Phage multifunctional affinity interface as well as preparation method and application thereof

Abstract

The invention discloses a phage multifunctional affinity interface as well as a preparation method and application thereof, and belongs to the field of functional biological materials. The preparation method of the phage multifunctional affinity interface comprises the following steps: modifying a phenylboronic acid group by using pVIII protein on the side wall of an M13 phage as a bracket; connecting histidine on the protein of the M13 bacteriophage pIII to the nickel magnetic microbead; the method comprises the following steps: carrying out fluorescence labeling on a biotinylated aptamer targeting EpCAM positive cells, hybridizing with a complementary chain of a labeled fluorescence quencher, and coupling with avidin according to a molar ratio of 4: 1 to obtain an avidin-biotinylated DNA switch; and then the M13 bacteriophage-nickel magnetic microbead containing the phenylboronic acid group is coupled with an avidin-biotinylated DNA (Deoxyribose Nucleic Acid) switch. The capture efficiency of the multifunctional affinity interface to MCF-7 cells reaches 94.2%, the release efficiency reaches 92.7%, and the multifunctional affinity interface has reproducibility and can be used for multiple rounds of cell separation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of functionalized biomaterials, and particularly relates to a phage multifunctional affinity interface as well as a preparation method and application thereof. BACKGROUND

[0002] Circulating tumor cell (CTC) is a malignant tumor cell that falls into the peripheral blood from a primary or metastatic solid tumor. As a key target of liquid biopsy, CTC has important clinical value in early diagnosis of tumor metastasis, prognosis evaluation and efficacy monitoring. However, CTC is extremely rare in peripheral blood, only a few CTCs (usually > 5) exist in 7.5 mL of blood, and the background is up to billions of normal blood cells (such as white blood cells and red blood cells). This extremely low abundance and high background noise constitute the core technical bottleneck of CTC enrichment.

[0003] Enriched CTCs usually need to be verified by standard immunofluorescence staining (immunocytochemistry, ICC). This scheme uses anti-keratin (CK) antibodies to label the epithelial properties of CTCs, anti-CD45 antibodies to exclude white blood cells, and relies on DAPI nuclear staining to confirm cell integrity. However, this process requires cell fixation for fluorescence microscopic observation, resulting in loss of cell activity, making it unsuitable for downstream applications such as genomic analysis, in vitro culture, and drug sensitivity testing. To overcome the above limitations, it is urgent to develop an efficient CTC separation and identification strategy that can achieve high capture efficiency and complete CTC recognition without the need for traditional immunostaining.

[0004] Currently, the separation and enrichment of CTCs are mainly based on two methods: one is based on the difference in physical properties: this method uses the difference in physical properties between CTCs and a large number of blood cells (mainly white blood cells and red blood cells) for separation, including but not limited to size, cell deformability, density, and surface charge. Typical physical separation techniques include microfiltration membrane filtration, density gradient centrifugation, dielectrophoresis, etc. The second is based on immunophilic enrichment: this method relies on tumor-associated antigens or biomarkers specifically expressed on the surface of CTCs, such as epithelial cell adhesion molecules (EpCAM), cytokeratin (CK), etc., to achieve targeted capture of CTCs through molecular probes (such as antibodies, peptides, nucleic acid aptamers, etc.) that specifically bind to them. Common platforms include immunomagnetic bead sorting (such as the CellSearch® system), microfluidic chips (such as EpCAM antibody-based chips), etc. However, the above existing technical solutions have significant defects and limitations in the application of efficient and high-purity enrichment of CTCs, especially in meeting the subsequent in vitro culture and functional analysis requirements: (1) Defects of the method based on physical properties: Lack of specificity and low purity: CTCs have significant heterogeneity in physical properties (e.g. size) and overlap with some blood cells (e.g. large lymphocytes, monocytes, activated leukocytes or megakaryocyte fragments). This leads to high background noise in physical sorting methods, with a large number of non-target blood cells mixed in the captured product, making it difficult to meet the purity requirements of high-sensitivity downstream analysis (e.g. single-cell sequencing) or in vitro culture.

