensing and detecting circulating tumor cells or extracellular vesicles in Example 2 of the present invention.

[0041] Fig.14 This is the standard curve of SortPhace-PNA used for MCF-7 cell sensing detection in Example 2 of the present invention. DETAILED DESCRIPTION

[0042] The present invention involves collecting venous blood samples from patients and volunteers for the isolation and analysis of circulating tumor cells (CTCs). All samples were signed with informed consent by patients and volunteers with full knowledge, agreeing that their blood samples would be used in this study. The research plan has passed the ethical review of the [Northeast University Biological and Medical Ethics Committee], with the approval number [NEU-EC-2024B035S] and the approval date [May 30, 2024]. During the research process, the relevant regulations on patient privacy protection and data anonymization processing were strictly observed, and the samples were only used for sample detection of the present invention and would not be used for any commercial purpose.

[0043] The raw materials and reagents used in the present invention are all commercially available products and can be obtained from conventional biochemical reagent suppliers.

[0044] Example 1 A preparation method and application of a universal renewable immunoaffinity magnetic bead SortPhace-TBP, the specific operation steps are as follows: 1. Preparation of genetically engineered phage: First, the wild-type M13KE phage vector (sequence shown in SEQ ID NO: 15) was subjected to site-directed mutagenesis by site-directed mutagenesis. Specifically, primers SDM1-F and SDM1-R (sequence shown in SEQ ID NO: 16 and SEQ ID NO: 17) were used to mutate base A at 1372 to T and base G at 1381 to C, and primers SDM2-F and SDM2-R (sequence shown in SEQ ID NO: 18 and SEQ ID NO: 19) were used to mutate base T at 6246 to A. Then, by homologous recombination, primers G5-F and G5-R (sequence shown in SEQ ID NO: 20 and SEQ ID NO: 21) were used to insert the exogenous gene fragment encoding 5Gly into the gpVIII region encoding pVIII of M13KE. Similarly, primers H6-F and H6-R (sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23) were used to insert the exogenous gene fragment encoding 6His into the gpIII region encoding pIII of M13KE. Finally, primers SDM3-F and SDM3-R (sequences shown in SEQ ID NO: 24 and SEQ ID NO: 25) were used to perform site-directed mutation on base 1371, from C to G, to ensure that 5Gly was located at the N-terminus of the M13 phage pVIII protein, and the sequence accuracy was verified by sequencing to construct the H6G5-M13KE phage vector. Then, the genetically modified H6G5-M13KE phage vector was transformed into Escherichia coli XL1-Blue, and the H6G5-M13 phage particles that were completely consistent with the theoretical sequence were screened and sequenced by the double-layer plate method, and were screened and amplified in large quantities. The construction process and amino acid sequence of the engineered phage H6G5-M13 are as follows: Figure 1 shown.

[0045] 2. Peptide ligation reaction mediated by transpeptidase A: Transpeptidase A first specifically recognizes and cuts the peptide bond between T and G in the C-terminal LPXTG motif of the polypeptide module to form a thioester intermediate, and then the 5Gly motif displayed at the N-terminus of the major capsid protein pVIII of the engineered phage H6G5-M13 attacks the thioester intermediate nucleophilically to anchor the polypeptide module on the surface of the engineered phage. Similarly, the generated ligation product also has the LPXTG motif, which can be recognized by transpeptidase A and continue to be cut and ligated, thereby realizing the renewal and replacement of the polypeptide module.

[0046] Specifically, in Example 1, a polypeptide TBP whose A part is a sequence targeting lysosomal four-transmembrane protein B (LAPTM4B) as shown in SEQ ID NO: 1, whose B part is a specific cleavage sequence of cathepsin B as shown in SEQ ID NO: 2, and whose C part is a LPETG sequence as a polypeptide module is designed, and whose sequence is shown in SEQ ID NO: 3. In 500 μL of transpeptidase A reaction buffer (Tris 50 mM, NaCl 150 mM, CaCl2 10 mM, pH 7.5), the engineered phage H6G5-M13 and the polypeptide TBP are put into the reaction at an optimized molar ratio (pVIII:TBP=1:10), and reacted at 37°C for 1 h under the action of transpeptidase A (20 μM). After the reaction, the excess components are removed by PEG / NaCl sedimentation or ultrafiltration centrifugation to obtain TBP-H6G5-M13.

