Bionic circulating tumor cell detection device as well as preparation method, use method and regeneration method thereof

Abstract

The invention belongs to the technical field of circulating tumor cell detection, and provides a bionic circulating tumor cell detection device. The device comprises a detection molecule, a function construction molecule and a bionic blocking molecule, wherein the molecular structure of the bionic blocking molecule is sulfydryl-A-B; the molecular structure of the function construction molecule is sulfydryl-C-D; the molecular structure of the detection molecule is E-F-G; the A group and the C group are hydrophobic chains; the group B is a bionic amphoteric group; the D group and the E group can be subjected to reversible reaction; the F group is a hydrophilic chain; the G group is a capturing group and can capture circulating tumor cells; the functional construction molecules and the bionic blocking molecules are fixedly connected with the surface gold coating through sulfydryl. According to the device provided by the invention, a biological orthogonal reaction is carried out on the surface of the device to accurately and nondestructively capture the MGE treatment labeled CTC, the captured CTC is nondestructively released through a reversible reaction, and accurate and nondestructive detection of the CTC is realized and cyclic regeneration of the detection device is realized through a detection process of a'labeling-capturing / releasing 'device.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of circulating tumor cell detection, and particularly relates to a biomimetic circulating tumor cell detection device and a preparation method, use and regeneration method thereof. BACKGROUND

[0002] Circulating tumor cell (CTC) is a tumor cell that falls off from a tumor primary site, enters and survives in the circulatory system. During the metastasis of CTC, the cell surface epithelial cell adhesion factor (EpCAM) level decreases, the invasion ability increases, thereby having strong fluidity and invasiveness, easily adhering to and penetrating the blood vessel wall, leading to cancer metastasis and spread. The presence of CTC is an early sign of cancer metastasis and spread. CTC detection has multiple important significance for early tumor micrometastasis discovery, treatment plan and prognosis evaluation, and individualized tumor treatment.

[0003] The main content and process of the CTC detection method based on bio-orthogonal metabolic sugar engineering labeling include: first, based on metabolic glyco-engineering (MGE), a special sugar unit is used to artificially label CTC; then a detection device integrating capture / release functions is designed and constructed, and CTC is captured by bio-orthogonal reaction between the surface groups of the device and the surface labels of CTC; finally, a release molecule is introduced to release the captured CTC through a reversible reaction. The detection device uses gold as the substrate, and the capture / release bifunctional molecules are fixed through S-Au bonds, and the unreacted sites are blocked by thiol polyethylene glycol. On the one hand, polyethylene glycol is biologically inert, which can avoid non-specific adsorption of cells due to the interaction between surface proteins and the gold substrate during the detection process. On the other hand, in the release step, the capture / release bifunctional molecules are released without damage through ligand exchange of S-Au bonds. However, small molecule polyethylene glycol is not good at resisting non-specific adsorption, which often leads to serious non-specific adsorption of unmarked blood cells. Large molecule polyethylene glycol is good at resisting non-specific adsorption, but it often coats the capture / release bifunctional small molecules on the surface of the device in the polymeric molecule layer, which affects the bio-orthogonal reaction of the bifunctional molecules to capture the labeled CTC, thereby reducing the detection accuracy. In addition, the polyethylene glycol blocking molecules are also fixed on the surface of the detection device through S-Au bonds, which affects the efficiency of the release process of the bifunctional molecules through S-Au ligand exchange, thereby affecting the non-destructiveness of the release process.

[0004] Therefore, there is an urgent need for a device and method for detecting circulating tumor cells to overcome the problems of insufficient detection accuracy and inability to non-destructively capture / release CTCs in existing detection methods. SUMMARY

[0005] In view of the problems of insufficient detection accuracy and inability to non-destructively capture / release CTCs in the prior art, the present application provides a bionic circulating tumor cell detection device.

[0006] The bionic circulating tumor cell detection device comprises a substrate, a surface gold coating layer and a surface molecule layer, wherein the surface molecule layer comprises detection molecules, functional construction molecules and bionic blocking molecules, and the molecular structure of the bionic blocking molecules is as follows:

[0007] thiol-A-B;

[0008] The molecular structure of the functional construction molecules is as follows:

[0009] thiol-C-D;

[0010] The molecular structure of the detection molecules is as follows:

[0011] E-F-G;

[0012] The A group and the C group are both hydrophobic chains;

[0013] The B group is a bionic amphiphilic group, and is selected from one or more of a trimethylamine oxide group (TMAO), a sulfobetaine group (SB), a carboxybetaine group (CB) and a phosphorylcholine group (PC);

[0014] The D group and the E group can undergo a reversible reaction;

[0015] The F group is a hydrophilic chain;

[0016] The G group is a capture group, and the capture group is capable of capturing circulating tumor cells;

[0017] The functional construction molecules and the bionic blocking molecules are fixedly connected to the surface gold coating layer through the thiol groups.

[0018] Further, the A group and the C group are independently selected from one or more of an alkyl chain (-(CH2) y ), an alkyl chain with an amide bond (-CONH-), an alkyl chain with an ether bond (-O-) and a thioether bond (-S-);

[0019] The F group is selected from one or more of a polyethylene glycol chain (-(C2H4O) z ) and a polyethylene glycol chain with an amide bond (-CONH-).

[0020] one of the D group and the E group is an α-cyclodextrin group and / or a β-cyclodextrin group, and the other is an azobenzene group.