[0005] High risk of cell activity damage: Some physical separation processes (e.g. high-pressure filtration, high centrifugal force field, high electric field strength dielectrophoresis) can exert excessive mechanical stress or shear force on fragile CTCs, damaging cell activity and integrity, which is fatal for applications aimed at in vitro culture amplification and drug sensitivity testing of living cells.

[0006] Incomplete coverage of heterogeneous CTC population: CTCs with stronger invasive and metastatic potential (e.g. CTCs undergoing epithelial-mesenchymal transition EMT) tend to be smaller in size and more deformed, easily lost in size-based filtration, resulting in a captured CTC population that does not fully represent the heterogeneity of the tumor.

[0007] (2) Defects of immunophilic method-based methods: Tumor heterogeneity and loss of markers: CTCs have high heterogeneity in antigen expression, and can undergo dynamic changes during primary and metastasis (e.g. EMT leading to down-regulation or even loss of epithelial markers such as EpCAM / CK). Relying solely on a single or limited number of surface markers for capture will inevitably result in missed detection of specific subpopulations (especially CTCs with low or no marker expression), failing to fully reflect the true situation of CTCs.

[0008] Non-specific adsorption: Antibodies and other molecular probes can non-specifically bind to non-target cells, also leading to a decrease in purity and an increase in background.

[0009] Complex pretreatment requirements: Red blood cells often need to be pre-lysed or certain blood cell components need to be removed, increasing the number of operation steps and the risk of sample loss.

[0010] Therefore, the development of a new separation and enrichment technology for CTCs that can overcome the above-mentioned defects, achieve high capture efficiency, high purity and high activity, and effectively compatible with subsequent in vitro culture and functional analysis, is of urgent need and great significance for fully exploiting the potential of CTCs in precise diagnosis and treatment of cancer (especially early diagnosis and individualized treatment). SUMMARY

[0011] In view of the problems in the prior art, the present application aims to provide a bacteriophage multifunctional affinity interface and a preparation method thereof, since EpCAM exists in the form of aggregates rather than monomers on the surface of CTC, local multivalent interaction is more effective in capturing EpCAM aggregates on the surface of CTC, the present application connects magnetic materials to M13 bacteriophage nanotang, enhances the flexibility of the interface, and then binds multivalent EpCAM aptamers to the phenylboronic acid groups modified on the side wall of the M13 bacteriophage, so as to construct a multifunctional affinity interface based on bacteriophage, realize the enhancement of the flexibility of the interface and the local multivalent interaction, and be applied to the specific enrichment of EpCAM-positive cells for subsequent analysis, thereby improving the affinity for target CTC and enhancing the capture effect of CTC, realizing the efficient capture of target CTC; based on DNA hybridization technology, specific targeting of EpCAM-positive cells is realized, and fast and accurate labeling and identification are realized; CTC with complete phenotypes are released mildly, so as to be used for downstream functional analysis. The released bacteriophage multifunctional affinity interface is renewable, and through reloading of aptamers, multiple rounds of cell separation and enrichment can be realized, and high-sensitivity and specific detection of blood samples of cancer patients is realized, which is highly consistent with the detection by a standard immunofluorescence method.

[0012] The object of the present application is realized by the following technical solutions. The present application provides a preparation method of a bacteriophage multifunctional affinity interface, comprising the following steps: (1) taking M13 bacteriophage as a scaffold, and modifying phenylboronic acid groups on 2700 pⅧ proteins on the side wall of the M13 bacteriophage; (2) connecting histidine on five pⅢ proteins of the M13 bacteriophage modified with phenylboronic acid groups to nickel magnetic microbeads; (3) fluorescently labeling biotinylated aptamers targeting EpCAM-positive cells, hybridizing the fluorescently labeled biotinylated aptamers with a complementary strand labeled with a fluorescence quencher to obtain a biotinylated DNA switch, and coupling the biotinylated DNA switch with avidin according to a molar ratio of 4:1 to obtain an avidin-biotinylated DNA switch; (4) coupling the M13 bacteriophage-nickel magnetic microbeads containing phenylboronic acid groups prepared in step (2) with the avidin-biotinylated DNA switch obtained in step (3) to obtain a bacteriophage multifunctional affinity interface, hereinafter referred to as NataFace.

