Preparation method of flexible dynamic bioactive antifouling interface and application of separating heterogeneous circulating tumor cells

Abstract

The application discloses a preparation method of a flexible dynamic bioactive antifouling interface and application of the interface in separation of heterogeneous circulating tumor cells. The steps are as follows: (1) preparation of TiO2 nanotubes; (2) preparation of TiO2 nanotube / silk fibroin composite film; and (3) preparation of the flexible dynamic bioactive antifouling interface. The application constructs a flexible base material and modifies the surface to graft two bioactive peptides, thereby preparing a novel flexible dynamic bio-interface capable of specifically capturing heterogeneous tumor cells and realizing controllable release of the cells, and to a certain extent, solving the problems of cell damage caused by rigidity of the base material and low specific capture efficiency caused by heterogeneity of tumor cells. Meanwhile, the interface can effectively inhibit non-specific adhesion of blood proteins and cells due to the joint action of bioactive peptides, non-bioactive PEG antifouling chains and bovine serum albumin.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of material preparation and circulating tumor cell separation, and particularly relates to a preparation method of a flexible dynamic bioactive antifouling interface and application of the flexible dynamic bioactive antifouling interface in separation of heterogeneous circulating tumor cells. BACKGROUND

[0002] Circulating tumor cells (CTCs) that fall off from primary tumors into the blood circulation or lymphatic system have become important biomarkers for "liquid biopsy", which is crucial for early diagnosis, prognosis and real-time efficacy detection of cancer. However, the rarity (a few to a few hundred per milliliter) and phenotypic heterogeneity of CTCs in the blood circulation system of patients lead to considerable technical challenges in detection. In order to overcome these problems, various methods have been developed to effectively and sensitively capture CTCs from patient blood, mainly using nanostructured substrates based on cancer cell surface biomarkers to isolate and enrich CTCs. In particular, nanowires (NWs) as unique nanostructures have been developed, such as multi-scale titanium dioxide nanorod arrays and electrospun titanium dioxide nanofiber substrates. However, these substrates are usually based on glass plates or metal plates, which do not have the soft action conditions similar to ECM that may have an impact on cell capture. In addition to this, the design of most in vitro cell capture materials focuses on the combination of chemistry (molecular recognition) and physics (micro / nano topological interaction), ignoring the mechanical (including softness) factor of cell-ECM interaction. Therefore, it is necessary to develop a high-quality cell capture platform that combines soft materials with chemical and physical properties to simulate the natural cell microenvironment. In addition, some CTCs will undergo phenotypic changes during blood circulation, such as epithelial-mesenchymal transition (EMT), leading to biological heterogeneity of CTCs in the same patient. Therefore, affinity methods based on a single antibody may reduce the ability to capture CTCs. In addition, antibodies have inherent disadvantages such as poor stability and high cost, which also limit their application. All these factors have brought great difficulties to the specific capture of CTCs from patient blood. Therefore, it is urgent to develop new strategies and specific affinity molecules to improve the efficient capture of cancer cells under different phenotypes. SUMMARY

[0003] In view of the problems existing in the prior art, the application first reports a new flexible dynamic bioactive antifouling interface based on multivalent molecular interaction for specific capture of tumor cells of different phenotypes. First, we prepared TiO2 nanotube composite film with a special structure similar to "bricks and cement" and "pearl and enamel", and used dopamine with excellent biocompatibility to modify the film, and finally obtained PDA@TiO2 nanotube composite film which is not only soft in texture, but also contains various reactive groups (such as catechol, quinone), which can be grafted with bioactive biomimetic peptide PBA-(PEG)8-X and NH2-(PEG)8-Y to specifically capture tumor cells. In addition, bovine serum albumin (BSA) is covalently grafted on the polydopamine layer as an antifouling molecule, which greatly inhibits the adhesion of blood proteins and cells to improve the capture purity of CTCs. In addition, the addition of fructose and glutathione to the system can release the cells by the molecular exchange mechanism of the catechol group and the disulfide bond breaking program, respectively.

