JP2008249434A - Biosensor chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biosensor chip having a large surface area and capable of improving sensitivity by being applied to an SPR sensor or the like, and a manufacturing method thereof.
[Solution]
A substrate 11, a metal film 12 provided on the surface, a fine porous polymer film 13 fixed on the metal film 12 via a thiol-based silane coupling agent 14 and having a hydrophilic functional group on the surface It comprises.
[Selection] Figure 1

Description

  The present invention relates to a biosensor chip applicable to a biosensor such as a surface plasmon resonance biosensor and a method for manufacturing the same.

  A biosensor is a general term for chemical sensors that use a molecular recognition mechanism of biological origin, and includes, for example, a surface plasmon resonance sensor (hereinafter referred to as an SPR sensor), a crystal oscillator microbalance sensor (hereinafter referred to as a QCM sensor), and the like. be able to. SPR sensor is a method of detecting mass change such as adsorption and desorption occurring near the surface of the organic functional film in contact with the metal film of the chip as a refractive index change and detecting it as a peak shift of reflected light wavelength or a change in reflected light quantity at a constant wavelength It is. The QCM sensor can detect the adsorption / desorption mass at the ng level from the change in the oscillation frequency of the oscillator due to the adsorption / desorption of a substance on the gold electrode (device) of the crystal oscillator.

  The structure of such a biosensor will be described using surface plasmon resonance (SPR) as an example. A commonly used measuring chip has a transparent substrate such as glass, a metal film deposited thereon, and a functional film that can immobilize a physiologically active substance provided on the metal film. It has a structure in which a physiologically active substance is immobilized on the surface of the membrane via its functional group. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the sample substance.

  As such a functional membrane, a physiologically active substance is immobilized using a functional group that binds to a metal, a linker having a chain length of 10 or more, and a compound that has a functional group that can bind to a physiologically active substance. A measuring chip has been reported (see Patent Document 1), and generally an alkanethiol monomolecular film is used. Such alkanethiol has a carboxyl group that reacts with a reagent to be added to a functional group that binds to a physiologically active substance while binding to a metal film via a thiol group.

  In such a biosensor, there is a demand for improving the sensitivity as much as possible. For this purpose, the test surface of the measuring chip needs to be large and highly wettable with respect to the physiologically active substance, but none has been realized.

Japanese National Patent Publication No. 4-502605

  An object of the present invention is to provide a biosensor chip that has a large surface area and can be applied to an SPR sensor or the like to improve sensitivity and a method for manufacturing the same.

  A first aspect of the present invention that achieves the above object is to provide a substrate, a metal film provided on the surface, a thiol-based silane coupling agent on the metal film, and a hydrophilic functional group on the surface. A biosensor chip comprising a microporous polymer film having a group.

  In the first aspect, since the microporous polymer film having a hydrophilic functional group on the surface is fixed to the surface of the metal film, the physiologically active substance is chemically modified through the hydrophilic functional group. Functionality that can be immobilized can be easily provided, and since the surface area is large, a biosensor chip with high sensitivity per unit area can be realized.

  According to a second aspect of the present invention, in the biosensor chip according to the first aspect, the microporous polymer membrane is a hydrophobic organic solvent solution containing an amphiphilic polymer at a relative humidity of 50 to 95%. The biosensor chip is a film formed by casting under high-humidity conditions.

  In the second aspect, a hydrophobic organic solvent solution in which an amphiphilic polymer is dissolved is cast under high humidity, so that water droplets are arranged on the casting film by self-assembly during the film formation, and the water droplets are vaporized. After that, a fine porous polymer film having fine pores formed on the surface is formed.

  According to a third aspect of the present invention, in the biosensor chip according to the second aspect, the hydrophobic organic solvent solution contains a hydrophobic polymer.

  In the third aspect, the hydrophobic organic solvent solution contains a hydrophobic polymer, whereby a microporous polymer film having high mechanical strength is formed.

  According to a fourth aspect of the present invention, in the biosensor chip according to the third aspect, the hydrophobic organic solvent solution contains a hydrophobic polymer and an amphiphilic polymer in a mass ratio of 50:50 to 99: 1. The biosensor chip is characterized by comprising at a ratio of

  In the fourth aspect, by setting the composition of the polymer in the hydrophobic organic solvent solution to be cast within a predetermined range, a microporous polymer film having high mechanical strength can be formed relatively easily.

