WO2023104059A1 - Circulating tumor cell detection material, detector and detection method - Google Patents

Abstract

Disclosed in the present invention are a circulating tumor cell detection material, detector and detection method. The circulating tumor cell detection material is fixed on a substrate. The detection material is a functionalized group modified chitosan. The functionalized group comprises a biological orthogonal reaction group; or comprises a biological orthogonal reaction group and a reversible reaction group. The circulating tumor cell detector comprises the substrate and the detection material fixed on the substrate. By means of the novel circulating tumor cell detection material in the present invention, according to different modification degrees, three functions of biological orthogonal / anti-adhesion bifunctional or biological orthogonal / anti-adhesion/reversible release can be achieved, chitosan is used for replacing gold to serve as a functionalized surface, and the cost is reduced; the surface of the biological orthogonal chitosan has a good anti-adhesion effect, the detection accuracy is improved, and the probability of false positive detection results is reduced.

Description

A detection material, detector and detection method for circulating tumor cells technical field

The invention belongs to the technical field of biological materials, and in particular relates to a detection material, a detector and a detection method for circulating tumor cells.

Background technique

In 1869, Australian doctor Ashworth first discovered and proposed the concept of Circulating Tumor Cell (CTC) in the blood of breast cancer patients. In 1976, Nowell revised the definition of CTC as: tumor cells originating from the primary tumor or metastatic tumor, gaining the ability to escape from the basement membrane and invade blood vessels through the tissue matrix. At present, CTC refers to the general designation of various tumor cells existing in peripheral blood, and its growth and dissemination run through all stages of tumor growth.

Liver cancer is one of the six most common tumors and ranks among the top three in terms of mortality among all cancers. Early diagnosis and treatment are the difficulties in clinical treatment of liver cancer. Early screening is beneficial to prolong the survival time of liver cancer patients, and early diagnosis is also conducive to the adoption of various treatment methods, thereby improving the prognosis. However, only about 35% of patients with liver cancer can receive effective treatment at an early stage, and only a few biomarkers can be used as evaluation indicators for liver cancer, among which alpha-fetoprotein (AFP) is the most widely used biomarker. landmark. However, AFP has some limitations, such as a high proportion of false positive results, lack of sufficient sensitivity and specificity.

The CTC detection method based on bio-orthogonal metabolic glycoengineering labeling (CN113358865A), through the "label-capture-release" detection process, realizes the accurate and non-destructive detection of CTC. In this method, a CTC labeling process based on metabolic glycoengineering is firstly designed, and samples are treated with labeling molecules based on sugar units, and the labeled molecules are artificially labeled by metabolic glycoengineering; secondly, a bioorthogonal group and reversible The capture molecule of the reactive group is immobilized on the device substrate (such as microfluidic chip, magnetic nanoparticle material, etc.) Capture CTCs; finally design and introduce release molecules, which can exchange capture molecules immobilized on the substrate of the detection device through a reversible reaction to release captured CTCs.

The existing bioorthogonal reaction-based circulating tumor cell detection materials are based on gold and directly undergo bioorthogonal modification of small molecules. However, due to the existence of tumor heterogeneity, the adhesion of liver cancer CTCs on the bioorthogonal gold surface is weak and the non-specific adhesion is strong. The golden surface is used to detect liver cancer, and its detection effect is poor and the false positive is high. Finding a functional surface suitable for liver cancer CTC detection is the key to overcome the technical bottleneck of liver cancer CTC detection.

Chitosan, the N-deacetylated product of chitin, is the second most abundant natural polysaccharide in nature, second only to cellulose. Chitosan has many useful properties, such as biocompatibility, biodegradability, antibacterial activity, wound healing properties, antitumor effect, etc.

In recent years, since chitosan coating has many modifiable sites, the research on its functional modification and its application in basic medicine, disease treatment, medical detection, etc. is increasing day by day.

Contents of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide a detection material, a detector and a detection method for circulating tumor cells. The invention provides a material for CTC detection, a preparation method and a use method thereof. A new type of CTC detector is mainly prepared by performing "substrate surface coating-functional modification" on commercially purchased chitosan, and finally applied to CTC detection. The surface coating of the substrate is prepared by spin coating, electrospinning and other methods to prepare a uniform chitosan coating suitable for downstream applications; based on the fact that chitosan molecules have regular amino groups and are easily modified, chemical methods are used to gradually Link the required reversible reaction linker and bio-orthogonal/anti-adhesion bifunctional group to the amino site of chitosan; in line with the principle of "high specific affinity, low non-specific affinity", so that the final Improve the accuracy of CTC detection.

