JP7816326B2 - Polymer-coated glass substrate - Google Patents

Description

The present invention relates to a polymer-coated glass substrate.

A technology has been proposed to coat the surface of a substrate with a special polymer in order to create devices that can capture specific cells in blood and body fluids (blood cells, cancer cells present in blood and body fluids, etc.).

However, there is a concern that when specific cells such as cancer cells are captured on the substrate surface, blood cells may also be captured. Therefore, there is a need for a polymer-coated substrate that can more selectively capture specific cells such as cancer cells while suppressing the capture of normal cells such as blood cells.

The present invention aims to solve the above-mentioned problems by providing a polymer-coated glass substrate that can suppress the capture of normal cells such as blood cells and selectively capture specific cells such as cancer cells.

The present invention relates to a polymer-coated glass substrate having a polymer layer formed on the surface of the glass substrate, wherein the surface of the polymer layer has an elastic modulus of 1.20 MPa or less in water or an aqueous solution.

According to the present invention, a polymer-coated glass substrate is provided in which a polymer layer is formed on the surface of the glass substrate. The surface of the polymer layer has an elastic modulus of 1.20 MPa or less in water or an aqueous solution. This makes it possible to suppress the capture of normal cells such as blood cells and selectively capture specific cells such as cancer cells. Therefore, the polymer-coated glass substrate is expected to improve the ability to capture specific cells such as cancer cells.

The present invention relates to a polymer-coated glass substrate in which a polymer layer is formed on the surface of the glass substrate, and the surface of the polymer layer has an elastic modulus of 1.20 MPa or less in water or an aqueous solution.

Tumor cells (e.g., cancer cells) that appear in bodily fluids, such as circulating tumor cells (several to several hundred cells per mL of blood), are very rare. Therefore, capturing as many tumor cells as possible in collected bodily fluids is considered important for testing. The polymer-coated glass substrate of the present invention has a polymer layer surface with an elastic modulus of 1.20 MPa or less in water or aqueous solution. Compared to normal cells such as blood cells, specific cells such as cancer cells are generally known to be softer. This is related to the fact that, when specific cells such as cancer cells metastasize, they significantly deform their shape and slip through gaps to migrate. Therefore, normal cells such as blood cells, which are resistant to deformation and are hard to capture, are difficult to capture on polymer substrates with soft surfaces. On the other hand, specific cells such as cancer cells that have acquired deformability are easily captured even on polymer substrates with soft surfaces. Therefore, measuring the number of tumor cells captured in the polymer layer of the polymer-coated glass substrate of the present invention can determine the number of tumor cells in bodily fluids, which can be expected to confirm the effectiveness of cancer treatment. In addition, by culturing the captured tumor cells and using these cultured cells to confirm the effectiveness of anticancer drugs, etc., it is possible to confirm the effectiveness of anticancer drugs outside the body before administering them, and this is also useful for selecting anticancer drugs, etc. Furthermore, genetic analysis of the captured tumor cells is useful for selecting anticancer drugs, etc.

The polymer-coated glass substrate has a polymer layer formed on the surface of the glass substrate, and the surface of the polymer layer has an elastic modulus of 1.20 MPa or less in water or an aqueous solution.
From the viewpoint of suppressing capture of normal cells such as blood cells and selectively capturing specific cells such as cancer cells, the elastic modulus is preferably 0.80 MPa or less, more preferably 0.50 MPa or less, and even more preferably 0.30 MPa or less. The lower limit is not particularly limited, but is preferably 0.01 MPa or more, more preferably 0.05 MPa or more, and even more preferably 0.07 MPa or more.

The elastic modulus in water or an aqueous solution can be adjusted by changing the molecular weight of the polymer forming the polymer layer or the film thickness. Specifically, the elastic modulus tends to increase as the molecular weight of the polymer increases, and the elastic modulus also tends to increase as the film thickness of the polymer layer increases.
The elastic modulus in water or an aqueous solution can also be adjusted by previously subjecting the glass substrate to a surface treatment such as primer treatment, silane coupling agent treatment, etc. Specifically, when the polymer layer is formed on the surface of a surface-treated glass substrate, the elastic modulus tends to increase.

