JP2005114468A - Manufacturing method of substrate for bioassay using lyophilic processing and substrate for bioassay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a manufacturing method of a substrate for bioassay capable of reducing the loss of a sample, capable of efficiently performing interaction such as hybridization or the like, and to provide the substrate for bioassay enhanced in the detection precission of interation by efficiently bringing about interaction such as hybridization or the like. <P>SOLUTION: The substrate for bioassay for detecting interaction such as hybridization or the like is manufactured by processing a substrate having a reaction region, which can provide an interaction field between substances, formed thereto by combining (1) a hydrophobicity imparting process for treating the surface of the substrate with a hydrophobic substance to form a hydrophobic surface, (2) a hydrophilizing process for converting a part of the hydrophobic surface to a hydrophilic surface, and (3) a detecting surface forming process for fixing a detection substance to the hydrophilic or hydrophobic surface to form a detection surface in the reaction region. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a bioassay substrate such as a DNA chip and a protein chip. More specifically, the present invention relates to a bioassay substrate subjected to amphiphilic processing (hydrophilicity, hydrophobicity, etc.).

  The main prior art of the present invention will be described below. Currently, integrated substrates for bioassays called DNA chips or DNA microarrays (hereinafter collectively referred to as “DNA chips”), in which predetermined DNA is finely arranged by microarray technology, are used for gene mutation analysis, SNPs (single nucleotides). Polymorphism) analysis, gene expression frequency analysis, etc., and has begun to be widely used in drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other fields.

  Since this DNA chip has a large number of DNA oligo chains and cDNA (complementary DNA) integrated on a glass substrate or silicon substrate, comprehensive analysis of intermolecular interactions such as hybridization becomes possible. It is characterized by dots.

  To briefly explain an analysis method using a DNA chip, a fluorescent probe dNTP is obtained by reverse transcription PCR reaction or the like by extracting mRNA extracted from cells, tissues, etc. with respect to a DNA probe solid-phased on a glass substrate or a silicon substrate. In this method, PCR amplification is carried out while incorporating, hybridization is performed on the substrate, and fluorescence is measured with a predetermined detector.

  Here, DNA chips can be classified into two types. The first type is one in which oligonucleotides are directly synthesized on a predetermined substrate using a photolithographic technique applying a semiconductor exposure technique, and a typical one is by Affymetrix (Affymetrix). (For example, refer to Patent Document 1). Although this type of chip has a high degree of integration, there is a limit to DNA synthesis on the substrate, and the length is about several tens of bases.

The second type is also referred to as “Stanford method”, and is prepared by dispensing and solidifying a prepared DNA on a substrate using a tip-breaking pin ( For example, see Patent Document 2). This type of chip is less integrated than the former, but has the advantage that a DNA fragment of about 1 kb can be immobilized.
Special table hei 4-505633 report Japanese National Patent Publication No. 10-503841

  However, the conventional techniques have the following problems to be solved.

  In the above-described conventional DNA chip technology and biosensor technology, in a narrow reaction part of a two-dimensional substrate, a detection nucleotide chain such as a DNA probe or protein is immobilized (immobilized), and a hybridization reaction or Because it is a technology that promotes interactions between substances such as antigen-antibody reactions, the degree of freedom of reaction products is limited spatially, and interactions such as hybridization are based solely on Brownian motion of the target nucleotide chain, etc. I was letting. For this reason, the conventional DNA chip technology or biosensor technology has a technical problem that the interaction efficiency is low and the reaction time is long.

  In addition, in a DNA chip or the like, since a detection nucleotide chain or the like is solid-phased on a flat surface, a droplet of a sample (nucleotide chain or the like) to be immobilized when a bioassay substrate such as a DNA chip is manufactured. There is a problem that it is difficult to keep the substrate at the target position on the substrate. For this reason, it has been difficult to efficiently perform the production work of the bioassay substrate in a short time, and there has been a problem that it is easy to lose the sample to be immobilized.

