JP2007192712A - Method of storing substrate, and substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vessel that is used in a chemical, biochemical, or biological field and has a reagent storage section where a reagent moves less, in the surface state holding method of a substrate and the substrate used for storing the reagent, detecting an antigen by antigen-antibody reaction, detecting DNA, or other chemical, biochemical, biological reaction or the like. <P>SOLUTION: The vessel has base material and one or more reagent storage sections in the base material, and has a recess in the bottom of at least one reagent storage section. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for maintaining a surface state of a substrate and a substrate used for containing reagents, detecting antigens by antigen-antibody reaction, detecting DNA, and other chemistry, biochemistry, biological reactions 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.
In addition, these wells can be used as a mixing place with other reagents such as a buffer solution after a very small amount of reagent is filled in a predetermined position.

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).

These all supply reagents to the cavity inside, but the amount of reagents such as enzymes and nucleic acids to be filled is extremely small.
However, the trace reagent easily moves due to the impact and inclination received during the transportation process and the like, and there is a problem that the composition of the mixed solution is different from the schedule.

For example, when a PCR reaction or a DNA reaction using an enzyme is performed on a reagent-filled chip in a well-shaped reagent container, the amount of DNA to be extracted from blood or tissue is suitably 0.1 ng to 50 ng, in order to amplify the DNA It is sufficient to use an extremely small amount of an enzyme such as a polymerase or an enzyme used in the reaction. However, since the enzyme cannot be mixed and filled with the reagent (because the enzyme is deactivated), the enzyme and the reagent need to be packed separately. In that case, it is most difficult to collect a small amount of enzyme, so it is most difficult to recover a reagent such as a buffer of several tens of μl, transfer it to a reagent container filled with a small amount of enzyme, and mix them. This is a method to increase the yield.
When a minute reagent such as an enzyme is placed in the well-shaped reagent container, and then another reagent is injected into the well and mixed, the minute reagent such as the enzyme that has been previously moved moves and partly later It may remain on the wall or the like without contacting with the added reagent, and desired mixing may not be possible.
Such a liquid residue is negligible in a system with a large amount of reagent. However, when an ng-unit sample such as DNA is used, the reagent becomes a very small amount of about 1 μl, and a slight liquid residue becomes a problem.

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 consideration of such circumstances, and an object of the present invention is to provide a container having a reagent container with little movement of a reagent in containers used in fields such as chemistry, biochemistry, and organisms. In particular, in a system using a trace reagent such as an enzyme, an object is to make the container without reagent transfer. It aims at high liquid recovery. Moreover, when using as a mixing field, it aims at mixing with a desired composition reliably.

  The invention of claim 1 is a container comprising a base material and one or a plurality of reagent storage parts on the base material, wherein the container has a recess at the bottom of at least one reagent storage part. .

  A second aspect of the present invention is the container according to the first aspect, wherein the volume of the recessed portion is within a range of 1/10 to 1/100 with respect to the total volume of the reagent storage unit.

  The invention according to claim 3 is characterized in that the total volume of the reagent container is in the range of 10 to 300 μl and the volume of the recessed portion is in the range of 0.1 to 5 μl. It is a container of description.

  In the invention of claim 4, the opening diameter of the reagent container is within the range of 1 to 50 mm, the opening diameter of the depression is within the range of 0.1 to 5 mm, and the opening diameter of the reagent container is the opening diameter of the depression. It is 1.5-5 times, The container in any one of Claims 1-3 characterized by the above-mentioned.

  A fifth aspect of the present invention is the container according to any one of the first to fourth aspects, wherein a portion excluding the depression at the bottom of the reagent containing portion is hemispherical.

  A sixth aspect of the present invention is the container according to any one of the first to fifth aspects, wherein the bottom of the recess is hemispherical.

  A seventh aspect of the present invention is the container according to any one of the first to sixth aspects, wherein a convex portion is provided around the opening of the reagent storage portion.

  The invention according to an eighth aspect is the container according to any one of the first to seventh aspects, wherein a film is laminated on the reagent container.

  A ninth aspect of the present invention is the container according to any one of the first to eighth aspects, wherein the substrate includes a reaction portion.

  The invention according to claim 10 is the container according to any one of claims 1 to 9, further comprising a second reaction part.