The first aspect of the present invention provides a detection material for circulating tumor cells, the detection material is fixed on a substrate, and the detection material is chitosan modified with a functional group;

(1) The functionalized group includes a bioorthogonal reactive group; or,

(2) The functional groups include bio-orthogonal reactive groups and reversible reactive groups.

Further, (1) when the functionalized group includes a bioorthogonal reactive group, the detection material has the structure shown in the following formula I,

The R is a bioorthogonal reactive group;

Preferably, the R has the structure shown in the following formula A, B, C or D,

(2) When the functionalized group includes a bioorthogonal reactive group and a reversible reactive group, the reversible reactive group is a disulfide bond;

Preferably, the reversible reactive group is used as a connecting arm to connect chitosan and a bioorthogonal reactive group;

Preferably, the detection material has the structure shown in the following formula II,

The R is a bioorthogonal reactive group;

Preferably, the R has the structure shown in the following formula A, B, C or D,

In the above technical solution of the present invention, the substrate has a hydrophilic surface;

Preferably, the substrate is selected from hydrophilized glass, silicon wafer or mica.

The second aspect of the present invention provides a detector for circulating tumor cells, the detector comprising a substrate and the detection material immobilized on the substrate.

In the above technical solution of the present invention, the substrate has a hydrophilic surface;

Preferably, the substrate is selected from microfluidic chip substrates, magnetic nanoparticle material substrates and other material substrates that can be hydrophilized.

The third aspect of the present invention provides a detection kit for circulating tumor cells, the detection kit includes the detection material or the detector and a sugar unit-based labeling molecule;

Preferably, the detection kit also includes a release molecule;

Preferably, the release molecule is dithiothreitol and/or borate;

Preferably, the sugar unit-based labeling molecule is selected from one or more of 2-aminomannose units, 2-galactosamine units, 2-glucosamine units and 6-azido-L-fucose units. kind;

The 2-aminomannose unit, 2-galactosamine unit, 2-glucosamine unit and 6-azido-L-fucose unit have the structure shown in the following formula,

Wherein, X=(CH ₂ ) _n . Preferably, n=1, 2, 3, 4, 5, 6.

The fourth aspect of the present invention provides the application of the circulating tumor cell detection material, the circulating tumor cell detector or the circulating tumor cell detection kit in the detection of circulating tumor cells;

Preferably, the circulating tumor cells are liver cancer circulating tumor cells.

The fifth aspect of the present invention provides a method for preparing the detection material of circulating tumor cells, comprising the following steps:

Preparation of chitosan coating: prepare chitosan coating on the surface of the base material, adjust the pH, so that the chitosan amino group is exposed;

Functional modification of chitosan coating: linking bio-orthogonal reactive groups or bio-orthogonal reactive groups and reversible reactive groups to chitosan molecules;

Preferably, the acetic acid solution of chitosan is evenly spread on the surface of the base material, and the pH is adjusted after drying to obtain the chitosan coating;

Preferably, the acetic acid solution of chitosan is evenly spread on the surface of the base material by spin coating or electrospinning;

Preferably, the chitosan has a deacetylation degree of 95%;

Preferably, the volume percentage of the acetic acid solution is 90%.

In the above technical scheme of the present invention, when the functionalized group includes a bioorthogonal reactive group, the functional modification method of the chitosan coating is that the chitosan coating is contained in ethanol containing 5% triethylamine React with NHS activated ester containing bio-orthogonal reactive groups for 24 hours; wash 5 times with ethanol containing 5% triethylamine, wash 3 times with water containing 5% triethylamine, and wash 3 times with water; dry at 60°C for 1 hour;

When the functionalized group includes a bio-orthogonal reactive group and a reversible reactive group, the functional modification method of the chitosan coating is that the chitosan coating is coupled with Molecular SPDP was reacted for 24 hours; washed 5 times with ethanol containing 5% triethylamine, washed 3 times with water containing 5% triethylamine, and washed 3 times with water; dried at 60°C for 1 hour; Molecular reaction of cross-reactive groups for 24 hours; wash with ethanol 5 times, wash with water 5 times; dry at 60°C for 1 hour.

