JP5015441B2 - Reaction chip and reaction method - Google Patents

Description

  The present invention relates to a reaction chip and a reaction method.

  In recent years, μ-Total Analysis System technology for performing chemical reaction, DNA reaction, protein reaction, etc. on a chip, Lab-on-Chip technology, and the like have been studied and realized. As a result, it has become possible to carry out reaction experiments, which have heretofore required a large experimental apparatus and a large amount of reaction reagent, with a small amount of reaction reagent using a reaction chip of several millimeters or less.

On this reaction chip, a plurality of minute holes and indentations called wells are formed and used as reaction vessels. The plurality of well-like reaction vessels are connected by a reagent solution flow path extending from the reagent solution reservoir (see, for example, Patent Document 1).
In order to perform a reaction using this reaction chip, first, a reaction reagent is arranged in a plurality of well-like reaction vessels. Next, by injecting the reaction reagent into the reagent storage part, the reaction reagent is supplied to the plurality of well-like reaction containers via the reagent flow path. Thereby, a reaction reagent and a reaction reagent solution contact and reaction starts. If necessary, the well-like reaction vessel is heated during the reaction.
Japanese translation of PCT publication No. 2002-503336

However, in the above-described reaction method, when the reaction reagent is supplied into the well-like reaction container via the reagent solution channel, the reaction reagent previously arranged in the well-like reaction container is transferred to the adjacent well-like reaction container. There is a risk of spillage. As a result, there is a problem that contamination occurs.
In addition, during the reaction in each well-like reaction container, there is a possibility that reaction reagents, reaction reagents, fluorescent substances for detection, etc. may diffuse into adjacent well-like reaction containers. As a result, there is a problem that accurate reaction data cannot be measured.

  The present invention has been made in consideration of such circumstances, and provides a reaction chip and a reaction method capable of preventing the occurrence of contamination and measuring accurate reaction data. With the goal.

In order to achieve the above object, in the reaction chip according to the invention of claim 1, a plurality of well-like reaction containers in which reaction reagents are arranged, and a reagent solution flow for sequentially supplying reaction reagent solutions to the plurality of well-like reaction containers A reaction chip provided with a channel, wherein the reaction reagent is covered with a hot-melt sealing agent disposed in the well-like reaction container, and the sealing agent includes the reaction reagent and the reaction reagent The sealant, which is made of a material having a lower specific gravity and is melted by heat, closes the opening of the well-like reaction vessel and the reagent solution flow path .

Further, in the reaction chip according to the invention of claim 2 , the reagent solution flow path is extended from the reagent solution storage part.

The reaction chip according to the invention of claim 3 is characterized in that a plurality of the reagent flow paths are extended from the reagent storage part.

The reaction chip according to the invention of claim 4 is characterized in that the reaction reagent includes a nucleic acid probe.

On the other hand, in the reaction method according to the invention of claim 5 , a reagent solution storage part formed on the substrate, a reagent solution channel extending from the reagent solution storage part, and a plurality of wells sequentially connected by the reagent solution channel A reaction chip comprising a reaction container, a reaction reagent disposed in the well reaction container, and a hot-melt sealant disposed in the well reaction container so as to cover the reaction reagent The sealing agent is made of a material having a specific gravity smaller than that of the reaction reagent and the reaction reagent, and by injecting the reaction reagent into the reagent reservoir, Supplying the reaction reagent solution above the sealant in the plurality of well-like reaction vessels; and heating and melting the sealant to open the well-like reaction vessel with the sealant. And block the reagent channel. One, characterized by having a the steps of contacting the reaction reagent and the reagent.

According to the first aspect of the present invention, the reaction reagent is supplied above the sealant covering the reaction reagent, so that the reaction reagent does not flow out to the adjacent well-like reaction container. Therefore, the occurrence of contamination can be prevented.
In addition , by using a sealant that melts at the reaction temperature, the reaction reagent and the sealant are exchanged at the reaction temperature, and while the reaction reagent and the reaction reagent are in contact and reacting with each other, the opening of the reaction vessel is opened. It will be in the state obstruct | occluded with the sealing agent. This prevents the reaction reagent and reaction reagent from diffusing into the adjacent well-like reaction vessel during the main reaction. Therefore, accurate reaction data can be measured.