In this specification, unless otherwise specified, the elastic modulus in water or an aqueous solution refers to the elastic modulus in water or an aqueous solution measured using an atomic force microscope (AFM).

An atomic force microscope (AFM) is a type of scanning probe microscope that detects the force acting between atoms in a sample and a probe. The probe is attached to the tip of a cantilever (cantilever spring). The force (deflection) acting on the cantilever is measured while changing the distance between the sample and the probe, and a curve (force curve) is obtained that plots the relationship between the two. Analyzing this force curve determines the elastic modulus (hardness) of the sample surface, allowing the elastic modulus to be measured at the nano level. The method of determining the elastic modulus of a sample surface by force curve measurement is known to those skilled in the art, and it is possible to determine the elastic modulus using such known methods.

One method for calculating the elastic modulus from a force curve is to fit the force curve using the Johnson-Kendall-Roberts (JKR) theory, where the force F applied to the cantilever and the sample deformation δ are expressed by the following equations (1) and (2), where w is the adhesion energy.
In the formula, a represents the radius of the contact line between the probe and the sample, R represents the radius of curvature of the probe tip, and K represents the elastic modulus.

The elastic modulus can be determined by fitting the F-δ curve obtained by force curve measurement with equations (1) and (2).

Here, the elastic modulus in water or an aqueous solution specifically means a measured value measured by the following method.
Measurements in water or an aqueous solution can be performed by dropping water or an aqueous solution onto the surface of a sample so that a droplet (a convex meniscus) is formed, and then measuring with an AFM.
As the aqueous solution, for example, phosphate buffered saline (PBS) can be suitably used.

The elastic modulus of the sample (surface of the polymer layer) can be determined, for example, by scanning a specified area on the sample surface to obtain force curves at multiple points within that specified area, determining the elastic modulus from each force curve, and then calculating the average value.

The type of glass constituting the glass substrate is not particularly limited, and examples thereof include soda-lime glass, alkali-free glass, borosilicate glass (SiO ₂ -B ₂ O ₃ -ZnO-based glass, SiO ₂ -B ₂ O ₃ -Bi ₂ O ₃ -based glass, etc.), potash glass, crystal glass (glass containing PbO, for example, SiO ₂ -PbO-based glass, SiO ₂ -PbO-B ₂ O _3- based glass, SiO ₂ -B ₂ O ₃ -PbO-based glass _, etc.), titanium crystal glass, barium glass, boron glass (B ₂ O ₃ -ZnO-PbO-based glass, _{B 2 O 3} -ZnO- _{Bi 2 O 3 -} based glass, B ₂ O ₃ Examples of the glass include soda-zinc glass, soda-barium glass (BaO—SiO _{2-based glass, etc.), strontium glass, alumina silicate glass, soda-zinc glass, and soda-barium glass (BaO—SiO 2} -based glass, etc.). These glasses may be used alone or in combination of two or more.

The thickness of the glass substrate is not particularly limited, but the average thickness is preferably 100 μm or more and 5000 μm or less, and more preferably 500 μm or more and 3000 μm or less. The average thickness is calculated by measuring the thickness at 10 random locations using a micrometer and averaging the measured values.

As the polymer constituting the polymer layer, any known polymer can be used.
Examples of the polymer include a homopolymer of one type of monomer and a copolymer of two or more types of monomers. The polymer may be used alone or in combination of two or more types. Among the polymers, a polymer having hydrophilic properties (hydrophilic polymer) is preferred.

The above polymer can be produced by known methods, for example, by using a solution of the monomers that make up the polymer and polymerizing the monomers by known methods. The solvent for the monomer solution is not particularly limited, and the solvents described below can be used, for example. Of these, toluene and methanol are preferred.

