CN112300993A

CN112300993A - Based on TiO2Nanofiber CTC (CTC) capturing and separating substrate as well as preparation method and application thereof

Info

Publication number: CN112300993A
Application number: CN201910670021.7A
Authority: CN
Inventors: 裴仁军; 陈昌冲
Original assignee: Beijing Sansheng Technology Co ltd; Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Beijing Sansheng Technology Co ltd; Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2019-07-24
Filing date: 2019-07-24
Publication date: 2021-02-02
Anticipated expiration: 2039-07-24
Also published as: CN112300993B

Abstract

The invention discloses a method based on TiO₂A nanofiber CTC capturing and separating substrate, and a preparation method and application thereof. The substrate comprises TiO₂Nano-fiber coated on TiO₂The nanofiber comprises a polymer layer on the surface of the nanofiber, anti-adhesion molecules connected to the surface of the polymer layer, and CTC affinity capture molecules connected with the anti-adhesion molecules. The CTC capturing and separating substrate has good biocompatibility, can realize high-efficiency capturing of target cells on the premise of low non-specific cell adhesion, has simple and convenient preparation process and low cost, can realize in-situ culture and separation of the captured cells, and is easy for large-scale implementation.

Description

Based on TiO2Nanofiber CTC (CTC) capturing and separating substrate as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of medical clinical CTC separation, relates to a CTC separation and purification method, and particularly relates to a separation and purification method based on TiO₂A nanofiber CTC capturing and separating substrate, and a preparation method and application thereof.

Background

Tumor metastasis is the leading cause of cancer recurrence and patient death. The early diagnosis and treatment of cancer can remarkably prolong the survival period of patients and improve the cure rate of cancer, so that detection technologies related to the early diagnosis of cancer are developed, and Circulating Tumor Cells (CTCs) become important markers for the early diagnosis of cancer because blood drawing is convenient and easy, repeated operation can be realized, no wound is generated, and tumor cells in blood carry all information of cancer. The content of CTCs in blood is very rare and the blood components are very complicated, so that it is very difficult to separate CTCs from a patient's blood sample with high sensitivity, efficiency and specificity. Most researchers have chosen biomacromolecules as recognition molecules for capturing tumor cells, but they are expensive and have false negative detection of EMT-transformed tumor cells. Therefore, small molecule affinity molecules with good stability, low price, strong affinity and tumor cell specific recognition are more and more favored by researchers. Therefore, it has been used as a specific recognition probe to label tumor cells in the fields of tumor detection and treatment.

In Cell Adhesion Molecules (CAM), integrins (. alpha.) are present_Ⅴβ₃) Are cell adhesion receptors for extracellular matrix (ECM) proteins, immunoglobulins, growth factors, cytokines and matrix degrading proteases. Integrin is mainly expressed in neovascular endothelial cells and tumor cells and, therefore, is targeted to tumor cells. The RGD sequence (Arg-Gly-Asp) was first discovered by e.ruoslahti in the early 70 s of the 20 th century as a cell attachment site for fibronectin. Later, this sequence was considered the minimal polypeptide sequence and integrin alpha_vβ₃And (4) protein combination. The aminopeptide N protease (CD13) can be used as a tumor cell marker. The small molecule peptide NGR has the ability to recognize aminopeptidase N (CD13) of tumor cells or tumor vascular endothelial cells. The peptide fragment containing asparagine-glycine-arginine (NGR) motif is one of the peptide fragments with highest specificity found by phage display technology.

The current research of nanostructured substrates based on CTC isolation has become a hot area and direction. The electrostatic spinning is a simple and multifunctional nanoRice structure technology for preparing very long and diameter controllable nano fiber with diameter range of several nanometers to several micrometers. CTC capture requires better cell compatibility of the substrate interface, and TiO is not used at present₂The three-dimensional structure of the nanofiber is subjected to biological interface modification to be applied to the research of CTC capture.

Disclosure of Invention

The invention mainly aims to provide a catalyst based on TiO₂A nanofiber CTC capture and separation substrate and a preparation method thereof, which overcome the defects of the prior art.

Another object of the present invention is to provide the above TiO-based alloy₂Use of a nanofibrous CTC capture and separation substrate for CTC capture and separation.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a catalyst based on TiO₂The CTC capture and separation substrate for the nanofiber comprises TiO with the diameter of 50-500 nm₂Nano-fiber coated on TiO₂The nanofiber comprises a polymer layer on the surface of the nanofiber, anti-adhesion molecules connected to the surface of the polymer layer, and CTC affinity capture molecules connected with the anti-adhesion molecules.