According to the invention of claim 2 , by injecting the reaction reagent into the reagent storage part, the reaction reagent can be continuously supplied to a plurality of well-like reaction vessels connected to the reagent solution flow path.

According to the invention of claim 3 , by injecting the reaction reagent into the reagent storage part, the reaction reagent can be continuously supplied to the plurality of well-like reaction vessels respectively connected to the plurality of reagent channels. .

According to the invention of claim 4 , a DNA detection reaction by hybridization can be realized.

According to the invention of claim 5 , since the reaction reagent is supplied above the sealing agent covering the reaction reagent, the reaction reagent does not flow out to the adjacent well-like reaction container. Therefore, the occurrence of contamination can be prevented. In addition, by using a sealant that melts at the reaction temperature, the reaction reagent and the sealant are exchanged at the reaction temperature, and the reaction container and the reaction reagent are in contact with each other while the reaction reagent is in contact with the reaction reagent. It will be in the state of being blocked by the stop agent. This prevents the reaction reagent and reaction reagent from diffusing into the adjacent well-like reaction vessel during the main reaction. Therefore, accurate reaction data can be measured.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a plan view of a reaction chip according to an embodiment. The reaction chip 1 includes a rectangular substrate 2 and a plurality of well-like reaction containers 10 in which reaction reagents are arranged, and a reagent solution channel 40 for sequentially supplying reaction reagent solutions to the plurality of well-like reaction containers 10. It is provided.

  The board | substrate 2 is exhibiting the substantially rectangular shape as a whole, and is formed with the thickness which does not bend easily during use. The substrate 2 is made of a resin material such as PP (polypropylene), PC (polycarbonate), acrylic resin (polymethyl methacrylate), PET (polyethylene terephthalate), PE (polyethylene), PV (polyvinyl chloride), PS (polystyrene). It is configured. It is preferable to produce the substrate 2 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 making the substrate 2 by making use of the characteristics of each resin, it is possible to make various substrates 2 according to the characteristics of the reaction reagent, the sample, and the like, which can be properly used for each application. For example, it is possible to divide the material between the upper half and the lower half of the substrate 2. Note that quartz glass may be used as the material of the substrate 2.

  The substrate 2 is provided with a reagent storage unit 3 that stores a reaction reagent, a reagent storage unit 4 that stores a reaction reagent after reaction, and a detection unit 5 that detects a specific substance in the reaction reagent. A reaction unit for performing a PCR reaction or the like may be provided between the reagent storage unit 3 and the reagent storage unit 4 or on the opposite side of the reagent storage unit 4 across the reagent storage unit 3. In the detection part 5, in order to detect the specific substance in the reaction reagent, a detection reaction using a reaction reagent is performed. Therefore, a plurality of well-like reaction vessels 10 are formed in the detection unit 5, and reaction reagents are arranged in each well-like reaction vessel. In order to supply the reaction reagent solution to the well-like reaction container 10, a plurality of reagent solution channels 40 are extended from the reagent solution storage unit 4 and a forced liquid delivery device (not shown) such as a micropump is provided. Yes. A plurality of well-like reaction vessels 10 are connected to each reagent solution channel 40. A lid member 50 is provided so as to cover the well-like reaction vessel 10 and the reagent solution flow path 40.

  FIG. 2A is an explanatory view of a well-like reaction vessel, and is a side cross-sectional view taken along the line AA in FIG. As shown in FIG. 2A, the well-like reaction vessel 10 is formed in a hemispherical shape (a bowl shape), an inverted truncated cone shape (a bowl shape), or the like. The diameter of the opening of the well-like reaction vessel 10 is desirably 0.01 mm or more and 5 mm or less. Thereby, supply of the reaction reagent with respect to the well-like reaction container 10 becomes easy, and mixing of bubbles can be prevented.