[0004] The application prepares a new flexible dynamic bioactive antifouling interface which can specifically capture tumor cells of different phenotypes and realize programmed cell release. The advantage of the application is that the method provides a new idea for constructing a dynamic biological interface in a soft ECM-like environment from a novel perspective, and to some extent, solves the problems of cell damage caused by the rigidity of the substrate material and low specific capture efficiency caused by tumor cell heterogeneity.

[0005] A preparation method of a flexible dynamic bioactive antifouling interface, comprising the following steps:

[0006] Step 1, preparation of TiO2 nanotubes:

[0007] The TiO2 nanotubes are prepared by a simplified one-step anodization method, wherein the anode is a high-purity titanium sheet, the cathode is a pure carbon rod electrode, and a direct current stabilized power supply is used as the power supply.

[0008] First, the titanium sheet is pretreated and ultrasonically cleaned with acetone, anhydrous ethanol and deionized water in sequence to remove surface impurities and dried with nitrogen for standby.

[0009] Citric acid and ammonium fluoride are uniformly dissolved in a mixed solvent of ethylene glycol / water to prepare an electrolyte.

[0010] A voltage is applied between the two electrodes for reaction. After the reaction is completed, the titanium sheet is cleaned with deionized water and dried with nitrogen, and TiO2 nanotubes are obtained.

[0011] Step 2, preparation of TiO2 nanotube / silk fibroin composite film:

[0012] The cocoon shell is put into Na2CO3 aqueous solution and heated to boil for degumming, and then cleaned with deionized water. The dried silk is dissolved in formic acid A containing CaCl2, the solution is poured into a culture dish and placed in a ventilated place until it is completely dried. The dried degummed silk protein film is soaked in circulating water to remove the residual CaCl2 in the film, and after drying, the film is cut and dissolved in formic acid B. The film solution is uniformly coated on the surface of the TiO2 nanotube prepared above, and is naturally dried in a ventilated place. After it is completely dried, the film is slowly peeled off from the titanium sheet and fixed above the HF solution, and etched at room temperature to obtain a TiO2 nanotube / silk fibroin composite film.

[0013] Step 3, preparation of a flexible dynamic bioactive antifouling interface:

[0014] The TiO2 nanotube / silk fibroin composite film is soaked in a freshly prepared dopamine solution, and the solution pH is adjusted with Tris-HCl, and the reaction is carried out at room temperature. Then, it is washed with deionized water and sequentially soaked in PBS solutions containing bioactive biomimetic peptides PBA-(PEG)8-X and NH2-S-S-(PEG)8-Y, and incubated at room temperature overnight. After washing with PBS, it is transferred to a BSA aqueous solution for further culture. Finally, a TiO2 nanotube / silk fibroin composite film grafted with bioactive peptides and antifouling molecules simultaneously is obtained, i.e. a flexible dynamic bioactive antifouling interface (TNTM).

[0015] In step 1, the size of the high-purity titanium sheet used is 0.1×30×70mm, and the ultrasonic cleaning time is 10 minutes;

[0016] In the electrolyte, the concentrations of citric acid and ammonium fluoride are 0.11 mol / L and 0.15 mol / L respectively, the volume ratio of ethylene glycol to water is 9:1, the voltage is 20V, the reaction temperature is 25℃-70℃, and the reaction time is 1.0h.

[0017] In step 2, the concentration of Na2CO3 aqueous solution is 1.5g / L, and the heating and boiling treatment time is 1.0h,

[0018] The mass ratio of silk, CaCl2 and formic acid A is 43:3:4;

[0019] The soaking time in circulating water is 24h,

[0020] The mass ratio of the film and formic acid B is 6:94;

[0021] The etching time is 5min.

[0022] In step 3,

[0023] The concentration of the dopamine solution is 2mg / mL;

[0024] pH 8.5, room temperature for 2 h,

[0025] PBA-(PEG)8-X concentration is 80-120 μg / mL, and the amino acid sequence of X is W-(D-Nle)-EAAYQrFL (remark: r is D-Arg).