  Furthermore, when supplying a droplet containing a target nucleotide chain or the like to a reaction region on a substrate where the interaction occurs, the droplet containing the target nucleotide chain or the like is moved to a target position (reaction). It is difficult to keep it in the region), and the loss of the target sample is large, which is one of the causes of poor efficiency. In addition, since only a small amount of a target sample such as a nucleic acid can be supplied to a DNA chip or the like, it has been difficult to increase the accuracy of detecting a target interaction.

  Accordingly, the main object of the present invention is to provide a method for producing a substrate for bioassay that can cause an interaction such as hybridization efficiently with little loss of a sample. It is another object of the present invention to provide a bioassay substrate that efficiently raises interactions such as hybridization and has improved interaction detection accuracy.

  In order to solve the above technical problem, the present invention provides the following means.

  First, a bioassay substrate for detecting an interaction is formed by processing a substrate on which a reaction region capable of providing an interaction field between substances is formed by combining the following steps (1) to (3). A method of manufacturing is provided.

  (1) A hydrophobization step of forming a hydrophobic surface by treating the substrate surface with a hydrophobic substance.

  (2) A hydrophilization step in which part of the hydrophobic surface is converted to a hydrophilic surface.

  (3) A detection surface forming step in which a detection substance is immobilized on a hydrophilic surface or a hydrophobic surface to form a detection surface in the reaction region.

  By appropriately combining these three steps, each region on the bioassay substrate can be made a hydrophilic surface or a hydrophobic surface depending on the purpose.

  For example, by converting the entire reaction region to a hydrophilic surface by the hydrophilization process of (2), the reaction region becomes a hydrophilic surface, and the other part (non-reactive region) on the substrate becomes a hydrophobic surface. Furthermore, a bioassay substrate can be manufactured.

  Since biological materials such as nucleotide chains and proteins used in bioassays are mostly hydrophilic, these biological materials are dropped and supplied to the substrate as an aqueous solution or the like. Therefore, by manufacturing a bioassay substrate so that the reaction region on the substrate is a hydrophilic surface and the non-reaction region is a hydrophobic surface, when an aqueous solution or the like in which a biological material is dissolved is dropped and supplied onto the substrate. The aqueous solution or the like can remain in the reaction region so that it does not spill into the non-reaction region on the substrate.

  In the above method, each part in the reaction region on the substrate can be appropriately made a hydrophilic surface or a hydrophobic surface depending on the purpose. For example, in the hydrophilization step (2), only the detection surface portion in the reaction region is converted to a hydrophilic surface, or conversely, only the portion other than the detection surface (non-detection surface) in the reaction region is parented. It can also be converted to the water surface. Thus, by providing a hydrophilic surface and a hydrophobic surface in the reaction region, the hydrophilic or hydrophobic solution used in the bioassay substrate manufacturing stage can be maintained at the target location, or the target region in the reaction region can be maintained. It becomes possible to immobilize a detection substance such as a nucleotide chain at a location.

  The hydrophobization step of forming the hydrophobic surface by treating the substrate surface of (1) with a hydrophobic substance is, for example, an alkylsilane (R—Si—X1, X2, X3, R: alkyl group (non-hydrophobic) that is a hydrophobic substance. (Which may contain a saturated group), Xn: Cl, OR) may be immobilized on the surface of the substrate.

  The hydrophilizing step (2) for converting a part of the hydrophobic surface into a hydrophilic surface can be easily performed by a reaction for forming a hydroxyl group on the hydrophobic surface. For example, when the hydrophobic surface is formed of the above-described alkylsilane, a hydroxyl group can be added to the alkyl group or silicon of the alkylsilane by irradiating the alkylsilane with ultraviolet light in the presence of oxygen.

  The detection surface forming step of immobilizing the detection substance on the hydrophilic surface or the hydrophobic surface in (3) to form a detection surface in the reaction region is appropriately performed according to the detection substance (nucleotide chain, protein, etc.). The reaction can be performed according to the purpose.

  For example, when the surface region for detection in the reaction region has a hydrophilic surface by adding a hydroxyl group to alkylsilane, a mercaptoalkylsilane (HS-R-Si-X1, X2, X3 in a hydrophilic solvent is added to the hydroxyl group. ), And reacting a mercapto group with a detection substance (such as a nucleotide chain) previously added with a mercapto group at the end to form a disulfide bond, and immobilizing the detection substance via the disulfide bond, A bioassay substrate having a detection substance immobilized thereon can be produced.

