CN110133267A - A carrier for adsorbing tumor cells, preparation method, kit and application - Google Patents

Abstract

The invention discloses a kind of for adsorbing the carrier of tumour cell, preparation method, kit and application, it is related to vitro diagnostic techniques field, silicon line, high-molecular compound and Avidin are modified on carrier, bridge joint has biotin antibody on Avidin, high-molecular compound is polyethylene glycol, any one in PLGA and carboxymethyl chitosan.Carrier provided by the invention strengthens the interaction with tumour cell by nanostructure, improves biocompatibility, effectively reduces the non-specific adsorption of haemocyte.

Description

A carrier for adsorbing tumor cells, preparation method, kit and application

technical field

The invention relates to the technical field of in vitro diagnosis, in particular to a carrier for adsorbing tumor cells, a preparation method, a kit and application.

Background technique

Clinical significance of circulating tumor cells (CTCs) in peripheral blood as a liquid biopsy for cancer diagnosis or prognosis. In the past decade, scientists and doctors have developed various devices or biochips to detect CTCs from patient blood, such as immunomagnetic separation or utilizing size, density, electrical properties, etc. But expensive equipment or complicated manufacturing processes (especially the microfluidics in these chips) have become huge obstacles for cheap blood testing CTCs. Recently, nanochips have shown high-efficiency capture of CTCs, with microscale topography providing suitable locations for cancer cells, and nanostructures matching the size of the cancer. However, the development of glass-based biochips and the provision of easy-to-fabricate and low-cost biochips are still technical problems that need to be solved urgently.

In addition, most of the existing biochips have poor biocompatibility and serious non-specific adsorption of blood cells, seriously affecting normal cancer diagnosis and prognosis.

In view of this, the present invention is proposed.

Contents of the invention

The first object of the present invention is to provide a carrier for adsorbing tumor cells. The carrier strengthens the interaction with tumor cells through the nanostructure, improves biocompatibility, and effectively reduces the non-specific adsorption of blood cells.

The second object of the present invention is to provide a method for preparing a carrier for adsorbing tumor cells. The preparation method has the advantages of simple process, low cost and cheap equipment, and has relatively wide application prospects.

The third object of the present invention is to provide a kit. The detection kit can be used to detect circulating tumor cells.

The fourth object of the present invention is to provide an application of a carrier for adsorbing tumor cells in detection of circulating tumor cells.

The present invention is achieved like this:

A carrier for adsorbing circulating tumor cells. The carrier has a spherical or polyhedral structure. The carrier is any one of glass beads, plastic beads, nano-gold beads, polymer spheres and polyhedrons. The carrier is modified to bind circulating tumor cells. Antibodies to cells;

The polyhedron is any one of tetrahedron, hexahedron, octahedron, dodecahedron, icosahedron and tetrahedron, and the polymer sphere is any one of PLGA and carboxymethyl chitosan.

In a preferred embodiment of the present invention, the carrier is modified with silicon wires, polymer compounds and avidin, biotin is bridged to the avidin, and antibodies for binding circulating tumor cells are linked to the biotin;

The avidin is any one of avidin and streptavidin, and the polymer compound is any one of polyethylene glycol, PLGA and carboxymethyl chitosan.

In a preferred embodiment of the application of the present invention, the above-mentioned antibody is a biotin EpCAM antibody.

A preparation method of a carrier, the preparation method specifically includes linking the antibody to the carrier through bridging.

In a preferred embodiment of the application of the present invention, the above preparation method specifically includes first grafting and growing silicon nanowires on the carrier, then modifying the polymer compound on the surface of the carrier, and then combining avidin with the polymer modified The carrier is mixed and finally the antibody-linked biotin is mixed.

In a preferred embodiment of the application of the present invention, the graft growth of silicon nanowires on the above-mentioned carrier specifically includes: first cleaning the carrier with a plasma cleaner, then adding PVP solution, absolute ethanol and citrate reagent to the carrier, and then adding Ammonia water, and finally TEOS was added and kept warm to obtain a carrier with silicon nanowires grown on it.

