JP2016205850A - Microplate containing reagent and method for forming the plate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microplate containing a reagent that can increase the work efficiency and wastes a less amount of reagent, and a method for forming the microplate.SOLUTION: A reagent-containing microplate 1 includes: a microplate 2 having a plurality of wells 3; a freeze-dried reagent 4 in the wells 3; and a cover material 5 on the microplate 2 sealing the openings of the wells 3.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a microplate used for qualitative inspection and quantitative inspection.

  In a chemical test method or a biological test method including a step of reacting a sample with a reagent, a microplate having a plurality of wells is used as a reaction container.

For example, in protein quantification by a colorimetric method, a sample and a reagent containing protein are placed in a well, the protein and reagent are reacted, the absorbance is measured, and the protein is quantified.
In a test using such a microplate, since a reaction is performed in each well, it is necessary to dispense a sample and a reagent into each well, which requires complicated work and time. In addition, when a reagent is dispensed from a vial or the like to each well, the reagent remaining in the vial or the like is wasted and the cost is increased. Therefore, there is a demand for improved work efficiency and efficient use of reagents.

  For example, Patent Document 1 proposes a method of adding a test material solution to a well of a microplate and freeze-drying it in a microplate used in a compoundability test for the purpose of labor saving and efficiency improvement.

JP 2005-249570 A

  Therefore, it is conceivable to inject a reagent solution into the well of the microplate and freeze-dry it. In this case, it is necessary to seal the microplate immediately after freeze-drying. However, Patent Document 1 has no detailed description on sealing the microplate.

  The present invention has been made in view of the above circumstances, and a main object of the present invention is to provide a reagent-containing microplate that can improve work efficiency and has little reagent waste, and a method for manufacturing the same.

  In order to achieve the above object, the present invention provides a microplate having a plurality of wells, a reagent housed in the well and freeze-dried, and disposed on the microplate, and sealing the opening of the well. And a reagent-containing microplate.

  According to the present invention, since the freeze-dried reagent is previously stored in the well of the microplate, it is only necessary to inject the sample into the well at the time of the inspection, and the inspection can be performed easily and quickly. is there. Further, since a minute amount of reagent can be accommodated in the well of the microplate, waste of the reagent can be reduced and the cost can be reduced.

  Moreover, in this invention, it is preferable that the said cover material has a base material and the sealant layer formed on the said base material. Such a lid material can be freeze-dried by placing a lid material on the microplate so that it can be ventilated by partially heat-sealing the sealant layer to the microplate. Can seal the well opening by heat-sealing the sealant layer to the microplate as a whole.

  In the above case, it is preferable that the lid member has an adhesive layer formed between the base material and the sealant layer. For example, when the cover material is peeled off when using the reagent-containing microplate of the present invention, if the adhesive layer is formed between the base material and the sealant layer, the heat seal part of the sealant layer may be broken. Since the adhesive layer is exposed, the well can be sealed again with the lid material by the exposed adhesive layer after the sample is injected into the well. Therefore, it is not necessary to use a microplate cover separately, and the cost can be reduced.

  The present invention also includes an injecting step of injecting a reagent solution in which a reagent is dissolved or dispersed in a solvent into a well of a microplate having a plurality of wells, and a lid member disposed on the microplate so as to allow ventilation. There is provided a method for producing a reagent-containing microplate, comprising: a freeze-drying step of freeze-drying the reagent solution; and a sealing step of sealing the opening of the well with the lid.

  According to the present invention, the sample solution in the well can be lyophilized using the lid material, and the opening of the well can be sealed after lyophilization. Can be manufactured.

  Moreover, in this invention, it is preferable that the said cover material has a base material and the sealant layer formed on the said base material. Such a lid material can be freeze-dried by placing a lid material on the microplate so that it can be ventilated by partially heat-sealing the sealant layer to the microplate. Can seal the well opening by heat-sealing the sealant layer to the microplate as a whole.

  In the above case, it is preferable that the lid member has an adhesive layer formed between the base material and the sealant layer. For example, when the lid is peeled off when using the reagent-containing microplate produced according to the present invention, if an adhesive layer is formed between the base material and the sealant layer, the heat seal part of the sealant layer is completely broken. Since the adhesive layer is exposed by the occurrence of the above, after the sample is injected into the well, the well can be sealed again with the lid material by the exposed adhesive layer. Therefore, it is not necessary to use a microplate cover separately, and the cost can be reduced.

  The present invention has an effect that the working efficiency can be improved and the reagent can be used efficiently.

It is a schematic sectional drawing which shows an example of the microplate with a reagent of this invention. It is the schematic plan view which shows the other example of the microplate containing a reagent of this invention, and the schematic sectional drawing which shows the other example of the cover material in this invention. It is a schematic sectional drawing which shows the other example of the microplate with a reagent of this invention. It is process drawing which shows an example of the usage method of the microplate containing a reagent of this invention. It is a schematic sectional drawing which shows the other example of the microplate with a reagent of this invention. It is the schematic sectional drawing which shows the other example of the microplate with a reagent of this invention, and the schematic plan view and side view which show the other example of the cover material in this invention. It is process drawing which shows an example of the manufacturing method of the microplate with a reagent of this invention. It is a schematic diagram which shows an example of the freeze-drying process in the manufacturing method of the microplate containing a reagent of this invention. It is process drawing which shows the other example of the manufacturing method of the microplate with a reagent of this invention. It is process drawing which shows the other example of the manufacturing method of the microplate with a reagent of this invention.

  Hereinafter, the reagent-containing microplate of the present invention and the production method thereof will be described in detail.

A. Reagent-containing microplate The reagent-containing microplate of the present invention is a microplate having a plurality of wells, a reagent housed in the well and lyophilized, and disposed on the microplate, with the opening of the well being opened. It has the lid | cover material to seal, It is characterized by the above-mentioned.

