CN110483683A - A kind of preparation method and purposes of target tumor nano artificial antibody - Google Patents

Abstract

The invention discloses a preparation method and application of a tumor-targeting nano-artificial antibody. Various functional monomers and cross-linking agents are polymerized in different components to form a gel nano-particle artificial antibody. Regulate the affinity and selectivity of artificial antibodies for tumor markers. Combined with molecular imprinting technology, the specificity and selectivity of nanoartificial antibodies to tumor markers can be improved by using the whole protein or stable polypeptide fragment of tumor markers as templates. After screening, the KD value of the affinity constant of the _{nanoartificial} antibody to the tumor marker reached 10 ^‑11 M, which was equivalent to that of the antibody. The obtained nano-artificial antibodies combined with magnetic nanoparticles, monolithic columns or microfluidic chips can realize the selective recognition and capture of tumor markers, circulating tumor cells and exosomes, through high-sensitivity Raman spectroscopy, fluorescence quantitative detection, ELISA reagents Kits, chemiluminescent kits and other methods to achieve high-sensitivity detection of tumor markers, circulating tumor cells and exosomes.

Description

A preparation method and application of nano-artificial antibody targeting tumor

technical field

The invention belongs to the technical field of biological nanomaterials, and in particular relates to a preparation method and application of a tumor-targeting nanoartificial antibody.

Background technique

Cancer is one of the diseases with the highest fatality rate in the world, and its early clinical manifestations are not very specific, so patients are often in the middle and late stages when they see a doctor, and early diagnosis and timely and effective treatment are closely related to the survival rate and quality of life of patients . Early cancer detection can detect precancerous lesions, early cancers and potential invasive cancers, improve the treatment effect of cancer patients, and finally achieve the purpose of prolonging the overall survival of cancer patients.

Tumor markers are produced by tumor tissue or the host's response to tumors, which can reflect and monitor the occurrence and development of tumors, and judge the prognosis and recurrence of tumors. Common tumor marker detection methods include immunological methods, biosensors, proteomics techniques, molecular biology methods, liquid biopsy, etc. The detection of biomarkers has received great attention in the early diagnosis of cancer, among them, epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), vimentin (Vimentin), tyrosinase, programmed death receptor-1 (PD-1) and programmed death receptor-ligand 1 (PD-L1) are common biomarkers , the detection of tumor markers has the advantages of non-invasive, simple operation, and easy acceptance by patients.

Natural antibodies are widely used in the diagnosis and treatment of tumors. However, natural antibodies are mainly obtained by animal immunization, which has the disadvantages of high cost, low preparation efficiency, long screening cycle, variability, difficulty in storage, and immunogenicity. Monoclonal antibodies also have the problem of large batch performance differences. In practical applications has great limitations.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for preparing a tumor-targeting nano-artificial antibody.

The technical solution is as follows:

A method for preparing a tumor-targeting nanoartificial antibody is: N-isopropylacrylamide, N-tert-butylacrylamide, sodium dodecylsulfonate, charged functional monomer, N,N'-methylene The bisacrylamide is used as a cross-linking agent, the tumor-targeting marker is used as a template molecule, and the template molecule is polymerized and eluted in an aqueous phase under the action of an initiator to obtain a tumor-targeting nanoartificial antibody.

Preferably, the targeted tumor markers include epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), vimentin ( Vimentin), tyrosinase, programmed death receptor-1 (PD-1) and programmed death receptor-ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), Lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain mucin domain protein-3 (TIM-3), alpha-fetoprotein (AFP), carbohydrate antigen 12-5 (CA125), carbohydrate antigen 19-9 (CA199), squamous cell carcinoma antigen (SCCA), prostate-specific antigen (PSA), human chorionic gonadotropin (HCG) or its polypeptide fragments, or precursor secretin releasing peptide (Pro-GRP) any one or more of them.

Preferably, the charged functional monomer includes N-(3-aminopropyl) methacrylic acid, acrylic acid, methacrylic acid, 1-vinylimidazole, N-(3-dimethylaminopropyl) methacrylamide , acrylamide, (3-acrylamidopropyl) trimethyl ammonium chloride, N-(2-aminoethyl) acrylamide any one or more.