Examples of the hydrophilic polymer include homopolymers and copolymers of one or more hydrophilic monomers, and copolymers of one or more hydrophilic monomers with one or more other monomers. These may be used alone or in combination of two or more.

The hydrophilic monomer is not particularly limited, and various monomers having a hydrophilic group can be used. Examples of the hydrophilic group include known hydrophilic groups such as an amide group, a sulfate group, a sulfonic acid group, a carboxylic acid group, a hydroxyl group, an amino group, and an oxyethylene group.

Specific examples of the hydrophilic monomer include (meth)acrylic acid, (meth)acrylic acid esters (alkoxyalkyl (meth)acrylates such as methoxyethyl (meth)acrylate, hydroxyalkyl (meth)acrylates such as hydroxyethyl (meth)acrylate), (meth)acrylamide, and (meth)acrylamide derivatives having a cyclic group ((meth)acryloylmorpholine, etc.). Of these, (meth)acrylic acid, (meth)acrylic acid esters, and alkoxyalkyl (meth)acrylates are preferred, alkoxyalkyl (meth)acrylates are more preferred, and 2-methoxyethyl acrylate is particularly preferred. These may be used alone or in combination of two or more types.

The other monomers may be selected appropriately as long as they do not interfere with the effects of the hydrophilic polymer. Specific examples of the other monomers include aromatic monomers such as styrene, vinyl acetate, and N-isopropylacrylamide, which can impart temperature responsiveness. These may be used alone or in combination of two or more.

Specific examples of the above-mentioned homopolymers and copolymers include homopolymers composed of one hydrophilic monomer such as polyacrylic acid, polyacrylic acid esters, polymethacrylic acid, polymethacrylic acid esters, polyacryloylmorpholine, polymethacryloylmorpholine, polyacrylamide, polymethacrylamide, polyalkoxyalkyl acrylates, and polyalkoxyalkyl methacrylates; copolymers composed of two or more of the above-mentioned hydrophilic monomers; and copolymers composed of one or more of the above-mentioned hydrophilic monomers and one or more of the above-mentioned other monomers. The above-mentioned hydrophilic polymers may be used alone or in combination.

Specific examples of homopolymers and copolymers of the above hydrophilic monomers include polyacrylic acid, polyacrylic acid esters, polymethacrylic acid, polymethacrylic acid esters, polyacryloylmorpholine, polymethacryloylmorpholine, polyacrylamide, polymethacrylamide, polyalkoxyalkyl acrylates, and polyalkoxyalkyl methacrylates.

Among these, the hydrophilic polymer is preferably a polymer represented by the following formula (I): These may be used alone or in combination of two or more kinds.
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, p represents 1 to 8, m represents 1 to 5, and n represents the number of repetitions.)

As the polymer represented by formula (I), for example, a polymer represented by the following formula (I-1) can be suitably used. These may be used alone or in combination of two or more kinds.
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, m represents 1 to 5, and n represents the number of repetitions.)

The number of carbon atoms in the alkyl group of R ⁵² is preferably 1 to 10, and more preferably 1 to 5. Of these, R ⁵² is particularly preferably a methyl group or an ethyl group. p is preferably 1 to 5, and more preferably 1 to 3. m is preferably 1 to 3. n (the number of repeating units) is preferably 15 to 1,500, and more preferably 40 to 1,200.

Copolymers of the compound represented by formula (II) below with other monomers can also be suitably used as the hydrophilic polymer. These may be used alone or in combination of two or more types.

(wherein R ⁵¹ , R ⁵² , p and m are the same as defined above.)

As the compound represented by formula (II), for example, a compound represented by formula (II-1) below can be suitably used. These may be used alone or in combination of two or more kinds.
(In the formula, R ⁵¹ , R ⁵² and m are the same as above.)