The reagent-containing microplate of the present invention will be described with reference to the drawings.
FIG. 1 is a schematic cross-sectional view showing an example of a reagent-containing microplate of the present invention. As illustrated in FIG. 1, a reagent-containing microplate 1 includes a microplate 2 having a plurality of wells 3, a reagent 4 housed in the well 3 and lyophilized, and a microplate 2 disposed on the microplate 2. 3 and a lid member 5 that seals the opening of 3. The lid member 5 includes a base material 11 and a sealant layer 12 formed on the base material 11, and the sealant layer 12 is heat-sealed with respect to the microplate 2.

In the present invention, a freeze-dried reagent is stored in advance in a well of a microplate, and a test can be performed by injecting a sample into the well and reacting the sample and the reagent. Therefore, by using the reagent-containing microplate of the present invention, it is possible to improve the working efficiency and perform the inspection simply and quickly.
Further, since a minute amount of reagent can be accommodated in the well of the microplate, waste of the reagent can be reduced and the cost can be reduced.

  Moreover, in this invention, it is possible to apply to various chemical test methods and biological test methods by selecting the reagent accommodated in a well suitably.

  Further, in the present invention, since the inspection process can be simplified, it is possible to prevent other samples from being mixed inadvertently during work and floating matters from outside.

  Hereinafter, each configuration of the reagent-containing microplate of the present invention will be described.

1. Lid Material The lid material used in the present invention is disposed on the microplate and seals the opening of the well of the microplate.

The lid material is not particularly limited as long as it can seal the well opening and can be disposed on the microplate so that it can be ventilated during lyophilization. A first laminated film having a sealant layer formed on a material, a second laminated film having a substrate, an adhesive layer formed on the substrate, and a fluororesin film formed on the adhesive layer And rubber plugs. Among these, the first laminated film or the second laminated film is preferable, and the first laminated film is more preferable. This is because the well opening can be easily sealed.
Hereinafter, the first laminated film, the second laminated film, and the rubber stopper will be described separately.

(1) 1st laminated film The 1st laminated film used for this invention has a base material and the sealant layer formed on the base material.
Hereinafter, each structure in the first laminated film will be described.

(A) Sealant layer The sealant layer in the present invention is a heat-sealable layer.

  The material for the sealant layer is not particularly limited as long as it has heat sealability, and is appropriately selected according to the material for the microplate. For example, when the material of the microplate is polystyrene, polycarbonate, polypropylene, or acrylic resin, the material of the sealant layer is a compound polyolefin such as polyethylene, unstretched polypropylene, CMPS (registered trademark), VMX (registered trademark), or the like. A polyolefin polymer alloy or the like is preferably used.

In the present invention, since the reagent in the well is freeze-dried, the material of the sealant layer is preferably one that can maintain adhesion to the microplate even at a low temperature, that is, a temperature at the time of freeze-drying.
Specifically, the peel strength of the sealant layer with respect to the microplate at −5 ° C. after heat sealing is preferably in the range of 0.5 N / 15 mm to 50 N / 15 mm. This is because, if the peel strength of the sealant layer is in the above range, the adhesion to the microplate can be maintained even at a low temperature. On the other hand, if the peel strength of the sealant layer is too high, it may be difficult to peel off the lid material when using the reagent-containing microplate of the present invention.
The peel strength is a value measured with Tensilon (tensile tester RTA-250, manufactured by Orientec) according to JIS-K-6854-3 1999 (T-type peel test method).
Examples of the material for the sealant layer include polyethylene and linear low density polyethylene (LLDPE) when the material of the microplate is polyethylene terephthalate (PET) or a nylon base material.

  In addition, when using the reagent-containing microplate of the present invention, optical measurements such as absorption, fluorescence, and luminescence are performed. Therefore, when the lid is used as it is even during the optical measurement, the material of the sealant layer is transparent. It is preferable to have properties. In this case, it is preferable to add a material capable of imparting antifogging properties to the material of the sealant layer. This is because it is possible to suppress the vapor generated in the well from adhering to the sealant layer as droplets and affecting the optical measurement. Examples of materials that can impart antifogging properties include ethylene vinyl acetate copolymer (EVA), ethylene methyl acrylate copolymer (EMA), saponified ethylene vinyl acetate copolymer, polyamide resin, and chlorination. Examples include polypropylene resins and urethane resins.

  The material of the sealant layer preferably has a non-adsorbing property that hardly adsorbs the reagent, and is appropriately selected according to the type of the reagent. For example, when the reagent is a protein, amino acid, primary or secondary amine reagent, the material for the sealant layer is a polyester such as polyacrylonitrile (PAN), cyclic olefin copolymer (COC), or polyethylene terephthalate (PET). System, ethylene vinyl alcohol copolymer (EVOH) or the like can be used.

  The thickness of the sealant layer is preferably in the range of 10 μm to 100 μm, for example. This is because if the thickness of the sealant layer is too thin, sufficient adhesive strength cannot be obtained, and if it is too thick, it is difficult to peel off the cover material, and the reagent in the well may scatter when the cover material is peeled off. .

  Although it does not specifically limit as a method of forming a sealant layer in a base material, For example, the method of pasting together by the coextrusion method, the dry lamination, and the method of extruding and laminating a sealant layer from T-die can be mentioned.

(B) Base material The base material used for this invention supports a sealant layer.
Examples of the substrate include resin films such as polyester resins such as polyethylene terephthalate and polyethylene naphthalate, polyamide resins such as nylon 6 and nylon 66, and polyolefin resins such as polypropylene. Moreover, metal foils, such as aluminum foil, can also be used as a base material.
Moreover, the sealant layer may also serve as the base material.

