阅读说明：本技术 一种筛选控冰材料的方法 (Method for screening ice control material ) 是由 王健君 金晟琳 严杰 乔杰 闫丽盈 李蓉 于 2019-04-09 设计创作，主要内容包括：本发明公开了一种筛选抗冻材料的方法,包括：测得所述控冰材料与水的亲和性；测得所述控冰材料在冰水界面的铺展性能,其中所述铺展性能由冰吸附实验测得。本发明首次提出控冰材料需同时兼具水和冰的亲和性,可在冰水界面吸附并铺展以更有效的抑制冰晶生长这一控冰机制,进而提出一种控冰材料筛选方法,建立水溶液体系并通过观察/表征冰晶生长以及材料在冰表面的吸附实现冰与控冰材料之间相互作用的定量化测量,全面评估不同控冰材料在不同浓度下对于冰和水的亲和性,为相应材料控冰性能的比较以及控冰材料在应用时所适用的浓度范围和控冰效果提供准确参考,改善了现有的通过纳升渗透压仪测定热滞后等实验方法操作复杂、难以定量的局限。(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.)

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.

Technical Field

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

Background

Cryopreservation refers to keeping the biological material at ultralow temperature to slow down or stop cell metabolism and division, and to continue to develop once normal physiological temperature is restored. Since the advent, this technique has become one of indispensable research methods in the field of natural science, and has been widely adopted. In recent years, with the increasing pressure of life, human fertility tends to decrease year by year, and fertility preservation is receiving more and more attention, and cryopreservation of human germ cells (sperm and oocyte), gonadal tissue, and the like is an important means for fertility preservation. In addition, as the world population ages, the need for cryopreservation of donated human-derived materials available for regenerative medicine and organ transplantation has also increased dramatically. Therefore, how to efficiently store precious cells, tissues and organ resources in a freezing way becomes a scientific and technical problem to be solved urgently.

The most common cryopreservation method currently used is vitrification freezing. The vitrification freezing technology adopts permeable or impermeable cryoprotectant, although liquid inside and outside cells can directly become glass state in the process of rapid freezing, thereby avoiding the damage caused by the formation of ice crystal in the freezing process. However, prior art cryopreservation reagents are not effective in controlling the growth of ice crystals during the rewarming process, thereby damaging the cells. Because the ice control mechanism of the antifreeze protein and the bionic ice control material on the molecular level is still controversial, the development of the bionic ice control material can only gradually try the ice control effect of a certain ice control material by a trial-and-error method, the workload is large, the success probability is poor, and the general principle and the screening method of the ice control mechanism of the bionic material need to be explored.

Disclosure of Invention

The invention provides a method for screening ice control materials, which comprises the following steps: (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.

As an embodiment of the present invention, the step (1) may be determined by measuring solubility, hydration constant, dispersion size, diffusion coefficient, etc. of the ice controlling material in water, and/or calculating the number of intermolecular hydrogen bonds formed by the ice controlling material and water molecules, etc.; specifically, for example, the number of intermolecular hydrogen bonds formed between the molecules of the ice control material and water molecules is measured by Molecular dynamics simulation (MD), or the dispersion size of the ice control material in an aqueous solution is measured by dynamic light scattering.

As an embodiment of the 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 number of intermolecular hydrogen bonds formed by the ice-controlling material and ice-water molecules, and the like to measure the affinity of the material with ice; specifically, for example, MD simulation is used to measure the number of intermolecular hydrogen bonds formed between the ice-controlling molecules and ice-water molecules, or ice adsorption experiment is used to measure the adsorption amount of the ice-controlling material molecules on the ice surface at the ice water interface.

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

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

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

s1, taking the ice control material with the mass of m2, preparing an aqueous solution of the ice control material, and cooling to the 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 aqueous solution and the temperature control rod at the supercooling temperature;

and S3, measuring the adsorption quantity of the ice control material on the ice surface.

According to an embodiment of the invention, the temperature control rod is a rod made of a heat conducting material. The rod may be solid or hollow. When the temperature control rod is hollow, the hollow inner cavity of the temperature control rod is provided with cooling liquid to flow, and the temperature of the temperature control rod can be controlled by controlling the temperature of the cooling liquid, so that the growth speed of ice blocks is controlled.

