JP2009047643A - Biochip, and temperature detection method of same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biochip capable of detecting heat applied to a reagent in processes such as manufacturing, transportation, storage, and reaction. <P>SOLUTION: This biochip 1 has a plurality of reaction chambers 3 on a substrate 2. A first temperature detection section 8 where a first temperature detection object 12 whose melting point is a predetermined first temperature is disposed in a recessed part 10 of the same size as the reaction chambers 3. Whether the reaction chambers 3 are in an environment of a first temperature or higher can be determined based on the shape or color of the first temperature detection object 12 in the first temperature detection section 8. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a biochip for performing a chemical reaction, a DNA reaction, a protein reaction, and the like, and a biochip temperature detection 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 that previously required a large experimental apparatus and a large amount of reaction reagent with a small amount of reaction reagent using a biochip of several millimeters square or less.

On this biochip, a plurality of minute holes and indentations called wells are formed, and each is used as an independent reaction vessel. 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 enclose the reagent in the biochip, a method of filling and storing the reagent in each well in advance, or a method of filling the reagent into each well from the reagent reservoir through a flow path can be considered.
Japanese translation of PCT publication No. 2002-503336

  However, reagents used in biochemistry are vulnerable to heat, and even at room temperature, they are susceptible to fatal damage such as reduced enzyme function and nucleic acid disruption. In particular, when reaction detection is performed on a biochip, even a slight decrease in reaction efficiency due to deterioration of the reagent is often not allowed. Also, from the viewpoint of quality assurance, it is necessary to grasp and record how much heat has been applied to the reagent during the manufacturing and transportation process. As a method for directly measuring the temperature, there are various conventional techniques including measurement with a thermocouple, but these are not desirable because of destructive inspection.

  Also, there is a method of nondestructive inspection such as incorporation of a dye layer that causes a chemical reaction depending on temperature, and applying a temperature indicating material on the outer wall of the storage container, or applying a temperature indicating material paint inside or outside the container. Although known, it is not necessarily appropriate from the viewpoint of mounting on a small chip such as a biochip. In order to accurately grasp the heat applied to the reagent, it is desirable to measure the temperature inside the reagent storage unit, not the substrate.

The present invention has been made in view of the above circumstances, and provides a biochip capable of detecting heat applied to a reagent in steps such as manufacturing, transportation, storage, and reaction, and a biochip temperature detection method. For the purpose.
Another object of the present invention is to provide a biochip temperature detection method capable of easily detecting the temperature applied to the reaction chamber.

  The present invention is a biochip having a plurality of reaction chambers on a substrate, and a temperature detector having a predetermined first temperature as a melting point is disposed in a recess having the same size as any of the plurality of reaction chambers. The temperature detection unit is provided, and it can be determined whether the reaction chamber is in an environment of the first temperature or higher by the shape or color of the temperature detection body in the temperature detection unit.

  According to the biochip of the present invention, it is possible to easily determine whether or not the biochip has been exposed to a temperature equal to or higher than the first temperature by looking at the shape and color of the temperature detector of the temperature detector.

  In the biochip of the present invention, a second temperature detection unit in which a second temperature detection body having a second temperature different from the first temperature as a melting point is disposed in a recess having the same size as any of the plurality of reaction chambers. May be further provided. In this case, it is possible to easily determine whether or not temperature management is appropriately performed in a plurality of environments such as during reaction, during production, and during transportation.

  A reagent may be disposed in at least one of the plurality of reaction chambers. In this case, it is possible to configure a biochip that can easily react and can easily grasp whether or not the reagent is under appropriate temperature control. The reagent may include an enzyme or a nucleic acid.

  The “reagent” refers to any substance used for reacting with a reaction solution on a biochip, and includes a substrate, a buffer, and the like.

  The biochip of the present invention may further include a lid member that covers the upper surfaces of the plurality of reaction chambers. In this case, contamination to the reaction chamber can be prevented.