  As shown in FIG. 1, the well-like reaction vessel 10 is formed by a method of cutting a substrate 2 made of a resin material, a method of injection molding a resin material in a mold, or the like. When the substrate 2 is made of a hard resin material such as PC (polycarbonate), the well-like reaction vessel 10 can be formed using a cutting method. When the substrate is made of a soft resin material such as PP (polypropylene), it is preferable to form the well-like reaction vessel 10 using a molding method. Further, the well-like reaction vessel 10 can be formed using a molding method with a PC.

  A reagent channel 40 is formed on the surface of the substrate 2 in order to supply the reaction reagent to the plurality of well-like reaction vessels 10 in order. A plurality of well-like reaction vessels 10 are connected by the reagent solution channel 40. Similar to the well-like reaction vessel 10, this reagent solution channel 40 is also formed by a cutting method, a molding method, or the like.

  In this manner, surface treatment for smoothly flowing the reaction reagent solution may be performed on the surface of the substrate 2 on which the well-like reaction vessel 10 and the reagent solution channel 40 are formed. Examples of the surface treatment include various hydrophilization treatments such as plasma treatment, corona treatment, and buff treatment. By subjecting the substrate 2 to the surface treatment, it becomes easier to supply the reaction reagent solution from the reagent solution storage unit 4 to the well-like reaction vessel 10 via the reagent solution channel 40, and the power consumption of the solution feeding device can be reduced. Furthermore, since air in the substrate 2 escapes well, it is possible to prevent bubbles from entering the reaction reagent.

(Sealing agent)
As shown in FIG. 2 (a), a solid reaction reagent 12 such as a nucleic acid probe is disposed inside the well-like reaction vessel 10. This reaction reagent is covered with a hot-melt sealant 20 disposed in the well-like reaction vessel 10. The hot-melt type sealing agent 20 is a sealing agent that is solid at room temperature and melts in the vicinity of the start temperature of the reaction between the reaction reagent and the reaction reagent (hereinafter referred to as “main reaction”). It is desirable for the melting point to be around 35-90 ° C. so that it dissolves at around 80-90 ° C. As the sealant 20, it is desirable to employ a sealant having a specific gravity smaller than that of the reaction reagent and the reaction sample solution. However, it is assumed that the main reaction is not inhibited. As a specific sealant, AmpliWax (registered trademark) PCR Gem 100/50 manufactured by Takara Bio Inc. is employed. This is a product devised as an alternative to mineral oil to dissolve and form a layer during PCR amplification to prevent evaporation of the reaction reagent. It is solid at room temperature and melts at 55-58 ° C. The reaction reagent 12 covered with the sealant 20 may be liquid or solid. In the case of a solid, it is advantageous because mixing with a reaction reagent is easier.

  In order to arrange the sealing agent 20 in the well-like reaction vessel 10, an appropriate amount of solid sealing agent is put into a well-like reaction vessel in which a reaction reagent has been arranged in advance. Next, the sealing agent is heated. As a result, the melted sealant wets and spreads on the bottom of the well-like reaction vessel 10. Thereafter, if the sealant is cooled, the sealant 20 can be disposed in the well-like reaction vessel 10 in a state of covering the reaction reagent 12. Thus, after arrange | positioning the sealing agent 20 in the well-like reaction container 10, the cover member 50 shown in FIG. 1 is mounted | worn.

(how to use)
Next, a method for using the above-described reaction chip will be described with reference to FIGS. 2B and 2C are process diagrams of the reaction method according to the embodiment, and are side cross-sectional views taken along line AA in FIG.
First, a reaction reagent solution is injected into the reagent solution storage unit 4 shown in FIG. Next, the liquid feeding device is operated to cause the reaction reagent solution to flow out from the reagent solution reservoir 4 to the reagent solution channel 40. The reaction reagent solution is sequentially supplied to the plurality of well-like reaction containers 10 through the reagent solution channel 40. The reaction reagent solution is supplied at room temperature or at a low temperature at which the liquid can be fed at or below room temperature.

