CN113358865A - Circulating tumor cell detection method - Google Patents

Circulating tumor cell detection method Download PDF

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CN113358865A
CN113358865A CN202010140696.3A CN202010140696A CN113358865A CN 113358865 A CN113358865 A CN 113358865A CN 202010140696 A CN202010140696 A CN 202010140696A CN 113358865 A CN113358865 A CN 113358865A
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reagent
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circulating tumor
tumor cells
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李伟
王怀雨
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Shenzhen Institute of Advanced Technology of CAS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
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    • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
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    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/50—Organo-phosphines
    • C07F9/505—Preparation; Separation; Purification; Stabilisation
    • C07F9/5054—Preparation; Separation; Purification; Stabilisation by a process in which the phosphorus atom is not involved
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract

The invention discloses a circulating tumor cell detection method, which accurately marks highly heterogeneous circulating tumor cells through metabolic sugar engineering independent of tumor cell types, captures the marked circulating tumor cells in a nondestructive way through bioorthogonal reaction, and releases the captured circulating tumor cells in a nondestructive way through reversible reaction, thereby realizing accurate and nondestructive detection of the circulating tumor cells.

Description

Circulating tumor cell detection method
Technical Field
The invention belongs to the technical field of circulating tumor cell detection methods, and particularly relates to a circulating tumor cell detection method based on bioorthogonal metabolic sugar engineering markers, namely 'marker-capture-release' three-stage CTC detection based on metabolic sugar engineering pathway markers, bioorthogonal reaction capture and reversible reaction release.
Background
Circulating Tumor Cells (CTCs) are cancer cells that fall off from a tumor site, enter and survive in the circulatory system, have strong fluidity and invasiveness, are easy to adhere to a blood vessel wall and cross, and cause metastasis and spread of the cancer cells, the presence of CTCs is an early marker event of cancer spread and metastasis, and detection of CTCs is of great significance for early detection of tumor micrometastasis, assessment of prognosis and curative effect, and individualized treatment of tumors.
The difficulty and the challenge of CTC detection are that cancer patients have small quantity and high heterogeneity of CTC in blood, 1mL of blood sample contains only 1-100 CTC, and about 10 CTC9Red blood cell and 106The CTCs of individual white blood cells are distributed in a small absolute quantity and low relative proportion, and in addition, the CTCs have high heterogeneity, and the CTCs of different tumor individuals have the differences and the diversity on genes and phenotypes, even if the CTCs in the same patient also always have heterogeneity and have diversified physiological and biochemical characteristic changes, and more than ten subtypes are classified according to CTC surface marker molecules, so that the accurate separation of the CTCs from the cells has great difficulty and challenge.
A series of CTC detection methods have been developed, and detection is mainly performed by using changes in the physiological and biochemical characteristics of CTCs, such as physical properties (cell size, density, deformability, and the like), tumor-related genes, surface specific antigens, and the like, and technologies such as nanomaterials and microfluidic chips are generally combined. In principle, the detection method based on the change of the physical properties of CTCs is most common in that the CTCs and the white blood cells are sorted by utilizing the cell size difference of the CTCs larger than the white blood cells through the separation processes of filtering, deterministic lateral displacement and the like, and the method is suitable for primary screening enrichment of the CTCs and cannot realize accurate detection due to the fact that the sizes of part of the CTCs and the white blood cells are overlapped; CTC detection based on tumor genes generally firstly cracks sample cells and extracts genetic materials, and then detects the genetic materials such as tumor-related DNR/RNA and the like through Polymerase Chain Reaction (PCR), wherein the cell cracking operation cannot count and capture cells to obtain complete CTC cells for downstream application expansion; CTC detection based on cell surface specific antigen is the mainstream detection method at present, and mainly utilizes specific antigen existing on the surface of CTC, fixes corresponding antibody on the substrate of a detection device, and captures CTC by immunological binding, for example, current CTC detection usually takes cell surface specific antigen Epithelial cell adhesion factor (EpCAM) as a detection target, CTC undergoes Epithelial-Mesenchymal Transition (EMT) during the process of entering blood circulation from the primary part of tumor, surface EpCAM level is reduced, invasion capability is enhanced, and enters blood circulation, and EpCAM molecule does not exist on the surface of leukocyte, so that EpCAM antibody can be used as capture molecule, and fixed on the surface of the detection device, CTC is captured and separated by immunological binding of antibody to the EpCAM on the surface of CTC, CTC apoptosis can be captured due to immunological reaction, and CTC has high heterogeneity as described above, there are more than ten subtypes classified according to their surface marker molecules, and detection targeting one or several cell surface antigens only covers a part of CTCs, often accompanied by high omission ratio and severe false negative interference, so detection of CTCs based on cell surface marker antigens can provide cell counting results, but is limited in terms of accuracy of detection results and non-destructive capture process.
The main reason for the insufficient detection precision and the inability to capture/release CTCs without damage in the current CTCs is that CTCs have high heterogeneity (heterogeneity), i.e., the CTCs of different tumor individuals have differences and diversity in gene and phenotype, and even in the same patient, the CTCs often show heterogeneity, so that a single physical or biochemical characteristic change index only covers a part of CTCs, and the precision and the damage of the current CTC detection method are limited. The key to developing accurate, non-destructive detection methods for CTCs is the development of broad-spectrum markers and/or capture methods that can satisfy CTC heterogeneity, and development of non-destructive capture/release methods based thereon.
The invention provides a brand-new CTC detection method based on bioorthogonal metabolic sugar engineering markers, which accurately marks highly heterogeneous CTCs through metabolic sugar engineering independent of tumor cell types, carries out lossless capture on the marked CTCs through bioorthogonal reaction, and carries out lossless release on the captured CTCs through reversible reaction, thereby realizing accurate and lossless detection of the CTCs.
Disclosure of Invention
The invention aims to provide a CTC detection method based on bioorthogonal metabolic sugar engineering markers, which realizes accurate and nondestructive detection of CTC mainly through a 'marker-capture-release' detection process. 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.
In order to achieve the above purpose, the invention provides the following technical scheme: a method for detecting circulating tumor cells, comprising the following steps:
1) marking the circulating tumor cells by metabolic sugar engineering;
2) capturing the labeled circulating tumor cells by bioorthogonal reaction;
3) the captured circulating tumor cells are released by a reversible reaction.
Further, the marking in the step 1) refers to a process of marking the circulating tumor cells by using marked sugar molecules to process the sample through metabolic sugar engineering;
the labeled sugar molecule is one of compounds shown in a formula I, a formula II, a formula III and a formula IV or a composition thereof,
Figure BDA0002398974280000031
in the formula I, R1is-H, R2Is composed of
Figure BDA0002398974280000032
R3is-OAc, R4is-H or R1Is composed of
Figure BDA0002398974280000033
R2is-H, R3is-OAc, R4is-H or R1Is composed of
Figure BDA0002398974280000034
R2is-H, R3is-H, R4is-OAc; x is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; r5is-N3 or
Figure BDA0002398974280000035
Figure BDA0002398974280000036
In the formula II, X is- (C)2H4O)m-, where m is 0,1,2,3,4,5,6, 7; r5is-N3 or
Figure BDA0002398974280000037
Figure BDA0002398974280000038
In the formula III, X is- (C)2H4O)m-, where m is 0,1,2,3,4,5,6, 7; r5is-N3 or
Figure BDA0002398974280000039
Figure BDA0002398974280000041
In the formula IV, X is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; r5is-N3 or
Figure BDA0002398974280000042
The sugar unit types used for marking in the invention comprise amino site substituted 2-amino mannose, 2-amino galactose, 2-amino glucose, 6-substituted-L fucose, sialic acid, 6-substituted-2-amino mannose and other sugar unit types, all of which can be metabolized and converted into tumor cell surface sialic acid units after being taken in by tumor cells, and corresponding R5 groups can be reserved.