  The biochip temperature detection method of the present invention is a temperature detection method for detecting a temperature applied to a reaction chamber of a biochip having a plurality of reaction chambers on a substrate, wherein the biochip includes the plurality of reaction chambers. A step of forming a recess having the same size as any of the above, and a step of disposing a temperature detector having a predetermined first temperature as a melting point in the recess.

According to the biochip of the present invention, it is possible to provide a biochip capable of detecting heat applied to a reagent in steps such as production, transportation, storage, and reaction.
Moreover, according to the biochip temperature detection method of the present invention, the temperature applied to the reaction chamber can be easily detected.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a plan view showing a biochip 1 of the present embodiment. The biochip 1 is configured by providing a plurality of reaction chambers 3 on a substantially rectangular substrate 2.

  The substrate 2 is a substantially rectangular plate member made of resin or the like. Examples of the resin used as a material include polypropylene (PP), polycarbonate (PC), acrylic resin (polymethyl methacrylate), polyethylene terephthalate (PET), polyethylene (PE), polyvinyl chloride (PV), polystyrene (PS), and the like. It is preferable because it is excellent in heat resistance, chemical resistance, molding processability and the like.

  The thickness of the substrate 2 is preferably set to a thickness that does not easily bend during use. The substrate 2 may be formed by bonding two or more kinds of resins. 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 physical properties 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. Further, quartz glass can be used as the material of the substrate 2.

As shown in FIG. 2A, the reaction chamber 3 is a concave portion having a hemispherical shape (a bowl shape) or an inverted conical shape (a bowl shape), and is aligned on the substrate 2. The reaction chamber 3 is formed by a method such as cutting the resin constituting the substrate 2 or injection molding the material resin of the substrate 2.
The diameter of the opening of the reaction chamber 3 is preferably 0.01 mm or more and 5 mm or less. If it does in this way, supply of the reaction reagent mentioned below becomes easy, and mixing of bubbles is prevented. In each reaction chamber 3, a first reagent (reagent) 4 necessary for an initial reaction with a sample containing DNA added at the time of use is arranged. The first reagent 4 may be disposed only in a part of the reaction chambers 3 so that the biochip 1 can perform a plurality of types of reactions.

  As shown in FIG. 1, a second reagent indwelling unit 6 for storing the second reagent 5 used for the reaction after the initial reaction in the reaction chamber 3 is provided at another part of the substrate 2. The second reagent indwelling portion 6 is formed by arranging the second reagent 5 in an indwelling chamber 7 having substantially the same shape as the reaction chamber 3 as shown in a cross section in FIG. The second reagent 5 is added to each reaction chamber 3 at a necessary timing using a quantitative dispenser or the like.

Furthermore, on the substrate 2 around the area where the reaction chamber 3 is provided, a first temperature detection unit (temperature detection unit) 8 and a second temperature detection unit 9 for detecting the temperature applied to the biochip 1. Is provided.
FIG. 2C is a cross-sectional view of the first temperature detector 8, and FIG. 2D is a cross-sectional view of the second temperature detector 9. Each of the temperature detectors 8 and 9 includes concave portions 10 and 11 having substantially the same shape as the reaction chamber 3, and a first temperature detector (temperature detector) 12 and a second temperature disposed in the concave portions 10 and 11, respectively. It has a detection body 13.

The 1st temperature detection part 8 is a part which detects whether the temperature which affects the quality of the 1st reagent 4 and the 2nd reagent 5 which were arrange | positioned at the time of manufacture of the biochip 1 and transportation. For example, a substance having a melting point of 35 ° C. (first temperature) is selected as the first temperature detector 12 to be arranged.
The second temperature detector 9 is a part that detects whether or not the reaction temperature is normally applied to the reagent in the reaction chamber 3 during the reaction on the biochip 1. For example, a substance having a melting point of 80 ° C. (second temperature) is selected.
In addition, both the 1st temperature detection part 8 and the 2nd temperature detection part 9 arrange | position several types of 1st temperature detection bodies 12 and 2nd temperature detection bodies 13 from which melting | fusing point differs in a respectively different recessed part, and determine about several temperature It may be configured to be able to.