  Further, a mercaptoalkylsilane in a hydrophobic solvent is brought into contact with the hydrophobic surface to add a mercapto group, and the mercapto group is reacted with a detection substance having a mercapto group in advance to form a disulfide bond, via a disulfide bond. By immobilizing the detection substance, a bioassay substrate on which the detection substance is immobilized can be produced.

  By the above method, a detection substance such as a nucleotide chain can be immobilized in the reaction region of the bioassay substrate. By dropping and supplying a target substance (such as a nucleotide chain) into the reaction region of the bioassay substrate, an interaction (such as hybridization) occurs, and the target substance can be detected and measured.

  Then, the board | substrate for bioassay manufactured using the said method suitably is provided.

  First, a substrate having at least a reaction region capable of providing an interaction field between substances and a detection surface formed in the reaction region, and the entire reaction region including the detection surface is hydrophilic. A substrate for bioassay is provided. This bioassay substrate has a hydrophilic sample solution (an aqueous solution in which nucleotide chains and the like are dissolved) because the reaction region including the detection surface is hydrophilic and the other part (non-reaction region) is hydrophobic. When the sample solution is dropped and supplied into the reaction region, the sample solution stays in the reaction region and does not spill into the non-reaction region. Therefore, the loss of the sample can be reduced, and the interaction efficiency can be increased.

  Further, the substrate includes at least a reaction region capable of providing an interaction field between substances and a detection surface formed in the reaction region, and the region excluding the detection surface of the reaction region is hydrophilic. A substrate for bioassay is provided. In this bioassay substrate, since the region (non-detection surface) except for the detection surface in the reaction region is hydrophilic, after causing an interaction (hybridization etc.), an unnecessary substance (free substance or / and It acts to adsorb misinteracting substances, etc.) to the non-detection surface. Therefore, unnecessary substances and the like can be prevented from returning again to the peripheral region of the detection surface, and the interaction detection efficiency can be increased.

  The substrate is further provided with a detection surface provided at a central position of the reaction region, a hydrophilic surface formed on both sides of the detection surface, and each of which is disposed outside each hydrophilic surface and sandwiches the reaction region. Thus, it can be configured to have at least a pair of electrodes facing each other. By installing this electrode, unnecessary substances existing in the vicinity of the detection surface can be attracted to the hydrophilic surface side by dielectrophoresis. As described above, the hydrophilic surfaces formed on both sides of the detection surface have an action of adsorbing unnecessary substances that have not interacted (such as free substances and / or erroneously interacting substances). By adsorbing free substances drawn to the side to the hydrophilic surface, it is possible to more efficiently prevent unnecessary substances from returning to the peripheral area of the detection surface again, thereby improving the interaction detection efficiency. Can be increased.

  Furthermore, the substrate comprises at least a reaction region capable of providing an interaction field between substances and a detection surface formed in the reaction region, and only the detection surface portion of the reaction region is hydrophilic, The other reaction region portion provides a bioassay substrate that is hydrophobic. Since the detection surface in the reaction region is hydrophilic and the other part (non-detection surface) in the reaction region is hydrophobic, the aqueous solution in which the sample such as nucleotide chain is dissolved stays only on the detection surface. It does not move to the non-detection surface. Therefore, since substances such as nucleotide chains remain around the detection surface, interaction (hybridization or the like) can be promoted and the detection efficiency of the interaction can be increased.

  In addition, a first substrate including at least a reaction region capable of providing an interaction field between substances, a detection surface formed in the reaction region, and a second substrate superimposed on the first substrate. Provided is a bioassay substrate in which the inner surface region facing the reaction region of the second substrate is hydrophilic. By superposing the second substrate on the first substrate, it is possible to cover the reaction region that is recessed from the front surface of the first substrate to the back surface, so that the buffer in which the sample in the reaction region is melted An aqueous solution such as a liquid can be reliably held in the reaction region. At the time of reaction such as interaction, since an aqueous solution in which a sample or the like is dissolved is often contained in the reaction region, the inner surface region facing the reaction region of the second substrate should be hydrophilic.