[0047] In addition to the above sequences, the A part of the polypeptide module can also be Anti-EpCAM targeting epithelial cell adhesion factor (EpCAM) protein, the sequence of which is shown in SEQ ID NO: 4, Anti-Ncadherin targeting neural cadherin (N-cadherin), the sequence of which is shown in SEQ ID NO: 5, and Anti-CD63 targeting extracellular vesicle marker protein (CD63), the sequence of which is shown in SEQ ID NO: 6. The B part of the polypeptide module can also be a sequence that enhances the water solubility of the polypeptide (preferably KKK), a thrombin-specific cleavage sequence (preferably LVPRGS) or a combination thereof as shown in SEQ ID NO: 8.

[0048] 3. Construction of magnetic beads: The 6His-tag displayed by the minor capsid protein of the engineered phage H6G5-M13 was fixed on Ni 2+ -IDA magnetic beads provide specific sites. 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 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.

[0049] Through the reaction mediated by transpeptidase A, the peptide TBP with FITC fluorescent label is connected to the major capsid protein of the engineered phage H6G5-M13. The fluorescence intensity of the engineered phage H6G5-M13 at 520nm is recorded, and the peptide TBP content on the surface of the engineered phage H6G5-M13 can be calculated by inserting it into the standard curve, and then the modification efficiency of the peptide TBP can be obtained.

[0050] The engineered phage H6G5-M13 that successfully linked the peptide TBP to Ni 2+ -IDA-coated magnetic beads were incubated at room temperature for 3 hours, and the centrifuge tube was placed on a magnetic stand for magnetic separation, and the supernatant and magnetic beads were collected separately. The phages in the supernatant that were not fixed on the magnetic beads were quantified by plating and measuring the titer, and then the fixation efficiency of the phages on the magnetic bead surface was obtained.

[0051] In Example 1, the modification efficiency of the peptide TBP on the surface of the engineered phage H6G5-M13 was 36.1% through the reaction mediated by transpeptidase A, and the efficiency of phage fixation on the magnetic bead surface was >90% through the action of Ni-6His ( Figure 2-Figure 3 ).

[0052] According to application needs, more SortPhace magnetic beads can be obtained by replacing the corresponding affinity peptides, such as SortPhace-Anti-EpCAM targeting epithelial cell adhesion factor (EpCAM), SortPhace-Anti-CD63 targeting extracellular vesicle marker protein (CD63), and SortPhace-Anti-Ncad targeting neural cadherin (N-cadherin), which are used for cell sensing detection or separation and enrichment of circulating tumor cells (CTCs) or tumor extracellular vesicles (tEVs) with high expression of EpCAM or high expression of N-Cadherin. The preparation method is the same as SortPhace-TBP magnetic beads.

[0053] 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 as 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.

[0054] The present invention also provides applications of different SortPhace magnetic beads in cell capture, release and detection.

[0055] (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, use a circulating tumor cell solid-state immunostaining kit (Cytointelligen, IFH-001, China) to immunostain the separated cells, count the cells under a microscope, and calculate the cell capture efficiency using the following formula: .

[0056] After separation, add 1 mL of release agent (transpeptidase A 20 μM, 3Gly peptide 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 which point the released circulating tumor cells are present in the supernatant. Collect the supernatant, identify and count under a fluorescence microscope, and calculate the cell capture efficiency and release efficiency using the following formula: .

[0057] The released cells were subjected to live and dead cell staining experiments, and the cell activity was calculated using the following formula: .