Preferably, the amount of N-isopropylacrylamide is 5-60wt%, the amount of N-tert-butylacrylamide is 5-50wt%, the amount of charged functional monomer is 0.5-20wt%, N,N'- The amount of methylenebisacrylamide is 0.5-10wt%.

Preferably, the polymerization method is any one of inverse emulsion polymerization, precipitation polymerization or free radical polymerization.

Preferably, the initiator is ammonium persulfate or azobisisobutyronitrile.

Preferably, the polymerization reaction temperature under nitrogen atmosphere is 20-70°C, and the polymerization reaction time is 3-36h.

Preferably, the template molecule elution solution is any one of NaCl, sodium citrate, glycine or sodium dodecylsulfonate, or the template molecule can be eluted by changing the temperature and pH value.

Preferably, the particle size of the nanoparticle artificial antibody is 10-3000nm.

The tumor-targeting nano-artificial antibody prepared by the method can be used in magnetic nanoparticles, monolithic columns or microfluidic chips to realize the selective recognition and capture of tumor markers, circulating tumor cells (CTC) and exosomes and the treatment of tumors, And through high-sensitivity Raman spectroscopy, fluorescence quantitative detection, ELISA kits, chemiluminescence kits, test strips, electrochemical sensors, immune turbidity, immunochromatography, ultrasonic detection, CT detection and nuclear magnetic detection and other methods to realize tumor detection Highly sensitive detection of markers, circulating tumor cells (CTCs) and exosomes.

The resulting nano-artificial antibody combined with photothermal therapy (PPT), photodynamic therapy (PDT) and immunotherapy can achieve effective cancer treatment.

The invention utilizes biofilm interferometry (BLI) to measure the affinity between the non-biological nano particle artificial antibody and the tumor marker, and the biosensor fixes the tumor marker. Then put the nanoparticles to be tested in the detection cell. When the artificial antibody interacts with the marker, the thickness of the biological layer increases; the binding and dissociation time is taken as the abscissa, and the signal intensity increase caused by the drift of the interference curve is the ordinate. coordinates, draw a standard curve, and fit the binding affinity K _D between the non-biological nanoparticle artificial antibody and the marker, as well as the binding and dissociation rate constants k _on and k _off .

The technical effect that the present invention obtains:

(1) The nano-artificial antibody used for tumor diagnosis and treatment provided by the present invention has high selectivity, and can replace biological antibodies in the detection and treatment of tumors; the artificial antibody is prepared by chemical methods and has high stability , long service life and strong ability to resist harsh environments, overcoming the shortcomings of traditional biological antibodies such as long preparation cycle, easy inactivation, and high cost.

(2) The present invention combines nanotechnology and biofilm interference technology to establish a high-throughput screening system for nanoartificial antibodies used in tumor diagnosis and treatment. The method can obtain the affinity K _D between the nanoparticle and the marker, as well as the binding and dissociation rates kon and koff within 20 minutes, which greatly shortens the screening time and is also suitable for high-throughput screening of artificial antibodies for other antigens.

(3) The artificial antibody prepared by the method can be used repeatedly, and the cost is greatly reduced; and the synthesis process and regeneration process are simple, and are suitable for the diagnosis and treatment of tumors.

(4) The artificial antibody prepared by this method has a wide range of applications, including the selective recognition and capture of tumor markers, circulating tumor cells (CTCs) and exosomes by combining magnetic nanoparticles, monolithic columns or microfluidic chips, and tumor The treatment can be performed through high-sensitivity Raman spectroscopy, fluorescence quantitative detection, ELISA kits, chemiluminescence kits, test strips, electrochemical sensors, immune turbidity, immune chromatography, ultrasonic detection, CT detection and nuclear magnetic detection, etc. The method realizes the highly sensitive detection of tumor markers, circulating tumor cells (CTCs) and exosomes. The resulting nanoartificial antibody combined with photothermal therapy (PPT), photodynamic therapy (PDT) and immunotherapy can achieve effective cancer treatment.