  The substrate may be transparent or opaque, and is appropriately selected depending on the presence or absence of peeling of the lid material and the type of optical measurement when using the reagent-containing microplate of the present invention. Even when the base material is opaque and the lid member is not peeled off, the lid member may be measured by providing a hole for optical measurement.

  In addition, when using the reagent-containing microplate of the present invention, when the sample is injected into the well by piercing the lid with a pipette or the like without peeling the lid, the substrate has a piercing opening It is preferable. In this case, as the base material, for example, a stretched polypropylene film, a metal foil such as an aluminum foil, or the like is preferably used.

  Moreover, when using the reagent-containing microplate of the present invention, in the case where the lid member is peeled off, it is preferable that the substrate has a linear cut property. In this case, the lid member can be easily peeled off, and the reagent in the well can be prevented from scattering when the lid member is peeled off. Further, only wells in a predetermined column or row can be opened, and a reagent-containing microplate can be used according to the number of inspection points. As a base material which has linear cut property, a uniaxially stretched film is mentioned, for example.

Moreover, when peeling a lid | cover material when using the microplate containing a reagent of this invention, it is also preferable that the base material 11 has the cutting line 15 so that it may illustrate in Fig.2 (a)-(d). 2 (a) to 2 (c) are schematic top views showing other examples of the reagent-containing microplate of the present invention, and FIG. 2 (d) is a schematic sectional view showing another example of the lid material of the present invention. is there. In this case, the lid member can be easily peeled along the cutting line, and the reagent in the well can be prevented from scattering when the lid member is peeled. Further, only predetermined wells can be opened, and a reagent-containing microplate can be used according to the number of inspection points.
The cutting line may be formed continuously or may be formed discontinuously in a perforated shape. As the formation position of the cutting line, the cutting line 15 may be formed linearly along the column or row so that the lid member can be peeled for each column or row as illustrated in FIG. As illustrated in FIG. 2B, the cutting lines 15 may be formed in a lattice shape so that the lid material can be peeled for each well 3. Further, as shown in FIG. 2C, a cutting line 15 may be formed so as to cross over the well 3 so that the lid on the well 3 can be partially opened. The cutting depth of the cutting line may be the same as the thickness of the substrate, for example, as long as the lid material can be cut, and may be smaller than the thickness of the substrate. Examples of the method for forming the cutting line include a laser processing method.

  The thickness of the base material is appropriately adjusted depending on whether or not the lid material is peeled off.

(C) Adhesive layer In this invention, it is preferable that the adhesive layer 13 is formed between the base material 11 and the sealant layer 12, as illustrated in FIG.
4A to 4D are process diagrams showing an example of a method of using the reagent-containing microplate of the present invention, and an example using the reagent-containing microplate shown in FIG. First, as shown to Fig.4 (a), in the microplate 1 containing a reagent, the part which is contacting the microplate 2 of the sealant layer 12 becomes the heat seal part 14. FIG. As shown in FIG. 4B, when the lid member 5 is peeled off, it is broken at the boundary between the heat seal part 14 that has received heat from the sealant layer 12 and a part other than the heat seal part 14 that has not received heat, so-called. A root break occurs. Therefore, the adhesive layer 13 is exposed at the heat seal part 14. Next, as shown in FIG. 4C, the sample 10 is injected into the well 3. Next, as shown in FIG. 4 (d), the lid 5 is covered again. At this time, the lid 5 can be brought into close contact with the microplate 2 by the exposed adhesive layer 13.
Therefore, when an adhesive layer is formed, the well can be sealed again with a lid, and it is possible to prevent other samples from being mixed inadvertently or from external floating substances. can do. Therefore, it is not necessary to separately use an expensive microplate cover, and the cost can be reduced.

The material of the pressure-sensitive adhesive layer (hereinafter referred to as pressure-sensitive adhesive) is not particularly limited as long as it has removability and resealability, and is appropriately selected depending on the material of the adherend and the required adhesive strength. Can do. Specific examples include rubber adhesives, acrylic adhesives, silicone adhesives, urethane adhesives, and the like.
Among these, it is excellent in heat deterioration suppressing action during heat sealing and durability under low temperature conditions, etc., and it effectively suppresses the occurrence of delamination by suppressing the decrease in lamination strength between the base material and the sealant layer. From the viewpoint, preferably, a silicone-based pressure-sensitive adhesive is used.
Moreover, the said adhesive can mix | blend suitably a tackifier, a crosslinking agent, a softener, a filler, an anti-aging agent, etc.

  The thickness of the adhesive layer is preferably in the range of 20 μm to 100 μm, for example.

  The method for forming the pressure-sensitive adhesive layer is not particularly limited. Specifically, the pressure-sensitive adhesive obtained by stirring the cross-linking agent in the above-mentioned pressure-sensitive adhesive main agent is applied onto the release paper, and the moisture and solvent are removed in a drying oven. After evaporating and removing, other methods may be used, such as a method of adding other materials and press-bonding, or a method of forming by irradiating ultraviolet rays.

(D) First laminated film The method for producing a reagent-containing microplate when the lid is the first laminated film is described in “B. Method for producing a reagent-containing microplate” described later. Description of is omitted.

(2) 2nd laminated film The 2nd laminated film used for this invention has a base material, the contact bonding layer formed on the base material, and the fluorine resin film formed on the contact bonding layer. .
FIG. 5 is a schematic sectional view showing another example of the reagent-containing microplate of the present invention. As shown in FIG. 5, in the reagent-containing microplate 1, the lid member 6 includes a base material 21, an adhesive layer 22 formed on the base material 21, and a fluororesin film 23 formed on the adhesive layer 22. And have.
Hereinafter, each structure in the second laminated film will be described.

(A) Fluorine-based resin film In the present invention, a fluorine-based resin film is preferably used because it exhibits low sliding resistance, that is, good surface slipperiness and low reactivity with various reagents.

