JP5929752B2 - Storage sensor substrate and storage sensor substrate and dry sensor substrate manufacturing method - Google Patents

Description

The present invention may be dried, stored sensor substrate not physiologically active substance is inactivated by a specific component contained in the preservation solution, and a method for manufacturing a storage sensor substrate and drying the sensor board.

  Currently, many measurements using intermolecular interactions such as immune reactions are performed in clinical tests, etc., but the conventional method requires complicated operations and labeling substances, so the change in the amount of binding of the measuring substance is highly sensitive. Several techniques are used that can be detected.

  For example, surface plasmon resonance [SPR] measurement technology, surface plasmon excitation enhanced fluorescence spectroscopy [SPFS] measurement technology, quartz crystal microbalance [QCM] measurement technology, measurement using functionalized surfaces from gold colloid particles to ultrafine particles Technology.

  The SPR measurement technology determines the refractive index change in the vicinity of the organic functional film in contact with the metal film of the chip by measuring the peak shift of the reflected light wavelength or the change in the amount of reflected light at a certain wavelength, thereby adsorbing and desorbing in the vicinity of the surface. It is a measurement technique to detect.

  In the SPFS measurement technique, the number of photons contained in the irradiated laser beam is calculated by generating a dense wave (surface plasmon) on the surface of the metal thin film under the condition that the irradiated laser beam is attenuated by total reflection [ATR] on the surface of the gold thin film. Increased by 10 to several hundred times (surface plasmon electric field enhancement effect), thereby enabling the detection of trace amounts and / or extremely low concentrations of analyte by efficiently exciting the fluorescent dye in the vicinity of the gold thin film. Measurement technology.

  The QCM measurement technique is a measurement technique that 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.

  In addition, by functionalizing the surface of gold ultrafine particles (nm level), immobilizing a physiologically active substance on the surface, and performing a specific recognition reaction between the physiologically active substances, it is possible to obtain a living body from the sedimentation and arrangement of gold fine particles. Related substances can be detected.

  In any of these measurement techniques, the surface on which the physiologically active substance is immobilized is analyzed in order to analyze the interaction between biomolecules by measuring the specific binding reaction between the physiologically active substance and the sample substance. It becomes important.

  The physiologically active substance is immobilized on the substrate surface by covalent bond, ligand bond, ionic bond, physical adsorption, inclusion method, etc., but the physiologically active substance on the substrate surface deteriorates with the passage of time, especially the physiologically active substance. When is a protein, there is a problem that it is inactivated and the specific binding reaction is lowered.

  Such deterioration over time is considered to be caused by a combination of various factors, but is particularly noticeable in a dry state.

  Therefore, in order to prevent drying, the average molecular weight is larger than 350 and smaller than 5 million and is hydrogen bonded to a physiologically active substance immobilized through a hydrophilic polymer layer formed from a hydrophilic polymer on the substrate surface. Attempts have been made to prevent deterioration by adding a compound having a residue capable of forming dextran (for example, dextran) (Patent Document 1). However, in this method, the compound in the preservation solution binds to a physiologically active substance. There was a problem inhibiting the reaction.

JP 2008-185494 A

  The present invention provides a storage sensor substrate having improved storage stability of a physiologically active substance immobilized on the sensor substrate surface, and a dry sensor substrate that facilitates removal of the storage solution before using the substrate. It is intended to provide further.

  As a result of intensive studies to solve the above problems, the present inventors have found that when a bioactive substance preservation solution containing a low molecular weight saccharide and a non-specific protein such as BSA is used, the bioactivity It has been found that the storage stability of the sensor substrate on which the substance is immobilized is improved, and that the storage solution before use of the substrate can be easily removed, and the present invention has been completed.

That is, storage sensor substrate of the present invention, possess a sensor substrate physiologically active substance is immobilized on the surface thereof, comprising a saccharide and non-protein, a preservative solution covering the physiologically active substance, the said saccharide but characterized by have a molecular weight of less than 350.