[0026] NH2-(PEG)8-Y concentration is 80-120 μg / mL, and the amino acid sequence of Y is GRQLFDNPDQALLDTANDG.

[0027] BSA mass fraction is 1%, and the sample is stored at 4 ℃.

[0028] The citric acid and the ammonium fluoride in the technical solution have the effect of electrolyte.

[0029] The cocoon shell in the technical solution has the effect of silk protein body.

[0030] The formic acid in the technical solution has the effect of solvent, and dissolves the degummed silk protein film.

[0031] The HF in the technical solution has the effect of etching liquid.

[0032] The TiO2 nanotube in the technical solution has the effect of providing a nanoscale topography to enhance the capture efficiency of cells.

[0033] The silk protein film in the technical solution has the effect of fixing the TiO2 nanotube and endowing the material with excellent flexibility.

[0034] The dopamine in the technical solution has the effect of surface modification of the TiO2 nanotube, endowing the surface with catechol and quinone reaction groups, and enhancing the biocompatibility and softness of the material.

[0035] The bioactive peptides PBA-(PEG)8-X and NH2-(PEG)8-Y in the technical solution have the effect of reacting with catechol and quinone, respectively, and being grafted onto the dopamine-modified TiO2 nanotube, endowing the material with the ability of specific recognition of CTCs.

[0036] The PBS in the technical solution has the effect of solvent, and dissolves the bioactive peptides.

[0037] The BSA in the technical solution has the effect of inhibiting the adhesion of blood proteins and non-specific cells, and improving the capture purity of CTCs.

[0038] The acetone, anhydrous ethanol and deionized water in the technical solution have the effect of non-solvent.

[0039] The application of the flexible dynamic bioactive antifouling interface prepared by the application for the purpose of selective separation of circulating tumor cells for non-diagnostic and therapeutic purposes.

[0040] Method and mechanism of interface modification:

[0041] Dynamic receptor-ligand interactions between cells and extracellular matrix (ECM) are crucial for cell processes such as cell adhesion and spreading, stem cell proliferation and differentiation. As a mimic of ECM, flexible dynamic bio-interface materials with low stiffness are significantly more conducive to cell behavior processes. In the field of cell-related, especially in rare cell separation, the softness of the material interacting with cells is extremely important. Therefore, the present application has carried out a series of modifications on TiO2 nanotube / silk fibroin composite film, and prepared a flexible dynamic bioactive antifouling interface (TNTM).

[0042] Firstly, based on the flexible interface of TiO2 nanotube / silk fibroin composite film, the surface of TiO2 nanotube / silk fibroin composite film is modified with dopamine which has good biocompatibility, and a variety of reactive groups (such as catechol and quinone) are introduced on its surface, so as to further combine with bioactive substances. At the same time, a bioactive biomimetic peptide PBA-(PEG)8-X is designed, which is composed of a cell-binding sequence (X) at the C-terminal, a non-bioactive PEG antifouling chain and an N-terminal with a phenylboronic acid group (PBA). Among them, the phenylboronic acid group can be anchored on the TiO2 nanotube / silk fibroin composite film by forming a dynamic catechol / PBA ester with the catechol group introduced by dopamine, while exposing the bioactive X sequence to interact with tumor cell membrane receptors to capture tumor cells with high expression of X receptors. In order to realize the multivalent interaction of the interface with cells, the bioactive peptide NH2-S-S-(PEG)8-Y is also synthesized, including a cell-binding sequence (Y), a non-bioactive PEG antifouling chain and an anchor group (NH2). Among them, the anchor group (NH2) can be anchored on the TiO2 nanotube / silk fibroin composite film by forming an imine dynamic covalent bond through Schiff base reaction with the quinone group on the dopamine-modified TiO2 nanotube / silk fibroin composite film, so that the bioactive Y sequence is exposed to the outside to interact with cells to capture tumor cells with high expression of Y receptors.