  According to a fifth aspect of the present invention, in the biosensor chip according to any one of the second to fourth aspects, the hydrophilic functional group is derived from a hydrophilic portion of the amphiphilic polymer. The biosensor chip is characterized by the above.

  In the fifth aspect, hydrophilic portions are unevenly distributed on the inner surface of the fine pores on the surface of the fine porous polymer film, and hydrophilic functional groups are unevenly present in the fine pores.

  According to a sixth aspect of the present invention, in the biosensor chip according to any one of the first to fifth aspects, the microporous polymer film is subjected to a surface treatment by plasma oxidation treatment or ozone oxidation treatment. It is in the biosensor chip characterized by being.

  In the sixth aspect, hydrophilicity is imparted to the surface of the microporous polymer film by plasma oxidation treatment or ozone oxidation treatment.

  According to a seventh aspect of the present invention, in the biosensor chip according to the sixth aspect, the hydrophilic functional group includes those formed by the surface treatment.

  In the seventh aspect, many hydrophilic functional groups formed by the surface treatment exist on the surface of the microporous polymer membrane.

  According to an eighth aspect of the present invention, in the biosensor chip according to any one of the first to seventh aspects, the hydrophilic functional group is a carboxyl group.

  In the eighth aspect, functionality can be easily imparted to the film surface by chemical modification using a carboxyl group.

  According to a ninth aspect of the present invention, in the biosensor chip according to any one of the first to eighth aspects, the biosensor chip is used for a surface plasmon resonance biosensor.

  The ninth aspect is useful as a surface plasmon resonance biosensor.

  According to a tenth aspect of the present invention, in the biosensor chip according to any one of the first to ninth aspects, the surface of the microporous polymer film is chemically modified. is there.

  In the tenth aspect, the surface of the biosensor chip is chemically modified to have a function capable of immobilizing a physiologically active substance.

  An eleventh aspect of the present invention includes a step of providing a metal film on the surface of a substrate, a step of treating the surface of the metal film with a thiol-based silane coupling agent, and a hydrophobic organic solvent solution containing an amphiphilic polymer Forming a fine porous polymer film by casting the metal film on the metal film under a high humidity condition of 50 to 95% relative humidity.

  In the eleventh aspect, by casting a hydrophobic organic solvent solution in which the amphiphilic polymer is dissolved under high humidity, water droplets are arranged on the casting film by self-assembly during the film formation, and the water droplets are vaporized. After that, a fine porous polymer film having a large surface area that is firmly fixed to the metal film surface via a thiol-based silane coupling agent can be formed relatively easily.

  According to a twelfth aspect of the present invention, there is provided the method for producing a biosensor chip according to the eleventh aspect, wherein the hydrophobic organic solvent solution contains a hydrophobic polymer. .

  In the twelfth aspect, the hydrophobic organic solvent solution contains a hydrophobic polymer, whereby a microporous polymer film having high mechanical strength can be obtained.

  According to a thirteenth aspect of the present invention, in the method for producing a biosensor chip according to the twelfth aspect, the hydrophobic organic solvent solution comprises a hydrophobic polymer and an amphiphilic polymer in a mass ratio of 50:50 to 50:50. The biosensor chip manufacturing method is characterized by comprising 99: 1.

  In the thirteenth aspect, a fine porous polymer film having high mechanical strength can be obtained relatively easily by setting the polymer contained in the hydrophobic organic solvent solution to a predetermined composition.

  A fourteenth aspect of the present invention is the biosensor chip manufacturing method according to any one of the first to thirteenth aspects, wherein the surface of the microporous polymer film is subjected to a surface treatment by plasma oxidation treatment or ozone oxidation treatment. In a method of manufacturing a biosensor chip, the method includes:

  In the fourteenth aspect, the surface hydrophilicity can be further improved by subjecting the surface of the microporous polymer film to plasma oxidation treatment or ozone oxidation treatment.

  According to the present invention, by applying a microporous polymer film by self-organization, a functional film having a hydrophilic functional group on the surface and a large surface area due to micropores can be obtained. A biosensor chip applicable to the above and a manufacturing method thereof can be provided.

  An example of the biosensor chip of the present invention is schematically shown in FIG. As shown in FIG. 1, the biosensor chip has a microporous polymer film 13 having a hydrophilic functional group on the surface and provided with a pore 13a on a metal film 12 provided on the surface of the substrate 11. It is provided. Further, the surface of the metal film 12 on which the microporous polymer film 13 is provided is provided with a surface treatment layer 14 made of a thiol-based silane coupling agent in order to improve the adhesion to the microporous polymer film 13. Is.

