JP2016183124A - Sample holding device, sample holding method and holding member - Google Patents

Abstract

PROBLEM TO BE SOLVED: To store a held object containing a projective agent for protecting a sample, in a dry state over a long period of time.SOLUTION: A sample holding device 10 comprises: a packing member 15; a holding member 12 stored in the packing member 15; and a held object 21 held to one face of the holding member 12, the held object 21 containing a protective agent for protecting the sample. The held object 21 is in a dry state, and inside of the packing member 15 is maintained to a vacuum state.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a sample holding device, a sample holding method, and a holding member.

  Conventionally, quantitative measurement of RNA (ribonucleic acid) and protein such as biomarkers contained in specimens such as blood used in clinical tests is an early detection of diseases such as cancer (cancer), individualized treatment Indispensable for realization. However, since such biomolecules deteriorate very rapidly when extracted outside the body, examinations using stored specimens in the past are not generalized in the medical field. Some molecules used in test kits such as protein chips are vulnerable to the environment and easily deteriorate.

  Therefore, a technique has been proposed in which biomolecules are frozen or dried and stored for a long period (see, for example, Patent Document 1).

JP 2008-44953 A

  However, in the conventional method, a long time of several tens of minutes or more is required to dry a sample such as a biomolecule by freeze drying or vacuum drying. Also, labor and time are required when the dried sample is rehydrated.

  The present invention solves the above-mentioned conventional problems, and a sample holding device, a sample holding method, and a holding member capable of storing an object to be held containing a protective agent for protecting the sample in a dry state for a long period of time The purpose is to provide.

  Therefore, in the sample holding device of the present invention, the packaging member, the holding member accommodated in the packaging member, and the object to be held held on one surface of the holding member, the protective agent protecting the sample A sample holding device comprising: a holding object including: the holding object is dried, and the inside of the packaging member is kept in a vacuum.

  In another sample holding device of the present invention, the object to be held is a thin film.

  In still another sample holding device according to the present invention, the object to be held is an amorphous product obtained by drying a thin liquid film of an aqueous solution formed on one surface of the holding member. .

  In still another sample holding device of the present invention, the holding member is housed in an auxiliary container housed in the packaging member, the one surface being hydrophilic.

  In still another sample holding device of the present invention, the packaging member is a bag made of a material having a gas barrier property, and includes an opening / closing portion that can be hermetically sealed.

  In still another sample holding device of the present invention, the object to be held includes a sample together with the protective agent.

  In still another sample holding device of the present invention, the sample is a biomolecule or a polymer, and the protective agent contains trehalose.

  In the sample holding method of the present invention, a holding member for holding a holding object containing a protective agent for protecting the sample on one side is accommodated in the packaging member, the holding object is dried, and the inside of the packaging member is evacuated. keep.

  In another sample holding method of the present invention, the object to be held can be made into an aqueous solution by releasing the vacuum in the packaging member and hydrating the object to be held.

  In still another sample holding method of the present invention, it is possible to dry the held object again as an aqueous solution again and keep the inside of the packaging member in a vacuum again.

  In the holding member of the present invention, the object to be held containing a protective agent for protecting the sample is held on one surface thereof, and the object to be held is in a thin film shape and becomes amorphous by drying. Has been.

  In another holding member of the present invention, the one surface is a hydrophilic surface.

  In still another holding member of the present invention, the object to be held includes a sample together with the protective agent.

  In still another holding member of the present invention, the sample is a biomolecule or a polymer, and the protective agent contains trehalose.

  ADVANTAGE OF THE INVENTION According to this invention, the sample holding device which can preserve | save the to-be-held thing containing the protective agent which protects a sample over a long period in the dry state can be provided.