Further, the preparation method of the compound of the formula I comprises the following steps: dissolving 2-aminosugar hydrochloride in ethanol, adding sodium bicarbonate, stirring for 5-30 min, adding an acylation reagent to form an amido bond, evaporating to remove the solvent, dissolving the residue in N, N-dimethylformamide, adding a substitution reagent, and stirring overnight (12 hours) at 50-70 ℃ to obtain an intermediate; dissolving the intermediate in pyridine, adding acetic anhydride, and reacting at room temperature for 3-12 hours to obtain a compound shown in a formula I; the hydrochloric acid 2-aminosugar is hydrochloric acid 2-amino mannose, hydrochloric acid 2-glucosamine, hydrochloric acid 2-galactosamine; the acylating agent is
Figure BDA0002398974280000043
Wherein LG is1Is Cl, Br, I, OTs; LG (Ligno-lead-acid)2Is Cl, Br, X is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; the substitution reagent is an azide reagent or R5-X-H/base composition, said base being sodium hydroxide, potassium hydroxide, sodium hydride, said azidation reagent being sodium azide, potassium azide, lithium azide, tetrabutylammonium azide, ammonium azide, trimethylsilane azide/MF composition, M being Li+,Na+，K+，Cs+，NH4+，Bu4N+(ii) a The molar ratio of the hydrochloric acid 2-aminosugar, the sodium bicarbonate, the acylation reagent and the substitution reagent is 1: 1-1.2: 1.1-2: 2-5, wherein the molar ratio of the intermediate to the acetic anhydride is 1: 5-20 parts of; the substituting reagent is R5In the case of-X-H/base combination, R5-the molar ratio of X-H to base is 1: 1-1.3;
the preparation method of the compound of the formula II comprises the following steps: adding L-galactose into acetone, and dehydrating under the action of a dehydrating agent to generate 1, 2; 3, 4-bis-O-isopropylidene- α -L-galactose; then dissolving the mixture in dichloromethane, and reacting the mixture with a sulfonylation reagent under the action of a catalyst to obtain a sulfonic ester intermediate; the substitution reagent is substituted with the sulfonate intermediateReacting to obtain 1 and 2; 3, 4-bis-O-isopropylidene-6-R5An X-alpha-L-galactose intermediate; suspending the intermediate in acetonitrile/water mixed solvent, and obtaining 6-R under the action of acid catalyst5An X-L-galactose intermediate; dissolving the intermediate in pyridine, adding acetic anhydride, and reacting for 3-12 hours to obtain a compound shown in a formula II; the dehydrating agent is concentrated sulfuric acid, copper sulfate, zinc chloride and phosphoric acid; the catalyst is pyridine, triethylamine, isopropyl diethylamine, diisopropyl ethylamine, tri-N-propylamine, tri-N-butylamine, N-methylmorpholine and tetramethyl ethylenediamine; the sulfonylation reagent is methanesulfonyl chloride, p-toluenesulfonyl chloride or trifluoromethanesulfonic anhydride; the substitution reagent is an azide reagent or R5-X-H/base composition, said base being sodium hydroxide, potassium hydroxide, sodium hydride, said azidation reagent being sodium azide, potassium azide, lithium azide, tetrabutylammonium azide, ammonium azide, trimethylsilane azide/MF composition, M being Li+,Na+，K+，Cs+，NH4+，Bu4N+(ii) a The acid catalyst is dilute hydrochloric acid, dilute sulfuric acid, p-toluenesulfonic acid, trifluoroacetic acid and methanesulfonic acid; the molar ratio of the L galactose to the dehydrating agent is 1: 3-10, said 1, 2; the mol ratio of the 3, 4-bi-O-isopropylidene-alpha-L-galactose to the catalyst to the sulfonylation reagent is 1: 2-4: 1.5-2, wherein the molar ratio of the sulfonate intermediate to the substitution reagent is 1:1.1-3, said 1, 2; 3, 4-bis-O-isopropylidene-6-R5The molar ratio of the X-alpha-L-galactose intermediate to the acid catalyst is 1: 0.01 to 0.1, said 6-R5The molar ratio of the X-L-galactose intermediate to the acetic anhydride is 1: 5-30; the substituting reagent is R5In the case of-X-H/base combination, R5-the molar ratio of X-H to base is 1: 1-1.3;
the preparation method of the compound of the formula III comprises the following steps: dissolving amino mannose hydrochloride in ethanol, adding sodium bicarbonate, stirring for 5-30 minutes, adding an acetylation reagent to form an amido bond, evaporating to remove the solvent, dissolving the residue in pyridine, adding trityl chloride, stirring for 0.5-4 hours, and continuously adding the acetylation reagent to obtain a 2-N-acetyl-1, 2, 4-tri-O-acetyl-6-O-trityl-amino mannose intermediate; dissolving the intermediate in methanol, adding acid catalyst, and stirring 15After about 60 minutes, evaporating to remove the solvent, dissolving the residue in dichloromethane, and reacting with a sulfonylation reagent under the action of an alkali catalyst to obtain a sulfonate intermediate; dissolving the substitution reagent and the sulfonate intermediate in tetrahydrofuran, and reacting overnight (12 hours) to obtain a compound shown in a formula III; the acetylation reagent is acetic anhydride, acetyl bromide and acetyl chloride; the acid catalyst is dilute hydrochloric acid, dilute sulfuric acid, p-toluenesulfonic acid, trifluoroacetic acid and methanesulfonic acid; the base catalyst is pyridine, triethylamine, isopropyl diethylamine, diisopropyl ethylamine, tri-N-propylamine, tri-N-butylamine, N-methylmorpholine and tetramethyl ethylenediamine; the sulfonylation reagent is methanesulfonyl chloride, p-toluenesulfonyl chloride or trifluoromethanesulfonic anhydride; the substitution reagent is an azide reagent or R5-X-H/base composition, said base being sodium hydroxide, potassium hydroxide, sodium hydride, said azidation reagent being sodium azide, potassium azide, lithium azide, tetrabutylammonium azide, ammonium azide, trimethylsilane azide/MF composition, M being Li+,Na+，K+，Cs+，NH4+，Bu4N+(ii) a The mol ratio of the amino mannose hydrochloride to the sodium bicarbonate to the acetylation reagent to the trityl chloride to the acetylation reagent is 1: 1-1.2: 1-1.5: 1.2-3: 5-30; the molar ratio of the 2-N-acetyl-1, 2, 4-tri-O-acetyl-6-O-trityl-amino mannose intermediate to the acid catalyst to the base catalyst to the sulfonating reagent is 1: 0.1-0.5: 1.3-2: 1-1.5; the molar ratio of the sulfonate intermediate to the substitution reagent is 1: 1.1-3; the substituting reagent is R5In the case of-X-H/base combination, R5-the molar ratio of X-H to base is 1: 1-1.3.
Further, the capturing in the step 2) refers to a process of capturing and fixing the circulating tumor cells by utilizing a bio-orthogonal reaction between detection molecules fixed on the substrate of the detection device and the surface marker groups of the circulating tumor cells;
the detection molecule is a compound shown as a formula V,
Figure BDA0002398974280000061
in the formula VR6is-N3Or
Figure BDA0002398974280000062
Figure BDA0002398974280000063
Wherein R is6And R5Correspond to when R5When it is azide, R6Is a cyclooctyne group, a dibenzocyclooctyne group, a triphenylphosphine group, when R is5When the group is cyclooctyne group, dibenzocyclooctyne group or triphenylphosphine group, R6Is azido, when R5When it is a tetrazole group, R6Is a cycloheptene group when R5When cycloheptene, R6Is a tetrazole group, when R5When it is a quinonyl group, R6Is a cyclooctyne group, when R5When it is a cyclooctyne group, R6Is a quinone group, when R5When it is propargyl, R6Is a nitrone group, when R5When it is a nitrone group, R6Is propargyl; r7is-SH; n1 is 0,1,2,3,4,5,6,7, 8.
The invention provides a biological orthogonal reaction based on SPAAC reaction, R5、R6Are paired groups in the SPAAC reaction, such as azido and cyclooctyne groups, azido and dibenzocyclooctyne groups; also given are 1,2,4, 5-tetraazaphenyl and trans-cycloheptenyl (iedd reaction), 1,2,4, 5-tetraazaphenyl and cyclopropenyl (iedd reaction), 1, 2-benzoquinonyl and cyclooctyne cyclopropyl (SPOQC reaction), azido and substituted triphenylphosphino (Staudinger-Bertozzi ligation), alkynyl and Nitrone groups (Nitrone-alkyne cycloaddition reaction), and the like.
