CN111795909A - A kind of method of screening ice control material - Google Patents

Abstract

The invention discloses a method for screening antifreeze materials, which comprises the following steps: measuring the affinity of the ice control material and water; and measuring the spreading performance of the ice control material at an ice-water interface, wherein the spreading performance is measured by an ice adsorption experiment. The invention provides an ice control material which needs to have affinity of water and ice at the same time for the first time, can be adsorbed and spread on an ice water interface to more effectively inhibit the growth of ice crystals, and further provides an ice control material screening method.

Description

A kind of method of screening ice control material

technical field

The invention belongs to the technical field of biomedical materials, and particularly relates to a method for screening ice-controlling materials.

Background technique

Cryopreservation refers to the preservation of biological materials under ultra-low temperature, which slows down or stops cell metabolism and division, and can continue to develop once the normal physiological temperature is restored. Since its inception, this technology has become one of the indispensable research methods in the field of natural sciences and has been widely used. In recent years, with the increase of life pressure, human fertility has been declining year by year, and fertility preservation has attracted more and more attention. The cryopreservation of human germ cells (sperm, oocytes), gonadal tissue, etc. important means of fertility. In addition, as the world's population ages, the need for cryopreservation of donated human-derived materials that can be used in regenerative medicine and organ transplantation is rapidly increasing. Therefore, how to efficiently cryopreserve precious cells, tissues and organ resources for emergencies has become an urgent scientific and technical problem to be solved.

The most commonly used cryopreservation method is vitrification. The vitrification technology uses permeable or non-permeable cryoprotectants, although the liquid inside and outside the cell can be directly turned into a glass state during the rapid freezing process to avoid the damage caused by the formation of ice crystals during the freezing process. However, during the rewarming process, the existing cryopreservation reagents cannot effectively control the growth of ice crystals, thereby damaging the cells. Since the ice control mechanism of antifreeze proteins and bionic ice control materials at the molecular level is still controversial, the research and development of bionic ice control materials can only rely on the "trial and error method" to gradually try the ice control effect of a certain ice control material, which requires a lot of work. The chances of success are slim, so it is still necessary to explore the general principles and screening methods for the ice control mechanism of biomimetic materials.

SUMMARY OF THE INVENTION

The present invention provides a method for screening ice-controlling materials, comprising the following steps: (1) measuring the affinity of the ice-controlling material with water; (2) measuring the spreading performance of the ice-controlling material at the ice-water interface .

As an embodiment of the present invention, the step (1) can be performed by measuring the solubility, hydration constant, dispersion size, diffusion coefficient, etc. of the ice-controlling material in water, and/or calculating the formation of the ice-controlling material with water molecules Methods such as the number of intermolecular hydrogen bonds to measure; Specifically, for example, adopt molecular dynamics simulation (Molecular dynamics simulation, MD) to measure the number of intermolecular hydrogen bonds formed by the ice control material molecule and water molecule, or adopt dynamic light scattering to measure The dispersion size of the ice control material in the aqueous solution.

As an embodiment of the present invention, the step (2) can obtain the spreading performance of the material at the ice-water interface by measuring the adsorption content of the ice-controlling material on the ice surface at the ice-water interface, and/or calculate the The affinity of the material and ice is determined by methods such as the number of intermolecular hydrogen bonds formed by the ice-controlling material and ice-water molecules; specifically, for example, MD simulation is used to determine the molecules formed by the ice-controlling molecules and ice-water molecules. The number of hydrogen bonds between the two, or the adsorption amount of the ice-controlling material molecules on the ice surface is measured at the ice-water interface using an ice adsorption experiment.

According to the present invention, the ice adsorption experiment includes measuring the adsorption amount of the ice control material on the ice surface.

According to the present invention, the adsorption amount of the ice control material on the ice surface=(mass m ₁ of the ice control material adsorbed on the ice surface/total mass m ₂ of the ice control material in the original solution containing the ice control material)╳100%.