  Subsequently, the substrate includes at least a reaction region capable of providing an interaction field between substances and a detection surface formed in the reaction region, and only the detection surface portion of the reaction region is hydrophilic. The bioassay method using the bioassay substrate in which the other reaction region portion is hydrophobic, and exhibits an interaction with the detection substance immobilized on the detection surface with respect to the hydrophobic region of the reaction region. There is provided a bioassay method characterized by dripping an aqueous solution containing a target substance. In the case of this bioassay method, the detection surface of the reaction region on the substrate surface is hydrophilic and the other part (non-detection surface) in the reaction region is hydrophobic, so the aqueous solution containing the target substance is not detected. Even if it is dropped on the surface for detection, it immediately moves to the surface for detection and is collected. Therefore, since the aqueous solution containing the target substance is easily held on the detection surface on which the detection substance is immobilized, the interaction can be efficiently detected and measured. In addition, since it is not necessary to drop the aqueous solution containing the target substance directly on the detection surface on which the detection substance is immobilized, the detection substance is damaged or the fixation is lost due to the physical force of the dropping. It is possible to eliminate harmful effects such as being washed away.

  In the present invention, the interaction means that the detection substance and the target substance cause some kind of reaction such as hybridization, antigen-antibody reaction, enzyme reaction and the like. The substance for detection refers to a substance immobilized in a reaction region of a bioassay substrate among a plurality of interacting substances such as a nucleotide chain such as DNA and a protein. A target substance refers to a substance that is dropped and supplied into a reaction region of a bioassay substrate on which a detection substance is immobilized, among a plurality of interacting substances such as a nucleotide chain such as DNA and a protein.

  The effects produced by the present invention are as follows.

  According to the present invention, the amphiphilicity (hydrophilicity and hydrophobicity) of the substrate surface can be controlled.

  In the manufacturing process of the bioassay substrate, the amphiphilicity of each region of the substrate can be appropriately set and changed according to the purpose. This makes it possible to use both hydrophilic and hydrophobic solutions in the manufacturing process of the bioassay substrate, and to cause a reaction only in the target region.

  By appropriately adjusting the surface treatment agent such as alkylsilane, which is a hydrophobic substance, and hydrophilic means such as ultraviolet irradiation, the surface for detection in the reaction region of the substrate can be arbitrarily set, and simple In the process, the detection substance can be immobilized.

  By controlling the amphiphilicity of each region, an aqueous solution in which biomolecules and the like are melted can be moved and held at a target location.

  The bioassay substrate according to the present invention can detect and measure a target interaction with a small number of samples with high accuracy.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the accompanying drawings.

  FIG. 1 is a diagram showing a series of steps until the nucleotide chain N is immobilized on the reaction region 12 of the substrate 1. As shown in FIG. 1A, reaction regions 12 that are recessed from the front surface 11 toward the back surface of the substrate 1 (downward in the drawing) are accumulated on the front surface 11 of the substrate 1. In the reaction region 12, there are a detection surface 13 for immobilizing the nucleotide chain N and another portion 14 (non-detection surface). The detection surface 13 does not need to be located at the center of the reaction region 12, and can be appropriately set according to the purpose as long as it is within the reaction region 12. A portion other than the reaction region 12 on the surface 11 of the substrate 1 is defined as a non-reaction region 15. As the substrate 1, a substrate having a surface made of silica, silicon oxide, metal having an oxide surface, or the like can be used.

  First, as shown in FIG. 1A, alkylsilane A is immobilized on the entire surface 11 of the substrate 1 using a solution of a coupling agent having an alkyl group such as alkylsilane (indicated by symbol A). Since alkylsilane A is a hydrophobic substance, the surface 11 of the substrate 1 forms a hydrophobic surface (hydrophobization).

  Next, after covering the region (the non-detection surface 14 and the non-reactive region 15) where the nucleotide chain N is not to be immobilized with the mask 2 made of a material that absorbs or reflects the ultraviolet ray U, the ultraviolet ray is exposed in the air or in an oxygen atmosphere U is irradiated, and only the portion of the surface 11 of the substrate 1 that is not covered with the mask 2 (detection surface 13) is hydroxylated. As a result, a hydroxyl group (—OH) is added to the R group or silicon of alkylsilane A, so that only the portion irradiated with ultraviolet U (detection surface 13) is converted to a hydrophilic surface as shown in FIG. 1B ( Hydrophilization).