  Here, the substrate 11 is not particularly limited and may be selected according to the biosensor to be used. However, when a biosensor chip for a surface plasmon resonance biosensor is used, a transparent substrate is used. As the transparent substrate, generally used is an optical glass such as BK7, or a synthetic resin, specifically, a material made of a material transparent to laser light such as polymethyl methacrylate, polyethylene terephthalate, polycarbonate, or cycloolefin polymer. it can. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  On the other hand, the metal film 12 is not particularly limited as long as it can cause surface plasmon resonance, for example, when considering use for a surface plasmon resonance biosensor. Preferred examples include free electron metals such as gold, silver, copper, aluminum, and platinum, with gold being particularly preferred. These metals can be used alone or in combination. In consideration of adhesion to the substrate 11, an intervening layer made of chromium or the like may be provided between the substrate 11 and the metal film 12.

  Although the thickness of the metal film 12 is arbitrary, for example, when considering the use for a surface plasmon resonance biosensor, it is preferably 0.1 nm to 500 nm, more preferably 0.5 nm to 500 nm, particularly 1 nm or more. It is preferably 200 nm or less. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 to 10 nm.

  The metal film 12 may be formed by a conventional method, for example, a sputtering method, a vapor deposition method, an ion plating method, an electroplating method, an electroless plating method, or the like.

  Further, a surface treatment layer 14 using a thiol-based silane coupling agent is provided on the surface of such a metal film 12. Here, the thiol-based silane coupling agent is a silane coupling agent having a thiol-based functional group that chemically bonds to the metal film 12 such as gold, and fixes the metal film 12 and the microporous polymer film 13. It is something to do firmly.

  The fine porous polymer film 13 of the present invention is a polymer film having a large number of fine pores on the surface, a so-called self-organized porous film, and is also referred to as a honeycomb structure film. Here, “self-organization” means that the system itself forms an ordered structure without giving information on the structure from the outside, and represents a state in which micropores themselves form an ordered structure. .

  In other words, when a hydrophobic organic solvent solution containing an amphiphilic polymer is cast under high humidity conditions, fine water droplets adhere to the surface in the process of evaporation of the solvent, and the water droplets evaporate. A fine porous polymer film having fine pores arranged by the formation is obtained.

  Specifically, a solution in which an amphiphilic polymer is dissolved in a hydrophobic organic solvent is applied on a substrate, and a gas having a predetermined relative humidity is substantially constant on the surface of the solution applied on the substrate. The organic solvent is evaporated and the water vapor is condensed on the surface of the solution, and the generated water droplets are evaporated, so that a large number of pores are arranged substantially continuously and regularly. A porous polymer film (honeycomb structure film) is formed.

  The formation mechanism of the microporous polymer film is estimated, for example, as follows, but is not intended to be any limitation. First, the water vapor in the gas is condensed by the latent heat when the solvent evaporates to form water droplets, which adhere to the surface of the solution applied on the substrate. At this time, the amphiphilic polymer reduces the surface tension between the water droplet and the hydrophobic organic solvent, thereby preventing the water droplet from fusing into a large mass. Water droplets are transported to the boundary of the solution by the convection and transverse capillary force generated in the solution and arranged in the closest packing, and the water droplet is fixed on the substrate by the receding of the solution surface as the solvent evaporates. . Finally, the water droplets evaporate to form a microporous polymer film in which a large number of pores are substantially continuously and regularly arranged using the water droplet array as a template.