As an embodiment of the present invention, the ice adsorption experiment includes the following steps:

S1: get the ice-controlling material that quality is m , prepare the aqueous solution of described ice-controlling material, be cooled to supercooling temperature;

S2: place the pre-cooled temperature control rod in the aqueous solution to induce the growth of an ice layer on the surface of the temperature control rod, continuously stir the aqueous solution, gradually adsorb the ice control material on the surface of the ice layer, and keep the temperature of the aqueous solution and the temperature control rod at a higher temperature than that of the temperature control rod. cold temperature;

S3: Measure the adsorption amount of the ice control material on the ice surface.

According to an embodiment of the present invention, the temperature control rod is a rod made of a thermally conductive material. The rod may be solid or hollow. When the temperature control rod is hollow, and a cooling liquid flows in the hollow cavity, the temperature of the temperature control rod can be controlled by controlling the temperature of the cooling liquid, thereby controlling the growth rate of the ice cubes.

According to an embodiment of the present invention, the temperature control rod can be pre-cooled by one of liquid nitrogen, dry ice, freezing in an ultra-low temperature refrigerator, and the like.

According to an embodiment of the present invention, during the ice adsorption experiment, the subcooling degree and the adsorption time are kept constant so that the surface area of the ice obtained on the surface of the temperature control rod remains constant within the allowable error range.

According to the embodiment of the present invention, by configuring aqueous solutions of ice-controlling materials with different concentrations, and performing ice adsorption experiments, the applicable concentration range of the same ice-controlling material in specific applications can be evaluated.

According to an embodiment of the present invention, the ice control material in the step S1 may be pre-fluorescently labeled, for example, labeled with fluorescein, and the fluorescein may be selected from fluorescein isothiocyanate (FITC), tetraethylrhodane At least one of rhodamine (RB200), tetramethylisothiocyanate (TRITC), propidium iodide (PI) and the like. Those skilled in the art should understand that the function of the fluorescent label is to measure the amount of the ice control material, therefore, if the adsorption amount of the ice control material can be accurately measured by other means, or if the material itself has an ultraviolet or fluorescence spectrum absorption properties, no fluorescent labeling is required.

According to an embodiment of the present invention, step S3 comprises:

S3a: take out the ice cubes that have been adsorbed, rinse the ice surface with pure water, and melt the ice cubes to obtain the ice-controlling material adsorption solution;

S3b: Measure the volume V of the melted ice-controlling material adsorption solution, measure the mass/volume concentration c of the ice-controlling material in the adsorption solution, and calculate the mass m1 of the ice-controlling material adsorbed on the ice surface by formula m ₁ =cV.

According to an embodiment of the present invention, in the S3b, the concentration c can be measured by methods known in the art, such as ultraviolet-visible spectroscopy, fluorescence spectroscopy, and the like.

According to the present invention, the method is used for the screening of controlled ice crystal growth materials, such as PVA, polyamino acids, antifreeze proteins, polypeptides, and the like.

According to the present invention, the method further includes step (3): evaluating the affinity of the material with water and the spreading performance at the ice-water interface, and the material with strong spreading ability has good ice control performance.

As a specific evaluation scheme of step (3) of the present invention, the smaller the amount of ice-controlling material needed to cover a certain ice surface area, the better the spreading performance, that is, the spreading coefficient S>0 is satisfied, where S=γ _IW −( γ _IRIA -I+γIRIA _-W ), γ _IW is a constant, that is, the ice-water interface energy γ _IW is greater than the sum of the interfacial energies between material and ice and material and water γ _IRIA-I +γ _IRIA-W (γ _IRIA-I : the interface energy between the material and ice; γ _IRIA-W : the interface energy between the material and water).

In the present invention, the supercooling temperature refers to a temperature lower than the freezing point of water but still not solidified or crystallized. When the room temperature is 25°C, the supercooling temperature is generally in the range of -0.01 to -0.5°C, such as -0.1°C. -

The invention also provides an ice adsorption experiment device, which includes a multi-layer liquid storage chamber, a temperature control rod and a temperature controller. The multi-layer liquid storage chamber sequentially includes an ice adsorption chamber, a warm bath chamber and a cooling liquid storage chamber from inside to outside , the temperature control rod is placed in the ice adsorption chamber, and the temperature of the temperature control rod and the liquid storage chamber is controlled by a temperature controller.