  In addition, by using the above steps, a hydrophobic surface and / or a hydrophilic surface can be appropriately formed in all the reaction regions 12 accumulated on the surface 11 of the substrate 1. For example, by appropriately changing the portion covered with the mask and performing ultraviolet ray U irradiation, the entire reaction region 12 is converted to a hydrophilic surface, and the non-reactive region 15 is made a hydrophobic surface (not shown). Only the non-detection surface 14 can be converted into a hydrophilic surface, and the detection surface 13 can be configured as a hydrophobic surface (not shown).

  Next, the substrate 1 whose surface has been partially hydroxylated by the above-described step is immersed in an aqueous mercaptoalkylsilane solution to bond the hydroxyl group (—OH) on the detection surface 13 to the mercapto group (—SH). Thereby, a mercapto group (-SH) is added only to the area | region (hydrophilic surface) to which the hydroxyl group (-OH) was added in the board | substrate 1 surface 11 (refer FIG. 1C).

  Then, when the substrate 1 with the mercapto group added to the surface 11 is immersed in a solution of the nucleotide chain N in which a mercapto group (—SH) is previously bonded to one end, a target nucleotide chain is obtained by disulfide bonding between the mercapto groups. N is fixed to the detection surface 13 of the substrate 1 surface 11 (see FIG. 1D).

  2 and 3 are schematic cross-sectional views showing the manufacturing process of the bioassay substrate. On the front surface 11 of the substrate 1, reaction regions 12 that are recessed in the rear surface direction (downward in the drawing) are accumulated.

  Hereinafter, a manufacturing process of a bioassay substrate using an aqueous solution of mercaptoalkylsilane will be described with reference to FIG. As in FIG. 1, the region in the reaction region 12 where the nucleotide chain N is immobilized is the detection surface 13, and the other region is the non-detection surface 14, and the reaction region on the surface 11 of the substrate 1. A portion other than 12 is defined as a non-reactive region 15.

  After the entire surface of the substrate 1 is hydrophobized with alkylsilane, the non-detection surface 14 and the non-reactive region 15 are covered with a mask in the same manner as described above, and irradiated with ultraviolet rays U, so that only the detection surface 13 has a hydroxyl group ( -OH) is added to make it hydrophilic. Thereby, the detection surface 13 becomes hydrophilic, and the non-detection surface 14 and the non-reactive region 15 become hydrophobic (see FIG. 2A).

  Next, when mercaptoalkylsilane dissolved in ethanol and an aqueous solvent is dropped into the reaction region 12, the mercaptoalkylsilane is bonded to the hydroxyl group (—OH) added to the detection surface 13.

  Next, after washing and removing the mercaptoalkylsilane solution, the detection surface 13 and the non-reactive region 15 are masked and irradiated with ultraviolet rays U, whereby hydroxyl groups (—OH) are formed on the non-detection surface 14. Add to make it hydrophilic. Through the above steps, the detection surface 13 is added with a mercapto group (-SH), the non-detection surface 14 becomes hydrophilic, and the non-reactive region 15 becomes hydrophobic (FIG. 2B).

  Next, a solution of a nucleotide chain N in which a mercapto group (—SH) is bonded to one end in advance is dropped into the reaction region 12. Thereby, the mercapto group (-SH) on the detection surface 13 and the mercapto group added to the nucleotide chain N are disulfide-bonded, and the nucleotide chain N is immobilized on the detection surface 13 (FIG. 2C).

  Thus, the nucleotide chain N is immobilized on the reaction region 12 accumulated on the surface 12 of the substrate 1, and the manufacturing process of the bioassay substrate (DNA chip) is completed. Then, by dropping and supplying an aqueous solution in which the target nucleotide chain N ′ is dissolved to the reaction region 12 of the bioassay substrate 1, the target becomes the nucleotide chain N immobilized on the detection surface 13 and the target. The nucleotide chain N ′ interacts (hybridizes), and the target nucleotide chain N ′ can be detected and measured.