  An amphiphilic polymer is used as the main component of the microporous polymer membrane of the present invention. Here, the “amphiphilic polymer” means that the main chain skeleton has a hydrophobic portion and / or a hydrophobic side chain (both are referred to as hydrophobic sites), a hydrophilic portion and / or a hydrophilic side chain (both are hydrophilic). A polymer having a sex site). In addition, what does not melt | dissolve in water is used for the amphiphilic polymer concerning this invention. This is because the amphiphilic polymer is added to prevent water droplets attached to the polymer solution applied on the substrate from fusing together as described above. The amphiphilic polymer that can be used is not particularly limited as long as it is a polymer having a hydrophobic part and / or a hydrophobic side chain and a hydrophilic part and / or a hydrophilic side chain in the main chain skeleton. However, for example, polyacrylamide as a main chain skeleton, and as a hydrophobic side chain, for example, a linear hydrocarbon group having 5 to 20 carbon atoms, a nonpolar linear group such as a phenylene group, In addition, an amphiphilic polymer having a hydrophilic group such as a hydroxy group, a carboxyl group, an amino group, a carbonyl group, or a sulfo group as the hydrophilic side chain, such as the following formula (Chemical Formula 1) (N-dodecylacrylamide) n- (N-5-carboxypentylacrylamide) m-block copolymer (n: m = 1: 4) and (styrene) p- (acrylic acid) q-block represented by the following formula (Chemical Formula 2) Obtained by polycondensation of a polymer, a block copolymer of polyethylene glycol and polypropylene glycol having a hydrophobic part and a hydrophilic part, or dichlorodiphenyl sulfone and a sodium salt of bisphenol A, And polysulfone having a diphenylenedimethylmethylene group which is a hydrophobic portion and a diphenylenesulfone group which is a hydrophilic portion. These amphiphilic polymers may be used alone or in combination of two or more.

  In addition to the amphiphilic polymer, which is the main component, a hydrophobic polymer may be used. Here, the “hydrophobic polymer” means a polymer exhibiting poor solvent resistance to water. The hydrophobic polymer that can be used is not particularly limited as long as it shows poor solvent resistance to water. For example, polyvinyl chloride, polyvinylidene chloride, polystyrene, poly-N-vinylcarbazole, Vinyl polymers such as polyvinyl acetate, polyacrylonitrile, polymethacrylonitrile, polymethyl vinyl ether; acrylic polymers such as polymethyl acrylate, polyethyl acrylate, polymethyl methacrylate, polyethyl methacrylate; polycaprolactone, polyethylene terephthalate, polybutylene terephthalate, Polyesters such as polycyclohexyldimethylene terephthalate, polyethylene naphthalate, polybutylene naphthalate; interfacial polycondensation of bisphenol A and phosgene, bisphenol A and di Polycarbonate obtained by transesterification with phenyl carbonate; polyarylate obtained by polycondensation of bisphenol A and aromatic dicarboxylic acid; polyimide obtained by polycondensation of pyromellitic anhydride and aromatic diamine, trimellitic acid and aromatic Polyamideimide obtained by polycondensation with diamine, polyimide such as polyetherimide obtained by polycondensation of 4,4 ′-[isopropylidenebis (p-phenyleneoxy)] diphthalic dianhydride and aromatic diamine; Polysulfone obtained by polycondensation of dichlorodiphenylsulfone and bisphenol A, polyethersulfone obtained by polycondensation of dichlorodiphenylsulfone and dihydroxydiphenylsulfone, obtained by oxidative polymerization of 2,6-dimethylphenol Polyphenylene ethers, polyether, such as polyether ether ketone obtained by polycondensation of a dihalogeno benzophenone and hydroquinone; polydimethylsiloxanes, polydiene propyl siloxane, polysiloxanes, such as polydiphenylsiloxane; and the like. Here, unless otherwise specified, the “polymer” is not only a single polymer, but also an alternating copolymer, a random copolymer, or a block copolymer obtained by copolymerizing 50 mol% or less of another copolymerizable monomer. And a graft copolymer. These polymers may be used alone or in combination of two or more. By using the hydrophobic polymer as described above, a fine porous polymer film having high mechanical strength can be formed.

  The molecular weights of the amphiphilic polymer and the hydrophobic polymer are not particularly limited as long as they are dissolved in the hydrophobic organic solvent described later. For example, the weight average molecular weight (Mw) or the number average molecular weight (Mn ), The lower limit is preferably 1,000, more preferably 5,000, still more preferably 10,000, and the upper limit is preferably 1,000,000, more preferably 500,000, still more preferably Is 100,000.

  Since the solvent for preparing the polymer solution needs to form water droplets on the polymer solution, a hydrophobic organic solvent is used. The usable hydrophobic organic solvent is not particularly limited as long as it dissolves the above-mentioned amphiphilic polymer and hydrophobic polymer. For example, aromatic hydrocarbons such as benzene, toluene, xylene, etc. Halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and methylene chloride; esters such as ethyl acetate and butyl acetate; hydrophobic ketones such as methyl isobutyl ketone; ethers such as diethyl ether; carbon disulfide And so on. These hydrophobic organic solvents may be used alone or in combination of two or more.