  As the fluorine resin film, a film showing surface slipperiness and low reactivity can be used. Examples of such a fluororesin film include polytetrafluoroethylene (PTFE), perfluoroalkoxy resin (PFA) made of a copolymer of tetrafluoroethylene and perfluoroalkyl vinyl ether, and tetrafluoroethylene and hexafluoropropylene. Copolymer (FEP), copolymer of tetrafluoroethylene and perfluoroalkyl vinyl ether and hexafluoropropylene (EPE), copolymer of tetrafluoroethylene and ethylene or propylene (ETFE), polychlorotrifluoroethylene resin (PCTFE), ethylene and chloro One kind of fluorine resin such as copolymer with trifluoroethylene (ECTFE), vinylidene fluoride resin (PVDF), vinyl fluoride resin (PVF) Others can be used films composed of two or more.

  Among these, fluororesin film made of vinyl fluoride resin (PVF), copolymer of tetrafluoroethylene and ethylene or propylene (ETFE) has excellent sliding properties, low reactivity with various reagents and solvents, heat resistance From the viewpoint of properties and durability.

  Also, for example, film processability, heat resistance, light resistance, weather resistance, mechanical properties, dimensional stability, antioxidant properties, slipperiness, mold release properties, flame retardancy, antifungal properties, electrical properties, etc. Various plastic compounding agents, additives and the like can be added for the purpose of improving and modifying the above. The addition amount can be arbitrarily set from a very small amount to several tens of percent depending on the additive or the like, depending on the purpose, with respect to the total amount of the fluororesin.

  Moreover, in order to improve adhesiveness, an epoxy-type silane coupling agent may be added, and in order to prevent a film blocking etc., an antiblocking agent may be added. These addition amounts are preferably about 0.1% by mass to 10% by mass.

The thickness of the fluororesin film is preferably in the range of 12 μm to 300 μm, and more preferably in the range of 50 μm to 150 μm. This is because manufacturing and handling becomes difficult when the thickness is too thin, and the cost increases as the thickness increases.
The thickness can be measured using, for example, a fluorescent X-ray analyzer (model name, RIX2000 type) manufactured by Rigaku Corporation.

  The method for forming the fluororesin film is not particularly limited. For example, a method for forming a film using a film forming method such as an extrusion method, a cast molding method, a T-die method, a cutting method, an inflation method, or the like. Alternatively, a method of forming a multilayer co-extrusion film using two or more kinds of fluorine-based resins, and further using two or more kinds of fluorine-based resins and mixing to form a film before forming a film. Methods and the like. Moreover, you may extend | stretch uniaxially or biaxially using a tenter system or a tubular system etc. as needed, for example.

The surface of the fluororesin film can be provided with a desired surface treatment layer in advance, if necessary, in order to improve adhesion and the like.
As the surface treatment, for example, treatment such as corona discharge treatment, ozone treatment, glow discharge treatment, oxidation treatment using chemicals, or the like can be used.
As a method for improving adhesiveness, for example, a primer coat agent layer, an undercoat agent layer, an anchor coat agent layer, an adhesive layer, or a vapor deposition anchor coat agent layer is previously formed on the surface of the fluororesin film. It can be arbitrarily formed to form a surface treatment layer. Examples of the pretreatment coating agent layer include polyester resins, polyamide resins, polyurethane resins, epoxy resins, phenol resins, (meth) acrylic resins, polyvinyl acetate resins, polyolefins such as polyethylene and polypropylene It is possible to use a resin composition whose main component is a vehicle resin or a copolymer or modified resin thereof, a cellulose resin or the like.

  In addition, as a method for forming the coating agent layer, for example, a solvent type, aqueous type, or emulsion type coating agent is used, and a coating method such as a roll coating method, a gravure roll coating method, or a kiss coating method is used. it can. The step of forming the coating agent layer may be carried out after the formation of the fluororesin film or as a subsequent step after the biaxial stretching treatment, or by film formation or in-line treatment of the biaxial stretching treatment. it can.

(B) Adhesive layer The adhesive layer used in the present invention is formed between the substrate and the fluororesin film.

  The material used for the adhesive layer is not particularly limited as long as it can adhere the substrate and the fluororesin film, and examples thereof include a silicone adhesive, and specifically an oxime type silicone adhesive. .

  The thickness of the adhesive layer is preferably in the range of 5 μm to 40 μm, for example.

  Examples of the method for forming the adhesive layer include a method using a spatula, a blade coating method, a spin coating method, a comma coating method, and a kiss coating method.

(C) Substrate The substrate used for the second laminated film in the present invention can be the same as the substrate used for the first laminated film.
Moreover, the base material laminated | stacked on a fluororesin film may be the same as the base material of a sealant layer, and may be laminated to a cover material, but heat-seal a base material directly to a fluororesin film. Since it may not be possible, it is preferable to bond an adhesive-processed or surface-treated material to the lid.

(D) Second laminated film The method for producing a reagent-containing microplate when the lid material is the second laminated film is described in “B. Method for producing a reagent-containing microplate” described later. Description of is omitted.

(3) Rubber plug The rubber plug in the present invention may be any one that can seal the well opening and can be disposed on the microplate so that it can be ventilated during freeze-drying. Those having leg portions having notches are preferably used.
6 (a) is a schematic cross-sectional view showing another example of the reagent-containing microplate of the present invention, and FIGS. 6 (b) and 6 (c) are schematic plan views showing other examples of the lid material according to the present invention. It is a side view. FIG. 6B is a schematic plan view seen from the leg side of the rubber plug. As shown in FIGS. 6A to 6C, the lid member 7 is a rubber plug, has a flange portion 31 and a leg portion 32, and the leg portion 32 has a notch portion 33.
With such a rubber stopper, the notched part can function as a vent by half-plugging during freeze-drying, and the well opening can be sealed by completely plugging after freeze-drying. .