Further, the TiO₂The nano-fibers are distributed on the surface of the base material to construct a three-dimensional nano-structure, and the aperture of holes contained in the three-dimensional nano-structure is larger than 0.01 mu m and smaller than 10 mu m.

Further, the anti-adhesion molecule includes bovine serum albumin, and is not limited thereto.

Further, the polymer forming the polymer layer is a polymer containing a phenolic hydroxyl group or a quinone group functional group.

Still further, the polymer is polydopamine.

Further, the CTC affinity capture molecules include, but are not limited to, NGR (asparagine-glycine-arginine) short peptides and/or RGD (arginine-glycine-aspartic acid) short peptides.

Further, the polymer layer is formed by polymerizing polymer monomers in TiO₂Nanofiber surfacesFormed by in situ polymerization.

Further, the CTC capturing and separating substrate has good biocompatibility and hydrophilicity, wherein the surface water contact angle is 10-50 degrees.

The embodiment of the invention also provides a preparation method of the CTC capturing and separating substrate, which comprises the following steps:

preparing TiO by using TBOT-PVP electrospinning solution through electrostatic spinning process₂Precursor of nano-fiber substrate, and calcining the precursor to obtain TiO₂A nanofiber substrate of said TiO₂The nanofiber substrate is made of TiO₂A three-dimensional nanostructure composed of nanofibers;

making polymer monomer compose the TiO₂Nanofiber-based TiO₂Self-polymerizing the surface of the nano-fiber to form a coating on the TiO₂A polymer layer on the surface of the nanofiber;

and coupling anti-adhesion molecules on said polymer layer, followed by cross-linking CTC affinity capture molecules with said anti-adhesion molecules, obtaining said CTC capture and separation substrate.

Further, the polymer monomer includes a small molecule containing a phenolic hydroxyl or quinonyl functionality.

Further, the polymer monomer is dopamine.

Further, the preparation of the TBOT-PVP electro-spinning solution comprises the following steps: adding TBOT and PVP into CaCl of 5-15 mmol/L₂Stirring the mixture for 0.5 to 5 hours at room temperature in an absolute ethyl alcohol solution to obtain a mixed solution of TBOT and PVP with the concentration of 5 to 20 wt%; adding 1-10 wt% of glacial acetic acid into the mixed solution, aging for 2-10 h, and continuously stirring for 0.5-2h at 25-60 ℃ after aging; then standing at room temperature for 5-12 h to obtain a homogeneous, transparent and viscous TBOT-PVP electrospinning solution, wherein M of PVP_WIs 500-2000 kDa.

In a more preferred embodiment, the preparation method specifically comprises: preparing TiO by using TBOT-PVP electrospinning solution through electrostatic spinning process₂The sample feeding speed of a micro sample injector in electrostatic spinning is 0.5-3 mL/h,the voltage of a high-voltage power supply is 10-20 kV, the distance from a needle to a negative plate is 10-20 cm, and then the precursor is calcined for 1-6 hours at the temperature of 400-600 ℃ to obtain TiO₂A nanofiber substrate.

Further, the substrate may be a glass sheet, a single-crystal silicon sheet, a metal aluminum sheet, a metal, or the like, and is not limited thereto.

Further, the TiO₂The nanofiber precursor fibers are uniform and consistent in appearance, and the fiber surfaces are smooth.

Further, the TiO₂The shapes of the fibers of the nano fibers are uniform and consistent, and the surfaces of the fibers are granular.

In a more preferred embodiment, the preparation method specifically comprises: subjecting the TiO to a reaction₂Immersing the nanofiber substrate in a Tris-HCl buffer solution with the polymer monomer concentration of 0-10 mg/mL, and reacting at room temperature for 6-24 hours to form a coating on TiO₂The polymer layer on the surface of the nanofiber is 1-50nm in thickness, the pH value of a Tris-HCl buffer solution is 7-9, and the concentration is 5-15 mmol/L.

In a more preferred embodiment, the preparation method specifically comprises: in TiO₂Wrapping polymer layer on the surface of the nanofiber, and then coating TiO₂The nanofiber substrate is immersed in a 1 xPBS buffer solution with bovine serum albumin concentration of 0.1-2 m/v%, and reacts for 6-24 hours at room temperature, so that the bovine serum albumin is coupled with the polymer layer.

