JP6026027B1 - Rapid detection of biomolecules using electric field agitation - Google Patents

Abstract

An object of the present invention is to provide a biomolecule detection method using electric field agitation that makes it possible to further save expensive reagents. In order to solve the above problems, the present inventors have intensively studied, and as a result, even if the amount of the reaction solution containing an expensive reagent is reduced, the oil coating liquid is used to increase the volume. It has been found that droplets can be formed. In order to apply a fluctuating electric field and vibrate the droplets at high speed, a low-viscosity oil-based coating solution of 0.7 to 18.6 mPa · s is used, and the droplet forming region and the outer periphery thereof are disposed. It was found that electric field agitation can be suitably performed by using a plate having an oil-repellent region, and the present invention has been completed. [Selection] Figure 1

Description

The present invention is an improved method for detecting a biomolecule contained in a solid biological sample, including an in situ hybridization (ISH) method and an immunohistochemistry (IHC) method. The present invention relates to a method characterized by electric field stirring of a droplet whose surface is coated with a coating liquid.
The present invention also relates to a biomolecule detection plate, a biomolecule detection kit and a biomolecule detection device used in the method, and a method for producing a biological sample specimen.

  Biomolecules (proteins, DNA, RNA, polysaccharides, etc.) contained in solid biological tissues such as biological tissue slices are detected while they are present in solid biological tissues without extracting the biomolecules, and their distribution is detected. There are various methods for imaging. For example, detection and imaging using molecules that bind specifically to biomolecules, fluorescent proteins (tags) linked to expressed proteins, and detection and imaging using them as markers, or enzymes For example, a method for detecting and imaging a protein using activity. Among these, detection and imaging using molecules that specifically bind to biomolecules are the most widely performed, in situ hybridization (ISH) method using complementary binding between nucleic acids, and specific The so-called immunohistochemistry (IHC, also called “immunostaining”) method using an antibody specific for a biomolecule of the above has become a common technique.

  By using immunohistochemistry (IHC) method or in situ hybridization (ISH) method, it is possible to microscopically observe the expression of specific proteins and genes related to specific diseases in tissues and cells collected from patients. Therefore, these techniques are often used for pathological diagnosis.

For example, the HER2 gene is an oncogene in which gene amplification and HER2 protein overexpression are observed in 15 to 25% of human breast cancer cases, but breast cancer patients with HER2 gene amplification / HER2 protein overexpression have a poor prognosis. There are reports that it shows treatment resistance to hormonal therapy and CMF therapy. In addition, for breast cancer in which HER2 gene amplification / protein overexpression has been confirmed, significant improvement in survival time and survival rate has been observed by administration of Herceptin (registered trademark, generic name trastuzumab).
Therefore, for breast cancer patients, examination of HER2 gene / HER2 protein by immunohistochemistry (IHC) method or in situ hybridization (ISH) method is important in prognosis prediction and treatment policy determination. .

  However, pathological diagnosis by immunohistochemistry (IHC) or in situ hybridization (ISH) requires a long time for antigen-antibody reaction and hybridization. In the case of the immunohistochemistry (IHC) method, it usually takes about 70 to 200 minutes in all steps, and in the case of the in situ hybridization (ISH) method, it usually takes about 1 to 2 days. Was necessary.

Therefore, the present inventors formed a dome-shaped droplet using a reaction solution containing an antibody or a labeled nucleic acid, and applied a varying electric field to the droplet to vibrate the droplet at high speed, thereby causing an antigen-antibody reaction. And methods for rapidly detecting biomolecules by promoting hybridization (Patent Documents 1 to 7 and Non-Patent Document 1). According to this method, it is possible to perform an antigen-antibody reaction and hybridization in a short time. For example, in the case of an immunohistochemistry (IHC) method, all the steps can be performed in about 15 to 30 minutes. Become. For this reason, it has become possible to quickly perform pathological diagnosis of tissues collected from patients during surgery (rapid pathological diagnosis during surgery) and to determine a surgical policy.
This method of applying a fluctuating electric field to a droplet and causing it to vibrate is also called “electric field agitation”, and if used in an immunohistochemistry (IHC) method or the like, the time for detecting biomolecules can be greatly shortened. Therefore, it is an extremely useful method, and further improvement and development have been desired.

By the way, in the immunohistochemistry (IHC) method, a technique for suppressing evaporation by covering a reaction solution with a medium-chain alkane oil or the like has been developed (Patent Document 8). However, this technique is not a technique used for electric field stirring and does not cover dome-shaped droplets with oil.
In addition, using a substrate having a hydrophilic region and a water-repellent region provided therearound, a droplet of a solution for nucleic acid amplification is formed, and the droplet is coated with oil to prevent nucleic acid from being evaporated. Has been developed (Patent Documents 9 to 12). However, these techniques are techniques aiming at nucleic acid amplification, and are not techniques used for electric field stirring.

JP 2010-119388 A JP 2012-013598 A JP 2014-160060 A Japanese Patent Laying-Open No. 2015-155811 JP 2015-219109 A Japanese Patent No. 5825618 Japanese Patent No. 5839526 JP 2000-046827 A JP 2009-136219 A JP 2009-171921 A JP 2009-207392 A JP 2010-158201 A Hiroshi Toda, Yoshihiro Nanya, 8 others, Acta Histochemica et Cytochemica (abbreviation ActaCytochem. Cytochem.), Japanese Society of Tissue Chemistry, Issued June 3, 2011, Vol. 3, No. 3 , P. 133-139

The conventional biomolecule detection method using electric field agitation needs to form a dome-shaped droplet that can vibrate, and therefore requires a predetermined amount or more of a reaction solution. For this reason, there is a limit to saving expensive reagents such as antibodies and labeled nucleic acids.
Therefore, in view of the above-described conventional situation, an object of the present invention is to provide a biomolecule detection method using electric field agitation that makes it possible to save an expensive reagent.

  In order to solve the above problems, the present inventors have intensively studied. As a result, even if the amount of the reaction solution containing an expensive reagent is reduced, it is increased in volume (increased) using an oily coating solution. It has been found that droplets can be formed. In order to apply a varying electric field and vibrate the droplets at high speed, an oily coating liquid having a low viscosity of 0.7 to 18.6 mPa · s is used instead of a normal oil such as vegetable oil. The inventors have found that electric field agitation can be suitably performed by using a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof, and the present invention has been completed.

  That is, the present invention relates to a first invention related to a biomolecule detection method, a second invention related to a biomolecule detection plate, a third invention related to a biomolecule detection kit, and a fourth invention related to a biomolecule detection apparatus. And a fifth invention relating to a method for producing a biological specimen, a sixth invention relating to an in situ hybridization method, and a seventh invention relating to an immunohistochemical method.

The first invention relates to a method of detecting a biomolecule in a solid biological sample by binding a biomolecule contained in the solid biological sample and a detection molecule specific to the biomolecule. This biomolecule detection method is:
A) A solid biological sample is placed on a droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof,
B) A droplet is formed in the droplet formation region so as to cover the biological sample with the solution containing the detection molecule,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) By performing electric field stirring that vibrates the droplet by applying a varying electric field to the droplet, the biomolecule contained in the biological sample and the detection molecule are combined,
E) The presence of a biomolecule is detected by a detection molecule bound to the biomolecule.
It is characterized by including.
In the biomolecule detection method of the first invention, the viscosity of the coating liquid is more preferably 0.7 to 5.5 mPa · s.
In any of the above biomolecule detection methods, liquid paraffin is preferably used as the coating liquid.
In any one of the biomolecule detection methods described above, it is preferable to use a plate having a water-repellent / lipophilic region at the boundary between the droplet-forming region and the oil-repellent region, and the droplet-forming region is hydrophilic.
Any of the above biomolecule detection methods can be a method for detecting a nucleic acid. In this case, a labeled probe having a sequence complementary to the nucleic acid to be detected is used as the detection molecule. Can do.
Moreover, it can also be set as the method of detecting protein, In that case, an antibody specific to the protein made into the detection object can be used as a detection molecule.