  Since the non-reactive region 15 is hydrophobic, the aqueous solution in which the nucleotide chain N or N ′ is dissolved stays in the hydrophilic reaction region 12 when dropped or supplied, and the hydrophobic non-reactive region Do not spill into area 15. Therefore, according to the present invention, it is possible to immobilize a nucleic acid and to detect and measure an interaction sufficiently and reliably with a smaller amount of a nucleic acid aqueous solution. In addition, by appropriately providing a hydrophilic region and a hydrophobic region in the substrate 1 or the reaction region 12 according to the purpose, various solutions can be guided to the region to be supplied. In addition, in order to maintain the hydrophilicity of the reaction region 12, a neutral surfactant may be used.

  Subsequently, a manufacturing process of a bioassay substrate using a hydrophobic solution of mercaptoalkylsilane such as toluene will be described below with reference to FIG.

  First, the entire surface of the substrate 1 is hydrophobized with alkylsilane, the detection surface 13 is masked, and the non-detection surface 14 and the non-reactive region 15 are irradiated with ultraviolet rays U to add a hydroxyl group (—OH). Then, the non-detection surface 14 and the non-reactive region 15 are made hydrophilic (FIG. 3A).

  Next, a toluene solution of mercaptoalkylsilane is dropped onto the reaction region 12, and the mercapto group (—SH) is added to the alkylsilane on the detection surface 13 (FIG. 3B). Since the toluene solution is hydrophobic, it repels the hydrophilic non-detection surface 14 and the non-reactive region 15 and tries to stay on the hydrophobic detection surface 13. Therefore, the mercaptoalkylsilane contacts the alkylsilane on the detection surface 13 and adds a mercapto group.

  Next, after the toluene solution of mercaptoalkylsilane is washed and removed, the entire substrate 1 is again treated with alkylsilane, and the non-reactive region 15 is coated again with alkylsilane (FIG. 3C). Thereby, the detection surface 13 is in a state where a mercapto group is bonded, the non-detection surface 14 is hydrophobic, and the non-reactive region 15 is also hydrophobic.

  Next, by the same method as described above, only the non-detection surface 14 is irradiated with ultraviolet rays U to add a hydroxyl group to make it hydrophilic. As a result, as in the case of using the aqueous solution of mercaptoalkylsilane described above, the detection surface 13 is in a state where mercapto groups are bonded, the non-detection surface 14 is hydrophilic, and the non-reactive region 15 is hydrophobic ( FIG. 3D).

  The following steps are the same as when the above-described aqueous solution of mercaptoalkylsilane is used. That is, a solution of a nucleotide chain N in which a mercapto group (—SH) is bonded to one end in advance is dropped into the reaction region 12. As a result, the mercapto group (—SH) on the detection surface 13 and the mercapto group added to the nucleotide chain N are disulfide-bonded, and the nucleotide chain N is immobilized on the detection surface 13 (see FIG. 2C).

  Thus, the nucleotide chain N is immobilized on the reaction region 12 accumulated on the surface 12 of the substrate 1, and the manufacturing process of the bioassay substrate (DNA chip) is completed. Then, by dropping and supplying an aqueous solution in which the target nucleotide chain N ′ is dissolved to the reaction region 12 of the bioassay substrate 1, the target becomes the nucleotide chain N immobilized on the detection surface 13 and the target. The nucleotide chain N ′ interacts (hybridizes), and the target nucleotide chain N ′ can be detected and measured.

  As in the case of using the aqueous solution of mercaptoalkylsilane, the non-reactive region 15 is hydrophobic. Therefore, when the aqueous solution in which the nucleotide chain N or N ′ is dissolved is dropped and supplied, It stays in the reaction region 12 that is hydrophilic and does not spill into the hydrophobic non-reaction region 15. Therefore, according to the present invention, it is possible to immobilize a nucleic acid and to detect and measure an interaction sufficiently and reliably with a smaller amount of a nucleic acid aqueous solution. In addition, by appropriately providing a hydrophilic region and a hydrophobic region in the substrate 1 or the reaction region 12 according to the purpose, various solutions can be guided to the region to be supplied. In addition, in order to maintain the hydrophilicity of the reaction region 12, a neutral surfactant may be used.