Among the hydrophilic polymers mentioned above, the hydrophilic polymer represented by formula (I) is preferred, and the hydrophilic polymer represented by formula (I-1) is particularly preferred, from the viewpoint of achieving better effects.

From the viewpoint of obtaining better effects, the number average molecular weight (Mn) of the polymer is preferably 8,000 to 150,000, more preferably 10,000 to 60,000, and even more preferably 10,000 to 39,000. When the polymer is a hydrophilic polymer, a similar number average molecular weight (Mn) is desirable.
In this specification, the number average molecular weight (Mn) and weight average molecular weight (Mw) can be determined in terms of standard polystyrene based on measured values obtained by gel permeation chromatography (GPC) (GPC-8000 series manufactured by Tosoh Corporation, detector: differential refractometer, column: TSKGEL SUPERMULTIPORE HZ-M manufactured by Tosoh Corporation).

The thickness of the polymer layer (layer formed of a polymer) is preferably 10 to 1000 nm, more preferably 30 to 700 nm, and even more preferably 50 to 350 nm. By adjusting the thickness within the above range, good low adsorption to proteins and cells and selective capture of cancer cells can be expected.
When the polymer layer is a hydrophilic polymer layer (a layer formed from a hydrophilic polymer), the same thickness (film thickness) is also desirable.
The thickness of the polymer layer can be measured by transmission electron microscopy (TEM).

At least a portion (part or all) of the surface of the polymer layer (the surface of the polymer layer in the polymer-coated glass substrate) preferably has a water contact angle of 60 degrees or less, more preferably 50 degrees or less. Furthermore, the water contact angle is preferably 25 degrees or more, more preferably 33 degrees or more.

The polymer layer can be produced by known methods, such as: (1) injecting a polymer solution or dispersion, prepared by dissolving or dispersing a polymer in various solvents, onto the surface of a glass substrate (e.g., into recesses in the substrate), holding the polymer for a predetermined period of time, and drying; or (2) applying (spraying) the polymer solution or dispersion onto the surface of the glass substrate and drying as needed. A polymer-coated glass substrate with a polymer layer formed on all or part of the surface can then be produced. By adding other components to the polymer-coated glass substrate as needed, a device capable of capturing, culturing, testing, etc., specific cells can be produced.

Conventionally known materials and methods can be used for the solvent, injection method, coating (spraying) method, and the like.
The holding and drying times of (1) and (2) may be set appropriately depending on the size of the substrate, the type of liquid introduced, etc. The holding time is preferably 10 seconds to 10 hours, more preferably 1 minute to 5 hours, and even more preferably 5 minutes to 2 hours. Drying is preferably carried out at room temperature (about 23°C) to 80°C, and more preferably room temperature to 60°C. Drying may also be carried out under reduced pressure. Furthermore, after a certain period of holding, excess polymer solution/dispersion may be appropriately discharged and dried.

The solvent is not particularly limited as long as it can dissolve the polymer, and can be selected appropriately depending on the polymer being used. Examples include water, organic solvents, and mixed solvents of these. Examples of organic solvents include alcohols such as methanol, ethanol, n-propanol, i-propanol, and methoxypropanol; ketones such as acetone and methyl ethyl ketone; tetrahydrofuran, acetonitrile, ethyl acetate, and toluene.

The concentration of the polymer solution or dispersion is not particularly limited and may be selected appropriately taking into consideration factors such as injectability, coatability, sprayability, and productivity. However, the concentration of the polymer in the polymer solution or dispersion (100% by mass) is preferably 0.1 to 10.0% by mass, and more preferably 0.2 to 5.0% by mass.

The polymer-coated glass substrate produced by the above-mentioned method has a soft surface, with the surface of the polymer layer having an elastic modulus of 1.20 MPa or less in water or an aqueous solution. Therefore, while the polymer-coated glass substrate has a low ability to capture normal cells such as blood cells, it has a high ability to capture specific cells such as cancer cells, making it excellent at selectively capturing specific cells.