  The sensor substrate includes: a transparent support; a metal thin film formed on one surface of the support; and a self-organized single film formed on the other surface of the metal thin film that is not in contact with the support. A plasmon excitation sensor comprising: a molecular film [SAM]; and the above-mentioned physiologically active substance immobilized on the other surface of the SAM that is not in contact with the metal thin film. Further includes a hydrophilic polymer layer formed on the other surface of the SAM that is not in contact with the metal thin film, and the physiologically active substance is in contact with the SAM of the hydrophilic polymer layer. It is preferably immobilized on the other surface that is not.

  The physiologically active substance may be a protein that specifically binds to the substance to be captured.

The saccharide is preferably at least one saccharide selected from monosaccharides, and disaccharides or Ranaru group.

  The saccharide is preferably contained in the preservation solution in an amount of 1 to 20% by weight.

  The non-specific protein is preferably bovine serum albumin [BSA].

  The non-specific protein is preferably contained in the preservation solution in an amount of 1 to 5% by weight.

  The storage solution preferably further contains phosphate buffered saline [PBS], Tris buffered saline [TBS] or HEPES buffered saline [HBS].

The method for producing a storage sensor substrate of the present invention includes at least the following step (i):
Step (i): A step of coating the physiologically active substance immobilized on the sensor substrate with the preservation solution.

  The dry sensor substrate of the present invention is obtained by drying the storage sensor substrate of the present invention described above.

Moreover, the manufacturing method of the dry sensor substrate of the present invention includes at least the following steps (i) and (ii):
Step (i): The step of obtaining the preserved sensor substrate of the present invention by coating the physiologically active substance immobilized on the sensor substrate with the preservation solution, and Step (ii): Removing the solvent contained therein by drying to obtain a dry sensor substrate;

  The present invention can provide a storage sensor substrate having improved storage stability of a physiologically active substance immobilized on the sensor substrate surface.

  Furthermore, in the present invention, since the saccharide contained in the preservation solution has a low molecular weight, it quickly dissolves in an aqueous solvent (for example, a washing solution that flows in a flow path before measurement) and is easily removed from the physiologically active substance. A dry sensor substrate can be provided.

FIG. 1 is a cross-sectional view schematically showing one embodiment of a plasmon excitation sensor used in the present invention.

  Next, the preservation | save sensor board | substrate of this invention, a dry sensor board | substrate, the manufacturing method of these sensor board | substrates, etc. are demonstrated concretely.

<Storage sensor board>
The preservation | save sensor board | substrate of this invention has a sensor board | substrate with which the physiologically active substance was fix | immobilized on the surface, and the preservation | save liquid which contains saccharides and a nonspecific protein, and coat | covers this physiologically active substance.

  The storage sensor substrate of the present invention is a substrate before the step of drying the solvent (or liquid component) contained in the storage solution or the like. Even if the liquid component coexists, Excellent storage stability.

[Sensor board]
The sensor substrate used in the present invention is only required to have a physiologically active substance immobilized on the substrate surface, and can be used for various measurements and detections as a biosensor.

  The sensor substrate used in the present invention comprises: a transparent support; a metal thin film formed on one surface of the support; and a self formed on the other surface of the metal thin film that is not in contact with the support. It may be a plasmon excitation sensor comprising an organized monolayer [SAM] and a physiologically active substance immobilized on the other surface of the SAM that is not in contact with the metal thin film.

  Such a plasmon excitation sensor further includes a hydrophilic polymer layer formed on the other surface of the SAM that is not in contact with the metal thin film, and the physiologically active substance is the hydrophilic polymer layer. It is preferably immobilized on the other surface not in contact with the SAM.

[Plasmon excitation sensor]
As shown in FIG. 1, the plasmon excitation sensor (5) used in the present invention includes a transparent support (1) having a metal thin film formed on one surface thereof; and on one surface of the support (1). A self-assembled monolayer [SAM] (2) formed; and a physiologically active substance (4) immobilized on one surface of the SAM (2).

  The plasmon excitation sensor (5) further includes a hydrophilic polymer layer (3) formed on one surface of the SAM (2), and the physiologically active substance (4) is hydrophilic. It may be immobilized on the other surface of the conductive polymer layer (3) that is not in contact with the SAM (2).