  The material used as the first temperature detector 12 and the second temperature detector 13 may be any material that causes a change in state when the temperature is raised above a certain temperature. Among them, waxes are preferable because they are easy to handle, and polyethylene wax is used when the set melting point is 100 ° C. or higher, microcrystalline wax is used when the set melting point is 80 to 100 ° C., and paraffin wax is used when the set melting point is 80 ° C. or less. be able to.

  It is preferable that the shape of each of the temperature detectors 12 and 13 can be easily determined to be different before and after melting. For example, if it is normally melted, it becomes a shape close to a sphere due to surface tension, and therefore, it is made into a square plate at the time of placement. On the other hand, from the viewpoint of accurately grasping the influence on the first reagent 4 disposed in the reaction chamber 3, the contact area of each temperature detector 12, 13 with respect to the bottom of the recesses 10, 11 is the first area in the reaction chamber 3. It is preferably substantially the same as reagent 4. Further, when the shapes of the first reagent 4 and the second reagent 5 are different, the one of the temperature detectors 12 and 13 of each of the temperature detectors 8 and 9 has a shape having a contact area substantially equal to that of the second reagent 5. Alternatively, as described above, a plurality of sets of these may be installed so that a plurality of temperatures can be detected.

  The temperature detection may be determined only by the shape change due to melting of the temperature detection bodies 12 and 13, or the dyes conjugated to the temperature detection bodies 12 and 13 and the dyes conjugated when melted are the concave portions 10. , 11 may be configured so as to be more easily discriminated.

  As shown in FIG. 1 again, on the right side of the reaction chamber 3, when the second reagent supplied to each reaction chamber 3 is a test solution, a test solution storage unit 14 is provided for storing the test solution. Yes. The reagent storage unit 14 can also be used when the amount of the second reagent is large.

  Before use, as shown in FIGS. 2 (a) to 2 (d), the upper surfaces of the substrate 2, the reaction chamber 3, the second reagent indwelling unit 6, the temperature detecting units 8, 9 and the reagent storage unit 14 are as shown in FIGS. In order to prevent contamination and the like, it is covered with a lid 15. The lid member 15 may be bonded to the substrate 2 by a method such as heat sealing or heat fusion using a laser. And the temperature detection body for judging whether the reagent was damaged by the heat | fever which takes in such a fixing process of a lid | cover material may be previously arrange | positioned at the chip | tip before the fixing process. The lid member 15 is preferably one that can be easily remounted after adding a necessary sample or the like to the reaction chamber.

  In addition, when a temperature detector having a melting point lower than the temperature at the time of heat sealing is disposed for the purpose of, for example, detecting the temperature environment at the time of transportation when the lid member 15 is adhered by heat sealing, the temperature The temperature detector may be disposed after heat sealing without covering the temperature detector where the detector is disposed with the lid 15. Since no sample is added to the temperature detector, there is no major problem with respect to contamination.

  Hereinafter, the biochip of the present invention will be further described using examples. The biochip 20 of the present embodiment is a biochip used for the polymerase chain reaction (PCR) method.

(Example 1)
First, a hemispherical reaction chamber 22 having a radius of 1.5 mm was formed by cutting at a position on a substrate 21 made of PP as shown in FIG. Moreover, in order to form a temperature detection part, the recessed parts 23 and 24 of the same shape as the reaction chamber 22 were formed (concave part formation process). The volume of the reaction chamber 22 is 7.7 μL (microliter) in theory. The inner surface of the reaction chamber 22 was lyophilic by plasma treatment.
In addition, an indwelling chamber 25 and a reagent solution storage unit 26 to be a second reagent indwelling unit were also formed on the substrate 21.

