JP2007190512A - Method for manufacturing container - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing a container having a well-shaped reaction part and/or a well-shaped reagent housing part and a protective film or a film-shaped cover material heat-sealed in the upper parts of the well-shaped reaction part and/or the well-shaped reagent housing part, the method by which the protective film or the film-shaped cover material can be heat-sealed without damaging a substrate and the content in a well and a reagent can be recovered at an excellent rate. <P>SOLUTION: The method for manufacturing the container is characterized in that the peripheries of the well-shaped reaction part and/or the well-shaped reagent housing part are raised, the protective film or the film-shaped cover material is formed in a roll shape and is supplied so as to cover the well-shaped reaction part and/or the well-shaped reagent housing part at the least, and then a heating member is brought into contact with or close to the the protective film or the film-shaped cover material from above. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a container for containing a reagent, a method for producing a reaction container used for detection of antigen by antigen-antibody reaction, detection of DNA, and the like.

In recent years, μ-Total Analysis System technology and Lab-on-Chip technology that perform biochemical reactions such as chemical reactions, DNA reactions, and protein reactions on a chip have been studied and realized, and until now, large-scale experimental devices and large quantities have been developed. It is now possible to carry out reaction experiments that require these reagents with a small amount of reagent on a chip of several millimeters or less.

Examples of biochemical reactions include DNA amplification reaction by enzymatic reaction, a method of detecting a sample DNA sequence by a hybridization method using a probe DNA having a known sequence, and SNP (single nucleotide sequence) in DNA sequencing. Type) detection method.
As a method for detecting SNP, a method using an invader method or a Tuckman PCR method in a typing process is known (see Patent Document 1).

In general, a detection reaction using DNA is performed by collecting and extracting blood or the like. However, in order to use a small amount of sample such as collected blood, a DNA amplification reaction based on an enzymatic reaction is used as a method for preparing a sample DNA. There are many cases.
Various methods are known for increasing a trace amount of DNA contained in a sample, and a PCR amplification reaction is known as a typical method. This method consists of three steps consisting of a denaturation step of double-stranded DNA in a sample (dissociation into single strands), an annealing step (binding single-stranded DNA and a primer), and an extension step (synthesize DNA from the primer). Is one cycle, and this cycle is repeated to increase the DNA in the sample. The denaturing step is performed at about 95 ° C., the annealing step is performed at 50 to 60 ° C., and the extension step is performed at 60 to 80 ° C. The PCR amplification reaction is performed by repeating this thermal cycle. The time required for one cycle is about several minutes at most, and this cycle is repeated to obtain a necessary amount of DNA.
In addition, before PCR reaction, it may heat for several minutes-5 to 6 minutes at 95 degreeC as pre-processing.

  The invader method, which is one of the methods for detecting SNPs, consists of two types of non-fluorescently labeled oligonucleotides (allele probe, invader probe), one type of fluorescently labeled oligonucleotide (FRET probe), and an endonuclease specific to the DNA structure ( Chestnut base). The allele probe is an oligonucleotide having a sequence (flap) unrelated to the sequence of the template DNA on the 5 ′ side and a complementary sequence specific to the template DNA on the 3 ′ side, and the 5 ′ end of the complementary sequence. Is a SNP site. On the other hand, the invader probe is designed to complementarily bind to the 3 'side of the template DNA from the SNP site. The FRET probe is an oligonucleotide having a fluorescent label, and has a fluorescent label (reporter) at its 5 'end, and a quencher is bound upstream thereof. The 3 'site from this reporter is self-hybridized to form a double strand. From this double strand to the 3' end, a single strand that is complementary to the allele probe flap. It has a part of. Cribase is an enzyme that recognizes a triplet overlapping site and cleaves and releases the 3 'side of the triplet nucleotide.

In this invader method, when the template DNA to be examined and the allele probe are first hybridized, the 3 ′ end of the invader probe enters the SNP site. For this reason, at this SNP site, the template DNA, the allele probe and the invader probe are overlapped to form a triple. Crybase recognizes the structure of this SNP site and cleaves / releases the flap of the allele probe. The free flap originating from the allelic probe is then hybridized with the FRET probe. This hybridization causes a triple at the intersection of the self-hybridization duplex and the free flap originating from the allele probe, and the chestnut base again recognizes this structure, cleaves the reporter of the FRET probe, and is released from the quencher. . Then, by irradiating with excitation light, the fluorescent label of the reporter released by cleavage emits fluorescence. If the base of the SNP site does not match the allele probe, the flap originating from the allele probe will not be cleaved / released, and therefore the fluorescence emission rate is extremely low. Therefore, by detecting this difference in fluorescence intensity, Can be inspected. In general, ultraviolet light or visible light is used as the excitation light.
These reactions are carried out by incubating at about 63 ° C. for about several tens of minutes to about 4 hours.