Further, R6 is-N3When the compound is a group, a cyclooctyne group, a dibenzocyclooctyne group, a nitrone group, a propargyl group, a cycloheptene group, a tetrazole group and a 1, 2-benzoquinone group, the general preparation method of the compound shown in the formula V comprises the following steps: TsO-PEGn1+1Preparation of — Ts (n1 ═ 0,1,2,3,4,5,6,7, 8): HO-PEGn1+1-H, PEG is- (C)2H5Dissolving O) -group in dichloromethane, adding p-toluenesulfonyl chloride, stirring at room temperature under the action of alkali catalystStirring for 4-12 hours; the base catalyst is triethylamine, isopropyl diethylamine, diisopropyl ethylamine, tri-N-propylamine, tri-N-butylamine, N-methylmorpholine and tetramethyl ethylenediamine; the HO-PEGn1+1-H, p-toluenesulfonyl chloride, base catalyst in a molar ratio of 1: 3-5: 2-5;
monosubstituted R6-(C2H4O)n1-C2H4-Ots intermediate preparation: r6-OH in tetrahydrofuran, R6Adding alkali into cyclooctyne group, nitrone group, propargyl group, cycloheptene group, tetrazole group and 1, 2-benzoquinone group, stirring at 50-70 ℃ for 30-120 minutes, and adding TsO-PEGn1+1-Ts, stirring for 2-12 hours to obtain an intermediate, wherein the added base is sodium hydroxide, potassium hydroxide or sodium hydride, and R is6-OH, base, TsO-PEGn1+1-Ts in a molar ratio of 1: 1-1.2: 1; alternatively, TsO-PEGn1+1-Ts in tetrahydrofuran and MR is added6M is Li+、Na+、K+、NH4 +、Bu4N+，R6is-N3Stirring the solution at 50-70 ℃ overnight, wherein the TsO-PEG is preparedn1+1-Ts、MR6In a molar ratio of 1: 1;
dissolving the intermediate in acetonitrile, adding potassium thioacetate, and stirring for 2-12h to obtain disubstituted R6-(C2H4O)n1-C2H4-an OSAc intermediate; dissolving the disubstituted intermediate in methanol, and obtaining a compound shown in a formula V under the action of a sodium methoxide catalyst; the R is6The molar ratio of-OH, potassium thioacetate and sodium methoxide is 1: 2-4: 0.01-0.05;
R6in the case of triphenylphosphino, the compound of formula V is prepared by: TsO-PEGn1+1Preparation of H (n1 ═ 0,1,2,3,4,5,6,7, 8): HO-PEGn1+1-H, PEG is- (C)2H5Dissolving an O) -group in dichloromethane, adding p-toluenesulfonyl chloride, and stirring at room temperature for 2-12 hours under the action of an alkali catalyst; the alkali catalyst is: triethylamine, isopropyl diethylamine, diisopropyl ethylamine, tri-N-propylamine, tri-N-butylamine, N-methylmorpholine, tetramethyl ethylene glycolAn amine; the HO-PEGn1+1-H, p-toluenesulfonyl chloride, base catalyst in a molar ratio of 1: 0.25-0.5: 0.5 to 1;
monosubstituted R6-(C2H4O)n1-C2H4-Ots intermediate preparation: TsO-PEGn1+1-H in pyridine, adding R6-LG3Stirring for 2-12 hours; LG (Ligno-lead-acid)3Is Cl, Br, TsO-PEGn1+1-H and R6-LG3In a molar ratio of 1: 1.2-2;
dissolving the intermediate in acetonitrile, adding potassium thioacetate, and stirring for 2-12h to obtain disubstituted R6-(C2H4O)n1-C2H4-an OSAc intermediate; dissolving the disubstituted intermediate in methanol, and obtaining a compound shown in a formula V under the action of a sodium methoxide catalyst; the R is6-(C2H4O)n1-C2H4-OTs, potassium thioacetate in a molar ratio of 1: 2-4.
Further, the detection device comprises a device substrate layer, a surface metal gold plating layer and a surface molecular layer, wherein the surface molecular layer consists of detection molecules and sulfhydryl polyethylene glycol blocking molecules, S-Au bonds are formed between sulfydryl and gold atoms on the surface molecular structure and are fixed on the surface of the surface metal gold plating layer, and the device substrate layer is selected from device substrates which have a modifiable metal gold layer surface structure and can be applied to single cell capture/release.
Further, the device substrate layer is selected from devices or materials commonly used for CTC detection, such as microfluidic chips, magnetic nanoparticles, gold nanorods, composite nanomaterials, and the like.
Further, the construction method of the detection device comprises the following steps: mixing detection molecules with mercaptopolyethylene glycol according to a molar ratio of 90: 10-1: 99, preparing an aqueous solution with a molar concentration of 0.1-10 mM, soaking the device substrate with the surface metal gold coating in the aqueous solution for 1-12 hours, or allowing the aqueous solution to flow through the device substrate with the surface metal gold coating at a flow rate of 0.1-0.5 mL/min for 15-90 minutes, and finally washing the device with PBS buffer solution for later use.
Further, the release in the step 3) refers to a process of releasing the circulating tumor cells by utilizing small molecule release molecules to exchange and fix the detection molecules on the substrate of the detection device through a reversible reaction;
the small molecule release molecules are reduced glutathione, dithioerythrol and dithiothreitol.
A detection kit for circulating tumor cells comprises a labeled sugar molecule, a cell culture solution, a density gradient separation solution, a PBS buffer solution, a detection device and a small molecule release molecule;
the labeled sugar molecule is one of compounds shown in a formula I, a formula II, a formula III and a formula IV or a composition thereof,
Figure BDA0002398974280000081
in the formula I, R1is-H, R2Is composed of
Figure BDA0002398974280000082
R3is-OAc, R4is-H or R1Is composed of
Figure BDA0002398974280000083
R2is-H, R3is-OAc, R4is-H or R1Is composed of
Figure BDA0002398974280000084
R2is-H, R3is-H, R4is-OAc; x is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; r5is-N3 or
Figure BDA0002398974280000085
Figure BDA0002398974280000086
In the formula II, X is- (C)2H4O)m-, where m is 0,1,2,3,4,5,6, 7; r5is-N3 or
Figure BDA0002398974280000087
Figure BDA0002398974280000088
In the formula III, X is- (C)2H4O)m-, where m is 0,1,2,3,4,5,6, 7; r5is-N3 or
Figure BDA0002398974280000089
Figure BDA0002398974280000091
In the formula IV, X is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; r5is-N3 or
Figure BDA0002398974280000092
The density gradient separation liquid is a lymph separation liquid;
the detection device comprises a device substrate layer, a surface metal gold plating layer and a surface molecular layer, wherein the surface molecular layer consists of detection molecules and sulfhydryl polyethylene glycol blocking molecules, an S-Au bond is formed between a sulfhydryl group on a surface molecular structure and a gold atom and is fixed on the surface of the surface metal gold plating layer, and the device substrate layer is selected from a device substrate which has a modifiable metal gold layer surface structure and can be applied to single cell capture/release;
the detection molecule is a compound shown as a formula V,
Figure BDA0002398974280000093
in the formula V, R6is-N3Or
Figure BDA0002398974280000094
Figure BDA0002398974280000095
Wherein R is6And R5Correspond to when R5When it is azide, R6Is a cyclooctyne group, a dibenzocyclooctyne group, a triphenylphosphine group, when R is5When the group is cyclooctyne group, dibenzocyclooctyne group or triphenylphosphine group, R6Is azido, when R5When it is a tetrazole group, R6Is a cycloheptene group when R5When cycloheptene, R6Is a tetrazole group, when R5When it is a quinonyl group, R6Is a cyclooctyne group, when R5When it is a cyclooctyne group, R6Is a quinone group, when R5When it is propargyl, R6Is a nitrone group, when R5When it is a nitrone group, R6Is propargyl; r7is-SH; n1 is 0,1,2,3,4,5,6,7, 8;
the small molecule release molecules are reduced glutathione, dithioerythrol and dithiothreitol.
