JP5799395B2 - Microchip that can capture suspended cancer cells in blood - Google Patents

Description

  The present invention relates to a functional microchip for isolating or detecting floating cancer cells in blood.

Although the importance of direct analysis of cancer cells is recognized in cancer treatment and research, the collection is generally not easy due to the great physical burden.
On the other hand, recent studies have revealed that cancer cells are present in peripheral blood from the initial stage of cancer, and cancer cells can be easily collected if such blood cells can be isolated.
However, since the concentration of cancer cells in the blood is extremely low, a large amount of blood is required by a normal separation technique, and at present, it is substantially impossible to isolate cancer cells.
Non-Patent Document 1 discloses a technique of capturing cancer cells by immobilizing an antibody in a flow path of a microchip processed from a silicon wafer and flowing a few ml of blood.
However, the microchip disclosed in this document is a silicon wafer processed, and the processing procedure is complicated and expensive, it is opaque and cannot be observed through transmission, and it is brittle, so the range of use is limited, so it is practical. It was inferior in nature.
Patent Document 1 discloses a microchip in which an antibody is immobilized on the surface of a thin gold film, but the structure is still complicated and expensive.

JP 2010-38628 A

Isolation of rare circulating tumour cells in cancer patients by microchip technology, Sunitha Nagrath et al, Nature Vol.450 (2007), P.1235

An object of the present invention is to provide a resin-made microchip that is excellent in isolation or detection of floating cancer cells in blood, has high productivity, and is inexpensive.
In particular, an object of the present invention is to provide a resin microchip effective for isolating and detecting blood floating cancer cells.

The microchip according to the present invention includes a monomer Z having one polymerizable double bond and a carboxyl group and / or an epoxy group, and a monomer Z having one polymerizable double bond. A microchip comprising a polymer composition obtained by polymerizing and solidifying a mixture of a monomer X, a monomer Y having two or more polymerizable double bonds capable of crosslinking reaction, and a polymer B, It has a channel having a micro structure that allows fluid to pass through the surface or inside of the polymer composition, and in the channel, an antibody that binds to an antigen present on the surface of cancer cells is fixed to at least a part of the surface. The monomer X has a glass transition temperature of 10 ° C. or lower when it is polymerized alone, the polymer B is a core-shell type polymer fine particle, and the flow path has a depth. Is 50 to 150 μm, and the height below the depth at the bottom of the channel A plurality of protrusions are provided, the minimum distance between adjacent protrusions is 50 to 100 μm so that the flow of blood cells is not hindered, and the protrusions arranged on the bottom surface of the flow path are micro pillars. Is characterized by having a circumscribed circle diameter of 50 to 200 μm capable of capturing floating cancer cells in blood.
In the present invention, an antibody that binds to an antigen present directly on the surface of a cancer cell is immobilized in a microchannel.

In the present invention, a water-soluble polymer that binds to an antibody may be immobilized (covalently bonded) to the surface of the microchip channel .
In the present invention, the expression “having a flow path on the surface or inside of the polymer composition” means that a micro flow path is formed on the surface of the polymer composition, and a transparent resin plate or glass cover is formed on the micro flow path. A flow channel for feeding blood may be formed by superimposing lids such as two-dimensional and three-dimensional channels by superposing two or more polymer compositions having micro-channels on the surface. It may be formed.
Such a microchip is light transmissive and allows transmission observation.

Water-soluble polymers with carboxyl groups for immobilizing antibodies that react with antigens present on the surface of cancer cells floating in the blood are polyacrylic acid, polyacrylic acid copolymer, polymethacrylic acid, polymethacrylic acid. An acid copolymer or the like is preferable.
Therefore, the polymer composition (resin) according to the present invention contains a monomer Z having a functional group that reacts with a carboxyl group.
The monomer Z preferably has one polymerizable double bond that is polymerized by free radical polymerization, and the monomer Z is preferably contained in an amount of 10 to 35% by mass in the polymer composition.
If the amount is less than 10%, a water-soluble polymer having a carboxyl group is not sufficiently attached. If the amount exceeds 35%, a problem arises in moldability due to an increase in hardness or an increase in adsorption force.

