CN113331174A - Nanoparticle antifreeze agent containing small molecule monolayer and preparation method thereof - Google Patents
Nanoparticle antifreeze agent containing small molecule monolayer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a nanoparticle antifreeze agent containing a small molecule monolayer, which comprises the following steps: step S1, preparing a micromolecule solution and a nanometer glue solution; step S2, stirring and mixing the micromolecule solution and the nanometer glue solution to obtain hydroxyl exposed nanoparticles which take the nanoparticles as inner cores and are coupled with micromolecule monomer substances on the surfaces of the nanoparticles; and S3, centrifuging and washing the hydroxyl exposed nanoparticles obtained in the step S2 to remove uncoupled micromolecules, and obtaining the nanoparticle antifreeze agent. The interface size effect of the nano particles and mercapto groups on the small molecules are utilized to form stable covalent bonds, the nano particles are used as inner cores, a layer of small molecule monomer substances is coupled on the surfaces of the nano particles, the nano particles with exposed hydroxyl groups and densely arranged are formed, the small molecules containing the hydroxyl groups are enriched on the surfaces of the nano particles, and the nano particles are better adsorbed on the surfaces of ice crystals to achieve high-efficiency anti-freezing activity.
Description
Technical Field
The application relates to the technical field of synthesis of anti-freezing bionic nano materials, in particular to a nano particle antifreeze agent containing a small molecular monolayer and a preparation method thereof.
Background
Cryopreservation is a fundamental and important leading technology that achieves long-term storage of organs, tissues, cells and other biological materials under cryogenic conditions (e.g., -80 ℃ or-196 ℃). Under low-temperature storage conditions, chemical and biological reactions are significantly reduced or even stopped in living cells, which is the most fundamental mechanism for achieving long-term storage of various biological samples. For example, with the development of stem cell-based medicine, the demand for stem cells has increased dramatically, and achieving high-quality, efficient stem cell storage is critical to overcome the current imbalance in supply and demand. Human assisted reproduction is another typical application area, the risk of infertility in young women is constantly increasing due to incidental diseases, overdose and external stress, and cryopreservation of materials such as egg cells, sperm, eggs and embryos is a key part of assisted reproduction. In summary, the science of cryopreservation of various biological specimens (e.g., cells, tissues, organs, vaccines, etc.) is of great importance for clinical applications and scientific research in the field of biomedical engineering.
Various chemical and physical insults that occur during the freeze-thaw process of cryopreservation are the major destructive mechanisms for cryopreservation of biological samples. Among these damages, the formation, growth and recrystallization of ice crystals are fatal to the cryopreservation of samples, which is a major problem and limitation in achieving efficient cryopreservation. The only reliable way to solve this problem is to cryopreserve cells using large amounts of cryoprotectants (e.g., DMSO), which is toxic in itself, and repeated use of DMSO can affect changes in the epigenetic profile, particularly the DNA methylation profile, of the cells, resulting in phenotypic changes.
In nature, many organisms adapted to extreme cold have evolved a specific protein, the antifreeze (glyco) protein AF (G) Ps. AF (G) Ps are proteins with the capability of improving biological freezing resistance, can prevent the formation and growth of ice nuclei in body fluid and maintain the non-freezing state of the body fluid so as to adapt to low-temperature living conditions. AF (G) Ps are rich in threonine, and hydroxyl groups in threonine form hydrogen bonds with water molecules during the recrystallization of ice, thereby increasing the growth curvature between ice crystals to achieve the effect of inhibiting ice crystal growth (Kelvin effect). In addition, af (g) Ps have some thermal hysteresis effect and dynamic ice formation effect. However, the extraction of natural af (g) Ps from organisms is typically a complex and low-yielding process, and although some af (g) Ps have been synthesized or expressed, they are generally expensive and have low thermal stability. In addition, some af (g) Ps are cytotoxic to mammalian cells and may even elicit an immune response.