Further, the using method comprises the following steps:
1) artificially marking circulating tumor cells, dissolving and diluting one compound or a combination of compounds in marked sugar molecules by using a cell culture solution to prepare a marked culture solution with the total concentration of the marked sugar molecules being 1-100 uM; taking peripheral blood to be detected, carrying out gradient density centrifugation to obtain erythrocytes, then incubating in a marking culture solution, washing to remove marker molecules which are not taken in, and obtaining an artificially marked circulating tumor cell sample;
2) capturing the circulating tumor cells based on bioorthogonal reaction, flushing the detection device with PBS buffer solution at the flow rate of 0.1-0.5 ml/min for 15-30 minutes, then enabling the artificially marked circulating tumor cell sample in the step 1) to flow through the detection device at the same flow rate, continuing flushing the device with PBS buffer solution at the same flow rate for 5-15 minutes, and obtaining the cells immobilized on the surface of the detection device after reaction as the captured circulating tumor cells;
3) based on the release of circulating tumor cells by reversible reaction, dissolving release molecules by PBS buffer solution to prepare 0.01-0.1M release solution, enabling the release solution to flow through the detection device at the flow rate of 0.02-0.1ml/min for 5-15 minutes, discarding the eluate, then flushing the detection device at the flow rate of 0.1-0.5 ml/min for 5-15 minutes, exchanging the detection molecules fixed on the surface of the detection device through reversible reaction, releasing the circulating tumor cells, and collecting the eluate, thus obtaining the released circulating tumor cells.
The invention carries out artificial marking on CTC through a metabolic sugar engineering (MGE) process, carries out nondestructive capture on the marked CTC through bioorthogonal reaction, carries out nondestructive release on the captured CTC through reversible reaction, and carries out accurate and nondestructive detection on the CTC through the detection process design of 'marking-capturing-releasing'. Compared with the prior art, the invention has the following advantages:
1) compared with the traditional mainstream 'capture-release' two-stage detection process for detecting CTC based on a cell surface natural marker target, the manual marking step can greatly improve the coverage rate of the detection target, so that the overall detection accuracy is greatly improved;
2) the method is characterized in that a CTC artificial labeling method is provided, a blood sample to be detected is processed by using a labeled sugar molecule according to an MGE principle, and the CTC is artificially labeled, and compared with the conventional mainstream detection method based on a CTC surface natural marker target, the artificial labeling method is a broad-spectrum labeling method independent of tumor cell types, can label and cover more types and more numbers of CTC, and thus the detection accuracy is greatly improved;
3) in the MGE marking process, micromolecule marked sugar molecules are used for carrying out metabolic sugar engineering marking on CTC, the micromolecule marked sugar molecules have structural diversity, and the marked molecules can be prepared in a large amount and high efficiency only through reaction in multiple steps, so that the availability is high, and the economical efficiency is good;
4) the marking process can be controlled by marking conditions, and the marking effect is effectively regulated and controlled, so that accurate marking of CTC is realized, and the artificial marking method has sufficient controllability;
5) the method for capturing the CTC in a nondestructive way is provided, the CTC is captured by using a bioorthogonal reaction, and compared with the capturing process of using CTC surface marker antigen molecule immune combination reaction in the current mainstream method, the method can keep the cell activity of the CTC before and after capturing and realize the nondestructive capturing of the CTC;
6) in the capturing process, the small molecular compound and a CTC surface marker group are used for carrying out bio-orthogonal reaction to capture CTC, and compared with the conventional mainstream method which uses a corresponding antibody biomacromolecule of a CTC surface antigen, the small molecular compound only needs to contain a specific reaction group, has structural diversity, can be controllably reacted in multiple steps, is efficient and prepared in large quantities, and has high availability and good economy;
7) the detection device utilized in the capturing process is prepared by simple reaction of small molecules and a device substrate, and can be controllably and efficiently constructed by controlling reaction conditions;
8) the method for nondestructive release of CTC utilizes commercial sulfhydryl-containing biological micromolecules to exchange detection molecules fixed on a device substrate through simple reversible reaction, and the biological micromolecules have no influence on cell activity, are commercialized, can be directly purchased, are very cheap and easily obtained, and have very good economy; the controllability of the involved reversible exchange reaction is high.
Drawings
FIG. 1 is a flow chart of a method for detecting circulating tumor cells;
FIG. 2 is a schematic diagram of a detection apparatus and a construction method;
FIG. 3 is a flowchart of preparation of a detecting unit according to example 8;
FIG. 4 is a flowchart of the preparation of a detecting unit in example 9.
Detailed Description
The invention provides a CTC detection method based on bio-orthogonal metabolic sugar engineering markers, which comprises the following general technical scheme: 1) according to the metabolic sugar engineering principle, the labeled sugar molecules are utilized to process the primarily enriched blood sample, and the CTC is artificially labeled; 2) designing a detection molecule, fixing the detection molecule on a detection device substrate, and capturing CTC by performing bio-orthogonal reaction on the detection molecule and a CTC surface marker group; 3) the design introduces release molecules, and exchanges the detection molecules fixed on the device substrate through a reversible reaction to release CTC. The design of a three-stage CTC detection process of 'mark-capture-release' is shown in figure 1, wherein 'mark' refers to the process of marking CTC by using a marked sugar molecule and processing a sample through MGE; "capture" refers to the process of capturing and immobilizing CTCs by bio-orthogonal reaction of the detection molecules immobilized on the substrate of the detection device with the CTC surface marker groups; "Release" refers to the process of releasing CTC by exchanging the detector molecules immobilized on the substrate of the detection device through a reversible reaction using small molecules to release the molecules.
The protocol for each step is detailed as follows:
1) metabolic glyco-engineering markers for CTC
Taking peripheral blood to be detected, removing red blood cells after gradient density centrifugation to obtain a primary enrichment sample, processing the sample by using a marked sugar molecule containing a bioorthogonal reaction group, and expressing the marked group on the surface of CTC through an MGE process after the CTC intakes the marked sugar molecule to obtain the CTC sample marked by the bioorthogonal reaction group.
2) Bio-orthogonal reaction capture of CTCs
Designing and synthesizing capture/release bifunctional detection molecules containing bio-orthogonal reaction groups and reversible reaction groups on the structure, and constructing a detection device integrating capture/release functions after the molecules are fixed on the surface of a device substrate (such as a microfluidic chip) through reversible reaction, wherein the detection device and the construction method are shown in figure 2; enabling the marked sample to flow through a detection device, and performing bio-orthogonal reaction on detection molecules on a detection device substrate and CTC surface marker groups to capture marked CTCs;
3) reversible reaction release of CTC
The step is mainly carried out according to a reversible reaction principle, release molecules structurally containing the same reversible reaction group with the detection molecules are introduced according to the reversible reaction types of the detection molecules and a device substrate in the construction process of the detection device, and the captured CTC is released through reversible reaction and exchange of the detection molecules fixed on the device substrate. The main process of the step is as follows: the introduction of commercial release small molecules, passing their aqueous solution through a capture device, through a reversible reaction, exchanges capture molecules immobilized on the device substrate, releasing the captured CTCs.
The invention is described in further detail below with reference to specific figures and examples.
EXAMPLE 1 preparation of tagged molecules
Figure BDA0002398974280000121
Dissolving amino mannose hydrochloride (1.0g,4.64mmol) in ethanol (10mL), adding sodium bicarbonate (0.4g,4.77mmol), stirring for 15min, adding chloroacetyl chloride (0.63g,5.56mmol), stirring at room temperature for 1 h, evaporating the solvent, dissolving the residue in N, N-dimethylformamide DMF (10mL), adding sodium azide (0.6g,9.28mmol), stirring in a 50 ℃ oil bath overnight, cooling the reaction solution to room temperature, removing the solvent, dissolving the residue in ethanol (10mL), filtering off insoluble substances, evaporating the filtrate to dryness to obtain an intermediate crude product (0.91g, 74.8%), ESI-MS m/z calcd for [ C ] C8H15N4O6]+(M+H)+:263.23；found:263.24；
Intermediate (0.91g,3.47mmol) was dissolved in pyridine (5mL), acetic anhydride (2mL) was added to the ice bath, the mixture was stirred overnight at room temperature, methanol (5mL) was added, the solvent was evaporated, and the residue was dissolved in dichloromethane DCM (20mL) and washed with 1N HClaq.、sat.NaHCO3The reaction mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the objective compound (1.48g, 99%).
