JP2021135141A - Device and method of preparing particle evaluation specimen - Google Patents

Abstract

To prepare a supercooled nanoparticle specimen liquid thin film in a manner simpler and enabling maintaining high quality stably for a long period of time to form a particle evaluation specimen for an electron microscope and an atomic force microscope.SOLUTION: A device of preparing a particle evaluation specimen according to the present invention includes: a flat substrate 5 to which a hydrophilization process is performed to make a contact angle 10° or less with respect to specimen liquid S in which particles for evaluation are mixed and dispersed, a sealed container 2 that stores the flat substrate 5 and shuts it off from outside air, a cooler 1 that cools the specimen liquid S dropped on the flat substrate 5 to a solidification temperature or lower to make it supercooled, and a vibrating device 3 that applies impact to the substrate, transitions it from the supercooled state and freezes the specimen liquid S. After freezing the specimen liquid S, outside air in the sealed container 2 is sucked by a vacuum pump 8, the frozen specimen liquid S is sublimated, and water content in the specimen liquid S is dried to prepare the particle evaluation specimen.SELECTED DRAWING: Figure 1

Description

The present invention relates to an apparatus and a method for preparing a particle evaluation sample for evaluating particle shape and the like by an electron microscope and an atomic force microscope, and particularly for evaluating the size and shape distribution of nanoparticles. The present invention relates to a sample preparation device and a preparation method for preparing a sample from a state in which the particles are mixed with a liquid such as water and fixed to an ice thin film.

The sample used in such particle evaluation is generally prepared by dropping a suspension in which nanoparticles are dispersed in water or an organic solvent onto a flat substrate such as a silicon substrate, and then air-drying. Since the nanoparticles move during the drying process of this natural drying method, a structure (coffee ring) in which a large number of nanoparticles are deposited in a ring shape is formed.
Therefore, the particle size, particle size distribution, and shape have been evaluated by observing a region in which nanoparticles inside the ring are thinly dispersed.
On the other hand, in the freeze-vacuum drying method, liquid nitrogen (-196 ° C) and a high thermal conductivity special flat substrate (gold-plated flat copper block) are used for the nanoparticle suspension to freeze at ultra-high speed for uniform dispersion. The method of obtaining is known.

In addition, a quick freezing method using an extremely low temperature is also used for observing cells and the like.
On the other hand, as another method of quick freezing used in biotechnology, a method using supercooling of a sample suspension as shown in Patent Document 1 has also been proposed.
On the other hand, in Patent Document 2, the inventors have proposed a method of colliding a sample-dispersed droplet with a silicon flat substrate at a low temperature to prepare an ice thin film in which nanoparticles are uniformly dispersed. ..

Further, as a similar method for producing an ice thin film, Patent Document 3 proposes a method of rapidly freezing a sample liquid sandwiched between two flat silicon substrates. In common with these methods, the key point is to instantly freeze the sample liquid film to prepare an ice thin film, and it is expected that the freezing rate will be improved by freezing from a thinner sample liquid film. Will be done.

Japanese Unexamined Patent Publication No. 2009-31272 WO2018 / 020877 Japanese Unexamined Patent Publication No. 2017-194307

The natural drying method is a simple method in practice, but the nanoparticle concentration varies greatly depending on the location on the substrate, and there is no guarantee that the diameter and shape of the nanoparticles will match depending on the evaluation position.
In addition, the conventional freeze-vacuum drying method requires liquid nitrogen (-196 ° C) and a special flat substrate with high thermal conductivity, which poses a problem in practical use.
On the other hand, in the freeze-vacuum drying method shown in Patent Document 3, a device for keeping a silicon flat substrate at a relatively low temperature (-30 ° C or lower) and for causing droplets to collide with the substrate at high speed is required. Therefore, there is a problem that the apparatus is complicated and the quality stability of the prepared sample becomes low.

As a method for producing an ice thin film, a method of sandwiching water between substrates and freezing has been proposed, but when it is difficult to control the thickness and distribution of the ice film thickness and it is not possible to form an ice film instantaneously depending on the conditions. Is also assumed.
Furthermore, it is generally known that supercooling becomes more difficult as the amount of impurities increases even in the conventional sample preparation method using supercooling, and when the concentration of nanoparticles in the sample liquid is high, a stable supercooled state is maintained. It was difficult to prepare an ice thin film in a controlled state from a supercooled state. Here, to produce an ice thin film in a controlled state means to momentarily freeze the thin film of the sample liquid by an artificial operation.