The sixth aspect of the present invention provides a method for detecting circulating tumor cells, comprising the following steps:

Samples are treated with marker molecules based on sugar units, and the marker molecules are artificially labeled with circulating tumor cells through metabolic glycoengineering;

Co-incubating the circulating tumor cells with the detection material or the detector, the bio-orthogonal reaction group and the marker molecule undergo a bio-orthogonal reaction, and the circulating tumor cells are captured;

Preferably, when the functional group includes a bio-orthogonal reaction group and a reversible reaction group, the detection method further includes releasing the captured circulating tumor cells, specifically: by injecting the captured circulating tumor cells Adding release molecules or regulating the electrical signal and/or temperature of the functionalized surface of the captured circulating tumor cells to break the reversible reactive group and release the captured circulating tumor cells;

Preferably, the release molecule is dithiothreitol and/or borate;

Preferably, the circulating tumor cells are liver cancer circulating tumor cells.

The beneficial effects of the present invention are:

1. The present invention provides a novel material that can be used for the detection of circulating tumor cells. The material is chitosan modified with functional groups. According to the degree of modification, it can play the dual functions of bio-orthogonal/anti-adhesion or Bioorthogonal/anti-adhesion/reversible release three functions, so as to meet different CTC detection purposes such as CTC counting and CTC downstream physiological and biochemical analysis. Its main advantages are as follows:

(1) Using chitosan instead of gold as the functionalized surface reduces the cost.

(2) The surface of bioorthogonal chitosan has a good anti-adhesion effect, which improves the detection accuracy and reduces the probability of false positive detection results.

(3) The chitosan coating can be evenly spread on almost all hydrophilic surfaces by means of dissolution and drying, and detectors with different physical shapes and properties can be prepared conveniently. Compared with the gold surface, the detector has mild preparation conditions, wide applicability and better effect.

2. The material for detecting circulating tumor cells provided by the present invention utilizes the physical and chemical properties of bio-orthogonal chitosan to improve the adhesion of liver cancer CTCs to it and reduce the non-specific adhesion of white blood cells thereon, thereby improving the detection effect , Reduce the false positive rate, and fill the gap in the detection technology of circulating tumor cells in liver cancer.

Description of drawings

Fig. 1 is the whole process of detection of liver cancer CTC in the present invention.

Figure 2 is a method for cleaving a reversibly reactive group.

Detailed ways

In order to understand the present invention more clearly, the present invention will now be further described with reference to the following examples and accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each original reagent material can be obtained commercially, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions suggested by the instrument manufacturer.

"Bioorthogonal" refers to having groups that can perform bioorthogonal reactions. The current bioorthogonal reactions are shown in Table 1. Any of the reactive groups can be used as a bioorthogonal group for the functional modification of chitosan coatings, including dibenzocyclohexyl used in the SPAAC reaction. Derivatives of alkyne, cyclooctene used in iEDDA reaction, cyclooctyne used in SPOQC reaction, ortho-substituted triphenylphosphine group used in Staudinger-Bertozzi ligation reaction and nitrone used in Nitrone-alkyne cycloloaddition reaction things.

Table 1

Example 1

This embodiment provides a detection material for circulating tumor cells, which is a bio-orthogonal/anti-adhesion bifunctional chitosan coating. Among them, "bioorthogonal" refers to groups that can undergo bioorthogonal reactions, including eight-membered ring octynes and ortho-substituted triphenylphosphine groups used in Staudinger-Bertozzi ligation. "Anti-adhesion" refers to a functionalized chitosan coating that has both a hydrophilic chitosan structure and a hydrophobic bioorthogonal structure. The preparation method of bio-orthogonal/anti-adhesion bifunctional chitosan coating comprises the preparation of chitosan coating and the functional modification of chitosan coating, specifically as follows:

Preparation of chitosan coating: Dissolve 95% deacetylated chitosan in 90% acetic acid solution; 4000 rpm spin coating on the substrate with hydrophilic surface to obtain chitosan coating, or use A chitosan coating was prepared on a substrate with a hydrophilic surface by electrospinning with a No. 7 needle, 30s, and 16kv; the chitosan coating was dried at 60°C for 72 hours; the pH was adjusted so that the chitosan Amino exposed. Among them, glass, silicon wafer, mica, etc. can be selected as the base material with a hydrophilic surface.