Example 2 preparation of tagged molecules
Figure BDA0002398974280000131
H2SO4(1mL) is added with Acetone (30mL) in a dropwise manner in an ice bath, L-galactose (1g,5.55mmol) is added in batches, the mixture is stirred for 4 hours at room temperature until the mixture is completely dissolved, saturated NaOH solution is added to adjust the solution to be neutral, insoluble substances are filtered out, and the filtrate is concentrated to obtain an intermediate (1.4g, 97%);
the intermediate compound (1.4g,5.38mmol) was dissolved in dichloromethane (10mL) and TsCl (1.54g,8.07mmol) was added followed by Et3N (1.09g,10.76mmol), stirring overnight at room temperature, adding methanol (1mL), washing the solution with saturated brine, drying over anhydrous sodium sulfate, concentrating, and purifying to obtain intermediate (2.1g, 94%);
dissolving the intermediate compound (2.1g,5.07mmol) in DMF (10mL), adding sodium azide (0.66g,10.14mmol), stirring overnight in an oil bath at 50 ℃, cooling the reaction solution to room temperature, adding DCM (50mL), washing with saturated saline, collecting the organic phase, drying with anhydrous sodium sulfate, concentrating and purifying to obtain an intermediate (1.3g, 90%);
the intermediate compound (1.3g,4.56mmol) was suspended in a mixed solvent of acetonitrile/water (v/v ═ 1:1,15mL), and catalyst p-toluenesulfonic acid TsOH was added, reacted overnight under reflux, Et was added3Neutralizing the reaction liquid to be neutral by N, and evaporating the solvent to obtain an intermediate (0.93g, 99%);
the intermediate compound (0.93g,4.53mmol) was dissolved in pyridine (5mL), acetic anhydride (2mL) was added slowly, stirred overnight at room temperature, methanol (2mL) was added, after stirring for 20 min, the solvent was evaporated, the residue was dissolved in DCM (15mL) and washed with 1N HClaq.、sat.NaHCO3The reaction mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the objective compound (1.7g, 99%).
Example 3 preparation of tagged molecules
Figure BDA0002398974280000132
Dissolving amino mannose hydrochloride (1.0g,4.64mmol) in ethanol (10mL), adding sodium bicarbonate (0.4g,4.77mmol), stirring for 15min, adding acetic anhydride (0.91g,5.10mmol), stirring at room temperature for 1 hr, evaporating solvent to obtain crude N-acetamido mannose intermediate (0.95g, 92.6%), ESI-MS m/z calcd for [ C ] C8H16NO6]+(M+H)+:222.09；found:221.10
Dissolving N-acetamidomanmannose intermediate (0.95g,4.30mmol) in pyridine (10mL), adding trityl chloride (1.8g,6.44mmol), stirring at room temperature for 3 hours, adding acetic anhydride (4.38g,42.95mmol) to react to obtain 2-N-acetyl-1, 2, 4-tri-O-acetyl-6-O-trityl-aminommannose intermediate (2.1g, 82.9%), ESI-MS m/z calcd for [ C-1, 2, 4-tri-O-acetyl-6-O-trityl-aminommannose intermediate (2.1g, 82.9%)33H36NO9]+(M+H)+:590.23；found:590.23
The intermediate obtained in the above step (2.1g,3.56mmol) was dissolved in methanol (20mL), trifluoroacetic acid (70mg, 0.71mmol) was added, and the reaction was stirred for 30 minutes to give 2-N-acetyl-1, 2, 4-tri-O-acetyl-aminomannnose intermediate (1.1g, 88.9%), ESI-MS m/z calcd for [ C14H22NO9]+(M+H)+:348.12；found:348.13；
The intermediate (1.1g,3.17mmol) obtained in the above step was dissolved in dichloromethane (15mL), pyridine (0.5g,6.33mmol) was added, trifluoromethanesulfonic anhydride (1.34g,4.75mmol) was added dropwise under ice bath, and reaction was carried out for 15 minutes to obtain a sulfonate intermediate (1.45g, 95.5%), ESI-MS m/z calcd for [ C15H21F3NO11S]+(M+H)+:480.07；found:480.08；
Dibenzocyclooctynol (0.8g,3.63mmol) was dissolved in tetrahydrofuran (10ml), sodium hydride (60%) (0.15g,3.78mmol) was added, and after reaction in an oil bath at 50 ℃ for 1 hour, the sulfonic acid ester intermediate (1.45g,3.03mmol) obtained in the above step was added, and the reaction was continued for 12 hours to obtain the objective compound (1.48g, 89%), ESI-MS m/z calcd for [ C ] C30H32NO9]+(M+H)+:550.20；found:549.21；
Example 4 preparation of test molecules
Figure BDA0002398974280000141
Tetraethylene glycol (1g, 5.15mmol) was dissolved in DCM (20mL) and TsCl (2.94g,15.45mmol) was added followed by Et3N (2.34g,23.17mmol), stirring overnight at room temperature, adding methanol (2mL), washing the solution with saturated brine, drying over anhydrous sodium sulfate, concentrating, purifying to obtain intermediate (2.5g, 96.7%);
dibenzocyclooctynol (0.55g,2.5mmol) was dissolved in tetrahydrofuran (15mL), sodium hydride (72mg, 3.0mmol) was added, the mixture was refluxed in an oil bath at 50 ℃ for 1 hour, the intermediate compound (2.5g,4.97mmol) was added, the reaction was allowed to stand overnight, the reaction mixture was cooled to room temperature, the solvent was distilled off, and the intermediate (2.3g, 84%) was obtained after purification by ESI-MS m/z calcd for [ C ] -31H35O7S]+(M+H)+:551.21；found:551.22；
The intermediate compound (2.3g,4.18mmol) was dissolved in acetonitrile (20mL), KSAc (0.96g,8.4mmol) was added, stirred for 2 hours, the solvent was evaporated off, the residue was dissolved in DCM (20mL), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give an intermediate (1.8g, 95%), ESI-MS m/z calcd for [ C ]26H31O5S]+(M+H)+:455.18；found:455.19；
Dissolving the intermediate compound (1.8g,3.96mmol) in methanol (10mL), adding sodium methoxide, adjusting the pH of the solution to 8-10, stirring for 30min, and adding H+Neutralizing the reaction solution with cationic resin to neutrality, filtering off resin, evaporating solvent to obtain target compound (1.6g, 99%), ESI-MS m/z calcd for [ C ]24H29O4S]+(M+H)+:413.18；found:413.17.
Example 5 preparation of test molecules
Figure BDA0002398974280000153
The procedure is as in example 4 except that the benzocyclooctynol reagent is replaced with a cyclooctyne-cyclopropanemethanol reagent, ESI-MS m/z calcd for [ C ]18H31O4S]+(M+H)+:343.19；found:343.19。。
Example 6 preparation of test molecules
Figure BDA0002398974280000151
The procedure is as in example 4 for detection of molecules except that tetraethylene glycol is replaced by triethylene glycol, the benzocyclooctynol/sodium hydride reagent combination is replaced by sodium azide reagent, ESI-MS m/z calcd for [ C ]6H14N3O2S]+(M+H)+:191.07；found:191.08。
Example 7 preparation of test molecules
Figure BDA0002398974280000152
Tetraethylene glycol (1g, 5.15mmol) was dissolved in DCM (20mL) and TsCl (0.24g,1.29mmol) was added followed by Et3N (0.26g,2.57mmol), stirred at room temperature overnight, methanol (2mL) was added, the solution was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified to give an intermediate (0.35g, 78%);
the intermediate (0.35g, 78%) obtained in the above step was dissolved in pyridine (5mL), triphenylphosphine oxide acid chloride (0.51g,1.51mmol) was added, stirring was carried out overnight, the solvent was distilled off, the residue was washed with 1N HCla. q. and then dried over anhydrous sodium sulfate, and concentrated to obtain an intermediate (0.63g, 96%), ESI-MS m/z calcd for [ C.sub.C. ]34H38O9PS]+(M+H)+:653.19；found:653.20；
The intermediate compound obtained in the above step was dissolved in acetonitrile (10mL), KSAc (0.22g,1.93mmol) was added, stirring was carried out for 2 hours, the solvent was distilled off, the residue was dissolved in DCM (20mL), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain an intermediate (0.5g, 93%), ESI-MS m/z calcd for [ C29H34O7PS]+(M+H)+:557.17；found:557.16；
Dissolving the intermediate compound obtained in the previous step in methanol (10mL), adding sodium methoxide, adjusting the pH of the solution to 8-10, stirring for 30min, and adding H+Neutralizing the reaction solution with cationic resin to neutrality, filtering off resin, evaporating solvent to obtain target compound (0.46g, 99%), ESI-MS m/z calcd for [ C ]27H32O6PS]+(M+H)+:515.16；found:515.17.