Description of drawings

Figure 1 is a transmission electron microscope representation of gold nanospheres.

Fig. 2 is a transmission electron microscope characterization diagram of gold nanorods.

Fig. 3 is a scanning electron microscope characterization diagram of a nano-artificial antibody targeting epidermal growth factor receptor (EGFR).

Fig. 4 is a scanning electron microscope characterization diagram of a nano-artificial antibody targeting epithelial cell adhesion molecule (EpCAM).

Fig. 5 is a scanning electron microscope characterization diagram of a nano-artificial antibody targeting programmed death receptor-1 (PD-1).

Fig. 6 is a transmission electron microscope characterization diagram of the nano-artificial antibody Raman probe targeting EGFR.

Fig. 7 is a transmission electron microscope characterization diagram of the nano-artificial antibody Raman probe targeting vimentin.

Figure 8 is the surface-enhanced Raman spectrum of the nanoartificial antibody Raman probe

Figure 9 shows the confocal laser microscope test results after 4T1 cells were co-incubated with the artificial antibody wrapped nano-gold rods for laser irradiation.

Fig. 10 is the photothermal imaging result of photothermal treatment on the tumor site of the experimental mice.

Figure 11 is the growth curve of tumor size in experimental mice.

Fig. 12 is a curve of body weight change of experimental mice.

Detailed ways

In order to enable those skilled in the art to better understand the technical solution of the present invention, the preparation method and application of a tumor-targeting nano-artificial antibody provided by the present invention will be described in detail below in conjunction with the examples. The following examples are only used to illustrate the present invention but not to limit the scope of the present invention.

Example 1: Preparation of nano-gold spheres and nano-gold rods

1. Preparation of gold nanospheres

Dissolve 0.0255g HAuCl4 in _260mL water, heat to boil and then add 15g of 1% sodium citrate solution to the solution. After the color of the solution remains unchanged, continue to boil for 15min, centrifuge to collect gold nanospheres, and store them for later use. The particle size and morphology of the synthesized gold nanoparticles were studied by transmission electron microscopy (TEM), as shown in Figure 1.

2. Preparation of gold nanorods

0.3553g of CTAB, 0.25mL of 0.1M HAuCl ₄ and 0.2277mg of NaBH ₄ were rapidly mixed for 2min, and the solution turned brown after the mixing was completed. This is the seed solution, which was left to stand at 28°C for 2 hours. Then 15mL of 0.1M HAuCl ₄ , 5.096g of AgNO ₃ , 0.55mL of HCl and 42.2712mg of ascorbic acid were sequentially added to 300mL of the solution containing 10.9535g of CTAB, and then 1.125mL of 10-fold diluted seed solution was added. overnight at 30°C, washed with water, centrifuged to collect gold nanorods, and stored for future use. The particle size and morphology of the synthesized gold nanorods were studied by transmission electron microscopy (TEM), as shown in Figure 2.

Example 2: Preparation of Nano-artificial Antibodies Targeting Epidermal Growth Factor Receptor (EGFR)

1. Design and synthesis of nanoparticles

N-isopropylacrylamide (58-X mol%), charged functional monomer (3-acrylamidopropyl) trimethylammonium chloride (X mol%), N-tert-butylacrylamide (35mol %), the crosslinker N,N'-methylenebisacrylamide (7 mol%) and sodium dodecylsulfonate (10 mg) were dissolved in water to give a total monomer concentration of 130 mM. After adding the initiator, polymerization was carried out at 65° C. for 3 hours with a magnetic stirrer under a nitrogen atmosphere. The polymerized solution was purified by dialysis against an excess of pure water, and polymer nanoparticles were obtained after freeze-drying.

