CN114478828A - Detection material, detector and detection method for circulating tumor cells - Google Patents
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Abstract
The invention discloses a detection material, a detector and a detection method of circulating tumor cells, wherein the detection material of the circulating tumor cells is fixed on a substrate, and the detection material is chitosan modified by functional groups; the functional group comprises a bio-orthogonal reactive group; or include bio-orthogonal reactive groups and reversible reactive groups. The detector for the circulating tumor cells comprises a substrate and the detection material fixed on the substrate. The novel material for detecting the circulating tumor cells can play a biorthogonal/anti-adhesion dual function or a biorthogonal/anti-adhesion/reversible release function according to different modification degrees, and the chitosan replaces gold to be used as a functionalized surface, so that the cost is reduced; the bioorthogonal chitosan surface has good anti-adhesion effect, the detection precision is improved, and the probability of false positive detection results is reduced.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a detection material, a detector and a detection method for circulating tumor cells.
Background
In 1869, the concept of Circulating Tumor Cells (CTCs) was first discovered and proposed by Ashworth, an australian physician, in the blood of breast cancer patients. In 1976 Nowell revised the definition of CTC to: derived from primary or metastatic tumors, acquire the ability to detach from the basement membrane and invade tumor cells that enter the blood vessels through the tissue matrix. CTC is now a generic term for the various types of tumor cells present in peripheral blood that grow and spread throughout the various stages of tumor growth.
Liver cancer is one of the six common tumors, with the first three fatality rates among all cancers. Early diagnosis and treatment are difficult points for clinically dealing with liver cancer treatment. Early screening is beneficial to prolonging the survival time of liver cancer patients, and early diagnosis is also beneficial to adopting various treatment modes, thereby improving prognosis. However, only about 35% of patients with liver cancer can be treated effectively in the early stage clinically, and only a few biomarkers can be used as the evaluation index of liver cancer, among which Alpha Fetoprotein (AFP) is the most widely used biomarker. However, AFP has certain limitations, such as a high proportion of false positive results, lack of sufficient sensitivity and specificity.
The CTC detection method (CN113358865A) based on the bioorthogonal metabolic sugar engineering marker realizes the accurate and nondestructive detection of CTC through the detection process of 'marker-capture-release'. The method comprises the steps of firstly designing a CTC marking process based on metabolic sugar engineering, processing a sample by utilizing a marker molecule based on a sugar unit, and artificially marking CTC by the marker molecule through the metabolic sugar engineering; secondly, designing and synthesizing capture molecules simultaneously containing bioorthogonal groups and reversible reaction groups, preparing a detection device integrating capture/release functions by fixing the capture molecules on a device substrate (such as a microfluidic chip, a magnetic nanoparticle material and the like) through reversible reaction, and capturing CTC by performing bioorthogonal reaction with CTC surface marker groups; finally, release molecules are designed to be introduced, and captured CTCs can be released by exchanging capture molecules immobilized on the substrate of the detection device through a reversible reaction.
The existing circulating tumor cell detection material based on the bioorthogonal reaction is obtained by directly carrying out small molecule bioorthogonal modification on gold serving as a substrate. However, due to the existence of tumor heterogeneity, the adhesion of the liver cancer CTC on the bio-orthorhombic gold surface is weak, the nonspecific adhesion is strong, and the detection effect of the bio-orthorhombic gold surface in the CTC detection method (CN113358865A) of the bio-orthorhombic glycoengineering marker is poor and the false positive is high when the liver cancer is detected. The key to overcoming the technical bottleneck of the detection of the liver cancer CTC is to find a functionalized surface suitable for the detection of the liver cancer CTC.
Chitosan is an N-deacetylation product of chitin, 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, anti-tumor effect, etc.