[0013] The present application provides a bacteriophage multifunctional affinity interface, which is prepared by the preparation method of the bacteriophage multifunctional affinity interface.

[0014] The application also provides the use of the phage multifunctional affinity interface for the separation and enrichment of EpCAM positive cells, efficient capture of EpCAM positive cells is realized through a large number of loaded EpCAM aptamers; the labeling and identification of the captured EpCAM positive cells are realized through complementary strand hybridization of the EpCAM aptamers; the addition of acidic fructose enables the mild release of the EpCAM positive cells with complete phenotypes, which is suitable for subsequent in vitro culture and functional analysis, and the released NataFace is renewable, so that multiple rounds of cell separation can be realized.

[0015] Further, in the use of the phage multifunctional affinity interface, the targeted EpCAM positive cells are MCF-7 cells. The biotinylated aptamer of the fluorescently labeled MCF-7 cells is 5'-biotin-CAC TAC AGA GGT TGCGTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3', hereinafter referred to as Bio-Apt-FAM, and the nucleotide sequence is shown as SEQ ID NO. 1; The complementary strand labeled with a fluorescence quencher is 5'-BHQ-CAGGCCAACCCCCCATGACAACGTGG-3', hereinafter referred to as C-Apt-BHQ, and the nucleotide sequence is shown as SEQ ID NO. 2.

[0016] Further, the phenylboronic acid group on the side wall of the M13 phage is modified by a chemical modification method, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are used as coupling agents, the carboxyl group of 4-carboxyphenylboronic acid (4-CPBA) is combined with the amino group of pⅧ protein on the side wall of the M13 phage to form a stable amide bond, and the phenylboronic acid group is modified on the M13 phage, and the specific steps are as follows: (1) EDC is dissolved in MES buffer solution as solution A, the concentration of the MES buffer solution is 0.1 M, and the pH value is 6.0-6.5; 4-CPBA and NHS are dissolved in dimethyl sulfoxide (DMSO) as solution B; then solution A is added to solution B in portions, and oscillation incubation is performed to obtain activated 4-CPBA; (2) M13 phage in PBS buffer solution is taken, and the concentration of the M13 phage is 1.0×10 11 -1.0×10 13pfu / mL, the PBS buffer concentration is 10 mM-100 mM, the pH is 6-8, the activated 4-CPBA solution is added to the stirring reaction, after the reaction, centrifugal filtration is performed to remove excess reactants, and the M13 bacteriophage with modified phenylboronic acid groups, hereinafter referred to as CPBA-M13, is obtained, which is collected and refrigerated for standby use, and the CPBA-M13 concentration is 1.0*10 10 -1.0*10 12 pfu / mL.

[0017] Further, the molar ratio of CPBA, EDC and NHS is 1:(1-3):(1.5-5); The volume ratio of solution A to solution B is 1:1, solution A is added to solution B in three times, the oscillation incubation temperature is 30-40℃, and the time is 25-30 min; The volume ratio of the activated 4-CPBA solution to the M13 in the phosphate buffer is 1:(0.5-1.5); The stirring reaction temperature is 0-10℃, the rotation speed is 100-200 rpm, and the time is 16-20 h; The filtration uses an ultrafiltration centrifuge tube, the centrifugal treatment temperature is 0-10℃, the rotation speed is 7000-8000 rpm, and the time is 0.5-1 h, and the centrifugal treatment is performed three times; The refrigeration temperature is 0-10℃.

[0018] Further, the His-Tag protein purified Ni-IDA magnetic bead suspension is washed with PBS buffer three times, the PBS buffer concentration is 5-20 mM, the pH is 6-8, then the CPBA-M13 is added, the volume ratio of the Ni-IDA magnetic bead suspension to the CPBA-M13 is 3:(1-5), and the incubation is performed at room temperature to obtain the CPBA-M13 connected Ni-IDA magnetic beads, hereinafter referred to as CPBA-M13@MBs, the CPBA-M13@MBs is washed with PBS to remove unbound CPBA-M13, and the CPBA-M13@MBs is resuspended in PBS buffer and stored at 0-10℃ for standby use.