2. Preliminary screening of artificial antibodies

The epidermal growth factor receptor (EGFR) was selected as the target, and the molecularly imprinted polymer nanoparticles were used as the biomimetic antibody. The biofilm interferometry (BLI) was used to measure the affinity between the nanoparticle artificial antibody and EGFR, and the biosensor was used to immobilize EGFR in the interacting molecule, forming biofilm layers. Then the nanoparticles to be tested are placed in the detection cell, and when the interaction between the artificial antibody and EGFR occurs, the thickness of the biological layer increases. Taking the binding and dissociation time as the abscissa, and the signal intensity increase caused by the drift of the interference curve as the ordinate, draw a standard curve to fit the binding affinity K _D between nanoparticles and EGFR and the binding and dissociation rates k _on and k _dis .

3. Improving the _KD value and selectivity of artificial antibodies by molecular imprinting technology

After preliminary screening of nanoparticles with high K _D value with EGFR, in order to further improve its K _D value and selectivity, this experiment uses the method of molecular imprinting to improve the specificity and selectivity of artificial antibodies. The synthetic method of imprinted polymer (MIP) is as follows: with N-isopropylacrylamide (28mol%), N-(3-aminopropyl) methacrylic acid (30mol%), N-tert-butylacrylamide (35mol%) %), N,N'-methylenebisacrylamide (7mol%), sodium dodecylsulfonate (10mg) and 20mg of EGFR polypeptide fragments were dissolved in water to obtain a total monomer concentration of 130mM. After adding the initiator ammonium persulfate, polymerization reaction was carried out at 45° C. for 12 hours under a nitrogen atmosphere with a magnetic stirrer. Then add 0.04 mol of NaCl and continue to stir at room temperature for 30 minutes to elute the template polypeptide. Finally, the polymerized solution is purified by dialysis (three times a day) with excess pure water, and the molecularly imprinted polymer nanoparticles are obtained after freeze-drying. The scanning electron microscope image of the nano-artificial antibody is shown in Fig. 3 .

Example 3: Preparation of Nano-artificial Antibodies Targeting Epithelial Cell Adhesion Molecule (EpCAM)

Dissolve 228.6 mg of N-isopropylacrylamide, 23 mg of acrylamide, 82.6 mg of N-tert-butylacrylamide, 50 mg of N,N'-methylenebisacrylamide and sodium dodecylsulfonate SDS (30 mg) In 50mL of water, the total monomer concentration was 65mM, and 2mg of epithelial cell adhesion molecule (EpCAM) polypeptide was added at the same time, the mixed solution was filtered, nitrogen gas was blown for 30min, ammonium persulfate was added, and reacted at 65°C for 10h. The polypeptide template was then eluted with 1 M sodium chloride solution to obtain nanoartificial antibodies against epithelial cell adhesion molecule (EpCAM). Then the reaction mixture was dialyzed for 3 days, and then freeze-dried to obtain the nano-artificial antibody.

The scanning electron microscope image of the nano-artificial antibody obtained by mixing the above monomer solution and gold nanoparticles is shown in Figure 4.

Example 4: Preparation of Nano-artificial Antibodies Targeting Programmed Death Receptor-1 (PD-1)

N-isopropylacrylamide 134.5mg, acrylic acid 30μL, acrylamide 23mg and N-tert-butylacrylamide 123.9mg, N,N'-methylenebisacrylamide 25mg and 30mg sodium dodecylsulfonate SDS Dissolve in 50mL water, the total monomer concentration is 65mM, add 2mg of programmed death receptor-1 (PD-1) polypeptide at the same time, filter the mixed solution, blow nitrogen gas for 30min, add ammonium persulfate, and react at 65 degrees for 10h . The polypeptide template was then eluted with 1M sodium chloride solution to obtain nanoartificial antibodies against programmed death receptor-1 (PD-1). Then the reaction mixture was dialyzed for 3 days, and then freeze-dried to obtain the nano-artificial antibody.

The scanning electron micrograph of the nano-artificial antibody obtained after adding nano-gold to the above reaction solution is shown in FIG. 5 .