Functional modification of chitosan coating: functionalized chitosan structure is shown in formula IA, IB, IC, ID, and its general synthesis method is: chitosan coating is mixed with ethanol containing 5% triethylamine NHS-activated esters containing A, B, C or D groups were reacted for 24 hours; washed 5 times with ethanol containing 5% triethylamine, 3 times with water containing 5% triethylamine, and 3 times with water; dried at 60°C for 1 Hour.

Wherein R has the structure shown in the following formula A, B, C or D,

In a specific embodiment, the functionalized chitosan structure is shown in formula IA, the bioorthogonal/anti-adhesion bifunctional group is dibenzocyclooctyne (DBCO), and the bio-orthogonal/anti-adhesion The reaction formula of bifunctional chitosan coating is as follows:

In a specific embodiment, the present invention provides a detector for circulating tumor cells, comprising a substrate and the above-mentioned detection material for circulating tumor cells immobilized on the substrate. The substrate is selected from microfluidic chip substrates, magnetic nanoparticle material substrates and other material substrates that can be hydrophilized.

Example 2

This embodiment provides a detection material for circulating tumor cells, which is a bio-orthogonal/anti-adhesion/reversible release trifunctional chitosan coating. Among them, "bioorthogonal" refers to groups that can undergo bioorthogonal reactions, including eight-membered ring octynes and ortho-substituted triphenylphosphine groups used in Staudinger-Bertozzi ligation. "Anti-adhesion" refers to a functionalized chitosan coating having both a hydrophilic chitosan structure and a hydrophobic bioorthogonal structure. "Reversible release" refers to having a reversible cleavage group, thereby releasing captured circulating tumor cells. The preparation method of bio-orthogonal/anti-adhesion/reversible release three-functional chitosan coating includes preparation of chitosan coating and functional modification of chitosan coating, specifically as follows:

Preparation of chitosan coating: Dissolve 95% deacetylated chitosan in 90% acetic acid solution; 2.4000 rpm spin coating on the substrate with hydrophilic surface to obtain chitosan coating, or use A chitosan coating was prepared on a substrate with a hydrophilic surface by electrospinning with a No. 7 needle, 30s, and 16kv; the chitosan coating was dried at 60°C for 72 hours; the pH was adjusted so that the chitosan Amino exposed. Among them, glass, silicon wafer, mica, etc. can be selected as the base material with a hydrophilic surface.

Functional modification of chitosan coating: functionalized chitosan structure is shown in formula IIA, IIB, IIC, IID, and its general synthetic method is: chitosan coating is mixed with ethanol containing 5% triethylamine The coupling molecule SPDP was reacted for 24 hours; washed 5 times with ethanol containing 5% triethylamine, washed 3 times with water containing 5% triethylamine, and washed 3 times with water; dried at 60°C for 1 hour; Molecules of groups A, B, C or D were reacted for 24 hours; washed 5 times with ethanol and 5 times with water; dried at 60°C for 1 hour.

Wherein, R has the structure shown in the following formula A, B, C or D,

In a specific embodiment, the functionalized chitosan structure is as shown in formula IIA, the reversible cleavage group is a disulfide bond, and the bioorthogonal/anti-adhesion bifunctional group is dibenzocyclooctyne (DBCO) , the reaction formula of the bio-orthogonal/anti-adhesion/reversible release trifunctional chitosan coating is as follows:

In a specific embodiment, the present invention provides a detector for circulating tumor cells, comprising a substrate and the above-mentioned detection material for circulating tumor cells immobilized on the substrate. The substrate is selected from microfluidic chip substrates, magnetic nanoparticle material substrates and other material substrates that can be hydrophilized.

Example 3

This example provides the whole process of liver cancer CTC detection based on bio-orthogonal/anti-adhesion dual-functional chitosan coating. The method is carried out step by step (Figure 1A). Specific steps are as follows:

Clinical blood sample pretreatment: Collect 5ml of patient blood from an anticoagulant tube, use density gradient centrifugation to initially enrich CTCs, and use red blood cell lysate to remove residual red blood cells.