[0021] Further, the G group captures the circulating tumor cell through a bio-orthogonal reaction;

[0022] The circulating tumor cell is labeled by metabolic sugar engineering, and the labeled sugar molecule has one or more of the following structures:

[0023]

[0024]

[0025] wherein the R1 group is selected from an H atom or an acetyl group Ac-;

[0026] one of the G group and the R2 group is selected from an azido group, and the other is selected from one or more of a bicyclo[6,1,0]nonyne group, a dibenzo cyclooctyne group, and a dibenzo cyclooctyne amine group, or one is selected from an azido tetrazine group, and the other is selected from a trans cyclooctene group;

[0027] X is selected from one or more of an alkyl chain (-(CH2) a ), a polyethylene glycol chain (-(C2H4O) b ), an alkyl chain with an amide bond (-CONH-), and a polyethylene glycol chain.

[0028] Further, the substrate is selected from one of a microfluidic chip, a magnetic nanoparticle, a gold nanoparticle, a gold nanorod, and a composite material.

[0029] The application also provides a preparation method of the biomimetic circulating tumor cell detection device.

[0030] The preparation method of the biomimetic circulating tumor cell detection device comprises the following steps:

[0031] S1: The biomimetic blocking molecule and the functional building molecule are mixed in a molar ratio of 95:5 to 30:70 to prepare an aqueous solution with a molar concentration of 0.1 to 10 mM;

[0032] S2: The surface gold coating is washed with an organic solvent and dried;

[0033] S3: The aqueous solution in step S1 flows through the surface gold coating at a flow rate of 0.1 to 0.5 mL / min for 15 to 90 minutes, is washed with deionized water, and is introduced into a detection molecule solution with the same concentration as the functional building molecule for 10 to 30 minutes to cause the positive reaction in the reversible reaction with the detection molecule, and is washed with deionized water and a PBS buffer, respectively.

[0034] Further, the organic solvent in step S2 is methanol.

[0035] The deionized water in step S3 is washed for 15-30 minutes, and the PBS buffer is washed for 15 minutes.

[0036] The application further provides a method for using and recycling the bionic circulating tumor cell detection device.

[0037] The method for using and recycling the bionic circulating tumor cell detection device comprises the following steps:

[0038] ⅰ: introducing a blood sample containing labeled circulating tumor cells into the bionic circulating tumor cell detection device as described in any one of the above;

[0039] ⅱ: introducing PBS buffer and controlling the conditions, i.e. flow rate and time, which can be achieved by a solvent pump;

[0040] ⅲ: the reverse reaction of the reversible reaction of the D-E system releases the circulating tumor cells.

[0041] Further, the condition for the reverse reaction of the reversible reaction is irradiation with 365 nm wavelength light.

[0042] Further, in step Sⅰ, the flow rate of the blood sample introduced into the bionic circulating tumor cell detection device is 0.02-0.1 mL / min.

[0043] In step Sⅱ, the flow rate of the PBS buffer introduced is 0.02-0.1 mL / min, and the time length is 30-60 minutes.

[0044] Advantages:

[0045] The application provides a CTC detection device with a biomimetic interface, comprising a substrate, a surface gold coating layer and a surface molecular layer, wherein the surface molecular layer comprises detection molecules, functional construction molecules and biomimetic blocking molecules. The biomimetic blocking molecules can effectively avoid non-specific adsorption of the existing CTC detection device and reduce detection interference; only small molecules are involved in the operation, the interface structure has good uniformity and is suitable for fine regulation; the biomimetic blocking molecules have good biocompatibility, extremely low immunogenicity and can effectively maintain the biological activity of CTC. The biological orthogonal reaction between the detection molecules and the functional construction molecules precisely and non-destructively captures the CTC labeled by MGE, the captured CTC is non-destructively released through a reversible reaction, the precise and non-destructive detection of CTC is realized through the "labeling-capture / release" device detection process, and the recycling of the detection device is realized; in the release step, the non-destructive release process can be realized through specific reversible reactions by condition control, and the introduction of additional release reagents is not involved. The precise sorting of CTC is realized through the fine regulation of the capture and release process between the biomimetic blocking molecules, the detection molecules and the functional construction molecules, and the detection interference of false positives and false negatives is effectively reduced. The construction process only involves several small molecules and corresponding labeled sugar molecules, and the construction process only needs several steps of reaction to prepare a large amount of efficient products, so that the availability is high and the economy is good. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 It is a preparation flow chart of the biomimetic blocking molecule I;

[0047] Figure 2 It is a preparation flow chart of the biomimetic blocking molecule II;

[0048] Figure 3 It is a preparation flow chart of the biomimetic blocking molecule III;

[0049] Figure 4 It is a preparation flow chart of the biomimetic blocking molecule IV;

[0050] Figure 5 It is a preparation flow chart of the functional construction molecule V;

[0051] Figure 6 It is a preparation flow chart of the functional construction molecule VI;

[0052] Figure 7 It is a preparation flow chart of the detection molecule VII;

[0053] Figure 8 It is a preparation flow chart of the detection molecule VIII;

[0054] Figure 9 It is a preparation flow chart of the labeled sugar molecule IX;

[0055] Figure 10 It is a preparation flow chart of the labeled sugar molecule X;

[0056] Figure 11 Preparation flow chart of the bionic circulating tumor cell detection device XI;

[0057] Figure 12 Preparation flow chart of the bionic circulating tumor cell detection device XII;

[0058] Figure 13 Preparation flow chart of the bionic circulating tumor cell detection device detection flow, use and regeneration method. DETAILED DESCRIPTION

[0059] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the accompanying drawings, but it should not be understood as limiting the scope of the present application.