In a more preferred embodiment, the preparation method specifically comprises: coupling bovine serum albumin on the polymer layer, and then coupling TiO₂Immersing the nanofiber substrate in 1 XPBS buffer solution with glutaraldehyde content of 1-10 wt%, reacting for 4-5 h at room temperature in a dark place, washing, drying, then contacting with CTC affinity capture molecule solution with concentration of 0.1-2 mg/mL, reacting for 4-8 h at room temperature in a dark place, then placing at the temperature of below 5 ℃ for overnight reaction, and finally sealing for 0.5-2h by ethanolamine solution with concentration of 0.5-10mmol/L to obtain the CTC capture and separation substrate.

The embodiment of the invention also provides any one of the TiO-based materials₂Nano-fiberThe use of the CTC capture and isolation substrate of (a) in the capture and isolation of CTCs.

For example, one of the application schemes may be: a device comprising any of the foregoing TiO-based₂The CTC of the nanofibers captures and separates the substrate.

For another example, one of the application schemes may be: a CTC isolation process, comprising: releasing or incubating the captured CTCs in situ on the CTC capture and separation substrate.

Wherein, due to the difference of the proliferation of CTC and blood cells, the purity of target cells can be further improved by culturing the captured cells in situ. The in-situ culture is as follows: the captured cells are "superior or inferior" in the subsequent culture process of the base, in other words: target Cells (CTCs) are active and continue to proliferate, while blood cells are eliminated gradually losing activity.

Compared with the prior art, the invention has the advantages that:

1) based on TiO₂The CTC capture and separation substrate of the nanofiber can provide a three-dimensional nano attachment substrate matched with a cell surface and an extracellular matrix nanostructure;

2) based on TiO₂The CTC capturing and separating substrate of the nanofiber can provide a three-dimensional nano interface 'soft' substrate with good cell compatibility by wrapping a polymer monomer, so that the damage of the substrate to the CTC is reduced;

3) on the basis of modifying anti-adhesion molecules, the CTC capturing and separating substrate introduces specific capturing molecules to ensure that target cells can be captured efficiently under the condition of low nonspecific cell adhesion precursors;

4) the CTC capturing and separating substrate can be used for in-situ culture of captured CTCs, so that the purification and proliferation culture of the captured CTCs are realized, and the subsequent research on the biological and physical characteristics of the CTCs is facilitated;

5) the preparation method can be used for preparing titanium dioxide fibers with the diameter of several nanometers to several micrometers and single-layer or multi-layer titanium dioxide fiber films;

6) the method is simple, convenient, low in cost, high in practicability, easy to implement in a large scale and universal for materials with different properties.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a diagram of an embodiment of the present invention utilizing a TiO-based catalyst₂Schematic illustration of the preparation of a nanofiber CTC capture and separation substrate and the capture and separation of CTCs;

FIG. 2 is an RGD-modified TiO-based material according to example 1 of the present invention₂Schematic of the CTC capture and separation substrate of nanofibers;

FIGS. 3a to 3f show TiO compounds in example 1 of the present invention₂The shape and hydrophilicity of the nanofiber before and after dopamine modification are tested to obtain a result graph;

FIGS. 4 a-4 b are schematic views of RGD-modified TiO-based nanoparticles of example 1 of the present invention₂Capture efficiency and fluorescence results plot of nanofiber CTC capture and isolation substrate capture and isolation of tumor cells at different incubation times;

FIGS. 5 a-5 d are different chemically modified TiO-based materials of example 1 of the present invention₂The capture efficiency of the nanofiber substrate on different positive cells and different negative cells and a fluorescence result chart are obtained;

FIGS. 6a to 6c are the RGD-modified TiO-based nanoparticles of example 1 of the present invention₂The capture efficiency, capture purity and fluorescence result of the CTC capture and separation substrate of the nanofiber on positive tumor cells in mixed cells are shown;

FIGS. 7 a-7 b are schematic views of RGD-modified TiO-based nanoparticles according to example 1 of the present invention₂A graph of the results of capture of rare numbers of tumor cells in PBS buffer and blood by nanofiber CTC capture and isolation substrates;

FIG. 8 is an NGR modified TiO-based material according to the invention in example 1₂Schematic of the CTC capture and separation substrate of nanofibers;

FIGS. 9 a-9 b are schematic diagrams of NGR-modified TiO-based nanoparticles of example 1 of the present invention₂Capture efficiency and fluorescence results plot of nanofiber CTC capture and isolation substrate capture and isolation of tumor cells at different incubation times;