It is a schematic perspective view which shows the structure of the sample holding device in embodiment of this invention. It is sectional drawing of the sample holding | maintenance apparatus in embodiment of this invention. It is a perspective view of the packaging member in embodiment of this invention. It is a photograph which shows the side surface of the droplet of the trehalose aqueous solution dropped on the upper surface of the glass plate in embodiment of this invention. It is a photograph of the holding member accommodated in the auxiliary container in embodiment of this invention. It is a perspective view of the apparatus for vacuum-drying the sample in embodiment of this invention. It is a figure which shows the result of having measured the residual effect of the enzyme activity of the sample in embodiment of this invention. It is a figure which shows the infrared absorption spectrum of a known trehalose. It is a figure which shows the relationship between a known bond sound peak and moisture content. It is a figure explaining the method of calculating a moisture content from a known bond sound peak. It is a 1st figure which shows the result of having measured the infrared absorption spectrum of the to-be-held object in embodiment of this invention. It is a 2nd figure which shows the result of having measured the infrared absorption spectrum of the to-be-retained object in embodiment of this invention. It is a 3rd figure which shows the result of having measured the infrared absorption spectrum of the to-be-held object in embodiment of this invention. It is a 4th figure which shows the result of having measured the infrared absorption spectrum of the to-be-retained object in embodiment of this invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  1 is a schematic perspective view showing a configuration of a sample holding device according to an embodiment of the present invention, FIG. 2 is a cross-sectional view of the sample holding device according to the embodiment of the present invention, and FIG. 3 is a packaging member according to the embodiment of the present invention. FIG.

  In the figure, reference numeral 10 denotes a sample holding device according to the present embodiment, which includes an object to be held 21 containing a protective agent for protecting a sample such as a nucleic acid, an enzyme, a hormone, and other biomolecules such as various proteins and polymers. It is a holding device. The sample is, for example, a substance contained in a specimen such as blood used in a clinical test, and is a target for various observations such as pathological tests and biological tests, detection, and measurement.

  As shown in the figure, the sample holding device 10 according to the present embodiment includes an auxiliary container 11, a holding member 12 accommodated in the auxiliary container 11, and a packaging member 15 covering the periphery of the auxiliary container 11. Is provided. In FIG. 1, the packaging member 15 is inflated for convenience of explanation, but when the inside is evacuated, the packaging member 15 is attached to the auxiliary container 11 as shown in FIG. 2. It will be in close contact with the surroundings.

  In the example shown in the figure, the auxiliary container 11 is a container having an open upper surface including a disk-shaped bottom plate 11a and a cylindrical side plate 11b, like a Petri dish, and the upper surface is a disk shape. However, the container may be a rectangular parallelepiped container or any shape. The lid member 14 may be formed separately from the auxiliary container 11 as in the example shown in the drawing, or may be formed integrally with the auxiliary container 11. . The material of the auxiliary container 11 and the lid member 14 is, for example, transparent polystyrene. However, the material is not necessarily transparent and may be any kind of material. Note that at least one of the auxiliary container 11 and the lid member 14 is formed with a vent hole 14a that communicates the inside and the outside. In the example shown in the figure, a plurality of vent holes 14 a are formed in the lid member 14.

  The packaging member 15 is a bag-shaped member made of a resin film such as polypropylene, and is an opening / closing portion 16 into which the auxiliary container 11 can be inserted and removed, and can be hermetically sealed and opened. At least one part 16 is provided. In the example shown in the figure, the packaging member 15 is a rectangular bag in a plan view, and three sides of the rectangle are permanently closed, and the remaining one side is an opening / closing part 16. The material of the packaging member 15 may be any kind of material as long as it has a gas barrier property, does not transmit moisture and oxygen, and has a certain degree of light shielding property. For example, an aluminum foil ( Foil), or a laminate of a plurality of types of resin films, aluminum foils, or the like.

  In the open state, as shown in FIG. 3, the opening / closing part 16 can be separated into an upper film part 16a and a lower film part 16b, and an opening 16c is formed therebetween. Further, in the closed state, the opening / closing part 16 is hermetically sealed by the seal part 17 formed by heating and fusing the upper film part 16a and the lower film part 16b, and moisture and oxygen cannot pass therethrough. It becomes. The seal portion 17 may be of any kind, for example, forming a ridge continuous with the upper film portion 16a, forming a ridge continuous with the lower film portion 16b, The chuck may be an airtightly sealable and openable / closable chuck formed by biting the concave stripes, but here, the upper film part 16a and the lower film part 16b are fused. It will be described as being.

  The holding member 12 is a disk-shaped plate member made of glass, and is accommodated in the auxiliary container 11 in such a posture that its lower surface faces the bottom plate 11a of the auxiliary container 11, and a thin film-like covering is formed on the upper surface thereof. The holding | maintenance thing 21 is provided. The to-be-held object 21 includes a protective agent for protecting the sample and does not necessarily include the sample, but here, for convenience of explanation, a mixed agent in which the sample and the protective agent are mixed. Only the case will be described.

  Further, the object to be held 21 is obtained by drying a thin liquid film of an aqueous solution formed on the upper surface of the holding member 12, is vitrified (amorphized), and does not contain a hydrate. Is. Although the method of drying the thin liquid film may be natural drying, only the case of vacuum drying will be described here for convenience of explanation.