[0060] In recent years, composite materials for constructing surface bionic structures on material substrates have made progress in many aspects. By referring to the monolayer structure formed by biomolecules such as phosphatidylcholine (PC), sulphobetaine (SB), carboxybetaine (CB), trimethyl oxide (TMAO) and the like, which has good anti-non-specific adsorption capacity, the functional materials are modified while the monolayer structure is constructed on the surface of the functional materials, so as to endow the functional materials with good anti-non-specific adsorption capacity. For example, an amphoteric ion monolayer is formed on the surface of the materials, so as to have excellent anti-bacterial and cell adsorption performance (Langmuir, 2001, 17, 6336-6343); a TMAO polymer layer is formed on the surface of the gold substrate, which can exhibit excellent anti-protein adsorption performance (Sci. Adv., 2019, 5, eaaw9562).

[0061] Based on the above background, the present application provides a CTC detection device with a bionic surface interface and a construction and use method thereof. By constructing a bionic structure on the surface interface of the device and designing and constructing a novel capture / release bifunctional molecule, the capture / release function of the device for sugar metabolism marker CTC is maintained, the bionic structure of the surface interface of the device achieves good anti-non-specific adsorption performance, and the controllability of the release process is improved, so as to improve the accuracy of CTC detection.

[0062] The present application provides a bionic circulating tumor cell detection device.

[0063] The biomimetic circulating tumor cell detection device comprises a substrate, a surface gold coating layer and a surface molecule layer, wherein the surface molecule layer comprises detection molecules, functional construction molecules and biomimetic blocking molecules, and the molecular structure of the detection molecules is as follows:

[0064] thiol-A-B;

[0065] The molecular structure of the functional construction molecules is as follows:

[0066] thiol-C-D;

[0067] The molecular structure of the biomimetic blocking molecules is as follows:

[0068] E-F-G;

[0069] The A group and the C group are both hydrophobic chains;

[0070] The B group is a biomimetic amphoteric group, which is selected from one or more of a trimethylamine oxide group (TMAO), a sulfobetaine group (SB), a carboxybetaine group (CB) and a phosphorylcholine group (PC);

[0071] The D group and the E group can undergo reversible reactions;

[0072] The F group is a hydrophilic chain;

[0073] The G group is a capture group, and the capture group can capture circulating tumor cells;

[0074] The functional construction molecules and the biomimetic blocking molecules are fixedly connected to the surface gold coating layer through the thiol groups.

[0075] The detection molecules have a three-segment structure of “release group-hydrophilic chain-capture group”, and the release group and the hydrophilic chain, and the hydrophilic chain and the capture group are connected through a connecting arm, and the connecting arm is represented by “-”.

[0076] The functional construction molecules have a three-segment structure of “thiol-hydrophobic chain-grafting group”, and the thiol and the hydrophobic chain, and the hydrophobic chain and the grafting group are connected through a connecting arm, and the connecting arm is represented by “-”.

[0077] The biomimetic blocking molecules have a three-segment structure of “thiol-hydrophobic chain-biomimetic zwitterion group”, and the thiol and the hydrophobic chain, and the hydrophobic chain and the biomimetic zwitterion group are connected through a connecting arm, and the connecting arm is represented by “-”.

[0078] The connecting arms in or between the detection molecules, the functional construction molecules and the biomimetic blocking molecules can be the same or different, and can be any structure that does not affect the function of the molecules, such as a chemical bond or a molecular chain.

[0079] In one embodiment, the D group and the E group, one of which is an alpha-cyclodextrin group and / or a beta-cyclodextrin group, the other of which is an azobenzene group. Under 450 nm wavelength light, the azobenzene group enters the cavity of the alpha-cyclodextrin group or the beta-cyclodextrin group, and the positive reaction in the reversible reaction, namely chelation, occurs, and under 365 nm wavelength light, the reverse reaction in the reverse reaction occurs, namely the azobenzene group comes out of the cavity of the alpha-cyclodextrin group or the beta-cyclodextrin group, and becomes a free group.

[0080] In one embodiment, the A group, the C group is independently selected from one or more of an alkyl chain, an alkyl chain with an amide bond, an alkyl chain with an ether bond and a thioether bond.

[0081] The F group is selected from one or two of a polyethylene glycol chain, a polyethylene glycol chain with an amide bond.

[0082] The G group captures the circulating tumor cells by a bio-orthogonal reaction;

[0083] The circulating tumor cells are labeled by metabolic sugar engineering, and the labeled sugar molecules have one or more of the following structures:

[0084]

[0085] Among them, the R1 group is selected from an H atom or an acetyl group Ac-;

[0086] One of the G group and the R2 group is selected from an azido group, and the other is selected from one or more of a bicyclo[6,1,0]nonyne group, a dibenzo cyclooctyne group and a dibenzo cyclooctyne amine group, or one is selected from an azido group tetrazine group, and the other is selected from a trans-cyclooctene group;

[0087] X is selected from one or more of an alkyl chain (-(CH2) a ), a polyethylene glycol chain (-(C2H4O) b ), an alkyl chain with an amide bond (-CONH-) and a polyethylene glycol chain.

[0088] The substrate is selected from one of a microfluidic chip, a magnetic nanoparticle, a gold nanoparticle, a gold nanorod and a composite material.

[0089] The application provides a preparation method of a biomimetic circulating tumor cell detection device.