When these reactions are performed using a chip, or when the sequence of DNA is determined, a method is known in which probe DNA is immobilized on a slide glass and a hybridization reaction is performed thereon.
It is also known that a minute hole or depression called a well provided on a chip is formed and used as a reaction field. The well has been formed by etching a semiconductor or glass or laminating a plate with holes.
In the case of using wells, it is not necessary to fix the reagent on the substrate, and it can be applied to a PCR reaction or the like.

As a well-type substrate, for example, a detection substrate having a large number of wells provided on the substrate surface is disclosed (see Patent Documents 2, 3, and 4).
An apparatus for PCR reaction is also known which has a flow path inside and has openings at both ends (see Patent Document 5).

  It is known that these chips are covered with a protective film or a film-like lid material in order to prevent the inside of the well from being contaminated with dust or other contaminants. Then, the reagent is peeled off during use, or the reagent is taken out by piercing from above the lid material using a needle-like dispenser such as an injection needle.

Such a film material may be bonded with an adhesive, but it is preferably formed by heat sealing from the viewpoint of influence of the adhesive on the reagent and reaction system.
However, when heat-sealing, it is usually heat-sealed by placing a film on the substrate and contacting and pressurizing the heating member. However, when the contact area is large, it is difficult to heat-press uniformly and heat-seal uniformly. It is difficult to stabilize the quality.

In addition, the amount of heat supplied during heat sealing is large, which may adversely affect the contents in the well. The substrate itself may be deformed such as warpage.
In order to avoid such a problem, it is conceivable to perform overheating and pressurization so as to avoid the well. In that case, it is necessary to improve the shape of the heating member and to add an alignment function when overheating. However, since the contact area is still large, there is a concern about the influence on the contents.

  Further, when a film is placed on a conventional substrate and the heating member is contact-pressed, the sealed film is somewhat bent. If the film is only used as a protective lid, this is not a problem. However, when the reagent contained in the well is pierced with something like an injection needle and collected, the piercing property is poor and the reagent is collected. Becomes difficult.

Japanese Patent Laid-Open No. 2002-300894 WO2003 / 031972 JP 09-99932 A JP 2003-70456 A Japanese Patent No. 2759071

The present invention has been made in view of such circumstances, and includes a well-like reaction part and / or a well-like reagent storage part, and is provided above the well-like reaction part and / or the well-like reagent storage part. In a container manufacturing method in which a protective film or a film-like lid material is provided by heat sealing, a method for manufacturing a container in which a film can be bonded by heat sealing without damaging the contents in the substrate and well is provided. The purpose is to do.
Another object of the present invention is to provide a production method for making a container having excellent recoverability when a recovery needle is pierced from such a film to recover a reagent.

The invention of claim 1 comprises a substrate provided with a well-like reaction part and / or a well-like reagent storage part, and a protective film or film-like lid material on the well-like reaction part and / or well-like reagent storage part. Is a method of manufacturing a container provided by heat sealing,
Around the well-like reaction part and / or well-like reagent storage part is raised,
In addition, the protective film or the film-like lid material is supplied in a roll shape so as to cover at least the well-like reaction part and / or the well-like reagent storage part, and then the heating member is brought into contact with or in close proximity from the upper part of the protective film or the film-like lid material It is a manufacturing method of the container characterized by making it carry out.

  The invention according to claim 2 is the method for producing a container according to claim 1, wherein the periphery of the well-like reaction part and / or the well-like reagent storage part is raised by 1.0 mm or more.

  The invention according to claim 3 is characterized in that the width of the raised portion around the well-like reaction part and / or well-like reagent storage part is in the range of 0.1 mm to 3 mm. It is a manufacturing method of the container as described in above.

  The invention according to claim 4 is the method for producing a container according to any one of claims 1 to 3, wherein a plurality of the well-like reaction parts and / or well-like reagent storage parts are provided.

  The invention according to claim 5 is characterized in that the tension of the protective film or film-like lid material when the heating member is brought into contact with or close to the upper part of the protective film or film-like lid material is in the range of 100 g to 1000 g. It is a manufacturing method of the container in any one of Claims 1-4.

  Invention of Claim 6 supplies the said roll-shaped protective film or film-like cover material so that it may orthogonally cross with respect to a board | substrate, The manufacturing method of the container in any one of Claims 1-5 characterized by the above-mentioned. It is.

  The invention according to claim 7 is the method for producing a container according to any one of claims 1 to 6, further comprising a second reaction part.

  According to the present invention, a film can be bonded uniformly without damaging a substrate and contents. In addition, the bonded film has less deflection and can prevent the volatilization and evaporation of the liquid with higher accuracy. Further, when the reagent is injected and collected using the needle, the needle piercing property is improved.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a diagram showing an embodiment of the present invention. In FIG. 1, a plurality of wells for reacting a sample and a reagent or storing a reagent are formed on a substantially rectangular plate-shaped substrate.