According to the ice adsorption experimental device of the present invention, the temperature control rod is a hollow structure made of thermally conductive material, and the hollow structure of the temperature control rod is provided with a liquid inlet and a liquid discharge port; the temperature controller is a fluid temperature control The temperature controller is provided with a cooling liquid outflow end and a return end; both ends of the cooling liquid storage cavity are provided with a liquid inlet and a liquid discharge port; the cooling liquid outflow end of the temperature controller, the temperature control rod The liquid inlet, the liquid discharge port of the temperature control rod, the liquid inlet of the cooling liquid storage tank, the liquid discharge port of the cooling liquid storage tank and the return end of the temperature controller are sequentially connected through a pipeline, and the cooling liquid flows in the pipeline.

According to the ice adsorption experimental device of the present invention, the multi-layer liquid storage chamber is provided with a cover.

When in use, the ice adsorption chamber is filled with an aqueous solution of the ice-controlling material, and the middle-layer temperature bath chamber is equipped with a temperature bath medium with a predetermined temperature, such as a water bath, an ice bath or an oil bath, etc.; It flows out and flows into the hollow structure of the hollow temperature control rod to control the temperature of the temperature control rod, and then flows out from the liquid outlet of the temperature control rod, and then flows into the outer cooling liquid storage cavity to keep the temperature of the temperature bath medium at a predetermined level, and then passes through the cooling liquid. The liquid outlet of the storage tank flows through the return end of the temperature controller and enters the temperature controller cycle.

beneficial effect

The invention proposes for the first time that the ice control material needs to have both affinity with water and ice, and can adsorb and spread at the ice-water interface to more effectively inhibit the growth of ice crystals. This is the ice control mechanism, and proposes a new ice control material. The screening method not only evaluates the properties of the compound itself, but also realizes the observation and/or characterization of the growth of ice crystals and the adsorption of materials on the ice surface through the establishment of an aqueous solution and an ice-water interface system, thereby realizing the relationship between ice and ice-controlling materials. Quantitative measurement of the interaction between different ice control materials, comprehensive evaluation of the affinity of different ice control materials for ice and water at different concentrations, comparison of the ice control performance of corresponding materials, and the applicable concentration range and control of ice control materials in specific applications. Ice effect provides accurate reference. The method of the invention has simple operation, can be quantitatively measured, and has high accuracy, and improves the limitations of the existing experimental methods such as measuring thermal hysteresis by a nanoliter osmometer, which are complicated in operation and difficult to quantify.

Description of drawings

Fig. 1: the dispersion size distribution of different concentrations of a-PVA and i-PVA in aqueous solution in Example 1;

Figure 2: Schematic diagram of ice adsorption experiment and its device;

Fig. 3: The ice adsorption amount of two kinds of PVA of embodiment 1 varies with concentration;

Figure 4: Optical micrographs of ice crystal growth of two PVA in DPBS solution, A is a-PVA and B is i-PVA.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. In addition, it should be understood that after reading the content disclosed in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the protection scope defined by the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, materials, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

a-PVA: The molecular weight is about 13-23kDa, and the syndiotacticity r (diad syndiotacticity) is about 55% (Sigma-Aldrich);

i-PVA: The molecular weight is about 14-26 kDa, and the isotacticity m (isotacticity) is about 84%.

(1) Determination of the affinity of the two PVAs with water

The particle size distributions of the two PVAs in aqueous solution at 25°C were measured by dynamic light scattering (DLS) experiments using Nano ZS (Malvern Instruments) with a constant temperature chamber and a 4mW He-Ne laser (λ=632.8nm). where the scattering angle is 173°. First, prepare a-PVA and i-PVA aqueous solutions with concentrations of 1.0 mg mL ^-1 , 4.0 mg mL ^-1 , 10 mg mL ^-1 , and 20 mg mL ^-1 respectively; about 1.0 mL of the PVA solution is loaded into a 12 mm disposable Measurements were made with polystyrene cuvettes.

