JP2008304356A - Reagent sealing structure of lab-on-chip, and lab-on-chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent sealing structure of a lab-on-chip capable of starting a reaction of a reagent with reaction liquid at an appropriate timing while the reagent is prevented from being affected by the flow of the reaction liquid in a reaction tub. <P>SOLUTION: A reagent sealing section 1 of the lab-on-chip 2 has a plurality of reaction tubs 4 filled with the reagent 7, the reaction liquid is added to the reaction tubs 4 and reaction is performed. The reagent sealing section 1 has sealing layers 8 that contain sealing material that is solid at normal temperature and is fused by heating and are fixed to the inside of respective reaction tubs 4. The reagent 7 is distributed and stored in the sealing layers 8 or covered with the sealing layers 8. The sealing layers 8 are heated together with the reaction liquid, thereby bringing the reagent 7 into contact with the reaction liquid to form a reactable state. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a sealing structure for sealing a reagent in a lab-on-chip reaction tank and a lab-on-chip.

  Lab-on-a-chip is a technology for conducting chemical and biological experiments on a substrate. This is because the reaction solution is added to the reagents, etc. that are pre-loaded in the reaction tank on the substrate, and the experimental system is integrated on the substrate, reducing the labor of operation and saving space and improving efficiency. It can be made to.

On the other hand, when a common reaction liquid is used in a plurality of reaction tanks, all reaction tanks are communicated with each other through a flow path, and the reaction liquid is injected from an inlet provided at one end, so that There is known a reaction vessel in which a reaction solution can be simply and efficiently placed in a reaction vessel (see, for example, Patent Document 1).
JP-T-2004-502164

  However, when trying to apply the structure of the reaction vessel of Patent Document 1 to a lab-on-chip, if the reagent or the like is simply loaded in the reaction vessel, the reaction solution comes into contact with the reagent when flowing into the reaction vessel, The reaction may start immediately. Therefore, there is a problem that it is difficult to control the timing of starting the reaction.

  In addition, when the reaction liquid flows out from a certain upstream reaction tank to the adjacent downstream reaction tank, the loaded reagents and the like flow to the downstream reaction tank together with the reaction liquid, so that the upstream reaction tank The reagent etc. of the inside may be lost or the amount may be insufficient. This is a problem because it causes no proper reaction.

  The present invention has been made in view of the above circumstances, and is a lab-on-a-chip capable of starting the reaction between the reagent and the reaction liquid at an appropriate timing without being affected by the flow of the reaction liquid in the reaction tank. It is an object to provide a reagent sealing structure and a lab-on-chip.

  The lab-on-chip reagent sealing structure of the present invention is a lab-on-chip reagent sealing structure in which a reaction liquid is added to the reaction tank and a reaction is performed. A sealing material that is solid at normal temperature and melts when heated, and includes a sealing layer fixed in each of the reaction vessels, and the reagent is dispersedly stored in the sealing layer, Or it is coat | covered with the said sealing layer, and when the said sealing layer is heated with the said reaction liquid, the said reagent and the said reaction liquid will contact and it will be in the state which can react.

  The “reagent” refers to any substance used for reacting with a reaction solution on a lab-on-chip, and includes nucleic acids, enzymes, substrates, buffers, and the like.

  According to the reagent sealing structure of the lab-on-chip of the present invention, the reagent is fixed in the reaction vessel so as not to come into contact with the reaction solution until the reaction vessel is melted by heating.

  The reagent sealing structure of the lab-on-chip of the present invention further includes a coating layer that contains the sealing material at a higher concentration than the sealing layer, and is arranged to cover the sealing layer. It may be distributed and stored in the sealing layer. In this case, since the sealing layer is covered with the coating layer, the elution of the reagent dispersedly stored near the upper surface of the sealing layer is effectively suppressed.

  The sealing layer may be formed in a film shape, and the reagent may be covered with the sealing layer. In this case, a reagent sealing portion with a small volume can be configured.

  The sealing material may be formed including any of agarose, gelatin, fats and oils, and paraffin. In this case, since the lattice structure is included in the sealing layer and the covering layer, the reagent is more efficiently dispersed and sealed.

  The lab-on-a-chip of the present invention is characterized by including the lab-on-a-chip reagent sealing structure of the present invention. In this case, the reaction on the lab-on-chip can be started at an appropriate timing.

  The lab-on-chip of the present invention is further provided with a flow path provided between the reaction tanks and communicating between the reaction tanks and an injection port into which the reaction solution is injected, and is injected from the injection port. The reaction solution may flow into each reaction tank through the flow path. In this case, the reaction solution can be placed in all the reaction vessels by injecting the reaction solution from the injection port. In this case, the reagent does not flow out to the downstream reaction tank.