FIGS. 10 a-10 d are TiO-based materials with different chemical modifications in example 1 of the present invention₂The capture efficiency of the nanofiber substrate on different positive cells and different negative cells and a fluorescence result chart are obtained;

FIGS. 11 a-11 c are NGR modified TiO-based materials of example 1 of the present invention₂The capture efficiency, capture purity and fluorescence result of the CTC capture and separation substrate of the nanofiber on positive tumor cells in mixed cells are shown;

FIGS. 12 a-12 b are separately NGR-modified TiO-based materials in example 1 of the present invention₂Results of capture of nanofiber CTC capture and isolation substrate in PBS buffer and blood for rare numbers of tumor cells.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions used in the examples can be further adjusted according to actual needs, and the conditions used in the conventional experiments are not generally indicated.

In view of the defects of the prior art, the inventor of the present invention provides a technical scheme of the present invention through long-term research and practice, namely, a titanium dioxide nanofiber three-dimensional substrate is constructed through electrostatic spinning, then biological interface modification is performed, and finally the titanium dioxide nanofiber three-dimensional substrate is used for capturing and separating CTC.

Provides a catalyst based on TiO₂A nanofiber CTC capturing and separating substrate, and a preparation method and application thereof.

Specifically, one aspect of the present invention includes: coating TiO by dopamine with good cell compatibility₂The nano-fiber constructs a three-dimensional nano-interface soft substrate, then introduces anti-adhesion molecules to reduce the nonspecific adhesion of cells on the interface,and realizing high specificity capture of the CTC cells by utilizing the CTC affinity capture molecules, and realizing high-efficiency capture of the target cells.

Another aspect of the invention includes: using electrospun TiO frameworks₂Nano-fiber, then dopamine self-polymerization wrapping on TiO₂The TiO-based nano-fiber is obtained by coupling anti-adhesion molecules, such as Bovine Serum Albumin (BSA), to the polydopamine surface through chemical reaction, and then crosslinking affinity capture molecules, such as NGR short peptide and/or RGD short peptide, with bovine serum albumin through glutaraldehyde₂The CTC of the nanofibers captures and separates a substrate capable of specifically recognizing the target cells, as shown in fig. 1.

The CTC capturing and separating substrate provided by the invention has better biocompatibility, can realize high-efficiency capturing of target cells on the premise of low non-specific cell adhesion, has simple and convenient preparation process and low cost, can realize in-situ culture and purification of the captured cells, and is easy for large-scale implementation.

Example 1 based on TiO₂The preparation method of the CTC capturing and separating substrate of the nanofiber comprises the following specific steps:

a) preparing a TBOT-PVP sol-gel electrospinning solution: TBOT and PVP were added to 10mmol/L CaCl₂Stirring for 2.5h at room temperature in absolute ethyl alcohol solution to obtain TBOT and PVP (M)_W500-2000kDa) are 7 wt% of the mixed solution; adding 4.5% glacial acetic acid into the mixed solution, aging for 4h, and stirring for 1h at 45 ℃; and then standing for 10 hours at room temperature to obtain the homogeneous, transparent and viscous TBOT-PVP sol-gel electrospinning solution.

b) Electrospinning of TiO using electrospinning apparatus₂Nanofibers, electrospun TiO₂The process of the nanofiber is as follows: sucking TBOT-PVP electrospinning liquid by using a 1mL syringe, installing a No. 22 stainless steel flat-head needle as a positive electrode, controlling sample conveying amount, and placing the syringe in a microsyringe; setting the sample feeding speed of a microsyringe to be 1.6mL/h, the voltage of a high-voltage power supply to be 18kV, and the distance from a needle to a negative plate to be 15 cm; then placing the glass sheet cleaned in advance on a negative plate to receive the nano-fibers, thus preparing the TiO₂A nanofiber precursor.

c) Putting the prepared TiO2 precursor nanofiber precursor into a program-controlled box-type electric furnace, calcining for 4 hours at the temperature of 450 ℃, and naturally cooling to room temperature to obtain TiO2 precursor nanofiber precursor₂The diameter of the nano fiber is 90-300 nm.

d) Dopamine in TiO₂Self-polymerization of the nanofiber surface₂Immersing the nanofiber substrate in a Tris-HCl buffer solution (10 mmol/L Tris-HCl with the pH of 8.5) with the dopamine concentration of 2mg/mL, polymerizing for 12h at room temperature, and then washing with deionized water for 4 times to obtain the polydopamine-coated TiO₂And (3) nano fibers.