The results are shown in Figure 1. The dispersion size of a-PVA in aqueous solution with the same concentration is much smaller than that of i-PVA. That is, i-PVA is more likely to exist in an aggregated state in an aqueous solution than a-PVA. It can be seen that the above a-PVA has better water affinity.

(2) Determination of the spreading properties of the two PVAs at the ice-water interface

The adsorption amount of PVA on the ice surface was determined by the ice adsorption experiment. The experimental setup is shown in Figure 2.

a. a-PVA and i-PVA were fluorescently labeled with FITC Isomer I.

b. The FITC-labeled PVA aqueous solutions (40 mL) of different concentrations were placed in a beaker, the beaker was placed in a circulating cold tank, and the solution temperature and the temperature control rod were cooled to -0.1°C.

c. Before inserting the temperature control rod into the pre-cooled FITC-labeled PVA aqueous solution, insert the temperature control rod into liquid nitrogen to pre-cool for 1.0 minutes. After that, the temperature control rod was quickly inserted into the pre-cooled FITC-labeled PVA aqueous solution to induce a very thin ice layer on the surface of the temperature control rod to induce ice growth.

d. The FITC-labeled PVA aqueous solution was continuously magnetically stirred at a supercooled temperature of -0.1 °C for 1.0 hours, until the PVA was gradually adsorbed to the surface of the ice. The subcooling degree and the adsorption time were kept constant for all adsorption experiments to ensure that the surface area of the resulting ice was almost constant within the error tolerance.

e. Remove the formed ice cubes from the solution and rinse the surface with pure water to remove the solution adhering to the surface. Melt the ice cubes.

f. The adsorption amount of PVA on the ice surface is obtained by the mass of the solute PVA in the ice cube to the mass of the solute PVA in the original solution. The concentration of the PVA solution is determined by UV-Vis spectrophotometry, and the volume is determined by a pipette gun and a graduated cylinder.

Ice adsorption experiments show that the adsorption amounts of a-PVA and i-PVA at various concentrations are shown in Figure 3. The adsorption amount of a-PVA on the ice surface increased from 16.3% at the concentration of 0.2 mg mL ^-1 to 1.0 mg mL ^-1 . 28.7%, and after the concentration was greater than 1.0 mg mL ^-1 , the adsorption amount of a-PVA on the ice surface reached saturation, and the adsorption amount was about 36.5% at saturation. When the concentration of i-PVA is less than 1.0 mgmL ^-1 , the ice adsorption capacity is 0%～19.3%, which is lower than the adsorption capacity of a-PVA on the ice surface at the same concentration. At low concentrations, the adsorption of ice on both PVAs was not saturated, and the ice surface area covered by i-PVA was lower than that of a-PVA.

When the concentration of i-PVA ≥ 1.2 mg mL ^-1 , the amount of adsorption on the ice surface is higher than that of a-PVA, and when the concentration of i-PVA is 2.0 mg mL ^-1 , the adsorption amount of i-PVA on the ice surface reaches saturation, and the adsorption amount when saturated was 56.5%. It is further explained that the amount of i-PVA required is much larger than that of a-PVA when the adsorption coverage of the two PVAs on the ice surface of the same size reaches saturation. That is to say, a-PVA can cover the ice surface more effectively.

(3) Ice crystal recrystallization inhibition (IRI) activity measurement

The ice crystal recrystallization inhibition (IRI) activity adopts the "sputter freezing method". The above two kinds of PVA are dissolved and dispersed in the DPBS solution respectively, and 10-30 μL of the solution is added dropwise at a height of 1.0 m or more to -60 ℃ pre-cooling The surface of the clean silicon wafer was slowly heated to -6°C at a rate of 10°C min ^-1 using a hot and cold stage, and annealed at this temperature for 30 min. The size of the ice crystals was observed and recorded with a polarizing microscope and a high-speed camera, and the hot and cold stage was sealed. Make sure the humidity inside is around 50%. Each sample was repeated at least three times, and the size of the ice crystals was counted using Nano Measurer 1.2. The error of the results was the standard deviation.