  According to the lab-on-chip reagent sealing structure and the lab-on-chip of the present invention, the reagent is not affected by the flow of the reaction liquid in the reaction vessel, and the reaction between the reagent and the reaction liquid can be started at an appropriate timing. Lab-on-chip reagent sealing structure and lab-on-chip

Hereinafter, a lab-on-chip 2 including the reagent sealing portion (reagent sealing structure) 1 according to the first embodiment of the present invention will be described with reference to FIGS. 1 to 7.
FIG. 1 is a plan view of the lab-on-chip 2, and FIG. 2 is a cross-sectional view of the lab-on-chip 2. As shown in FIGS. 1 and 2, the lab-on-chip 2 is configured by forming a plurality of recesses as a reaction tank 4 on a substrate 3 made of resin or the like. Each reaction tank 4 is communicated with a groove-like channel 5, and an inlet 6 through which a reaction solution is injected is provided at one end of the channel 5.

  Inside each reaction tank 4, a reagent sealing portion 1 in which a reagent 7 used for the reaction is sealed is fixed. The reagent sealing portion 1 includes a reagent 7 and a sealing layer 8 made of a sealing member (sealing material) 8A that seals the reagent 7.

  The sealing member 8A that forms the sealing layer 8 is a solid phase (solid) at room temperature, and is fixed to the reaction tank 4 so that the reaction liquid described later is supplied from the flow path 5 when the reaction liquid is supplied into the reaction tank 4. It is necessary not to leak. Further, those having a lattice-like part (matrix) in the molecular structure and not affecting the chemical reaction between the reaction solution and the reagent 7 are preferable. Furthermore, it is necessary that the material melts into a liquid phase when heated to a predetermined temperature.

Specific examples of the sealing member 8A include agarose, gelatin, fats and oils, and paraffin. An appropriate melting point is appropriately selected from these materials in consideration of the heat resistance of the reagent 7 to be sealed.
The sealing layer 8 may include materials other than the sealing member 8A. For example, a preservative such as sodium azide may be added to maintain the quality of the reagent 7 to be sealed.

As the reagent 7, one or more kinds of nucleic acids, enzymes, substrates, buffers and the like necessary for the reaction are appropriately selected based on a chemical reaction performed on a lab-on-chip. These reagents 7 are held in the matrix of the sealing member 8 </ b> A in a state of being dispersed in the sealing layer 8 to constitute the reagent sealing portion 1.
In the case where the polymerase chain reaction method (PCR method) is performed using a combination of a plurality of probes, a different reagent may be arranged for each reaction tank.

  The reagent 7 to be sealed may be subjected to a treatment such as freeze-drying as necessary. If it does in this way, the volume of the reagent 7 can be made small and the volume of the whole reagent sealing part 1 can be made small. Moreover, the stability of the reagent 7 can be improved and the usable period of the lab-on-chip 2 can be lengthened.

  In the reagent sealing part 1 configured as described above, the mixture in which the reagent 7 is dispersed in advance in the sealing member 8A is placed in each reaction tank 4 using a quantitative dispenser 100 or the like as shown in FIG. Formed by dispensing and loading. Instead of dispersing the reagent 7, as shown in FIG. 4 (a), the reagent 7 dried by lyophilization or the like is filled in the reaction tank 4, and as shown in FIG. Then, the reagent sealing portion 1 may be formed by injecting and covering the sealing member 8A.

The operation at the time of using the lab-on-chip 2 configured as described above will be described below with reference to FIGS. 5 (a) to 5 (c).
First, as shown in FIG. 5 (a), with the sealing layer 8 of the reagent sealing part 1 holding a solid phase, the reaction solution 9 containing purified genomic DNA or the like is dispensed with a dispenser 101 or the like. It injects, applying pressure from the inlet 6 using.

  The injected reaction solution 9 is disposed in each reaction tank 4 through the flow path 5 as shown in FIG. At this time, since the reagent sealing portion 1 is fixed to the reaction vessel 4, it does not move downstream. Moreover, since it is a solid phase, the reagent 7 and the reaction liquid 9 do not contact, and reaction is not started.

Subsequently, when the lab-on-chip 2 is installed in a reactor equipped with a heat block and heated to a predetermined temperature, the sealing member 8A of the sealing layer 8 is melted as shown in FIG. 5 (c). It changes from a solid phase to a liquid phase. Then, the reagent 7 sealed in the reagent sealing portion 1 is mixed with and brought into contact with the reaction liquid 9, and a predetermined reaction is advanced by being placed under a predetermined temperature condition.
In addition, if the communication between the reaction tanks 4 is interrupted by plastically deforming the flow path 5 before the lab-on-chip 2 is heated, each of the sealing layers 8 is in a liquid phase. The contents of the reaction vessel are not mixed, which is preferable.