[0090] Referring to Figures 11-12 The preparation method of the biomimetic circulating tumor cell detection device comprises the following steps:

[0091] S1: the biomimetic blocking molecule and the functional building molecule are mixed in a molar ratio of 95:5 to 30:70 to prepare an aqueous solution with a molar concentration of 0.1-10 mM;

[0092] S2: the surface gold coating is washed with methanol and dried;

[0093] S3: the aqueous solution in step S1 is flowed through the surface gold coating at a flow rate of 0.1-0.5 mL / min for 15-90 min, deionized water is used for flushing for 15-30 min, a detection molecule solution with the same concentration as the functional building molecule is introduced under 450 nm wavelength light for 10-30 min, and deionized water and PBS buffer are used for flushing for 15-30 min and 15 min, respectively.

[0094] Referring to Figure 13 , the application provides a method for using a biomimetic circulating tumor cell detection device and recycling.

[0095] The method for using a biomimetic circulating tumor cell detection device and recycling comprises the following steps:

[0096] i: a blood sample containing labeled circulating tumor cells is introduced into the biomimetic circulating tumor cell detection device according to any one of the above at a flow rate of 0.02-0.1 mL / min;

[0097] ii: PBS buffer is introduced and the flow rate is controlled to be 0.02-0.1 mL / min for 30-60 min;

[0098] iii: under 365 nm wavelength light irradiation, the D group and the E group undergo reversible reaction to release the circulating tumor cells.

[0099] Example 1

[0100] Preparation of biomimetic blocking molecule I

[0101] Referring to Figure 1 , the method for preparing biomimetic blocking molecule I comprises the following steps:

[0102] Commercial reagent 30% H2O2 aqueous solution is diluted with water to prepare 3% H2O2 aqueous solution (1M), commercial raw material N,N-dimethylaminopropylamine I-1 (10g, 97.9mmol) is slowly added dropwise into 3% H2O2 aqueous solution (200mL, 200mmol), stirring is performed at room temperature for 72 hours, the solvent is evaporated, the residue is dissolved in methanol, spin-drying is performed, and the operation is repeated three times to obtain compound I-2 (11.3g, 98%), ESI-MS m / z calcd for [C5H 15 N2O] + (M+H) +: 119.11, found: 119.12;

[0103] Thiol-11-alkanoic acid (1 g, 4.58 mmol) was dissolved in dichloromethane (20 mL), N-hydroxysuccinimide (0.79 g, 6.87 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) (1.76 g, 9.16 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC. The reaction mixture was washed with water and dried over anhydrous sodium sulfate to give the intermediate HS-C 10 H 20 -CO-NHS (1.3 g, 4.34 mmol) was added, and the intermediate was dissolved in a mixture of dichloromethane / methanol (V / V = 3:1, 20 mL). Compound I-2 (487 mg, 4.12 mmol) and triethylamine (0.9 mL, 6.5 mmol) were added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was directly concentrated and purified by column chromatography to give the compound of formula I (1.25 g, 95%). 16 H 35 N2O2S] + (M+H) + : 318.52, found: 318.51.

[0104] Example 2

[0105] Preparation of biomimetic blocking molecule II

[0106] Reference Figure 2 The method for preparing the biomimetic blocking molecule II comprises the following steps:

[0107] Commercially available starting material I-1 (5 g, 48.9 mmol) and sodium bicarbonate (8.2 g, 97.8 mmol) were dissolved in tetrahydrofuran (THF) (100 mL), and di-tert-butyl dicarbonate (Boc2O) (12.8 g, 58.7 mmol) was added portionwise in an ice bath. The mixture was stirred at room temperature for 8 h. The reaction mixture was poured into water, and dichloromethane was added for extraction. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated to give the compound of formula II-2 (8.4 g, 84.9%).

[0108] The compound of formula II-2 (8.4 g, 41.5 mmol) was dissolved in acetonitrile (100 mL), and 4-bromo-butyl acetate (12.2 g, 62.3 mmol) was added. The reaction was carried out at reflux for 12 h. The solvent was evaporated, and the residue was dissolved in dichloromethane. Ethyl ether was added to precipitate the compound of formula II-3 (16.1 g, 97.6%), which was filtered. 16 H 33 N2O4] +(M-Br) + : 317.24, found: 317.25;

[0109] Compound of formula II-4 (9.5 g, 99%) was prepared by stirring compound of formula II-3 (16.1 g, 50.72 mmol) in methanol, adjusting the pH of the reaction solution to ~1 by adding 4M methanolic hydrochloric acid, stirring at room temperature for 30 minutes, adjusting the pH of the solution to ~14 by adding saturated sodium hydroxide solution, and stirring the reaction at room temperature for 1 hour. The reaction solution was directly concentrated, and column chromatography was performed to obtain the compound of formula II-4 (9.5 g, 99%). 21 N2O2] + (M+H) + : 189.15, found: 189.16;

[0110] Compound of formula II was prepared by the process for preparing compound of formula I-2, except that compound of formula I-2 was replaced by compound of formula II-4, ESI-MS m / z calcd for [C 20 H 41 N2O3S] + (M+H) + : 389.28, found: 389.28.