  According to the present invention, when the reagent is accommodated, the bottom of the reagent accommodating portion has a recess, and therefore the liquid does not move to the wall surface and the lid surface due to vibration applied during the experiment and transportation. Therefore, when used as a mixing field, there is no liquid residue of the moved liquid on the wall surface and lid surface due to vibration and the like, and mixing can be performed with a desired liquid composition. For the same reason, a desired amount of liquid can be recovered even if the amount of liquid contained is very small.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a diagram showing an embodiment of a substrate of the present invention. FIG. 1 shows a chip in which a plurality of recesses for 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 by providing a reaction part, it is necessary not to adversely affect the reaction system. Furthermore, when a reaction part is provided and the reaction in the reaction part is optically detected 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 size of the substrate is preferably about 10 to 150 mm × 10 to 150. If it is this range, it is preferable at the point of handling.

The substrate includes a reagent container having a recess at the bottom. An example is shown in FIG. In FIG. 1, eight reagent storage portions 3 are provided on a substrate 2, and one of them has a recess 4.
The 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, and in this case, it is sufficient that at least one reagent storage unit has a depression at the bottom. 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 shape of the reagent container is not particularly limited, but a concave shape (well shape) is preferable. Among these, a hemispherical or cylindrical shape with a hemispherical bottom is preferable. 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 the case of a reagent container having a depression, it is only necessary that the bottom of the portion excluding the depression is hemispherical. It is preferable that the bottom of the recess is also hemispherical. If a small amount of reagent droplets are supplied in the form of a ball when stored, the reagent droplets are just held in a hemispherical depression. Even if the amount of the reagent is larger than the capacity of the depression, a part of the reagent enters the depression and is retained if it is hemispherical.
In addition, the opening may be circular or polygonal, and the cross section may be triangular, quadrangular, or trapezoidal. The same applies to the depressions.

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 extracted from blood is usually about 0.1 ng to 50 ng, and a reagent containing DNA is about 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. Further, when the amount of the reagent is several hundred μl or more, it may be divided into two and accommodated.
In addition, the reagent storage part which has a hollow and the reagent storage part which does not have a hollow are also in the above-mentioned range.

Moreover, it is preferable that the capacity | capacitance of the hollow part of a reagent storage part exists in the range of 0.1-5 microliters.
If it is about this, for example, when a reagent of about several tens to several hundreds of μl as described above is accommodated, the liquid is held triggered by the depression.
In addition, as described above, DNA extracted from blood is usually about 0.1 ng to 50 ng, a reagent containing DNA is about several hundred nl to several μl, and several hundreds of reagents such as enzymes used for the reaction are similarly used. nl to several μl. In this way, when a very small amount of reagent is accommodated, the reagent is just fixed in the recess.

It is preferable that the capacity | capacitance of a hollow part exists in the range of 1/10-1/100 with respect to the capacity | capacitance of the whole reagent storage part. Within this range, when a reagent of about several tens to several hundreds of μl is accommodated, the reagent is held around the depression triggered by the depression.
In addition, when a very small amount of reagent of several hundreds nl to several μl is accommodated, it can be used as a mixing field by holding a very small amount of reagent in the recess and adding another reaction reagent later.

The opening diameter of the reagent container is within a range of 1 to 50 mm, the opening diameter of the depression is within a range of 0.1 to 5 mm, and the opening diameter of the reagent container is 1.5 to 5 times the opening diameter of the depression. Preferably there is.
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.
When the amount of the reagent is about several tens to several hundreds of μl, if the opening diameter of the dent is in the range of 0.1 to 5 mm, the reagent is held around the dent triggered by the dent. Moreover, if the amount of the reagent is about several hundred nl to several μl, a very small amount of reagent can be fixed in the recess.
Further, if the opening diameter of the reagent container is 1.5 to 5 times the opening diameter of the recess, the above-described reagent holding is effective.

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

  In addition, the reagent container may be formed by hollowing out a substrate as shown in FIG. 6B, or the back surface of the substrate is directed downward along the shape of the reaction portion as shown in FIG. 6A. It may be convex.

Moreover, you may provide a cover material on a reagent storage part. By providing the cover material, it is possible to prevent contamination by dust, contaminants, and the like.
A film-like thing can be used as a cover material. 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 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.