The present invention will be explained in detail below based on examples, but the present invention is not limited to these examples.

Example 1
(Preparation of Polymer)
2-Methoxyethyl acrylate (2.5 wt % toluene) was thermally polymerized at 60° C. for 7 hours using a 12.5 mg/ml toluene solution of AIBN (azobisisobutyronitrile) to prepare poly(2-methoxyethyl acrylate) (PMEA).

145 μL of a 0.155% PMEA methanol solution was injected into a two-chamber chamber slide (made of soda-lime glass, average thickness 1300 μm). It was then immediately vacuum-dried (coated) in an oven at 40°C for 5 minutes to produce a polymer-coated glass substrate.

Example 2
(Preparation of Polymer)
2-Methoxyethyl acrylate (2.5 wt % methanol) was thermally polymerized at 60° C. for 7 hours using a 12.5 mg/ml methanol solution of AIBN (azobisisobutyronitrile) to prepare poly(2-methoxyethyl acrylate) (PMEA).

145 μL of a 0.155% PMEA methanol solution was injected into a two-chamber chamber slide (made of soda-lime glass, average thickness 1300 μm). It was then immediately vacuum-dried (coated) in an oven at 40°C for 5 minutes to produce a polymer-coated glass substrate.

<Comparative Example 1>
(Preparation of Polymer)
2-Methoxyethyl acrylate (5.0 wt % methanol) was thermally polymerized at 60° C. for 7 hours using a 12.5 mg/ml methanol solution of AIBN (azobisisobutyronitrile) to prepare poly(2-methoxyethyl acrylate) (PMEA).

145 μL of a 0.155% PMEA methanol solution was injected into a two-chamber chamber slide (made of soda-lime glass, average thickness 1300 μm). It was then immediately vacuum-dried (coated) in an oven at 40°C for 5 minutes to produce a polymer-coated glass substrate.

For the polymer-coated glass substrates prepared in the above examples and comparative examples, the thickness of the polymer layer and the elastic modulus in PBS (phosphate buffer solution) were measured using an AFM using the following methods. The results are shown in Table 1.

[Polymer layer thickness]
The thickness (film thickness) of the polymer layer formed on the polymer-coated glass substrate was measured by TEM under the condition of an acceleration voltage of 200 kV (JEM-2800, manufactured by JEOL).
The film thicknesses of Examples 1 and 2 and Comparative Example 1 are 78 nm, 88 nm, and 90 nm, respectively.

[Elastic modulus measurement in PBS (phosphate buffer solution) by AFM]
Phosphate buffered saline was dropped onto the surface of the polymer layer of the polymer-coated glass substrate so that a droplet (a convex meniscus) was formed, and the elastic modulus was measured using the following apparatus (AFM) and the following method. The obtained elastic modulus is the elastic modulus measured in water or an aqueous solution.
When measuring the elastic modulus, the obtained force curve was analyzed based on the JKR contact theory to determine the elastic modulus.
<Method for measuring elastic modulus>
Apparatus (AFM): Oxford Instruments MFP-3D-SA
Measurement mode: AFM force curve mapping Cantilever: Material: Si, tip curvature radius R = 150 nm, spring constant 0.67 N/m
Measurement range: 20 μm x 20 μm scan, elastic modulus calculated using the JKR two-point method Scan speed: 1 Hz
Measurement atmosphere: in PBS Measurement temperature: 23°C

Comparative Example 1 has an elastic modulus of 2 MPa or more, which is thought to reduce the adhesiveness of certain cells such as cancer cells. It is also thought to have high adhesiveness for white blood cells.

In contrast, in Example 2, the elastic modulus is 0.50 MPa or less, which is thought to improve the adhesiveness of certain cells such as cancer cells. It is also thought to reduce the adhesiveness of white blood cells.

