阅读说明：本技术 一种模拟微重力条件的晶体生长方法 (Crystal growth method for simulating microgravity condition ) 是由 戴国亮 史建平 于 2019-09-24 设计创作，主要内容包括：本发明实施例涉及一种模拟微重力条件的晶体生长方法,所述方法包括：制备PDMS多孔膜,将所制备的PDMS多孔膜放入样品池中,并密封样品池；利用泵在样品池一端开口位置抽出样品池中的气体,同时在样品池另一端开口位置注入生长溶液,以使所制备的PDMS多孔膜的小腔中充满生长溶液；取下样品池中上层盖片,将预先准备的晶体转移至所制备的PDMS多孔膜中充满生长溶液的小腔中；将上层盖片重新覆盖至样品池上方,倒置样品池；在倒置样品池之后,控制样品池温度为设定温度,晶体在充满生长溶液的小腔中生长,对生长的晶体进行原位观测。(The embodiment of the invention relates to a crystal growth method for simulating microgravity conditions, which comprises the following steps: preparing a PDMS porous membrane, putting the prepared PDMS porous membrane into a sample cell, and sealing the sample cell; pumping gas in the sample cell at the opening position of one end of the sample cell by using a pump, and injecting a growth solution at the opening position of the other end of the sample cell so as to fill the small cavity of the prepared PDMS porous membrane with the growth solution; taking down the upper cover plate in the sample pool, and transferring the prepared crystal to a small cavity filled with a growth solution in the prepared PDMS porous membrane; covering the upper cover plate above the sample pool again, and inverting the sample pool; and after inverting the sample cell, controlling the temperature of the sample cell to be a set temperature, growing the crystal in a small cavity filled with a growth solution, and carrying out in-situ observation on the grown crystal.)

1. A method of crystal growth simulating microgravity conditions, the method comprising:

Preparing a PDMS porous membrane, putting the prepared PDMS porous membrane into a sample cell, and sealing the sample cell;

Pumping gas in the sample cell at the opening position of one end of the sample cell by using a pump, and injecting a growth solution at the opening position of the other end of the sample cell so as to fill the small cavity of the prepared PDMS porous membrane with the growth solution;

Taking down the upper cover plate in the sample pool, and transferring the prepared crystal to a small cavity filled with a growth solution in the prepared PDMS porous membrane;

the upper cover plate is covered above the sample pool again, and then the sample pool is inverted;

And after inverting the sample cell, controlling the temperature of the sample cell to be a set temperature, growing the crystal in a small cavity filled with a growth solution, and carrying out in-situ observation on the grown crystal.

2. The method of claim 1, wherein the preparing the PDMS porous membrane comprises:

Mixing the PDMS prepolymer and the curing agent according to a preset proportion, and putting the mixture into a container;

sequentially adding xylene and template particles with preset weight into a container to form a mixture in the container;

Solidifying the mixture in a vessel to form a solid mixture;

Dissolving the template particles of the solid mixture, and airing to obtain the PDMS porous membrane.

3. the method of claim 2, wherein solidifying the mixture in the vessel forms a solid mixture comprising:

Uniformly stirring the mixture until no bubbles exist;

After stirring until no bubbles are formed, the mixture is solidified in a container to form a solid mixture.

4. the method of claim 2, wherein the dissolving the template particles of the solid mixture and air drying to obtain the PDMS porous membrane comprises:

dissolving template particles of the solid mixture and washing for a plurality of times;

and after multiple times of washing, drying to obtain the PDMS porous membrane.

5. The method according to any one of claims 1 to 4, wherein the sample well is composed of two cover sheets, an upper cover sheet and a lower cover sheet, and two plastic strips, which are arranged between the upper cover sheet and the lower cover sheet and enclose a square area by the two plastic strips, wherein the square area has two openings at opposite corners.

6. the method of claim 5, wherein any of the openings is sealed with a setting glue.

7. the method of claim 5, wherein the placing the prepared PDMS porous membrane into a sample cell comprises:

the prepared PDMS porous membrane was placed in the square area.

Technical Field

The embodiment of the invention relates to the technical field of crystal growth, in particular to a crystal growth method for simulating microgravity conditions.

Background

Crystal growth in a space microgravity environment is an emerging discipline that has developed with the development of high technology in aerospace. The research aims at exploring the principle, method and rule of crystal growth in the space microgravity environment, seeking a way for improving the crystal characteristics, discovering new crystal varieties and expanding the application prospect of the crystal. Microgravity conditions are helpful in growing better-performing crystals for two reasons.