EXAMPLE 8 preparation of detection device
The compound prepared in example 4 and commercial mercaptotetraethylene glycol were mixed in a molar ratio of 1:2, dissolved in ultrapure water to prepare a solution with a total concentration of 1mM, flowed through a microfluidic chip with a microfluidic channel at a flow rate of 0.5ml/min for 15min, then the chip was rinsed with ultrapure water at the same flow rate for 30min, and then rinsed with PBS buffer for 30min to prepare a detection chip device with a microfluidic channel surface modified with the compound prepared in example 3, with the flow chart shown in fig. 3.
EXAMPLE 9 preparation of detection device
The compound prepared in example 5 and commercial mercaptotetraethylene glycol were mixed in a molar ratio of 1:2, dissolved in ultrapure water to prepare a solution having a total concentration of 1mM, flowed through a microfluidic chip having a microcolumn at a flow rate of 0.5ml/min for 15min, then the chip was rinsed with ultrapure water at the same flow rate for 30min, and then the chip was rinsed with PBS buffer for 30min to prepare a detection chip device having a surface of the microcolumn modified with the compound prepared in example 4, as shown in fig. 4.
Example 10 CTC sample assay
1) Taking 2ml of peripheral blood of a cancer patient (left lung cancer, male, 60 years old and IV stage) by using an EDTA (ethylene diamine tetraacetic acid) anticoagulant tube, fully and uniformly mixing the peripheral blood with sterile PBS according to a ratio of 1:1, slowly superposing the peripheral blood on a layered liquid surface of a centrifuge tube containing lymphocyte separating liquid Ficoll, keeping a clear interface, horizontally centrifuging the mixture for 400g for 30 minutes, dividing the mixture into three layers in the centrifuge tube, and taking a narrow band component of a white cloud layer at the interface of an upper layer and a middle layer to obtain a primary enrichment sample;
the labeled molecules prepared in example 1 were dissolved in cell culture media and prepared into 50uM labeled media, the collected primary enrichment samples were incubated in the labeled media for 30 minutes, the labeled media were removed by centrifugation, and the cells were redispersed in the cell culture media to obtain MGE labeled samples;
2) the labeled sample was passed through the detection device prepared in example 9 at a flow rate of 0.2ml/min, and the device was washed with PBS buffer at the same flow rate for 15 minutes, and the cells fixed to the interface of the device were labeled cells containing highly labeled CTC and lowly labeled leukocytes;
3) using reduced glutathione GSH as a release molecule, dissolving the release solution with PBS buffer solution to prepare 0.1M release solution, flowing the release solution through the detection device at the flow rate of 0.05mL/min for 10 minutes, discarding a part of eluate (the part of eluate contains low-labeled leukocytes), then flowing the part of eluate through the detection device at the flow rate of 0.2mL/min for 10 minutes, collecting the part of eluate, and counting to obtain 22 cells, wherein the released cells are captured CTCs, and the CTCs detected in the patient are 22/2 mL.
EXAMPLE 11 partial examination of different samples
The invention is applied to detect the blood of cancer patients with different cancer types, sexes and stages, and the detection result is as follows according to the general detection flow by taking the blood of healthy people as a contrast:
numbering Sex Age (year of old) Type (B) Labeling molecules Detection device Results (2/2 mL)
1 For male 35 Healthy person Example 1 Example 8 0
2 Woman 42 Healthy person Example 2 Example 9 0
3 For male 56 Stage II Lung cancer Example 1 Example 8 13
4 For male 60 Stage IV of left-sided Lung cancer Example 2 Example 8 22
5 Woman 55 Stage IIa breast cancer Example 1 Example 8 9
6 Woman 68 Stage IIIc of colon cancer Example 1 Example 8 8
7 Woman 73 Stage IIb cervical cancer Example 1 Example 9 10
8 For male 70 Stage IIIb of colon cancer Example 1 Example 9 9
9 Woman 43 Breast cancer stage IV Example 1 Example 9 18
10 For male 72 Stage IV Lung cancer Example 2 Example 9 20
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims (10)

1. A method for detecting circulating tumor cells, comprising the steps of:
1) marking the circulating tumor cells by metabolic sugar engineering;
2) capturing the labeled circulating tumor cells by bioorthogonal reaction;
3) the captured circulating tumor cells are released by a reversible reaction.
2. The method for detecting circulating tumor cells according to claim 1, wherein the labeling in step 1) refers to a process of labeling circulating tumor cells by metabolic sugar engineering of the sample using labeled sugar molecules;
the labeled sugar molecule is one of compounds shown in a formula I, a formula II, a formula III and a formula IV or a composition thereof,
Figure FDA0002398974270000011
in the formula I, R1is-H, R2Is composed of
Figure FDA0002398974270000012
R3is-OAc, R4is-H or R1Is composed of
Figure FDA0002398974270000013
R2is-H, R3is-OAc, R4is-H or R1Is composed of
Figure FDA0002398974270000014
R2is-H, R3is-H, R4is-OAc; x is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; r5is-N3Or
Figure FDA0002398974270000015
Figure FDA0002398974270000016
In the formula II, X is- (C)2H4O)m-, where m is 0,1,2,3,4,5,6, 7; r5is-N3Or
Figure FDA0002398974270000017
Figure FDA0002398974270000021
In the formula III, X is- (C)2H4O)m-, where m is 0,1,2,3,4,5,6, 7; r5is-N3Or
Figure FDA0002398974270000022
Figure FDA0002398974270000023
In the formula IV, X is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; r5is-N3Or
Figure FDA0002398974270000024
3. The method for detecting circulating tumor cells of claim 2, wherein the compound of formula I is prepared by: dissolving 2-aminosugar hydrochloride in ethanol, adding sodium bicarbonate, stirring for 5-30 min, adding an acylation reagent to form an amido bond, evaporating to remove the solvent, dissolving the residue in N, N-dimethylformamide, adding a substitution reagent, and stirring for 12 hours at 50-70 ℃ to obtain an intermediate; dissolving the intermediate in pyridine, adding acetic anhydride, and reacting at room temperature for 3-12 hours to obtain a compound shown in a formula I; the saltThe acid 2-aminosugar is hydrochloric acid 2-amino mannose, hydrochloric acid 2-glucosamine, hydrochloric acid 2-galactosamine; the acylating agent is
Figure FDA0002398974270000025