As shown in FIG. 2B, a solid sealing agent 20 is disposed inside the well-like reaction vessel 10 so as to cover the reaction reagent 12. Therefore, the reaction reagent solution 42 supplied to the well-like reaction vessel 10 is disposed on the surface of the sealant 20 without contacting the reaction reagent 12.
As described above, in the reaction chip of this embodiment, the reaction reagent is supplied above the sealing agent covering the reaction reagent, so that the reaction reagent does not flow out to the adjacent well-like reaction container. Therefore, the occurrence of contamination can be prevented.

  Next, as shown in FIG. 2C, the well-like reaction vessel 10 is heated to melt the sealant 20. Since the sealant 20 has a specific gravity smaller than that of the reaction reagent 42, the reaction reagent 42 and the upper and lower sides are interchanged. As a result, the reaction reagent 12 comes into contact with the reaction reagent 42. When the reaction start temperature of the main reaction is equal to or lower than the melting point of the sealant 20, the main reaction starts simultaneously with the contact between the reaction reagent 12 and the reaction reagent 42. If the reaction start temperature of the main reaction is higher than the melting point of the sealant 20, the well-like reaction vessel 10 is further heated to start the main reaction.

  Here, the opening of the well-like reaction container 10 and the reagent solution flow path 40 are closed by the sealant 20 whose top and bottom are exchanged with the reaction reagent solution 42. Thereby, in the reaction chip of this embodiment, a reaction reagent, a reaction reagent, etc. do not diffuse into an adjacent well-like reaction container during the main reaction. Therefore, the occurrence of contamination can be prevented, and accurate reaction data can be measured.

  Next, as an example of the reaction chip and reaction method according to the present invention, a DNA detection method by hybridization will be described. In each drawing of FIG. 3, the upper stage is a comparative example, and the lower stage is an example.

  As shown in FIG. 3A, a simple reaction chip was prepared by forming a plurality of well-like reaction vessels 10 on a polypropylene resin substrate having a thickness of 2 mm. The well-shaped reaction vessel 10 has an opening diameter of 3 mm, a bottom diameter of 1 mm, and a well volume of 7.7 μL theoretically. A reagent solution channel 40 was formed on the substrate so as to connect a plurality of well-like reaction vessels 10. The test solution flow path 40 had a width of 0.5 mm and a depth of 0.5 mm, and was formed by cutting.

  Of the plurality of well-like reaction vessels 10 arranged in a row, the probe DNA and the fluorescent substance were placed as the reaction reagent 12 in the odd-numbered well-like reaction vessels 10a and dried under reduced pressure. Nothing was placed in the even-numbered well-like reaction vessel 10b.

  Next, AmpliWax (registered trademark) PCR Gem 50 (manufactured by Takara Bio Inc.) was introduced into all well-like reaction vessels 10 as a sealant. Note that the amount of sealing agent introduced into one well-like reaction vessel 10 is 2 to 4 μL. This sealant was heated to 80 ° C. to melt and solidified again at room temperature. Thereby, in the odd-numbered well-like reaction vessel 10a, the reaction reagent 12 was covered with the sealant. Next, a lid member was disposed on the surface of the reaction chip, and the portions other than both ends of the reagent solution channel 40 were almost sealed.

  Next, DNA extracted from blood or the like was supplied as a reaction reagent. The reaction reagent solution was supplied at normal temperature from the end of the reagent solution channel 40 and was distributed to all the well-like reaction vessels 10. The supply amount of the reaction reagent for one well-like reaction vessel 10 is 3 to 5 μL. Next, the reaction chip was heated to melt the sealing agent, and the reaction reagent and the reaction reagent were reacted. Finally, the fluorescent substance in the well-like reaction container was detected.