The substrate used in the present invention may be any substrate that does not adversely affect the reagent. Further, when the reaction is performed in the well, it is necessary that the reaction system is not adversely affected. Furthermore, when detecting the reaction in the well, when optical detection is performed from below the substrate, it is preferable that the transparency is high.
As such, for example, PC (polycarbonate), PP (polypropylene), cycloolefin-based polymer, methylpentene-based resin, fluoropolymer, silicone resin, and the like can be used.
PP is preferably used from the viewpoints of transparency, heat resistance, chemical resistance and influence on the reaction system.

It is preferable to form a substrate using such a synthetic resin because it is excellent in heat resistance, chemical resistance, moldability, and the like. Further, two or more kinds of resins may be joined and used. In this case, by creating a substrate by taking advantage of the characteristics of each resin, it becomes possible to make various substrates according to the characteristics of the reagent, the sample, and the like, which can be used for each application. For example, it is possible to divide the material between the upper half and the lower half of the substrate. In addition, the material can be divided into parts such as a reagent storage section and a PCR reaction section described later.
In addition, you may use glass as a raw material of a board | substrate.

  The substrate is provided with a well-like reaction part or reagent storage part.

The well-like reagent container can be formed by cutting or molding if the substrate is plastic or synthetic resin. If it is glass, it can be formed by cutting. In addition, a plurality of reagent storage units can be provided. When a plurality of reagent storage units are provided, the sizes may be different. The number of reagent storage units can be appropriately set according to the purpose.
In addition, the reagent container can be used as a mixing field in which one reagent is put in advance and another reagent is put and mixed later.

The reagent container may be concave (well shape). The shape is not particularly limited, but is preferably hemispherical or cylindrical with a hemispherical bottom. If hemispherical, when the reagent is filled, the reagent can be prevented from scattering and bubbles from being mixed. Moreover, it becomes excellent in the taking-out property at the time of taking out the accommodated reagent.
In addition, the opening may be circular or polygonal, and the cross section may be triangular, quadrangular, or trapezoidal.

  The volume of the reagent storage unit is preferably in the range of 10 to 300 μl. In particular, in biochemical reactions dealing with DNA, the reaction amount is very small and the reagents used are often expensive. In addition, DNA collected from blood is usually about 0.1 ng to 50 ng, and a reagent containing DNA is about several hundred nl to several μl, several hundred nl to several μl. Therefore, the number of reagents, diluents, etc. used in the reaction is at most several hundred μl, and is preferably within the above-mentioned range. In addition, when the reagent to be used is several hundred μl or more, it may be stored in two.

It is preferable that the opening diameter of the reagent container is in the range of 1 to 50 mm.
The amount of the reagent is from a very small amount of about several hundred nl to about several hundred μl, and the diameter of a general dispensing needle is about several tens of μm to several mm. Then, it is preferable that the opening diameter of a reagent accommodating part exists in the range of 1-50 mm.

  The depth is preferably in the range of 1 to 50 mm.

  In addition, the reagent container may be formed by hollowing out the substrate as shown in FIG. 11B, or the back surface of the substrate is directed downward along the shape of the reaction portion as shown in FIG. 11A. It may be convex.

Further, a film-like lid material is provided on the reagent storage unit. By providing the cover material, it is possible to prevent contamination by dust, contaminants, and the like.
Examples of the film-like cover material include polyolefin films such as polyethylene, polypropylene and polymethylpentene, acrylic films such as polymethyl acrylate and polymethyl methacrylate, polystyrene films, polyacetal films, polyamide films, polyacrylonitrile films, polycarbonate films, Examples include olefin-based films, silicon resin-based films, and fluorine-based resin films.
Moreover, you may use metal foil, such as aluminum, and the laminated film of metal foil and the above-mentioned resin film.

These film-like cover materials can be bonded together using an adhesive. As the adhesive, a heat-resistant curable adhesive can be used.
Further, it may be bonded by heat sealing. A heat seal is preferable because it is not necessary to consider the influence of the adhesive on the reagent.

  In the case where a film-like lid member is provided, the reagent may be collected by piercing with a needle-like collecting tool such as a syringe from above the lid member.

  Note that when the reagent is recovered without removing the lid, the lid does not need to be peeled off.

Protrusions (convex portions) are provided around the opening of the reagent storage unit. By providing the convex portion, the lid material can be easily bonded. As a convex part, a thing as shown in FIG. 2 is mentioned, for example.
In the case of pasting together by heat sealing, the portion to be heated is not the entire base material, but only the raised portion (convex portion), so that the thermal influence on the reagent in the reagent storage portion can be reduced.