  The polymer concentration of the amphiphilic polymer and the hydrophobic polymer in the polymer solution (the total polymer mass (%) with respect to the polymer solution mass) is the thickness of the microporous polymer membrane, It may be appropriately adjusted according to the pore diameter, porosity, etc., and is not particularly limited. For example, the lower limit is preferably 0.001% by mass, more preferably 0.005% by mass, and still more preferably 0. The upper limit is preferably 10% by mass, more preferably 5% by mass, and still more preferably 1% by mass. When the polymer concentration is less than 0.001% by mass, the strength of the microporous polymer film may be reduced, and the pore size and arrangement may be irregular. On the other hand, when the polymer concentration exceeds 10% by mass, vacancies may not be sufficiently formed.

  In particular, in the case of a microporous polymer membrane having high mechanical strength, the amount of the hydrophobic polymer used is 50% by mass or more based on the total mass of the amphiphilic polymer and the hydrophobic polymer. The upper limit is preferably 99% by mass. By setting the content to 50% by mass or more, a fine porous polymer film having high mechanical strength can be formed relatively easily. If the amount of the hydrophobic polymer used is more than 99%, that is, if the amount of the amphiphilic polymer used is less than 1% by mass, it is difficult to grow and transport water droplets on the surface of the polymer solution. The array may become irregular. A fine porous polymer film can also be formed without using a hydrophobic polymer.

  In order to apply the polymer solution on the substrate (actually on the metal film, it is expressed in this way, the same applies hereinafter), a normal coating technique may be used. The application amount of the polymer solution may be appropriately adjusted according to the size and thickness of the film, and is not particularly limited, but is, for example, in the range of 1 mL or more and 50 mL or less.

  The gas sprayed onto the surface of the polymer solution applied on the substrate is not particularly limited as long as it does not inhibit the formation of the microporous film, and examples thereof include air, oxygen, carbon dioxide, nitrogen, and argon. It is done. If a large amount of dust is present, film formation may be affected. Therefore, it is preferable to remove the dust by passing a gas through a filter, and the film formation is preferably performed in a clean room.

  When preparing a microporous polymer film by self-assembly, a gas having a relative humidity of 50% or more and 95% or less may be sprayed at a substantially constant flow rate onto the surface of the polymer solution coated on the substrate. is important. If the relative humidity is less than 50%, sufficient water vapor does not condense on the surface of the polymer solution, and the desired pore size and porosity may not be obtained. On the other hand, if the relative humidity exceeds 95%, excessive water vapor condenses on the surface of the polymer solution, and vacancies may not be sufficiently formed. The gas flow rate may be appropriately adjusted according to the thickness, pore diameter, porosity and the like of the microporous polymer film to be formed, and is not particularly limited. 01 m / s, more preferably 0.05 m / s, still more preferably 0.1 m / s, and the upper limit is preferably 1 m / s, more preferably 0.8 m / s, still more preferably 0.7 m. / S. When the flow rate is less than 0.01 m / s, it takes time to form a film, and the productivity may decrease. On the other hand, when the flow rate exceeds 1 m / s, the surface of the polymer solution is disturbed, and the thickness of the microporous polymer film may become non-uniform.

  The direction in which the gas is blown onto the surface of the polymer solution coated on the substrate may be any direction from a direction perpendicular to the substrate to a horizontal direction. The angle is preferably within the range of 30 degrees or more and 60 degrees or less with respect to the direction perpendicular to the substrate. In practice, a funnel sized to cover the entire substrate is held in a down position above the substrate on which the polymer solution has been applied, and the gas is supplied from the capillary tube of the funnel. Just spray it. In this case, the distance between the substrate and the funnel may be adjusted as appropriate according to the flow rate of the gas, and is not particularly limited, but is, for example, in the range of 2 cm to 10 cm.

  The lower limit of the porosity of the microporous polymer membrane by self-assembly is preferably 30%, more preferably 35%, still more preferably 40%, and the upper limit is preferably 80%, more preferably. Is 75%, more preferably 70%. Here, the “porosity” means the ratio of the total opening area of the pores existing in a predetermined region of the microporous polymer membrane. The porosity is a value obtained by observing the microporous polymer film with an optical microscope and obtaining a region having a predetermined area randomly extracted from the actual use region excluding the peripheral portion. When the porosity is less than 30%, when used in a display device, the non-display portion increases, and the display quality may deteriorate. On the contrary, when the porosity exceeds 80%, the strength of the film may be lowered.