  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.

  Further, when a lid member is provided, the reagent may be collected by piercing with a needle-like collection 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.

Moreover, you may provide a convex part around an opening part. By providing the convex portion, the lid material can be easily bonded. An example is shown in FIG. In FIG. 2, eight well-like reagent storage portions 3 are provided on a substrate 2, and one of them has a recess 4. The reagent container 3 has a convex portion 5 around the opening.
In particular, when pasting together by heat sealing, the portion to be heated is not only the entire base material but the convex portion, so that the thermal influence on the reagent in the reagent storage portion can be reduced.

As such a convex part, it is preferable that the width is within a range of 0.1 to 2 mm, preferably 0.3 to 0.7 mm. If it is smaller than this range, it is impossible to heat-seal at the convex portion, and if it is larger than this range, the influence (damage) on the base material and contents will increase.
Moreover, you may increase the capacity | capacitance of an accommodating part by providing a convex part. In that case, in consideration of the strength of the housing portion and heat sealability, the convex portion may be formed in two stages as shown in FIG. 8B, for example. That is, it is possible to achieve both strength and heat sealability by providing a two-stage structure in which a convex portion having a certain thickness is provided in order to increase strength and a convex portion having a small width is provided thereon.

  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 a 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 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.

Moreover, you may provide a reaction part on a board | substrate. The reaction part can be formed in a concave shape (well shape). An example is shown in FIG. In FIG. 3, three well-like reagent storage portions 3 are provided on a substrate 2, and one of them has a recess 4. Twenty well-like reaction units 6 are provided.
The number of reaction parts 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, a small amount of reagent is often used, and in order to perform the reaction efficiently, it is preferably within the above range.
Moreover, the reagent required for reaction may be accommodated in the reaction part in advance.

The shape of the well-like reaction detection unit is not particularly limited, but is preferably a truncated cone shape in which the bottom of the well-like reaction unit is flat and the wall surface is inclined 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.

  Further, the reaction part may be formed by hollowing out the substrate as shown in FIG. 7B, or the back surface of the substrate is directed downward along the shape of the reaction part as shown in FIG. 7A. It may be convex.

Moreover, you may provide a protective film 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 can be bonded using an adhesive. As the adhesive, a heat-resistant curable adhesive can be used.
Further, it may be bonded 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.

  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.

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

Moreover, you may provide a convex part around an opening part. By providing the convex portion, the lid material can be easily bonded. 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.

As such a convex part, it is preferable that the width is within a range of 0.1 to 2 mm, preferably 0.3 to 0.7 mm. If it is smaller than this range, it is impossible to heat-seal at the convex portion, and if it is larger than this range, the influence (damage) on the base material and contents will increase.
Moreover, you may increase the capacity | capacitance of a reaction part by providing a convex part. In that case, in consideration of the strength of the reaction part and heat sealability, the convex part may be formed in two stages. That is, it is possible to achieve both strength and heat sealability by providing a two-stage structure in which a convex portion having a certain thickness is provided in order to increase strength and a convex portion having a small width is provided thereon.
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.

  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 a 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. 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 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.

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 a flow path may be provided to carry out the reaction in the flow path. An example is shown in FIG. In FIG. 4, three well-like reagent storage portions 3 are provided on a substrate 2, and one of them has a recess 4. 20 well-like reaction parts 6 are provided. A flow path-shaped second reaction unit 7 is provided between the reaction unit and the reagent storage unit.
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 0.1 cm to 5 cm.

The opening may have a shape protruding upward from the base material in order to increase the capacity.
When the reaction is performed in the internal flow path, an evaporation inhibitor such as mineral oil may be added to the openings at both ends in order to prevent evaporation of the reaction solution. In that case, if the depth of the opening is increased, the amount of evaporation inhibitor to be added can be increased, and evaporation can be reliably prevented. The opening may be formed by increasing the thickness of the substrate. However, when the thickness of the substrate is small, a structure in which a part of the opening protrudes from the substrate in a chimney shape may be used to deepen the opening.

  Moreover, you may provide a cover part on a 2nd reaction part. The material and shape of the lid are not particularly limited, and a desired lid can be provided according to the purpose.