  The rubber plug has only to have a flange portion and a leg portion having a notch, and the structure of the rubber plug can be the same as a general freeze-drying rubber plug.

As a material for the rubber plug, for example, a rubber composition obtained by kneading any compounding agent as necessary using any synthetic rubber or natural rubber as a main raw material can be used.
Synthetic rubbers include, for example, butyl rubber such as butyl rubber, chlorinated butyl rubber, brominated butyl rubber, divinylbenzene copolymer butyl rubber, isoprene rubber, isoprene-isobutylene rubber, butadiene rubber, styrene butadiene rubber, ethylene propylene rubber, nitrile rubber, etc. Is mentioned.
Among them, chlorinated butyl rubber or brominated butyl rubber is preferably used in order to maintain good gas impermeability, ozone resistance, aging resistance, electrical properties, chemical resistance, etc., and to exhibit excellent heat resistance. be able to.

  Moreover, the fluorine resin film may be arrange | positioned through the contact bonding layer on the surface of the rubber stopper. This is because the reagent can be made difficult to adsorb.

  The fluorine resin film can be the same as the fluorine resin film used for the second laminated film.

  The adhesive layer is a layer for adhering a rubber plug to a fluororesin film by thermocompression bonding, and is a silica film formed such that at least one of a methyl group and an ethyl group is present on the surface, and an organosilicon compound It is the vapor deposition film formed by the plasma CVD method using the gas composition for vapor deposition containing this. When at least one of the methyl group and the ethyl group is present on the surface of the adhesive layer, the adhesion to the rubber stopper is improved.

  The amount of methyl groups and ethyl groups present on the surface can be adjusted by changing the ratio of the vapor deposition monomer gas to the oxygen supply gas in the vapor deposition gas composition during film formation.

  The thickness of the adhesive layer is preferably in the range of 5 nm to 200 nm, more preferably in the range of 20 nm to 100 nm. If the thickness of the adhesive layer is too thin, the adhesive layer does not exist as a continuous film, and fluorine of the fluororesin film is exposed on the surface, which may impair the adhesion with the rubber plug. On the other hand, if the thickness is too thick, the productivity is inferior, the rigidity is increased, and cracks are easily generated.

The surface of the adhesive layer preferably has a contact angle with water as a polar solvent in the range of 35 ° to 85 °, more preferably in the range of 35 ° to 70 °. Moreover, it is preferable that the contact angle with respect to diiodomethane which is a nonpolar solvent exists in the range of 10 degrees-70 degrees, More preferably, it exists in the range of 10 degrees-40 degrees. When the contact angle is too large, the fluorine on the surface inhibits adhesion, and good adhesion cannot be obtained.
The contact angle can be obtained from the angle of the straight line connecting the left and right end points and the vertex of the droplet with respect to the solid surface by the θ / 2 method according to JIS K 6768. The contact angle is a value measured by a fully automatic contact angle meter Drop Master 700 manufactured by Kyowa Interface Science Co., Ltd. under the conditions of 20 ° C. and 50% RH.

  Examples of the organosilicon compound used for forming the adhesive layer include organosilicon compounds containing a methyl group directly bonded to a silicon atom, such as hexamethyldisiloxane (HMDSO), tetramethyldisiloxane (TMDSO), octamethylcyclotetra Siloxane, methylsilane, dimethylsilane, trimethylsilane, tetramethylsilane, ethylsilane and the like are preferably used. Among these, HMDSO, TMDSO, and octamethylcyclotetrasiloxane are preferable. These organosilicon compounds are stable in the film because the methyl group that expresses adhesiveness is directly bonded to the silicon atom, not through the Si—O bond or the O—C bond, which is easily broken. It is because it becomes easy to be taken in.

  The conditions of the plasma CVD method are detailed in JP2013-107961A.

  In addition, the surface of the adhesive layer may be subjected to plasma processing again using a non-reactive gas such as argon gas or helium gas in a plasma CVD apparatus or an arbitrary plasma processing apparatus. By the plasma treatment, the surface of the adhesive layer can be modified to further improve the adhesion with the rubber stopper.

  As a method of disposing a fluororesin film on the surface of the rubber plug through an adhesive layer, the adhesive layer surface of the laminated film in which the adhesive layer is formed on the fluororesin film is overlapped so as to face the rubber substrate. And a method of thermocompression bonding. Thereby, extremely strong adhesion between the laminated film and the rubber base material is achieved, and the slipperiness and solvent resistance of the surface can be improved while maintaining the elasticity of the rubber base material. For example, a rubber sheet is used as a rubber base material, and a composite rubber sheet is produced by superimposing it on the surface of the adhesive layer of the laminated film and thermocompression bonding. The composite rubber sheet is punched into an arbitrary shape, and a rubber plug is attached. Can be manufactured. Also, a rubber plug is manufactured by vulcanizing and molding an unvulcanized rubber sheet into a desired shape, and overlaying the resulting vulcanized molded product on the adhesive layer surface of the laminated film, followed by thermocompression laminating. You can also In addition, an unvulcanized rubber sheet is used as a rubber base material, which is superimposed on the surface of the adhesive layer of the laminated film, placed in an arbitrary molding die, vulcanized into a desired shape, and heated at the same time. Crimp lamination can also be performed. In addition, the unvulcanized rubber sheet is semi-vulcanized and molded into the desired shape, and the resulting semi-vulcanized molded product is used as a rubber base material, which is superimposed on the adhesive layer surface of the laminated film, and the remaining vulcanized molding At the same time as performing thermocompression laminating.