The second invention relates to a biomolecule detection plate used for detection of biomolecules using electric field stirring. This biomolecule detection plate has a droplet formation region and an oil-repellent region disposed on the outer periphery thereof, and can be used for the biomolecule detection method of the first invention.
In the biomolecule detection plate according to the second aspect of the invention, it is preferable to use a plate having a water-repellent / lipophilic region at the boundary between the droplet-forming region and the oil-repellent region, and the droplet-forming region is hydrophilic.

The third invention relates to a biomolecule detection kit used for detection of biomolecules using electric field stirring. This biomolecule detection kit includes any one of the above biomolecule detection plates, a solution containing a detection molecule that can specifically bind to a biomolecule, and an oily coating solution having a viscosity of 0.7 to 18.6 mPa · s. It is characterized by including. This biomolecule detection kit can be used in the biomolecule detection method of the first invention.
In the biomolecule detection kit of the third invention, it is preferable that the viscosity of the coating liquid is 0.7 to 5.5 mPa · s.
In any of the biomolecule detection kits described above, liquid paraffin is preferably used as the coating liquid.
Any of the above biomolecule detection kits can be used for nucleic acid detection or in situ hybridization. In this case, a sequence complementary to the nucleic acid to be detected is used as the detection molecule. Labeled probes can be used. Moreover, it can also be set as the kit for protein detection or immunohistochemistry, In that case, an antibody specific to the protein made into the detection object can be used as a detection molecule.

  The fourth invention is an invention relating to a biomolecule detection apparatus used for detection of biomolecules using electric field stirring. This biomolecule detection apparatus includes any one of the above-described biomolecule detection plates, a heating and heat-retaining apparatus that heats a droplet formed on the plate to maintain a constant temperature, and a variation electric field application that applies a variation electric field to the droplet. And a device. This biomolecule detection apparatus can be used in the biomolecule detection method of the first invention.

The fifth invention relates to a method for producing a biological sample specimen for detecting a biomolecule obtained by binding a biomolecule contained in a solid biological sample and a detection molecule specific to the biomolecule. The method of manufacturing the biological sample specimen is as follows:
A) A solid biological sample is placed on a droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof,
B) Using the solution containing the detection molecule, a droplet is formed in the droplet formation region so as to cover the biological sample,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) The biomolecule contained in the biological sample and the detection molecule are combined by performing electric field stirring that oscillates the droplet by applying a varying electric field to the droplet.
It is characterized by including.
In the method for producing a biological sample specimen of the fifth invention, the viscosity of the coating liquid is more preferably 0.7 to 5.5 mPa · s.
In any one of the above biological sample specimen manufacturing methods, it is preferable to use liquid paraffin as the coating liquid.
In any one of the above-described methods for producing a biological sample specimen, it is preferable to use a plate having a water-repellent / lipophilic region at the boundary between the droplet forming region and the oil-repellent region, and the droplet forming region being hydrophilic. .
In any one of the above-described methods for producing a biological sample specimen, a labeled probe having a sequence complementary to a nucleic acid to be detected is used as a detection molecule, and a method for producing a biological specimen specimen for nucleic acid detection or in situ hybridization can do. In addition, an antibody specific for a protein to be detected can be used as a detection molecule, and a method for producing a biological specimen for protein detection or immunohistochemistry can be obtained.

In a sixth aspect of the present invention, in situ high detection of nucleic acid contained in a biological sample is performed by hybridizing a nucleic acid contained in a solid biological sample with a labeled probe containing a sequence complementary to the nucleic acid. The invention relates to a hybridization method. This in situ hybridization method is
A) A solid biological sample is placed on a droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof,
B) A droplet is formed in the droplet formation region so as to cover the biological sample with the hybridization solution containing the labeled probe,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) Hybridizing the nucleic acid contained in the biological sample with the labeled probe by performing electric field stirring that oscillates the droplet by applying a varying electric field to the droplet,
E) detecting the label of the labeled probe hybridized to the nucleic acid,
It is characterized by including.
In the in-situ hybridization method of the sixth invention, it is more preferable that the viscosity of the coating solution is 0.7 to 5.5 mPa · s.
In any of the above in situ hybridization, liquid paraffin is preferably used as the coating solution.
In any one of the above-mentioned in situ hybridization methods, it is preferable to use a plate having a water-repellent / lipophilic region at the boundary between the droplet-forming region and the oil-repellent region, and the droplet-forming region is hydrophilic.

The seventh invention relates to an immunohistochemical method for detecting a biomolecule contained in a biological sample by binding a biomolecule contained in a solid biological sample and an antibody specific for the biomolecule. This immunohistochemistry method
A) A solid biological sample is placed on a droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof,
B) A droplet is formed in the droplet formation region so as to cover the biological sample with the solution containing the antibody,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) By binding the biomolecule contained in the biological sample and the antibody by performing electric field stirring that oscillates the droplet by applying a varying electric field to the droplet,
E) detecting antibodies bound to biomolecules,
It is characterized by including.
In the immunohistochemical method of the seventh invention, it is more preferable that the viscosity of the coating liquid is 0.7 to 5.5 mPa · s.
In any of the above immunohistochemical methods, liquid paraffin is preferably used as the coating solution.
In any of the above immunohistochemical methods, it is preferable to use a plate having a water-repellent / lipophilic region at the boundary between the droplet-forming region and the oil-repellent region, and the droplet-forming region is hydrophilic.

The method for detecting a biomolecule of the first invention, the method for producing a biological sample specimen of the fifth invention, the in situ hybridization method of the sixth invention, and the immunohistochemistry method of the seventh invention, Since the bulk is increased by the coating solution, even if the amount of the solution containing the detection molecules is reduced, droplets large enough for electric field stirring can be formed, and the effect of saving expensive detection molecules can be achieved. . In addition, since these inventions use an oily coating liquid having a low viscosity of 0.7 to 18.6 mPa · s, the liquid droplets can be vibrated at high speed even when the liquid droplets are coated. The droplets can be coated over a wide area to efficiently suppress the evaporation of the droplets. For this reason, the droplets are efficiently stirred, the reaction between the biomolecule and the detection molecule is promoted, and evaporation is suppressed, so that the biomolecule can be detected quickly and reproducibly. Furthermore, these inventions use a plate having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof to form a droplet in the droplet formation region and not exceed the surrounding oil-repellent region. In the range, the droplets are coated with an oily coating solution. As a result, the oil-repellent region surrounds the outer periphery of the oil-based coating liquid, and the droplets can be prevented from collapsing, so that sufficient electric field agitation can be performed, and biomolecules are detected quickly and reproducibly. There is an effect that can be.
The biomolecule detection plate of the second invention and the biomolecule detection device of the fourth invention have a droplet formation region and an oil-repellent region disposed on the outer periphery thereof on the plate. The droplets can be formed and coated with the oil-based coating liquid as long as the surrounding oil-repellent region is not exceeded. For this reason, since the oil-repellent region surrounds the outer periphery of the oil-based coating liquid and the droplets can be prevented from collapsing, sufficient electric field agitation can be performed, and biomolecules can be detected quickly and reproducibly. There is an effect that can be.
Since the biomolecule detection kit of the third invention includes the oil-based coating liquid, the droplets can be increased in volume with the oil-based coating liquid, and the effect of saving expensive detection molecules can be achieved. In addition, since the biomolecule detection kit of the third invention uses a low-viscosity oil-based coating solution of 0.7 to 18.6 mPa · s, the droplet can be vibrated at high speed even when the droplet is coated. In addition, the droplets can be covered over a wide area and the evaporation of the droplets can be efficiently suppressed, so that the biomolecules can be detected quickly and reproducibly. Furthermore, since the biomolecule detection kit of the third invention uses a plate having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof, a droplet is formed in the droplet formation region, The droplets can be coated with the oil-based coating liquid as long as the oil-repellent region is not exceeded. As a result, the oil-repellent region surrounds the outer periphery of the oil-based coating liquid, and the coated droplets can be prevented from collapsing, so that sufficient electric field agitation can be performed, and biomolecules can be rapidly and reproducibly reproduced. There is an effect that can be detected.