In recent years, because the chitosan coating has more modifiable sites, the chitosan coating is functionally modified, and thus, researches on application of the chitosan coating to aspects of basic medicine, disease treatment, medical detection and the like are increasing.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a detection material, a detector and a detection method for circulating tumor cells. The invention provides a material for CTC detection and a preparation method and a using method thereof. The novel CTC detector is mainly prepared by performing substrate surface coating-functionalized modification on commercially available chitosan, and is finally applied to CTC detection. The chitosan coating which is uniform and suitable for downstream application is prepared on the surface coating of the substrate by methods of spin coating, electrostatic spinning and the like; based on the characteristic that chitosan molecules have amino groups which exist regularly and are easy to modify, the required reversible reaction connecting arm and the bioorthogonal/anti-adhesion bifunctional group are gradually connected to the amino site of the chitosan through a chemical method; based on the principle of 'high specificity affinity and low non-specificity affinity', the accuracy of CTC detection is finally improved.
The invention provides a detection material for circulating tumor cells, which is fixed on a substrate and is chitosan modified by functional groups;
(1) the functional group comprises a bio-orthogonal reactive group; or the like, or, alternatively,
(2) the functional groups include bio-orthogonal reactive groups and reversible reactive groups.
Further, (1) when the functional group comprises a bio-orthogonal reaction group, the detection material has a structure shown in the following formula I,
the R is a bio-orthogonal reactive group;
preferably, R has a structure represented by formula A, B, C or D,
(2) when the functional group comprises a bio-orthogonal reactive group and a reversible reactive group, the reversible reactive group is a disulfide bond;
preferably, the reversible reactive group serves as a linking arm to link the chitosan and the bio-orthogonal reactive group;
preferably, the detection material has a structure shown in the following formula II,
the R is a bio-orthogonal reactive group;
preferably, R has a structure represented by 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 wafers or mica.
In a second aspect, the invention provides a detector of 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 a microfluidic chip substrate, a magnetic nanoparticle material substrate and other material substrates capable of being subjected to hydrophilization treatment.
In a third aspect, the invention provides a detection kit for circulating tumor cells, which comprises the detection material or the detector and a sugar unit-based labeling molecule;
preferably, the detection kit further comprises a release molecule;
preferably, the releasing molecule is dithiothreitol and/or a borate;
preferably, the sugar unit-based labeling molecule is selected from one or more of a 2-amino mannose unit, a 2-amino galactose unit, a 2-amino glucose unit and a 6-azide-L-fucose unit;
the 2-amino mannose unit, the 2-amino galactose unit, the 2-amino glucose unit and the 6-azido-L-fucose unit have the following structures,
wherein X is (CH)2)n. Preferably, n is 1, 2, 3, 4, 5, 6.
The fourth aspect of the invention provides the use of the material for detecting circulating tumor cells, the detector for circulating tumor cells or the detection kit for circulating tumor cells in circulating tumor cell detection;
preferably, the circulating tumor cell is a circulating tumor cell of liver cancer.
The fifth aspect of the present invention provides a method for preparing the material for detecting circulating tumor cells, comprising the following steps:
preparing a chitosan coating: preparing a chitosan coating on the surface of a substrate material, and adjusting the pH value to expose chitosan amino;
functional modification of the chitosan coating: connecting a bio-orthogonal reaction group or a bio-orthogonal reaction group and a reversible reaction group on a chitosan molecule;
preferably, uniformly spreading acetic acid solution of chitosan on the surface of the substrate material, drying and adjusting pH to obtain a chitosan coating;
preferably, the acetic acid solution of chitosan is uniformly spread on the surface of the substrate material by means of spin coating or electrostatic spinning;
preferably, the chitosan has a degree of deacetylation of 95%;
preferably, the volume percentage of the acetic acid solution is 90%.