e) Dopamine-modified TiO coupled with anti-adhesion molecules₂The nanofibers were immersed in 1 XPBS buffer solution with bovine blood protein concentration (BSA) of 1% (m/v), reacted at room temperature for 12 hours, and then washed with 1 XPBS buffer solution.

f) Cross-linking of CTC affinity capture molecule and bovine blood protein by coupling polydopamine modified TiO coupled with bovine serum protein₂Immersing the nano-fibers in 1 XPBS buffer solution with the glutaraldehyde content of 2.5 wt%, reacting for 4 hours at room temperature in a dark place, then washing with the 1 XPBS buffer solution, and drying by blowing with nitrogen; then, 30 mu L of CTC affinity capture molecule solution with the concentration of 1mg/mL is dripped on the surface of the substrate, the substrate is protected from light for reaction for 4h at room temperature, then the substrate is placed in a refrigerator at 4 ℃ for overnight reaction, and finally ethanolamine solution with the concentration of 1mmol/L is used for sealing for 1h to obtain RGD (or NGR) modified TiO₂Nanofiber substrates, i.e. based on TiO₂The nanofiber CTC captures and separates the substrates. The schematic diagrams of the substrates modified by RGD and NGR are shown in FIG. 2 and FIG. 8, respectively.

Example 2TiO₂Characterization graph of shape and hydrophobicity of nanofiber before and after dopamine modification

The TiO obtained by electrospinning in example 1₂Calcining the nanofiber precursor in an oven, modifying dopamine, and detecting the surface morphology and hydrophilicity and hydrophobicity of the nanofiber precursor before and after modifying dopamine, wherein the result is shown in fig. 3, fig. 3a is an electron microscope image before modification of dopamine, and fig. 3b is an enlarged electron microscope image of an area marked by a box in fig. 3 a; FIG. 3c is dopamine modificationFIG. 3d is an enlarged electron micrograph of the area indicated by the box in FIG. 3 c; fig. 3e and 3f are surface water contact angle results before and after dopamine modification, respectively. TiO2₂The nanofiber precursor is still fibrous after being calcined for 4 hours at 450 ℃, and the diameter of the nanofiber precursor is 90-300 nm. TiO modified by dopamine₂The nanofiber is wrapped by polydopamine, the surface biocompatibility is improved, the flexibility is improved, the water contact angle of an interface is reduced to 22.3 degrees from the original 54.7 degrees, and the substrate obtains better hydrophilicity after polydopamine modification.

Example 3 detection of CTC affinity Capture molecule RGD modified TiO₂Effect of nanofiber substrate in capturing CTC at different incubation times

The capture efficiency of the basal surface to cancer cells under different incubation time conditions is systematically investigated by taking the integrin alphavbeta 3 protein positive prostate cancer cell strain MDA-MB-231 as a model cell. Digesting and stripping MDA-MB-231 cells which are cultured for two days and have good growth state by using 0.25 percent of pancreatin, then discarding the trypsin liquid, adding fresh culture solution to blow and beat the cells uniformly, counting the cells, and adjusting the cell suspension to 0.5 multiplied by 10⁵And/ml. The substrate was placed in a 24-well plate and 1ml of the stained PC-3 cell suspension was injected into the well, and after incubation in a cell incubator for 10-60min, washed with PBS, the captured cells were observed with a fluorescence microscope and counted, as shown in fig. 4 b. The experimental result shows that the capture amount of the substrate surface to the positive target cells basically reaches saturation when the substrate surface is incubated for 40min, and the capture efficiency reaches 77.01%, and the result is shown in fig. 4 a.

Example 4 Capture Effect of different chemically modified interfaces on different types of tumor cells

The capture behaviors of different modified nanofiber surfaces on yin and yang cancer cells are systematically investigated by taking integrin alpha v beta 3 protein positive breast cancer MDA-MB-231 and lung cancer H460 cell strains as positive model cells and taking integrin alpha v beta 3 receptor negative prostate cancer PC-3 and kidney epithelial 293T cell strains as negative model cells. FIG. 5a shows 3 different modified interfaces (TiO)₂、TiO₂-PDA-BSA、TiO₂PDA-BSA-RGD) on MDA-MB-231 and H460 cellsDifferent capture results; FIG. 5b shows 3 different modified interfaces (TiO)₂、TiO₂-PDA-BSA、TiO₂-PDA-BSA-RGD) different capture results for PC-3 and 293T cells; FIG. 5c is a captured fluorescence plot of MDA-MB-231 and H460 from FIG. 5 a; FIG. 5d is a graph of the captured fluorescence of FIG. 5b for 293T and PC-3. Experimental results show that the BSA-RGD modified surface has high capture efficiency of more than 75% for positive cells and has good anti-adhesion property for negative cells.