The first reason is as follows: the buoyant convection generated by gravity is suppressed or eliminated. When the crystal is grown, a thin layer of solution around the crystal is known as a "dissipation layer". Solute molecules (including molecules constituting the crystal, impurity molecules, etc.) in the growth solution enter the dissipation layer by diffusion or convection, and finally adsorb to the crystal surface to cause a process in which the crystal is continuously grown. In solution, when convection is present together with diffusion, the effect of convection on the entry of solute molecules into the dissipative layer is dominant, since the diffusion coefficient of solute molecules is much smaller than the convection velocity, and the effect of diffusion is somewhat negligible. The impurity molecules can thus enter the dissipation layer together with the crystal molecules by the action of convection, the partition coefficient of the impurity molecules in the dissipation layer corresponding to its partition coefficient in the growth solution. When there is substantially no convection in the growth solution, the influence of diffusion dominates and solute molecules enter the dissipation layer by diffusion. Since the impurity molecules are generally much larger than the crystal molecules, and the diffusion coefficient of the impurity molecules is much smaller than that of the crystal molecules, the distribution coefficient of the crystal molecules and the impurity molecules in the dissipation layer is changed compared with that in the growth solution, the distribution coefficient of the crystal molecules is increased, the distribution coefficient of the impurity molecules is reduced, and therefore fewer impurity molecules are adsorbed to the surface of the crystal, and the content of impurities in the grown crystal is reduced. In a space microgravity environment, buoyancy convection caused by gravity is almost nonexistent, and the transport of substances is mainly dependent on the diffusion of solutes, so that crystals with higher quality can be grown under the growth conditions of the crystals. In the ground experimental environment, buoyancy convection caused by gravity is difficult to avoid, so that the transport process of solutes and the interface characteristics between crystals/solutions are greatly influenced, and the size and the quality of the crystals are further influenced to a certain extent.

The second reason is that: gravity settling disappears. In the space microgravity environment, the gravity settling phenomenon caused by the gravity field disappears, the aggregation of particles is inhibited, and the number of spontaneously nucleated crystal nuclei is greatly reduced, so that crystals with larger sizes are easier to grow.

of the two reasons, the main reason is that the influence on the crystal growth process is significant. Research to simulate microgravity techniques has focused on this reason. The correctness of the theory is verified by the results of a plurality of space microgravity crystal growth experiments which are completed.

There are two experimental approaches to crystal growth under microgravity conditions: firstly, growing crystals in a space microgravity environment; secondly, simulating microgravity conditions on the ground to grow crystals. However, the second approach is more often used because of the inexhaustible and expensive opportunities for conducting space experiments in space.

Four related techniques for growing crystals under simulated microgravity conditions on the ground have been disclosed as follows. One, Hitoshi Wada et al (Application of High-Field Superconducting Magnet to protein crystallization, Physics Procedia,2012) discloses a technique that utilizes a magnetic Field to simulate microgravity conditions. They have implemented the weakening of gravity settling and buoyancy convection in the growth solution when the protein crystal grows by using the technology of adding a magnetic field to the growth solution. However, the technology has the disadvantages that the magnetic field slows down the nucleation and growth rate of protein crystals, which hinders the protein crystallization, and in addition, the strong magnetic field is needed to obtain the required simulated microgravity condition, so that the technology has high requirements on relevant equipment required by experiments. Second, Thiessen KJ et al (The use of two novel methods to growth proteins crystals by micro-catalysis and vapor diffusion in an agar gel, actaCrystallogr D,1994) disclose techniques for simulating microgravity conditions using a gel as The medium. They mixed the crystal solution with agarose and finally formed a gel, and the precipitant solution required for crystal growth was diffused into the gel outside the gel, so that the crystal was grown in the gel and the interface formed by the gel and the precipitant solution. The technology utilizes the characteristic that buoyancy convection is basically eliminated when Grashof number (the Grashof number is a dimensionless constant for representing fluid convection, and the smaller the Grashof number is, the smaller the fluid convection) is very small in small pores formed by gel, so that the buoyancy convection in the crystallization process is greatly inhibited, but the defect that a part of protein crystals and the gel are mixed together and cannot be separated is overcome, and meanwhile, the in-situ observation of the crystal growth is hindered due to the existence of the gel. Third, wang jing et al (preliminary studies on collagen fibrosis and Hydroxyapatite (HAP) crystallization under microgravity, proceedings of space science 2015) discloses a technique for simulating microgravity conditions using a rotary controller. The growth solution is fixed on the cantilever of the rotation controller, and the cantilever is controlled to randomly rotate in a rotating space, so that the crystal is in a gravity field with constantly changing directions in the growth process, and the gravity settling effect on integral disappears. However, the technology has the defects that only gravity settlement is eliminated, but the inhibition on buoyancy convection is limited, and the effect of better simulating microgravity conditions cannot be achieved. Ismagiov et al (A Draplet-Based, composite PDMS/Glass Capillary Microfluidic System for Evaluating protein crystallization Conditions by Microbatch and Vapor-Diffusion Methods with On-Chip X-Ray Diffraction, Crystal Growth,2004) disclose techniques for simulating microgravity Conditions using Microfluidic technology. The characteristics that Grashof number is very small after the size of a microfluidic system is reduced are utilized, so that buoyancy convection generated by gravity in the crystal growth process is inhibited or reduced, and the protein crystal growth under the condition of simulating microgravity is realized, but the technology has some defects. Because the change of the pressure of the injector to the propulsion speed of the micro pump usually has a certain hysteresis, the rapid and accurate regulation and control of the sample liquid drop ratio can be realized by using the common micro pump and the injector to regulate the flow rate of the fluid, and the realization is almost impossible, so the technology has higher requirements on instruments and equipment. In addition, it should be noted that, when the crystals are grown by the micro-fluidic technology, the positions of the crystals appearing in the micro-channel are random, and long-time continuous optical microscopic observation cannot be performed on the designated positions in the micro-channel, because the observed regions are likely not to have crystals, or the sizes and shapes of the appearing crystals are not suitable for optical observation to obtain crystal growth kinetic data.