It is drawing which shows one Embodiment of the detection method of the biomolecule of this invention. 1A to 1C are schematic diagrams drawn corresponding to the steps A) to C) of the biomolecule detection method of the present invention. It is drawing which shows the continuation of FIG. (D) and (E) of FIG. 2 are schematic diagrams drawn corresponding to the steps D) and E) of the biomolecule detection method of the present invention. 2 is a diagram illustrating an embodiment in which droplets vibrate periodically in the present invention. FIG. 3A shows a state in which the positive voltage supplied to the upper electrode (9) is maximum. FIG. 3B shows a state in which the voltage supplied to the upper electrode (9) is minimized. The liquid droplets periodically reciprocate between the states shown in FIGS. 3A and 3B. It is the top view and sectional drawing which show embodiment of the plate for biomolecule detection of this invention. FIG. 4 (A) shows a plan view of one embodiment of the biomolecule detection plate of the present invention, and FIG. 4 (B) is a cross-sectional view of the same plate as FIG. 4 (A) and an upper part of the plate. The state of the formed droplet is schematically shown. FIG. 4C schematically shows a sectional view of another embodiment of the biomolecule detection plate of the present invention and the state of a droplet formed on the top. It is the top view and sectional drawing which show another embodiment of the biomolecule detection plate of this invention. FIG. 5A shows a plan view, and FIGS. 5B and 5C schematically show how droplets are formed on the upper part of the plate by cross-sectional views. It is a graph which shows the result of the electric field stirring transpiration experiment at the time of using several types of liquid paraffin from which a viscosity differs. FIG. 6A is a graph showing the change over time in the weight (transpiration amount), and FIG. 6B is a graph showing the relationship between the transpiration amount and the viscosity. It is a graph which shows the measurement result of the area coverage rate at the time of using several types of liquid paraffin from which a viscosity differs. It is a graph which shows the result of having measured the difference (vibration width) of the maximum height and minimum height of the vibrating droplet at the time of using several types of liquid paraffin from which a viscosity differs.

1. 1. Detection method of biomolecule of the present invention 1-1. Outline of Biomolecule Detection Method of the Present Invention The biomolecule detection method of the present invention is a solid biological sample obtained by binding a biomolecule contained in a solid biological sample and a detection molecule specific to the biomolecule. The present invention relates to a method for detecting biomolecules therein.
Here, the term “solid biological sample” means a solid biological sample such as a biological organ, biological tissue, a part thereof, or a section thereof, and is a solution such as protein or nucleic acid extracted from the living organism. Does not mean. As a solid biological sample, not only those that maintain the form of the biological tissue like a section of biological tissue, but also those that are cultured cells, blood, microorganisms etc. in the form of blocks or sheets with embedding agents, An artificially solidified biological sample may be used.

In the present invention, the “biomolecule” refers to a molecule existing in a living body such as protein, nucleic acid, polysaccharide, lipid and the like, and includes those in which these biomolecules are bound to each other like glycoprotein.
In the present invention, the “detection molecule” specific to a biomolecule is not limited to these. For example, an antibody specific for a protein to be detected, a nucleic acid probe having a sequence complementary to a nucleic acid Furthermore, low molecular compounds having affinity for biomolecules, toxins, lectins that are proteins having the ability to bind to sugar chains, and the like can be used.
By using an antibody as a detection molecule, protein can be detected by immunohistochemistry (IHC). By using a labeled probe as a detection molecule, nucleic acid can be detected by in situ hybridization (ISH). Can do.

The biomolecule detection method of the present invention includes the following A) to E).
A) Placing a solid biological sample on the droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof. B) Covering the biological sample with a solution containing detection molecules. C) forming a droplet in the droplet formation region C) using an oil-based coating liquid having a viscosity of 0.7 to 18.6 mPa · s, covering the droplet within a range not exceeding the oil-repellent region, Dome-shaped droplets D) Binding biomolecules contained in a biological sample and detection molecules by performing electric field agitation that oscillates the droplets by applying a varying electric field to the droplets E ) To detect the presence of a biomolecule by a detection molecule bound to the biomolecule.

One embodiment of the biomolecule detection method of the present invention is shown in FIGS.
1A to 1C and FIGS. 2D and 2E correspond to one embodiment of the biomolecule detection method of the present invention corresponding to A) to E) of the present invention. It is the drawn schematic diagram.
First, as shown in FIG. 1 (A), a plate (3) having a droplet formation region (1) and an oil-repellent region (2) disposed on the outer periphery thereof is used, and the droplet formation region (1) A solid biological sample (4) such as a slice of biological tissue is placed thereon. The solid biological sample (4) contains various biomolecules, and this kind of biomolecule (5) is schematically shown by a triangular figure.

Next, as shown in FIG. 1B, a solid biological sample (4) and / or a solution (7) containing a detection molecule (6) capable of specifically binding to the biomolecule (5) is used. By injecting the solution (7) onto the droplet formation region (1), droplets are formed.
Further, as shown in FIG. 1 (C), the droplet formed in FIG. 1 (B) is covered with an oily coating liquid (8) to form a dome-shaped droplet. Here, the oil-based coating liquid (8) may contact the oil-repellent region (2), but injects an amount that does not exceed the oil-repellent isomeric region (2) to cover the droplets. Thereby, since the outer periphery of the oil-based coating liquid (8) is surrounded by the oil-repellent region (2), the oil-based coating liquid (8) tends to be retained on the inner peripheral side of the oil-repellent region (2). The force works to prevent the dome-shaped droplet from collapsing. Although the oil-based coating liquid has a surface tension smaller than that of water, it can prevent the liquid droplet from collapsing in this way, so that sufficient electric field agitation can be performed, and biomolecules can be detected quickly and reproducibly. There is an effect that can be.
Here, since the viscosity of the oil-based coating liquid (8) is as low as 0.7 to 18.6 mPa · s, the droplets can be coated over a wide range as shown in Example 2 below. This produces an effect that the evaporation of the droplets can be efficiently suppressed.

Next, as shown in FIG. 2 (D), the plate (3) on which the droplets are formed is set in a varying electric field applying device. The fluctuating electric field applying device includes an upper electrode (9), a lower electrode (10), and a fluctuating voltage supply device (11). By setting the plate (3), the upper electrode (9 ) And is located on the lower electrode (10) under the plate (3). Then, by supplying the variable voltage by the variable voltage supply device (11), a variable electric field is generated between the electrodes (9, 10). Since the droplet is polarized, a negative charge (12) is present on the surface of the droplet, and the detection molecule (6) present in the droplet may be negatively charged. Therefore, when a fluctuating electric field is generated by the fluctuating electric field applying device, the droplets vibrate periodically according to the frequency of the fluctuating electric field.
Due to the vibration of the droplet, the solution (7) containing the detection molecule (6) is agitated, the chance of collision between the detection molecule (6) and the biomolecule (5) increases, and the detection molecule (6) and the biomolecule ( It becomes possible to carry out the reaction in which 5) is bound in a short time.

When normal oil, for example, vegetable oil, was used as the coating liquid for the droplets, the droplets did not vibrate even when a varying electric field was applied. However, as shown in Example 2 below, in the present invention, By using a low-viscosity oil-based coating liquid having a viscosity of 0.7 to 18.6 mPa · s, the droplets can be vibrated at high speed.
Thus, if a low-viscosity oil-based coating liquid having a viscosity of 0.7 to 18.6 mPa · s is used, the solution containing the detection molecule (6) is bulked to form a oscillating droplet. Therefore, it is possible to save expensive detection molecules.