Example 5: Preparation of Nano-artificial Antibody Raman Probe Targeting EGFR

Mix 195 mg of N-isopropylacrylamide, 10 μL of acrylic acid, 11.5 mg of acrylamide and 90.9 mg of N-tert-butylacrylamide, 50 mg of N,N'-methylenebisacrylamide and sodium dodecylsulfonate SDS ( 30mg) was dissolved in 50mL of water, the total monomer concentration was 65mM, and 2mg of epidermal growth factor receptor (EGFR) polypeptide was added at the same time, the mixed solution was filtered, nitrogen gas was blown for 30min, ammonium persulfate was added, and reacted at 65°C for 10h. The polypeptide template was then eluted with 1M sodium chloride solution to obtain a Raman probe for the nanometer artificial antibody detected by the epidermal growth factor receptor (EGFR). Then the reaction mixture was dialyzed for 3 days, and then freeze-dried to obtain the nano-artificial antibody.

The nano-artificial antibody obtained by adding nano-gold balls to the above reaction solution was characterized by a transmission electron microscope (TEM) and the synthetic nano-artificial antibody Raman probe was shown in FIG. 6 .

Example 6: Preparation of Nano-artificial Antibody Raman Probe Targeting Vimentin

Mix 195 mg of N-isopropylacrylamide, 10 μL of acrylic acid, 11.5 mg of acrylamide, 90.9 mg of N-tert-butylacrylamide, 50 mg of N,N'-methylenebisacrylamide and sodium dodecylsulfonate SDS ( 30mg) was dissolved in 50mL water, the total monomer concentration was 65mM, and 2mg vimentin polypeptide was added at the same time, the mixed solution was filtered, nitrogen gas was blown in for 30min, ammonium persulfate was added, and reacted at 65°C for 10h. Then, the peptide template was eluted with 1M sodium chloride solution to obtain a Raman probe of nano-artificial antibody for vimentin detection. Then the reaction mixture was dialyzed for 3 days, and then freeze-dried to obtain the nano-artificial antibody.

Nano-gold rods were added to the above reaction solution, and the obtained nano-artificial antibody was characterized by a transmission electron microscope (TEM) to characterize the synthesized nano-artificial antibody Raman probe, as shown in FIG. 7 . Carry out Raman depth scanning to determine the characteristic peaks (Figure 8).

Example 7: Characterization of the affinity and selectivity of nanoartificial antibodies to tumor markers

Biomembrane interferometry (BLI) was used to determine the affinity constant K _D value of molecularly imprinted artificial antibody (MIP) and non-imprinted polymer (NIP) on epidermal growth factor receptor (EGFR) adsorption, as well as the adsorption value of impurity proteins (human serum albumin and The affinity constant K _D value of cytochrome c) adsorption, the results are shown in Table 1, show that nano-artificial antibody has very high affinity and selectivity to epidermal growth factor receptor (EGFR), which is equivalent to natural antibody.

Table 1

Biomembrane interferometry (BLI) was used to determine the affinity constant K _D value of molecularly imprinted artificial antibody (MIP) and non-imprinted polymer (NIP) for vimentin adsorption and the adsorption of miscellaneous proteins (human serum albumin and cytochrome c). The affinity constant K _D value, the results are shown in Table 2, indicating that the nano-artificial antibody has high affinity and selectivity to vimentin, which is comparable to natural antibodies.

Table 2

Biomembrane interferometry (BLI) was used to measure the affinity constant K _D value of molecularly imprinted artificial antibody (MIP) for the adsorption of carcinoembryonic antigen CEA, EpCAM, and tyrosinase, and the results were 8.46×10 ^-9 M and 5.68×10 ^{- 9} M, 7.89×10 ^-10 M, indicating the high affinity of the nano-artificial antibody, which is comparable to the natural antibody.