  Moreover, the pore diameter of the microporous polymer film by self-organization is in the range of 10 nm to 100 μm, and is not particularly limited, and may be set according to the application.

  In general, in order to reduce the pore size, for example, an organic solvent having a low boiling point may be used as the hydrophobic organic solvent, the temperature of the substrate may be increased, or the amount of the polymer solution applied on the substrate may be reduced.

  The thickness of the microporous polymer membrane varies depending on the pore size and is not particularly limited. For example, the lower limit is preferably 10 μm, more preferably 15 μm, still more preferably 20 μm, The upper limit is preferably 200 μm, more preferably 180 μm, and even more preferably 150 μm.

  The film forming process of such a microporous polymer film is shown in FIG. When a polymer solution is cast under high humidity, water is supplied to the surface of the cast solution, the water droplets are condensed, then the water droplets grow, and then the water droplets aggregate due to capillary force in the process of evaporation of the solvent. Finally, the water droplets evaporate and pores 13a are formed where the water droplets were present.

  It is presumed that hydrophilic portions of the amphiphilic polymer are arranged on the inner wall of the pores 13a of the microporous polymer membrane 13 formed in this way by the action of water droplets. Will exist. On the other hand, the surface between the pores 13a is presumed to be more hydrophobic due to the hydrophobic portion.

  Thus, hydrophilic functional groups exist in the pores on the surface of the microporous polymer membrane of the present invention. Here, the hydrophilic functional group is not particularly limited as long as it can be chemically modified according to the use, and examples thereof include a carboxyl group, an amino group, and a hydroxyl group. Groups are preferred.

  In addition, the biosensor chip of the present invention may be any as long as it has such a fine porous polymer film on its surface. However, when the hydrophilic functional group is insufficient, or the partition walls between the pores In order to further improve the sensitivity by providing a hydrophilic functional group, surface treatment such as plasma oxidation treatment or ozone oxidation treatment may be performed. By such surface treatment, the organic substance is oxidized, a carboxyl group is formed, and the hydrophilic functional group can be increased.

  Furthermore, the fine porous polymer film having such a hydrophilic functional group can be chemically modified to provide a functional site for fixing a physiologically active substance on the surface. Examples of the chemical modification include maleimide modification.

(Example)
First, a cover glass of 16 mm × 16 mm was used as a substrate, and a gold thin film having a thickness of 45 nm was provided thereon. The substrate having the gold thin film was immersed in a 1% aqueous acetic acid solution of 3-mercapto-propyl-trimethoxysilane having a concentration of 1.0% for 60 minutes to couple the gold thin film.

  Next, in order to coat a microporous polymer film by self-assembly, a polystyrene-polyacrylic acid copolymer chloroform as a polymer compound having a concentration of 0.5 mg / mL on a gold thin film of a cover glass. When the solution is cast and 90% humidity air is blown from the top at a flow rate of 5 mL / min, the chloroform evaporates and the water droplets form a mold, forming a self-assembled microporous polymer film on the gold thin film It was done.

  Furthermore, the microporous polymer film provided on the gold thin film on the cover glass was subjected to plasma oxidation using a plasma apparatus. This condition is that the high frequency oscillator frequency is 13.56 MHz, the output is 100 W, the oxygen gas flow rate is 50 mL / min, the degree of vacuum is 1.0 to 1.2 Torr, and the processing time is 60 seconds.

  The water contact angle before the treatment was 150 deg. However, when the plasma oxidation treatment was performed, it became 68 deg, and it was confirmed that the hydrophilicity was improved.

  Further, when the film surface was measured by Fourier transform infrared spectroscopy (FT-IR), an increase in carboxyl groups (COOH groups) was confirmed.

(Comparative example)
Similar to the examples, a 16 mm × 16 mm cover glass having a gold thin film with a thickness of 45 nm was immersed in an ethanol solution of 10-carboxy-1-decanethiol having a concentration of 1 mmol / L for 24 hours, and then with ethanol. The monolayer of alkanethiol was prepared by washing.

(Test example)
The biosensor chips of Examples and Comparative Examples produced as described above were fixed on the prism of the SPR measurement device, a dedicated measurement mount was set, and the following operation was performed using a flow method.