Next, as shown in FIG. 2 (E), the plate (3) on which the droplet is formed is taken out of the fluctuation electric field applying device, and after the droplet is washed away, the detection molecule (6) bound to the biomolecule (5). The presence of the biomolecule (5) is detected by the color development (fluorescence or reflected light). In order to color the detection molecule (6), for example, a labeling enzyme (chromogenic enzyme), a fluorescent dye, a fluorescent protein, a dye or the like is linked to the detection molecule (6), or these color labels are linked. The developed antibody (secondary antibody) can be colored by binding to the detection molecule (6).
The color development (fluorescence or reflected light) of the detection molecule (6) can be observed with a microscope (15) or the like, and the localization position and expression level of a biomolecule in a solid biological sample such as a biological tissue can be determined by color. Can be imaged.

As described above, according to the present invention, an oily coating liquid having a low viscosity of 0.7 to 18.6 mPa · s is used, so that it is possible to vibrate the droplet at high speed even when the droplet is coated. At the same time, the droplets can be covered over a wide area and the evaporation of the droplets can be efficiently suppressed. For this reason, the droplets are efficiently stirred, the reaction between the biomolecule and the detection molecule is promoted, and evaporation is suppressed, so that the biomolecule can be detected quickly and reproducibly.
In addition, according to the present invention, a plate having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof is used to form a droplet in the droplet formation region and to exceed the surrounding oil-repellent region. As long as there is no oil, the droplets are coated with an oily coating solution. For this reason, since the oil-repellent region surrounds the outer periphery of the oil-based coating liquid and the droplets can be prevented from collapsing, sufficient electric field agitation can be performed, and biomolecules can be detected quickly and reproducibly. There is an effect that can be.
In order to perform electric field stirring, it is necessary to form a dome-shaped droplet, and therefore a predetermined amount or more of a reaction liquid containing an expensive detection molecule is required. According to the present invention, the viscosity is 0.7 to By using an oily coating liquid of 18.6 mPa · s, it is possible to increase the volume of the solution containing the detection molecule and form a oscillating liquid droplet, and thus it is possible to save expensive detection molecules. Play.

1-2. Detailed Description of Biomolecule Detection Method of the Present Invention Hereinafter, the biomolecule detection method of the present invention will be described in more detail.
The biomolecule detection method of the present invention includes: A) placing a solid biological sample on a droplet formation region of a plate having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof. .
Here, the “plate” serves as a base for providing a droplet formation region and an oil-repellent region disposed on the outer periphery thereof, and may be any shape without being limited to a flat plate shape.

  In the present invention, the “droplet formation region” is a region on the plate and may be any region as long as a droplet can be formed. The material constituting the droplet formation region may be a hydrophilic material or a water-repellent material, and is not limited to these materials. For example, glass, polyethylene, polystyrene, etc. Plastic or the like can be used. The material for the droplet formation region may be the same material as the plate, or a material different from the plate may be applied to the plate surface to form the droplet formation region. The cross-sectional shape of the droplet formation region may be any shape as long as the droplet can be formed, and may be a flat plate shape without unevenness or a shape having a depression or a step. The planar shape of the droplet formation region may be any shape as long as the droplet can be formed, but a circular or elliptical shape is preferable.

In the present invention, the “oil repellent region” refers to a region formed of an oil repellent material on the plate surface, and preferably a region formed of a material different from the droplet formation region. The oil-repellent material may be a hydrophilic oil-repellent material or a water- and oil-repellent material, and is not limited to these, but for example, an oil-repellent plastic such as polyvinyl alcohol or silicone resin, or a fine surface Depending on the structure, a material that exhibits oil repellency can be used.
The oil repellent area is disposed on the outer periphery of the droplet forming area. For this reason, when the planar shape of the droplet formation region is circular or elliptical, the oil-repellent region is also circular or elliptical, and droplets having a circular planar shape can be formed. A circular droplet is a preferred shape because it can be efficiently stirred in an electric field.

  In the present invention, it is preferable that a water-repellent / lipophilic region is further provided at the boundary between the droplet forming region and the oil-repellent region on the plate surface, and the droplet forming region is hydrophilic. When such a plate is used, when a droplet is formed with a solution containing a detection molecule, the droplet is surrounded by the water-repellent / lipophilic region, and the droplet remains in the hydrophilic droplet formation region. The force to try acts, and it becomes easy to form a droplet. Then, when the droplet is coated with the oil-based coating liquid, the coating liquid is surrounded by the oil-repellent region, and a force to stay in the water-repellent / lipophilic region works to maintain the droplet more stably. There is an effect.

  In the step A) of the present invention, a solid biological sample is placed on the droplet formation region of the plate. Here, as described above, the “solid biological sample” means a solid biological sample such as a biological organ, biological tissue, part thereof, or a section thereof. Solid biological samples are not only those that maintain the form of biological tissues, such as slices of biological tissues, but also those that are cultured cells, blood, microorganisms, etc. that are made into blocks or sheets with embedding agents. Alternatively, an artificially solidified biological sample may be used.

In the present invention, as the “solid biological sample”, a chemically or physically fixed sample may be used, or a non-fixed sample may be used. Examples of chemical fixation include, but are not limited to, for example, formaldehyde, glutaraldehyde, dimethyl suberimidate dihydrochloride, osmium tetroxide, and the like infiltrated into living tissue or cells. There is a method of immobilization by cross-linking the polymer substance inside. In addition, the physical fixation is not limited to these, but there is, for example, a method of immobilizing a macromolecular substance in a living tissue or cell by boiling or thermal coagulation by microwave irradiation or freezing. In addition, a combination of these may be used, for example, a solid biological sample may be rapidly frozen and replaced and fixed in formalin / acetone. In addition, a solid biological sample may be subjected to a treatment such as antigen activation.
Usually, when a part of an organ or biological tissue is used as a solid biological sample, it is difficult to make a microscopic observation with the same size, so a slice with a certain thickness is prepared. The method for preparing the section is not limited to these. For example, a method in which a solid biological sample is embedded in a frozen tissue embedding agent such as an OCT compound, frozen, and sliced with a frozen microtome, There are a method of slicing a solid biological sample as it is with a vibrating microtome without freezing, a method of embedding a solid biological sample in paraffin, cooling and hardening, and then slicing with a microtome.

The biomolecule detection method of the present invention includes B) forming a droplet in a droplet formation region so as to cover the biological sample with a solution containing the detection molecule.
Here, the “detection molecule” refers to a molecule that can specifically bind to a biomolecule to be detected. “Biomolecules” for detection purposes refer to molecules existing in living organisms such as proteins, nucleic acids, polysaccharides, lipids, etc., and include those in which these biomolecules are bound together such as glycoproteins.
In the present invention, the “detection molecule” is not limited to these, but includes, for example, an antibody specific for a protein to be detected, a nucleic acid probe having a sequence complementary to a nucleic acid, and an affinity for a biomolecule. A low molecular weight compound, a toxin, a lectin that is a protein having an ability to bind to a sugar chain, or the like can be used.

In the biomolecule detection method of the present invention, C) an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s is used, and the liquid droplets are coated within a range not exceeding the oil-repellent region. Including dropping.
Here, “viscosity” refers to the viscosity at room temperature and can be measured with a rotary rheometer (viscosity measuring device) under the condition of 23.8 ° C.
The viscosity of the oil-based coating liquid needs to be higher than the viscosity of water 0.7 mPa · s in order to sufficiently vibrate the droplets, and as shown in Example 2 described later, In order to sufficiently vibrate and cover the surface of the droplet, it is necessary to set it to 18.6 mPa · s or less.
The viscosity of the oil-based coating liquid is more preferably 0.7 to 13.5 mPa · s, and still more preferably 0.7 to 5.5 mPa · s.