In the technical scheme of the invention, when the functionalized groups comprise bio-orthogonal reactive groups, the chitosan coating is functionally modified by reacting the chitosan coating with NHS activated ester containing the bio-orthogonal reactive groups in ethanol containing 5% of triethylamine for 24 hours; 5 times of ethanol washing with 5% triethylamine, 3 times of water washing with 5% triethylamine, and 3 times of water washing; drying for 1 hour at 60 ℃;
when the functional groups comprise bio-orthogonal reaction groups and reversible reaction groups, the functional modification method of the chitosan coating is that the chitosan coating reacts with coupling molecules SPDP in ethanol containing 5% of triethylamine for 24 hours; 5 times of ethanol washing with 5% triethylamine, 3 times of water washing with 5% triethylamine, and 3 times of water washing; drying for 1 hour at 60 ℃; reacting with thiol-modified bio-orthogonal reactive group-containing molecules in ethanol for 24 hours; washing with ethanol for 5 times, and washing with water for 5 times; drying for 1 hour at 60 ℃.
The sixth aspect of the present invention provides a method for detecting circulating tumor cells, comprising the steps of:
processing a sample by using a marker molecule based on a sugar unit, and artificially marking the circulating tumor cells by the marker molecule through metabolic sugar engineering;
co-incubating the circulating tumor cells with the detection material or the detector, wherein a bio-orthogonal reaction group and a marker molecule generate a bio-orthogonal reaction, and the circulating tumor cells are captured;
preferably, when the functionalized groups include bio-orthogonal reactive groups and reversible reactive groups, the detection method further comprises releasing the captured circulating tumor cells, in particular: cleaving the reversible reactive group to release the captured circulating tumor cell by adding a release molecule to the captured circulating tumor cell or by regulating an electrical signal and/or temperature of the functionalized surface of the captured circulating tumor cell;
preferably, the releasing molecule is dithiothreitol and/or a borate;
preferably, the circulating tumor cell is a circulating tumor cell of liver cancer.
The invention has the beneficial effects that:
1. the invention provides a novel material for detecting circulating tumor cells, which is chitosan modified by functional groups, and can play the roles of bioorthogonality/anti-adhesion or bioorthogonality/anti-adhesion/reversible release according to different modification degrees, thereby meeting the aim of detecting different CTC (cytotoxic T cell) such as CTC counting, CTC downstream physiological and biochemical analysis and the like. Its main advantage is as follows:
(1) the chitosan replaces gold to be used as the functionalized surface, so that the cost is reduced.
(2) The bioorthogonal chitosan surface has good anti-adhesion effect, the detection precision is improved, and the probability of false positive detection results is reduced.
(3) The chitosan coating can be uniformly spread on almost all hydrophilic surfaces in a dissolving and drying mode, and detectors with different physical shapes and properties can be conveniently prepared. Compared with a gold surface, the detector has the advantages of mild preparation conditions, wide universality and better effect.
2. The material for detecting the circulating tumor cells provided by the invention utilizes the physicochemical property of bioorthogonal chitosan to improve the adhesion of the liver cancer CTC to the material and reduce the non-specific adhesion of leukocytes on the liver cancer CTC, thereby improving the detection effect, reducing the false positive rate and filling the blank of a liver cancer circulating tumor cell detection technology.
Drawings
FIG. 1 is a diagram of the overall process of the detection of CTC in liver cancer according to the present invention.
FIG. 2 is a method of cleaving a reversibly reactive group.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
"bioorthogonal" refers to having groups that can undergo bioorthogonal reactions. At present, bioorthogonal reactions are shown in table 1, wherein any one of the reactive groups can be used as a bioorthogonal group for the functional modification of chitosan coating, including dibenzocyclooctyne used in SPAAC reaction, cyclooctene used in iedd reaction, cyclooctyne used in SPOQC reaction, ortho-substituted triphenylphosphine group used in Staudinger-bertozizi ligation reaction, and derivatives of Nitrone used in Nitrone-alkyne cyclic addition reaction.