[0019] Further, the Bio-Apt-FAM and its complementary strand C-Apt-BHQ are annealed and hybridized at 95-105℃, the molar ratio of the Bio-Apt-FAM to the complementary strand C-Apt-BHQ is 1:(0.8-1.2), a stable structure of the biotinylated DNA switch is obtained, the biotinylated DNA switch and the avidin are mixed and incubated in a TBS solution at a molar ratio of 4:1, the TBS solution pH value is 7.5-8.5, then the CPBA-M13@MBs is added for incubation, and finally the PBS buffer is used for washing and resuspension to obtain the NataFace.

[0020] Further, the annealing time is 6min-10min, the incubation temperature of the biotinylated DNA switch and avidin is 30℃-40℃, and the incubation time is 1.5h-2.5h; the incubation in the CPBA-M13@MBs is carried out at a temperature of 30℃-40℃ for 1h-4h.

[0021] Further, the CTC-containing body fluid sample is incubated in the NataFace dispersion, the CTC is captured, the mixed liquid after the CTC capture is subjected to magnetic separation, and the supernatant after the magnetic separation is removed; in the capturing process, the EpCAM aptamer on the NataFace is combined with the EpCAM protein on the surface of the CTC, and then the complementary strand with the fluorescence quencher pre-hybridized on the biotinylated DNA switch is competed down, so that the CTC is labeled; the captured NataFace is incubated in the PBS buffer containing acidic fructose, so that the CTC with an intact phenotype is released from the surface of the NataFace, and then subjected to magnetic separation, so that the enriched CTC is obtained from the supernatant.

[0022] Advantages and effects of the present application: 1. The phage multifunctional affinity interface of the present application realizes the enhancement of interface flexibility and local multivalent interaction by combining the M13 phage nanotactile with the borate affinity technology, improves the affinity to the target CTC, realizes the efficient capture of the CTC, and the capture efficiency of the MCF-7 cells is more than 84.5%.

[0023] 2. After the CTC is captured, the biotinylated DNA switch is used for selectively "luminescent" labeling of the CTC, and the precise identification can be carried out without complicated and destructive immunostaining, and the labeling efficiency of the MCF-7 cells is 80%, which reflects the applicability to subsequent analysis.

[0024] 3. The chemical reversibility of the borate is used for the mild release of the captured CTC, which lays a foundation for the downstream analysis, the release efficiency is more than 91.8%, the activity of the released cells is 98.6%, and the phage multifunctional affinity interface can be regenerated efficiently, is used for loading the aptamer of other biomarkers, can realize multiple rounds of cell separation, and has the potential of a universal cell separation platform. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 a flowchart for preparing Example 1; Figure 2The following are fluorescence microscopy images used to verify cell recognition capability in Example 2, wherein: (a) is a DAPI channel image; (b) is a DiI channel image; (c) is a NataFace channel image; and (d) is a multi-channel overlay image. Figure 3 This is a cell viability graph used in Example 2 to verify the activity of released cells; Figure 4 This is a capture efficiency graph from Example 2, showing the regenerative test of the released NataFace. Figure 5 This is a comparison chart of the number of CTCs captured from 1 ml blood samples of cancer patients and healthy individuals in Example 3; Figure 6 This is a fluorescence microscopy image of CTCs isolated from the blood of a cancer patient after staining using standard immunocytochemistry in Example 3. Figure 7 This is a fluorescence microscopy image of CTCs isolated from the blood of the same cancer patient using the NataFace illumination strategy in Example 3. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the embodiments.