(Transparent support)
The “transparent support” used in the present invention may be made of quartz or glass, or may be made of plastic such as polycarbonate [PC] or cycloolefin polymer [COP], and has a refractive index [nd]. Preferably, it is 1.40 to 2.20, and the thickness (length × width) is not particularly limited as long as the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm.

  In addition, the transparent support made of glass is commercially available from “BK7” (refractive index [nd] 1.52) and “LaSFN9” (refractive index [nd] 1.85), ( “K-PSFn3” (refractive index [nd] 1.84), “K-LaSFn17” (refractive index [nd] 1.88) and “K-LaSFn22” (refractive index [nd]) manufactured by Sumita Optical Glass Co., Ltd. 1.90) and “S-LAL10” (refractive index [nd] 1.72) manufactured by OHARA INC. Are preferred from the viewpoint of optical properties and detergency.

  The transparent support is preferably cleaned with acid and / or plasma before forming a metal thin film on the surface.

  As the cleaning treatment with an acid, it is preferable to immerse in 0.001-1N hydrochloric acid for 1 to 3 hours.

  Examples of the plasma cleaning treatment include a method of immersing in a plasma dry cleaner (“PDC200” manufactured by Yamato Scientific Co., Ltd.) for 0.1 to 30 minutes.

(Metal thin film)
The “metal thin film” formed on one surface of the transparent support is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably gold. It is desirable to consist of these, and the alloy of these metals may be sufficient. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.

  Only when a flat glass substrate is used as the transparent support, it is preferable to form a thin film of chromium, nickel chromium alloy, or titanium in advance because glass and a metal thin film can be bonded more firmly.

  Examples of the method for forming the metal thin film on the transparent support include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Since it is easy to adjust the thin film formation conditions, it is preferable to form a chromium thin film and / or a metal thin film by sputtering or vapor deposition.

  The thickness of the metal thin film is preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 to 500 nm, and alloys thereof: 5 to 500 nm. The thickness of the thin film is preferably 1 to 20 nm.

  From the viewpoint of the electric field enhancing effect, gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20-70 nm, and alloys thereof: 10-70 nm are more preferable, and chromium The thickness of the thin film is more preferably 1 to 3 nm.

  When the thickness of the metal thin film is within the above range, surface plasmons are easily generated, which is preferable. Moreover, if it is a metal thin film which has such thickness, a magnitude | size (length x width) will not be specifically limited.

(SAM)
SAM (Self-Assembled Monolayer) is formed on the surface of the metal thin film opposite to the transparent support, and is used for fixing a physiologically active substance, preferably a hydrophilic polymer, to the metal thin film. It has a role as a foundation.

  In the present invention, the SAM contains a carboxyalkanethiol having about 4 to 20 carbon atoms (for example, available from Dojindo Laboratories Co., Ltd., Sigma Aldrich Japan Co., Ltd.), in particular. Preferably 10-carboxy-1-decanethiol is used. Carboxyalkanethiol having 4 to 20 carbon atoms has properties such as little optical influence of SAM formed using it, that is, high transparency, low refractive index, and thin film thickness. Therefore, it is preferable.

  The method for forming the SAM is not particularly limited, and a conventionally known method can be used. As a specific example, there is a method of immersing a glass transparent support having a metal thin film formed on its surface in an ethanol solution containing 10-carboxy-1-decanethiol (manufactured by Dojindo Laboratories). It is done. In this way, the thiol group of 10-carboxy-1-decanethiol binds to the metal and is immobilized, and self-assembles on the surface of the gold thin film to form a SAM.

(Hydrophilic polymer layer)
The hydrophilic polymer layer is preferably provided so as to intervene the bond between the SAM and the physiologically active substance. The hydrophilic polymer constituting the hydrophilic polymer layer is preferably dextran such as carboxymethyldextran [CMD] as the polysaccharide and polyacrylic acid as the synthetic resin. These macromolecules have a hydroxyl group [—OH] and a carboxyl group [—COOH] as functional groups highly effective in reducing non-specific adsorption, and prevent adsorption of non-specific proteins by the inclusion of water molecules by hydrogen bonding. Is preferable.