[0058] like Figure 4 In the experiment, A549 cell line expressed Ncadherin and TBPR; MCF-7 cell line expressed EpCAM and TBPR; MDA-MB-231 cell line expressed Ncadherin and TBPR; uEV (exosomes from urine of healthy people) expressed CD63; 16 HBE cell line was used as the control group and did not express the above markers. SortPhace-TBP was able to capture cells expressing TBPR (A549, MCF-7, MDA-MB-231), but had no capture effect on 16 HBE cell line that did not express TBPR. SortPhace-Anti-EpCAM was able to effectively capture MCF-7 cell line that expressed EpCAM, but had no capture effect on other cell lines. SortPhace-Anti-Ncad was able to capture A549 and MDA-MB-231 cell lines that expressed Ncadherin, but had no capture effect on MCF-7 and 16HBE cell lines that did not express Ncadherin. SortPhace-Anti-CD63 was able to effectively capture uEVs expressing CD63, but had no capture effect on other cell lines.

[0059] The results of the fluorescence microscope observations are as follows: after capture using SortPhace magnetic beads, different colors of fluorescent markers were observed under a fluorescence microscope, indicating the capture effects of different cell lines. Figure 4 As shown, after A549 cells pre-stained with DAPI (blue under fluorescence microscopy) were captured using SortPhace-TBP (red under fluorescence microscopy), red and blue fluorescent labels were visible; similarly, after being captured using SortPhace-Anti-Ncad, red and blue fluorescent labels were visible. Figure 4 The specific capture effects of different SortPhace magnetic beads on different cell lines are demonstrated, verifying the high efficiency and specificity of these magnetic beads in cell separation and capture.

[0060] After releasing the cells, the SortPhace magnetic beads are digested with 0.25% EDTA-trypsin for 3-5 minutes and washed 3-5 times with PBS to remove other residual cells on the surface. The targeted peptides can then be reconnected for a new round of capture and release of the target.

[0061] (2) High efficiency and renewable stability of magnetic beads: Figure 5 As shown, according to the test results of the dynamic equilibrium assay (ELISA saturation concentration method), the affinity of SortPhace-TBP for target cells (Kd = 1.93pM) was increased by four orders of magnitude (4.54×10 4 ), which means that SortPhace-TBP can bind to target cells more efficiently. This significantly improved affinity has been verified in practical applications: Figure 6 As shown in (a), the capture efficiency of SortPhace-TBP for circulating tumor cells (CTC) reached 85.5%-91.6%, and the release efficiency reached 79.1%-85.6%. This shows that SortPhace-TBP not only has a significant improvement in affinity, but also shows high efficiency in the capture and release process of CTC, which is of great significance for the detection and analysis of CTC. In addition, Figure 6 Other types of SortPhace beads mentioned in (a) (such as SortPhace-Anti-EpCAM, SortPhace-Anti-Ncad) also showed high capture and release efficiency. Figure 6 As shown in (b), the viability of cells separated by SortPhace magnetic beads reached 96.7%, further demonstrating the high efficiency and low damage of this technology in cell separation and processing. Figure 6As shown in (c), the SortPhace-TBP in Example 1 was recycled 14 times and its performance remained stable, indicating that it can still maintain a high capture and release efficiency after multiple cycles.

[0062] (3) SortPhace-TBP is used to assist in clinical evaluation of the effectiveness of treatment options; 1 mL of venous blood sample was collected from the patient, and CTC was isolated by SortPhace-TBP within 24 hours, and CTC immunostaining identification and downstream analysis were completed. The study subjects were patients aged >18 years, pathologically diagnosed with colorectal cancer or benign gastrointestinal diseases, and met the inclusion criteria of this study. 81 clinical samples (53 cases of colorectal cancer, 18 cases of benign gastrointestinal polyps, and 10 healthy volunteers) were analyzed. The number of CTCs in 1 mL of venous blood was correlated with the occurrence and development of malignant tumors (see Figure 7-Figure 9 , significant difference *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%) ( Fig.10 ).