In a preferred embodiment of the application of the present invention, the above-mentioned modification of the polymer compound on the surface of the carrier specifically includes the following steps: mixing the carrier modified with silicon wires with the polymer compound and a bridging agent, or mixing the carrier with the polymer compound and The mixed reagents composed of the bridging agent are mixed and reacted in the dark to prepare a carrier modified with a polymer compound;

Preferably, the macromolecular compound is any one of polyethylene glycol, PLGA and carboxymethyl chitosan, and the bridging agent is any one of silane and pentanediol, a mixture of the high molecular compound and the bridging agent The reagent is a silane-polyethylene glycol-carboxyl reagent, the concentration of the silane-polyethylene glycol-carboxyl reagent is 10-30 mg/ml, and the silane-polyethylene glycol-carboxyl reagent and the carrier modified with silicon wires are added in equal volumes.

In a preferred embodiment of the application of the present invention, the above-mentioned antibody is a biotin EpCAM antibody, and the amount of the antibody added is equal to that of the carrier, and the concentration is 10-50 ug/ml.

A kit for detecting circulating tumor cells, the kit includes a carrier for adsorbing circulating tumor cells.

In a preferred embodiment of the present invention, the above kit further includes a packed column, the two ends of the filled column are respectively provided with a liquid inlet and a liquid outlet, and the packed column is provided with a chamber for filling with multiple carriers.

Application of a carrier for adsorbing circulating tumor cells in detecting circulating tumor cells.

In a preferred embodiment of the present invention, a method for preparing antibody glass beads is also provided. The preparation method specifically includes the following steps: first graft and grow silicon nanowires on the glass beads, and then modify the polymer compound onto the glass beads. After being placed on the surface of the beads, EDC and streptavidin were mixed with glass beads modified by polymer compounds, and after 6-12 hours at room temperature in the dark, they were mixed with biotin antibody for 1.5-3 hours to prepare antibody glass beads.

In a preferred embodiment of the application of the present invention, the above-mentioned graft growth of silicon nanowires on the glass beads specifically includes: first cleaning the glass beads with a plasma cleaner, and then adding PVP solution, absolute ethanol and citrate reagent to the glass beads, Mix and shake for 5-7min, then add 100-120ul ammonia water, shake for 1-1.5min, finally add 200-250ul of TEOS, shake for 1-1.5min, then keep warm at 45-50°C for 6-12h to prepare grown silicon Glass beads of nanowires;

The amount of PVP solution added is 5-7mL of 0.1-0.12g/mL PVP solution per 0.1g of glass beads, the amount of absolute ethanol accounts for 8%-10% of the amount of PVP solution added, and the amount of citrate added It is 50-60ul, the concentration is 0.15-0.18M.

In a preferred embodiment of the application of the present invention, the above-mentioned mixing of EDC and streptavidin with the glass beads modified by the polymer compound specifically includes: first cleaning the glass beads modified by the polymer compound, and then using the addition amount of 500 - Treat glass beads with 600ul of EDC and NHS for 25-30min or use 500-600ul of EDC to treat glass beads for 25-30min. The concentration of EDC and NHS is 4-4.5mg/ml. 10-12mg/ml EDC 500-550ul, 45-50ug/ml streptavidin 500-550ul, react at room temperature in the dark for 6-12h, and prepare streptavidin-modified glass beads.

The present invention has the following beneficial effects:

The invention firstly modifies the silicon wire on the surface of the carrier, adds interaction with tumor cells through the nanostructure, and uses high molecular compounds to modify the surface of the carrier, greatly improving the biocompatibility of the carrier and effectively reducing the non-specific adsorption of blood cells. Modification of the antibody to the carrier by streptavidin and biotin is beneficial to increase the amount of modification of the antibody, further enhancing the amount of the antibody that can capture circulating tumor cells.