[0111] Example 3

[0112] Preparation of biomimetic blocking molecule III

[0113] Reference Figure 3 The preparation method of biomimetic blocking molecule III includes the following flow:

[0114] Compound of formula II-2 (5 g, 24.7 mmol) was dissolved in acetone (50 mL), 1,3-propane sulfonic acid lactone (3.17 g, 25.9 mmol) was added, and the reaction was stirred at room temperature for 12 hours to produce a precipitate. The precipitate was filtered, and the insoluble part was washed with acetone to obtain compound of formula III-1 (7.8 g, 97.3%). 13 H 29 N2O5S] + (M+H) + : 325.17, found: 325.16;

[0115] Compound of formula III-1 (7.8 g, 24 mmol) was dissolved in methanol (40 mL), 4M methanolic hydrochloric acid solution was added to adjust the pH of the reaction solution to about 1, the reaction was stirred at room temperature for 30 minutes, saturated sodium hydroxide solution was added to adjust the pH of the solution to about 14, the reaction solution was concentrated, and column chromatography was used to separate to obtain compound of formula III-2 (5.2 g, 96.4%), ESI-MS m / z calcd for [C8H 21 N2O3S] + (M+H) + :225.11, found: 225.12;

[0116] The process of preparing compound of formula III from compound of formula III-2 is the same as that of preparing compound of formula I from compound of formula I-2, except that compound of formula I-2 is replaced by compound of formula III-2, ESI-MS m / z calcd for [C 19 H 41 N2O4S2] + (M+H) + :425.24, found: 425.24.

[0117] Example 4

[0118] Preparation of biomimetic blocking molecule IV

[0119] Referring to Figure 4 The preparation process of biomimetic blocking molecule IV includes the following steps:

[0120] The process of preparing compound of formula IV-2 from compound of formula IV-1 is the same as that of preparing compound of formula II-1 from compound of formula I-1, except that compound of formula I-1 is replaced by compound of formula IV-1;

[0121] Phosphorus oxychloride (9.5 g, 62 mmol) was mixed in chloroform (120 mL), and a mixed solution of compound of formula IV-2 (1 g, 6.2 mmol) and triethylamine (0.63 g, 6.2 mmol) in chloroform (12 mL) was added dropwise, and stirred at room temperature for 3 hours, and the solvent was evaporated, and the residue was dissolved in pyridine (50 mL), and choline p-toluenesulfonic acid salt (8 g, 31 mmol) was added under vigorous stirring at 0°C, and the reaction was placed at room temperature for 24 hours, and water (20 mL) was added and stirred for 6 hours. The solvent was evaporated, the residue was suspended in 5% hydrochloric acid solution, extracted with chloroform, dried over anhydrous sodium sulfate, concentrated and column chromatography to obtain compound of formula IV-3 (0.65 g, 32.1%), ESI-MS m / z calcd for [C 12 H 28 N2O6P] + (M+H) + :327.16, found: 327.16;

[0122] The process for preparing compound of formula IV-4 from compound of formula IV-3 is the same as that for preparing compound of formula III-2 from compound of formula III-1, except that compound of formula III-1 is replaced by compound of formula IV-3, ESI-MS m / z calcd for [C7H 20 N2O4P] + (M+H) + :227.11, found: 227.12;

[0123] The process for preparing compound of formula IV from compound of formula IV-4 is the same as that for preparing compound of formula I from compound of formula I-2, except that compound of formula I-1 and thiol-11-alkanoic acid reagent are replaced by compound of formula IV-4 and thiol-16-alkanoic acid reagent, respectively, ESI-MS m / z calcd for [C 23 H 50 N2O5PS] + (M+H) + :497.31, found: 497.31.

[0124] Example 5

[0125] Preparation of functional building block V

[0126] Referring to Figure 5 , the method for preparing functional building block V includes the following scheme:

[0127] The process for preparing V from commercial starting material V-1 is the same as that for preparing compound of formula I from compound of formula I-2, except that the intermediate of formula I-2 is replaced by commercial starting material mono-6-amino-6-deoxy-β-cyclodextrin, ESI-MS m / z calcd for [C 53 H 92 NO 35 S] + (M+H) + :1334.51, found: 1334.52.

[0128] Example 6

[0129] Preparation of functional building block VI

[0130] Referring to Figure 6 , the method for preparing functional building block VI includes the following scheme:

[0131] The process for preparing compound of formula VI from commercial starting material of formula VI-1 is the same as that for preparing compound of formula IV from intermediate of formula IV-4, except that intermediate IV-4 is replaced by commercial starting material of formula VI-1, mono-6-amino-6-deoxy-α-cyclodextrin, ESI-MS m / z calcd for [C 52 H 92NO 30 S] + (M+H) + : 1242.53, found: 1242.53.

[0132] Example 7

[0133] Preparation of detection molecule VII

[0134] Referring to Figure 7 The preparation method of the detection molecule VII includes the following steps:

[0135] The compound of formula VII-1 (1 g, 5.12 mmol) was dissolved in dichloromethane (20 mL), 1,6-hexanedioic acid (2.99 g, 20.48 mmol), EDC (1.47 g, 7.68 mmol) and triethylamine (1 g, 10.24 mmol) were added, and the reaction was stirred at room temperature for 1 hour. The reaction solution was washed with brine, dried over anhydrous sodium sulfate, concentrated, and column chromatography to obtain the compound of formula VII-2 (1.42 g, 85.7%).