  As a method for forming the hydrophilic polymer layer on the SAM surface, for example, 1- (3-dimethylaminopropyl) -3ethylcarbodiimide [EDC] alone or EDC and N-hydroxysuccinimide [NHS] which is a water-soluble carbodiimide is used. ], A method of activating the carboxyl group of the hydrophilic polymer and reacting with the amino group of the SAM is included, but the present invention is not limited to this method, and a known method is appropriately used. Can do.

(Bioactive substance)
The physiologically active substance may be a protein that specifically binds to the substance to be captured, such as an enzyme that specifically binds to an antigen, an enzyme for a substrate / coenzyme, a receptor for a hormone, or a protein A for an antibody. -Protein G, avidins for biotin, calmodulin for calcium, and the like. Among these, an antibody is preferable as the "protein that specifically binds to the target substance to be captured".

  The method for immobilizing a physiologically active substance is a known method, for example, by activating the carboxyl group of SAM or hydrophilic polymer by EDC alone or in combination with EDC and immobilizing the physiologically active substance having an amino group. The method of making it. When a protein is used as a preferable physiologically active substance, the present invention is not limited to these as long as the protein is not inactivated.

[Preservation solution]
The preservation solution used in the present invention contains a saccharide and a non-specific protein, and further includes phosphate buffered saline [PBS], Tris buffered saline [TBS], or HEPES buffered saline [HBS], and It is preferable to contain a trace amount of sodium azide.

(Sugar)
The saccharide is preferably at least one saccharide selected from the group consisting of monosaccharides, disaccharides, and oligosaccharides composed of 3 to 10 monosaccharides, and has a molecular weight of less than 350. More preferred.

  Examples of monosaccharides include glucose (Mw: 180), fructose (Mw: 180), ribose (Mw: 150), xylose (Mw: 150), mannose (Mw: 180), galactose (Mw: 180), and talose. (Mw: 180).

  Examples of the disaccharide include sucrose, maltose, lactose, cellobiose, and trehalose. Both of these disaccharides have a molecular weight of about 342.

  Examples of oligosaccharides composed of 3 to 10 monosaccharides include raffinose (Mw: 504), panose (Mw: 504), melezitose (Mw: 504), gentianose (Mw: 504), stachyose (Mw: 667).

  The saccharide preferably has a molecular weight [Mw] of less than 350. If the molecular weight [Mw] is less than 350, the saccharide is easily dissolved in a water-soluble solvent such as a washing solution and can be easily removed from the physiologically active substance. Therefore, it is preferable.

  The saccharide is preferably contained in the preservation solution in an amount of 1 to 20% by weight, more preferably 5 to 12% by weight. When the saccharide content is within the above range, it is preferable from the viewpoint of storage stability of the physiologically active substance and washing.

(Non-specific protein)
The non-specific protein means a protein used for blocking non-specific adsorption at the time of immunostaining, and examples thereof include, but are not limited to, bovine serum albumin [BSA] and casein.

  The non-specific protein is preferably contained in the preservation solution in an amount of 0.1 to 5% by weight, more preferably 0.5 to 3% by weight. It is suitable from a viewpoint of blocking that content of nonspecific protein exists in the said range.

(Buffer solution)
As a buffer solution that may be contained in the preservation solution, phosphate buffered saline [PBS], Tris buffered saline [TBS] or HEPES buffered saline [HBS] can be used, and among these, Is PBS.

(Trace component)
The preservation solution may contain other components as trace components. For example, it may contain about 0.01 to 0.1% by weight of sodium azide.

<Dry sensor substrate>
The dry sensor substrate of the present invention is obtained by drying the sensor substrate of the present invention described above. Here, “drying” may be freeze-drying or natural drying.

  It is preferable to store and transport the dry sensor substrate as a dry sensor substrate between the production and use of the dry sensor substrate.