As shown in a cross section in FIG. 4B, granular crystal wax having a melting point of 98 ° C. (trade name “Hi-Mic-2095” manufactured by Nippon Seiwa Co., Ltd.) is used as the first temperature detector 27 in the recess 23. The first temperature detector 28 was formed by arranging (temperature detector assembly step). The first temperature detector 27 may be deformed by heat when a PCR denaturation temperature (96 ° C.) is applied for 30 seconds in a state where the first temperature detector 27 is disposed in the recess 23, and may not be deformed when a temperature 3 degrees lower than the PCR denaturation temperature is applied for the same time. It has been confirmed.
In Example 1, only the first temperature detection unit was provided, and the second temperature detection unit was not provided.

  Subsequently, as shown in a cross section in FIG. 4A, 1.0 μL of a primer (first reagent) 29 having a different amplification site is placed on the bottom of each reaction chamber 22 and dried and fixed. A biochip 20 was manufactured by attaching a lid material (ABI PRISM Optical Adhesive Cover) 32 manufactured by ABI.

  Two such biochips 20 are prepared, a buffer is added to template DNA, DNA polymerase, and dNTP to prepare a test solution 30, the lid 32 is peeled off, and once stored in the test solution storage unit 26, the primer 3.5 μL was dispensed into each reaction chamber 22 in which 29 was placed. Further, 3 μL of mineral oil 31 was dispensed to prevent evaporation during heating. Thereafter, the lid 32 was remounted to cover the upper surface.

  A thermal history was applied to the biochip 20 after completion of the operation using plate PCR. Here, one biochip 20 is regarded as a temperature-controlled normal sample B, and optimal for nucleic acid amplification according to the denaturation process (90-98 ° C.), annealing process (40-60 ° C.), and extension process (around 70 ° C.). Temperature adjustment was performed.

  On the other hand, the other biochip 20 was used as a comparative temperature-control deficient sample A. For the temperature control-deficient sample A, the temperature applied in the denaturation process with the highest temperature was set 3 ° C. lower than the temperature control normal sample, and the other processes were the same as the temperature control normal sample B. This temperature control deficient sample A assumes a case where a temperature malfunction has occurred so as to lower the reaction efficiency during the PCR method.

  Table 1 shows a comparison result between the normal temperature control sample B and the temperature control inadequate sample A. After completion of the PCR reaction, the first temperature detection unit of each sample was visually confirmed. As shown in FIG. 5, the first temperature detection unit 28 of the normal temperature control normal sample B detected the first temperature in the recess 23. The body 27 was melted so as to spread toward the edge, and it was changed to a shape that can be easily distinguished from that at the time of placement. On the other hand, in the 1st temperature detection part of the temperature control deficient sample A, as shown in FIG. 6, it was confirmed that the 1st temperature detection body 27 is the same shape as the time of arrangement | positioning, and has not melt | dissolved.

(Example 2)
Next, an example in which the invader method, which is one of the single gene polymorphism (SNP) detection methods, is performed using the biochip of the present invention will be described.
In order to manufacture the biochip 40 of the present embodiment, first, the reaction chamber 22, the recesses 23 and 24, the holding chamber 25, and the reagent solution storage section 26 are formed on the substrate 21 in the same procedure as in the first embodiment.

Next, as shown in FIG. 3, a highly purified paraffin wax having a melting point of 62 ° C. (trade name “HNP-5” manufactured by Nippon Seiwa Co., Ltd.) is disposed as the second temperature detector 41 in the recess 24. A two-temperature detector 42 was formed. It was confirmed in advance that the second temperature detector 41 was completely melted at 63 ° C.
In the recess 23, the above-described high-purity purified paraffin wax is diluted 26 times with mineral oil (trade name “Mineral oil, Light oil” manufactured by Sigma) and the melting point is adjusted to about room temperature. The first temperature detector 44 was formed as a detector 43. It was confirmed beforehand that the first temperature detector 43 started melting and changed its shape when left at a temperature of about 25 ° C. for several hours.