[0063] Figure 7 Demonstrated the use of SortPhace magnetic bead technology to capture and release circulating tumor cells (CTCs) from whole blood, which were further identified by immunofluorescence and gene mutation analysis.

[0064] Figure 8 The number of CTCs in the peripheral blood of clinical patients and healthy volunteers with different TNM stages (tumor staging system) is shown. It can be observed that the number of CTCs increases significantly with the increase of TNM stage (from stage I / II to stage IV). The number of CTCs in healthy / benign individuals is very low, which is significantly different from that in cancer patients. These results suggest that CTC counts can be used as an indicator of cancer progression, helping to assess the severity of the disease and monitor the effectiveness of treatment.

[0065] Fig. 9 The results of immunofluorescence staining of CTCs and white blood cells (WBCs) were shown, and different markers, DAPI, EpCAM, CK, and CD45, were used to distinguish CTCs from WBCs: DAPI was used to stain the cell nucleus and show all cells; EpCAM was an epithelial cell adhesion molecule, which was usually expressed on the surface of cancer cells; CK (cytokeratin) was a marker for epithelial cells, and CTCs usually expressed CK; CD45 was a marker for white blood cells, which was used to distinguish WBCs from CTCs. Fig. 9As can be seen in the figure, CTCs show positive expression of EpCAM and CK, but negative CD45, which is consistent with the characteristics of cancer cells. WBCs mainly show positive CD45, but negative EpCAM and CK, which is consistent with the characteristics of white blood cells. These results further verify the effectiveness of SortPhace technology in accurately isolating and identifying CTCs.

[0066] Fig.10 The receiver operating characteristic curve (ROC curve) of SortPhace magnetic beads in the clinical diagnosis of colorectal cancer was demonstrated. The ROC curve shows the performance of SortPhace magnetic beads in distinguishing colorectal cancer patients from non-patients, with AUC=0.981, close to 1, indicating that this method has high sensitivity and specificity. The isolated CTCs were recultured and gene amplified, and gene sequencing was used to analyze whether colorectal cancer-related genes (TP53, KRAS, APC) were mutated. The results showed that SortPhace-TBP separated CTCs from clinical samples and performed whole genome amplification, detecting common mutation sites in colorectal cancer (such as KRASG12C). Compared with clinical genetic diagnosis reports, the accuracy rate reached 93.94% (31 / 33) ( Fig.11 ), × indicates that it is inconsistent with the clinical genetic diagnosis.

[0067] Patients who received different treatments were followed up. Three patients received phase 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 ( Fig.12 ), proving the effectiveness of SortPhace-TBP in clinical auxiliary evaluation of treatment options.

[0068] Example 2 The application of universal and renewable immunoaffinity SortPhace-PNA in cell sensing detection is as follows: SortPhace-PNA was prepared by the method of preparing SortPhace-TBP in Example 1. First, an engineered phage H6G5-M13 was constructed, and then PNA (polypeptide module, the sequence of part A is shown in SEQ ID NO: 7, the sequence of part C is LPETG, and the sequence of the polypeptide module is shown in SEQ ID NO: 12) was coupled to the engineered phage H6G5-M13. Finally, the engineered phage was directionally coupled to Ni through the action of Ni-6His. 2+ -SortPhace-PNA was obtained on IDA magnetic beads.