  The sample may be of any kind as long as it is a biomolecule or polymer as described above and is hydrated, that is, can be an aqueous solution. The person used alcohol dehydrogenase (ADH), which is a kind of enzyme sold by Tokyo Chemical Industry Co., Ltd., as a sample and performed an experiment described later. The protective agent may be of any kind as long as it can protect the above-described biomolecules and macromolecules and can form an aqueous solution. Although it is a disaccharide such as sugar, the inventor of the present invention sells trehalose (Trhose) dihydrate sold by Hayashibara Co., Ltd. or the trehalose dihydrate and Sigma-Aldrich Japan LLC. A mixture with dextran (Dextran) was used.

The holding member 12 may be of any type as long as the upper surface is a hydrophilic substrate. However, the inventor of the present invention has a diameter of 20 mm and a thickness of 2 sold by Sigma Koki Co., Ltd. A [mm] calcium fluoride (CaF ₂ ) glass plate (product name: OPCF-20C02-P) having a surface treated was used. The surface treatment was performed for 5 minutes using a desktop plasma surface treatment apparatus (product name: YHS-R) sold by Sakai Semiconductor Co., Ltd. By applying this surface treatment, the upper surface of the glass plate became hydrophilic.

  Next, a method for drying the object 21 provided on the upper surface of the holding member 12 will be described. Here, for convenience of explanation, only the method of vacuum drying will be described.

  4 is a photograph showing a side surface of a droplet of trehalose aqueous solution dropped on the upper surface of a glass plate in the embodiment of the present invention, and FIG. 5 is a photograph of a holding member accommodated in an auxiliary container in the embodiment of the present invention. FIG. 6 is a perspective view of an apparatus for vacuum drying a sample according to the embodiment of the present invention. 4A is a photograph when the surface treatment is not performed on the upper surface of the glass plate, and FIG. 4B is a photograph when the surface treatment is performed on the upper surface of the glass plate.

  First, in a room temperature environment, 5 [μl] of the aqueous solution 21 to be held is plucked using a micropipette on the hydrophilic upper surface of the holding member 12 which is the upper surface of the surface-treated glass plate. In addition, the said to-be-held object 21 is previously given water | moisture content, and is an aqueous solution. Then, as shown in FIG. 4 (b), the droplet of the object to be held 21 spreads thinly on the upper surface of the holding member 12, and as shown in FIG. 5, a thin liquid film having a diameter of 10 [mm]. Formed. Then, the holding member 12 on which the thin liquid film is formed is placed on the bottom plate 11 a of the auxiliary container 11. In addition, the thin liquid film can also be formed by dropping the aqueous solution of the object to be held 21 on the upper surface of the holding member 12 previously placed on the bottom plate 11 a of the auxiliary container 11.

  For comparison, FIG. 4 (a) shows a droplet of an aqueous solution of an object 21 to be held on the upper surface of a glass plate not subjected to surface treatment. In FIG. 5, the holding member 12 is placed on the bottom plate 11 a of the auxiliary container 11. A plurality of black dots on the upper surface of the holding member 12 are oil paint markers, and indicate a circumference having a diameter of 10 mm.

In the example shown in FIGS. 4 and 5, the object to be retained 21 is a mixture of alcohol dehydrogenase as a sample and a mixture of trehalose and dextran as protective agents. In addition, it is known that a thin liquid film is formed when a trehalose aqueous solution is plucked on the upper surface of a surface-treated glass plate, and vitrification occurs when the thin liquid film is rapidly dried. (See, for example, Non-Patent Document 1). And the aqueous solution of the to-be-held object 21 which mixed the alcohol dehydrogenase with the mixture of trehalose and dextran is also picked on the upper surface of the glass plate which surface-treated like the trehalose aqueous solution, and a thin liquid film is formed.
Kiyoshi Takano, Ryo Shirasagi, “Measurement of Infrared Absorption Spectrum and Moisture Content of Aqueous Rapidly Drying Trehalose Aqueous Membrane”, Journal of Low Temperature Biotechnology, Vol.60, No.2, pp.115-118,2014.

  Subsequently, the upper surface of the auxiliary container 11 on which the holding member 12 on which the thin liquid film of the article to be held 21 is formed is placed on the bottom plate 11a is closed by the lid member 14 in a room temperature environment. Then, the auxiliary container 11 whose upper surface is closed by the lid member 14 is stored in a bag-shaped packaging member 15.