  FIG. 4 is a schematic cross-sectional view of the periphery of the reaction region 12 of the substrate 1 provided with the electrodes 3 and 3 facing each other so as to sandwich the reaction region 12.

  This substrate 1 is manufactured by the above-described manufacturing process of the bioassay substrate. The nucleotide surface N is immobilized on the detection surface 13, the non-detection surface 14 is hydrophilic, and the non-reactive region 15 is Is hydrophobic. As shown in FIG. 4, when an aqueous solution in which the target nucleotide chain N ′ is dissolved is dropped and supplied to the reaction region 12, the immobilized nucleotide chain N and the target nucleotide chain N ′ interact with each other (hybridization). The target nucleotide chain N ′ can be detected and measured.

  At that time, as described above, since the non-detection surface 14 is hydrophilic by adding a hydroxyl group (—OH), the nucleotide chain N ′ that has not caused the interaction is removed from the hydroxyl group of the non-detection surface 14. And can be removed to some extent from the region of the detection surface 13.

  The reaction region 12 shown in FIG. 4 includes the electrodes 3 and 3 as described above. After dripping and supplying the target nucleotide chain N ′ to cause an interaction (hybridization), an alternating electric field was applied to the electrode 3 to cause a non-interacting nucleotide chain N ′ or mishybridization ( The nucleotide chain N ′ (weakly bonded) can be dielectrophoresed in the direction of the electrode 3 (arrow in the figure), and the nucleotide chain N ′ can be adsorbed to the hydroxyl group (—OH) of the hydrophilic non-detection surface 14. Therefore, by providing the electrode 5, the nucleotide chain N ′ and the like that did not interact can be removed from the peripheral region of the detection surface 13 by an electric field, and the nucleotide chain N ′ induced by the electric field can be removed. It can be adsorbed on the non-detection surface 14 and prevented from diffusing again into the peripheral region of the detection surface 13.

  The present invention is a method for producing a substrate for bioassay by controlling the amphiphilicity (hydrophilicity, hydrophobicity), in that the detection substance can be fixed in the reaction region of the substrate by a simple process. Industrially useful.

  According to the present invention, a bioassay substrate can be efficiently produced with a smaller amount of a detection substance, which is industrially useful.

  The bioassay substrate and bioassay method according to the present invention have industrial applicability in that the interaction can be detected and measured with high accuracy with a smaller amount of the target substance.

It is a cross-sectional schematic diagram of the reaction region (12) of the substrate (1) and its periphery, showing a series of steps until the nucleotide chain (N) is immobilized on the reaction region (12) of the substrate (1). It is a figure which shows the process which hydrophobized the surface. In the said process, it is a figure which shows the process which hydrophilized the detection surface (13) by ultraviolet-ray U irradiation. It is a figure which shows the process which substituted the hydroxyl group (-OH) by the mercapto group (-SH) in the said process. It is a figure which shows the process which fix | immobilized the nucleotide chain N in the said process. The reaction area (12) of a board | substrate (1) which showed the manufacturing process of the board | substrate for bioassays, and the cross-sectional schematic diagram of the periphery (when using the aqueous solution of a mercaptoalkylsilane). It is a figure which shows the process of hydrophilizing the detection surface (13). It is a figure which shows the process which substituted the hydroxyl group (-OH) by the mercapto group (-SH) in the said process, and hydrophilized the non-detection surface (14). It is a figure which shows the process which fix | immobilized the nucleotide chain N in the said process. The reaction area | region (12) of a board | substrate (1) which showed the manufacturing process of the board | substrate for bioassay, and the cross-sectional schematic diagram of the periphery (when using hydrophobic solutions, such as toluene of a mercaptoalkylsilane). It is a figure which shows the process which hydrophilized the non-detection surface (14) and the non-reaction area | region (15). It is a figure which shows the process which added the mercapto group (-SH) to the surface for detection (13) in the said process. It is a figure which shows the process which hydrophobized the non-detection surface (14) and the non-reaction area | region (15) in the said process. It is a figure which shows the process which hydrophilized the surface for non-detection (14) in the said process. It is a cross-sectional schematic diagram of the periphery of the reaction region (12) of the substrate (1) provided with the electrodes (3, 3) opposed so as to sandwich the reaction region (12).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 12 Reaction area 13 Detection surface 14 Non-detection surface 15 Non-reaction area 2 Mask 3 Electrode A Alkylsilane