[0043] In summary, a new type of interface modification method is designed: on the dopamine-modified flexible substrate, different bioactive peptides are covalently grafted onto the flexible substrate by forming catechol / PBA ester and imine dynamic covalent bonds, so that a flexible dynamic bioactive antifouling interface with cancer targeting activity is obtained. In addition, dopamine, which is called a universal adhesive, can adhere to the surface of almost all materials, so that any bioactive peptide designed can be grafted onto the dopamine-modified substrate material by using the grafting method of the dynamic covalent bond we proposed. Moreover, the bioactive peptide is not limited to X and Y, but can be any cell recognition peptide as long as the polypeptide meets the modification condition of phenylboronic acid / amino group. Therefore, the new type of interface modification method we designed can be universal without limitation of substrate material and targeting target.

[0044] Technical advantages of the present application:

[0045] (1) By dopamine modification of flexible substrate material to graft two-section bioactive peptides PBA-(PEG)8-X and NH2-(PEG)8-Y, a new type of flexible dynamic soft biological interface capable of specifically capturing heterogeneous tumor cells and realizing controllable release of cells is prepared, which realizes specific capture of biologically heterogeneous tumor cells and to some extent solves the problems of cell damage caused by the rigidity of the substrate material and low specific capture efficiency caused by tumor cell heterogeneity. At the same time, thanks to the joint action of bioactive peptides, non-bioactive PEG antifouling chains and bovine serum albumin, the interface can effectively inhibit the non-specific adhesion of blood proteins and cells to improve the purity of tumor cell capture. Finally, the addition of fructose and glutathione to the system can release the cells through the molecular exchange mechanism of catechol group and the disulfide bond breaking program.

[0046] (2) The captured tumor cells are released from the interface in a programmable controllable manner, which is beneficial to the downstream analysis of tumor cells.

[0047] (3) The TiO2 nanotube-based flexible composite film obtained by the present application has obvious nanoscale topographic effect, excellent tensile property and high-efficiency tumor cell separation capacity.

[0048] (4) The preparation steps of TNTM in the present application are simple and easy to operate, and only formic acid is used in the solution process of silk (i.e. step two) in the whole modification process, and the formic acid is completely removed in the subsequent steps. No chemical reagent is used in other links, which belongs to the green and environment-friendly category. This method breaks the drawbacks of traditional interface material preparation (mostly using chemical reagent polymerization grafting method), has excellent biocompatibility, and is expected to be applied in the future in the direction of biomedical detection. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 SEM and AFM images of TiO2 nanotube-based flexible composite film;

[0050] Figure 2 Physical images of TiO2 nanotube-based flexible composite film;

[0051] Figure 3 Proliferation of SK-BR-3 cells and MCF-7 cells on quartz plate and TNTM.

[0052] Figure 4 Fluorescence images of PRP adhered on TNTM (BSA + and BSA - ).

[0053] Figure 5 TNTM selectively separates tumor cells. a) Fluorescence image of mixed cells before TNTM capture; b) Fluorescence image of cells on the interface after TNTM capture.

[0054] Figure 6 a) Fluorescence image before cell release; b) Fluorescence image of cells after fructose treatment; c) Fluorescence image of cells after glutathione treatment. Specific embodiments

[0055] The application will be further described below with reference to specific examples.

[0056] Example 1

[0057] A method for preparing a flexible dynamic bioactive antifouling interface, comprising the following steps:

[0058] (1) Preparation of TiO2 nanotubes:

[0059] TiO2 nanotubes were prepared by a simplified one-step anodization method. The anode was a high-purity titanium sheet (0.1 x 30 x 70 mm), and the cathode was a pure carbon rod electrode. A direct current stabilized power supply was used. First, the titanium sheet was pretreated by ultrasonic cleaning with acetone, anhydrous ethanol, and deionized water for 10 minutes to remove surface impurities, and then dried with nitrogen. Citric acid (0.11 mol / L) and ammonium fluoride (0.15 mol / L) were uniformly dissolved in a mixed solvent of ethylene glycol / water (v / v, 9 / 1) to prepare the electrolyte. A voltage of 20 V was applied between the two electrodes, and the reaction was carried out at 25°C for 1.0 h. After the reaction, the titanium sheet was cleaned with deionized water and dried with nitrogen, and TiO2 nanotubes were obtained.