Example 5 the specificity of the short peptides RGD was tested.

An integrin alphavbeta 3 protein positive cell strain MDA-MB-231 and white blood cells are mixed in equal proportion, and a cell capture experiment is carried out. The results of capture of the RGD-modified substrate by MDA-MB-231 are summarized in FIGS. 6 a-6 c. FIG. 6a is a graph showing the results of the capture efficiency of the substrate on the positive cell line MDA-MB-231 in the mixed cells and the capture efficiency of leukocytes on the surface of the substrate, FIG. 6b is a graph showing the results of the capture purity of the substrate on the MDA-MB-231 cells, FIG. 6c is a graph showing the fluorescence of the capture results of the substrate on the leukocytes, and green is a graph showing the fluorescence of the capture results of the substrate on the MDA-MB-231 cells; the experimental result shows that the RGD modified TiO₂The nanofiber substrate has higher capture specificity higher than 78% and better capture purity, namely higher than 92.4%, on the positive cell strain MDA-MB-231.

Example 6 based on TiO₂Capture sensitivity of CTC capture and separation substrate of nanofibers on small amounts of MDA-MB-231 cells in PBS buffer

1cm multiplied by 2cm of modified TiO₂The nanofiber substrate was placed in a 4-well chamber, and 1mL of a cell suspension prepared from 10, 20, 50, 100, 200 MDA-MB-231 cells previously stained with DiO was added to each well using PBS as a buffer. Standing at 37 deg.C for 5% CO₂After incubation for 40min under conditions, the cells were washed with PBS. The captured cells were observed using a fluorescence microscope and counted.

TiO₂The capture results of the nanofiber substrate on target cell samples with different proportions show that the substrate has higher sensitivity on the capture of target cells, and the capture efficiency is higher than 70%. Especially when the target cell content is extremely low, the surfaceThe capture rate of MDA-MB-231 cells was as high as 80%, and the results are shown in FIG. 7 a.

Example 7 is based on TiO₂Capture sensitivity of nanofiber CTC capture and separation substrate on a small number of cell lines in blood

1cm multiplied by 2cm of modified TiO₂The nanofiber substrate was placed in a 4-well chamber, fresh blood of healthy people was used as a buffer, and 1mL of mixed cell blood sample prepared from 10, 20, 50, 100, 200 MDA-MB-231 cells which had been previously stained with DiO was added to each well. Standing at 37 deg.C for 5% CO₂After incubation for 40min under conditions, the cells were washed with PBS. The captured cells were observed using a fluorescence microscope and counted.

TiO₂The capture results of the nanofiber substrate on target cell samples with different proportions show that the substrate has certain sensitivity on the capture of the target cells. Especially when the target cell content is only 10, the capture rate of MDA-MB-231 cells by the surface is as high as 60%, and the experimental result is shown in FIG. 7 b.

Example 8 testing the Effect of affinity capture molecule NGR-modified substrates on capturing CTC at different incubation times

The capture efficiency of the basal surface on cancer cells under different incubation time conditions is systematically examined by using the PC-3 model cell of the prostate cancer cell line with positive CD 13. Digesting and stripping PC-3 cells which are cultured for two days and have good growth state by using 0.25 percent of pancreatin, then discarding the trypsin liquid, adding fresh culture solution to blow and beat the cells uniformly, counting the cells, and adjusting the cell suspension to 0.5 multiplied by 10⁵And/ml. The substrate was placed in a 24-well plate and 1ml of the stained PC-3 cell suspension was injected into the well, and after incubation in a cell incubator for 10-60min, washed with PBS, and the captured cells were observed with a fluorescence microscope and counted, see fig. 9 b. The experimental results show that the capture amount of the substrate to the positive target cells basically reaches saturation when the substrate is incubated for 40min, as shown in FIG. 9 a.