Therefore, an object of the present invention is to prepare a sample liquid thin film in a supercooled state, which is simpler and can stably maintain high quality for a long period of time, and to prepare an ice thin film in the state of maintaining the state, to prepare particles. It is to form an evaluation sample.

In order to solve this problem, the sample preparation device for particle evaluation of the present invention was supercooled so that the contact angle was 10 ° or less with respect to the sample liquid mixed in a state in which the particles to be evaluated were dispersed. A flat substrate, a closed container that stores the substrate and shuts it out from the outside air, a cooling device that cools the sample liquid dropped on the substrate below the solidification temperature to make it supercooled, and an impact on the substrate. Consists of a phase transition device that transitions from the cooled state and freezes or solidifies the sample liquid, and a vacuum drying device that sucks the outside air in the closed container to sublimate the phase-transitioned sample liquid and dry the moisture in the sample liquid. Will be done.
Further, in the method for preparing a sample for particle evaluation of the present invention, a substrate that has been supercooled so that the contact angle is 10 ° or less with respect to the sample liquid mixed in a state in which the particles to be evaluated are dispersed is prepared. The first step, the second step of dropping a limited amount of the sample liquid onto the substrate, and the third step of cooling the substrate and maintaining the dropped sample liquid in a supercooled state below the solidification temperature. The process, the fourth step in which the substrate is impacted by a phase transition device to transition from the supercooled state, and the sample liquid is frozen (solidified) to undergo a phase transition, and the sample liquid that has undergone a phase transition to become a liquid phase It consists of a fifth step of instantly drying using a vacuum drying device.

According to the present invention, it is possible to obtain a uniform and thin ice film that cannot be obtained by the prior art with a practical and simple device and manufacturing conditions, and in the field of material development, a specimen for microscopic evaluation such as nanoparticles can be obtained at low cost. It can be manufactured in a short time.

FIG. 1 is an overall configuration diagram showing an example of a particle evaluation sample preparation device based on the present invention. FIG. 2 is a camera image of a supercooled droplet spreading and standing still. FIG. 3 is a camera image when the sample liquid in the supercooled state on the silicon flat substrate is instantly frozen. FIG. 4 is a camera image of observing light scattering from the silicon surface after vacuum drying. FIG. 5 is a cross-sectional graph showing the estimated distribution of ice film thickness with respect to the sample.

FIG. 1 shows an example of a sample preparation device for particle evaluation based on the present invention.
A closed container 2 is housed in the recess of the cooler 1, and a vibration exciter 3 is installed inside the closed container 2. A heat storage plate 4 such as a copper plate having a predetermined heat capacity is installed on the surface of the vibration exciter 3, and a flat sample liquid S mixed with nanoparticles or the like is dropped on the surface of the heat storage plate 4. The substrate 5 is placed. Further, a vacuum pump 8 is connected to the upper part of the closed container 2 via a heat insulating cap 6 and a valve 7 for blocking the outside air.
The sample liquid S contains water containing a dispersant for dispersing nanoparticles as a main component. Further, depending on the material of the flat substrate 5 and the heat capacity of itself, it is not always necessary to provide the heat storage plate 4.

The flat substrate 5 is, for example, a so-called preparation formed from a silica flat substrate or ordinary flat glass (made of soda glass or the like). The sample dropping surface is hydrophilized using an existing ozone / UV surface treatment device or the like so that the contact angle with the sample liquid S is 10 ° or less (hereinafter, this is referred to as “superhydrophilic treatment”). Has been made.

When preparing a sample for evaluating nanoparticles mixed in the sample liquid S at a predetermined concentration, first, the sample is cooled in advance by a cooler 1 in a closed container 2 opened to the atmosphere, as will be described later. A predetermined amount of the sample liquid S is dropped onto the surface of the flat substrate 5 (-10 ° C. to -20 ° C.) by a pipette or a dropping device connected to the closed container 2. Then, the closed container 2 is sealed in an atmospheric pressure state.
Since the surface of the flat substrate 5 is superhydrophilicized, the sample liquid spreads widely on the surface immediately after dropping and the temperature drops. Finally, the film thickness in the liquid phase state becomes 100 μm or less, becomes the same temperature as the flat substrate 5, and reaches a supercooled state. It has been confirmed that this supercooled state is maintained stably for 10 minutes or more.