[0027] Example 1 The present invention provides a method for preparing a multifunctional affinity interface for bacteriophages, such as... Figure 1 As shown, the targeted EpCAM-positive cells are MCF-7 cells, and the specific steps include: (1) Using M13 phage as a scaffold, CPBA-M13 was synthesized by modifying phenylboronic acid groups with 2700 pVIII proteins on the sidewall of M13 phage. First, 63.26 mg EDC was dissolved in 0.1 M MES buffer (2.5 mL, pH 6.0) as solution A; 49.80 mg 4-CPBA and 86.30 mg NHS were dissolved in DMSO as solution B (2.5 mL). Then, solution A was added to solution B in three portions, with a molar ratio of CPBA, EDC, and NHS of 1:1:1.5, and the mixture was incubated at 37°C with shaking for 30 min. Next, 500 μL of activated 4-CPBA solution was added to 500 μL of PBS buffer containing M13 phage (10 mM, pH 7.4, M13 phage concentration 1.0 × 10⁻⁶). 12 The reaction was carried out at 4℃ and 200 rpm for 18 h. After the reaction was completed, excess reactants were removed by ultrafiltration centrifuge tubes (100 kDa). The mixture was then centrifuged at 4℃ and 7300 rpm for 45 min. After three centrifugations, the obtained CPBA-M13 (1.0 × 10⁻⁶ pfu / mL) was collected.11 pfu / mL) were collected and stored in a 4 °C refrigerator for later use.

[0028] (2) The histidine on the five pIII proteins of the M13 bacteriophage modified with the phenylboronic acid group was connected to the nickel magnetic microbeads; first, 300 μL of His-Tag protein purified Ni-IDA magnetic bead suspension was washed three times with PBS buffer (10 mM pH 7.4), then 200 μL of CPBA-M13 was added and incubated at room temperature for 3 h to obtain CPBA-M13@MBs, which was then washed three times with PBS to remove unbound CPBA-M13, and finally resuspended in PBS buffer to obtain CPBA-M13@MBs, which was stored at 4 °C for later use.

[0029] (3) The biotinylated aptamer of MCF-7 cells was fluorescently labeled to obtain Bio-Apt-FAM: 5'-biotin-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3', which was hybridized with the complementary strand C-apt-BHQ labeled with a fluorescence quencher: 5'-BHQ-CAGGCCAACCCCCCATGACAACGTGG-3' at 95 °C for 6 min, the molar ratio of Bio-Apt-FAM to its complementary strand C-Apt-BHQ was 1:1, and then slowly cooled to room temperature to obtain a biotinylated DNA switch with a stable structure. The biotinylated DNA switch was mixed with avidin in TBS (10 mM, pH 8.5) solution at a molar ratio of 4:1 and incubated at 37 °C for 1 h to obtain an avidin-biotinylated DNA switch.

[0030] (4) The CPBA-M13@MBs prepared in step (2) were added to the avidin-biotinylated DNA switch obtained in step (3) and incubated for 2 h, washed with PBS buffer (10 mM pH 7.4) and resuspended to obtain NataFace.

[0031] Example 2 The NataFace prepared in Example 1 was used for the capture, labeling and release of MCF-7 cells, and the MCF-7 cells were counted under a microscope by a hemocytometer; 500 μL of cell suspension was obtained by diluting the cells, 500 μL of NataFace was added and mixed evenly, and the reaction was carried out at 37 °C for 40 min on a shaking bed; then the product was magnetically separated, the supernatant was collected and counted, and the efficiency of the captured cells was calculated by the following formula:

[0032]

[0033] The capture efficiency of different amounts of MCF-7 cells in whole blood samples was calculated, and the number of MCF-7 cells in each mL of whole blood sample was 1000, 100 and 50, and the capture efficiency was 84.5%, 92.7 and 94.2%, respectively.

[0034] During the capture process, the EpCAM aptamer on the NataFace binds to the EpCAM protein on the surface of the MCF-7 cells, and then competes with the complementary strand C-Apt-BHQ with a fluorescence quencher pre-hybridized on the biotinylated DNA switch, the fluorescence of the FAM group on the NataFace is restored, and the precise labeling of the MCF-7 cells is achieved.

[0035]

[0036] The number of MCF-7 cells introduced into the whole blood sample was 1000, and the labeling efficiency was 80.5%.

[0037] After cell capture, the cells were released by adding PBS buffer containing 60 mM fructose (0.2 M, pH 6.7) and incubating at 37°C, 150 rpm for 30 min; the released cells were separated from the NataFace, and the NataFace was washed three times with PBS buffer at pH 6.7, the supernatant was collected and counted under a microscope.