Such a bulge (convex portion) preferably has a height of 1 mm or more. When the thickness is 1 mm or less, it becomes difficult to perform heat sealing uniformly, and the influence (damage) on the base material and contents is increased.
Further, the ridge (convex portion) width is preferably in the range of 0.1 to 2 mm, preferably 0.3 to 0.7 mm. If it is smaller than this range, the bulges (convex parts) may be deformed at the time of sealing and the reliability of the seal may be lowered. If this range is larger and larger, pressure will be dispersed and uniform sealing will not be possible. The problem (such as damage) becomes large.
Moreover, you may increase the capacity | capacitance of an accommodating part by providing a protrusion (convex part). In that case, in consideration of the strength of the housing portion and heat sealability, the bulge (convex portion) may be formed in two stages as shown in FIGS. 2B and 2C, for example. In other words, the strength and heat seal suitability are achieved by providing a two-stage structure in which a bulge (convex portion) having a certain thickness is provided to increase the strength, and a ridge (convex portion) having a small width is provided thereon. May be)

  The heat sealing conditions are preferably a temperature of 140 ° C. to 220 ° C., a pressure of 1 kg to 3 kg, and a time of 0.3 seconds to 2.0 seconds, and pressure bonding. If the temperature, pressure, and time are more than this, the substrate tends to be deformed. Further, if the temperature, pressure and time are less than this, sufficient adhesive strength cannot be obtained and the film is easily peeled off. Moreover, when raising temperature, it is good to shorten time in consideration of the thermal deterioration of the contents.

  Moreover, you may perform a surface treatment in a reagent accommodating part. When the well-shaped reagent container is filled with a solution such as a reagent or specimen, the solution can be injected without mixing bubbles if a surface treatment is performed in the well-shaped reagent container. Also, a high recovery rate can be expected when recovering a solution from a well-like reagent container.

In addition, as a surface treatment, when the solution is aqueous, it is preferable to perform a hydrophilic treatment. Specifically, the contact angle with pure water is 70 ° or less, preferably 40 ° or less.
The contact angle is measured using a known contact angle meter. In addition, since it is difficult to measure the contact angle in the well, the measurement may be performed using the surface of the substrate in the same surface state.

As the surface treatment method, ultraviolet irradiation treatment, plasma treatment, corona treatment or the like can be used.
The processing may be performed in the atmosphere, but a processing gas may be flowed between the substrate and the upper electrode. Examples of the processing gas include inert gases such as nitrogen, argon, and helium.
Among these, plasma treatment using argon gas is preferable.

The well-like reaction part can be formed by cutting or molding if the substrate is plastic or synthetic resin. If it is glass, it can be formed by cutting. In addition, a plurality of reaction units can be provided and can be appropriately set according to the purpose.
The opening diameter of the well-like reaction part is preferably in the range of 0.1 to 5 mm and the depth is preferably in the range of 0.1 to 5 mm. As described above, in the life science field, strict temperature control is often performed using a trace amount of reagent, and it is preferably within the above range in order to perform the reaction efficiently.
Moreover, the reagent required for reaction may be accommodated in the reaction part in advance.

The shape of the well-like reaction part is not particularly limited as long as it is a concave shape, but it is preferably a truncated cone shape having a flat bottom and an inclined wall surface from the well opening to the bottom. A frustoconical shape having a flat bottom and an inclined wall surface from the well opening to the bottom is advantageous for optical detection from below. For example, when a fluorescent material is irradiated into the reaction part from below and the fluorescence is detected from below, if the fluorescent material is spherical or other complicated shape, the ultraviolet light that is the excitation source of the fluorescent material is refracted and scattered to the fluorescent material. The amount of irradiation will decrease. In addition, the generated fluorescence is also refracted and scattered, resulting in a decrease in the detected fluorescence intensity and false detection.
In addition, the opening may be circular or polygonal, and the cross section may be hemispherical, U-shaped, triangular, or quadrangular. The opening may be polygonal and the cross section may be trapezoidal.

  In addition, the reaction part may be formed by hollowing out the substrate as shown in FIG. 11B, or the back surface of the substrate is directed downward along the shape of the reaction part as shown in FIG. 11A. It may be convex.

Moreover, a protective film is provided on the reaction part. By providing the protective film, it is possible to prevent contamination by dust, contaminants, and the like.
A film-like thing can be used as a protective film. Examples of such films include polyolefin films such as polyethylene, polypropylene, and polymethylpentene, acrylic films such as polymethyl acrylate and polymethyl methacrylate, polystyrene films, polyacetal films, polyamide films, polyacrylonitrile films, polycarbonate films, cyclohexane films, and the like. Examples include olefin-based films, silicon resin-based films, and fluorine-based resin films.
Moreover, you may use metal foil, such as aluminum, and the laminated film of metal foil and the above-mentioned resin film.

  These protective films are bonded together by heat sealing. When the reagent is stored in the reaction part in advance, it is preferable to use a heat seal because it is not necessary to consider the influence of the adhesive on the reagent.

  Since it is necessary to peel off the protective film before use, it is preferable that the protective film is easily peelable.