  In general, physiologically active substances such as proteins maintain a three-dimensional structure by coordinating water molecules in an aqueous solution. However, when dried, they cannot maintain the three-dimensional structure and are deactivated. In addition, when the substrate is supported in a hydrophilic polymer on the substrate surface, the physiologically active substances come close to each other and aggregate due to drying. Saccharides / non-specific proteins that are contained in the preservation solution used in the present invention and remain after drying can suppress inactivation by retaining the three-dimensional structure of the physiologically active substance instead of the water molecule, or It can coat | cover and can suppress aggregation by a three-dimensional effect.

<Method for manufacturing storage sensor substrate / dry sensor substrate>
The storage sensor substrate can be manufactured according to the manufacturing method of the present invention characterized in that it includes at least the following step (i). On the other hand, the dry sensor substrate further includes the following step (ii) in the storage sensor substrate manufacturing method. ).

  Step (i): A step of obtaining the preserved sensor substrate of the present invention by coating the physiologically active substance immobilized on the sensor substrate with the preservation solution.

  Step (ii): A step of removing the solvent contained in the storage sensor substrate by drying to obtain a dry sensor substrate.

  That is, the dry sensor substrate of the present invention can be manufactured by manufacturing the storage sensor substrate of the present invention and then subjecting the storage sensor substrate to the step (ii).

[Step (i)]
Step (i) is a step of obtaining the storage sensor substrate of the present invention by coating the physiologically active substance immobilized on the sensor substrate with the storage solution.

  As a method of coating the physiologically active substance using the preservation solution, the preservation solution may be filled or applied to the sensor substrate surface.

  Since the physiologically active substance can be coated on the sensor substrate without any gap, a method of filling the storage solution on the substrate is more preferable.

The amount of the preservation solution used per unit area of the sensor substrate can be appropriately adjusted according to the amount of the physiologically active substance immobilized and the concentration of the saccharide / non-specific protein contained in the preservation solution. 5 to 5 μL / mm ² is preferable.

  In addition, as a preservation solution, a preservation solution in which saccharides are dissolved and a preservation solution in which non-specific proteins are dissolved are prepared separately. It is also possible to further coat the physiologically active substance using the other after coating.

[Step (ii)]
The step (ii) is a step of obtaining a dry sensor substrate by removing the solvent contained in the storage sensor substrate obtained in the step (i) by drying.

  The solvent (or liquid component) is removed by drying, and the drying method may be freeze-dried or natural-dried as described above, and is not particularly limited in the present invention.

<How to use>
In the dry sensor substrate of the present invention, a washing solution is usually flowed before measurement to remove saccharides and non-specific proteins contained in the preservation solution, but low molecular weight saccharides are easily dissolved in the sample solution. The sample solution can also be used by flowing.

  Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these.

[Example 1]
(1) Preparation of sensor substrate Step (a): Plasma cleaning of a glass transparent support (“S-LAL 10” manufactured by OHARA INC.) Having a refractive index [nd] of 1.72 and a thickness of 1 mm, A chromium thin film was formed on one surface of the support by sputtering, and a gold thin film was further formed on the surface by sputtering. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

  Step (b1): The support obtained in step (a) was immersed in an ethanol solution containing 1 mM 10-carboxy-1-decanethiol for 24 hours or more to form SAM on one side of the gold thin film. The support was removed from the solution, washed with ethanol and isopropanol, and then dried with an air gun.

  Step (d): MES [2 containing 25 mg / mL of N-hydroxysuccinimide [NHS] and 25 mg / mL of water-soluble carbodiimide [EDC] on the surface of the support obtained in step (b1). -morpholinoethanesulfonic acid] buffered saline (pH 5.0) was added dropwise in an amount of 0.8 mL and allowed to react for 20 minutes, followed by anti-α-fetoprotein [AFP] monoclonal antibody (1D5, 2.5 mg / mL; Nippon Medical Co., Ltd.) The primary antibody was solid-phased on SAM by reacting 0.8 mL of an Acetate solution (pH 6.0) containing Laboratory Laboratory) for 30 minutes.