[0136] The compound of formula VII-2 (1.42 g, 4.39 mmol) was dissolved in dichloromethane (20 mL), and the commercial raw material dibenzo cyclooctyne amine (0.95 g, 4.61 mmol), EDC (1.26 g, 6.59 mmol) and triethylamine (0.88 g, 8.78 mmol) were added. The reaction was stirred at room temperature for 1 hour. The reaction solution was washed with brine, dried over anhydrous sodium sulfate, concentrated, and column chromatography to obtain the compound of formula VII (2.1 g, 93.7%), ESI-MS m / z calcd for [C 35 H 31 N2O2] + (M+H) + : 511.23, found: 511.23.

[0137] Example 8

[0138] Preparation of detection molecule VIII

[0139] Referring to Figure 8 The preparation method of the detection molecule VIII includes the following steps:

[0140] Commercially available 6-amino hexanoic acid (5 g, 38.12 mmol) and sodium bicarbonate (6.4 g, 76.23 mmol) were dissolved in water (50 mL), Boc20 (9.98 g, 45.74 mmol) was added in an ice bath, the reaction was stirred at room temperature for 12 hours, the reaction solution was extracted with dichloromethane, the organic phase was washed with brine, and then concentrated to obtain 6-Boc-amino hexanoic acid intermediate (8.4 g, 36.32 mmol), which was mixed with the commercially available compound VII-1 (6.5 g, 33.29 mmol) in dichloromethane (60 mL), EDC (9.57 g, 49.93 mmol) and triethylamine (6.74 g, 66.58 mmol) were added, the reaction was stirred at room temperature for 2 hours, the reaction solution was washed with brine, dried over anhydrous sodium sulfate, concentrated, and then column chromatography to obtain the compound of formula VIII-1 (12.8 g, 94.1%);

[0141] The compound of formula VIII-1 (12.8 g, 31.3 mmol) was mixed in a 10% trifluoroacetic acid solution in dichloromethane (50 mL), the reaction was stirred at room temperature for 15 minutes, the solvent was evaporated to obtain the compound of formula VIII-2 (9.5 g, 98.3%);

[0142] The commercially available compound of formula VIII-3 (1 g, 6.66 mmol) was dissolved in dichloromethane (20 mL), p-nitrophenyl chloroformate (1.61 g, 7.99 mmol) and triethylamine (1.35 g, 13.3 mmol) were added, the reaction was stirred at room temperature for 2 hours, the reaction solution was washed with brine, dried over anhydrous sodium sulfate, and concentrated to obtain the compound of formula VIII-4 (1.8 g, 85.7%), which was directly used in the next step reaction;

[0143] The compound of formula VIII-2 (1 g, 3.24 mmol) and the compound of formula VIII-4 (1.02 g, 3.24 mmol) were mixed in dichloromethane (20 mL), triethylamine (0.39 g, 3.89 mmol) was added, the reaction was stirred at room temperature for 3 hours, the reaction solution was washed with brine, dried over anhydrous sodium sulfate, concentrated, and then column chromatography to obtain the compound of formula VIII (1.5 g, 95.5%), ESI-MS m / z calcd for [C 31 H 37 N2O3] + (M+H) + :485.27, found:485.28.

[0144] Example 9

[0145] Preparation of the labeled sugar molecule IX

[0146] Reference Figure 9 The preparation method of the labeled sugar molecule IX comprises the following steps:

[0147] Dissolve amino mannose hydrochloride (1.0 g, 4.64 mmol) in ethanol (10 mL), add sodium bicarbonate (0.4 g, 4.77 mmol), stir for 15 min, then add chloroacetyl chloride (0.63 g, 5.56 mmol), stir at room temperature for 1 h, evaporate the solvent, dissolve the residue in N,N-dimethylformamide (DMF) (10 mL), add sodium azide (0.6 g, 9.28 mmol), stir at 50 °C in an oil bath overnight, cool the reaction solution to room temperature, evaporate the solvent, dissolve the residue in ethanol (10 mL), filter off the insoluble material, evaporate the filtrate to obtain the crude intermediate of formula IX-1 (0.91 g, 74.8%);

[0148] Dissolve the intermediate of formula IX-1 (0.91 g, 3.47 mmol) in pyridine (5 mL), add acetic anhydride (2 mL) in an ice bath, transfer to room temperature and stir overnight, add methanol (5 mL), evaporate the solvent, dissolve the residue in dichloromethane (DCM) (20 mL), wash with 1 N HCI aq. , sat. NaHCO3, saturated brine, dry over anhydrous sodium sulfate, and concentrate to obtain the compound of formula IX (1.48 g, 99%).

[0149] Example 10

[0150] Preparation of the labeled sugar molecule X

[0151] Reference Figure 10 The method for preparing the labeled sugar molecule X comprises the following steps:

[0152] Dissolve L-galactose (1 g, 5.55 mmol) in acetone (30 mL) dropwise with H2SO4(1 mL) in an ice bath, transfer to room temperature and stir for 4 h until complete dissolution, add saturated NaOH solution to adjust the solution to neutral, filter off the insoluble material, concentrate the filtrate to obtain the intermediate X-1 (1.4 g, 97%);

[0153] Dissolve the compound X-1 (1.4 g, 5.38 mmol) in dichloromethane (10 mL), add TsCl (1.54 g, 8.07 mmol) and Et3N (1.09 g, 10.76 mmol) successively, stir at room temperature overnight, add methanol (1 mL), wash the solution with saturated brine, dry over anhydrous sodium sulfate, concentrate and purify to obtain the intermediate of formula X-2 (2.1 g, 94%);

[0154] Compound X-2 (2.1 g, 5.07 mmol) was dissolved in DMF (10 mL), sodium azide (0.66 g, 10.14 mmol) was added, and the reaction was stirred in an oil bath at 50 °C overnight. The reaction was cooled to room temperature, DCM (50 mL) was added, and the organic phase was collected after washing with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The intermediate of formula X-3 (1.3 g, 90%) was obtained after purification.