Claims (16)

A substrate having a reaction region capable of providing a field of interaction between substances is manufactured by combining the following steps (1) to (3) to produce a bioassay substrate for detecting the interaction. how to.
(1) A hydrophobization process in which the substrate surface is treated with a hydrophobic substance to form a hydrophobic surface.
(2) A hydrophilization step in which part of the hydrophobic surface is converted to a hydrophilic surface.
(3) A detection surface forming step of forming a detection surface in the reaction region by immobilizing a detection substance on the hydrophilic surface or the hydrophobic surface. The method for producing a bioassay substrate according to claim 1, wherein the hydrophilization step is performed on the entire reaction region. The method for producing a bioassay substrate according to claim 1, wherein the hydrophilization step is performed only on a detection surface portion in the reaction region. The method for producing a bioassay substrate according to claim 1, wherein the hydrophilization step is performed only on a reaction region portion other than the detection surface. The method for producing a bioassay substrate according to claim 1, wherein the hydrophilization step is a step of forming a hydroxyl group on the hydrophobic surface. The hydrophilization step is a step of forming a hydroxyl group by oxidizing the alkylsilane, which is the hydrophobic substance, with ultraviolet irradiation in the presence of oxygen to oxidize an alkyl group of the alkylsilane. A method for producing a substrate for bioassay. A mercaptoalkylsilane in a hydrophilic solvent is brought into contact with the hydroxyl group, a detection substance having the same mercapto group is reacted with the mercapto group to form a disulfide bond, and the detection substance is immobilized through the disulfide bond. The method for producing a bioassay substrate according to claim 6. A mercaptoalkylsilane in a hydrophobic solvent is brought into contact with the hydrophobic surface, a detection substance having the same mercapto group is reacted with the mercapto group to form a disulfide bond, and the detection substance is immobilized through the disulfide bond. The method for producing a bioassay substrate according to claim 6. The method for producing a bioassay substrate according to claim 1, wherein the detection substance is a nucleotide chain. The method for producing a bioassay substrate according to claim 1, wherein the interaction is hybridization. A substrate comprising at least a reaction region capable of providing an interaction field between substances, and a detection surface formed in the reaction region,
A bioassay substrate, wherein the entire reaction region including the detection surface is hydrophilic. A substrate comprising at least a reaction region capable of providing an interaction field between substances, and a detection surface formed in the reaction region,
A bioassay substrate, wherein a region excluding the detection surface of the reaction region is hydrophilic. The detection surface provided at the central position of the reaction region and the hydrophilic surfaces formed on both sides of the detection surface are arranged outside the hydrophilic surfaces and face each other so as to sandwich the reaction region. At least a pair of electrodes, and the electrodes are means for attracting free substances and / or misinteracting substances present in the vicinity of the detection surface to the hydrophilic surface side by dielectrophoresis. The bioassay substrate according to claim 12. A substrate comprising at least a reaction region capable of providing an interaction field between substances, and a detection surface formed in the reaction region,
A bioassay substrate, wherein only the detection surface portion of the reaction region is hydrophilic, and the other reaction region portion is hydrophobic. A first substrate comprising at least a reaction region capable of providing an interaction field between substances, and a detection surface formed in the reaction region;
A second substrate overlaid on the first substrate,
A bioassay substrate, wherein an inner surface region facing the reaction region of the second substrate is hydrophilic. A substrate having at least a reaction region capable of providing an interaction field between substances and a detection surface formed in the reaction region, wherein only the detection surface portion of the reaction region is hydrophilic; A reaction region portion of the bioassay method using a bioassay substrate that is hydrophobic,
A bioassay method, wherein an aqueous solution containing a target substance that interacts with a detection substance immobilized on the detection surface is dropped onto the hydrophobic area of the reaction area.