TABLE 1
Example 1
This example provides a circulating tumor cell detection material that is a bioorthogonal/anti-adhesion bifunctional chitosan coating. Wherein "bioorthogonal" refers to groups having bioorthogonal reactivity, including eight-membered cyclooctyne 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. The preparation method of the bioorthogonal/anti-adhesion bifunctional chitosan coating comprises the preparation of the chitosan coating and the functional modification of the chitosan coating, and comprises the following specific steps:
preparing a chitosan coating: dissolving chitosan with deacetylation degree of 95% in acetic acid solution of 90%; spin-coating the substrate with the hydrophilic surface at 4000 rpm to obtain a chitosan coating, or preparing the chitosan coating on the substrate with the hydrophilic surface by using a 7-gauge needle, and performing electrostatic spinning under the conditions of 30s and 16 kv; drying the chitosan coating for 72 hours at 60 ℃; and adjusting the pH value to expose the amino group of the chitosan. Among them, the substrate material having a hydrophilic surface may be selected from glass, silicon wafer, mica, and the like.
Functional modification of the chitosan coating: the structure of the functionalized chitosan is shown as formulas IA, IB, IC and ID, and the general synthesis mode is as follows: reacting the chitosan coating with NHS activated ester containing A, B, C or D group in ethanol containing 5% triethylamine for 24 hours; 5 times of ethanol washing with 5% triethylamine, 3 times of water washing with 5% triethylamine, and 3 times of water washing; drying for 1 hour at 60 ℃.
Wherein R has a structure represented by formula A, B, C or D,
in a specific embodiment, the functionalized chitosan has the structure shown in formula IA, the bio-orthogonal/anti-adhesion bifunctional group is Dibenzocyclooctyne (DBCO), and the reaction formula of the bio-orthogonal/anti-adhesion 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 fixed on the substrate. The substrate is selected from a microfluidic chip substrate, a magnetic nanoparticle material substrate and other material substrates capable of being subjected to hydrophilic treatment.
Example 2
The embodiment provides a detection material for circulating tumor cells, which is a bioorthogonal/anti-adhesion/reversible release tri-functional chitosan coating. Wherein "bioorthogonal" refers to groups having bioorthogonal reactivity, including eight-membered cyclooctyne 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. "reversibly released" refers to having a reversible cleavage group, thereby releasing the captured circulating tumor cells. The preparation method of the bioorthogonal/anti-adhesion/reversible release three-functional chitosan coating comprises the preparation of the chitosan coating and the functional modification of the chitosan coating, and comprises the following specific steps:
preparing a chitosan coating: dissolving chitosan with deacetylation degree of 95% in acetic acid solution of 90%; spin-coating the substrate with the hydrophilic surface at 2.4000 rpm to obtain a chitosan coating, or preparing the chitosan coating on the substrate with the hydrophilic surface by using a 7-gauge needle, and carrying out electrostatic spinning under the conditions of 30s and 16 kv; drying the chitosan coating for 72 hours at 60 ℃; and adjusting the pH value to expose the amino group of the chitosan. Among them, the substrate material having a hydrophilic surface may be selected from glass, silicon wafer, mica, and the like.
Functional modification of the chitosan coating: the structure of the functionalized chitosan is shown in formulas IIA, IIB, IIC and IID, and the general synthesis mode is as follows: reacting the chitosan coating with coupling molecules SPDP in ethanol containing 5% of triethylamine for 24 hours; 5 times of ethanol washing with 5% triethylamine, 3 times of water washing with 5% triethylamine, and 3 times of water washing; drying for 1 hour at 60 ℃; reacting with a sulfhydryl-modified molecule containing A, B, C or D groups in ethanol for 24 hours; washing with ethanol for 5 times, and washing with water for 5 times; drying for 1 hour at 60 ℃.