[0038]

[0039] The release efficiency of the cells was calculated, and the number of cells in each mL of whole blood sample was 1000, 100 and 50, and the release efficiency was 91.8%, 92.0% and 92.7%, respectively.

[0040] The recognition ability of the NataFace prepared in Example 1 for MCF-7 cells was verified, as shown in Figure 2 The whole blood sample was pre-stained with DAPI, and the MCF-7 cells were pre-stained with DiI to distinguish them, 1x10 3 The pre-stained MCF-7 cells were mixed with the pre-stained whole blood, and the NataFace was co-incubated at 37°C for 40 min to capture the CTC, after capturing the CTC, the NataFace was collected and imaged under a fluorescence microscope, and it was observed that the NataFace captured and recognized the white blood cells (WBCs) and MCF-7 cells from the whole blood sample, and 80.5% of the MCF-7 cells were lit up, which indicated that the NataFace could accurately recognize the CTC cells without the need for tedious and destructive immunostaining.

[0041] The captured NataFace was incubated with acidic fructose PBS buffer to release the CTCs with intact phenotype from the NataFace surface, and then the enriched CTCs were obtained from the supernatant by magnetic separation. The activity of the released cells and the regeneration performance of the NataFace prepared in Example 1 were verified. The released MCF-7 cells were stained with Annexin V-FITC, and the same number of cells before capture were used as a positive control, and alcohol-treated dead cells were used as a negative control. The cells were detected by flow cytometry (FITC channel), as shown in Figure 3 The activity of the MCF-7 cells treated with the NataFace prepared in Example 1 was 98.6%, the activity of the positive control was 99.5%, and there were no surviving cells in the negative control. This indicates that the MCF-7 cells captured and released by the NataFace can maintain very good activity. The released NataFace was tested for regeneration. The released NataFace was reconnected to the avidin-biotinylated DNA switch, captured MCF-7 cells, and then released again, as shown in Figure 4 After 12 cycles of capture-release-regeneration, the average capture efficiency of the NataFace for MCF-7 cells was still more than 80%, which indicates that the interface has good regenerability.

[0042] Example 3 The NataFace prepared in Example 1 was used to separate and enrich CTCs in clinical samples. Whole blood samples from 19 volunteers were analyzed, including 16 cancer patients and 3 healthy volunteers.

[0043] 500 μL of NataFace was mixed with 500 μL of blood samples from volunteers, and the mixture was reacted at 37°C for 40 min on a shaker. Then the product was magnetically separated. The captured NataFace was incubated with acidic fructose PBS buffer to release the CTCs from the NataFace surface, and then the enriched CTCs were obtained from the supernatant by magnetic separation and counted. The labeling effect of the CTCs was verified by observing under a fluorescence microscope. The released cells were fixed on a slide with 4% paraformaldehyde and blocked with blocking solution, then stained with DAPI, anti-CD45, and CK and EpCAM proteins as markers of CTCs. The captured CTCs were stained with antibodies, and observed under a fluorescence microscope. When DAPI+ / CD45+ / CK- was identified as a white blood cell, and DAPI+ / CD45- / CK+ was identified as a CTC.

[0044] As shown in Figure 5As shown, the middle horizontal line represents the median of the number of CTCs, unpaired t-test: **p<0.01, 3 to 24 CTCs per 1 ml blood were found in the blood of cancer patients, while no CTCs were found in the blood of healthy blood donors, indicating that the interface has good diagnostic function for cancer patients. As shown in Figure 6 As shown, the captured cells were identified by standard immunocytochemistry (ICC), in which DAPI nuclear staining was used to confirm cell integrity, CD45-positive cells were leukocytes (WBCs), and CK and EpCAM-positive cells were CTC cells. Figure 7 As shown, the NataFace prepared in Example 1 was used to light up the strategy for CTC identification, in which leukocytes could not bind to the DNA switch on the NataFace and thus could not be lighted up; the EpCAM protein on the surface of CTCs could bind to the EpCAM aptamer on the NataFace, thereby competing down the complementary strand with a fluorescence quencher pre-hybridized on the biotinylated DNA switch, restoring the fluorescence of FAM, emitting green fluorescence, and achieving the labeling of CTCs. The CTC identification results of the NataFace prepared in Example 1 were consistent with the results of standard immunocytochemistry (ICC), indicating that the NataFace of the application has good clinical applicability.