In addition, the column is filled with the carrier, which effectively utilizes the three-dimensional structure of the column, increases the surface area of the carrier in contact with blood, and is conducive to improving the overall adsorption efficiency of the column.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Fig. 1 is the scanning electron microscope picture of the antibody glass bead prepared through silicon wire modification;

Fig. 2 is the scanning electron microscope picture of the antibody glass beads prepared without silicon wire modification;

Figure 3 is an electron micrograph of the captured tumor cells after the packed column prepared in Figure 1 passes through the column;

Figure 4 is an electron micrograph of the captured tumor cells after the packed column prepared in Figure 2 passes through the column;

Fig. 5 is the electron micrograph of the PLGA ball of embodiment 3 capturing tumor cells;

Fig. 6 is the electron microscope picture that the carboxymethyl chitosan sphere of embodiment 4 captures tumor cell;

Figure 7 is a microscopic examination of the eluate after PEG modification in Example 5 of the present invention;

Fig. 8 is a microscopic image of the eluate without PEG modification in Example 5 of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

Example 1

This embodiment provides a method for preparing antibody glass beads, which specifically includes the following steps in sequence:

(1) Prepare 0.1g/ml PVP with 1-pentanol and dissolve overnight in the dark. Dissolving overnight in the dark can ensure that the active ingredients of PVP are fully dissolved. In other embodiments, it can also be dissolved in the dark for 8-12 hours. PVP (polyvinylpyrrolidone) is a non-ionic polymer compound. In this example, its excellent solubility and physiological compatibility were used to modify the surface of the glass beads. The molecular weight of PVP is 40,000.

(2) Take 0.1g glass beads, the particle diameter of glass beads is 200um-1mm, after cleaning the glass beads with a plasma cleaner for 25min, add 5mL of PVP solution in step (1), 0.5mL of absolute ethanol, and 140ul of water , 50ul of 0.18M trisodium citrate, shake and mix for 5min. Treating the surface of the glass beads with a plasma cleaner can clean the surface of the glass beads, and then modify and activate the surface groups of the glass beads, which is beneficial to the subsequent surface modification of the glass beads. Mixing and vibrating the glass beads with absolute ethanol and trisodium citrate reagent is conducive to the formation of small molecular emulsion droplets of the mixed reagents, which are attached to the surface of the glass beads. In other embodiments, the cleaning method of the glass beads may also be acid cleaning or ultrasonic cleaning.

(3) Add 100ul of ammonia water, and then continue shaking for 1min.

(4) Silicon wire modification: In step (3), 200ul of TEOS (chemical formula: Si(OC ₂ H ₅ ) ₄ ) was added, shaken for 1min, and heated in an oven at 50°C overnight. TEOS can accelerate hydrolysis under alkaline conditions, and silicon nanowires can be grafted and grown on glass beads through steps (1)-(4).

(5) Wash the glass beads with absolute ethanol the next day. This operation can effectively remove excess TEOS.

(6) Add 30 mg/ml of silane-polyethylene glycol-carboxyl reagent (the silane-polyethylene glycol-carboxyl reagent is prepared with 95% ethanol), and react in the dark for 48 hours. In this embodiment, PEG is modified to the surface of glass beads by silane. In addition, in other embodiments, PEG can also be modified to the surface of glass beads by other bridging agents such as pentanediol through hydroxyl bridging. The polymer compound PEG The molecular weight can be several hundred to tens of thousands. PEG can also be replaced with other biocompatible materials such as PLGA, carboxymethyl chitosan, and also polymers.

(7) Use PBS to wash the glass beads to remove excess reagents that are not bound to the glass beads, and then add 500ul 4mg/ml of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 500ul 4mg/ml NHS (N-hydroxysuccinimide) to activate the carboxyl groups on the glass beads for 30min. EDC is used as an activating reagent for carboxyl groups in amide synthesis, and the coupling efficiency can be improved by using it in combination with NHS. In other embodiments, EDC can also be used in combination with N-hydroxysulfosuccinimide. In other embodiments, the carboxyl group can also be activated by EDC/NHS alone or EDC.

(8) Discard the supernatant of the solution obtained in step (7), add 500 ul of 10 mg/ml EDC and 500 ul of 50 ug/ml streptavidin respectively, and overnight at room temperature in the dark. Streptavidin is modified onto glass beads via carboxyl and amino groups.