The amount of the sample liquid S to be dropped is set to, for example, 1 μL, and is adjusted to a size suitable for a sample when it is stretched. Further, the concentration of nanoparticles is selected so that the particle size, particle size distribution, and shape of individual particles can be optimally evaluated without overlapping the particles on the surface of the flat substrate 5 after vacuum drying.
In the experiment, in the case of nanoparticles with a particle diameter of 50 to ¹⁰⁰ nm, about 1x10 10 particles per cc was a concentration suitable for observation with an electron microscope and an atomic force microscope.

As another method, the sample liquid S was dropped onto the flat substrate 5 at room temperature, the sample liquid was sufficiently extended by utilizing the superhydrophilicity of the flat substrate 5, and then the flat substrate 5 was cooled in advance. The thin film of the sample liquid and the flat substrate may be cooled at the same time by being housed in the closed container 2. Even in this case, it is possible to obtain a thinly stretched film of the sample solution as described above.
The cooling temperature by the cooler 1 is preferably lowered as it is lowered from the viewpoint of increasing the freezing rate, but if the cooling temperature is excessively low, the supercooled state is changed to the solidified state with a slight impact. The optimum temperature is around ° C.

When the sample liquid S that extends on the surface of the flat substrate 5 and is in a supercooled state is vibrated by the vibrating device 3 with a certain magnitude or more, a phase transition occurs in the sample liquid S in an instant. And coagulate or freeze. That is, the vibrating device 3 functions as a phase transition device, and instantly freezes the thin film of the sample liquid S while maintaining the state in which the nanoparticles are dispersed at an appropriate density.
At this stage, when the cooler 1 is stopped, the valve 7 is opened, the vacuum pump 8 is operated, and the pressure in the closed container 2 is rapidly reduced, the water content in the frozen thin film of the sample liquid S is sublimated. dry. As a result, a specimen in a state in which the nanoparticles are fixed to the flat substrate 5 while maintaining a state in which the nanoparticles are dispersed at an appropriate density is produced.

In this embodiment, a vibration device 3 composed of an electric motor is installed on the lower surface of the heat storage plate 4 as a phase transition device, but a vibration device 3 that gives vibration from the upper surface side of the flat substrate 5 may be provided or a closed container. A pulse pressure wave generator installed in 2, for example, a speaker may give a maximum pulse pressure of about ± 2000 Pa.
Further, by using the vacuum pump 8 to rapidly open the valve 7, the pressure in the closed container 2 can be rapidly reduced, which also functions as a phase transition device. A general refrigerator is used as the cooler 1, but a Stirling refrigerator, a Perche refrigerator, or the like can also be used. In particular, the Perche refrigerator is suitable as the cooler 1 because it has less vibration and is excellent in temperature controllability.

Next, the experimental results will be described.
As the sample liquid, in order to evaluate the thickness distribution of the ice film and examine the quality as a nanoparticle dispersion sample, commercially available polystyrene latex spheres (Micromer, manufactured by Micromod) with an average diameter of 100 nm are used for supercooled pure water. A mixture of 1 × 10 ¹⁰ particles / cm ^{3 in} concentration was used.

As the flat substrate 5, a silicon flat substrate with a thermal oxide film (for electronic devices, thickness 0.5 mm) that has been thoroughly cleaned with alcohol or the like is used and placed in a commercially available UV ozone surface modifier (ASM401OZ, manufactured by Asumi Giken). , The surface of the substrate was superhydrophilicized by UV irradiation for 10 minutes.

The flat substrate 5 was attached to a copper plate 2 (diameter 25 mm, thickness 2 mm) to be a heat storage plate 4, and cooled to -15 ° C. using a commercially available small refrigerator 1 (CryoPorter, CS-80CP, manufactured by Sinix). ..
Then, 1 μL of the sample solution was separated using a micropipette and dropped onto the flat substrate 5.

Next, as a phase transition device, a speaker (F100A123, manufactured by Tokyo Corn Paper Mfg. Co., Ltd.) was installed on the upper part of the cooler 1 facing the upper surface of the flat substrate 5, and was in a supercooled state with a maximum sound pressure of about ± 2000 Pa. A pulsed pressure wave was applied to the sample solution.

The thin film of the sample liquid dropped as described above and the ice thin film after freezing were observed with a camera installed on the upper part of the refrigerator. Further, the silicon flat substrate with the sample solution which became an ice thin film was transferred to a vacuum chamber, and the dispersion state of the nanoparticles remaining after the ice was sublimated by vacuum drying was observed with an optical microscope.

When 1 μL of the sample solution was dropped on the flat substrate 5 cooled to -15 ° C., it was visually measured that the droplets spread to a diameter of about 5 mm in a few seconds and were stationary.
In the camera image shown in FIG. 2, due to the refractive index of the sample liquid and the flatness of the liquid surface, the surface of the flat substrate 5 (silicon substrate) transmitted through the droplets is projected by the camera, and the state of the liquid phase is changed. Not always clear.