The lab-on-chip 2 after completion of the reaction is sent to a post-process such as typing reaction or fluorescence measurement.
Next, the reagent sealing structure of this embodiment will be further described using an actual example.

[Example 1]
In order to examine the influence of agarose as a sealing member on the PCR reaction efficiency, the following experiment was conducted.
First, as reagent 7, a mixture of PCR buffer (buffer: 10X, 1 μL), dNTP (substrate: 10X, 1 μL), primer 1 (CCAATACCGCCAACAGT, 10 pmol / μL, 1 μL), primer 2 (AGGAGTCCCATCACCAGGT, 10 pmol / μL, 1 μL) 12 samples were prepared.

  Among the 12 samples, as shown in FIG. 6, 10 samples were reagents to which polymerase (HS Ex Taq: manufactured by Takara Bio Inc., 5 U / μL, 0.5 μL) was further added. Five samples with and without polymerase were subjected to lyophilization, and all 12 samples were loaded into 200 μL PCR microtubes.

  Subsequently, a 2% agarose solution melted by heat was dropped onto each sample in the amount shown in FIG. 6 and allowed to stand at 4 ° C. for 10 minutes to gel the agarose solution. A purified genomic DNA solution (reaction solution: 200 ng / μL, 1 μL) is added to this, and distilled water is further added so that the volume of the sample becomes 10 μL, followed by heating to a predetermined temperature cycle to perform gene amplification by the PCR method. It was.

  FIG. 7 is a band diagram showing an amplification product obtained by the PCR method. As shown in FIG. 7, in 10 samples to which polymerase was added, substantially the same amplification product could be obtained regardless of whether the dripping amount of the agarose solution or the lyophilization treatment was performed. Therefore, it was confirmed that the reaction of the PCR method was not inhibited by agarose, and that the PCR method could be performed normally even if the reagent was sealed after lyophilization.

  According to the reagent sealing part 1 of the present embodiment, the reagent 7 is fixed to each reaction tank 4 in a state of being dispersed inside the sealing member 8A of the sealing layer 8 before heating. Even when the injected reaction liquid 9 flows in, the reagent 7 does not flow out into the flow path 5 on the downstream side. Therefore, reagents necessary for the reaction are held in each reaction tank 4 and can be appropriately reacted on the lab-on-chip 2.

  Further, since the reagent 7 before heating is dispersedly stored in the sealing layer 8, even if the reaction liquid 9 flows into the reaction tank, it does not come into contact immediately but is heated and the sealing layer 8 is melted. As a result, the reagent 7 and the reaction liquid 9 come into contact with each other, and the reaction becomes possible. Therefore, the reaction start timing can be controlled and the reaction can proceed appropriately.

  Next, a second embodiment of the present invention will be described with reference to FIGS. 8 (a) to 8 (c). In addition, about the component similar to the above-mentioned embodiment, the same code | symbol is attached | subjected and the overlapping description is abbreviate | omitted.

FIG. 8A to FIG. 8C are diagrams showing a procedure for forming the reagent sealing portion 11 of this embodiment and arranging it on the lab-on-chip.
First, two sealing films (sealing layers) 12 formed into a film shape using an agarose gel having a low water content were prepared, and the reagent 7 subjected to freeze-drying treatment as shown in FIG. Is sandwiched and covered with the sealing film 12 from above and below.

  When the peripheral part not touching the reagent 7 is bonded by a method such as welding or heat sealing, the reagent sealing part 11 in which the reagent 7 is sealed inside the sealing film 12 as shown in FIG. Is obtained. When the reagent sealing part 11 is fixed inside the reaction tank 13, the lab-on-chip 14 of this embodiment is completed.

In the reagent sealing part 11 and the lab-on-chip 14 configured as described above, the same effects as those of the reagent sealing part 1 and the lab-on-chip 2 of the first embodiment can be obtained.
Further, since the reagent 7 can be sealed without applying heat, the reagent 7 can be sealed without being denatured even when the reagent 7 is a substance that is weak against heat.

  Moreover, since the reagent 7 is freeze-dried, the volume of the reagent 7 can be reduced. Therefore, the whole reagent sealing part 11 can be configured smaller. Therefore, it can be applied to a smaller reaction tank, and the reaction tank of the lab-on-chip can be downsized and arranged more efficiently.

  Furthermore, if a sealing film 12 having a particularly low water content is used, the reagent sealing part 11 can be manufactured in large quantities at low cost by using a known roll-to-roll manufacturing machine or the like.