  Subsequently, using a vacuum device 30 as shown in FIG. 6 under a room temperature environment, the inside of the packaging member 15 is depressurized and evacuated, and the thin liquid film of the object 21 is vacuum dried. Here, “vacuum” is a pressure range corresponding to a low vacuum (100 [Pa] or more) according to JIS classification.

  The vacuum device 30 may be any type of device as long as the inside of the packaging member 15 can be evacuated and evacuated, but the inventor of the present invention is a tabletop sold by Takato Technica Co., Ltd. A vacuum packaging machine (product name: VP-300) was used.

  The vacuum apparatus 30 includes a chamber 33 formed in the upper portion of the main body 31, an open / close lid 32 attached to the upper portion of the main body 31 via a hinge 32a, and a heating member 35 disposed in the chamber 33. The operation panel 36 disposed on the front surface of the main body 31 and a vacuum pump (not shown) disposed inside the main body 31 are provided. The chamber 33 is a shallow concave portion having a rectangular upper surface opened in a plan view of about 380 mm in length and about 330 mm in width, and the upper surface is hermetically closed by the opening / closing lid 32. . Then, the inside of the chamber 33 that is hermetically closed is decompressed by the vacuum pump. Further, the heating member 35 is heated when energized, and a part of the opening / closing part 16 of the packaging member 15 can be heated and fused.

  Specifically, first, the packaging member 15 is placed in the chamber 33 in such a posture that the opening / closing part 16 is placed on the heating member 35 with the opening / closing lid 32 in the open posture. Subsequently, after opening and closing the open / close lid 32 and locking it, the inside of the chamber 33 closed in an airtight manner is decompressed by operating a vacuum pump. The inventor of the present invention reduced the pressure in the chamber 33 to 42 [Torr] or less in a room temperature environment, and maintained this reduced pressure state for 99 [s]. Thereby, the thin liquid film of the article 21 to be held formed on the holding member 12 is rapidly vacuum-dried.

  Then, when a predetermined pressure reduction time (for example, 99 [s]) has elapsed, the vacuum device 30 stops the vacuum pump and stops the pressure reduction, but immediately before that, the heating member 35 is automatically energized. A seal portion 17 is formed on the opening / closing portion 16. Subsequently, after the opening / closing lid 32 is set in the open posture, when the packaging member 15 is taken out from the chamber 33, as shown in FIG. 2, the sample holding device 10 in which the packaging member 15 is evacuated can be obtained. it can.

  The thin film-like object 21 in the packaging member 15 of the sample holding device 10 obtained as described above is in a state as it is, that is, in a state where it is held in the vacuum-dried packaging member 15 for a long time ( For example, it can be stored in a room temperature environment for several months or more. Similarly, a sample such as a biomolecule or a polymer contained in the thin liquid film of the object to be held 21 can be stored in an inactive state in a room temperature environment for a long period of time. This is presumably because the mixture of trehalose and dextran contained in the article to be held 21 is vitrified and does not contain hydrate and encloses the sample. It is also considered that the sample itself is vitrified.

  In addition, the held object 21 held in the vacuum-dried packaging member 15 in this way opens the opening / closing part 16 by cutting and removing the seal part 17, for example, in the packaging member 15. After releasing the vacuum, it is rehydrated by applying moisture, thereby returning to the state of the aqueous solution, that is, the initial state. Then, the sample contained in the object to be held 21 also returns to the active state, that is, the initial state. Thereby, the sample can be used as an object for various observations, detections, measurements, and the like.

  Furthermore, when the object to be held 21 thus returned to the initial state is again vacuum-dried by the same method and the inside of the packaging member 15 is maintained in a vacuum, it is stored again in a room temperature environment for a long period of time. It becomes possible. In this case, the sample contained in the thin liquid film of the object 21 can be stored again in an inactive state in a room temperature environment for a long time.

  And the to-be-held object 21 of the state vacuum-dried again in this way and hold | maintained in the vacuum packaging member 15 opens | releases the opening-and-closing part 16, releases moisture in the packaging member 15, and gives water | moisture content Then, it returns to the initial state again by hydration. Similarly, the sample contained in the object 21 is again returned to the initial state, and can be used as a target for various observations, detections, measurements, and the like.

  That is, the object to be held 21 that has been vacuum-dried and held in the vacuum packaging member 15 and the sample contained in the object to be held 21 can then be returned to the initial state and stored a plurality of times. Return to the correct state.