[0060] (2) Preparation of TiO2 nanotube / silk fibroin composite film:

[0061] The cocoon shell was heated and boiled in 1.5 g / L Na2CO3 boiling water for degumming, and the silk was washed with deionized water after heating and boiling for 1.0 h. The dried silk was dissolved in formic acid containing 6% (wt) CaCl2, the solution was poured into a culture dish and placed in a ventilated place until it was completely dried. The dried degummed silk protein film was soaked in circulating water for 24 h to remove the residual CaCl2 in the film, and the film was cut and dissolved in 8% (wt) formic acid after drying. 500 μL of film solution was uniformly coated on the surface of the TiO2 nanotube prepared above, and was placed in a ventilated place for natural drying. After complete drying, the film was slowly removed from the titanium sheet and fixed on the HF solution, and etched at room temperature for 5 min to obtain a TiO2 nanotube / silk fibroin composite film.

[0062] (3) Preparation of flexible dynamic bioactive antifouling interface:

[0063] The TiO2 nanotube / silk fibroin composite film was soaked in a freshly prepared 2 mg / mL dopamine solution, and the solution pH was adjusted to 8.5 with Tris-HCl, and reacted at room temperature for 2 h. The dopamine-modified TiO2 nanotube / silk fibroin composite film was washed with deionized water, and then soaked in PBS solutions containing bioactive biomimetic peptides PBA-(PEG)8-X and NH2-(PEG)8-Y (pH = 8.5, 80 μg / mL) in turn, and incubated at room temperature overnight. After washing with PBS, it was transferred to a 1% BSA solution. Finally, the TiO2 nanotube / silk fibroin composite film grafted with bioactive peptides and antifouling molecules, i.e. flexible dynamic bioactive antifouling interface (TNTM), was obtained and stored at 4°C for use.

[0064] The SEM image (a), water contact angle image (inset of a) and AFM image (b) of TNTM are shown in Figure 1 , which has a three-dimensional topological rough structure and hydrophilicity (contact angle of 46.1°) on the surface, which is conducive to the adhesion of tumor cells.

[0065] As shown in Figure 2 , TNTM has excellent flexibility and can be bent and folded without being damaged.

[0066] The TNTM prepared in Example 1 was used to separate heterogeneous circulating tumor cells:

[0067] (1) Advantages of flexible substrate material

[0068] SK-BR-3 cells and MCF-7 cells were used in the proliferation experiment to explore the effect of soft substrate material TNTM and hard substrate material quartz sheet. It can be concluded that SK-BR-3 cells ( Figure 3 a, b) and MCF-7 cells ( Figure 3c、d) The proliferation on soft ground material TNTM is significantly higher than that on rigid quartz plate, indicating that the cells tend to adhere and grow in soft biological interface materials.

[0069] (2) Anti-fouling performance

[0070] Platelet adhesion experiment was used to evaluate the anti-fouling performance of the samples. First, platelet-rich plasma (PRP) was extracted by centrifugal separation, and then the platelet-rich plasma was incubated with TNTM soaked in BSA solution (BSA + ) and TNTM not soaked in BSA solution (BSA - ) in BSA solution at 37°C, respectively. After incubation, 1% glutaraldehyde was used for fixation, and DiI C18 was used for staining. Finally, the PRP adhered on the samples was observed by inverted fluorescence microscope. As Figure 4 , the red fluorescence intensity on TNTM soaked in BSA solution (a) was significantly lower than that on TNTM not soaked in BSA solution (b) after incubation with platelet-rich plasma, which indicated that the presence of BSA could reduce the adhesion of platelets to a certain extent and play a certain anti-fouling effect.