Further, a convex portion is provided around the opening. By providing the convex portion, the lid material can be easily bonded. As a convex part, a thing as shown in FIG. 2 is mentioned, for example. Especially when pasting together by heat sealing, the heating part only needs to be at the convex part, not the whole base material, so when the reagent is stored in the reaction part in advance, the thermal influence on the reagent is reduced. be able to.
Also, when peeling, the contact is not on the whole substrate excluding the opening but only on the convex part, so that peeling can be easily performed.

Such a bulge (convex portion) preferably has a height of 1 mm or more. When the thickness is 1 mm or less, it becomes difficult to perform heat sealing uniformly, and the influence (damage) on the base material and contents is increased.
Further, the ridge (convex portion) width is preferably in the range of 0.1 to 2 mm, preferably 0.3 to 0.7 mm. If it is smaller than this range, the bulges (convex parts) may be deformed at the time of sealing and the reliability of the seal may be lowered. If this range is larger and larger, pressure will be dispersed and uniform sealing will not be possible. The problem (such as damage) becomes large.
Moreover, you may increase the capacity | capacitance of a reaction part by providing a protrusion (convex part). In that case, in consideration of the strength of the reaction part and heat sealability, the bulge (convex part) may be formed in two stages as shown in FIGS. 2B and 2C, for example. In other words, the strength and heat seal suitability are achieved by providing a two-stage structure in which a bulge (convex portion) having a certain thickness is provided to increase the strength, and a ridge (convex portion) having a small width is provided thereon. May be.
Moreover, you may connect convex parts with the same height as a convex part. By doing so, when peeling, there is no catch and it can peel smoothly.

  The heat sealing conditions are preferably a temperature of 140 ° C. to 220 ° C., a pressure of 1 kg to 3 kg, and a time of 0.3 seconds to 2.0, with pressure being applied. If the temperature, pressure, and time are more than this, the substrate tends to be deformed. Further, if the temperature, pressure and time are less than this, it is difficult to bond. Moreover, when raising temperature, it is good to shorten time in consideration of the thermal deterioration of the contents.

  Moreover, you may surface-treat in a reaction part. When a well-like reaction part is filled with a solution such as a reagent or a specimen, the solution can be injected without mixing bubbles if surface treatment is performed in the well-like reaction part. Furthermore, when the reaction is carried out by heating in the well, the liquid enrollment position becomes stable and is difficult to evaporate.

In addition, as a surface treatment, when the solution is aqueous, it is preferable to perform a hydrophilic treatment. Specifically, the contact angle with water is 70 ° or less, preferably 40 ° or less.
The contact angle is measured using a known contact angle meter. In addition, since it is difficult to measure the contact angle in the well, the measurement may be performed using the surface of the substrate in the same surface state.

As the surface treatment method, ultraviolet irradiation treatment, plasma treatment, corona treatment or the like can be used.
The processing may be performed in the atmosphere, but a processing gas may be flowed between the substrate and the upper electrode. Examples of the processing gas include inert gases such as nitrogen, argon, and helium.
Among these, plasma treatment using argon gas is preferable.

In the present invention, a protective film or a film-like lid material can be supplied in a roll shape and heat-sealed to a substrate that is continuously conveyed. Specifically, for example, as shown in FIG. 4, a roll-shaped protective film or a film-like lid material is supplied to the substrate to be transported so as to be orthogonal, and a heating member is brought close to or in contact with the heat-sealed portion. And heat sealing can be performed. In addition, the direction of the base material to convey is not specifically limited, In FIG. 4, although the longitudinal direction of a base material and a roll-shaped protective film or a film-like cover material are orthogonal, the longitudinal direction of a base material You may make a base material convey horizontally so that a direction and a roll-shaped protective film or a film-like cover material may become parallel.
Moreover, when heat-sealing, it is preferable to contact and pressurize a heating member.
The conditions for heat sealing are as described above.

Moreover, it is preferable that the tension | tensile_strength is in the range of 100g-1000g, when the roll-shaped protective film or film-shaped cover material to supply is heat-sealed. If it is smaller than 100 g, the film will bend, and when the reagent is injected and collected by piercing a needle or the like, the piercing property is not excellent. On the other hand, if the size is larger than 1000 g, a gap may be formed by the force of the stretched film to return after heat sealing, so it is not preferable to store a liquid reagent inside. If it is in the said range, a heat seal can be performed to a protruding part (convex part) uniformly in a short time.
Moreover, after bonding, there is little deflection and it is excellent in the puncture property at the time of collect | recovering and inject | pouring a reagent using a needle-shaped instrument like an injection needle.

For example, as shown in FIG. 6, the container of the present invention may be provided with a well-like reagent storage part and a well-like reaction part on the same substrate.
Reagents necessary for the reaction can be put in the well-like reagent container, and the reagent can be dispensed into the well-like reagent container and reacted in the well-like reaction part. In this case, a part of the reagent may be arranged in advance in the well-like reaction part.