Claims

1. A method for the production of a phage multifunctional affinity interface, characterized in that, Comprising the following steps: (1) Using M13 phage as a scaffold, 2700 pVIII protein on the side wall of M13 phage is used to modify phenylboronic acid group; (2) Using the five pIII proteins of the M13 phage modified with phenylboronic acid group to connect to nickel magnetic microbeads; (3) Biotinylated aptamer targeting EpCAM positive cells is fluorescently labeled, and then hybridized with complementary strands labeled with fluorescent quencher to obtain a biotinylated DNA switch, which is coupled with avidin according to a molar ratio of 4:1 to obtain an avidin-biotinylated DNA switch; (4) The M13 phage-nickel magnetic microbead containing the phenylboronic acid group prepared in step (2) is coupled with the avidin-biotinylated DNA switch obtained in step (3) to obtain a phage multifunctional affinity interface, hereinafter referred to as NataFace.

2. A phage multifunctional affinity interface, characterized in that, The preparation method of the phage multifunctional affinity interface of claim 1 is used for preparation.

3. Use of a bacteriophage multifunctional affinity interface according to claim 2, characterized in that, For the separation and enrichment of EpCAM positive cells, a large number of loaded EpCAM aptamers are used to achieve efficient capture of EpCAM positive cells; by hybridizing complementary strands to the EpCAM aptamer, labeling and identifying the captured EpCAM positive cells are achieved; the addition of acidic fructose allows the gentle release of the complete phenotype of the EpCAM positive cells, which is suitable for subsequent in vitro culture and functional analysis, and the released NataFace is renewable, realizing multiple rounds of cell separation.

4. Use of a phage multifunctional affinity interface according to claim 3, wherein the phage multifunctional affinity interface is a phage multifunctional affinity interface according to any one of claims 1 to 3. The targeted EpCAM positive cells are MCF-7 cells; The biotinylated aptamer of the fluorescently labeled MCF-7 cells is 5'-biotin-CAC TAC AGA GGT TGC GTCTGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3', hereinafter referred to as Bio-Apt-FAM, and its nucleotide sequence is shown as SEQ ID NO. 1; The complementary strand labeled with a fluorescent quencher is 5'-BHQ-CAGGCCAACCCCCCATGACAACGTGG-3', hereinafter referred to as C-Apt-BHQ, and its nucleotide sequence is shown as SEQ ID NO.

2.

5. Use of a phage multifunctional affinity interface according to claim 4, wherein the phage multifunctional affinity interface is a phage multifunctional affinity interface according to any one of claims 1 to 3. The chemical modification method is used to modify the phenylboronic acid group on the side wall of the M13 phage, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are used as coupling agents, the carboxyl group of 4-carboxyphenylboronic acid is made into a stable amide bond with the amino group of the pVIII protein on the side wall of the M13 phage, and the phenylboronic acid group is modified to the M13 phage, and the specific steps are as follows: (1) Dissolve EDC in MES buffer as solution A, the concentration of the MES buffer is 0.1 M, and the pH value is 6.0-6.5; dissolve 4-CPBA and NHS in dimethyl sulfoxide as solution B; then add solution A to solution B in portions, shake and incubate to obtain activated 4-CPBA; (2) Take M13 phage in PBS buffer, the concentration of M13 phage is 1.0×10 11 -1.0×10 13 pfu / mL, the concentration of PBS buffer is 10mM-100mM, pH is 6-8, add activated 4-CPBA solution to stirring reaction, remove excess reactants by centrifugal filtration after reaction, obtain M13 phage modified by phenylboronic acid group, hereinafter referred to as CPBA-M13, collect and refrigerate for standby, the concentration of CPBA-M13 is 1.0×10 10 -1.0×10 12 pfu / mL.