  In the invader method, two types of non-fluorescently labeled oligonucleotides (allele probe, invader probe), two types of fluorescently labeled oligonucleotides (FRET probe), and an endonuclease (chrybase) specific to the DNA structure are used. The invader reaction is performed by incubating at about 63 ° C. for about several tens of minutes to about 4 hours.

In this example, a biochip 40 was produced according to the configuration of an invader assay kit (manufactured by Third Wave Technologies).
First, 0.15 μL each of different invader probes and corresponding allele probes was added to each bottom of the reaction chamber 22 and dried. On top of that, 0.75 μL of a FRET probe bound with a FAM label or a RED label as a fluorescent substance was added to each reaction chamber 22 and dried. Then, 0.15 μL of chestnut was placed in each reaction chamber 22, and these were used as the first reagent (reagent) 46. And the biochip 40 was produced by mounting | wearing with the cover material 32 similarly to Example 1. FIG.
Three biochips 40 having the above-described configuration are prepared, and one of them (hereinafter referred to as chip A) is left at room temperature for 4 hours (incomplete storage state), and the remaining two (hereinafter referred to as chips B and C). ) Was stored refrigerated (good storage).

  After the preservation, the lid 32 of the biochip 40 was peeled off, and an invader buffer was added to the prepared PCR product (using QIAGEN's Multiprex PCR kit) to prepare a total amount of 60 μL as a test solution 47. This test solution 47 is once stored in the test solution storage unit 26, and then, as shown in FIG. 4A, 3.5 μL is dispensed into each reaction chamber 22 of each biochip 40 on which the first reagent 46 is arranged. Further, 3.0 μL of mineral oil 48 was dispensed in order to prevent evaporation during heating.

  Thereafter, the lid 32 was remounted on the upper surface of the substrate 21 to cover it, and then a thermal history was applied to each biochip 40 by plate PCR. At this time, chips A and C were heated at a reaction temperature of 63 ° C. (set reaction temperature), and chip B was heated at 59 ° C. (insufficient reaction temperature).

  That is, chip A is incompletely stored and sample is reacted at the set temperature, chip B is in good condition but sample is inadequate in reaction temperature, and chip C is in preserved state and reaction. It is set and adjusted as a sample with no problem with temperature. Table 2 shows the comparison results of the chips A, B, and C.

  Before starting the invader reaction, when the first temperature detector 43 installed in the first temperature detectors 44 of the chips A, B, and C is visually observed, the first temperature detector 43 is deformed in the chip A. Therefore, it was easily determined visually that the storage state is incomplete. On the other hand, there was no change in the first temperature detectors 43 of the chips B and C that had been refrigerated and stored, and it was determined that there was no problem in the stored state.

  After the invader reaction, when the second temperature detector 41 installed in the second temperature detector 42 of the chips A, B, and C was visually observed, the second temperature detector 41 was deformed in the chips A and C. Thus, it was easily determined visually that the reaction was performed at a temperature higher than the set temperature. On the other hand, there was no change in the second temperature detector 41 of the chip B heated at 59 ° C., and it was determined that the reaction temperature was insufficient.

  Each chip A, B, C was measured using a fluorescence image analyzer (trade name “Molecular Imager FX466” manufactured by Bio-Rad Laboratories Co., Ltd.). Both chips A and B, which are deficient in either one, had extremely low fluorescence values, and the reaction efficiency was lowered as expected. It was inferred that chip A had inadequate storage conditions and thus reduced reaction efficiency due to reagent degradation, and chip B had inadequate temperature control during the invader reaction, which resulted in decreased reaction efficiency.

  According to the biochip of the present invention, the first and second temperature detectors are configured by disposing the first and second temperature detectors having a predetermined temperature as the melting point in the concave portion having substantially the same shape as the reaction chamber. Therefore, the heat applied to the reagent in the reaction chamber acts on each temperature detector almost in the same manner. Therefore, it can be easily determined visually what kind of temperature environment the reagent in the reaction chamber was in.