  Vulcanization molding and thermocompression bonding of the laminated film and the rubber substrate can be performed by a conventionally known method. Moreover, the reaction conditions in that case can be suitably set according to the kind of rubber base material to be used, a vulcanization | cure state, melting | fusing point of a fluororesin film, thickness, etc. For example, when laminating a vulcanized rubber base material and a laminated film, thermocompression laminating can be performed at a temperature of 150 ° C. to 250 ° C., a pressure of 0.1 Pa to 20 Pa, and a time of 10 seconds to 300 seconds. Moreover, when laminating an unvulcanized or semi-cured rubber base material and a laminated film, thermoforming and thermocompression laminating at a temperature of 150 ° C. to 250 ° C., a pressure of 0.1 Pa to 20 Pa, and a time of 10 seconds to 600 seconds. It can be performed.

2. Reagent The reagent used in the present invention is contained in a well of a microplate and freeze-dried.
Any reagent can be used as long as it is used in a chemical test method or a biological test method including a step of reacting a sample with a reagent, and is appropriately selected according to the test method. For example, proteins, peptides, amino acids, nucleic acids and nucleic acid-related substances, sugars, lipids, vitamins, hormones, and other substrates, and chromogenic synthetic substrates in which chromophores are bound to enzymes, amino acids, and peptides used for quantification of the above substrates , Colorimetric reagents, redox reagents, polymerization reagents and the like. Specifically, in the endotoxin test method, examples of the reagent include a lysate reagent and a chromogenic synthetic substrate (colorimetric reagent) in which a chromogenic group is bound to an amino acid (peptide).
In addition, polyols such as glycerol, pH adjusters, chelating agents, and the like may be added for the purpose of preventing protein denaturation during lyophilization.

3. Microplate The microplate used in the present invention has a plurality of wells.

  The number of wells can be in a general range, and examples thereof include 6, 12, 24, 48, 96, 384, and the like.

  When the microplate containing the reagent of the present invention is used, optical measurement such as absorption, fluorescence, and luminescence is performed. Therefore, the microplate material may be any material as long as it has transparency. Among them, the material of the microplate is preferably one that does not easily adsorb the reagent and the sample, and is appropriately selected according to the kind of the reagent and the sample. Specific examples include polystyrene, polycarbonate, acrylic resin, and the like.

  Moreover, it is preferable to add a modifier to the material of the microplate from the viewpoint of resin flowability, antifogging property, and antistatic control. Examples of the modifier include ethylene vinyl acetate copolymer (EVA), ethylene methyl acrylate copolymer (EMA), saponified ethylene vinyl acetate copolymer, polyamide resin, chlorinated polypropylene resin, urethane resin, and the like. Is mentioned. In addition, the modifying agent may be appropriately changed in order to make it difficult to adsorb the reagent and the sample.

  In order to make it difficult to adsorb the reagent and the sample, the surface of the microplate may be subjected to a hydrophilic treatment such as plasma treatment.

4). Reagent-containing microplate The reagent-containing microplate of the present invention may be hermetically packaged. This is because the sealing performance can be improved.
As a package, what has an aluminum foil or an aluminum vapor deposition film, for example can be used.

5. Method of Use The reagent-containing microplate of the present invention can be used for a chemical test method or a biological test method including a step of reacting a sample with a reagent, and can be used for various tests. For example, endotoxin test method, protein quantification, preconditioning of hybridization method for PCR amplified gene identification, preconditioning of staining method for protein identification, nucleotides, nucleosides, amino acids, various sugars, lipids, vitamins, hormones by enzymatic method And the like.

B. Manufacturing method of reagent-containing microplate The manufacturing method of the reagent-containing microplate of the present invention includes an injection step of injecting a reagent solution in which a reagent is dissolved or dispersed in a solvent into the well of a microplate having a plurality of wells; A method comprising: placing a lid material on the microplate so as to allow ventilation, and freeze-drying the reagent solution; and a sealing step of sealing the well opening with the lid material. is there.

A method for producing a reagent-containing microplate of the present invention will be described with reference to the drawings.
FIGS. 7A to 7D are process diagrams showing an example of the method for producing the reagent-containing microplate of the present invention, in which the lid material is the first laminated film. First, as shown in FIG. 7A, the reagent solution 4 a is injected into the well 3 of the microplate 2. Next, as shown in FIG. 7 (b), the lid 5 having the base material 11 and the sealant layer 12 formed on the base material 11 is placed in the microplate so that the sealant layer 12 faces the microplate 2. 2 is arranged. At this time, for example, as shown in FIG. 8A, only the four corners of the sealant layer 12 are heat-sealed to form the heat-seal portion 14, and the lid material is attached to the microplate. Since the portions other than the heat seal portion 14 are not heat sealed, the gas in the well 3 can be circulated and can be ventilated. Next, as shown in FIGS. 7B to 7C, the reagent solution 4 a is lyophilized to obtain the lyophilized reagent 4. Next, as shown in FIG. 7 (d), the opening of the well 3 is sealed with a lid 5. At this time, the sealant layer 12 can be hermetically sealed with the lid member 5 by heat-sealing the sealant layer 12 as a whole.

FIGS. 9A to 9D are process diagrams showing another example of the method for producing a reagent-containing microplate according to the present invention, in which the lid material is a second laminated film. First, as shown in FIG. 9A, the reagent solution 4 a is injected into the well 3 of the microplate 2. Next, as shown in FIG. 9B, a lid 6 having a base material 21, an adhesive layer 22 formed on the base material 21, and a fluororesin film 23 formed on the adhesive layer 22. The fluororesin film 23 is disposed on the microplate 2 so as to face the microplate 2.
The lid member 6 is designed to have a slight gap with the microplate 2, and the inside of the well 3 can be evacuated from the slight gap during freeze-drying.
Next, as shown in FIGS. 9B to 9C, the reagent solution 4 a is lyophilized to obtain the lyophilized reagent 4. Next, as shown in FIG. 9 (d), the opening of the well 3 is sealed with a lid member 6.
Usually, after freeze-drying, it is preferably sealed with a lid, and then stored in a desiccator or the like, or moisture-proof packaged. If there is an invisible pinhole or gap, the freeze-dried reagent may absorb moisture and deteriorate, so it is better to perform the above moisture-proof treatment.