  Moreover, the to-be-held object 21 may not include a sample, that is, include only a protective agent. In this case as well, the thin liquid film of the object to be held 21 can be stored in a room temperature environment for a long period of time while being vacuum dried and held in the vacuum packaging member 15. In addition, the return to the initial state and the return to the storable state can be repeated a plurality of times. And when it becomes necessary to preserve | save a sample, the thin liquid film of the to-be-held thing 21 containing only a protective agent is returned to an initial state, the sample aqueous solution was provided there, and the sample and the protective agent were mixed. Since the object 21 to be held, which is a mixture, can be obtained, the thin liquid film of the object 21 is vacuum-dried as described above.

  Furthermore, the method for drying the object to be held 21 is not necessarily limited to vacuum drying, and any method may be used, for example, natural drying.

  Next, the contents and results of the experiment conducted by the inventor of the present invention will be described. First, an experiment for measuring the residual effect of the enzyme activity of a sample will be described.

  FIG. 7 is a diagram showing the results of measuring the residual effect of the enzyme activity of the sample in the embodiment of the present invention. In addition, in the figure, (a) is a figure which shows the measurement result in the case of vacuum drying, (b) is a figure which shows the measurement result in the case of natural drying.

  The inventor of the present invention measured the residual activity of the enzyme activity of alcohol dehydrogenase, which is a sample contained in the object to be held 21 held in the sample holder 10 vacuum-dried according to the above-described method.

As shown in the following formula (1), alcohol dehydrogenase functions as a catalyst for dehydrating ethanol to acetaldehyde and incidentally reducing NAD ⁺ to NADH.

  And the enzyme activity of alcohol dehydrogenase was measured by the fluorescence spectroscopic analysis method according to following Formula (2) using the corona absorption microplate reader (product name: MTP-310) which Hitachi High-Tech Science Co., Ltd. sells.

  Here, A is the absorbance, V is the total amount of the analyte [ml], D is the dilution factor, d is the optical path length, and v is the amount of the aqueous enzyme solution [ml]. In addition, 6.3 is a 1000 molar extinction coefficient of β-NADH at 340 [nm].

  The sample alcohol dehydrogenase is dissolved in ultrapure water (18.3 [MΩ · cm]) to give an aqueous solution with a final concentration of 10 mg / ml, and is a trehalose dihydrate / dextran mixture. Mixed with protective agent. The volume ratio of the protective agent to the sample is 10: 1. The final concentration of the protective agent is 9.377 [wt%].

  The protective agent which is a trehalose dihydrate / dextran mixture was prepared with mass ratios of 2: 1 (T: D = 2: 1) and 1: 2 (T: D = 1: 2). Furthermore, as the protective agent, those containing only trehalose dihydrate (T: D = 1: 0) and those containing only dextran (T: D = 0: 1) were also prepared.

  Then, the inventors of the present invention used four kinds of protective agents (T: D = 0: 1, T: D = 1: 2, T: D = 2: 1 and T: D as samples for experiments). = 1: 0) to prepare four types of objects 21 to be held. Moreover, the sample 21 was not mixed with the protective agent (T: D = 0: 0), that is, the article 21 to be held without the protective agent was also prepared. In addition, the various to-be-held objects 21 prepared 3 each. Subsequently, these objects to be held 21 were applied to the upper surface of the holding member 12 according to the above-described method to form a thin liquid film, and then vacuum-dried.

  Subsequently, the inventor of the present invention stores the sample holding device 10 that holds various vacuum-dried objects to be held 21 in a temperature environment of 65 [° C.], and each time a predetermined time elapses. In addition, the enzyme activity of alcohol dehydrogenase contained in various objects to be held 21 was measured by fluorescence spectroscopy.

  The reason why the sample holding device 10 is stored in a temperature environment of 65 [° C.] is that if it is stored in a room temperature environment, it takes too much time for the enzyme activity of alcohol dehydrogenase to change, which is not suitable for experiments. . That is, by setting the temperature higher than room temperature, the enzyme activity of alcohol dehydrogenase is promoted and the experiment is completed in a relatively short time. The sample holding device 10 has a humidity of about 0 [%] and a temperature of 65 [inside a dry sterilizer (product name: MOV-112) sold by Panasonic Healthcare Co., Ltd. (former: Sanyo Electric Co., Ltd.). C.] environment.