  Further, as the first temperature detector, a substance having a melting point at a temperature at which the influence on the reagent or the like is concerned at the time of manufacture or transportation is selected, and as the second temperature detector, a substance having a preset temperature at the time of reaction as the melting point Since it is selected, it is possible to monitor two points of the state of the reagent before the test and the temperature adjustment during the reaction. Therefore, the quality of measurement can be further improved.

  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, an example in which each reaction chamber is independent has been described, but instead of this, each reaction chamber 51 has a groove shape like a biochip 50 of a modified example shown in FIG. The flow path 52 may be configured to be connected to the reagent solution storage unit 53. If it does in this way, a test solution can be collectively supplied to all the reaction chambers 51 by applying a pressure to the test solution storage part 53. FIG.

  In addition, when only one type of temperature detection unit is provided as the first temperature detection unit, any indicator of the set temperature during reaction, the temperature during manufacturing, and the temperature during transportation may be used. Which one is provided can be appropriately determined according to the characteristics of the reagent or the like to be arranged.

  Furthermore, in the above-described embodiment, an example in which the reaction chambers are all the same size has been described, but instead, a plurality of reaction chambers having different sizes may be formed on the substrate. In this case, if a temperature detector is provided with a recess having the same size as each reaction chamber, a temperature detector corresponding to each reaction chamber can be configured.

  In addition, in each of the above-described embodiments, the example applied to the biochip used in the PCR method or the invader method has been described, but the present invention is not limited to this. The present invention can be applied to any biochip in which a reagent or the like requiring appropriate temperature control is arranged in a reaction chamber.

It is a top view which shows the biochip of one Embodiment of this invention. (A) is a reaction chamber of the biochip, (b) is a second reagent storage chamber of the biochip, (c) is a first temperature detector of the biochip, and (d) is a second temperature of the biochip. It is sectional drawing which shows a detection part, respectively. It is a top view which shows the biochip of one Example of this invention. (A) is a reaction chamber of the biochip, (b) is a cross-sectional view showing a first temperature detection unit of the biochip. It is a figure which shows the state which the 1st temperature detection body in the 1st temperature detection part of the biochip deform | transformed. It is a figure which shows the state which the 1st temperature detection body in the 1st temperature detection part of the biochip did not deform | transform. It is a top view which shows the modification of the biochip of this invention.

Explanation of symbols

1, 20, 40, 50 Biochip 2, 21 Substrate 3, 22, 51 Reaction chamber 4, 46 First reagent (reagent)
8, 28, 44 First temperature detector (temperature detector)
9, 42 Second temperature detector 10, 11, 23, 24 Recess 12, 27, 43 First temperature detector (temperature detector)
13, 41 Second temperature detector 15, 32 Lid 29 Primer (reagent)

Claims (6)

A biochip having a plurality of reaction chambers on a substrate,
A temperature detection unit in which a temperature detection body having a predetermined first temperature as a melting point is disposed in a recess having the same size as any of the plurality of reaction chambers;
The biochip according to claim 1, wherein the reaction chamber can be determined based on a shape or a color of the temperature detection body in the temperature detection unit.   And a second temperature detector having a second temperature detector having a second temperature different from the first temperature as a melting point in a recess having the same size as any of the plurality of reaction chambers. The biochip according to claim 1.   The biochip according to claim 1 or 2, wherein a reagent is disposed in at least one of the plurality of reaction chambers.   The biochip according to claim 3, wherein the reagent contains an enzyme or a nucleic acid.   The biochip according to any one of claims 1 to 4, further comprising a lid member that covers upper surfaces of the plurality of reaction chambers. A temperature detection method for detecting a temperature applied to a reaction chamber of a biochip having a plurality of reaction chambers on a substrate, wherein a recess having the same size as any of the plurality of reaction chambers is formed on the biochip. Process,
Disposing a temperature detector having a predetermined first temperature as a melting point in the recess;
A temperature detection method for a biochip, comprising:

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