Example 9 Capture Effect of different chemically modified substrates on different types of tumor cells

Takes CD13 positive prostate cancer PC-3 and cervical carcinoma HeLa cell strains as positive model cellsThe cell, CD13 negative breast cancer MDA-MB-231 and kidney epithelial 293T cell lines are used as negative model cells, and capture behaviors of different modified nanofiber surfaces on yin and yang cancer cells are systematically examined. FIG. 10a shows 3 different modified interfaces (TiO)₂、TiO₂-PDA-BSA、TiO₂PDA-BSA-NGR) different capture results for PC-3 and HeLa cells; FIG. 10b shows 3 different modified interfaces (TiO)₂、TiO₂-PDA-BSA、TiO₂PDA-BSA-NGR) differential capture of MDA-MB-231 and 293T cells; FIG. 10c is a captured fluorescence plot of PC-3 and MDA-MB-231 from FIG. 10 a; FIG. 10d is a captured fluorescence plot of PC-3 and MDA-MB-231 from FIG. 10 b. Experimental results show that the BSA-NGR modified substrate has high capture efficiency of more than 80% for positive cells and has good anti-adhesion property for negative cells.

Example 10 testing of specificity of short peptide NGR

A CD13 positive cell strain PC-3 and white blood cells are mixed in equal proportion and cell capture experiments are carried out. The results of capture of PC-3 by the NGR-modified substrate are summarized in FIGS. 11 a-11 c. FIG. 11a is a graph showing the substrate capture efficiency of positive cell lines PC-3 and leukocytes in mixed cells, FIG. 11b is a graph showing the substrate surface capture purity of PC-3 cells and leukocytes, FIG. 11c is a graph showing green fluorescence and red fluorescence respectively showing the substrate capture of leukocytes, and the cell line PC-3; the experimental result shows that NGR modified TiO₂The substrate of the nanofiber has higher capture specificity higher than 84% and better capture purity higher than 95% for the positive cell strain PC-3.

Example 11 affinity Capture molecule NGR modified TiO₂Capture sensitivity examination of nanofiber substrate on a few PC-3 cells in PBS buffer

1cm multiplied by 2cm of modified TiO₂The nanofiber substrate was placed in a 4-well chamber, and 1mL of a cell suspension containing 10, 20, 50, 100, 200 PC-3 cells previously stained with DiO was added to each well using PBS as a buffer. Standing at 37 deg.C for 5% CO₂After incubation for 40min under conditions, the cells were washed with PBS. The captured cells were observed using a fluorescence microscope and counted.

TiO₂The capture results of the nanofiber substrate on target cell samples with different proportions show that the substrate has higher sensitivity on the capture of target cells, and the capture efficiency is higher than 80%. Especially when the target cell content is extremely low, the capture rate of the surface to PC-3 cells is as high as 85%, and the experimental result is shown in FIG. 12 a.

Example 12 Capture sensitivity of affinity Capture molecule NGR-modified substrates on a few cell lines in blood

1cm multiplied by 2cm of modified TiO₂The nanofiber substrate was placed in a 4-well chamber, fresh blood of healthy people was used as a buffer, and 1mL of a mixed cell blood sample prepared by previously staining 10, 20, 50, 100, 200 PC-3 cells with DiO was added to each well. Standing at 37 deg.C for 5% CO₂After incubation for 40min under conditions, the cells were washed with PBS. The captured cells were observed using a fluorescence microscope and counted.

Based on TiO₂The capture results of the nanofiber substrate on target cell samples with different proportions show that the substrate has better sensitivity on the capture of the target cells. Especially when the target cell content is extremely low, the capture rate of the substrate to PC-3 cells is as high as 75%, and the experimental result is shown in FIG. 12 b.

In conclusion, the TiO with better appearance is constructed by the invention₂The nanofiber substrate has good cell compatibility through self-polymerization coating of dopamine, and after different short peptides are selected as affinity capture molecules and chemically modified, the interface has high cell capture specificity and sensitivity on circulating tumor cells. Efficient capture and separation of CTCs is achieved.

In addition, the present inventors have also conducted experiments with other materials and conditions and the like listed in the present specification with reference to the manner of example 1, and obtained the same results.

It should be understood that the above-mentioned embodiments are merely illustrative of the technical concepts and features of the present invention, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and therefore, the protection scope of the present invention is not limited thereby. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. Based on TiO₂A nanofibrous CTC capture and separation substrate characterized by comprising: from TiO₂Three-dimensional nano structure composed of nano fibers wrapped in TiO₂The three-dimensional nano structure comprises a polymer layer on the surface of a nanofiber, an anti-adhesion molecule connected to the surface of the polymer layer, and a CTC affinity capture molecule connected with the anti-adhesion molecule, wherein the pore diameter of a pore contained in the three-dimensional nano structure is larger than 0.01 mu m and smaller than 10 mu m.