The results are shown in Figure 4. The ice crystal size of a-PVA is significantly smaller than that of i-PVA at the same concentration, indicating that the ability of a-PVA to inhibit the growth of ice crystals is much better than that of i-PVA.

According to the results of Example 1, it can be seen that the affinity of i-PVA with water is weaker than that of a-PVA. Therefore, i-PVA tends to exist in an aggregated state in aqueous solution and ice-water interface, while a-PVA can spread well in aqueous solution and ice-water interface. The amount of i-PVA required is much higher than that of a-PVA when the adsorption coverage of the two PVAs on the ice surface of the same size reaches saturation. Therefore, compared with i-PVA, a-PVA is a better ice control material, and a lower concentration can play a better effect on inhibiting the growth of ice crystals.

Application experiment example 1

100 mL of cryopreservation solution: 2.0 g of the above-mentioned a-PVA was heated in a water bath at 80°C and dissolved in 25 mL of DPBS with magnetic stirring. After the a-PVA was completely dissolved and cooled to room temperature, the pH was adjusted to 7.0, which was solution 1; 17 g ( 0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5 mol L ^-1 ) was dissolved in 25 mL of DPBS by ultrasound, and after the sucrose was completely dissolved, 10 mL of ethylene glycol and 7.5 mL of DMSO were added to obtain solution 2. Return solution 1 and solution 2 to room temperature, then mix the two solutions, adjust the pH value and make up the balance to 80% of the total volume, and store 20 mL of serum separately until the preservation solution is used.

100 mL of frozen equilibration solution: dissolve 7.5 mL of ethylene glycol and 7.5 mL of DMSO in 65 mL of DPBS, mix well, and add serum to make the final concentration 20% (v/v) during use.

The thawing solution used for thawing after cryopreservation is a commercial formula commonly used by medical institutions at present: Thawing Solution I (containing 1.0mol L ^-1 sucrose, 20% serum, and the balance is DPBS); Thawing Solution II (containing 0.5mol L-1 sucrose, the balance is DPBS); L ^-1 sucrose, 20% serum, the balance is DPBS); Thawing Solution III (containing 0.25mol L ^-1 sucrose, 20% serum, the balance is DPBS); Thawing Solution IV (20% serum, the balance is for DPBS).

Cryopreservation of mouse oocytes was carried out using the cryopreservation solution and freezing balance solution prepared by the above formula. Specifically, the oocyte cryopreservation method used in the present invention is that the oocyte is first placed in the cryopreservation solution for equilibration for 5 minutes, then placed in the cryopreservation solution prepared by the above formula for 45 seconds, and the oocyte that has been equilibrated in the cryopreservation solution is placed in the cryopreservation solution. Place the oocytes on the frozen carrier rod, and then quickly put them into liquid nitrogen (-196°C), and seal the carrier rod for further storage; place the oocytes in 37°C Thawing Solution I for 3 minutes, and then place the oocytes in Thawing Solution II- Equilibrate in IV for 3 minutes each; thawed oocytes were cultured for 2 hours and found to be 100% viable. However, using the existing conventional formula (balance solution: 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum per 1 mL, the remaining The amount is DPBS; cryopreservation solution: each 1mL contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, The balance is DPBS), and mouse oocyte viability is approximately 95%. It is shown that the cryopreservation solution containing a-PVA can still achieve cryopreservation of oocytes and have a higher survival rate after resuscitation under the premise of reducing the content of DMSO by 50%. It also confirms that a-PVA is an excellent cryopreservation. Material.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (12)

1. A method of screening ice control materials, comprising the steps of: (1) measuring the affinity of the ice control material and water; (2) and measuring the spreading performance of the ice control material at an ice-water interface.

2. The method of screening for ice control materials of claim 1, wherein step (1) is determined by a method that determines the solubility of the ice control material in water, the hydration constant, the dispersion size, and/or the number of intermolecular hydrogen bonds formed by the ice control material molecules with water molecules.