Wherein LG is1Is Cl, Br, I, OTs; LG (Ligno-lead-acid)2Is Cl, Br, X is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; the substitution reagent is an azide reagent or R5-X-H/base composition, said base being sodium hydroxide, potassium hydroxide, sodium hydride, said azidation reagent being sodium azide, potassium azide, lithium azide, tetrabutylammonium azide, ammonium azide, trimethylsilane azide/MF composition, M being Li+,Na+，K+，Cs+，NH4+，Bu4N+(ii) a The molar ratio of the hydrochloric acid 2-aminosugar, the sodium bicarbonate, the acylation reagent and the substitution reagent is 1: 1-1.2: 1.1-2: 2-5, wherein the molar ratio of the intermediate to the acetic anhydride is 1: 5-20 parts of; the substituting reagent is R5In the case of-X-H/base combination, R5-the molar ratio of X-H to base is 1: 1-1.3;
the preparation method of the compound of the formula II comprises the following steps: adding L-galactose into acetone, and dehydrating under the action of a dehydrating agent to generate 1, 2; 3, 4-bis-O-isopropylidene- α -L-galactose; then dissolving the mixture in dichloromethane, and reacting the mixture with a sulfonylation reagent under the action of a catalyst to obtain a sulfonic ester intermediate; carrying out substitution reaction on a substitution reagent and a sulfonate intermediate to obtain 1, 2; 3, 4-bis-O-isopropylidene-6-R5An X-alpha-L-galactose intermediate; suspending the intermediate in acetonitrile/water mixed solvent, and obtaining 6-R under the action of acid catalyst5An X-L-galactose intermediate; dissolving the intermediate in pyridine, adding acetic anhydride, and reacting for 3-12 hours to obtain a compound shown in a formula II; the dehydrating agent is concentrated sulfuric acid, copper sulfate, zinc chloride and phosphoric acid; the catalyst is pyridine, triethylamine, isopropyl diethylamine, diisopropyl ethylamine, tri-N-propylamine, tri-N-butylamine, N-methylmorpholine and tetramethyl ethylenediamine; the sulfonylation reagent is methanesulfonyl chloride, p-toluenesulfonyl chloride or trifluoromethanesulfonic anhydride; the substitution reagent is azideReagent or R5-X-H/base composition, said base being sodium hydroxide, potassium hydroxide, sodium hydride, said azidation reagent being sodium azide, potassium azide, lithium azide, tetrabutylammonium azide, ammonium azide, trimethylsilane azide/MF composition, M being Li+,Na+，K+，Cs+，NH4+，Bu4N+(ii) a The acid catalyst is dilute hydrochloric acid, dilute sulfuric acid, p-toluenesulfonic acid, trifluoroacetic acid and methanesulfonic acid; the molar ratio of the L galactose to the dehydrating agent is 1: 3-10, said 1, 2; the mol ratio of the 3, 4-bi-O-isopropylidene-alpha-L-galactose to the catalyst to the sulfonylation reagent is 1: 2-4: 1.5-2, wherein the molar ratio of the sulfonate intermediate to the substitution reagent is 1:1.1-3, said 1, 2; 3, 4-bis-O-isopropylidene-6-R5The molar ratio of the X-alpha-L-galactose intermediate to the acid catalyst is 1: 0.01 to 0.1, said 6-R5The molar ratio of the X-L-galactose intermediate to the acetic anhydride is 1: 5-30; the substituting reagent is R5In the case of-X-H/base combination, R5-the molar ratio of X-H to base is 1: 1-1.3;
the preparation method of the compound of the formula III comprises the following steps: dissolving amino mannose hydrochloride in ethanol, adding sodium bicarbonate, stirring for 5-30 minutes, adding an acetylation reagent to form an amido bond, evaporating to remove the solvent, dissolving the residue in pyridine, adding trityl chloride, stirring for 0.5-4 hours, and continuously adding the acetylation reagent to obtain a 2-N-acetyl-1, 2, 4-tri-O-acetyl-6-O-trityl-amino mannose intermediate; dissolving the intermediate in methanol, adding an acid catalyst, stirring for 15-60 minutes, evaporating to remove the solvent, dissolving the residue in dichloromethane, and reacting with a sulfonylation reagent under the action of an alkali catalyst to obtain a sulfonic ester intermediate; dissolving a substitution reagent and a sulfonate intermediate in tetrahydrofuran, and reacting for 12 hours to obtain a compound shown in a formula III; the acetylation reagent is acetic anhydride, acetyl bromide and acetyl chloride; the acid catalyst is dilute hydrochloric acid, dilute sulfuric acid, p-toluenesulfonic acid, trifluoroacetic acid and methanesulfonic acid; the base catalyst is pyridine, triethylamine, isopropyl diethylamine, diisopropyl ethylamine, tri-N-propylamine, tri-N-butylamine, N-methylmorpholine and tetramethyl ethylenediamine; the sulfonylation reagent is methanesulfonyl chloride, p-toluenesulfonyl chloride, trifluoromethanesulfonic anhydride(ii) a The substitution reagent is an azide reagent or R5-X-H/base composition, said base being sodium hydroxide, potassium hydroxide, sodium hydride, said azidation reagent being sodium azide, potassium azide, lithium azide, tetrabutylammonium azide, ammonium azide, trimethylsilane azide/MF composition, M being Li+,Na+，K+，Cs+，NH4+，Bu4N+(ii) a The mol ratio of the amino mannose hydrochloride to the sodium bicarbonate to the acetylation reagent to the trityl chloride to the acetylation reagent is 1: 1-1.2: 1-1.5: 1.2-3: 5-30; the molar ratio of the 2-N-acetyl-1, 2, 4-tri-O-acetyl-6-O-trityl-amino mannose intermediate to the acid catalyst to the base catalyst to the sulfonating reagent is 1: 0.1-0.5: 1.3-2: 1-1.5; the molar ratio of the sulfonate intermediate to the substitution reagent is 1: 1.1-3; the substituting reagent is R5In the case of-X-H/base combination, R5-the molar ratio of X-H to base is 1: 1-1.3.
4. The method for detecting circulating tumor cells according to claim 1, wherein the capturing in step 2) is a process of capturing and fixing circulating tumor cells by using bio-orthogonal reaction between detection molecules fixed on the substrate of the detection device and surface marker groups of circulating tumor cells;
the detection molecule is a compound shown as a formula V,
Figure FDA0002398974270000041
in the formula V, R6is-N3Or
Figure FDA0002398974270000042
Figure FDA0002398974270000043
Wherein R is6And R5Correspond to when R5is-N3When R is6Is composed of
Figure FDA0002398974270000044
Figure FDA0002398974270000045
When R is5Is composed of
Figure FDA0002398974270000046
When R is6is-N3When R is5Is composed of
Figure FDA0002398974270000047
When R is6Is composed of
Figure FDA0002398974270000048
When R is5Is composed of
Figure FDA0002398974270000049
When R is6Is composed of
Figure FDA00023989742700000410
When R is5Is composed of
Figure FDA00023989742700000411
When R is6Is composed of
Figure FDA00023989742700000412
When R is5Is composed of
Figure FDA00023989742700000413
When R is6Is composed of
Figure FDA00023989742700000414
When R is5Is composed of
Figure FDA00023989742700000415
When R is6Is composed of
Figure FDA00023989742700000416
When R is5Is composed of
Figure FDA00023989742700000417
When R is6Is composed of
Figure FDA00023989742700000418
R7is-SH; n1 is 0,1,2,3,4,5,6,7, 8.