  In addition, since the moisture content of the sealing film 12 is small and the reagent 7 is freeze-dried, the moisture content is small as a whole and the storage stability is excellent. Then, by arranging the reagent sealing part of this embodiment in each reaction tank of a normal reaction container in which a plurality of reaction tanks are communicated with each other through a flow path, the reaction container can be used as a lab-on-chip. it can.

  Although the embodiments of the present invention have been described above, the technical scope of the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the spirit of the present invention. .

  For example, in the above-described embodiment, the example in which the reagent 7 is distributed and stored in the sealing layer 8 has been described, but instead, as in the modification examples illustrated in FIGS. 9A and 9B. On the reagent sealing part 1, sealing members made of the same or different materials may be arranged in layers using the dispenser 101 or the like. When the reagent sealing structure is configured as the reagent sealing portion 1A including the coating layer 10 formed in this manner, the elution of the reagent 7 on the surface of the reagent sealing portion 1 can be more effectively suppressed. it can.

  In this case, if the concentration of the sealing member in the coating layer 10 is set higher than that of the sealing layer 8, the matrix density of the coating layer becomes higher and the elution of the reagent 7 can be prevented more reliably.

  Further, in the above-described embodiment, an example is described in which each reaction tank is applied to a lab-on-chip communicated with a flow path, but the present invention is not limited to this and is applied to a lab-on-chip that does not have a flow path. May be. Also in this case, the reagent can be sealed in the reaction vessel in a stable state by the reagent sealing structure of the present invention.

  Furthermore, when a plurality of types of substances are sealed as the reagent 7, a reagent sealing part may be formed for each substance, and these reagent sealing parts may be stacked in the reaction tank. Furthermore, the stacked reagent sealing portions may be isolated by the covering layer 10 or the sealing film 12. If it does in this way, it can seal in the state where each substance in a reagent does not contact. Therefore, it is possible to form a sealed structure that can be stored well even with a reagent combined with substances that are not desired to be mixed from the viewpoint of storage stability.

  In addition, in the above-described embodiment, the example applied to the lab-on-chip for performing the PCR method has been described. However, the application of the present invention is not limited to this, for example, fluorescence detection using the FRET effect Can also be used.

It is a top view which shows the lab-on-chip provided with the reagent sealing structure of 1st Embodiment of this invention. It is sectional drawing of the same lab on chip. It is a figure which shows the state by which a reagent sealing part is arrange | positioned in the reaction tank of the same lab on chip. It is a figure which shows the other method by which a reagent sealing part is arrange | positioned. It is a figure which shows the operation | movement at the time of use of the same lab on chip. It is a figure which shows the structure of the reagent sealing part of each sample in the experiment example of the embodiment. It is a band figure which shows the amplification product after PCR reaction in the experiment example. It is a figure which shows the reagent sealing structure of 2nd Embodiment of this invention, and the arrangement | positioning process to a lab-on-chip. It is a figure which shows the reagent sealing part and its arrangement | positioning process in the modification of the lab-on-a-chip of this invention.

Explanation of symbols

1,11 Reagent sealing part (reagent sealing structure)
2, 14 Lab-on-chip 4, 13 Reaction tank 5 Channel 6 Inlet 7 Reagent 8 Sealing layer 8A Sealing member (sealing material)
9 reaction liquid 10 coating layer 12 sealing film (sealing layer)

Claims (6)

A reagent-sealed structure of a lab-on-chip that has a plurality of reaction tanks loaded with reagents, and a reaction liquid is added to the reaction tank to perform a reaction,
It includes a sealing material that is solid at normal temperature and melts when heated, and includes a sealing layer that is fixed in each of the reaction vessels.
The reagent is dispersedly stored in the sealing layer, or is covered with the sealing layer, and the reagent and the reaction liquid come into contact with each other by heating the sealing layer together with the reaction liquid, A lab-on-chip reagent sealing structure characterized by being in a reactive state. Containing the sealing material in a higher concentration than the sealing layer, further comprising a coating layer arranged to cover the sealing layer;
The lab-on-chip reagent sealing structure according to claim 1, wherein the reagent is distributed and stored in the sealing layer.   The lab-on-chip reagent sealing structure according to claim 1, wherein the sealing layer is formed in a film shape, and the reagent is covered with the sealing layer.   The lab-on-chip reagent sealing structure according to any one of claims 1 to 3, wherein the sealing material includes any one of agarose, gelatin, fats and oils, and paraffin.   5. A lab-on-chip comprising the lab-on-chip reagent sealing structure according to any one of claims 1 to 4. A flow path provided between the reaction vessels and communicating between the reaction vessels;
An inlet through which the reaction solution is injected;
Further comprising
The lab-on-chip according to claim 5, wherein the reaction liquid injected from the injection port flows into each of the reaction tanks through the flow path.

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