[0069] like Fig.13As shown, an aptamer probe Anti-EpCAM Apt (3'FAM modified) and a competitive probe C-Apt (5'BHQ1 modified) with specific recognition effect on 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, the fluorescent aptamer probe Anti-EpCAM Apt was reacted with SortPhace-PNA at 45°C for 15 min according to the molar ratio of aptamer to PNA of 1:1.2, and then slowly cooled to room temperature to complete the coupling to obtain the SortPhace-PNA-DNA complex. In the complex, Anti-EpCAM Apt and C-Apt carry the fluorescent group FAM and the quenching group BHQ1, respectively. When the two are hybridized, the FAM group on Anti-EpCAM Apt is quenched by the BHQ1 group due to the close distance between the two groups; when the complex binds to the CTC in the system, C-Apt is competed off, and the fluorescence of the FAM group on Anti-EpCAM Apt is restored. Based on this principle, an off-on nucleic acid probe SortPhace-PNA was constructed in Example 2. In order 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 minutes. The C-Apt in the SortPhace-PNA-DNA complex was competed off by the 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 MCF-7 cells. Fig.14 As shown, the linear range of MCF-7 cell detection is 50-10000 cells / mL, and the calculated linear regression equation is: FF 0=1.048x-21.23, R 2 =0.9965, and the detection limit was 30 cells / mL (3σ / s, n=11). Fig.14 This provides strong evidence for the application of immunoaffinity SortPhace-PNA in cell sensing detection, demonstrates its advantages in sensitivity, accuracy, specificity and rapid response, and lays the foundation for future research and clinical applications.

[0070] Example 3 The specific method for the isolation and labeling of tEVs is as follows: SortPhace-PNA-DNA was constructed in the same manner as in Example 2 and co-incubated with tumor-derived extracellular vesicles (tEVs) secreted by MCF-7 cells. At this time, tEVs were captured, and the fluorescence of Anti-EpCAM Apt on SortPhace-PNA was restored. The surface of the microspheres was photographed by fluorescence microscopy, and the fluorescence intensity was quantitatively analyzed. By capturing and labeling tEVs with the SortPhace-PNA-DNA complex, the role of tEVs in tumor development, metastasis, and treatment resistance can be further studied, which not only helps to improve the sensitivity and specificity of tEVs detection, but also provides new possibilities for future research and clinical applications.

[0071] Table 1 Primers and sequences used in the present invention:

[0072] Those skilled in the art should understand that the above embodiments are only intended to help understand the present invention and should not be regarded as specific limitations of the present invention.

Claims

1. A method for preparing universal renewable immunoaffinity magnetic beads, characterized in that: The following steps are involved: First, an engineered phage H6G5-M13 was constructed, and then a polypeptide module was coupled to the engineered phage H6G5-M13. The polypeptide module comprised three parts, A, B, and C, from the N-terminus to the C-terminus. Part A was a target recognition sequence located at the N-terminus, part B was a selectively added linker sequence, and part C was a LPXTG substrate sequence located at the C-terminus, wherein X was any amino acid. Finally, the engineered phage was coupled to Ni through Ni-6His. 2+ -IDA beads.

2. The method for preparing a universal renewable immunoaffinity magnetic bead according to claim 1, characterized in that: The engineered phage H6G5-M13 was constructed. The specific steps included: performing site-directed mutagenesis on a wild-type M13KE phage vector by a site-directed mutagenesis method, wherein the mutation sites included bases 1372, 1381 and 6246, and then inserting an exogenous gene fragment encoding 5Gly into the gpVIII region of the M13KE pVIII protein encoding protein, and inserting an exogenous gene fragment encoding 6His into the gpIII region of the M13KE pIII protein encoding protein, wherein 5Gly was located at the N-terminus of the M13 phage pVIII protein, and the sequence accuracy was verified by sequencing to construct an H6G5-M13KE phage vector, and then the gene-modified H6G5-M13KE phage vector was transformed into Escherichia coli XL1-Blue.

3. The method for preparing a universal renewable immunoaffinity magnetic bead according to claim 1, characterized in that: The polypeptide module is coupled to the engineered phage H6G5-M13, and the specific steps include: under the action of transpeptidase A, the polypeptide module with the 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.