  A plurality of well-shaped reagent containers can be provided depending on the type of reagent used. For example, the reagent container has a solution containing a detection substance and a plurality of reagents for detection, and when performing a multi-step reaction, a solution containing one of the reagents for detection, or other buffers, diluents, etc. Can be put in.

  In the reagent storage unit, a plurality of reagents may be mixed. After mixing the reagent in the reagent storage unit, the reaction can be performed in a reaction unit provided separately.

  A plurality of well-like reaction units may be received. By receiving a plurality, a plurality of reactions can be performed simultaneously.

  Moreover, when performing 2 or more types of reaction, you may provide a 2nd reaction part.

For example, when the reaction is used for a DNA detection reaction, the second reaction part can be a PCR reaction part.
By providing a PCR reaction section, sample preparation and DNA detection can be performed on the same chip.
As the PCR reaction part, a well-like reaction part may be provided, or, for example, as shown in FIG. In the case of providing a flow path, for example, a structure having openings at both ends and a flow path inside is given as an example. The opening diameter is about 5 cm or less, and the opening depth is about 1 cm to 5 cm. When the surface treatment is performed on the opening, the treatment method of the present invention is effective similarly to the above-described treatment in the well.

When the second reaction part has a flow path shape, specifically, for example, a shape as shown in FIG. 9 may be used.
The flow path-like second reaction part is provided with through holes penetrating the base material at both ends, and provided with a groove part connecting both through holes on the back surface of the base material. A channel-like reaction part is formed by bonding a bottom forming film on the groove part.
At this time, it is preferable that the width and height of the groove are within a range of 1 mm to 5 mm, respectively.

  The groove portion may be formed by connecting both through holes with a straight line, or may have a bent shape in order to prevent evaporation of a reagent or a test object.

A film-like film can be used as the bottom forming film. Examples of such films include polyolefin films such as polyethylene, polypropylene, and polymethylpentene, acrylic films such as polymethyl acrylate and polymethyl methacrylate, polystyrene films, polyacetal films, polyamide films, polyacrylonitrile films, polycarbonate films, cyclohexane films, and the like. Examples include olefin-based films, silicon resin-based films, and fluorine-based resin films.
Moreover, you may use metal foil, such as aluminum, and the laminated film of metal foil and the above-mentioned resin film.

The bottom forming film can be bonded using an adhesive. Moreover, you may bond together by heat sealing. A heat seal is preferable because it is not necessary to consider the influence of the adhesive in the reaction part.
Moreover, if the film for bottom part formation is a shape which bites into a part groove part, it is preferable. This is because there is no gap between the base material and the bottom forming film, and there is no leakage of reagents and test objects.

  For example, as shown in FIG. 9, the through-hole opening may have a shape protruding upward from the base material in order to increase the capacity.

  Moreover, you may provide a cover material in an opening part.

  Moreover, you may provide another reaction part.

  Moreover, you may provide the flow path which connects well-like reaction parts (refer FIG. 8). Further, a flow path connecting the well-like reaction part and the reagent containing hole part, the PCR reaction part, and other reaction parts may be provided. By forming these flow paths, it is possible to perform a continuous reaction.

  Further, side ribs may be provided on the substrate in order to reduce deformation and the like. As the rib, a member having a width of about 0.1 to 3 mm and a height of about 0.1 to 3 mm may be provided at the lower part and / or the upper part of the end part of the substrate. Moreover, you may provide the leg part for support so that it may be stabilized when putting.

In the present invention, it can be used for various biochemical reactions, for example, for detection of antigen-antibody reaction and DNA reaction.
In the case of antigen detection by antigen-antibody reaction, for example, a sample containing the antigen is previously placed in each well-like reaction part, a reagent containing the antibody is added later as a specimen, and a labeling substance is attached to either the antigen or the antibody. The presence or absence of reaction can be detected. As the labeling substance, a luminescent substance such as fluorescence is generally used. In this case, a reagent storage unit may be provided on the substrate to store the specimen.

  In the case of detecting DNA, for example, a nucleic acid probe is prepared in advance in a well-like reaction part. Thereafter, the sample DNA is supplied to the well-like reaction section, and the DNA can be detected by a hybridization reaction between the nucleic acid probe and the sample DNA. At this time, if a labeling substance is attached to the sample DNA, detection can be performed by detecting the presence or absence of the labeling substance. The sample DNA can be prepared by adjusting DNA extracted from blood or the like by the PCR method, the LAMP method, or the like. In addition, by preparing a plurality of nucleic acids having different sequences as nucleic acid probes, it is possible to detect the sequence of the sample DNA as the detection substance. In this case, a reagent storage unit may be provided on the substrate to store the detection substance.