3. The method for screening ice control material according to claim 1 or 2, wherein the step (2) is measuring the adsorption amount of the ice control material on the ice surface by using an ice adsorption experiment,

the adsorption amount of the ice control material on the ice surface (mass m of the ice control material adsorbed on the ice surface)₁Total mass m of ice control material in stock solution containing ice control material₂)╳100％。

4. The method of screening ice control materials of any of claims 1-3, wherein the ice adsorption experiment comprises:

s1, preparing an aqueous solution of the ice control material, and cooling to a supercooling temperature;

s2, placing the pre-cooled temperature control rod in the aqueous solution to induce the ice layer to grow on the surface of the temperature control rod, continuously stirring the aqueous solution to enable the ice control material to be gradually adsorbed on the surface of the ice layer, and keeping the temperature of the temperature control rod and the aqueous solution at the supercooling temperature;

s3, measuring the adsorption quantity of the ice control material on the ice surface;

preferably, the temperature control rod is pre-cooled by any one of liquid nitrogen, dry ice or ultra-low temperature refrigerator freezing.

5. The method of screening ice control materials of any of claims 1-4, wherein the degree of supercooling and the adsorption time are kept constant during the ice adsorption experiment to ensure that the surface area of the resulting ice remains constant within a tolerance.

6. The method for screening ice control material according to any one of claims 4-5, wherein the ice control material in step S1 is pre-fluorescently labeled, such as with fluorescein.

7. The method for screening ice control material of any one of claims 4-6, wherein step S3 includes:

s3a, taking out the ice blocks after adsorption, washing with pure water, and melting to obtain an adsorption solution of the ice control material;

s3b, measuring the volume V of the adsorption solution of the melted ice control material, measuring the mass/volume concentration c of the ice control material in the adsorption solution, and obtaining the mass/volume concentration c of the ice control material through a formula m₁Calculating the mass m of the ice control material adsorbed on the ice surface according to the cV₁。

8. The method of screening for ice control materials of claim 7, wherein in said S3b, said concentration c is measured by uv-vis spectroscopy.

9. The method of screening ice control materials of any one of claims 1-8, wherein the method is used to control the screening of ice crystal growth materials.

10. The method of screening for ice control material of any one of claims 1-9, further comprising step (3): the affinity of the material with water and the affinity with ice were evaluated, and a material having strong water affinity and ice affinity had good ice control performance.

11. The utility model provides an ice adsorbs experimental apparatus, includes multilayer stock solution chamber, accuse temperature stick and temperature controller, multilayer stock solution chamber is by interior to outer including ice absorption chamber, warm bath chamber, coolant liquid storage chamber in proper order, the ice absorption intracavity is arranged in to accuse temperature stick, the temperature of accuse temperature stick and stock solution chamber is controlled by temperature controller.

12. The ice adsorption experimental facility of claim 11, wherein the temperature control rod is a hollow structure made of a heat conducting material, and the hollow structure of the temperature control rod is provided with a liquid inlet and a liquid outlet; the temperature controller is a fluid temperature controller and is provided with a cooling fluid outflow end and a reflux end; a liquid inlet and a liquid outlet are formed in two ends of the cooling liquid storage cavity; the cooling liquid outflow end of the temperature controller, the liquid inlet of the temperature control rod, the liquid outlet of the temperature control rod, the liquid inlet of the cooling liquid storage box, the liquid outlet of the cooling liquid storage box and the reflux end of the temperature controller are sequentially communicated through pipelines, and the pipelines are cooled in a flowing mode;

preferably, the multilayer liquid storage cavity is provided with a cover;

preferably, when the ice adsorption experimental device is used, an aqueous solution of an ice control material is contained in the ice adsorption cavity, and a temperature bath medium with a preset temperature, such as a water bath, an ice bath or an oil bath, is contained in the middle temperature bath cavity; after the temperature of the cooling liquid reaches the set temperature, the cooling liquid flows out through the temperature controller, flows into the hollow temperature control rod hollow structure, controls the temperature of the temperature control rod, then flows out from the liquid outlet of the temperature control rod, flows into the outer cooling liquid storage cavity to keep the temperature of the temperature bath medium at the preset level, and then flows through the backflow end of the temperature controller through the liquid outlet of the cooling liquid storage box to enter the temperature controller for circulation.

Images