Wherein R has a structure represented by formula A, B, C or D,
in a specific embodiment, the functionalized chitosan has the structure shown in formula IIA, the reversible cleavage group is a disulfide bond, the bio-orthogonal/anti-adhesion bifunctional group is Dibenzocyclooctyne (DBCO), and the bio-orthogonal/anti-adhesion/reversible release trifunctional chitosan coating has the following reaction formula:
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 fixed on the substrate. The substrate is selected from a microfluidic chip substrate, a magnetic nanoparticle material substrate and other material substrates capable of being subjected to hydrophilic treatment.
Example 3
This example provides the whole detection process of hepatoma CTC based on bioorthogonal/anti-adhesion bifunctional chitosan coating, which is performed step by step in the manner of "pretreatment of clinical blood sample-artificial labeling of metabolic sugar engineering-co-incubation of detector and sample" (FIG. 1A). The method comprises the following specific steps:
pretreatment of clinical blood samples: blood of 5ml of a patient is collected by an anticoagulation tube, CTC is primarily enriched by using a density gradient centrifugation mode, and residual erythrocytes are removed by using erythrocyte lysate.
Artificial marking of metabolic sugar engineering: ac with 100uM4ManAz is subjected to 48-hour metabolic sugar engineering treatment, and the treatment method refers to Chinese patent CN 113358865A.
Detector and sample co-incubation: the metabolic glycoengineering samples were incubated with approximately 200mm in 1ml of DMEM medium2Detector co-incubation (standing or slow flow-through) washes away other blood components that do not adhere to the detector, resulting in CTCs adhering to the detector surface.
The method comprises the following steps of (1) performing functionalized chitosan biological coating treatment by using a hydrophilized vitreous micro-fluidic chip as a substrate, wherein the structure of the functionalized chitosan is shown as formula IA, preparing a bioorthogonal/anti-adhesion bifunctional detector, and capturing CTC of a liver cancer patient according to the results shown in Table 2:
TABLE 2
Example 4
The whole detection process is gradually carried out in a mode of 'clinical blood sample pretreatment-metabolic sugar engineering artificial marking-detector and sample co-incubation-liver cancer CTC reversible release' (fig. 1B). The method comprises the following specific steps:
pretreatment of clinical blood samples: blood of 5ml of a patient is collected by an anticoagulation tube, CTC is primarily enriched by using a density gradient centrifugation mode, and residual erythrocytes are removed by using erythrocyte lysate.
Artificial marking of metabolic sugar engineering: ac with 100uM4ManAz was subjected to a 48 hour metabolic glycoengineering treatment. The treatment method of metabolic sugar engineering refers to Chinese patent CN 113358865A.
Detector and sample co-incubation: the metabolic glycoengineering samples were incubated with approximately 200mm in 1ml of DMEM medium2Detector co-incubation (standing or slow flow-through) washes away other blood components that do not adhere to the detector, resulting in CTCs adhering to the detector surface.
Reversible release of liver cancer CTCs: the detector was soaked with 10mM dithiothreitol in PBS for 30 minutes, and then the detector surface was rinsed with PBS to obtain released hepatoma CTCs.
In another specific embodiment, the reversibly reactive groups can be cleaved by modulating the electrical signal and/or temperature of the functionalized surface of the captured circulating tumor cell, or by selecting boronates as releasing molecules, as shown in FIG. 2.
The functional chitosan coating treatment is carried out by taking the glass micro-fluidic chip subjected to hydrophilic treatment as a substrate, the structure of the functional chitosan is shown as formula IIA, the bioorthogonal/anti-adhesion/reversible release three-function detector is prepared, and the results of capturing and releasing CTC of a liver cancer patient are shown in Table 3:
TABLE 3
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. The detection material for the circulating tumor cells is characterized in that the detection material is fixed on a substrate and is chitosan modified by functional groups;
(1) the functional group comprises a bio-orthogonal reactive group; or the like, or, alternatively,
(2) the functional groups include bio-orthogonal reactive groups and reversible reactive groups.