(9) Wash the glass beads in step (8) with PBS, add 10 ug/ml biotin EpCAM antibody, and react at room temperature for 2 hours. One streptavidin (SA) can bind 4 biotins, and by modifying streptavidin on glass beads, more biotin binding can be achieved, thereby increasing the amount of antibody modification and further amplifying the effect. Enhances the antibody's ability to capture tumor cells. This setting is beneficial to when the blood flows through the glass beads, the antibodies on the surface of the glass beads can fully contact the circulating tumor cells in the blood, adsorb and retain the circulating tumor cells in the blood, and increase the capture ability of the antibody glass beads. Through the step (9), the streptavidin reacts with the biotin, and the antibody on the biotin is modified onto the glass beads, finally forming the glass bead-PEG-SA-biotin antibody. In other embodiments, instead of linking the antibody through streptavidin, the glass beads can be directly PEG-modified and then directly linked to the antibody.

(10) Wash the glass beads with PBS to remove excess unbound biotin-EpCAM antibody.

Example 2

The antibody glass beads prepared in Example 1 were used to prepare a packed column. Both ends of the packed column were open, one end was the liquid inlet end and the other end was the liquid outlet end, and the blood flow rate was controlled by a peristaltic pump. Pass the sample blood through the column, and the circulating tumor cells can be captured when they pass through the antibody glass beads, and stay in the column, while the normal blood cells flow out from the liquid outlet. By adjusting the gap between the glass beads, the contact area with blood can be adjusted, and the flow of tumor cells can also be restricted.

The antibody glass beads prepared by silicon wire modification in Example 1 were scanned by electron microscope, and the electron microscope picture was shown in Figure 1; at the same time, the antibody glass beads prepared without silicon wire modification were used as the control test group to scan the antibody glass beads by electron microscope, and the electron microscope scan Refer to Figure 2 for the diagram. It can be seen from Figure 1 and Figure 2 that the outer surface of the antibody glass beads prepared by silicon wire modification is rougher, and the outer surface of the antibody glass beads in Figure 2 is relatively smooth. Prepare packed columns with antibody glass beads prepared by silicon wire modification and glass beads without silicon wire modification respectively, take equal amount and concentration of blood samples and put them on the column, then observe the capture effect of glass beads with electron microscope, refer to Figure 3 As shown in Figure 4, it can be seen from Figure 3 that more tumor cells are attached around the silicon wire-modified antibody glass beads, while less tumor cells are attached around the antibody glass beads without silicon wire modification in Figure 4 cell. It can be concluded that the outer surface of the glass beads modified by silicon wires is rougher and easier for tumor cells to attach. Modification of nano-silicon wire structures on glass beads can enhance the interaction with tumor cells.

Example 3

This embodiment provides that PLGA balls are directly made into PLGA balls, and streptavidin and biotin antibodies are modified on the surface of the PLGA balls to finally form PLGA balls-SA-biotin antibodies. The modified PLGA spheres were loaded into the packed column, and after the blood sample was passed through the column, the effect of PLGA spheres binding to tumor cells was observed under the electron microscope. The results are shown in FIG. 5 . From FIG. 5 , the PLGA spheres can bind more tumor cells.

Example 4

The present embodiment provides that carboxymethyl chitosan balls are directly made into carboxymethyl chitosan balls, and streptavidin and biotin antibodies are modified on the surface of carboxymethyl chitosan balls to finally form Carboxymethyl chitosan spheres-SA-biotin antibody. The modified carboxymethyl chitosan spheres were loaded into a packed column, and after blood samples were taken and passed through the column, the effect of carboxymethyl chitosan spheres binding to tumor cells was observed under an electron microscope. The results are shown in FIG. 6 . From FIG. 6 , carboxymethyl chitosan spheres can bind more tumor cells.

Example 5

This example provides comparative experiments to prove that the PEG-modified antibody glass beads can effectively enhance the ability to capture tumor cells. The PEG-modified antibody glass beads prepared in Example 1 were used to prepare packed columns. The control group was not modified with PEG, and the rest of the treatment was the same as in Example 1. The same whole blood was used to load the column, and the eluted glass beads captured The number of cells, microscopic examination under a microscope, and microscopic examination results under a 10x objective lens are shown in Figure 7 and Figure 8.