  Further, when measuring the enzyme activity of alcohol dehydrogenase, ultrapure water is applied to various vacuum-dried objects 21 to rehydrate the objects 21 and the concentration of alcohol dehydrogenase is increased. An aqueous solution of 5 [mg / ml] was obtained. Then, the enzyme activity of alcohol dehydrogenase in the aqueous solution was measured by fluorescence spectroscopy. The measurement result is shown in FIG.

  As in the case of vacuum drying, the inventor of the present invention naturally dried the object 21 at room temperature, and measured the enzyme activity of alcohol dehydrogenase contained in the various objects 21 by fluorescence spectroscopy. did. FIG. 7B shows a measurement result when the object 21 is naturally dried without being vacuum dried.

  In addition, the Aqueous solution shown in FIGS. 7 (a) and 7 (b) is for reference purposes. For reference, the aqueous solution of alcohol dehydrogenase is not mixed with a protective agent, and is not vacuum-dried or naturally dried. The sample was stored under a temperature environment of 4 [° C.].

  As shown in FIG. 7, it can be seen that the enzyme activity of alcohol dehydrogenase significantly decreases with time when not mixed with a protective agent. In addition, when the protective agent is T: D = 0: 1, that is, when the protective agent does not contain trehalose, the enzyme activity of alcohol dehydrogenase decreases gradually with time. I understand. When trehalose is included in the protective agent, it can be seen that the higher the ratio of trehalose to dextran, the less likely the enzyme activity of alcohol dehydrogenase decreases over time.

  Further, when FIG. 7A is compared with FIG. 7B, the enzyme activity of alcohol dehydrogenase is lower when the object 21 is vacuum-dried than when the object 21 is naturally dried. It is difficult to do.

  Next, an experiment for measuring the state of moisture contained in the dried object 21 will be described.

  FIG. 8 is a diagram showing an infrared absorption spectrum of a known trehalose, FIG. 9 is a diagram showing a relationship between a known bond sound peak and a water content, and FIG. 10 is a method for calculating a water content from the known bond sound peak. FIG. 11 is a first diagram showing the result of measuring the infrared absorption spectrum of the object to be held in the embodiment of the present invention, and FIG. 12 is the infrared absorption spectrum of the object to be held in the embodiment of the present invention. FIG. 13 is a third diagram showing the result of measuring the infrared absorption spectrum of the object to be held in the embodiment of the present invention, and FIG. 14 is the diagram in the embodiment of the present invention. It is a 4th figure which shows the result of having measured the infrared absorption spectrum of the to-be-held object. 8A is a diagram showing an infrared absorption spectrum of trehalose dihydrate, and FIG. 8B is a diagram showing an infrared absorption spectrum of vitrified trehalose. In FIG. ) Is a diagram showing the infrared absorption spectrum of vitrified trehalose, (b) is an enlarged view of the bond sound peak in (a), in FIGS. 11-14, (a) is a diagram in the case of vacuum drying, (B) is a figure in the case of natural drying.

  The inventor of the present invention confirms that the held object 21 in a state of being dried and held in the vacuum packaging member 15 does not contain a hydrate and is in a vitrified state. Further, an infrared absorption spectrum of the object to be held 21 was measured.

The infrared absorption spectra of trehalose dihydrate and vitrified trehalose are known (see, for example, Non-Patent Document 2), as shown in FIGS. 8 (a) and 8 (b).
Gniadecka, Monika, et al., “Water and protein structure in photoaged and chronically aged skin”, Journal of investigative dermatology 111.6 (1998) pp.1129-1132.

As shown in FIG. 8A, in the case of trehalose dihydrate, there is a sharp peak due to moisture at a wavelength of 3500 [cm ⁻¹ ], and CH ₃ and a wavelength of 2800 to 3000 [cm ⁻¹ ]. serrated peaks due to CH ₂ are present. On the other hand, as shown in FIG. 8B, in the case of vitrified trehalose, there is no peak at a wavelength of 3500 [cm ⁻¹ ], and the shape of the peak at a wavelength of 2800 to 3000 [cm ⁻¹ ] is different. ing.

Therefore, the inventor of the present invention measures the infrared absorption spectrum of the dried article 21 to be held, the presence or absence of a peak at a wavelength of 3500 [cm ⁻¹ ], and the shape of a peak at a wavelength of 2800 to 3000 [cm ⁻¹ ]. Based on the above, it was determined whether or not all of the objects 21 were vitrified. The infrared absorption spectrum was measured by a transmission method using a Fourier transform infrared spectrophotometer (product name: FT / IR-6100) sold by JASCO Corporation.