  First, maleimide modification treatment was performed in the following manner as chemical modification for binding antibody protein by antigen-antibody reaction. That is, a mixture of 400 mmol / L 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) and 100 mmol / L N-Hydrosulfosuccimide aqueous solution was flowed over the sensor for 7 minutes at a flow rate of 20 μL / min. Was processed by.

  Furthermore, in order to immobilize the antibody, a phosphate buffer prepared at a concentration of 20 μg / mL of human IgG antibody of MP BIOMEDICALS was flowed over the sensor for 7 minutes at a flow rate of 20 μL / min over the sensor.

  By such treatment, the biosensor chip of the example can be used as a biosensor chip for an SPR sensor.

Next, in order to examine the sensitivity with the SPR sensor, a phosphate buffer adjusted to a concentration of 1 × 10 ⁻⁸ mol / L of immunoglobulin G (human IgG) of MP BIOMEDICALS was flowed as an antigen, and subsequently the concentration A phosphate buffer solution of 1 × 10 ⁻⁷ mol / L was allowed to flow, and changes in the surface plasmon resonance signal were measured.

The results are shown in Table 1 and FIG. 3 (Examples only).
As a result, the change in the resonance angle as the output of the SPR sensor using the biosensor chip provided with the microporous polymer film of the example is the biosensor chip base material using the alkanethiol of the comparative example according to the prior art It turned out to be superior to the one.

  The biosensor chip of the present invention can be applied to various biosensors in addition to the SPR sensor.

It is a figure which shows typically the biosensor chip | tip of this invention. It is a figure which shows typically the film forming process of the microporous polymer film of the biosensor chip of this invention. It is a figure which shows the change of the surface plasmon resonance signal of the SPR sensor using the biosensor chip of the Example of this invention.

Explanation of symbols

11 Substrate 12 Metal film 13 Microporous polymer film

Claims (14)

  A substrate, a metal film provided on the surface, and a microporous polymer film fixed on the metal film via a thiol-based silane coupling agent and having a hydrophilic functional group on the surface A biosensor chip characterized by   2. The biosensor chip according to claim 1, wherein the microporous polymer film is formed by casting a hydrophobic organic solvent solution containing an amphiphilic polymer under a high humidity condition of a relative humidity of 50 to 95%. A biosensor chip characterized in that the film is a coated film.   The biosensor chip according to claim 2, wherein the hydrophobic organic solvent solution contains a hydrophobic polymer.   4. The biosensor chip according to claim 3, wherein the hydrophobic organic solvent solution contains a hydrophobic polymer and an amphiphilic polymer in a mass ratio of 50:50 to 99: 1. Biosensor chip.   The biosensor chip according to any one of claims 2 to 4, wherein the hydrophilic functional group includes one derived from a hydrophilic portion of the amphiphilic polymer.   The biosensor chip according to any one of claims 1 to 5, wherein the fine porous polymer film has been subjected to a surface treatment by plasma oxidation treatment or ozone oxidation treatment. .   The biosensor chip according to claim 6, wherein the hydrophilic functional group includes those formed by the surface treatment.   The biosensor chip according to any one of claims 1 to 7, wherein the hydrophilic functional group is a carboxyl group.   The biosensor chip according to any one of claims 1 to 8, wherein the biosensor chip is used for a surface plasmon resonance biosensor.   The biosensor chip according to any one of claims 1 to 9, wherein the surface of the microporous polymer film is chemically modified.   A step of providing a metal film on the surface of the substrate, a step of treating the surface of the metal film with a thiol-based silane coupling agent, and a hydrophobic organic solvent solution containing an amphiphilic polymer on the metal film with a relative humidity of 50 And a step of forming a microporous polymer film by casting under a high humidity condition of ˜95%.   12. The method of manufacturing a biosensor chip according to claim 11, wherein the hydrophobic organic solvent solution contains a hydrophobic polymer.   The method for producing a biosensor chip according to claim 12, wherein the hydrophobic organic solvent solution contains the hydrophobic polymer and the amphiphilic polymer in a mass ratio of 50:50 to 99: 1. A biosensor chip manufacturing method.   14. The biosensor chip manufacturing method according to claim 11, wherein the surface of the fine porous polymer film is subjected to a surface treatment by plasma oxidation treatment or ozone oxidation treatment. Manufacturing method.

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