2. The detection material of claim 1, wherein (1) when the functionalized group comprises a bio-orthogonal reactive group, the detection material has a structure represented by formula I,
the R is a bio-orthogonal reactive group;
preferably, R has a structure represented by formula A, B, C or D,
(2) when the functional group comprises a bio-orthogonal reactive group and a reversible reactive group, the reversible reactive group is a disulfide bond;
preferably, the reversible reactive group serves as a linking arm to link the chitosan and the bio-orthogonal reactive group;
preferably, the detection material has a structure shown in the following formula II,
the R is a bio-orthogonal reactive group;
preferably, R has a structure represented by 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 wafers or mica.
4. A detector of circulating tumor cells, comprising a substrate and the detection material according to any one of claims 1 to 3 immobilized on the substrate.
5. The detector of claim 4, wherein the substrate has a hydrophilic surface;
preferably, the substrate is selected from a microfluidic chip substrate, a magnetic nanoparticle material substrate and other material substrates capable of being subjected to hydrophilization treatment.
6. A detection kit for circulating tumor cells, comprising a detection material according to any one of claims 1 to 3 or a detector according to claim 4 or 5 and a sugar unit-based labeling molecule;
preferably, the detection kit further comprises a release molecule;
preferably, the releasing molecule is dithiothreitol and/or a borate;
preferably, the sugar unit-based labeling molecule is selected from one or more of a 2-amino mannose unit, a 2-amino galactose unit, a 2-amino glucose unit and a 6-azide-L-fucose unit;
the 2-amino mannose unit, the 2-amino galactose unit, the 2-amino glucose unit and the 6-azido-L-fucose unit have the following structures,
wherein X is (CH)2)n。
7. Use of the circulating tumor cell detection material according to any one of claims 1 to 3, the circulating tumor cell detector according to claim 4 or 5, or the circulating tumor cell detection kit according to claim 6 for circulating tumor cell detection;
preferably, the circulating tumor cell is a circulating tumor cell of liver cancer.
8. A method for preparing a circulating tumor cell detecting material according to any one of claims 1 to 3, comprising the steps of:
preparing a chitosan coating: preparing a chitosan coating on the surface of a substrate material, and adjusting the pH value to expose chitosan amino;
functional modification of the chitosan coating: connecting a bio-orthogonal reaction group or a bio-orthogonal reaction group and a reversible reaction group on a chitosan molecule;
preferably, uniformly spreading acetic acid solution of chitosan on the surface of the substrate material, drying and adjusting pH to obtain a chitosan coating;
preferably, the acetic acid solution of chitosan is uniformly spread on the surface of the substrate material by means of spin coating or electrostatic spinning;
preferably, the chitosan has a degree of deacetylation of 95%;
preferably, the volume percentage of the acetic acid solution is 90%.
9. The method according to claim 8, wherein when the functionalized group comprises a bio-orthogonal reactive group, the chitosan coating is functionally modified by reacting the chitosan coating with the NHS activated ester containing the bio-orthogonal reactive group in ethanol containing 5% triethylamine for 24 hours; 5 times of ethanol washing with 5% triethylamine, 3 times of water washing with 5% triethylamine, and 3 times of water washing; drying for 1 hour at 60 ℃;
when the functional groups comprise bio-orthogonal reaction groups and reversible reaction groups, the functional modification method of the chitosan coating is that the chitosan coating reacts with coupling molecules SPDP in ethanol containing 5% of triethylamine for 24 hours; 5 times of ethanol washing with 5% triethylamine, 3 times of water washing with 5% triethylamine, and 3 times of water washing; drying for 1 hour at 60 ℃; reacting with thiol-modified bio-orthogonal reactive group-containing molecules in ethanol for 24 hours; washing with ethanol for 5 times, and washing with water for 5 times; drying for 1 hour at 60 ℃.