When the second reaction part has a flow path shape, specifically, for example, a shape as shown in FIG. 6 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.

  Further, 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. An example is shown in FIG. In FIG. 4, four well-like reagent storage portions 3 are provided on a substrate 2, and one of them has a recess 4. 20 well-like reaction parts 6 are provided. The reagent storage units and the reagent storage unit and the well-like reaction unit are connected by a flow path 8.
Further, a flow path connecting the well-like reaction part, the reagent storage 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, a rib 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 as a chip for various biochemical reaction, 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 non-volatile liquid having a specific gravity lighter than that of the reaction liquid such as mineral oil may be added to the well-like reaction part and the PCR reaction part in order to prevent the reaction liquid 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.

<Example 1>
The well-shaped detection chip 1 shown in FIG. 6 is formed 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.).

Reagent storage part 3 is formed with six opening diameters of 6 mm, depth of 5.2 mm and hemispherical tip, and two recesses 4 having a diameter of 6 mm, depth of 5.2 mm and hemispherical tip. did. The opening diameter of the recess 4 was 1.5 mm, the depth was 0.5 mm, and the tip was hemispherical.
The reaction part 6 had a trapezoidal cross section with a diameter of 3 mm, a depth of 2 mm, a side surface inclined, and a bottom part flat. The bottom diameter was 1 mm. The base material had a thickness of 1 mm, and the bottoms of the well-shaped reagent container and the well-shaped reaction part protruded downward from the substrate. Moreover, 20 reaction parts were provided.

  The volume of the reagent container 3 having a depression is 30 μl, and the volume of the depression 4 is 0.6 μl.

  The PCR reaction part which is the second reaction part 7 had an opening diameter of 13 mm, a depth of 6 mm and a hemispherical tip.

  The reagent container 3, the second reaction unit 7 (PCR reaction unit), and the reaction unit 6 were provided in this order from the end of the substrate.

Next, as a surface treatment, Ar 10 L / min: O ₂ 0.5 L / min is supplied between a pair of electrodes, and a voltage is applied between the electrodes while the substrate is conveyed at a conveyance speed of 50 mm / sec. went. This process was performed three times.

Each reagent container contains 15 μl of sample DNA (0.1 ng / μl)
・ Mineral oil 120μl
20 μl of PCR Mixture containing PCR buff and primer mix and dNTP
・ 0.6 μl of 2-fold diluted polymerase
-37 μl of invader reagent containing invader buffer, fret fluorescent reagent and sterilized water
・ Enzyme (kuribase) 5μl
To accommodate.
In addition, a polymerase and an enzyme (kuribase) are accommodated in the reagent accommodating part which has a hollow. The polymerase is 0.6 μl, and is just in a state where it is held in a dent. The enzyme (kuribase) is not completely accommodated in the depression, but is held by the depression.
0.75 μl of allele probe (5-fold diluted solution) was supplied to each reaction part to make it dry.
Two reagent storage units were left open.

  Next, a lid material made of PET / aluminum / sealant film as a lid material was pasted on the reagent storage part by heat sealing, and a protective film made of PET / sealant film as a protective film was pasted on the reaction part by heat sealing. .

  Next, assuming the vibration applied to the container at the time of experiment and transportation, vibration was applied using a vibration generator (VORTEX GENIE2 manufactured by S / I). As the vibration conditions, the chip was pressed against the vibration generator from directly above, and vibration was applied for 30 seconds at a strength of 7.

  Next, using a pipette (dispensing needle) with an inner diameter of 0.9 mm, 11 μl of PCR Mixture is dispensed into a reagent container having a recess in which 0.6 μl of 2-fold diluted polymerase is accommodated, and pipetting (pumping) three times. And mixed. Thereafter, 8 μl of the sample DNA was extracted using the same pipette, dispensed into a reagent containing part having a depression containing a 2-fold diluted polymerase, and mixed by pipetting (pumping) three times to obtain a PCR reaction solution.

  The PCR reaction solution mixed with the same pipette was dispensed into the PCR reaction part. Thereafter, 7.5 μl of mineral oil was supplied to each of the two openings using the same pipette, and the PCR reagent was sandwiched between the mineral oil in the flow path.