Example 8: Preparation of PD-L1/PD-1 checkpoint blockade inhibitors

1. Mix 170.5 mg of N-isopropylacrylamide, 144 mg of N-tert-butylacrylamide, 73.4 μL of (3-acrylamidopropyl) trimethylammonium chloride, N-(3-dimethylaminopropyl) 10mg of methacrylamide, 10mg of bisacrylamide and 35mg of SDS, 20mg of the N-terminal polypeptide fragment of PD-L1 or PD-1 were dissolved in 50mL of water, the total monomer concentration was 65mM, and the above monomer solution was mixed with nano gold rods, The mixed solution was filtered, nitrogen gas was blown in for 30 minutes, ammonium persulfate was added, and the reaction was carried out at 65 degrees for 10 hours. The peptide template was then eluted with 1M sodium chloride solution to obtain nanoartificial antibodies targeting PD-1 and PD-L1. Then the reaction mixture was dialyzed for 3 days, and then freeze-dried to obtain the nano-artificial antibody.

2. Biomembrane interferometry (BLI) was used to measure the affinity constant K _D value of the molecularly imprinted artificial antibody (MIP) for the adsorption of PD-1 and PD-L1, and the results were 1.80×10 ^-9 M and 9.78x10 ^-11 M, respectively, indicating that The high affinity of nano-artificial antibodies is comparable to natural antibodies.

3. Nano-artificial antibodies are selective and specific for PD-1/PD-L1, and are used in immunotherapy. Combining the photothermal properties of the inner core nano-gold rods with nano-artificial antibodies, it can be applied to photothermal therapy.

Incubate the nano-artificial antibody with the tumor cells for 24 hours, then irradiate the tumor cells with an 808nm laser at a power of 2 W/cm ² for 3 minutes, stain the cells with Calcein AM live cell staining agent, and conduct laser confocal microscope testing , as shown in Figure 9, it can be observed that most of the cells incubated with nano-artificial antibodies and irradiated with laser light died.

Next, BALB/c mice can be used to establish tumor models, and nanoartificial antibodies can be used for combined photothermal-immune therapy. 106 cells of 4T1 cells were injected under the armpit of mice, and 14 days after modeling, they were injected with samples such as nano-artificial antibodies, and were grouped for photothermal treatment. The 808nm laser was used at a power of 2W/ ^cm2 After 3 minutes of photothermal therapy on the tumor, as shown in the figure, the local temperature of the tumor rises, and the killing effect on the tumor is obvious. As shown in Figure 10, long-term observation can clearly see the therapeutic effect, the tumor size of the mice in the treatment group has been greatly improved, greatly prolonging the life quality and time of the mice, and having a good cancer treatment effect ( Figure 10-Figure 12).

The invention discloses a method for preparing a tumor-targeting nano-artificial antibody. Various functional monomers and cross-linking agents are polymerized in different components to form a gel nano-particle artificial antibody, and the artificial antibody is regulated by changing the ratio of functional monomers. Antibody affinity and selectivity for tumor markers. Combined with molecular imprinting technology, the specificity and selectivity of nanoartificial antibodies to tumor markers can be further improved by using the whole protein or stable polypeptide fragment of tumor markers as templates. After screening, the affinity constant K _D value of nanoartificial antibodies to tumor markers reached 10 ^-11 M, which was comparable to that of antibodies, and had good selectivity. The controllable preparation method of artificial antibody nanoparticles was studied, and the artificial antibody gel nanoparticles with uniform particle size and controllable size and shape were obtained. By adjusting the ratio and type of functional monomers, the high affinity and high selectivity of nanoartificial antibodies to tumor markers can be realized. The obtained nano-artificial antibodies combined with magnetic nanoparticles, monolithic columns or microfluidic chips can realize the selective recognition and capture of tumor markers, circulating tumor cells (CTC) and exosomes, as well as the treatment of tumors, and can be pulled with high sensitivity. Mann spectroscopy, fluorescence quantitative detection, ELISA kits, chemiluminescence kits, test strips, electrochemical sensors, immune turbidity, immunochromatography, ultrasonic detection, CT detection and nuclear magnetic detection and other methods to achieve tumor markers, circulating tumor cells (CTC) and high-sensitivity detection of exosomes. The resulting nano-artificial antibody combined with photothermal therapy (PPT), photodynamic therapy (PDT) and immunotherapy can achieve effective cancer treatment.