The monomer Z may be any monomer having one polymerizable double bond, having a carboxyl group and / or an epoxy group, or having a functional group that reacts with a carboxyl group.
Examples of such monomers include glycidyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 4-vinylcyclohexane monoepoxide, 2-aminoethyl vinyl ether, dimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, diethylaminoethyl acrylate, diethylaminoethyl. Methacrylate, t-butylaminoethyl methacrylate, N-methylol acrylamide, N-methylol methacrylamide and the like can be mentioned.

The monomer Y is for causing a crosslinking reaction during polymerization.
The content of the monomer Y in the polymer composition is preferably 5 to 35% by mass.
If it is less than 5%, the adhesive strength of the polymer composition becomes too high, and if it exceeds 35%, the polymer composition becomes too hard and brittle.
Examples of monomer Y include ethylene glycol di (meth) acrylate, butanediol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, 1,10-di (meth) acrylate Examples include decanediol, polyethylene glycol di (meth) acrylate, and allyl (meth) acrylate.

The polymer composition of monomers Z and Y may be inferior in flexibility.
Therefore, in addition to the monomer Z and the monomer Y, the glass transition temperature for uniformly mixing the monomers Z and Y to soften the polymer composition is 10 ° C. or less, The monomer X having a polymerizable double bond may be contained.
Examples of the monomer X include (meth) acrylates in which an alkyl group or a cycloalkyl group is bonded to an ester moiety.
Examples of such monomers include methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, lauryl acrylate, nonyl acrylate, cyclohexyl acrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl Examples include methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, lauryl methacrylate, nonyl methacrylate, and cyclohexyl methacrylate.

The polymer composition composed of the monomers X, Y and Z may have excessive shrinkage during polymerization or may be inferior in elasticity.
Therefore, a polymer B that is uniformly mixed or dispersed in the monomers X, Y, and Z is contained, and the polymer B suppresses polymerization shrinkage of the polymer composition and improves elasticity.
The polymer B is not particularly limited as long as it is a polymer that can be dissolved or colloidally dispersed in a mixed liquid of the monomers X, Y, and Z. Poly (meth) acrylates, vinyl polymers, diene polymers Further, condensation polymers can be used.
In the present invention, the polymer B may be core / shell type polymer fine particles.
When the polymer B is a core / shell type polymer fine particle, the fine structure according to the present invention is dissolved or dispersed in a mixture of the monomers X, Y and Z and cured by photopolymerization. In this case, the increase in viscosity is suppressed and handling becomes easy.
The core-shell type polymer fine particles of the polymer B preferably have a generated stress of 100 MPa or less, more preferably 20 MPa or less, in a stretched 100% strain state.
As described above, the polymer composition constituting the fine structure needs to be given elasticity in order to improve the releasability, and therefore the polymer B is preferably flexible.
The particle diameter of the core / shell particles is not particularly limited, but considering that it is easy to use emulsion polymerization as the production method, particles having a particle diameter in the range of 0.01 to 10 μm are easy to use.
The polymer used for the shell of the core-shell particle is not particularly limited, but considering the ease of using emulsion polymerization as a production method and the generation of fluorescence, (meth) acrylate polymers such as polymethyl methacrylate and Examples thereof include a copolymer thereof, polyvinyl acetate and a copolymer thereof.
The polymer used for the core of the core / shell particle is not particularly limited, but considering the ease of using emulsion polymerization as a production method and the generation of fluorescence, (meth) acrylate polymers such as polybutyl acrylate and Examples thereof include a copolymer thereof, polyvinyl acetate and a copolymer thereof.
The core / shell type polymer fine particles are preferably cross-linked at least in order to maintain the shape.
As a result, the polymer B can stably form a dispersed phase in the polymer composition constituting the microstructure, and can function well as an elastic body when flexible.
There is no restriction | limiting in particular about the monomer which forms bridge | crosslinking, Divinyl monomers, di (meth) acrylates, allyl (meth) acrylate, etc. are mentioned.