In the present invention, “oiliness” refers to the property of separating from water. The “oil-based coating liquid” is not limited to these, but for example, liquid paraffin, squalene, fatty acids, esters and the like can be used. Among these, liquid paraffin having high stability is preferably used.
Liquid paraffin is also called mineral oil or baby oil, and is a low-viscosity oil containing a large amount of alkanes with a small number of carbon atoms. Various kinds of liquid paraffin having different compositions and viscosities are commercially available. In the present invention, those having a viscosity of 0.7 to 18.6 mPa · s are used.

  In C) of the present invention, the oil-based coating liquid is used to cover the droplets within the range not exceeding the oil-repellent region. The “range not exceeding the oil-repellent region” means that the oil-based coating liquid is oil-repellent. Although it may contact the region, it means a range in which the outer edge of the coating liquid does not reach the outer periphery of the oil-repellent region and is surrounded by the oil-repellent region.

In the biomolecule detection method of the present invention, D) the biomolecule contained in the biological sample and the detection molecule are combined by performing electric field agitation that oscillates the droplet by applying a varying electric field to the droplet. Including.
Here, the variable electric field means an electric field whose intensity varies periodically. Due to this fluctuating electric field, the droplets vibrate due to the Coulomb force that periodically changes on the surface or inside of the droplets.

One embodiment in which the droplets periodically oscillate is described with reference to FIG.
FIG. 3A shows the same state as FIG. In the state shown in this figure, the positive voltage supplied to the upper electrode (9) is maximized. The lower electrode (10) is grounded and the potential is fixed at zero. In this state, an electric field indicated by lines of electric force directed from the upper electrode (9) to the lower electrode (10) is generated, and the negative charge (12) of the droplet is directed to the positive charge (13) side of the upper electrode. Coulomb force works and the droplet is attracted to the upper electrode (9).

On the other hand, FIG. 3B shows a state in which the voltage supplied to the upper electrode (9) is the minimum zero. In this state, the action of the electric field disappears, the force that attracts the droplet to the upper electrode disappears, the solution above the droplet drops due to gravity, and the droplet is crushed by the slide glass with that momentum. Become.
The variable voltage supply device periodically reciprocates between the state shown in FIG. 3A and the state shown in FIG. 3B in order to periodically change the voltage supplied to the upper electrode (9). As a result, the droplets vibrate periodically. The droplets crushed on the slide glass in the state of FIG. 3B bounce due to the reaction of the slide glass, and can smoothly return to the state of FIG. 3A in accordance with the change in the electric field.
The application of the fluctuating electric field is not limited to this embodiment, and may be performed by supplying a negative voltage that periodically fluctuates to the upper electrode, and fluctuations having a sign opposite to that of the fluctuating voltage supplied to the upper electrode. You may carry out by supplying a voltage to a lower electrode.

The biomolecule detection method of the present invention includes E) detecting the presence of a biomolecule with a detection molecule bound to the biomolecule.
The detection in E) can be detected using, for example, color development or radiation of the detection molecule. The method for coloring the detection molecule is not limited to these methods. For example, a method in which a labeling enzyme (chromogenic enzyme), a fluorescent dye, a fluorescent protein, a dye or the like is linked to the detection molecule in advance, or detection is performed. After the molecule and the biomolecule are bonded, the detection molecule can be colored by a method in which an antibody or the like linked with a coloring label is bonded to the detection molecule. In addition, as a method of emitting radiation from the detection molecule, a method of labeling the detection molecule by incorporating a radioisotope in advance can be used.

In the biomolecule detection method of the present invention, detection by immunohistochemistry (IHC) can be performed using an antibody as a detection molecule. In addition, detection by in situ hybridization (ISH) can be performed using a labeled probe having a sequence complementary to the target nucleic acid as a detection molecule. These methods will be described in detail later.
Examples of methods other than these include, but are not limited to, a method of detecting by binding a lectin to a sugar chain of a glycoprotein, and detecting by binding a toxin called phalloidin to a protein called F-actin. And a method of detecting a protein that binds to a nucleic acid with a nucleic acid labeled with a fluorescent dye (Southwestern).

2. Biomolecule Detection Plate of the Present Invention The biomolecule detection plate of the present invention is characterized by having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof.
Here, the “droplet formation region” and the “oil repellency region” are the same as those described in “1. Biomolecule Detection Method of the Present Invention”. The biomolecule detection plate of the invention will be further described.

FIG. 4 is a plan view and a cross-sectional view schematically showing an embodiment of the biomolecule detection plate of the present invention. FIG. 4 (A) is a plan view showing one embodiment of the biomolecule detection plate of the present invention, and FIG. 4 (B) is the same plate as FIG. 4 (A) and the liquid formed thereon. It is sectional drawing which shows the state of a drop typically.
As shown in the plan view of FIG. 4A, a droplet forming region (1) is provided on the surface of the plate (3), and an oil repellent region (2) is provided on the outer periphery thereof. As shown in the cross-sectional view of FIG. 4B, the oil-based coating liquid (8) is surrounded by the oil-repellent region (2) and tries to stay on the inner peripheral side of the oil-repellent region (2). It is possible to prevent the dome-shaped droplet from collapsing.

  FIG. 4C is a cross-sectional view schematically showing another embodiment of the biomolecule detection plate of the present invention. In the embodiment shown in FIG. 4B, the droplet formation region (1) and the oil-repellent region (2) are in the same plane, but in the embodiment shown in FIG. 4C, the droplet formation region. A step is provided between (1) and the oil-repellent region (2), and the oil-repellent region (2) is higher. By providing such a step, the dome-shaped droplet can be further stabilized.

  As described above, the biomolecule detection plate of the present invention has a droplet formation region on the plate and an oil-repellent region disposed on the outer periphery thereof. The droplets can be coated with the oil-based coating liquid as long as the oil-repellent region is not exceeded. For this reason, since the oil-repellent region surrounds the outer periphery of the oil-based coating liquid and the droplets can be prevented from collapsing, sufficient electric field agitation can be performed, and biomolecules can be detected quickly and reproducibly. There is an effect that can be.

FIG. 5 is a plan view and a cross-sectional view showing another embodiment of the biomolecule detection plate of the present invention. As shown in the plan view of FIG. 5A, a water / lipophilic / oleophilic region (16) is provided at the boundary between the droplet forming region (1) and the oil-repellent region (2). In this embodiment, the droplet formation region (1) is hydrophilic.
FIGS. 5B and 5C are cross-sectional views schematically showing the same plate as FIG. 5A and a state in which droplets are formed thereon. As shown in FIG. 5B, when the liquid (7) is injected into the hydrophilic droplet formation region to form a droplet, the droplet is surrounded by the water-repellent / lipophilic region (16), A force to stay in the hydrophilic droplet formation region (1) works, and droplets can be easily formed. Then, as shown in FIG. 5C, when the droplet is coated with the oil-based coating liquid (8), the oil-based coating liquid (8) is surrounded by the oil-repellent region (2), A force to stay in the water-repellent / lipophilic region (16) works, and the dome-shaped droplet can be prevented from collapsing.
By using the plate having such a structure, the dome-shaped droplet can be more stably maintained.

3. Biomolecule detection kit of the present invention The biomolecule detection kit of the present invention comprises a biomolecule detection plate, a solution containing a detection molecule that can specifically bind to a biomolecule, and a viscosity of 0.7 to 18.6 mPa · s oily coating liquid.
Here, as the “biomolecule detection plate”, the same plate as described in “2. The biomolecule detection plate of the present invention” can be used. The “solution containing a detection molecule capable of specifically binding to a biomolecule” and the “oil-based coating solution having a viscosity of 0.7 to 18.6 mPa · s” are described in “1. Detection of Biomolecule of the Present Invention”. The same as described in “Method” can be used.