Therefore, how to grow the crystal under the microgravity condition can be simulated by using the characteristic that the Grashof number is very small and buoyancy convection generated by gravity basically disappears without special requirements on instruments and equipment on the ground, and the problem that the in-situ observation research on the crystal growth in a specified area can be performed is a problem which is urgently needed to be solved at present.

disclosure of Invention

in view of this, in order to solve the problems in the prior art, embodiments of the present invention provide a crystal growth method simulating microgravity conditions.

in a first aspect, an embodiment of the present invention provides a crystal growth method for simulating microgravity conditions, where the method includes:

Preparing a PDMS porous membrane, putting the prepared PDMS porous membrane into a sample cell, and sealing the sample cell;

Pumping gas in the sample cell at the opening position of one end of the sample cell by using a pump, and injecting a growth solution at the opening position of the other end of the sample cell so as to fill the small cavity of the prepared PDMS porous membrane with the growth solution;

Taking down the upper cover plate in the sample pool, and transferring the prepared crystal to a small cavity filled with a growth solution in the prepared PDMS porous membrane;

After the upper cover plate is covered above the sample pool again, controlling the temperature of the sample pool to be the set temperature;

the crystal grows in a small chamber filled with growth solution, and the grown crystal is observed in situ.

In one possible embodiment, the preparing the PDMS porous membrane includes:

Mixing the PDMS prepolymer and the curing agent according to a preset proportion, and putting the mixture into a container;

Sequentially adding xylene and template particles with preset weight into a container to form a mixture in the container;

solidifying the mixture in a vessel to form a solid mixture;

dissolving the template particles of the solid mixture, and airing to obtain the PDMS porous membrane.

In one possible embodiment, the solidifying the mixture in the vessel forms a solid mixture comprising:

Uniformly stirring the mixture until no bubbles exist;

After stirring until no bubbles are formed, the mixture is solidified in a container to form a solid mixture.

in one possible embodiment, the dissolving the template particles of the solid mixture and air drying to obtain the PDMS porous membrane, which comprises:

dissolving template particles of the solid mixture and washing for a plurality of times;

And after multiple times of washing, drying to obtain the PDMS porous membrane.

In one possible embodiment, the sample cell is composed of an upper cover sheet, a lower cover sheet and two plastic belts, wherein the two plastic belts surround a square area between the upper cover sheet and the lower cover sheet, and the square area has two openings at opposite corners.

In one possible embodiment, any of the openings is sealed with a setting glue.

in one possible embodiment, the placing the prepared PDMS porous membrane into a sample cell includes:

The prepared PDMS porous membrane was placed in the square area.