According to the embodiment of the invention, the temperature control rod can be precooled in one of the modes of liquid nitrogen, dry ice, ultralow temperature refrigerator freezing and the like.

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

According to the embodiment of the invention, the ice adsorption experiment is carried out by preparing the water solutions of the ice control materials with different concentrations, so that the applicable concentration range of the same ice control material in specific application can be evaluated.

According to an embodiment of the present invention, the ice control material in step S1 may be pre-fluorescently labeled, for example, labeled with fluorescein, which may be selected from at least one of Fluorescein Isothiocyanate (FITC), tetraethylrhodamine (RB200), tetramethylrhodamine isothiocyanate (TRITC), Propidium Iodide (PI), and the like. It will be appreciated by those skilled in the art that the fluorescent label functions to measure the amount of the ice control material, and thus, if the amount of the ice control material adsorbed can be accurately measured by other means, or the material itself has ultraviolet or fluorescent spectral absorption characteristics, no fluorescent label is required.

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

s3a, taking out the ice blocks after adsorption, rinsing the ice surfaces with pure water, and melting the ice blocks 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,determining the mass/volume concentration c of ice control material in the adsorption solution by formula m₁The mass m1 of ice control material adsorbed on the ice surface was calculated as cV.

According to an embodiment of the present invention, in the S3b, the concentration c can be measured by a method known in the art, such as uv-vis spectroscopy, fluorescence spectroscopy, and the like.

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

According to the invention, the method further comprises a step (3): the affinity of the material with water and the spreading performance of the material at an ice-water interface are evaluated, and the material with strong spreading capability has good ice control performance.

As a specific evaluation scheme of step (3) of the present invention, the smaller the amount of ice control material required to cover a given ice surface area, the better the spreading performance, i.e., the spreading factor S > 0 is satisfied, where S ═ γ_I-W-(γ_IRIA-I+γIRIA_-W)，γ_I-WIs constant, i.e. ice water interfacial energy gamma_I-WGreater than gamma which is the sum of the interfacial energies of the material and ice and the material and water_IRIA-I+γ_IRIA-W(γ_IRIA-I: the interfacial energy of the material and ice; gamma ray_IRIA-W: the interfacial energy of the material with water).

In the present invention, the supercooling temperature means a temperature lower than the freezing point of water but not solidified or crystallized, and is generally in the range of-0.01 to-0.5 ℃, for example-0.1 ℃ at room temperature of 25 ℃. -

The invention also provides an ice adsorption experimental device which comprises a multilayer liquid storage cavity, a temperature control rod and a temperature controller, wherein the multilayer liquid storage cavity sequentially comprises an ice adsorption cavity, a warm bath cavity and a cooling liquid storage cavity from inside to outside, the temperature control rod is arranged in the ice adsorption cavity, and the temperature of the temperature control rod and the temperature of the liquid storage cavity are controlled by the temperature controller.

According to the ice adsorption experimental device, the temperature control rod is of a hollow structure made of heat conducting materials, 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 discharge port of the temperature control rod, the liquid inlet of the cooling liquid storage box, the liquid discharge port of the cooling liquid storage box and the backflow end of the temperature controller are communicated in sequence through pipelines, and cooling liquid flows in the pipelines.

According to the ice adsorption experimental device, the multi-layer liquid storage cavity is provided with the cover.

When in use, the ice adsorption cavity is internally filled with an aqueous solution of an ice control material, and the middle-layer warm bath cavity is filled with a warm bath medium with a preset temperature, such as a water bath, an ice bath or an oil bath; 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.

Advantageous effects

The invention provides an ice control material which has affinity with water and ice at the same time, can be adsorbed and spread on an ice water interface to more effectively inhibit the ice crystal growth, and provides a novel ice control material screening method. The method disclosed by the invention is simple to operate, can be used for quantitative measurement, is high in accuracy, and overcomes the limitations that the existing experimental methods for determining thermal hysteresis through a nano-liter osmometer are complex in operation and difficult to quantify.

Drawings

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

FIG. 2: schematic diagram of ice adsorption experiment and its device;

FIG. 3: example 1 plots of ice adsorption versus concentration for two PVAs;

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

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that various changes or modifications can be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents also fall within the scope of the invention.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.