[0071] (3) Specific capture of circulating tumor cells

[0072] MCF-7 cells (ck high expression, Dio pre-stained), SK-BR-3 cells (Her 2 high expression, DAPI pre-stained) and HL 60 cells (control, DiI pre-stained) were used to test the cell separation performance of the prepared material. The specific operation is as follows:

[0073] First, TNTM was placed in a 24-well plate, and then 1 mL of MCF-7 cell, SK-BR-3 cell and HL 60 cell mixture was added to the well plate, and the cell density (1 x 10 5 cells / mL) of the three cells in the mixture was the same. After placing the well plate in a 37°C incubator with 5% CO2 for a period of time, the TNTM surface was gently washed with PBS solution. Finally, the fluorescence images of the captured cells on the TNTM were observed under a fluorescence microscope, as shown in Figure 5 , a is the initial fluorescence image of the three cells added to the system, respectively, and b is the fluorescence image of the three cells captured on the TNTM after cell capture. It can be seen that TNTM has good capture ability for targeted tumor cells MCF-7 cells (green fluorescence) and SK-BR-3 cells (blue fluorescence), and basically does not capture non-targeted cells HL 60 cells (red fluorescence). This indicates that the prepared TNTM has good selective separation effect on target tumor cells.

[0074] (4) Programmed cell release

[0075] After cell capture was completed, fructose-containing medium was added to the 24-well plate and the plate was incubated on a low speed shaker for 30 minutes. The TNTMs were then observed under a fluorescent microscope to determine the amount of cells remaining on the TNTMs. The above steps were repeated with the addition of glutathione-containing medium to the system. Figure 6 , a represents the fluorescent image of the captured cells on the TNTMs after cell capture, b represents the fluorescent image of the cells remaining on the TNTMs after the majority of the MCF-7 cells (green fluorescence) were released after the addition of fructose to the system, and c represents the fluorescent image of the cells remaining on the TNTMs after the majority of the SK-BR-3 cells (blue fluorescence) were released after the addition of glutathione to the system. As can be seen, there is essentially no cell fluorescence on the TNTMs after treatment with fructose and glutathione, indicating that both types of cells were released from the TNTMs.

[0076] Example 2

[0077] A method for preparing a flexible dynamic bioactive antifouling interface, comprising the following steps:

[0078] (1) Preparation of TiO2 nanotubes:

[0079] TiO2 nanotubes were prepared by a simplified one-step anodization method. The anode was a high-purity titanium sheet (0.1 x 30 x 70 mm), and the cathode was a pure carbon rod electrode. A direct current stabilized power supply was used. First, the titanium sheet was pretreated by ultrasonic cleaning with acetone, anhydrous ethanol, and deionized water for 10 minutes to remove surface impurities, and then dried with nitrogen. Citric acid (0.11 mol / L) and ammonium fluoride (0.15 mol / L) were uniformly dissolved in a mixed solvent of ethylene glycol / water (v / v, 9 / 1) to prepare the electrolyte. A voltage of 20 V was applied between the two electrodes, and the reaction was carried out at 50°C for 1.0 h. After the reaction was completed, the titanium sheet was washed with deionized water and then dried with nitrogen, and TiO2 nanotubes were obtained.

[0080] (2) Preparation of TiO2 nanotube / silk fibroin composite film:

[0081] The cocoon shell was heated in boiling water with 1.5 g / L Na2CO3 to degum it. After 1.0 h of heating, the silk was washed with deionized water. The dried silk was dissolved in formic acid with 6% (wt) CaCl2, the solution was poured into a petri dish and placed in a ventilated place until it was completely dry. The dried degummed silk protein film was soaked in circulating water for 24 h to remove the residual CaCl2 in the film, and after drying, the film was cut and dissolved in 8% (wt) formic acid. 500 μL of the film solution was uniformly coated on the surface of the TiO2 nanotube prepared above, and was placed in a ventilated place to dry naturally. After it was completely dry, the film was slowly peeled off from the titanium sheet and fixed on top of the HF solution, and etched at room temperature for 5 min to obtain a TiO2 nanotube / silk fibroin composite film.