5. The method of claim 4, wherein R is6is-N3A group,
Figure FDA00023989742700000419
Figure FDA0002398974270000051
The general preparation method of the compound of formula V is: TsO-PEGn1+1Preparation of-Ts: HO-PEGn1+1-H, PEG is- (C)2H5Dissolving an O) -group in dichloromethane, adding p-toluenesulfonyl chloride, and stirring at room temperature for 4-12 hours under the action of an alkali catalyst; the base catalyst is triethylamine, isopropyl diethylamine, diisopropyl ethylamine, tri-N-propylamine, tri-N-butylamine, N-methylmorpholine and tetramethyl ethylenediamine; the HO-PEGn1+1-H, p-toluenesulfonyl chloride, base catalyst in a molar ratio of 1: 3-5: 2-5; monosubstituted R6-(C2H4O)n1-C2H4-Ots intermediate preparation: r6-OH in tetrahydrofuran, R6-is of
Figure FDA0002398974270000052
Figure FDA0002398974270000053
Adding alkali, stirring for 30-120 minutes at 50-70 ℃, and adding TsO-PEGn1+1-Ts, stirring for 2-12 hours to obtain an intermediate, the base is hydrogen hydroxideSodium, potassium hydroxide, sodium hydride, said R6-OH, base, TsO-PEGn1+1-Ts in a molar ratio of 1: 1-1.2: 1; alternatively, TsO-PEGn1+1-Ts in tetrahydrofuran and MR is added6M is Li+、Na+、K+、NH4 +、Bu4N+，R6is-N3Stirring the solution at 50-70 ℃ overnight, wherein the TsO-PEG is preparedn1+1-Ts、MR6In a molar ratio of 1: 1; dissolving the intermediate in acetonitrile, adding potassium thioacetate, and stirring for 2-12h to obtain disubstituted R6-(C2H4O)n1-C2H4-an OSAc intermediate; dissolving the disubstituted intermediate in methanol, and obtaining a compound shown in a formula V under the action of a sodium methoxide catalyst; the R is6The molar ratio of-OH, potassium thioacetate and sodium methoxide is 1: 2-4: 0.01-0.05;
R6is composed of
Figure FDA0002398974270000054
The preparation method of the compound of the formula V comprises the following steps: TsO-PEGn1+1Preparation of H: HO-PEGn1+1-H, PEG is- (C)2H5Dissolving an O) -group in dichloromethane, adding p-toluenesulfonyl chloride, and stirring at room temperature for 2-12 hours under the action of an alkali catalyst; the alkali catalyst is: triethylamine, isopropyl diethylamine, diisopropyl ethylamine, tri-N-propylamine, tri-N-butylamine, N-methylmorpholine, tetramethyl ethylenediamine; the HO-PEGn1+1-H, p-toluenesulfonyl chloride, base catalyst in a molar ratio of 1: 0.25-0.5: 0.5 to 1; monosubstituted R6-(C2H4O)n1-C2H4-Ots intermediate preparation: TsO-PEGn1+1-H in pyridine, adding R6-LG3Stirring for 2-12 hours; LG (Ligno-lead-acid)3Is Cl, Br, TsO-PEGn1+1-H and R6-LG3In a molar ratio of 1: 1.2-2; dissolving the intermediate in acetonitrile, adding potassium thioacetate, and stirring for 2-12h to obtain disubstituted R6-(C2H4O)n1-C2H4-an OSAc intermediate; the disubstituted intermediate is dissolved in methanolIn the presence of sodium methoxide as catalyst to obtain compound of formula V; the R is6-(C2H4O)n1-C2H4-OTs, potassium thioacetate in a molar ratio of 1: 2-4.
6. The method for detecting circulating tumor cells according to claim 4, wherein the detection device comprises a device substrate layer, a surface metal gold plating layer and a surface molecular layer, the surface molecular layer is composed of detection molecules and thiol-polyethylene glycol blocking molecules, the detection molecules and the thiol-polyethylene glycol blocking molecules form S-Au bonds through thiol groups on surface molecular structures and gold atoms, the S-Au bonds are fixed on the surface of the surface metal gold plating layer, and the device substrate layer is selected from device substrates which have modifiable metal gold layer surface structures and can be applied to single cell capture/release.
7. The method for detecting circulating tumor cells according to claim 6, wherein the detecting device is constructed by: mixing detection molecules with mercaptopolyethylene glycol according to a molar ratio of 90: 10-1: 99, preparing an aqueous solution with a molar concentration of 0.1-10 mM, soaking the device substrate with the surface metal gold coating in the aqueous solution for 1-12 hours, or allowing the aqueous solution to flow through the device substrate with the surface metal gold coating at a flow rate of 0.1-0.5 mL/min for 15-90 minutes, and finally washing the device with PBS buffer solution for later use.
8. The method for detecting circulating tumor cells according to claim 1, wherein the releasing in step 3) refers to a process of releasing circulating tumor cells by exchanging detection molecules fixed on the substrate of the detection device with small molecule release molecules through a reversible reaction;
the small molecule release molecules are reduced glutathione, dithioerythrol and dithiothreitol.
9. A detection kit for circulating tumor cells is characterized by comprising a labeled sugar molecule, a cell culture solution, a density gradient separation solution, a PBS buffer solution, a detection device and a small molecule release molecule;
the labeled sugar molecule is one of compounds shown in a formula I, a formula II, a formula III and a formula IV or a composition thereof,
Figure FDA0002398974270000061
in the formula I, R1is-H, R2Is composed of
Figure FDA0002398974270000062
R3is-OAc, R4is-H or R1Is composed of
Figure FDA0002398974270000063
R2is-H, R3is-OAc, R4is-H or R1Is composed of
Figure FDA0002398974270000064
R2is-H, R3is-H, R4is-OAc; x is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; r5is-N3 or
Figure FDA0002398974270000071
Figure FDA0002398974270000072
In the formula II, X is- (C)2H4O)m-, where m is 0,1,2,3,4,5,6, 7; r5 is-N3 or
Figure FDA0002398974270000073
Figure FDA0002398974270000074
In the formula III, X is- (C)2H4O)m-, where m is 0,1,2,3,4,5,6, 7; r5is-N3 or
Figure FDA0002398974270000075
Figure FDA0002398974270000076
In the formula IV, X is-CnH2n-or- (C)2H4O)m-, where n is 1,2,3,4,5,6, m is 1,2,3, 4; r5is-N3 or
Figure FDA0002398974270000077
The density gradient separation liquid is a lymph separation liquid;
the detection device comprises a device substrate layer, a surface metal gold plating layer and a surface molecular layer, wherein the surface molecular layer consists of detection molecules and sulfhydryl polyethylene glycol blocking molecules, an S-Au bond is formed between a sulfhydryl group on a surface molecular structure and a gold atom and is fixed on the surface of the surface metal gold plating layer, and the device substrate layer is selected from a device substrate which has a modifiable metal gold layer surface structure and can be applied to single cell capture/release;
the detection molecule is a compound shown as a formula V,
Figure FDA0002398974270000081
in the formula V, R6is-N3Or
Figure FDA0002398974270000082
Figure FDA0002398974270000083
Wherein R is6And R5Correspond to when R5is-N3When R is6Is composed of
Figure FDA0002398974270000084
Figure FDA0002398974270000085
When R is5Is composed of
Figure FDA0002398974270000086
When R is6is-N3When R is5Is composed of
Figure FDA0002398974270000087
When R is6Is composed of
Figure FDA0002398974270000088
When R is5Is composed of
Figure FDA0002398974270000089
When R is6Is composed of
Figure FDA00023989742700000810
When R is5Is composed of
Figure FDA00023989742700000811
When R is6Is composed of
Figure FDA00023989742700000812
When R is5Is composed of
Figure FDA00023989742700000813
When R is6Is composed of
Figure FDA00023989742700000814
When R is5Is composed of
Figure FDA00023989742700000815
When R is6Is composed of
Figure FDA00023989742700000816
When R is5Is composed of
Figure FDA00023989742700000817
When R is6Is composed of
Figure FDA00023989742700000818
R7is-SH; n1 is 0,1,2,3,4,5,6,7, 8;
the small molecule release molecules are reduced glutathione, dithioerythrol and dithiothreitol.
10. The kit for detecting circulating tumor cells according to claim 9, wherein the method of use comprises:
1) artificially marking circulating tumor cells, dissolving and diluting one compound or a combination of compounds in marked sugar molecules by using a cell culture solution to prepare a marked culture solution with the total concentration of the marked sugar molecules being 1-100 uM; taking peripheral blood to be detected, carrying out gradient density centrifugation to obtain erythrocytes, then incubating in a marking culture solution, washing to remove marker molecules which are not taken in, and obtaining an artificially marked circulating tumor cell sample;
2) capturing the circulating tumor cells based on bioorthogonal reaction, flushing the detection device with PBS buffer solution at the flow rate of 0.1-0.5 ml/min for 15-30 minutes, then enabling the artificially marked circulating tumor cell sample in the step 1) to flow through the detection device at the same flow rate, continuing flushing the device with PBS buffer solution at the same flow rate for 5-15 minutes, and obtaining the cells immobilized on the surface of the detection device after reaction as the captured circulating tumor cells;
3) based on the release of circulating tumor cells by reversible reaction, dissolving release molecules by PBS buffer solution to prepare 0.01-0.1M release solution, enabling the release solution to flow through the detection device at the flow rate of 0.02-0.1ml/min for 5-15 minutes, discarding the eluate, then flushing the detection device at the flow rate of 0.1-0.5 ml/min for 5-15 minutes, exchanging the detection molecules fixed on the surface of the detection device through reversible reaction, releasing the circulating tumor cells, and collecting the eluate, thus obtaining the released circulating tumor cells.
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