2. The CTC capture and separation substrate of claim 1, wherein the anti-adhesion molecules comprise bovine serum albumin.

3. A CTC capture and separation substrate according to claim 1, wherein the polymer forming the polymer layer is a polymer containing phenolic hydroxyl or quinonyl functional groups; preferably, the polymer is polydopamine.

4. The CTC capture and separation substrate of claim 1, wherein the TiO is₂The diameter of the nanofiber is 50-500 nm;

and/or the polymer layer is formed by polymerizing polymer monomers in TiO₂The surface of the nano fiber is formed by in-situ polymerization, and the thickness of the nano fiber is 1-50 nm.

5. The CTC capture and isolation substrate of claim 1, wherein the CTC affinity capture molecules comprise NGR short peptides and/or RGD short peptides.

6. The CTC capture and separation substrate of claim 1, wherein the CTC capture and separation substrate is hydrophilic and biocompatible, wherein surface water contact angle is 10-50 degrees.

7. A process for the preparation of a CTC capture and isolation substrate of any one of claims 1-6, characterized by comprising:

8. The method of preparing a CTC capture and separation substrate of claim 7, wherein the polymer monomers comprise small molecules containing phenolic hydroxyl or quinonyl functionalities; preferably, the polymer monomer is dopamine.

9. The process for the preparation of a CTC capture and isolation substrate of claim 7, comprising: adding TBOT and PVP into CaCl of 5-15 mmol/L₂Stirring the mixture for 0.5 to 5 hours at room temperature in an absolute ethyl alcohol solution to obtain a mixed solution of TBOT and PVP with the concentration of 5 to 20 wt%; adding 1-10 m/v% of glacial acetic acid into the mixed solution, aging for 2-10 h, and continuously stirring for 0.5-2h at 25-60 ℃ after aging; then standing at room temperature for 5-12 h to obtain a homogeneous, transparent and viscous TBOT-PVP electrospinning solution, wherein M of PVP_WIs 500-2000 kDa.

10. The process for the preparation of a CTC capture and isolation substrate of claim 7, specifically comprising: preparing TiO on the surface of a base material by using TBOT-PVP electrospinning liquid through an electrostatic spinning process₂The precursor of the nanofiber substrate, wherein the sample feeding speed of a micro-sample injector in electrostatic spinning is 0.5-3 mL/h, and the sample feeding speed is high pressureThe voltage of a power supply is 10-20 kV, the distance from a needle to a negative plate is 10-20 cm, and then the precursor is calcined for 1-6 hours at the temperature of 400-600 ℃ to obtain TiO₂A nanofiber substrate.

11. The method for preparing a CTC capture and separation substrate of claim 10, wherein the substrate comprises any one of a glass sheet, a monocrystalline silicon sheet, a metallic aluminum sheet.

12. The process for the preparation of a CTC capture and isolation substrate of claim 7, specifically comprising: subjecting the TiO to a reaction₂Immersing the nanofiber substrate in a Tris-HCl buffer solution with the polymer monomer concentration of 0-10 mg/mL, and reacting at room temperature for 6-24 hours to form a coating on TiO₂The polymer layer on the surface of the nanofiber is 1-50nm in thickness, the pH value of a Tris-HCl buffer solution is 7-9, and the concentration is 5-15 mmol/L.

13. The process for the preparation of a CTC capture and isolation substrate of claim 7, specifically comprising: in TiO₂Wrapping polymer layer on the surface of the nanofiber, and then coating TiO₂The nanofiber substrate is immersed in a 1 xPBS buffer solution with bovine serum albumin concentration of 0.1-2 m/v%, and reacts for 6-24 hours at room temperature, so that the bovine serum albumin is coupled with the polymer layer.

14. The process for the preparation of a CTC capture and isolation substrate of claim 7, specifically comprising: coupling bovine serum albumin on the polymer layer, and then coupling TiO₂Immersing the nanofiber substrate in 1 XPBS buffer solution with glutaraldehyde content of 1-10 wt%, reacting for 4-5 h at room temperature in a dark place, washing, drying, then contacting with CTC affinity capture molecule solution with concentration of 0.1-2 mg/mL, reacting for 2-8 h at room temperature in a dark place, then reacting for 6-12 h at 0-8 ℃, and finally sealing for 0.5-2h by ethanolamine solution with concentration of 0.5-10mmol/L to obtain the CTC capture and separation substrate.

15. The TiO-based material according to any one of claims 1 to 6₂Use of a nanofibrous CTC capture and separation substrate for CTC capture and separation.