[0082] (3) Preparation of a flexible dynamic bioactive antifouling interface:

[0083] The TiO2 nanotube / silk fibroin composite film was soaked in a freshly prepared 2 mg / mL dopamine solution, and the solution pH was adjusted to 8.5 with Tris-HCl, and reacted at room temperature for 2 h. The dopamine-modified TiO2 nanotube / silk fibroin composite film was washed with deionized water, and then soaked in PBS solutions containing bioactive biomimetic peptides PBA-(PEG)8-X and NH2-(PEG)8-Y (pH = 8.5, 100 μg / mL), respectively, and incubated at room temperature overnight. After washing with PBS, it was transferred to a 1% BSA solution. Finally, the TiO2 nanotube / silk fibroin composite film grafted with bioactive peptides and antifouling molecules, i.e. a flexible dynamic bioactive antifouling interface (TNTM), was obtained and stored at 4°C for later use.

[0084] Example 3

[0085] A method for preparing a flexible dynamic bioactive antifouling interface, the steps are as follows:

[0086] (1) Preparation of TiO2 nanotubes:

[0087] TiO2 nanotubes were prepared by a simplified one-step anodization method, in which the anode was a high-purity titanium sheet (0.1 x 30 x 70 mm), and the cathode was a pure carbon rod electrode, and a direct current stabilized power supply was used. First, the titanium sheet was pretreated and ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 10 minutes to remove surface impurities and dried with nitrogen for standby. Citric acid (0.11 mol / L) and ammonium fluoride (0.15 mol / L) were uniformly dissolved in a mixed solvent of ethylene glycol / water (v / v, 9 / 1) to prepare the electrolyte. A voltage of 20 V was applied between the two electrodes, and the reaction was carried out at 70°C for 1.0 h. After the reaction was completed, the titanium sheet was washed with deionized water and dried with nitrogen, and TiO2 nanotubes were obtained.

[0088] (2) Preparation of TiO2 nanotube / silk fibroin composite membrane:

[0089] The cocoon shell was degummed by heating and boiling in 1.5 g / L Na2CO3 boiling water. After 1.0 h of heating and boiling treatment, the silk was washed with deionized water. The dried silk was dissolved in formic acid containing 6% (wt) CaCl2, the solution was poured into a petri dish and placed in a ventilated place until it was completely dry. The dried degummed silk fibroin membrane was soaked in circulating water for 24 h to remove the residual CaCl2 in the membrane, and after drying, the membrane was cut and dissolved in 8% (wt) formic acid. 500 μL of membrane solution was uniformly coated on the surface of the TiO2 nanotube prepared above, and placed in a ventilated place to dry naturally. After completely dried, the membrane was slowly peeled off from the titanium sheet and fixed on the HF solution, and etched at room temperature for 5 min to obtain the TiO2 nanotube / silk fibroin composite membrane.

[0090] (3) Preparation of flexible dynamic bioactive antifouling interface:

[0091] The TiO2 nanotube / silk fibroin composite membrane was soaked in a freshly prepared 2 mg / mL dopamine solution, and the solution pH was adjusted to 8.5 with Tris-HCl, and reacted at room temperature for 2 h. The dopamine modified TiO2 nanotube / silk fibroin composite membrane was washed with deionized water, and then soaked in PBS solution containing bioactive biomimetic peptide PBA-(PEG)8-X and NH2-(PEG)8-Y (pH = 8.5, 120 μg / mL), respectively, and incubated at room temperature overnight. After washing with PBS, it was transferred to a 1% BSA solution. Finally, the TiO2 nanotube / silk fibroin composite membrane grafted with bioactive peptides and antifouling molecules, i.e. flexible dynamic bioactive antifouling interface (TNTM), was obtained and stored at 4°C for later use.