The examples of the present invention have been described in detail above in conjunction with the embodiments, but the present invention is not limited to the above-mentioned examples. Within the scope of knowledge possessed by those of ordinary skill in the art, various modifications can also be made without departing from the gist of the present invention. Changes should also be regarded as the protection scope of the present invention.

Claims (10)

1. A preparation method for targeting tumor nanoartificial antibodies, characterized in that, N-isopropylacrylamide, N-tert-butylacrylamide, sodium dodecylsulfonate, charged functional monomers, N, N The '-methylenebisacrylamide is used as a cross-linking agent, the tumor-targeting marker is used as a template molecule, and the template molecule is polymerized and eluted in an aqueous phase under the action of an initiator to obtain a tumor-targeting nano-artificial antibody. 2. according to the preparation method of the described targeting tumor nanoartificial antibody of claim 1, it is characterized in that, described targeting tumor marker comprises epithelial cell adhesion molecule, epidermal growth factor receptor, carcinoembryonic antigen, neuron specificity Enolase, vimentin, tyrosinase, programmed death receptor-1 and programmed death receptor-ligand 1, cytotoxic T lymphocyte-associated antigen 4, lymphocyte activation gene-3, T cell immunoglobulin Protein domain mucin domain protein-3, alpha-fetoprotein, carbohydrate antigen 12-5, carbohydrate antigen 19-9, squamous cell carcinoma antigen, prostate-specific antigen, human chorionic gonadotropin or a polypeptide fragment thereof, or Any one or more of secretin releasing peptide precursors. 3. according to the preparation method of the described tumor-targeting nano-artificial antibody of claim 1, it is characterized in that, the charged functional monomer comprises N-(3-aminopropyl) methacrylic acid, acrylic acid, methacrylic acid, 1- Vinylimidazole, N-(3-dimethylaminopropyl)methacrylamide, acrylamide, bisacrylamide, (3-acrylamidopropyl)trimethylammonium chloride, N-(2-aminoethyl base) any one or more of acrylamide. 4. according to the preparation method of the described targeting tumor nanoartificial antibody of claim 1, it is characterized in that, the consumption of N-isopropylacrylamide is 5-60wt%, the consumption of N-tert-butylacrylamide is 5-50wt% %, the amount of charged functional monomer is 0.5-20wt%, and the amount of N,N'-methylenebisacrylamide is 0.5-10wt%. 5. The method for preparing the tumor-targeting nano-artificial antibody according to claim 1, wherein the polymerization method is any one of inverse emulsion polymerization, precipitation polymerization or free radical polymerization. 6. The method for preparing the tumor-targeting nanoartificial antibody according to claim 1, wherein the initiator is ammonium persulfate or azobisisobutyronitrile. 7 . The method for preparing the tumor-targeting nano-artificial antibody according to claim 5 , wherein the polymerization reaction temperature under nitrogen atmosphere is 20-70° C., and the polymerization reaction time is 3-36 hours. 8. according to the preparation method of the described tumor-targeting nano-artificial antibody of claim 1, it is characterized in that, template molecule eluting solution is any one in NaCl, sodium citrate, glycine or sodium dodecylsulfonate, or Elution of template molecules is achieved by changing temperature and pH. 9. The method for preparing the tumor-targeting nano-artificial antibody according to claim 1, wherein the particle size of the nano-particle artificial antibody is 10-3000 nm. 10. The use of a nano-artificial antibody targeting tumors, characterized in that the artificial nano-antibodies targeting tumors prepared according to any one of the methods described in claims 1 to 9 are used for magnetic nanoparticles, monolithic columns or microfluidic chips Realize the selective recognition and capture of tumor markers, circulating tumor cells and exosomes, and can use high-sensitivity Raman spectroscopy, fluorescence quantitative detection, ELISA kits, chemiluminescent kits, test strips, electrochemical sensors, immunoturbidity High-sensitivity detection of tumor markers, circulating tumor cells and exosomes can be achieved by using high-precision, immunochromatography, ultrasonic detection, CT detection and nuclear magnetic detection methods.