  Step (e): Next, 0.8 mL of 50 mM Tris (pH 7.4) is reacted for 15 minutes to block unreacted activated ester groups, and a TBS buffer containing 1 wt% bovine serum albumin [BSA]. By reacting with physiological saline for 30 minutes, nonspecific adsorption prevention treatment was performed.

(2) Manufacture of dry sensor substrate On the sensor substrate obtained in (1), 0.8 mL of PBS containing 3% by weight of sucrose (SIGMA) and 1% by weight of BSA as a preservation solution was dropped for 1 hour. After standing, it was removed using a pipette and allowed to air dry.

(3) Measurement of antibody activity remaining rate after 4 weeks 2 sheets of dry sensor substrate obtained in (2) were produced, and the antigen binding activity of the antibody was sealed immediately after the production, the other was sealed in a sealed container. After storing at 40 ° C. for 4 weeks, the remaining antibody activity was measured after 4 weeks as follows.

  First, it was washed with 0.8 mL of TBS containing 0.05% by weight of Tween 20 three times. Next, 800 μL of 1% BSA / PBS solution containing 0.1 ng / mL AFP as a target antigen was contacted for 25 minutes. After washing 3 times with 0.8 mL of TBS containing 0.05% by weight of Tween 20, secondary antibody labeled with Alexa Fluor (registered trademark) 647 (1% BSA / PBS solution prepared to be 1,000 ng / mL) ) Was added and allowed to react for 20 minutes.

  Then, it was washed 3 times with 0.8 mL of TBS containing 0.05% by weight of Tween20. Laser light (633 nm, 10 μW) is transmitted to each dry sensor substrate via the prism (manufactured by Sigma Kogyo Co., Ltd.) from the other surface of the glass transparent support on which the gold thin film is not formed. The signal value when the amount of fluorescence emitted from the irradiated and excited fluorescent dye was observed from a CCD image sensor was measured and used as an “assay measurement signal”.

  The SPFS measurement signal when AFP was 0 ng / mL was defined as “blank signal”. The assay signal was evaluated by the following formula from the assay measurement signal and the blank signal.

Assay signal = | (assay measurement signal) − (blank signal) |
The obtained results are shown in Table 1.

[Example 2]
(1) Production of sensor substrate Step (b2): Example 1 (1) The support obtained in step (a) is immersed in an ethanol solution containing 1 mM 11-amino-1-undecanethiol for 24 hours or more. A SAM was formed on one side of the gold thin film. The support was removed from the solution, washed with ethanol and isopropanol, and then dried with an air gun.

  Step (c): Next, in order to form a hydrophilic polymer layer composed of CMD (hereinafter also referred to as “CMD layer”) on the SAM, 1 mg / mL of CMD-500-06I4 (Meito Sangyo Co., Ltd.) EDC and NHS were added to 25 mM MES buffered saline containing 10 million average molecular weight and substitution degree 0.51) and 10 mM NaCl solution (pH 6.0) to a final concentration of 0.5 mM respectively. Immediately after stirring, the solution was dropped on the surface of the support on which SAM was formed, and reacted at room temperature for 1.5 hours. Then, 0.8 mL of 1N NaOH aqueous solution was added, it was made to react for 30 minutes, and the CMD surface support body was manufactured by wash | cleaning 3 times by 0.8 mL of ultrapure waters.

  Step (d): In step (d) of Example 1 (1), the CMD surface obtained in Step (c) of Example 2 (1) instead of the support obtained in Step (b1) The primary antibody was immobilized on the CMD layer in the same manner except that the support was used.

  Step (e): Non-specific adsorption prevention treatment was performed in the same manner as in Step (e) of Example 1 (1).

(2) Production of dry sensor substrate In Example 1, Example 1 (except that the sensor substrate obtained in Example 2 (1) was used instead of the sensor substrate obtained in Example 1 (1). A dry sensor substrate was produced in the same manner as 2).