FIGS. 10A to 10D are process diagrams showing another example of the method for producing the reagent-containing microplate of the present invention, in which the lid member is a rubber stopper. First, as shown in FIG. 10A, the reagent solution 4 a is injected into the well 3 of the microplate 2. Next, as shown in FIG. 10 (b), a lid member 7 that is a rubber plug is disposed on the microplate 2. As shown in FIGS. 6A to 6C, the lid member 7 is a rubber plug having a flange portion 31 and a leg portion 32 having a notch portion 33. At this time, the notch 33 of the leg 32 can function as a vent by half-plugging the rubber stopper. Next, as shown in FIGS. 10B to 10C, the reagent solution 4 a is lyophilized to obtain the lyophilized reagent 4. Next, as shown in FIG. 10 (d), the opening of the well 3 is sealed with a lid member 7. At this time, it is possible to seal with the lid material 7 by completely plugging the rubber stopper.
Normally, when the rubber plugs are fitted (sealed) by completely plugging them, unlike the method illustrated in FIG. 9, the sealing performance is very high, and generally the above-described moisture-proof treatment may not be required. However, moisture-proof treatment may be performed as appropriate.

  In the present invention, the sample solution in the well can be lyophilized using the lid material, and the well opening can be sealed after lyophilization, and a microplate containing a reagent can be manufactured in a simple process. can do.

  Hereafter, each process in the manufacturing method of the microplate with a reagent of this invention is demonstrated.

1. Injection Step The injection step in the present invention is a step of injecting a reagent solution in which a reagent is dissolved or dispersed in a solvent into the well of a microplate having a plurality of wells.

  Since the microplate and the reagent are described in the above “A. Microplate with reagent”, description thereof is omitted here.

  The solvent is appropriately selected depending on the type of reagent, and examples thereof include water and a buffer solution.

  The method for injecting the reagent solution into the well is not particularly limited, but it is preferably dispensed by a dispenser robot from the viewpoint of cost.

2. Freeze-drying step The freeze-drying step in the present invention is a step in which a lid material is disposed on the microplate so as to allow ventilation, and the reagent solution is freeze-dried.

  In addition, since it described in the said "A. Microplate containing a reagent" about the lid | cover material, description here is abbreviate | omitted.

  The method for arranging the lid material on the microplate so as to allow ventilation is appropriately selected according to the type of the lid material.

For example, when the lid is a first laminated film having a base material and a sealant layer formed on the base material, the lid can be vented by partially heat-sealing the sealant layer to the microplate. Material can be placed. When the sealant layer is partially heat-sealed, the formation position of the heat-seal portion is not particularly limited as long as the lid material can be disposed so as to allow ventilation. For example, as shown in FIG. The heat seal portion 14 may be formed only at the four corners of the layer 12, or the heat seal portion 14 may be formed on two sides of the sealant layer 12 as shown in FIG. 8B, as shown in FIG. As shown, the heat seal portions 14 may be formed in a dot shape, or the heat seal portions 14 may be formed in a stripe shape as shown in FIG. The area of the heat seal part is such that the cover material is not detached during transport and freeze-drying, the adhesive strength of the heat seal part, the strength of the first laminate film, the first laminate film during transport and freeze-drying It adjusts suitably according to the force etc. which apply to. In FIGS. 8A to 8D, the base material is omitted.
The heat sealing conditions can be the same as general heat sealing conditions for pharmaceutical applications and the like, and are appropriately selected according to the material of the sealant layer.

For example, when the lid is a second laminated film having a base material, an adhesive layer formed on the base material, and a fluororesin film formed on the adhesive layer, the microplate of the fluororesin film By forming irregularities on the surface on the side (hereinafter referred to as the inner surface), a slight gap is formed between the inner surface of the lid member and the microplate, so that the lid member can be disposed so as to allow ventilation. Moreover, the said unevenness | corrugation may be formed in the whole inner surface of a fluororesin film, and may be formed only in the peripheral part of a cover material.
The unevenness can be melted by heat sealing to seal the microplate.

  Further, for example, when the lid member is a rubber plug having a flange portion and a leg portion having a notch portion, the lid member can be disposed so as to be ventilated by half-plugging the rubber plug.

  When the sample solution is freeze-dried, it is preferable to freeze the sample solution and vacuum-dry it by a conventionally known method. Keeping the frozen sample solution at a temperature where it cannot be melted and reducing the pressure to a vacuum state can lower the boiling point of water and sublimate the water from the ice state to the water vapor state, so that the protein is not denatured. This is because it can be dried.

  The lyophilization method and conditions can be appropriately selected and applied from general methods and conditions for uses such as pharmaceuticals. For example, after lyophilization at -3 ° C for 1 hour to 2 hours, the temperature is further lowered and lyophilized at -15 ° C to -10 ° C for 20 hours to obtain a dry reagent.

  Examples of the method for freezing the reagent solution before lyophilization include a method of placing the reagent solution in a freezer and a method of contacting with liquid nitrogen.

3. Sealing Step The sealing step in the present invention is a step of sealing the opening of the well with the lid material.

  The method of sealing with the lid material is appropriately selected according to the type of the lid material.

For example, when the lid material is a first laminated film having a base material and a sealant layer formed on the base material, the sealant layer is hermetically sealed with the lid material by heat-sealing the entire sealant layer to the microplate. can do.
The heat sealing conditions can be the same as general heat sealing conditions for pharmaceutical applications and the like, and are appropriately selected according to the material of the sealant layer.