Since the diameter of the droplet hardly changes even after several minutes have passed in this state, it can be said that the supercooled state exists stably.
One minute after the dripping, the above-mentioned speaker was brought close to the silicon flat substrate, and a pulse voltage was applied to generate a pressure wave. It was observed.
The diameter of this ring structure is about 5 mm, which is about the same as the droplets stretched on the substrate, which indicates that the droplets have a frozen structure.
In this structure, a flat silicon substrate exists stably at -15 ° C, and the average film thickness is estimated to be 51 μm from the volume of the sample solution of 1 μL and the diameter of the ice film of 5 mm.

Next, in order to confirm the uniformity of the film, after vacuum-drying the sample formed as an ice thin film as described above, white light is irradiated from diagonally above according to the method described in Patent Document WO2018 / 070324, and the sample is directly above. The light scattering intensity distribution from the substrate surface was observed under a microscope.
As a result, as shown in FIG. 4, it was confirmed that the scattered light intensity distribution was almost uniform, the central portion was strong, and the edge portion was weak.
Since the scattered light intensity is almost proportional to the concentration of the particles, a large number of particles are deposited at a high concentration near the center of the thick droplet of the ice thin film, while the thin film is deposited at the end. It indicates that the concentration of the number of particles is low. In this way, the thickness distribution of the ice film can be estimated from this scattered light intensity.

FIG. 5 shows a graph in which the position dependence of the scattered light intensity was acquired at a position almost passing through the center of the droplet trace in FIG. 4 and converted into the ice film thickness. As shown by the arrow in FIG. 4, the horizontal axis of the X-direction graph in FIG. 5 is the distance in mm units with respect to the left end in the horizontal direction, and the horizontal axis of the Y-direction graph is with reference to the upper end in the vertical direction. Distance in mm.
For conversion to ice film thickness, the volume of the dropped sample liquid is 1 μL, and the distribution of ice film thickness is estimated based on the above concept. From this result, it was confirmed that the maximum film thickness near the center was 92 μm, and an appropriate thickness was obtained as a sample for particle evaluation.
It can be seen that both the X-direction graph and the Y-direction graph have the same shape, and the symmetry of the film thickness distribution is high. In particular, it can be seen that a region having a constant film thickness is formed within a range of 2 mm in diameter near the center.

1: Cooler 2: Sealed container 3: Vibration device 4: Heat storage plate 5: Flat substrate 6: Insulation cap 7: Valve 8: Vacuum pump

Claims (6)

A flat substrate that has been superhydrophilized so that the contact angle is 10 ° or less with respect to the sample liquid mixed with the particles to be evaluated dispersed.
A closed container that stores the substrate and shuts it out from the outside air.
A cooling device that cools the sample liquid dropped on the substrate below the solidification temperature to bring it into a supercooled state.
A phase transition device that freezes or solidifies the sample liquid by giving an impact to the substrate to transition from the supercooled state.
A sample preparation device for particle evaluation, which comprises a vacuum drying device that sucks the outside air in the closed container, sublimates the sample liquid that has undergone a phase transition, and dries the water content in the sample liquid. The particle evaluation specimen preparation apparatus according to claim 1, wherein the substrate is placed inside the closed container via a heat storage plate. The particle evaluation sample preparation device according to claim 1 or 2, wherein a vibration device installed inside the closed container is used as the phase transition device. The particle evaluation specimen preparation apparatus according to claim 1 or 2, wherein a pulse pressure wave generator installed inside the closed container is used as the phase transition apparatus. The particle evaluation sample preparation device according to claim 1 or 2, wherein as the early phase transition device, a vacuum pump and a valve that rapidly reduce the pressure inside the closed container in the previous period are used. The first step of preparing a substrate that has been superhydrophilized so that the contact angle is 10 ° or less with respect to the sample liquid mixed with the particles to be evaluated dispersed.
A second step of dropping a limited amount of the sample liquid onto the substrate, and
A third step of cooling the substrate and maintaining the dropped sample liquid in a supercooled state below the solidification temperature.
A fourth step in which the substrate is impacted by a phase transition device to transition from the supercooled state, and the sample liquid is frozen (solidified) to undergo a phase transition.
A method for preparing a sample for particle evaluation, which comprises a fifth step of instantly drying a sample solution that has undergone a phase transition to become a liquid phase using a vacuum dryer.

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