The microchip according to the present invention can be solidified by photopolymerization by pouring a mixture of monomers X, Y, Z and polymer B into a mold.
There is no restriction | limiting in particular about a photoinitiator, It can select and use freely from photoinitiators, such as a benzoin ether type | system | group, a ketal type | system | group, an acetophenone type, a benzophenone type, and a thioxanthone type.
Moreover, a monomer, a polymer, an inorganic filler, etc. may be freely mix | blended with a photocurable resin in the range which does not inhibit the effect of this invention.
The light irradiation device used for photopolymerization is not particularly limited, and a device equipped with a high-pressure mercury lamp, a metal halide lamp, a UV discharge tube, or the like can be used freely.
It is more preferable that the photocurable resin is cured in a nitrogen atmosphere from the viewpoint of polymerizability.

The purpose of the microchip according to the present invention is to capture floating cancer cells in blood.
Therefore, it is preferable that the microchannel has a large surface area while ensuring the blood flow.
Therefore, in the microchip according to the present invention, the depth of the flow path is 50 to 150 μm, and a plurality of protrusions having a height equal to or less than the depth are arranged on the bottom surface of the flow path, and the minimum distance between adjacent protrusions Is preferably 50 to 100 μm.
The reason why the height of the protrusion is set to be equal to or less than the depth of the channel is to prevent interference with the upper portion of the protrusion when a transparent lid such as a resin plate or a glass cover is superimposed on the upper portion of the microchannel. is there.
The number of protrusions is preferably at least 100, and the protrusion shape itself is not limited.
In addition, if the protrusion arranged on the bottom surface of the flow path is a micro pillar, and the diameter of the circumscribed circle is 50 to 200 μm, it is easy to capture the suspended cancer cells in the blood on the side surface of the micro pillar. By setting the minimum distance between them to 50 to 100 μm, the flow of blood cells having a diameter of several μm to several tens of μm is not inhibited.

  Since the microchip according to the present invention is made of a resin, the productivity is high and the cost is low, and the antibody can be immobilized in the flow path, so that the antibody that binds to the antigen present on the surface of the cancer cell can be used in the fluid. Airborne cancer cells can be isolated and detected.

The flow-path enlarged image of the microchip which concerns on this invention is shown. The whole image of the microchip concerning the present invention is shown. The example of the state which mounted | wore the microchip with the holder is shown. The cancer cell capture | acquisition test result using the microchip based on this invention is shown.

A manufacturing example of the microchip according to the present invention will be described.
As monomer Z, glycidyl methacrylate manufactured by Wako Pure Chemical Industries, Ltd. was used.
As monomer X, n-butyl acrylate (glass transition temperature of polymer: −50 ° C.) manufactured by Wako Pure Chemical Industries, Ltd. was used.
As monomer Y, Blemmer PDE-100, which is diethylene glycol dimethacrylate manufactured by Nippon Oil & Fats, was used.
As the polymer B, Kuraray Parapet SA-NW201 (core-shell type polymer fine particles, generated stress of 100% strain of 11 MPa) was used.
As a photopolymerization initiator, a photopolymerization initiator DAROCUR 1173 (2-hydroxy-2-methyl-1-phenyl-propan-1-one) manufactured by Ciba Specialty Chemicals was used.
Monomer Z: 40 parts by weight of glycidyl methacrylate, monomer Y: 40 parts by weight of Blemmer PDE-100, monomer X: 40 parts by weight of n-butyl acrylate, polymer B: Parapet PET SA-NW201 20 parts by weight, photopolymerization initiator: DAROCUR 1173 is uniformly mixed at a blending ratio of 1 part by weight, the mixture is poured onto a mold of a microchip, and a glass plate is placed thereon to place a UV surface processing apparatus (Sun Energy) UV irradiation was performed with MDC15001Y-03 / SKC1102Y-01 (manufactured by Co., Ltd.) (ultraviolet irradiation was performed on a conveyor moving at a conveyance speed of 0.3 m / min).
FIG. 1 shows an enlarged photograph of the channel portion of the microchip taken out after curing, and FIG. 2 shows the whole image.
The microchip was dipped in a 10% polyacrylic acid methanol solution, evaporated at room temperature, and then heated at 150 ° C. for 1 hour. After heating, the microchip was washed with distilled water to remove unreacted polyacrylic acid.
It was confirmed by infrared absorption spectrum measurement (ATR method) that polyacrylic acid could be fixed in the channel.
Thus, the anti-mouse IgG antibody PBS solution (antibody concentration 20 μg / ml) and 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide meth- A solution prepared by mixing 1: 1 a solution of p-toluenesulfonate in PBS (concentration 20 mg / ml) was placed for 1 hour, and then the microchip was washed with PBS.
Here, the PBS solution refers to phosphate buffered saline that is generally used.
An anti-human EpCAM antibody (immunized animal: mouse) PBS aqueous solution (antibody concentration 20 μg / ml) was placed on the surface of the microchip on which the anti-mouse IgG antibody was fixed in this manner for 1 hour, and then the microchip was washed with PBS. Washed.