  Since the biomolecule detection kit of the present invention contains an oil-based coating liquid, the droplets can be increased in volume with the oil-based coating liquid, and the effect of saving expensive detection molecules can be achieved. In addition, the biomolecule detection kit of the present invention uses a low-viscosity oil-based coating solution of 0.7 to 18.6 mPa · s, so that the droplet can be vibrated at high speed even when the droplet is coated. In addition, since the droplets can be coated over a wide area and the evaporation of the droplets can be efficiently suppressed, the biomolecules can be detected quickly and reproducibly. Furthermore, since the biomolecule detection kit of the present invention uses a plate having a droplet formation region and an oil repellency region disposed on the outer periphery thereof, a droplet is formed in the droplet formation region and the oil repellency around it is formed. The liquid droplets can be coated with an oily coating liquid as long as the area is not exceeded. As a result, the oil-repellent region surrounds the outer periphery of the oil-based coating liquid, and the coated droplets can be prevented from collapsing, so that sufficient electric field agitation can be performed, and biomolecules can be rapidly and reproducibly reproduced. There is an effect that can be detected.

4). Biomolecule detection device of the present invention The biomolecule detection device of the present invention includes a biomolecule detection plate, a heating and heat-retaining device that heats a droplet formed on the plate to maintain a constant temperature, and a fluctuation electric field in the droplet. And a fluctuating electric field applying device for applying.
Here, as the “biomolecule detection plate”, the same plate as described in “2. The biomolecule detection plate of the present invention” can be used.
The “heat insulation device” in the biomolecule detection apparatus of the present invention is based on a heater that heats a droplet or its surroundings, a thermometer that measures the temperature of the droplet or its surroundings, a measurement result of the thermometer, and a set temperature. And a control circuit for controlling the heater.
The “fluctuating electric field applying device” in the biomolecule detection apparatus of the present invention is the same as that described in “1. Biomolecule detection method of the present invention”.

  Since the biomolecule detection device of the present invention uses a plate having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof, a droplet is formed in the droplet formation region, and the oil-repellent region around it is formed. The liquid droplets can be coated with an oily coating liquid as long as it does not exceed the range. For this reason, since the oil-repellent region surrounds the outer periphery of the oil-based coating liquid and the droplets can be prevented from collapsing, sufficient electric field agitation can be performed, and biomolecules can be detected quickly and reproducibly. There is an effect that can be.

5. Method for Producing Biological Sample Specimen of the Present Invention The method for producing a biological sample specimen of the present invention is a biological sample obtained by binding a biomolecule contained in a solid biological sample and a detection molecule specific to the biomolecule. The present invention relates to a method for producing a biological sample specimen for molecular detection.
The method for producing a biological sample specimen of the present invention comprises:
A) A solid biological sample is placed on a droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof,
B) A droplet is formed in the droplet formation region so as to cover the biological sample with the solution containing the detection molecule,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) The biomolecule contained in the biological sample and the detection molecule are combined by performing electric field stirring that oscillates the droplet by applying a varying electric field to the droplet.
It is characterized by including.

  According to the method for producing a biological sample specimen of the present invention, it is possible to increase the volume of a droplet with an oil-based coating liquid, and thus it is possible to save expensive detection molecules. Moreover, according to the method for producing a biological sample specimen of the present invention, since a low-viscosity oil-based coating liquid of 0.7 to 18.6 mPa · s is used, the droplet is vibrated at high speed even when the droplet is coated. In addition, since it is possible to efficiently cover the droplets by covering the droplets in a wide range and efficiently suppress the evaporation of the droplets, it is possible to produce a biological sample specimen quickly and with good reproducibility. Furthermore, according to the method for producing a biological sample specimen of the present invention, a plate having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof is used to form a droplet in the droplet formation region and to surround it. The liquid droplets are coated with an oil-based coating solution within a range not exceeding the oil-repellent region. As a result, the oil-repellent region surrounds the outer periphery of the oil-based coating liquid, and the coated droplets can be prevented from collapsing, so that sufficient electric field agitation can be performed, and biomolecules can be rapidly and reproducibly reproduced. There is an effect that can be detected.

6). In Situ Hybridization Method of the Present Invention In the in situ hybridization method of the present invention, a nucleic acid contained in a solid biological sample and a labeled probe containing a sequence complementary to the nucleic acid are hybridized to each other in the biological sample. It is invention regarding the method of detecting the nucleic acid contained in.
The in situ hybridization method of the present invention comprises:
A) A solid biological sample is placed on a droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof,
B) A droplet is formed in the droplet formation region so as to cover the biological sample with the hybridization solution containing the labeled probe,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) Hybridizing the nucleic acid contained in the biological sample with the labeled probe by performing electric field stirring that oscillates the droplet by applying a varying electric field to the droplet,
E) detecting the label of the labeled probe hybridized to the nucleic acid,
It is characterized by including.

  In the in situ hybridization method of the present invention, as described in “1. Biomolecule Detection Method of the Present Invention”, A) to E) are the detection target biomolecules as nucleic acids and the detection molecules as the detection molecules. In this method, a labeled probe having a sequence complementary to a nucleic acid is used, and the presence of a biomolecule (nucleic acid) is detected by labeling the labeled probe.

Hereinafter, the in situ hybridization method of the present invention will be described in more detail.
As the “nucleic acid” to be detected in the in situ hybridization of the present invention, DNA present on the chromosome and mRNA present in the nucleus and cytoplasm can be targeted. By using a labeled probe having a sequence complementary to these DNA and mRNA, DNA on the chromosome and mRNA present in the nucleus and cytoplasm can be detected.
In the method for detecting DNA on a chromosome, it is possible to determine where the DNA fragment is located on the chromosome and to know the copy number. In addition, in the method for detecting mRNA, the expression distribution and expression intensity of mRNA in the cell can be known.

For detection by in situ hybridization, FISH (Fluorescence in situ hybridization) using a fluorescent label and CISH (Chromogenic in situ hybridization) using an enzyme label are known, and a radioactive label can also be used.
The nucleic acid used for the probe is not limited to these, and for example, synthetic oligo DNA, single-stranded RNA, double-stranded cDNA, single-stranded cDNA, and the like can be used.

A labeled probe can be obtained by directly linking a fluorescent label, an enzyme label, a radioactive label or the like to a probe nucleic acid, or can be obtained by linking a hapten substance to a probe nucleic acid as a label.
When a hapten substance is linked to a probe nucleic acid, the probe nucleic acid can be indirectly labeled using a hapten-recognizing antibody linked with a fluorescent label, an enzyme label, or the like.
As the hapten substance, digoxigenin, TT dimer, dinitrophenyl and the like can be used. By labeling multiple types of nucleic acid probes with different hapten substances, it is possible to detect multiple types of genes simultaneously.

According to the in situ hybridization method of the present invention, it is possible to increase the volume of a droplet with an oily coating solution, so that it is possible to save an expensive labeled probe. In addition, according to the in situ hybridization method of the present invention, an oily coating solution having a low viscosity of 0.7 to 18.6 mPa · s is used, so that even when the droplet is coated, the droplet can be vibrated at high speed. In addition, it is possible to coat the droplets over a wide area and efficiently suppress the evaporation of the droplets, so that the nucleic acid can be detected quickly and reproducibly. Furthermore, according to the in situ hybridization method of the present invention, a droplet having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof is used to form droplets in the droplet formation region and the surrounding repellency. The droplets are coated with an oil-based coating liquid within a range not exceeding the oil-based region. As a result, the oil-repellent region surrounds the outer periphery of the oil-based coating liquid, and the coated droplets can be prevented from collapsing, so that sufficient electric field agitation can be performed, and nucleic acids can be rapidly and reproducibly reproduced. There is an effect that it can be detected.
When the in situ hybridization method of the present invention is used, for example, as shown in Example 1 described later, in situ hybridization that requires about 20 to 22 hours in the normal method can be shortened to 6 hours. .