Metabolic glycoengineering artificial labeling: use 100uM Ac ₄ ManAz for 48 hours of metabolic glycoengineering, and the treatment method refers to Chinese patent CN113358865A.

Co-incubation of the detector and the sample: In 1ml of DMEM medium, co-incubate the metabolic glycoengineering sample with a detector of about ^200mm2 (stand still or flow slowly), and wash away other blood that does not adhere to the detector components, resulting in CTCs adhering to the detector surface.

The hydrophilized glass microfluidic chip is used as the substrate, and the functionalized chitosan bio-coating is carried out. The functionalized chitosan structure is shown in formula IA, and a bio-orthogonal/anti-adhesion dual-functional detection device is obtained. The results of capturing CTCs of patients with liver cancer are shown in Table 2:

Table 2

serial number	gender	age)	type	Capture result (piece/5ml)
1	male	32	healthy	0
2	male	44	healthy	0
3	female	26	healthy	0
4	male	54	Phase IIa	twenty three

5	female	63	Phase IIIc	46
6	female	49	Phase IV	57
7	male	58	Phase IV	61

Example 4

The whole process of liver cancer CTC detection based on bio-orthogonal/anti-adhesion/reversible release chitosan coating, the whole detection process is based on "clinical blood sample pretreatment-artificial labeling of metabolic sugar engineering-co-incubation of detector and sample-reversible release Liver cancer CTC" approach was carried out step by step (Fig. 1B). Specific steps are as follows:

Clinical blood sample pretreatment: Collect 5ml of patient blood from an anticoagulant tube, use density gradient centrifugation to initially enrich CTCs, and use red blood cell lysate to remove residual red blood cells.

Glycoengineering artificial labeling: use 100uM Ac ₄ ManAz for 48 hours of glycoengineering. Metabolic sugar engineering treatment, the treatment method refers to Chinese patent CN113358865A.

Co-incubation of the detector and the sample: In 1ml of DMEM medium, co-incubate the metabolic glycoengineering sample with a detector of about ^200mm2 (stand still or flow slowly), and wash away other blood that does not adhere to the detector components, resulting in CTCs adhering to the detector surface.

Reversible release of liver cancer CTCs: the detector was soaked in 10 mM dithiothreitol in PBS solution for 30 minutes, and then the surface of the detector was washed with PBS to obtain released liver cancer CTCs.

In another specific embodiment, the reversible reactive group can be cleaved by modulating the electrical signal and/or temperature of the functionalized surface of the trapped circulating tumor cells, or by selecting boronate as the release molecule, as shown in Figure 2. Show.

The hydrophilized glass microfluidic chip is used as the substrate, and the functionalized chitosan coating is carried out. The functionalized chitosan structure is shown in formula IIA, and the bioorthogonal/anti-adhesion/reversible release three Functional function detector, the results of capturing and releasing CTCs of patients with liver cancer are shown in Table 3:

table 3

serial number	gender	age)	type	Release result (piece/5ml)
1	male	32	healthy	0
2	male	44	healthy	0
3	female	26	healthy	0
4	male	54	Phase IIa	19
5	female	63	Phase IIIc	43
6	female	49	Phase IV	54

7

male

58

Phase IV

59

Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims (10)

1. A detection material for circulating tumor cells, characterized in that the detection material is fixed on a substrate, and the detection material is chitosan modified by a functional group;

   (1) The functionalized group includes a bioorthogonal reactive group; or,

   (2) The functional groups include bio-orthogonal reactive groups and reversible reactive groups.
2. The detection material according to claim 1, wherein (1) when the functionalized group includes a bio-orthogonal reactive group, the detection material has the structure shown in the following formula I,

   

   The R is a bioorthogonal reactive group;

   Preferably, the R has the structure shown in the following formula A, B, C or D,

   

   (2) When the functionalized group includes a bioorthogonal reactive group and a reversible reactive group, the reversible reactive group is a disulfide bond;

   Preferably, the reversible reactive group is used as a connecting arm to connect chitosan and a bioorthogonal reactive group;

   Preferably, the detection material has the structure shown in the following formula II,

   

   The R is a bioorthogonal reactive group;