The technical scheme provided by the embodiment of the invention has the following three advantages. Firstly, crystals are introduced into a small cavity in the prepared PDMS porous membrane for growth, and the Grashof number (the Grashof number is a dimensionless constant for representing fluid convection, and the smaller the Grashof number is, the smaller the fluid convection is) is very small due to the reduced space, so that buoyancy convection can be eliminated, and the purpose of growing the crystals under the condition of simulating microgravity is realized; secondly, special requirements on instruments and equipment are not required; and thirdly, long-time, continuous and in-situ observation can be carried out on the crystal at a specified position (or area) in the crystal growth process.

drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the embodiments of the present invention, and it is also possible for a person skilled in the art to obtain other drawings based on the drawings.

FIG. 1 is a schematic flow chart of a crystal growth method for simulating microgravity conditions according to an embodiment of the present invention;

FIG. 2 is a schematic view of a sample cell according to an embodiment of the present invention;

FIG. 3 is a schematic view of gas extraction and growth solution injection according to an embodiment of the present invention;

FIG. 4 is a schematic view of an inverted sample cell according to an embodiment of the present invention;

FIG. 5 is a schematic illustration of in situ observation of a growing crystal according to an embodiment of the present invention;

FIG. 6 is a photograph of optical microscopy imaging of in situ observation of lysozyme crystals in accordance with an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

For the convenience of understanding of the embodiments of the present invention, the following description will be further explained with reference to specific embodiments, which are not to be construed as limiting the embodiments of the present invention.

polydimethylsiloxane (PDMS) is a polyorganosiloxane with a chain structure of different degrees of polymerization, and has excellent heat resistance, electrical insulation, weather resistance, hydrophobicity, physiological inertia and small surface tension. PDMS can be prepared into a porous material with good stability by a template method, the most prominent characteristic of the template method is that the template has good controllability, templates with different sizes can be adopted according to the researched crystal size, and the commonly adopted templates comprise sodium chloride, cane sugar and the like.

The crystal is introduced into a small cavity in the prepared PDMS porous membrane for growing, and the space is reduced, so that the Grashof number (the Grashof number is a dimensionless constant for representing fluid convection, and the smaller the Grashof number is, the smaller the fluid convection is) is very small, the purpose of growing the crystal under the condition of simulating microgravity can be realized by eliminating buoyancy convection, special requirements on instruments and equipment are not required, and in addition, the crystal can be observed in situ in the crystal growth process by utilizing a reflection type optical microscopic observation method.

compared with the existing method for growing crystals under the condition of simulating microgravity, the embodiment of the invention, in particular to the microfluidic technology, has the following differences: 1, the characteristics of small space scale and Grashof number can be utilized without special requirements on instruments and equipment, buoyancy convection is eliminated, and the microgravity condition is simulated. 2, the crystal can be observed in situ at the designated position to obtain the crystal growth kinetic data.

Based on the above, as shown in fig. 1, an implementation flow diagram of a crystal growth method for simulating microgravity conditions provided in an embodiment of the present invention is shown, where the method specifically includes the following steps:

S101, preparing a PDMS porous membrane, putting the prepared PDMS porous membrane into a sample cell, and sealing the sample cell;

the preparation method of the PDMS porous membrane comprises the following specific steps:

Mixing the PDMS prepolymer and the curing agent according to a preset proportion, and putting the mixture into a container; sequentially adding xylene and template particles with preset weight into a container to form a mixture in the container; uniformly stirring the mixture until no bubbles exist; after stirring uniformly until no bubbles exist, solidifying the mixture in a container to form a solid mixture; dissolving template particles of the solid mixture and washing for a plurality of times; and after multiple times of washing, drying to obtain the PDMS porous membrane.

In the embodiment of the invention, the sample cell consists of an upper layer cover plate, a lower layer cover plate and two plastic belts, wherein the two plastic belts surround a square area between the upper layer cover plate and the lower layer cover plate, two openings are formed in the opposite corners of the square area, and any opening is sealed by using solidified glue.

and putting the prepared PDMS porous membrane into a sample pool, specifically putting the prepared PDMS porous membrane into the square area.

For example, as shown in fig. 2, a plastic tape is adhered to a transparent optical glass sheet, an opening is left at each of the left and right ends, the opening is sealed with a setting adhesive (a syringe needle can be inserted after the gel is set, so as to facilitate the next step of injecting a growth solution), a PDMS porous membrane is placed in a space surrounded by the plastic tape, then a layer of glass sheet is covered above the plastic tape to form a sealed space, 1 points to the plastic tape, 2 points to the transparent optical glass sheet, and 3 and 4 point to two openings, respectively.