(3) Measurement of remaining antibody activity after 4 weeks In Example 1, the dry sensor substrate obtained in Example 2 (2) was used instead of the dry sensor substrate obtained in Example 1 (2). The assay measurement signals were measured immediately after the production of the dry sensor substrate and after 4 weeks in the same manner as in Example 1 (3) except for the above. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Example 3]
In Example 1, except that the concentration of sucrose contained in the preservation solution was changed from 3% by weight to 10% by weight, the assay measurement signals were measured immediately after production of the dry sensor substrate and after 4 weeks in the same manner as in Example 1. did. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Example 4]
In Example 2, except that the concentration of sucrose contained in the stock solution was changed from 3% by weight to 10% by weight, assay measurement signals were measured immediately after production of the dry sensor substrate and after 4 weeks in the same manner as in Example 2. did. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Example 5]
In Example 1, except that the concentration of sucrose contained in the preservation solution was changed from 3% by weight to 20% by weight, the assay measurement signals were measured immediately after production of the dry sensor substrate and after 4 weeks in the same manner as in Example 1. did. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Example 6]
In Example 2, except that the concentration of sucrose contained in the preservation solution was changed from 3% by weight to 20% by weight, the assay measurement signals were measured immediately after production of the dry sensor substrate and after 4 weeks in the same manner as in Example 2. did. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Example 7]
In Example 1, except that 10% by weight of trehalose was used instead of 3% by weight of sucrose contained in the preservation solution, the assay measurement signal immediately after the production of the dry sensor substrate and after 4 weeks, as in Example 1. Was measured. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Example 8]
In Example 2, except that 10% by weight of trehalose was used instead of 3% by weight of sucrose contained in the preservation solution, the assay measurement signal immediately after the production of the dry sensor substrate and after 4 weeks, as in Example 2. Was measured. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Comparative Example 1]
In the production of the dry sensor substrate of Example 1 (2), the assay measurement signals were measured immediately after the production of the dry sensor substrate and after 4 weeks in the same manner as in Example 1 except that the sensor substrate was not coated with the preservation solution. . Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Comparative Example 2]
Example 2 (2) In the production of a dry sensor substrate, assay measurement signals were measured immediately after the production of the dry sensor substrate and after 4 weeks in the same manner as in Example 2 except that the sensor substrate was not coated with the storage solution. did. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Comparative Example 3]
In Example 1, except that 10% by weight of CMD (molecular weight 40,000) was used instead of 3% by weight of sucrose contained in the preservation solution, immediately after the production of the dry sensor substrate and for 4 weeks, as in Example 1. Subsequent assay measurement signals were measured. Immediately after production of the dry sensor substrate, the assay measurement signal was almost the same as the blank signal, and no assay signal was obtained. Therefore, the antibody activity remaining rate cannot be obtained after 4 weeks, and the result is represented by (-) in Table 1.

[Comparative Example 4]
In Example 2, except that 10% by weight of CMD (molecular weight 40,000) was used instead of 3% by weight of sucrose contained in the preservation solution, immediately after the production of the dry sensor substrate and for 4 weeks, as in Example 2. Subsequent assay measurement signals were measured. As in Comparative Example 3, no assay signal was obtained. The results of the remaining antibody activity after 4 weeks are represented by (-) in Table 1.

[Comparative Example 5]
In Example 3, the assay measurement signal was measured immediately after the production of the dry sensor substrate and after 4 weeks in the same manner as in Example 3 except that the preservation solution containing no BSA was used. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Comparative Example 6]
In Example 4, assay measurement signals were measured immediately after the production of the dry sensor substrate and after 4 weeks in the same manner as in Example 4 except that the preservation solution containing no BSA was used. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Comparative Example 7]
In Example 1, the assay measurement signal was measured immediately after the production of the dry sensor substrate and after 4 weeks in the same manner as in Example 1 except that a storage solution containing no sucrose was used. Table 1 shows the results of the remaining antibody activity after 4 weeks.

[Comparative Example 8]
In Example 2, the assay measurement signal was measured immediately after the production of the dry sensor substrate and after 4 weeks in the same manner as in Example 2 except that a storage solution containing no sucrose was used. Table 1 shows the results of the remaining antibody activity after 4 weeks.