10. A method for detecting circulating tumor cells, comprising the steps of:
processing a sample by using a marker molecule based on a sugar unit, and artificially marking the circulating tumor cells by the marker molecule through metabolic sugar engineering;
incubating circulating tumor cells with the detection material of any one of claims 1-3 or the detector of claim 4 or 5, wherein the bio-orthogonal reactive group is bio-orthogonally reactive with the labeling molecule, and the circulating tumor cells are captured;
preferably, when the functionalized groups include bio-orthogonal reactive groups and reversible reactive groups, the detection method further comprises releasing the captured circulating tumor cells, in particular: cleaving the reversible reactive group to release the captured circulating tumor cell by adding a release molecule to the captured circulating tumor cell or by modulating an electrical signal and/or temperature of the functionalized surface of the captured circulating tumor cell;
preferably, the releasing molecule is dithiothreitol and/or a borate;
preferably, the circulating tumor cell is a circulating tumor cell of liver cancer.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111494317.1A CN114478828B (en) | 2021-12-08 | 2021-12-08 | Detection material, detector and detection method for circulating tumor cells |
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| WO2023104059A1 (en) * | 2021-12-08 | 2023-06-15 | 深圳先进技术研究院 | Circulating tumor cell detection material, detector and detection method |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2503009A1 (en) * | 2011-03-25 | 2012-09-26 | Massimo Zollo | Biomarker for detecting circulating tumor cells and related detecting methods and kit |
| US20130197296A1 (en) * | 2012-01-13 | 2013-08-01 | Karl-Heinz Ott | Removing Cells from an Organism |
| US20150125879A1 (en) * | 2013-09-25 | 2015-05-07 | Massachusetts Institute Of Technology | Biodegradable Layer-by-Layer (LbL) Films for Cell Capture and Release |
| CN104619856A (en) * | 2012-05-29 | 2015-05-13 | 加利福尼亚大学董事会 | Systems, methods and components for isolating cells from a fluid sample |
| CN106148315A (en) * | 2015-04-14 | 2016-11-23 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of CTC based on chitin nanometer capture and purification substrate and preparation method thereof |
| CN106170304A (en) * | 2015-03-23 | 2016-11-30 | 北京大学深圳研究生院 | Bio-orthogonal reagent, activator and precursor and bio-orthogonal test kit |
| CN107641618A (en) * | 2016-07-22 | 2018-01-30 | 中国科学院苏州纳米技术与纳米仿生研究所 | The application of nanofiber substrate and preparation method thereof, nanofiber substrate |
| CN109187978A (en) * | 2018-08-10 | 2019-01-11 | 北京莱尔生物医药科技有限公司 | It is a kind of detect circulating tumor cell HER2, ER, PR immunofluorescent reagent box and its application |
| CN110101684A (en) * | 2019-05-15 | 2019-08-09 | 深圳先进技术研究院 | A kind of cellular membrane biomimetic nano particle and its preparation method and application of bio-orthogonal targeting |
| CN110133267A (en) * | 2019-06-18 | 2019-08-16 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | It is a kind of for adsorbing the carrier of tumour cell, preparation method, kit and application |
| CN110514489A (en) * | 2019-06-05 | 2019-11-29 | 南开大学 | A kind of biological coating and preparation method thereof of the specific whole blood capture circulating tumor cell for cancer surveillance |
| CN111093708A (en) * | 2017-04-07 | 2020-05-01 | 坦伯公司 | Bioorthogonal compositions |
| CN112011513A (en) * | 2020-09-04 | 2020-12-01 | 四川大学 | Method for capturing high-purity circulating tumor cells based on bioorthogonal chemical method |
| CN112014563A (en) * | 2020-08-27 | 2020-12-01 | 武汉大学 | Molecular beacon transmission system for directly detecting circulating tumor cells in blood, preparation method and application thereof |