In order to send the liquid to the microchip, the resin plate is stacked on the microchip body processed as described above, attached to the holder and connected to the liquid supply tube (see FIG. 3), and the syringe is connected to the liquid supply tube. Then, the cell suspension was fed with a syringe pump.
As the cell suspension, KYSE220, which is an esophageal cancer cell line, was used, and a PBS suspension with a concentration of 400,000 cells / ml was used.
FIG. 4 shows the result of observing a chip subjected to a capture test by feeding about 1.5 ml of the cell suspension at a flow rate of 1 ml / h and then discharging the suspended matter with PBS.
When the anti-EpCAM antibody is fixed, a large number of cells are captured around the microcolumn (FIG. 4a), whereas when there is no anti-EpCAM antibody (until the anti-mouse IgG is fixed), no cells are observed. (FIG. 4b).
In addition, when the capture test was performed in the same manner using the blood of a healthy person on the microchip on which the anti-EpCAM antibody was immobilized, and the chip was observed, no cells were observed as in FIG. 4b.

Next, the same monomer, polymer and photopolymerization initiator as those described above were used and mixed at the same ratio as described above, and a microchip was produced using a UV surface processing apparatus.
On the surface of the obtained microchip, a PBS solution of an anti-mouse IgG antibody (antibody concentration 10 μg / ml) was placed for 1 hour, and then the microchip was washed with PBS.
An anti-human EpCAM antibody (immunized animal: mouse) PBS aqueous solution (antibody concentration 20 μg / ml) was placed on the surface of the microchip on which the anti-mouse IgG antibody was fixed in this manner for 1 hour, and then the microchip was washed with PBS. Washed.
The obtained microchip was mounted on a holder in the same manner as described above, and a cell suspension was fed with a syringe pump to perform a capture test.
When the microchip was observed after the capture test, it was observed that the cells were captured around the micropillars as in FIG. 4a.
Thus, it was confirmed that cancer cells could be captured by a microchip in which an antibody and a microchannel were combined.

Claims (1)

A monomer Z having one polymerizable double bond and having a carboxyl group and / or an epoxy group, and a monomer X other than the monomer Z having one polymerizable double bond, and a crosslinking reaction A microchip comprising a polymer composition obtained by polymerizing and solidifying a mixture of a monomer Y having two or more polymerizable double bonds and a polymer B,
Having a flow path having a microstructure that allows fluid to pass through the surface or inside of the polymer composition;
An antibody that binds to an antigen present on the surface of a cancer cell is fixed to at least a part of the surface of the channel,
The monomer X has a glass transition temperature of 10 ° C. or lower when polymerized alone.
The polymer B is a core-shell type polymer fine particle,
The flow path has a depth of 50 to 150 μm, and a plurality of protrusions having a height equal to or less than the depth are arranged on the bottom face of the flow path, and the minimum distance between adjacent protrusions does not inhibit the flow of blood cells. 50 to 100 μm, and the protrusion arranged on the bottom of the flow path is a micro pillar, and the micro pillar has a circumscribed circle diameter capable of capturing floating cancer cells in the blood of 50 to 200 μm. Microchip for capture of floating cancer cells.