  For example, when the lid material is a second laminated film having a base material, an adhesive layer formed on the base material, and a fluorine-based resin film formed on the adhesive layer, the lid is attached to the microplate. By heat-sealing the material as a whole, the concavities and convexities attached to the inner surface of the lid material can be fused and sealed.

  For example, when the lid member is a rubber plug having a flange portion and a leg portion having a notch, the lid member can be hermetically sealed by completely plugging the rubber plug.

After sealing with a lid, the reagent-containing microplate may be sealed and packaged. This is because the sealing performance can be improved.
In addition, since it described in the said "A. Microplate containing a reagent" about the package, description here is abbreviate | omitted.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has any configuration that has substantially the same configuration as the technical idea described in the claims of the present invention and that exhibits the same effects. Are included in the technical scope.

Hereinafter, the present invention will be specifically described with reference to examples.
[Example 1]
(Preparation of cover material)
An adhesive surface of a polyester film adhesive tape (631S # 100, manufactured by Teraoka Seisakusho Co., Ltd.) and CMPS (017C, manufactured by Mitsui Chemicals, Inc.) 30 μm were bonded together and cut according to the size of a 96-well microplate.
(Freeze drying of lysate reagent and buffer)
The lysate reagent (powder) of the endotoxin test reagent set (Endospecy ES-24S, manufactured by Seikagaku Corporation) was dissolved in 0.5 mL of water for injection and dispensed in a volume of 0.2 mL into a 96-well microplate.
Next, 0.3 mL of β-glucan inhibition buffer for dissolving lysate reagent was dispensed into a 96-well microplate.
The lysate reagent and β-glucan inhibition buffer dissolved in the water for injection were dispensed into different wells.
The reagent and buffer dispensed in the 96-well microplate were frozen in a freezer, and then placed so that the CMPS surface of the lid faced the microplate side, and lightly sealed so as to surround the well in a cross. Specifically, the top of the lid material is laid on the film with the solder claws laid down, and the lid material is pressure-bonded to the circular protrusions around the wells. Then, the solder claws are placed along the vertical and horizontal wells. It was slid in an upright state, and the lid was crimped so as to draw a cross, thereby sealing it to the extent that it was not sealed.
Thereafter, the microplate was freeze-dried overnight with a freeze dryer.
After visually confirming the drying, the entire lid material on the microplate was pressure-bonded with a seal tester heated to 180 ° C., and the circular protrusion around the well was completely sealed.
(Preparation of endotoxin standard solution)
A 2.0 EU / L endotoxin standard solution was prepared with injection water using a Japanese Pharmacopoeia standard endotoxin standard (manufactured by Seikagaku Corporation). The preparation method followed a general endotoxin standard solution preparation method.
(Color development evaluation test of freeze-dried reagent and buffer)
An endotoxin test was performed using a reagent obtained by re-dissolving and mixing the lysate reagent prepared by a normal preparation method, the lyophilized lysate reagent and the β-glucan inhibition buffer solution with water for injection. .
First, the lysate reagent (powder) of Endospecy ES-24S was dissolved in 0.5 mL of β-glucan inhibition buffer, and then 0.2 mL was dispensed into a 96-well microplate (hereinafter referred to as standard lysate reagent). .
Next, 0.2 mL of the buffer obtained by dissolving the lyophilized β-glucan inhibition buffer in 0.3 mL of injection water was added to the well containing the lyophilized lysate reagent and dissolved ( Hereinafter referred to as re-prepared lysate reagent).
To the standard lysate reagent and the reconstituted lysate reagent, 0.2 mL of 2.0 EU / L of standard endotoxin was dispensed and stirred, and the absorbance was measured at a wavelength of 405 nm.

[Evaluation]
(Evaluation of titer of re-prepared lysate reagent)
In order to evaluate the titer of the standard lysate reagent and the re-prepared lysate reagent, a color development evaluation test was performed by the method described in Example 1, and an equivalent color development was obtained.
(Evaluation of resealability of lid material)
After lyophilizing the reagent and buffer, the 96-well microplate was completely sealed with a lid using a seal tester and allowed to cool until the lid returned to room temperature. Then, when the said cover material was partially peeled off, the seal | sticker residue was confirmed only on the circular protrusion part around a well.
Next, when the lid member was returned to the initial position, re-adhesion between the circular protrusion and the lid member was confirmed.

DESCRIPTION OF SYMBOLS 1 ... Microplate with a reagent 2 ... Microplate 3 ... Well 4 ... Reagent 5, 6, 7 ... Cover material 10 ... Sample 11 ... Base material 12 ... Sealant layer 13 ... Adhesive layer 14 ... Heat seal part 15 ... Cutting line 21 ... Base material 22 ... Adhesive layer 23 ... Fluorine resin film

Claims (6)

A microplate having a plurality of wells;
A reagent contained in the well and lyophilized;
A reagent-containing microplate, comprising: a lid disposed on the microplate and sealing the opening of the well.   The reagent-containing microplate according to claim 1, wherein the lid member has a base material and a sealant layer formed on the base material.   The reagent-containing microplate according to claim 2, wherein the lid member has an adhesive layer formed between the base material and the sealant layer. Injecting a reagent solution in which a reagent is dissolved or dispersed in a solvent into the well of a microplate having a plurality of wells;
A lyophilization step of laying a lid on the microplate so as to allow ventilation, and lyophilizing the reagent solution;
And a sealing step of sealing the opening of the well with the lid.   The method for producing a microplate containing a reagent according to claim 4, wherein the lid member has a base material and a sealant layer formed on the base material.   The method for producing a reagent-containing microplate according to claim 5, wherein the lid member has an adhesive layer formed between the base material and the sealant layer.