7). Immunohistochemical method of the present invention The immunohistochemical method of the present invention is a method in which a biomolecule contained in a solid biological sample and a biomolecule contained in the biological sample are combined with an antibody specific to the biomolecule. The present invention relates to a method for detecting molecules.
The immunohistochemical method of the present invention comprises:
A) A solid biological sample is placed on a droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof,
B) A droplet is formed in the droplet formation region so as to cover the biological sample with the solution containing the antibody,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) By binding the biomolecule contained in the biological sample and the antibody by performing electric field stirring that oscillates the droplet by applying a varying electric field to the droplet,
E) detecting antibodies bound to biomolecules,
It is characterized by including.

In the immunohistochemical method of the present invention, A) to E) are methods in which an antibody is used as a detection molecule, as described in “1. Biomolecule detection method of the present invention”.
Hereinafter, the immunohistochemical method of the present invention will be described in more detail.
In the step E) of the immunohistochemical method of the present invention, as a technique for detecting an antibody, an enzyme antibody method for detecting an antibody by coloring with an enzyme using an antibody linked with an enzyme such as peroxidase or alkaline phosphatase as a label. And a fluorescent antibody method in which an antibody in which a fluorescent dye is linked as a label and an antibody is detected by fluorescence is a two-column technique.

Of these two methods, the enzyme antibody method is suitable for detecting biomolecules in tissue sections for pathological diagnosis and the like. The enzyme antibody method will be described in detail. Examples of the enzyme used as a label include, but are not limited to, peroxidase (horseradish peroxidase: HRP), alkaline phosphatase (alkaline).
Enzymes such as phosphatase (AP) and glucose oxidase (β-D galactosidase: GAL) can be used.
As a substrate for these enzymes, for example, 3,3'-diaminobenzidine (DAB) can be used to develop a brown color, or 3-amino-9-ethylcarbazole (AEC) can be used as a red color for peroxidase. Can develop color.

  Enzyme antibody methods are roughly classified into direct methods and indirect methods. In the direct method, an antibody is directly linked to an antibody and labeled, and then reacted with an antigen. On the other hand, the indirect method is a method in which the antibody (primary antibody) against the antigen to be detected is not enzyme-labeled but the antibody against the antibody (secondary antibody) is enzyme-labeled and detected. The indirect method has more reactions than the direct method, but if the primary antibody is made from the same species of animal, all kinds of immunostaining can be performed with one type of secondary antibody, and the sensitivity is also excellent. Yes. Therefore, indirect methods are often used in enzyme antibody methods.

  According to the immunohistochemical method of the present invention, the droplets can be increased in volume with the oil-based coating solution, so that it is possible to save expensive antibodies. In addition, according to the immunohistochemical method of the present invention, since a low viscosity oily coating solution of 0.7 to 18.6 mPa · s is used, the droplets can be vibrated at high speed even when the droplets are coated. In addition, it is possible to cover the liquid droplets over a wide area and efficiently suppress the evaporation of the liquid droplets, so that the biomolecules can be detected quickly and reproducibly. Furthermore, according to the immunohistochemical method of the present invention, a droplet having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof is used to form droplets in the droplet formation region and the surrounding repellency. The droplets are coated with an oil-based coating liquid within a range not exceeding the oil-based region. As a result, the oil-repellent region surrounds the outer periphery of the oil-based coating liquid, and the coated droplets can be prevented from collapsing, so that sufficient electric field agitation can be performed, and biomolecules can be rapidly and reproducibly reproduced. There is an effect that can be detected.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

(Example 1: In situ hybridization by the method of the present invention)
A) Using the kit reagent of ZytoDot 2C SPEC HER2CEN 17 Probe Kit (manufactured by ZytoVision), the protocol was improved and in situ hybridization was performed. A specimen of a breast cancer tissue that had been pretreated (deparaffinized and activated) for 1 hour according to the protocol was used as a test sample. In order to prepare a plate to be used for in situ hybridization, an oil-repellent sheet 3M label cover (manufactured by 3M) was used, the center of the sheet was cut out in a circle, and this was pasted on a slide glass. A section of breast cancer tissue was placed on a circular area surrounded by a sheet on a slide glass.
B) 10 μl of the DNA probe solution of the kit was heat-treated at 80 ° C. and then dropped on a section of breast cancer tissue. The DNA probe solution is a cocktail of a probe for HER2 gene labeled with digoxigenin (DIG) and a probe for chromosome centromere 17 (CEN17) labeled with dinitrophenyl (DNP).
C) A droplet of the DNA probe solution was covered with 40 μl of liquid paraffin (Hicol K-140N manufactured by Kaneda) to form a dome-shaped droplet. The viscosity of the liquid paraffin used was 5.5 mPa · s at 23.8 ° C.
D) A hybridization reaction was performed while stirring the electric field for 3 hours by supplying a fluctuating electric field by applying a fluctuating voltage of 15 Hz and 4,5 kV using a fluctuating electric field applying device.
E) After hybridization, washing was performed, and 30 μl of a cocktail solution of mouse anti-DIG antibody and rabbit anti-DNP antibody was dropped to form a droplet, which was then covered with 30 μl of liquid paraffin to form a dome-shaped droplet. The antigen-antibody reaction was carried out while the droplets were stirred for 20 minutes by applying a varying electric field by applying a varying voltage of 15 Hz and 4,5 kV using a varying electric field application device. After washing, an antigen-antibody reaction was performed for 30 minutes by the same method using a cocktail solution of HRP-labeled anti-mouse antibody and ALP-labeled anti-rabbit antibody. Next, a coloring reaction with a labeling enzyme was performed for 15 minutes using HRP-Green, and the HER2 gene was stained green and visualized. Further, a color development reaction with a labeling enzyme was performed for 15 minutes using AP-Red, and CEN17 was stained in red and visualized.
(Result of Example 1)
Staining was successful in 85 of 87 samples, and the success rate was higher than that of the protocol method. In addition, the entire process of in situ hybridization was successfully shortened to about 6 hours.

(Comparative Example 1)
In-situ hybridization was performed according to the protocol using a kit reagent of ZytoDot 2C SPEC HER2CEN 17 Probe Kit (manufactured by ZytoVision).
An ordinary slide glass was used as a plate, coating with liquid paraffin and electric field stirring were not performed, hybridization was performed for 16 to 18 hours, and antigen-antibody reaction was performed for 30 minutes each, and was performed in the same manner as in Example 1.
(Results of Comparative Example 1)
Staining was successful in 82 of 87 samples. In addition, it took about 20-22 hours for the whole process of in situ hybridization.

(Comparative Example 2)
Using the kit reagent of ZytoDot 2C SPEC HER2CEN 17 Probe Kit (manufactured by ZytoVision), the protocol was improved and in situ hybridization was performed.
The same treatment as in Example 1 was performed, except that 40 μl of water was added to 10 μl of the DNA probe solution to increase the bulk by forming the dome-shaped droplet and stirring the electric field without coating with liquid paraffin. .
(Results of Comparative Example 2)
All processing steps of in situ hybridization were performed, but staining was not successful.

(Comparative Example 3)
Using a kit reagent of ZytoDot 2C SPEC HER2CEN 17 Probe Kit (manufactured by ZytoVision), the protocol was improved and in situ hybridization was performed.
The same treatment as in Example 1 was performed except that the oil-repellent sheet was not used.
(Result of Comparative Example 3)
When electric field stirring was performed, the droplets collapsed, and subsequent processing could not be performed.