As shown in Table 1, in Examples 1 to 8 where the preservation solution containing both sucrose or trehalose and BSA is coated, immediately after the dry sensor substrate is manufactured and after the dry sensor substrate is manufactured and stored at 40 ° C. for 4 weeks. Antibody activity was equivalent, that is, the residual rate was 100%.

  In Examples 1-8, when BSA was used, only the mode in which the content was fixed at 1% by weight was shown, but when the content of BSA was changed to 3% by weight or 5% by weight, Table 1 also shows. The remaining 4-week antibody activity rate was also 100%.

  On the other hand, in Comparative Examples 1 and 2 that were not coated with the preservation solution, the antibody activity decreased to 10% or less after 4 weeks. In Comparative Examples 3 and 4, which were coated with a storage solution containing 10% by weight of CMD having a molecular weight of 40,000 and 1% by weight of BSA, the antibody activity was reduced immediately after the production of the sensor substrate. Furthermore, in Comparative Examples 5 to 8, each of which used a stock solution containing no BSA or sucrose in the stock solutions used in Examples 1 and 2, as in Comparative Examples 1 and 2, the antibody activity was 10% after 4 weeks. It decreased to the following.

  Since the dry sensor substrate of the present invention obtained by drying the sensor substrate of the present invention is excellent in storage stability, it can be easily commercialized.

1. Transparent substrate with a metal thin film formed on one surface 2. Self-assembled monolayer [SAM]
3 .... Hydrophilic polymer layer 4 .... Bioactive substance 5 ... Plasmon excitation sensor

Claims (11)

A sensor substrate having a physiologically active substance immobilized on its surface;
Include sugars and non protein, have a, a storage solution for coating the physiologically active substance,
Save sensor substrate on which the saccharide, characterized in that it have a molecular weight of less than 350. The sensor substrate is
A transparent support;
A metal thin film formed on one surface of the support;
A self-assembled monolayer [SAM] formed on the other surface of the metal thin film not in contact with the support;
The preservation | save sensor board | substrate of Claim 1 which is the plasmon excitation sensor which comprises the said bioactive substance fix | immobilized on the other surface which is not in contact with this metal thin film of this SAM.   The plasmon excitation sensor further includes a hydrophilic polymer layer formed on the other surface of the SAM that is not in contact with the metal thin film, and the physiologically active substance comprises the hydrophilic polymer layer, The preservation | save sensor board | substrate of Claim 1 or 2 currently fixed by the other surface which is not in contact with this SAM.   The storage sensor substrate according to claim 1, wherein the physiologically active substance is a protein that specifically binds to the substance to be captured. The saccharide, saving sensor substrate according to claim 1 is at least one saccharide selected from monosaccharides, and disaccharides or Ranaru group. The preservation | save sensor board | substrate in any one of Claims 1-5 in which the said saccharides are contained in the said preservation | save liquid 1-20 weight%. The storage sensor substrate according to any one of claims 1 to 6 , wherein the non-specific protein is bovine serum albumin [BSA]. The non-specific protein, saving sensor substrate according to any one of claims 1 to 7 included 1-5 wt% in the preservation solution. The preservation | save sensor board | substrate in any one of Claims 1-8 in which the said preservation | save liquid further contains phosphate buffered saline [PBS], Tris buffered saline [TBS], or HEPES buffered saline [HBS]. . Method of manufacturing a storage sensor substrate according to any one of claims 1 to 9, characterized in that it comprises at least the following steps (i);
Step (i): A step of coating the physiologically active substance immobilized on the sensor substrate with the preservation solution. Method of manufacturing Drying sensor substrate you, characterized in that it comprises at least the following steps (i) and (ii);
Step (i): A step of obtaining the storage sensor substrate according to any one of claims 1 to 9 by coating the physiologically active substance immobilized on the sensor substrate with the storage solution, ii): A step of removing the solvent contained in the storage sensor substrate by drying to obtain a dry sensor substrate.