In addition, a gene amplification reaction part is provided on the substrate, DNA extracted from blood or the like is continuously amplified on the chip by a gene amplification reaction, which is used as a sample, and the presence or absence of reaction with the nucleic acid probe in the reaction part May be detected. Specifically, for example, DNA extracted from blood or the like is stored as a sample in a well-shaped reagent storage unit, dispensed to the gene amplification reaction unit by a dispensing operation, and the sample prepared by the gene amplification reaction is in a well state To the reaction part. The fluid may be sent from the well-like reagent storage part to the gene amplification reaction part and the well-like reaction part using a flow path.
In addition, a gene here means DNA, RNA, etc. Examples of the gene amplification reaction method include the PCR method and the LAMP method.

  It can also be used to analyze single nucleotide gene polymorphisms (SNPs). Note that there may be a plurality of reagents for detecting the specimen, and if the specimen is not fluorescently labeled, one of the reagents only needs to be labeled.

  In addition, a labeling substance that acts specifically on the reaction product can be added after the reaction. Such an example includes an intercalator for detecting DNA. Further, the labeling substance here includes indirect substances. That is, a substance that binds to a fluorescent substance or the like may be bound to a reagent for detecting a specimen such as a specimen or a probe nucleic acid as a labeling substance, and the fluorescent substance may be added later.

Alternatively, SNP or DNA may be detected by performing a multistep reaction.
For example, an invader assay method (Third Wave Technologies, Inc. (Madison, Wisconsin, USA)) may be used, thereby enabling realization of SNP analysis.

  In this case, a plurality of types of reagents for detecting the sample may be used, and at least one type of reagent may be previously placed in the well-shaped reaction part, and then the sample and the reagent may be injected simultaneously or sequentially to perform the reaction. .

In addition, a solution having a lighter specific gravity than the reaction solution such as mineral oil may be added to the well-like reaction unit and the PCR reaction unit in order to prevent the reaction solution from drying.
In addition, the reagent for detecting the specimen may be fixed in the well-like reaction part or may be held without being fixed.

  The well-like reaction part, reagent storage part, and PCR reaction part may be covered with a lid material such as a film.

<Example 1>
The well-shaped detection chip shown in FIG. 10 is produced by molding. As the resin used for molding, a molded product having dimensions of 2.5 cm in length and 12 cm in width was prepared using polypropylene (manufactured by Prime Polymer Co., Ltd.).

The reagent storage portion was 13 mm in diameter and 6 mm in depth and the tip was hemispherical, and the reaction portion was 3 mm in diameter and 2 mm in depth, the side surface was inclined and the bottom surface was flat, and the shape was trapezoidal in cross section.
Around the opening of the reagent container, a raised portion having a two-stage structure including a convex portion having a width of 0.5 mm and a height of 0.3 mm was provided on a convex portion having a width of 1 mm and a height of 2 mm.
Around the opening of the reaction part, a raised part having a two-stage structure including a convex part having a width of 0.5 mm and a height of 0.3 mm was provided on a convex part having a width of 1 mm and a height of 2 mm.
The PCR reaction part, which is the second reaction part, has an opening diameter of 6 mm, a depth of 4 mm, and a tip that is hemispherical. The shape protruded.
Four reagent storage units and 20 reaction units were provided.

  The reagent container, the PCR reaction part, and the reaction part were provided in this order from the end of the substrate.

Next, a sealant film made of polyethylene terephthalate / aluminum / polypropylene was supplied in a roll shape so as to be orthogonal to the longitudinal direction of the base material as a film-like lid material for the reagent container. At this time, the tension of the film-like lid material was 200 g.
Then, an aluminum block having a size of 30 mm × 60 mm to which a heat source was connected from the upper part of the reagent container was contact-pressed so as to cover the reagent container, and heat sealing was performed.
The heat sealing conditions at this time were performed under a load of 3 kg and 190 ° C. for 0.5 seconds.

Next, a sealant film made of polyethylene terephthalate / polypropylene was supplied as a protective film for the reaction part in a roll shape so as to be orthogonal to the longitudinal direction of the substrate. At this time, the tension of the protective film was 200 g.
Then, an aluminum block having a size of 30 mm × 60 mm to which a heat source was connected from the upper part of the reaction part was contact-pressed so as to cover the reagent storage part, and heat sealing was performed.
The heat sealing conditions at this time were performed under a load of 3 kg and 220 ° C. for 0.5 seconds.

<Example 2>
Implemented except that the periphery of the opening of the reagent container is a raised portion having a two-stage structure consisting of a convex portion having a width of 0.5 mm and a height of 0.3 mm on a convex portion having a width of 1 mm and a height of 1.5 mm. Performed as in Example 1.

<Example 3>
The periphery of the opening of the reagent container is a two-tiered raised part consisting of a convex part having a width of 0.5 mm and a height of 0.3 mm on a convex part having a width of 1 mm and a height of 1.5 mm. The same operation as in Example 1 was performed except that the tension for supplying the upper film-like lid material was 500 g.