[0155] Compound X-3 (1.3 g, 4.56 mmol) was suspended in a mixture of acetonitrile / water (v / v = 1:1, 15 mL), and a catalytic amount of p-toluenesulfonic acid (TsOH) was added. The reaction was carried out at reflux overnight, and the reaction was neutralized with Et3N to neutral. The solvent was removed by evaporation, and the intermediate of formula X-4 (0.93 g, 99%) was obtained.

[0156] Compound X-4 (0.93 g, 4.53 mmol) was dissolved in pyridine (5 mL), and acetic anhydride (2 mL) was slowly added. The reaction was stirred at room temperature overnight, and methanol (2 mL) was added. After stirring for 20 minutes, the solvent was removed by evaporation, and the residue was dissolved in DCM (15 mL). The solution was washed with 1N HCl aq. , sat. NaHCO3, and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the compound of formula X (1.7 g, 99%).

[0157] Example 11

[0158] Preparation of the biomimetic circulating tumor cell detection device XI

[0159] Reference Figure 11 The preparation method of the biomimetic circulating tumor cell detection device XI includes the following steps:

[0160] 1) Preparation of a modified solution: biomimetic blocking molecule III and functional building molecule V were mixed at a molar ratio of 3:1, dissolved in ultrapure water, and configured into a solution with a total concentration of 1 mM. Detection molecule VII was dissolved in anhydrous methanol and configured into a 0.25 mM solution; 2) Modification of the device: anhydrous methanol was passed into the detection device at a flow rate of 0.5 mL / min for 30 minutes, and ultrapure water was continuously passed in for 15 minutes. After drying, the mixed solution of biomimetic blocking molecule III and functional building molecule V was passed through the microfluidic chip with a microfluidic channel at a flow rate of 0.5 mL / min for 15 minutes; 3) Functional construction: the chip was flushed with ultrapure water and anhydrous methanol at the same flow rate for 15 minutes, and then the methanol solution of detection molecule VII was passed in under 450 nm wavelength light for 15 minutes, followed by passing in anhydrous methanol solution for 15 minutes. Finally, the chip was flushed with PBS buffer for 30 minutes, and the detection chip device XI with a biomimetic treated microfluidic channel surface was prepared.

[0161] Example 12

[0162] Preparation of biomimetic circulating tumor cell detection device XII

[0163] Reference Figure 12 The preparation method of the biomimetic circulating tumor cell detection device XII includes the following steps:

[0164] The biomimetic blocking molecule IV and the functional building molecule VI are mixed at a molar ratio of 2:1, dissolved in ultrapure water, and configured into a solution with a total concentration of 0.9 mM. The detection molecule VIII is dissolved in anhydrous methanol and configured into a 0.3 mM solution. Anhydrous methanol is passed through the detection device at a flow rate of 0.5 mL / min for 30 minutes, and ultrapure water is continuously passed through for 15 minutes. After drying, the mixed solution of biomimetic blocking molecule IV and functional building molecule VI is passed through the microfluidic chip with a microcolumn at a flow rate of 0.5 ml / min for 15 min. The chip is washed with ultrapure water and anhydrous methanol at the same flow rate for 15 min, respectively. Then, the detection molecule VIII solution is passed through under 450 nm light for 15 minutes. Anhydrous methanol is continuously passed through at the same flow rate for 15 minutes. Finally, the chip is washed with PBS buffer for 30 min. The detection chip device XII with a biomimetic processed microcolumn surface is prepared.

[0165] Example 13

[0166] Reference Figure 13 The general biomimetic circulating tumor cell detection device detection process, use and regeneration method includes the following steps:

[0167] 1) Take 2 ml of peripheral blood from a cancer patient (left lung cancer, male, 63 years old, stage IV) with an EDTA anticoagulant tube, mix it with sterile PBS according to 1:1, and slowly stack it on the interface of the centrifuge tube containing the lymphocyte separation medium Ficoll. The volume ratio of peripheral blood, PBS and lymphocyte separation medium is 1:1:1. After horizontal centrifugation at 400g for 30 minutes, the centrifuge tube is divided into three layers. The narrow band of white cloud layer at the interface of the upper and middle layers is collected to obtain the initial treatment sample.

[0168] 2) Dissolve the labeled sugar molecule IX with DMSO solvent into a 50 mM solution, and then dilute it with cell culture medium to prepare a 50 uM labeled culture medium. Incubate the collected initial enrichment sample in the labeled culture medium for 8 hours, centrifuge to remove the labeled culture medium, and disperse the cells in the cell culture medium to obtain the MGE labeled sample.

[0169] 3) Pass the labeled sample through the detection device XI at a flow rate of 0.05 ml / min. After the passage is completed, continue to flush the device with PBS buffer at the same flow rate for 30 minutes. The cells fixed on the interface of the device are the labeled cells, which contain high labeled CTC and a small amount of white blood cell contamination.

[0170] 4) Flow PBS buffer through the detection device at a flow rate of 0.1 ml / min for 10 minutes, discard the part of the eluate (the part of the eluate contains white blood cell contamination), continue to flow PBS buffer into the detection device at a flow rate of 0.1 ml / min for 20 min under the irradiation of ultraviolet light at a wavelength of 365 nm, collect the part of the eluate, count 94 cells, the part of the released cells are captured CTCs, the CTCs detected in the patient are 94 / 2 mL.