(Example 2: Examination of viscosity of liquid paraffin)
Using six kinds of liquid paraffin having different viscosities, the optimum viscosity when used as a coating liquid for electric field stirring was investigated. The names of the six types of liquid paraffin and the viscosities measured using HAAKE RheoStress 1 (registered trademark, manufactured by Thermo Scientific) under the condition of 23.8 ° C. are as follows.
・ K-140N (manufactured by Kaneda) Viscosity = 5.5 mPa · s
-K-160 (manufactured by Kaneda) Viscosity = 13.5 mPa · s
・ K-230 (manufactured by Kaneda) Viscosity = 18.6 mPa · s
・ K-290 (manufactured by Kaneda Co., Ltd.) Viscosity = 57.8 mPa · s
・ K-350 (manufactured by Kaneda) Viscosity = 171.0 mPa · s
E-7 (manufactured by Kaneda) Viscosity = 143.0 mPa · s
(Field stirring transpiration experiment)
The oil-repellent sheet cut out in the center was pasted on a slide glass. After washing with a washing solution (0.05% addition of Tween 20 to DakoWashing buffer), 10 μl of TE buffer was injected into a circular area surrounded by the sheet on the slide glass to form droplets. Furthermore, 40 μl of liquid paraffin is injected to form a dome-shaped droplet, and a variable electric field is applied to stir the electric field, and the weight at the lapse of 60 minutes, 90 minutes, 120 minutes, and 180 minutes is measured. The amount of transpiration was measured. The result is shown in FIG.
FIG. 6A is a graph showing the change over time in the weight (transpiration amount), and FIG. 6B is a graph showing the relationship between the transpiration amount and the viscosity.
From this result, it became clear that the lower the viscosity, the less the transpiration amount.
(Measurement of area coverage rate)
For the dome-shaped droplets on the slide glass used in the electric field stirring transpiration experiment, a planar image was taken, and the ratio of the area where the TE buffer was covered with liquid paraffin (area coverage) was measured. The result is shown in FIG.
As shown in FIG. 7, the lower the viscosity of liquid paraffin, the larger the area coverage, and when using three liquid paraffins of K-140N, K-160 and K-230 having a viscosity of 18.6 mPa · s or less. In particular, the area coverage was large.
(Droplet behavior observation)
The dome-shaped droplet on the slide glass subjected to the electric field agitation transpiration experiment was photographed from the side, and the difference (vibration width) between the maximum height and the minimum height of the vibrating droplet was measured. The result is shown in FIG.
As shown in FIG. 8, when the viscosity of liquid paraffin is lower, the vibration width is larger, and three liquid paraffins of K-140N, K-160 and K-230 having a viscosity of 18.6 mPa · s or less are used. In particular, it was confirmed that the vibration width was particularly large and sufficient electric field stirring was performed. On the other hand, when the viscosity of liquid paraffin was large, sufficient electric field stirring was not performed. Although no data is shown, a fluctuating electric field was applied using vegetable oil with high viscosity instead of liquid paraffin as a reference experiment, but electric field stirring could not be performed.

  The biomolecule detection method, biomolecule detection plate, biomolecule detection kit, biomolecule detection apparatus, biosample preparation method, in situ hybridization method, and immunohistochemistry method of the present invention are rapid and reproducible. It is useful in the field of clinical testing and clinical laboratory reagents because it can detect molecules and save expensive reagents.

DESCRIPTION OF SYMBOLS 1 Droplet formation area 2 Oil-repellent area 3 Plate 4 Solid biological sample 5 Biomolecule 6 Detection molecule 7 Solution 8 Oil-based coating liquid 9 Upper electrode 10 Lower electrode 11 Fluctuating voltage supply device 12 Charge of droplet 13 Charge of upper electrode 15 Microscope 16 Water repellent / lipophilic area

Claims (14)

In a method of detecting the biomolecule in the solid biological sample by binding a biomolecule contained in the solid biological sample and a detection molecule specific to the biomolecule,
A) placing the solid biological sample on the droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof;
B) Forming a droplet in the droplet formation region so as to cover the biological sample with the solution containing the detection molecule,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) combining the biomolecules contained in the biological sample with the detection molecules by performing electric field agitation to vibrate the droplets by applying a varying electric field to the droplets;
E) detecting the presence of the biomolecule by the detection molecule bound to the biomolecule;
A method for detecting a biomolecule.   The method for detecting a biomolecule according to claim 1, wherein the coating liquid is a coating liquid having a viscosity of 0.7 to 5.5 mPa · s.   The method for detecting a biomolecule according to claim 1 or 2, wherein the coating liquid is liquid paraffin.   The said plate has a water-repellent lipophilic area | region in the boundary part of the said droplet formation area and the said oil-repellent area, The said droplet formation area is any one of Claims 1-3. Biomolecule detection method.   The method for detecting a biomolecule according to any one of claims 1 to 4, wherein the biomolecule is a nucleic acid, and the detection molecule is a labeled probe having a sequence complementary to the nucleic acid.   The method for detecting a biomolecule according to any one of claims 1 to 4, wherein the biomolecule is a protein, and the detection molecule is an antibody specific for the protein. In the plate used for detection of biomolecules using electric field stirring,
It possesses a droplet formation region and oil repellent region arranged on its periphery, between the droplet formation region the oil repellent region, the step that is higher towards the oil repellent region is provided, Plate for biomolecule detection. In the plate used for detection of biomolecules using electric field stirring,
A biomolecule detection plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof by providing a plate obtained by cutting out an oil-repellent sheet on the plate. In the plate used for detection of biomolecules using electric field stirring,
A droplet forming region and an oil-repellent region disposed on the outer periphery thereof;
The droplet formation region and has a water-repellent oleophilic areas in the boundary portion of the oil repellent region, the droplet formation region is hydrophilic, BIOLOGICAL molecule detecting plate. In a kit used for detection of biomolecules using electric field stirring,
A biomolecule detection plate having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof ;
A solution containing a detection molecule capable of specifically binding to a biomolecule;
A biomolecule detection kit comprising an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s. In an apparatus used for detection of biomolecules using electric field stirring,
A biomolecule detection plate having a droplet formation region and an oil-repellent region disposed on the outer periphery thereof ;
A heating and heat retention device that heats the droplets formed on the plate and maintains a constant temperature;
A biomolecule detection apparatus comprising: a fluctuation electric field application device that applies a fluctuation electric field to the droplet. In a method for producing a biological sample specimen for biomolecule detection obtained by binding a biomolecule contained in a solid biological sample and a detection molecule specific to the biomolecule,
A) placing the solid biological sample on the droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof;
B) A droplet is formed in the droplet formation region so as to cover the biological sample with the solution containing the detection molecule,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) The biomolecule contained in the biological sample and the detection molecule are combined by performing electric field agitation to vibrate the droplet by applying a varying electric field to the droplet.
A method for producing a biological sample specimen. In an in situ hybridization method for detecting the nucleic acid contained in the biological sample by hybridizing a nucleic acid contained in the solid biological sample and a labeled probe containing a sequence complementary to the nucleic acid,
A) placing the solid biological sample on the droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof;
B) A droplet is formed in the droplet formation region so as to cover the biological sample with a hybridization solution containing the labeled probe,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) Hybridizing the nucleic acid and the labeled probe contained in the biological sample by performing electric field stirring that oscillates the droplet by applying a varying electric field to the droplet,
E) detecting the label of the labeled probe hybridized to the nucleic acid;
An in situ hybridization method. In an immunohistochemical method for detecting the biomolecule contained in the biological sample by binding a biomolecule contained in the solid biological sample and an antibody specific to the biomolecule,
A) placing the solid biological sample on the droplet forming region of a plate having a droplet forming region and an oil-repellent region disposed on the outer periphery thereof;
B) A droplet is formed in the droplet formation region so as to cover the biological sample with the solution containing the antibody,
C) Using an oily coating liquid having a viscosity of 0.7 to 18.6 mPa · s, coating the liquid droplets within a range not exceeding the oil-repellent region to form dome-shaped liquid droplets;
D) by binding the biomolecule contained in the biological sample and the antibody by performing electric field stirring that vibrates the droplet by applying a varying electric field to the droplet,
E) detecting the antibody bound to the biomolecule;
Including immunohistochemistry.