Moreover, the peak which exists in wavelength 2000-2200 [cm < ^-1 >] is based on the bending motion of HO-H, and is known as a coupling sound peak ((nu) 2 bend + libration). Furthermore, as shown in FIG. 9, it is also known that the combined sound peak has a logarithmic relationship with the residual moisture (see, for example, Non-Patent Document 1). The combined sound peak can be calculated using a program (product name: SSE-4000) sold by JASCO Corporation.

  Further, FIG. 10A shows an example of an infrared absorption spectrum of vitrified trehalose, and FIG. 10B shows an enlarged view of a combined sound peak in FIG. 10A.

  The residual moisture amount per unit area of the object 21 can be calculated by substituting the measured area of the combined sound peak for x in the equation shown in FIG. The area of the object 21 in the present embodiment is considered to have a diameter of 10 [mm] as shown in FIG. Accordingly, the residual moisture content of the object 21 is calculated according to the following equation (3).

Here, W _water is the residual water content, and is calculated by a relational expression between the measured area of the combined sound peak and the residual water content per unit area. W _solid is the total weight of the protective agent and alcohol dehydrogenase.

  Also in this experiment, the inventor of the present invention used four types of protective agents (T: D = 0: 1, T) as experimental samples, as in the experiment for measuring the residual effect of the enzyme activity of the sample. : D = 1: 2, T: D = 2: 1 and T: D = 1: 0) to prepare four types of objects 21 to be held. In addition, the various to-be-held objects 21 prepared 2 each. Subsequently, these objects to be held 21 were applied to the upper surface of the holding member 12 according to the above-described method to form a thin liquid film, and then vacuum-dried. Similarly, two kinds of holding objects 21 were prepared two by two, and these holding objects 21 were applied to the upper surface of the holding member 12 to form a thin liquid film, and then naturally dried at room temperature.

  Subsequently, the inventor of the present invention stores the sample holding device 10 that holds the various types of objects to be held 21 that are vacuum-dried and naturally dried in a temperature environment of 65 [° C.], and 1 hour has passed. Each time, the infrared absorption spectra of various objects to be held 21 were measured. The measurement results are shown in FIGS. Specifically, the measurement result of the protective agent (T: D = 1: 0) is shown in FIG. 11, the measurement result of the protective agent (T: D = 2: 1) is shown in FIG. The measurement result of T: D = 1: 2) is shown in FIG. 13, and the measurement result of the protective agent (T: D = 0: 1) is shown in FIG. From the measurement results shown in FIGS. 11 to 14, it can be seen that the dried object 21 is in a vitrified state.

  As described above, in the present embodiment, the sample holding device 10 includes the packaging member 15, the holding member 12 accommodated in the packaging member 15, and the upper surface of the holding member 12, that is, the object to be held held on one surface. 21 to be held 21 containing a protective agent for protecting the sample. And the to-be-held thing 21 is dried, and the inside of the packaging member 15 is maintained by the vacuum. Therefore, the to-be-held thing 21 containing the protective agent which protects a sample can be preserve | saved for a long time in the dried state.

  Moreover, the dried to-be-held thing 21 is a thin film form. Furthermore, the to-be-held object 21 is obtained by drying a thin liquid film of an aqueous solution formed on one surface of the holding member 12 and is amorphized. Therefore, it is possible to suppress the molecular motion of water that is a medium for the deterioration reaction of the sample. Moreover, water molecules themselves can be evaporated.

  Further, the holding member 12 is a plate member having a hydrophilic surface, and is accommodated in the auxiliary container 11 accommodated in the packaging member 15. Therefore, the aqueous solution of the object to be held 21 can be easily thinned.

  Furthermore, the packaging member 15 is a bag made of a material having a gas barrier property, and includes an opening / closing portion 16 that can be hermetically sealed. Therefore, opening and closing of the opening / closing part 16 can be repeated, and evacuation and release of the vacuum in the packaging member 15 can be repeated.

  Furthermore, the to-be-held object 21 contains a sample with a protective agent. The sample is a biomolecule or a polymer, and the protective agent contains trehalose. As a result, the sample can be dried in a short time, and the sample can be stored for a long period of time.

  Further, in the sample holding method in the present embodiment, the holding member 12 holding the holding object 21 containing the protective agent for protecting the sample on one side is accommodated in the packaging member 15, the holding object 21 is dried, and the packaging is performed. The inside of the member 15 is kept in a vacuum. Therefore, the to-be-held thing 21 containing the protective agent which protects a sample can be preserve | saved for a long time in the dried state.