In Example 1, the elastic modulus is 0.30 MPa or less, so it is thought that the adhesiveness of certain cells, such as cancer cells, will be improved compared to Example 2. It is also thought that the adhesiveness of white blood cells will be lower compared to Example 2.

The present invention (1) provides a polymer-coated glass substrate having a polymer layer formed on the surface of the glass substrate,
The surface of the polymer layer is a polymer-coated glass substrate having an elastic modulus of 1.20 MPa or less in water or an aqueous solution.

The present invention (2) is a polymer-coated glass substrate according to the present invention (1), in which the elastic modulus is 0.50 MPa or less.

The present invention (3) is a polymer-coated glass substrate according to the present invention (1), in which the elastic modulus is 0.30 MPa or less.

The present invention (4) is a polymer-coated glass substrate in any combination with any of the present inventions (1) to (3), in which the elastic modulus is 0.05 MPa or more.

The present invention (5) is a polymer-coated glass substrate in any combination with any of the present inventions (1) to (4), in which the polymer layer is formed from a polymer represented by the following formula (I):
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, p represents 1 to 8, m represents 1 to 5, and n represents the number of repetitions.)

The present invention (6) is a polymer-coated glass substrate in any combination with any of the present inventions (1) to (4), in which the polymer layer is formed from a polymer represented by the following formula (I-1):
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, m represents 1 to 5, and n represents the number of repetitions.)

The present invention (7) is a polymer-coated glass substrate in any combination with any of the present inventions (1) to (4), wherein the polymer layer is formed from a copolymer of a compound represented by the following formula (II) and another monomer:
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, p represents 1 to 8, and m represents 1 to 5.)

The present invention (8) is a polymer-coated glass substrate in any combination with any of the present inventions (1) to (4), wherein the polymer layer is formed from a copolymer of a compound represented by the following formula (II-1) and another monomer:
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, and m represents 1 to 5.)

The present invention (9) is a polymer-coated glass substrate in any combination with any of the present inventions (5) to (8), in which the number average molecular weight (Mn) of the polymer forming the polymer layer is 10,000 to 39,000.

The present invention (10) is a polymer-coated glass substrate in any combination with any of the present inventions (1) to (9), in which the thickness of the polymer layer is 10 to 1,000 nm.

The present invention (11) is a polymer-coated glass substrate in any combination with any of the present inventions (1) to (10), in which the elastic modulus is a value measured using an atomic force microscope.

Claims (7)

A polymer-coated glass substrate having a polymer layer formed on the surface of the glass substrate,
A polymer-coated glass substrate, wherein the surface of the polymer layer has an elastic modulus in water or an aqueous solution of 0.05 MPa or more and 0.30 MPa or less,
A polymer-coated glass substrate, wherein the number average molecular weight (Mn) of the polymer forming the polymer layer is 10,000 to 39,000. 2. The polymer-coated glass substrate according to claim 1, wherein the polymer layer is formed from a polymer represented by the following formula (I):
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, p represents 1 to 8, m represents 1 to 5, and n represents the number of repetitions.) The polymer-coated glass substrate according to claim 1, wherein the polymer layer is formed from a polymer represented by the following formula (I-1):
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, m represents 1 to 5, and n represents the number of repetitions.) 2. The polymer-coated glass substrate according to claim 1, wherein the polymer layer is formed from a copolymer of a compound represented by the following formula (II) and another monomer:
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, p represents 1 to 8, and m represents 1 to 5.) The polymer-coated glass substrate according to claim 1, wherein the polymer layer is formed from a copolymer of a compound represented by the following formula (II-1) and another monomer:
(In the formula, R ⁵¹ represents a hydrogen atom or a methyl group, R ⁵² represents an alkyl group, and m represents 1 to 5.) The polymer-coated glass substrate according to claim 1, wherein the polymer layer has a thickness of 10 to 1,000 nm. 2. The polymer-coated glass substrate according to claim 1, wherein the elastic modulus is a value measured using an atomic force microscope.