[0171] 5) After the CTCs are released in the above step, the device obtained is subjected to the functional construction process of step 3) in Example XI to realize the recycling of the detection device XI.

[0172] CTC detection results of multiple clinical blood samples:

[0173] The blood of cancer patients of different cancer types, genders and stages is detected by using the device, and the blood of healthy people is used as a control, and the detection results are summarized as follows according to the general detection process:

[0174]

[0175] It can be known from the CTC detection results that the detection of circulating tumor cells by using the bionic circulating tumor cell detection device has good accuracy, which indicates that the bionic circulating tumor cell detection device can play the roles of anti-non-specific adsorption, improvement of controllability of the release process by precisely regulating the detection molecules, the functional construction molecules and the bionic blocking molecules.

Claims

1. A biomimetic circulating tumor cell detection device comprising a substrate, a surface gold coating, and a surface molecule layer, the surface molecule layer comprising detection molecules, characterized in that, The surface molecule layer further comprises a functional building molecule and a biomimetic blocking molecule, and the molecular structure of the biomimetic blocking molecule is as follows: Thiol-A-B; The molecular structure of the functional building molecule is as follows: Thiol-C-D; The molecular structure of the detection molecule is as follows: E-F-G; The A group and the C group are both hydrophobic chains; The B group is a biomimetic amphiphilic group, and is selected from one or more of trimethylamine oxide group, sulfobetaine group, carboxybetaine group and phosphorylcholine group; The D group and the E group can undergo reversible reactions; The F group is a hydrophilic chain; The G group is a capture group, and the capture group can capture circulating tumor cells; The functional building molecule and the biomimetic blocking molecule are fixedly connected to the surface gold coating through the thiol groups.

2. The biomimetic circulating tumor cell detection device according to claim 1, wherein: The A group and the C group are independently selected from one or more of an alkyl chain, an alkyl chain with an amide bond, an alkyl chain with an ether bond and a thioether bond; The F group is selected from one or both of a polyethylene glycol chain and a polyethylene glycol chain with an amide bond; One of the D group and the E group is an α-cyclodextrin group and / or a β-cyclodextrin group, and the other is an azobenzene group.

3. The bionic circulating tumor cell detection device of claim 1, wherein, The G group captures the circulating tumor cells through a bio-orthogonal reaction; The circulating tumor cells are labeled by metabolic sugar engineering, and the labeled sugar molecule has one or more of the following structures: Wherein, the R1 group is selected from an H atom or an acetyl group Ac-; One of the G group and the R2 group is selected from an azido group, and the other is selected from one or more of a bicyclo[6,1,0]nonyne group, a dibenzo cyclooctyne group and a dibenzo cyclooctyne amine group, or one is selected from an azido tetrazine group, and the other is selected from a trans-cyclooctene group; X is selected from one or more of an alkyl chain, a polyethylene glycol chain, an alkyl chain with an amide bond and a polyethylene glycol chain.

4. The bionic circulating tumor cell detection device of claim 1, wherein, The substrate is selected from one of a microfluidic chip, a magnetic nanoparticle, a gold nanoparticle, a gold nanorod and a composite material.

5. A method of preparing a bionic circulating tumor cell detection device according to any one of claims 1-4, characterized in that, The method comprises the following steps: S1: The biomimetic blocking molecule and the functional building molecule are mixed at a molar ratio of 95:5 to 30:70 to prepare an aqueous solution with a molar concentration of 0.1 to 10 mM; S2: The surface gold coating is washed with an organic solvent and dried; S3: The aqueous solution in step S1 is flowed through the surface gold coating at a flow rate of 0.1 to 0.5 mL / min for 15 to 90 minutes, and then deionized water is used for flushing, and a detection molecule solution with the same concentration as the functional building molecule is introduced for 10 to 30 minutes to allow the detection molecule to undergo a positive reaction in a reversible reaction, and then deionized water and PBS buffer are used for flushing, respectively.

6. The method for preparing the biomimetic circulating tumor cell detection device as described in claim 5, characterized in that, The condition for the positive reaction in the reversible reaction is irradiation at a wavelength of 450 nm.

7. The method for preparing the biomimetic circulating tumor cell detection device as described in claim 5, characterized in that, The organic solvent in step S2 is methanol; In step S3, the deionized water flushing is performed for 15 to 30 minutes, and the PBS buffer flushing is performed for 15 minutes.

8. A method for using and recycling the bionic circulating tumor cell detection device, wherein the device is as claimed in claim 1. The method comprises the following steps: ⅰ: Introducing a blood sample containing labeled circulating tumor cells into the biomimetic circulating tumor cell detection device according to any one of claims 1 to 4; ⅱ: passing PBS buffer and controlling conditions; ⅲ: the reverse reaction of the reversible reaction between the D group and the E group, releasing the circulating tumor cells.

9. The method of using and recycling the biomimetic circulating tumor cell detection device according to claim 8, wherein, The condition for the reverse reaction of the reversible reaction is irradiation with 365 nm wavelength light.

10. The method of using and recycling the biomimetic circulating tumor cell detection device according to claim 8, wherein, In step S i, the flow rate of the blood sample passing into the bionic circulating tumor cell detection device is 0.02-0.1 mL / min. In step S ii, the flow rate of the PBS buffer passing in is 0.02-0.1 mL / min, and the time length is 30-60 minutes.

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