  Further, the object to be held 21 can be made into an aqueous solution by releasing the vacuum in the packaging member 15 and hydrating the object to be held 21. Therefore, the sample can be used as an object for various observations, detections, measurements, and the like.

  Furthermore, it is possible to dry the to-be-held thing 21 made into aqueous solution again, and to keep the inside of the packaging member 15 in a vacuum again. Therefore, the sample can be used as an object for various observations, detections, measurements, and the like a plurality of times while being stored for a long period of time.

  In this embodiment, while the protective agent is adsorbed around a sample such as a biomolecule, the protective agent contained in the thin liquid film is vitrified with drying, so that water serving as a medium for deterioration reaction is obtained. It suppresses molecular motion or evaporates water molecules themselves. By such a mechanism, it is possible to maintain a high enzymatic reaction of alcohol dehydrogenase as compared with the conventional technique.

  In particular, if RNA, protein, etc. that deteriorate rapidly can be stored, the expression of the gene and the state of the disease at the time when the sample was collected retrospectively (by going back) by the test using the stored sample. Since it is precisely understood, the present invention can provide time-series data of the physiochemical state of a patient, which is indispensable for tailor-made drug discovery, which is one of advanced medical treatments.

  In addition, if the quality of the stored specimen is improved, it will be possible to use biomarkers that could not be clinically applied so far, so early detection of diseases that could not be captured so far And individual differences can be classified precisely, which greatly contributes to early diagnosis and individualized medical care.

  Furthermore, the realization of long-term, high-quality dry storage enables human physiological states to be accumulated as hard (specimens) at extremely low storage costs. It becomes possible to construct a library of physiological states corresponding to. These assets are expected to significantly improve the accuracy of disease prevention and treatment monitoring.

  In other words, the present invention can ultimately provide basic technology effective in curbing medical expenses related to treatment and the health management of the public, and is therefore meaningful in the welfare aspect of an aging society.

  In addition, this invention is not limited to the said embodiment, It can change variously based on the meaning of this invention, and does not exclude them from the scope of the present invention.

  The present invention can be applied to a sample holding device, a sample holding method, and a holding member.

DESCRIPTION OF SYMBOLS 10 Sample holding apparatus 11 Auxiliary container 12 Holding member 15 Packaging member 16 Opening-closing part 21 To-be-held object

Claims (14)

A sample holding apparatus comprising: a packaging member; a holding member housed in the packaging member; and a held object that is held on one surface of the holding member and includes a protective agent that protects the sample. Because
The sample holding apparatus is characterized in that the object to be held is dried and the inside of the packaging member is kept in a vacuum.   The sample holding device according to claim 1, wherein the object to be held is a thin film.   The sample holding device according to claim 2, wherein the object to be held is an amorphous material obtained by drying a thin liquid film of an aqueous solution formed on one surface of the holding member.   The sample holding device according to any one of claims 1 to 3, wherein the holding member has a hydrophilic surface and is accommodated in an auxiliary container accommodated in the packaging member.   The sample holding device according to any one of claims 1 to 4, wherein the packaging member is a bag made of a material having a gas barrier property, and includes an opening / closing portion that can be hermetically sealed.   The sample holding device according to claim 1, wherein the object to be held includes a sample together with the protective agent.   The sample holding device according to any one of claims 1 to 6, wherein the sample is a biomolecule or a polymer, and the protective agent includes trehalose.   A sample holding method characterized in that a holding member for holding a held object containing a protective agent for protecting a sample on one side is accommodated in a packaging member, the object to be held is dried, and the inside of the packaging member is kept in vacuum. .   The sample holding method according to claim 8, wherein the object to be held can be made into an aqueous solution by releasing the vacuum in the packaging member and hydrating the object to be held.   The sample holding method according to claim 9, wherein the object to be held in an aqueous solution is dried again, and the inside of the packaging member can be kept in a vacuum again. A holding member containing a protecting agent that protects the sample is held on one side,
A holding member, wherein the object to be held is a thin film and is made amorphous by drying.   The holding member according to claim 11, wherein the one surface is a hydrophilic surface.   The holding member according to claim 11 or 12, wherein the object to be held includes a sample together with the protective agent.   The holding member according to claim 11, wherein the sample is a biomolecule or a polymer, and the protective agent includes trehalose.