s102, pumping gas in the sample cell at an opening position at one end of the sample cell by using a pump, and injecting a growth solution at an opening position at the other end of the sample cell so as to fill the small cavity of the prepared PDMS porous membrane with the growth solution;

as shown in fig. 3, a pump is used to pump out the gas in the sample cell at the opening at one end of the sample cell, and simultaneously, a growth solution is injected into the opening at the other end of the sample cell, so that the small cavity of the prepared PDMS porous membrane is filled with the growth solution, 5 represents a pump, and 6 represents the growth solution.

s103, taking down the upper cover plate in the sample pool, and transferring the prepared crystal to a small cavity filled with a growth solution in the prepared PDMS porous membrane;

S104, covering the upper cover plate above the sample pool again, and inverting the sample pool;

and taking down the upper cover plate in the sample pool, transferring the prepared crystal to a small cavity filled with a growth solution in the prepared PDMS porous membrane, covering the upper cover plate above the sample pool again, and inverting the sample pool.

For example, as shown in fig. 4, the glass plate above the plastic tape in the sample cell is removed, at this time, the small cavity in the PDMS porous membrane is filled with the growth solution, then a crystal is transferred to the small cavity filled with the growth solution in the PDMS porous membrane, the glass plate is covered and sealed above the small cavity, and the small cavity where the crystal is located is inverted to form a closed cavity, 7 is directed to the small cavity filled with the growth solution in the PDMS porous membrane, 8 is directed to the crystal, and 9 is directed to the glass plate.

s105, after inverting the sample cell, controlling the temperature of the sample cell to be a set temperature, growing the crystal in a small cavity filled with a growth solution, and carrying out in-situ observation on the grown crystal.

after the sample cell is inverted, on one hand, a small cavity 7 filled with growth solution in the PDMS porous membrane and a glass sheet 9 form a closed small cavity, and crystals grow in the closed small cavity filled with the growth solution, so that natural convection is eliminated, and the purpose of growing the crystals under the condition of simulating microgravity is realized.

alternatively, the grown crystal may be observed in situ, for example, by reflective optics, as shown in FIG. 5 with 10 pointing to the objective lens.

in order to explain the technical scheme provided by the embodiment of the invention in detail, the following embodiment is combined for explanation:

1. Preparation of PDMS porous membranes of approximately 1mm thickness: according to the following steps of 10: 1, putting the PDMS prepolymer and a curing agent into a beaker, then sequentially adding a certain amount of dimethylbenzene and sodium chloride particles with required sizes, uniformly stirring until no bubbles exist, curing the mixture, then eliminating the sodium chloride particles in the solid mixture, washing the mixture for multiple times, and airing to obtain the PDMS porous membrane with the required pore size.

2. Putting the prepared PDMS porous membrane into a sample cell;

the schematic diagram of the sample cell is shown in fig. 2, a plastic tape 1 is adhered on a 24 × 32mm transparent optical glass sheet 2, the plastic tape has a width of 1mm, a length of 40mm and a thickness of 1mm, openings 3 and 4 are respectively reserved at the left end and the right end, the openings are sealed by solidification glue (which is convenient for injecting growth solution in the next step), a PDMS porous membrane is placed in a space enclosed by the plastic tape, and then a layer of glass sheet cover is used for sealing above the plastic tape to form a sealed space.

3. The gas in the sample cell is pumped out at one end opening by a pump 5, and the growth solution 6 is introduced into the sample cell at the other end opening, so that the small cavity of the PDMS porous membrane is filled with the growth solution, as shown in FIG. 3.

4. And (3) taking down the glass sheet above the plastic belt in the sample cell, filling the small cavity 7 in the PDMS porous membrane with the growth solution, transferring a crystal 8 into the PDMS porous membrane filled with the growth solution in the small cavity 7, covering the glass sheet 9 above the small cavity, sealing and inverting to form a closed cavity in the small cavity where the crystal is positioned, as shown in FIG. 4.

5. After inversion, the crystal grows in a closed small cavity, buoyancy convection is eliminated, the purpose of growing the crystal under the microgravity condition is achieved, and the grown crystal can be observed in situ by adopting a reflection type microscopic observation optical method. As shown in FIG. 6 below, a small cavity was made using NaCl as a template (400X 400 μm), and lysozyme crystals were placed and observed in situ, and some of the small crystals in the cavity were newly grown 2 days after observation.

those of skill would further appreciate that the various illustrative components and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both, and that the various illustrative components and steps have been described above generally in terms of their functionality in order to clearly illustrate this interchangeability of hardware and software. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the implementation. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.

the steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied in hardware, a software module executed by a processor, or a combination of the two. A software module may reside in Random Access Memory (RAM), memory, Read Only Memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art.

the above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.