  Next, the PCR reaction section was heated at 95 ° C. for 5 minutes, and then the cycle of 95 ° C. (60 seconds), 58 ° C. (60 seconds), and 72 ° C. (60 seconds) was repeated 30 times. This was done to obtain a PCR reaction product.

  Using the same pipette, the entire PCR reaction product was dispensed into the reagent container containing the invader reagent, and mixed by pipetting (pumping) three times. Thereafter, 3 μl of the enzyme (krivase) was extracted using the same pipette, dispensed into the reagent container containing the invader reagent, and mixed three times by pipetting (pumping) to obtain a reaction solution.

  The obtained reaction solution was dispensed into each of 20 reaction portions in a volume of 2 μl using the same pipette. Thereafter, 4 μl of mineral oil for preventing evaporation was dispensed into each of the 20 reaction parts using a similar pipette.

  Invader reaction / detection was performed by denaturing DNA at 95 ° C. for 5 minutes, followed by incubation at 61 ° C. for 40 minutes, irradiation and detection. ABI7300 (Applied biosystem) was used as an apparatus for reaction and detection.

<Comparative Example 1>
The same procedure as in Example 1 was performed except that the reagent container having a depression was not provided with a depression. That is, all of the eight reagent storage units had an opening diameter of 6 mm, a depth of 5.2 mm, and a hemispherical tip, and six types of reagents including polymerase and enzyme (chrybase) were inserted in the same manner as in Example 1.

As a result of the invader detection, the sample of Example 1 showed good results similar to the reaction performed in the conventional tube for both the homo and hetero samples. That is, it was shown that the reaction efficiency was good. Since the reaction efficiency depends on the amount of the PCR product, it is presumed that the reaction proceeded with the optimal reaction solution composition by the presence of the polymerase in the depression.
Enzymes are generally dissolved in glycerol and other high-boiling and high-viscosity solvents. If a trace amount of enzyme adheres to the side of the container or the top surface of the lid, it spreads thinly and usually cannot be centrifuged. The solution cannot be returned to the bottom. However, in Example 1, the reagent containing part with the depression can be used to firmly stabilize the reagent containing the enzyme and can be reliably mixed with other reagents, so that the above result is obtained. I think it was. In addition, since it was possible to reliably collect a small amount of enzyme, it seems that a reaction solution having a desired composition could be mixed.

  The sample of Comparative Example 1 did not give very good results. That is, it was shown that the reaction efficiency was lower than that of Example 1. The reason for the low reaction efficiency is that the polymerase and enzyme (kuribase) moved from the central part to the top surface from the side by applying vibrations, and the optimal PCR reaction solution composition and invader reaction solution composition were lost. It is speculated that it was caused by this.

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Explanation of symbols

1 Container (chip)
2 Substrate 3 Reagent storage part 4 Dimple 5 Convex part 6 Reaction part 7 Second reaction part (PCR reaction part (channel type))
7 (a) protruding portion 7 (b) lower film 8 flow path

Claims (10)

  A container comprising a base material and one or a plurality of reagent storage parts on the base material, wherein the container has a recess at the bottom of at least one reagent storage part.   The container according to claim 1, wherein the volume of the hollow portion is within a range of 1/10 to 1/100 with respect to the volume of the entire reagent storage unit.   3. The container according to claim 1, wherein the volume of the entire reagent container is in the range of 10 to 300 μl, and the volume of the recessed portion is in the range of 0.1 to 5 μl.   The opening diameter of the reagent container is within a range of 1 to 50 mm, the opening diameter of the depression is within a range of 0.1 to 5 mm, and the opening diameter of the reagent container is 1.5 to 5 times the opening diameter of the depression. The container according to any one of claims 1 to 3, wherein the container is provided.   The container according to any one of claims 1 to 4, wherein a portion of the reagent storage unit excluding the bottom is hemispherical.   The container according to any one of claims 1 to 5, wherein the bottom of the depression is hemispherical.   The container according to claim 1, further comprising a convex portion around the opening of the reagent storage portion.   The container according to any one of claims 1 to 7, wherein a film is laminated on the reagent storage unit.   The container according to claim 1, wherein the substrate is provided with a reaction part.   Furthermore, it has a 2nd reaction part, The container in any one of Claims 1-9 characterized by the above-mentioned.

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