In Figures 7 and 8, the large dots are tumor cells, and the dots smaller than the tumor cells are blood cells. Fig. 7 is a micrograph modified with PEG. Comparing Fig. 7 and Fig. 8, it can be seen that Fig. 7 has less blood cell content and more tumor cell content, and Fig. 8 has more blood cell content and less tumor cell content. Therefore, the ability to capture tumor cells with PEG modification is stronger than that without PEG modification, and the ability to non-specifically bind blood cells with PEG modification is weaker than that without PEG modification. According to statistics, the capture rate of tumor cells in Figure 7 is 45%, and that in Figure 8 is 35%.

Therefore, by modifying the surface of the glass beads with PEG, the biocompatibility is effectively improved, and the non-specific adsorption of blood cells is effectively reduced.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (11)

1. A carrier for adsorbing circulating tumor cells, characterized in that, the carrier is in a spherical or polyhedral structure, and the carrier is any one of glass beads, plastic beads, nano-gold beads, polymer spheres and polyhedrons species, the carrier is modified with an antibody for binding to circulating tumor cells; The polyhedron is any one of tetrahedron, hexahedron, octahedron, dodecahedron, icosahedron and tetrahedron, and the polymer sphere is any one of PLGA and carboxymethyl chitosan kind. 2. The carrier for adsorbing circulating tumor cells according to claim 1, characterized in that, the carrier is modified with silicon wires, polymer compounds and avidin, and the avidin is bridged with biotin, so The biotin is linked with an antibody for binding to circulating tumor cells; The avidin is any one of avidin and streptavidin, and the polymer compound is any one of polyethylene glycol, PLGA and carboxymethyl chitosan. 3. The carrier for adsorbing circulating tumor cells according to claim 1, wherein the antibody is a biotin EpCAM antibody. 4. A preparation method of the carrier according to any one of claims 1-3, characterized in that the preparation method specifically comprises linking the antibody to the carrier through bridging. 5. The preparation method according to claim 4, characterized in that, the preparation method specifically comprises first grafting and growing silicon nanowires on the carrier, then modifying the polymer compound on the surface of the carrier, and then combining avidin with The carrier modified by the polymer compound is mixed, and finally mixed with the biotin linked to the antibody. 6. The preparation method according to claim 5, characterized in that, grafting and growing silicon nanowires on the carrier specifically comprises: first cleaning the carrier with a plasma cleaning machine, then adding PVP solution, absolute ethanol and lemon to the carrier. Acid salt reagent, then add ammonia water, finally add TEOS, keep warm, and prepare the carrier with silicon nanowires grown. 7. The preparation method according to claim 5, characterized in that, modifying the polymer compound on the surface of the carrier specifically comprises the following steps: mixing the carrier modified with silicon wires with the polymer compound and a bridging agent, or mixing the carrier Mix with a mixed reagent composed of a polymer compound and a bridging agent, and react in the dark to prepare a carrier modified with a polymer compound; Preferably, the polymer compound is polyethylene glycol, any one of PLGA and carboxymethyl chitosan, and the bridging agent is any one of silane and pentylene glycol, and the polymer compound The mixed reagent composed of bridging agent is silane-polyethylene glycol-carboxyl reagent, the concentration of silane-polyethylene glycol-carboxyl reagent is 10-30mg/ml, and the silane-polyethylene glycol-carboxyl reagent is modified with Add equal volumes of the carrier for the silicon wires. 8 . The preparation method according to claim 5 , wherein the antibody is a biotin EpCAM antibody, and the amount of the antibody added is equal to that of the carrier, and the concentration is 10-50 ug/ml. 9. A kit for detecting circulating tumor cells, characterized in that the kit comprises the carrier for adsorbing circulating tumor cells according to claim 1. 10. The kit according to claim 9, characterized in that, the kit also includes a packed column, the two ends of the packed column are respectively provided with a liquid inlet and a liquid outlet, and a useful for chambers filled with multiple carriers. 11. The application of the carrier for adsorbing circulating tumor cells according to claim 1 in detecting circulating tumor cells.