6. Use of a phage multifunctional affinity interface according to claim 5, wherein the phage multifunctional affinity interface is a phage multifunctional affinity interface according to any one of claims 1 to 4. The molar ratio of CPBA, EDC and NHS is 1:(1-3):(1.5-5); The volume ratio of solution A to solution B is 1:1, solution A is added to solution B in three times, the oscillation incubation temperature is 30-40℃, and the time is 25-30min; The volume ratio of activated 4-CPBA solution to M13 in phosphate buffer is 1:(0.5-1.5); The stirring reaction temperature is 0-10℃, the rotation speed is 100-200rpm, and the time is 16-20h; The centrifugal treatment temperature is 0-10℃, the rotation speed is 7000-8000rpm, and the time is 0.5-1h, and the centrifugal treatment is performed three times; The refrigeration temperature is 0-10℃.

7. Use of a phage multifunctional affinity interface according to claim 4, wherein the phage multifunctional affinity interface is a phage multifunctional affinity interface according to any one of claims 1 to 3. First, the His-Tag protein purified Ni-IDA magnetic bead suspension is washed three times with PBS buffer, the PBS buffer concentration is 5-20mM, and the pH is 6-8, then CPBA-M13 is added, the volume ratio of Ni-IDA magnetic bead suspension to CPBA-M13 is 3:(1-5), and the incubation is carried out at room temperature to obtain CPBA-M13 linked Ni-IDA magnetic beads, which is abbreviated as CPBA-M13@MBs, and CPBA-M13@MBs is washed with PBS to remove unbound CPBA-M13, and then resuspended in PBS buffer and stored at 0-10℃ for standby.

8. Use of a phage multifunctional affinity interface according to claim 4, wherein the phage multifunctional affinity interface is a phage multifunctional affinity interface according to any one of claims 1 to 3. Bio-Apt-FAM and its complementary strand C-Apt-BHQ are annealed and hybridized at 95-105℃, the molar ratio of Bio-Apt-FAM to its complementary strand C-Apt-BHQ is 1:(0.8-1.2), and a stable structure of biotinylated DNA switch is obtained, then the biotinylated DNA switch is mixed with avidin in TBS solution at a molar ratio of 4:1, the pH value of TBS solution is 7.5-8.5, then CPBA-M13@MBs is added for incubation, finally PBS buffer is used for washing and resuspension to obtain NataFace.

9. Use of a phage multifunctional affinity interface according to claim 8, characterized in that, The annealing time is 6-10min, the incubation temperature of biotinylated DNA switch and avidin is 30-40℃, and the incubation time is 1.5-2.5h; CPBA-M13@MBs is added for incubation, the incubation temperature is 30-40℃, and the time is 1-4h.

10. Use of a phage multifunctional affinity interface according to claim 3, characterized in that, The body fluid sample containing circulating tumor cells is added to the NataFace dispersion for incubation to capture the circulating tumor cells, and then the mixed liquid after capturing the circulating tumor cells is subjected to magnetic separation, and the supernatant after magnetic separation is removed; In the process of capturing, the EpCAM aptamer on NataFace binds to the EpCAM protein on the surface of circulating tumor cells, and then the complementary strand with fluorescence quencher pre-hybridized on the biotinylated DNA switch is competed down, realizing the labeling of circulating tumor cells; the captured NataFace is incubated in acid fructose PBS buffer, so that the circulating tumor cells with complete phenotype are released from the surface of NataFace, and then subjected to magnetic separation to obtain enriched circulating tumor cells from the supernatant. In the process of capturing, the EpCAM aptamer on NataFace binds to the EpCAM protein on the surface of circulating tumor cells, and then the complementary strand with fluorescence quencher pre-hybridized on the biotinylated DNA switch is competed down, realizing the labeling of circulating tumor cells; the captured NataFace is incubated in acid fructose PBS buffer, so that the circulating tumor cells with complete phenotype are released from the surface of NataFace, and then subjected to magnetic separation to obtain enriched circulating tumor cells from the supernatant.

Images