   Preferably, the R has the structure shown in the following formula A, B, C or D,

   
3. The detection material according to claim 1 or 2, wherein the substrate has a hydrophilic surface;

   Preferably, the substrate is selected from hydrophilized glass, silicon wafer or mica.
4. A detector for circulating tumor cells, characterized in that the detector comprises a base and the detection material according to any one of claims 1-3 fixed on the base.
5. The detector according to claim 4, wherein the substrate has a hydrophilic surface;

   Preferably, the substrate is selected from microfluidic chip substrates, magnetic nanoparticle material substrates and other material substrates that can be hydrophilized.
6. A detection kit for circulating tumor cells, characterized in that the detection kit includes the detection material according to any one of claims 1-3 or the detector according to claim 4 or 5 and a sugar unit-based labeling molecule;

   Preferably, the detection kit also includes a release molecule;

   Preferably, the release molecule is dithiothreitol and/or borate;

   Preferably, the sugar unit-based labeling molecule is selected from one or more of 2-aminomannose units, 2-galactosamine units, 2-glucosamine units and 6-azido-L-fucose units. kind;

   The 2-aminomannose unit, 2-galactosamine unit, 2-glucosamine unit and 6-azido-L-fucose unit have the structure shown in the following formula,

   

   Wherein, X=(CH ₂ ) _n .
7. The detection material for circulating tumor cells according to any one of claims 1-3, the detector for circulating tumor cells according to claim 4 or 5, or the detection kit for circulating tumor cells according to claim 6 in the detection of circulating tumor cells application;

   Preferably, the circulating tumor cells are liver cancer circulating tumor cells.
8. A method for preparing a detection material for circulating tumor cells according to any one of claims 1-3, characterized in that it comprises the following steps:

   Preparation of chitosan coating: prepare chitosan coating on the surface of the base material, adjust the pH, so that the chitosan amino group is exposed;

   Functional modification of chitosan coating: linking bio-orthogonal reactive groups or bio-orthogonal reactive groups and reversible reactive groups to chitosan molecules;

   Preferably, the acetic acid solution of chitosan is evenly spread on the surface of the base material, and the pH is adjusted after drying to obtain the chitosan coating;

   Preferably, the acetic acid solution of chitosan is evenly spread on the surface of the base material by spin coating or electrospinning;

   Preferably, the chitosan has a deacetylation degree of 95%;

   Preferably, the volume percentage of the acetic acid solution is 90%.
9. The preparation method according to claim 8, wherein when the functionalized group includes a bioorthogonal reactive group, the functional modification method of the chitosan coating is that the chitosan coating contains 5% Reaction of NHS activated ester containing bio-orthogonal reactive groups in ethanol of triethylamine for 24 hours; washing with ethanol containing 5% triethylamine for 5 times, washing with water containing 5% triethylamine for 3 times, washing with water for 3 times; 60 ℃ drying for 1 hour;

   When the functionalized group includes a bio-orthogonal reactive group and a reversible reactive group, the functional modification method of the chitosan coating is that the chitosan coating is coupled with Molecular SPDP was reacted for 24 hours; washed 5 times with ethanol containing 5% triethylamine, washed 3 times with water containing 5% triethylamine, and washed 3 times with water; dried at 60°C for 1 hour; Molecular reaction of cross-reactive groups for 24 hours; wash with ethanol 5 times, wash with water 5 times; dry at 60°C for 1 hour.
10. A method for detecting circulating tumor cells, comprising the steps of:

    Samples are treated with marker molecules based on sugar units, and the marker molecules are artificially labeled with circulating tumor cells through metabolic glycoengineering;

    Co-incubating the circulating tumor cells with the detection material described in any one of claims 1-3 or the detector described in claim 4 or 5, the bio-orthogonal reaction group and the marker molecule undergo a bio-orthogonal reaction, and the circulating tumor cells are captured ;

    Preferably, when the functional group includes a bio-orthogonal reaction group and a reversible reaction group, the detection method further includes releasing the captured circulating tumor cells, specifically: by injecting the captured circulating tumor cells Adding release molecules or regulating the electrical signal and/or temperature of the functionalized surface of the captured circulating tumor cells to break the reversible reactive group and release the captured circulating tumor cells;

    Preferably, the release molecule is dithiothreitol and/or borate;

    Preferably, the circulating tumor cells are liver cancer circulating tumor cells.

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