(Comparative example)
Similar to the above example, a plurality of well-like reaction vessels 10 and a reagent solution channel 40 were formed on the substrate. Moreover, the reaction reagent 12 was arrange | positioned to the odd-numbered well-like reaction container 10a, and nothing was arrange | positioned to the even-numbered well-like reaction container 10a. In the comparative example, the lid member was arranged on the surface of the reaction chip without introducing the sealant into all the well-like reaction vessels 10. Next, the reaction reagent solution was supplied from the end of the reagent solution channel 40 at room temperature. In addition, the supply amount of the reaction reagent with respect to one well-like reaction vessel 10 is 7 to 8 μL. Next, the reaction chip was heated to react the reaction reagent with the reaction reagent, and the fluorescent substance in the well-like reaction vessel was detected.

  FIG. 3B is a plan view of the reaction chip at the stage where the reaction reagent solution is supplied. In the upper comparative example, a part of the reaction reagent arranged in the odd-numbered well-like reaction container 10a flows out into the even-numbered well-like reaction container 10b by the supply of the reaction reagent. On the other hand, in the lower example, it was confirmed that the reaction reagent did not flow out to the even-numbered well-like reaction vessel 10b. This is presumably because the reaction reagent was supplied to the surface of the sealant covering the reaction reagent.

  FIG. 3C is an explanatory diagram of the fluorescence detection result after the main reaction. In the upper comparative example, fluorescence was confirmed in the even-numbered well-like reaction vessel 10b in addition to the odd-numbered well-like reaction vessel 10a. This is considered to be because a part of the reaction reagent arranged in the odd-numbered well-like reaction vessel 10a diffused into the even-numbered well-like reaction vessel 10b during the main reaction. On the other hand, in the lower example, no fluorescence was confirmed in the even-numbered well-like reaction vessel 10b. This is presumably because the reaction reagent did not diffuse during the main reaction because the opening of the well-like reaction vessel 10a was blocked by the sealant.

  The technical scope of the present invention is not limited to the above-described embodiments, and includes various modifications made to the above-described embodiments without departing from the spirit of the present invention. That is, the specific materials and configurations described in the embodiments are merely examples, and can be changed as appropriate.

It is a top view of the reaction chip concerning an embodiment. It is explanatory drawing of a well-like reaction container and process drawing of the reaction method which concerns on embodiment. It is explanatory drawing of an Example and a comparative example.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Reaction chip 4 ... Test solution storage part 10 ... Well-like reaction container 12 ... Reaction reagent 20 ... Sealing agent 40 ... Test solution flow path 42 ... Reaction test solution

Claims (5)

A reaction chip comprising a plurality of well-like reaction containers in which reaction reagents are arranged, and a reagent solution channel for sequentially supplying reaction reagent solutions to the plurality of well-like reaction containers,
The reaction reagent is covered with a hot-melt sealant disposed in the well-like reaction vessel ,
The sealant is composed of a material having a specific gravity smaller than that of the reaction reagent and the reaction reagent,
The reaction chip , wherein the heat-melted sealing agent closes the opening of the well-like reaction vessel and the reagent solution flow path . The reaction chip according to claim 1, wherein the reagent solution flow path is extended from a reagent solution storage part. The reaction chip according to claim 2 , wherein a plurality of the test solution flow paths are extended from the test solution storage unit. The reaction chip according to any one of claims 1 to 3 , wherein the reaction reagent includes a nucleic acid probe. A reagent storage part formed on the substrate, a reagent solution channel extending from the reagent solution storage part, a plurality of well-like reaction vessels sequentially connected by the reagent solution channel, and disposed in the well-like reaction vessel A reaction chip comprising a reaction chip, and a hot melt sealing agent that covers the reaction reagent and is disposed in the well-like reaction container,
The sealant is composed of a material having a specific gravity smaller than that of the reaction reagent and the reaction reagent,
Supplying the reaction reagent solution above the sealant in the plurality of well-like reaction containers via the reagent solution channel by injecting the reaction reagent solution into the reagent solution storage unit;
Heating and sealing the sealant to bring the reaction reagent and the reaction reagent solution into contact with each other while closing the opening of the well-like reaction container and the reagent solution channel with the sealant ;
The reaction method characterized by having.

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