4. The method for preparing a universal renewable immunoaffinity magnetic bead according to claim 1, characterized in that: Engineered phages are directed to couple to Ni via Ni-6His 2+ -IDA magnetic beads, the specific steps include: in the reaction system: 1×PBS, containing 3% BSA, pH7.4, the anchor product of the peptide module on the surface of the engineered phage is combined with Ni 2+ -IDA-coated magnetic beads were co-incubated at room temperature, followed by magnetic separation, and the supernatant was removed to obtain universal renewable immunoaffinity magnetic beads.

5. The method for preparing a universal renewable immunoaffinity magnetic bead according to claim 1, characterized in that: Part A of the polypeptide module includes: a sequence targeting lysosomal tetraspanin B as shown in SEQ ID NO: 1, an Anti-EpCAM sequence targeting epithelial cell adhesion factor protein as shown in SEQ ID NO: 4, an Anti-Ncadherin sequence targeting neural cadherin as shown in SEQ ID NO: 5, an Anti-CD63 sequence targeting extracellular vesicle marker protein as shown in SEQ ID NO: 6, and a peptide nucleic acid sequence as shown in SEQ ID NO: 7; Part B of the polypeptide module includes: a specific cleavage sequence of cathepsin B as shown in SEQ ID NO: 2, a sequence that enhances the water solubility of the polypeptide, a thrombin-specific cleavage sequence, or a combination thereof as shown in SEQ ID NO:

8.

6. The method for preparing a universal renewable immunoaffinity magnetic bead according to claim 5, characterized in that: The universal renewable immunoaffinity magnetic beads are: SortPhace-TBP targeting lysosomal tetraspanin B, SortPhace-Anti-EpCAM targeting epithelial cell adhesion factors, SortPhace-Anti-Ncad targeting neural cadherin, SortPhace-Anti-CD63 targeting extracellular vesicle marker proteins, and SortPhace-PNA targeting nucleic acids; In SortPhace-TBP, part A of the polypeptide module is a sequence targeting lysosomal tetraspanin B as shown in SEQ ID NO: 1, part B is a specific cleavage sequence of cathepsin B as shown in SEQ ID NO: 2, part C is a LPETG sequence, and the polypeptide module sequence is shown in SEQ ID NO: 3; 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 as 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 sequence of the polypeptide module 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 sequence of the polypeptide module is shown in SEQ ID NO: 11; In SortPhace-PNA, the sequence of part A of the polypeptide module is shown in SEQ ID NO: 7, the sequence of part C is LPETG, and the sequence of the polypeptide module is shown in SEQ ID NO:

12.

7. Use of a universal renewable immunoaffinity magnetic bead prepared by the method for preparing a universal renewable immunoaffinity magnetic bead according to any one of claims 1 to 6 in preparing an immunoaffinity material.

8. Use of a universal renewable immunoaffinity magnetic bead prepared by the method for preparing a universal renewable immunoaffinity magnetic bead according to any one of claims 1 to 6 in the capture, release and sensing detection of cells and extracellular vesicles.

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

10. The use of a universal renewable immunoaffinity magnetic bead according to claim 8 in the capture, release and sensing detection of cells and extracellular vesicles, characterized in that: The cell sensing detection process is as follows: A fluorescent aptamer probe Anti-EpCAM Apt and a competitive probe C-Apt with specific recognition effects on EpCAM are 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 minutes, the fluorescent aptamer probe Anti-EpCAM Apt is reacted with the SortPhace-PNA described in claim 6 at a molar ratio of aptamer to PNA of 1:1.2 at 45°C for 10 minutes to 15 minutes, and then slowly cooled to room temperature to complete the coupling to obtain SortPhace-PNA-DNA; cells of different concentrations are incubated with SortPhace-PNA-DNA at 37°C, C-Apt in SortPhace-PNA-DNA is competed off by EpCAM on the cell surface, and the fluorescence of Anti-EpCAM Apt is restored. The restored fluorescence intensity is measured at Ex / Em = 484nm / 530nm, and a standard curve is drawn for quantitative analysis of cells.

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