Claims

1. A method of making a flexible dynamic bioactive antifouling interface, characterized by: The steps are as follows: Step 1, preparation of TiO2 nanotubes: TiO2 nanotubes are prepared by one-step anodic oxidation method, in which the anode is high-purity titanium sheet, the cathode is pure carbon rod electrode, citric acid and ammonium fluoride are dissolved in a mixed solvent of ethylene glycol / water as electrolyte, and a direct current stabilized power supply is used as power supply; after the reaction is completed, the titanium sheet is cleaned with deionized water and dried with nitrogen, and TiO2 nanotubes are obtained; Step 2, preparation of TiO2 nanotube / silk fibroin composite film: The cocoon shell is placed in Na2CO3 aqueous solution and heated and boiled for degumming, then washed with deionized water and dried. The silk is dissolved in formic acid A containing CaCl2, the solution is poured into a culture dish and placed in a ventilated place until it is completely dried. The dried degummed silk protein film is soaked in circulating water to remove residual CaCl2, and after drying, the film is cut and dissolved in formic acid B. The film solution is uniformly coated on the surface of the prepared TiO2 nanotubes and naturally dried in a ventilated place. After complete drying, the film is slowly removed from the titanium sheet and fixed above the HF solution, and etched at room temperature to obtain the TiO2 nanotube / silk fibroin composite film; Step 3, preparation of flexible dynamic bioactive antifouling interface: The TiO2 nanotube / silk fibroin composite film is soaked in freshly prepared dopamine solution, the pH of the solution is adjusted with Tris-HCl, and the reaction is carried out at room temperature. Then, it is washed with deionized water and sequentially soaked in PBS solutions containing bioactive biomimetic peptide PBA-(PEG)8-X and NH2-S-S-(PEG)8-Y, and incubated at room temperature overnight. After washing with PBS, it is transferred to a BSA aqueous solution for further culture. Finally, the TiO2 nanotube / silk fibroin composite film grafted with bioactive peptides and antifouling molecules simultaneously is obtained, which is a flexible dynamic bioactive antifouling interface TNTM. The amino acid sequence of X is W-(D-Nle)-EAAYQrFL, wherein r is D-Arg. The amino acid sequence of Y is GRQLFDNPDQALLDTANDG.

2. The production method according to claim 1, characterized by: In step 1, the size of the high-purity titanium sheet used is 0.1×30×70 mm. The titanium sheet needs to be pretreated before use: ultrasonic cleaning with acetone, anhydrous ethanol and deionized water for 10 min to remove surface impurities, and then dried with nitrogen for standby.

3. The production method according to claim 1, wherein: In step 1, the concentration of citric acid and ammonium fluoride in the electrolyte is 0.11 mol / L and 0.15 mol / L respectively, and the volume ratio of ethylene glycol to water is 9:

1.

4. The production method according to claim 1, wherein: In step 1, the voltage is 20 V, the reaction temperature is 25-70℃, and the reaction time is 1.0 h.

5. The production method according to claim 1, wherein: In step 2, the concentration of Na2CO3 aqueous solution is 1.5 g / L, and the heating and boiling treatment time is 1.0 h; The mass ratio of silk, CaCl2 and formic acid A is 43:3:4; The soaking time in circulating water is 24 h, The mass ratio of film to formic acid B is 6:94; The etching time is 5 min.

6. The production method according to claim 1, wherein: In step 3, the concentration of dopamine solution is 2 mg / mL, the pH value is 8.5, and the reaction is carried out at room temperature for 2 h.

7. The production method according to claim 1, wherein: In step 3, PBA-(PEG)8-X concentration is 80 ~ 120 μg / mL.

8. The production method according to claim 1, wherein: In step 3, NH2-(PEG)8-Y concentration is 80 ~ 120 μg / mL.

9. The production method according to claim 1, wherein: In step 3, BSA mass fraction is 1%, and the sample is stored at 4℃.

10. The use of the flexible dynamic bioactive antifouling interface prepared by the method of any one of claims 1-9 for the selective separation of circulating tumor cells for non-diagnostic and therapeutic purposes.

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