<Example 4>
The periphery of the opening of the reagent container is a two-tiered raised part consisting of a convex part having a width of 0.5 mm and a height of 0.3 mm on a convex part having a width of 1 mm and a height of 1.5 mm. The same operation as in Example 1 was performed except that the tension at the time of supplying the upper film-like lid material was 1300 g.

<Comparative Example 1>
Cut out the film-like lid material of the reagent container (necessary to cover the reagent container), place it on the reagent container, and cover the reagent container with a 30 mm x 60 mm aluminum block connected to a heat source In this way, heat sealing is performed by contact pressure, and a protective film for the reaction part is cut off as much as necessary (to the extent that the reaction part can be covered), placed on the reagent storage part, and a size to which a heat source is connected 30 mm × 30 mm This was performed in the same manner as in Example 1 except that heat sealing was performed by contacting and pressurizing the aluminum block so as to cover the reagent container.

<Comparative example 2>
It was created in the same manner as in Example 1 except that the raised portions were not provided around the openings of the reagent storage unit and the reaction unit, and the heat seal conditions were as follows.
The film-like lid material of the reagent container was heat-sealed in the same manner as in Example 1 except that the conditions were a load of 3 kg and 190 ° C. for 2 seconds.
The protective film of the reaction part was heat-sealed in the same manner as in Example 1 except that the conditions were a load of 3 kg and 220 ° C. for 5 seconds.

<Evaluation>
(Wrinkle)
The wrinkles of the film-like lid material on the reagent storage parts of Examples and Comparative Examples were visually observed. The results are shown in Table 1.
(Weight reduction)
Prior to heat sealing, pure water was put into the reagent container, the weight was measured immediately after the heat sealing, and the weight was measured after being allowed to stand at 60 ° C. for 24 hours for comparison. The results are shown in Table 1.
(Puncture)
A dispensing tip (dispensing needle) of a disposable tip (ART 20P, manufactured by Molecular Bio Products) was stabbed in a vertical direction with a load of 1 kgf, and the tearing was visually confirmed to perform the following evaluation.
○: Good △: Tip tip slides ×: Cannot be pierced

Since the samples of Examples 1 to 3 had no wrinkle on the film and no weight reduction, the adhesion was good. And the piercing property was also good. The sample of Example 4 had no wrinkles and excellent piercing characteristics, but a slight weight reduction was observed. This seems to be because the tension at the time of heat sealing was too high, and after the heat sealing, a slight gap was generated due to the shrinkage force of the film.
In the sample of Comparative Example 1, the film was bent and wrinkles were generated. Although there was no weight loss, the piercing property was not so good. In the sample of Comparative Example 2, the film was bent and wrinkles were generated. There was also a decrease in weight, and the piercing property was not so good.

It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the heat seal method of this invention. It is the schematic which shows an example of the heat seal method of this invention. It is the schematic which shows an example of the conventional heat seal method. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention.

Explanation of symbols

1 Container 2 Substrate 3 Well 4 Heater (also trimmer)
5 Film (Protective film or film-like lid material)
5b Film (part removed by heat sealing)
6 Well-like reagent storage part 7 Well-like reaction part 8 Second reaction part (PCR reaction part)
8 (a) Through-hole opening protrusion 8 (b) Lower film 9 Channel 10 Plasma generating part of remote type plasma processing apparatus

Claims (7)

The substrate is provided with a well-like reaction part and / or a well-like reagent storage part, and a protective film or a film-like lid material is provided by heat sealing on the well-like reaction part and / or well-like reagent storage part. A method of manufacturing a container,
Around the well-like reaction part and / or well-like reagent storage part is raised,
In addition, the protective film or the film-like lid material is supplied in a roll shape so as to cover at least the well-like reaction part and / or the well-like reagent storage part, and then the heating member is brought into contact with or in close proximity from the upper part of the protective film or the film-like lid material A method for producing a container, characterized in that   The method for producing a container according to claim 1, wherein the periphery of the well-like reaction part and / or the well-like reagent storage part is raised by 1.0 mm or more.   The method for producing a container according to claim 1 or 2, wherein a width of a protruding portion around the well-like reaction part and / or the well-like reagent storage part is in a range of 0.1 mm to 3 mm. .   The method for producing a container according to any one of claims 1 to 3, comprising a plurality of the well-like reaction parts and / or well-like reagent storage parts.   The tension of the protective film or the film-like lid material when the heating member is brought into contact with or close to the upper part of the protective film or the film-like lid material is in the range of 100 g to 1000 g. A method for producing the container according to claim 1.   The method for producing a container according to any one of claims 1 to 5, wherein the roll-shaped protective film or film-shaped lid material is supplied so as to be orthogonal to the substrate.   Furthermore, it has a 2nd reaction part, The manufacturing method of the container in any one of Claims 1-6 characterized by the above-mentioned.

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