| CN113358865A (en) * | 2020-03-03 | 2021-09-07 | 深圳先进技术研究院 | Circulating tumor cell detection method |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1243013C (en) * | 2004-03-19 | 2006-02-22 | 华东师范大学 | Adherence peptide specific to tissue of liver cancer, screen method combined between in vivo and in vitro, and usage |
| US11112410B2 (en) * | 2016-04-13 | 2021-09-07 | President And Fellows Of Harvard College | Methods for capturing, isolation, and targeting of circulating tumor cells and diagnostic and therapeutic applications thereof |
| CN112370537B (en) * | 2020-11-15 | 2023-08-11 | 大连理工大学 | Double-targeting magnetic fluorescent nanoparticle, preparation method thereof and application thereof in liver cancer circulating tumor cells |
| CN114478828B (en) * | 2021-12-08 | 2023-03-31 | 深圳先进技术研究院 | Detection material, detector and detection method for circulating tumor cells |
-
2021
- 2021-12-08 CN CN202111494317.1A patent/CN114478828B/en active Active
-
2022
- 2022-12-06 WO PCT/CN2022/137032 patent/WO2023104059A1/en unknown
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2503009A1 (en) * | 2011-03-25 | 2012-09-26 | Massimo Zollo | Biomarker for detecting circulating tumor cells and related detecting methods and kit |
| US20130197296A1 (en) * | 2012-01-13 | 2013-08-01 | Karl-Heinz Ott | Removing Cells from an Organism |
| CN104619856A (en) * | 2012-05-29 | 2015-05-13 | 加利福尼亚大学董事会 | Systems, methods and components for isolating cells from a fluid sample |
| US20150125879A1 (en) * | 2013-09-25 | 2015-05-07 | Massachusetts Institute Of Technology | Biodegradable Layer-by-Layer (LbL) Films for Cell Capture and Release |
| CN106170304A (en) * | 2015-03-23 | 2016-11-30 | 北京大学深圳研究生院 | Bio-orthogonal reagent, activator and precursor and bio-orthogonal test kit |
| CN106148315A (en) * | 2015-04-14 | 2016-11-23 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of CTC based on chitin nanometer capture and purification substrate and preparation method thereof |
| CN107641618A (en) * | 2016-07-22 | 2018-01-30 | 中国科学院苏州纳米技术与纳米仿生研究所 | The application of nanofiber substrate and preparation method thereof, nanofiber substrate |
| CN111093708A (en) * | 2017-04-07 | 2020-05-01 | 坦伯公司 | Bioorthogonal compositions |
| CN109187978A (en) * | 2018-08-10 | 2019-01-11 | 北京莱尔生物医药科技有限公司 | It is a kind of detect circulating tumor cell HER2, ER, PR immunofluorescent reagent box and its application |
| CN110101684A (en) * | 2019-05-15 | 2019-08-09 | 深圳先进技术研究院 | A kind of cellular membrane biomimetic nano particle and its preparation method and application of bio-orthogonal targeting |
| CN110514489A (en) * | 2019-06-05 | 2019-11-29 | 南开大学 | A kind of biological coating and preparation method thereof of the specific whole blood capture circulating tumor cell for cancer surveillance |
| CN110133267A (en) * | 2019-06-18 | 2019-08-16 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | It is a kind of for adsorbing the carrier of tumour cell, preparation method, kit and application |
| CN113358865A (en) * | 2020-03-03 | 2021-09-07 | 深圳先进技术研究院 | Circulating tumor cell detection method |
| CN112014563A (en) * | 2020-08-27 | 2020-12-01 | 武汉大学 | Molecular beacon transmission system for directly detecting circulating tumor cells in blood, preparation method and application thereof |
| CN112011513A (en) * | 2020-09-04 | 2020-12-01 | 四川大学 | Method for capturing high-purity circulating tumor cells based on bioorthogonal chemical method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023104059A1 (en) * | 2021-12-08 | 2023-06-15 | 深圳先进技术研究院 | Circulating tumor cell detection material, detector and detection method |
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| WO2023104059A1 (en) | 2023-06-15 |
| CN114478828B (en) | 2023-03-31 |
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