Currently, the theoretical explanation of the mechanism of freeze resistance is at issue. The main points of view include: 1. it has been reported that the coordination of hydrophobic interaction and hydrogen bond formation is key to the recognition and binding of molecules to ice, such as threonine in AFP, that hydrophobic groups (such as methyl) harden ice binding sites, slowing the hydrodynamics of ice binding surfaces, and that hydrogen bond formation can increase the growth curvature of ice crystals; 2. it has been reported that only hydrophobic interactions are required during antifreeze processes, such as hydrophobic groups in AFGP and type I AFP; 3. it has been reported that only hydrogen bonding is required in antifreeze processes, such as polyvinyl alcohol; 4. it has been reported that lattice matching is only required for antifreeze processes, such as oxidized quasi-carbon nitride quantum dots, whose distance between adjacent three nitrogen atoms matches the repeat spacing of oxygen atoms along the c-axis of the crystal's major prism plane, so that the quantum dots preferentially bind to the crystal. With the development of molecular dynamics simulations, there was further knowledge of the antifreeze mechanism (Fabinene, et al. Nature communications.2021,12(1):1323), which indicates that the lattice match between the functional groups of PVA and ice does not play a role, since PVA is a very flexible polymer that can bind to ice in any conformation. This is a very important result because it eliminates a long-standing design constraint, i.e., lattice matching, from the rational design of ice recrystallization inhibiting living polymers. In contrast, the effective volume of PVA and its contact area with the ice surface determine its ice recrystallization inhibiting strength. Researchers have also found that entropy contributions, possibly playing a role in ice-PVA interactions, and that researchers have demonstrated that small blocks of copolymers (heretofore thought to be without ice recrystallisation inhibition activity) may exhibit significant ice recrystallisation inhibition potential.
In recent years, inspired by AF (G) Ps and well understood anti-freezing mechanism, many AF (G) P biomimetic anti-freezing materials have been shown to be effective in inhibiting the recrystallization of ice crystals, mainly including macromolecular polymers and nanomaterials. Little has been reported on small molecule anti-freeze, since very high concentrations of anti-freeze effect are required, such as 10% (v/v) DMSO. The synthesis and application of Nanoparticles (NPs) have advanced greatly in the last two decades, and simple and general synthetic routes and NPs with highly uniform size and morphology are receiving wide attention. The ability to precisely control the morphology and size of NPs greatly improves nanoscale interfacial interactions between ice crystals and antifreeze molecules. It has been reported in the literature that oligopeptides consisting of amino acid sequences are coupled to the surface of gold nanoparticles of the same size but different morphology to control the growth of ice crystals (Lee, et al.j.am.chem.soc.2019,141,47,18682 18693). The paper mainly studies ice recrystallization inhibitory activity of four amino acids, threonine, serine, alanine and glycine (which are different in whether hydroxyl and methyl groups are present individually or simultaneously in the amino acid structure) constituting oligopeptides and sequence lengths (2, 5 and 7, respectively) of oligopeptides. The results indicate that: firstly, only threonine in four amino acids shows obvious ice recrystallization inhibition activity, namely, hydroxyl and methyl exist simultaneously; ② only 5 oligopeptides formed by threonine show obvious ice recrystallization inhibition activity; and the ice recrystallization inhibition activity of the gold nanocubes is superior to that of the gold nanospheres and the gold nano octahedrons. Therefore, a nanoparticle antifreeze agent containing a small molecule monolayer and a preparation method thereof are provided.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present patent application aims to provide a nanoparticle antifreeze agent containing a monolayer of small molecules and a method for preparing the same, which solves the above-mentioned problems of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of a nanoparticle antifreeze agent containing a small molecule monolayer comprises the following steps:
step S1, preparing a micromolecule solution and a nanometer glue solution;
step S2, stirring and mixing the micromolecule solution prepared in the step S1 and the nanometer glue solution to obtain hydroxyl exposed nanoparticles which take the nanoparticles as cores and are coupled with micromolecule monomer substances on the surfaces of the nanoparticles;
and S3, centrifuging and washing the hydroxyl exposed nanoparticles obtained in the step S2 to remove uncoupled micromolecules, and obtaining the nanoparticle antifreeze agent.
Further, the preparation method of the small molecule solution in step S1 is as follows: weighing 0.0010-0.0018g of micromolecule, dissolving in 1-10ml of deionized water, and fully stirring and mixing to obtain a micromolecule solution with the concentration of 10-100 mu M;
the preparation method of the nano-gel solution comprises the following steps: and (3) dispersing the nano particles in a water phase after centrifugal concentration to form a nano glue solution, wherein the concentration of the prepared nano glue solution is 0.1-10nM, and the pH of the prepared nano glue solution is adjusted to 4.5-5.5 by using a 50-200mM NaOH solution.
Further, the small molecules in the small molecule solution are monomer small molecule substances, and the structural general formula of the monomer small molecules is as follows: one end of the hydroxyl-containing polymer contains hydroxyl, and the hydroxyl-containing polymer is connected with a coupling group through a carbon skeleton; the carbon skeleton has a structure of one of alkane, alkene, alkyne, aromatic ring or nitrogen-containing heterocycle; the structure of the coupling group is one of sulfydryl, carboxyl, amino or silane group.
Further, the hydroxyl end of the small molecule contains a methyl group.
Further, the small molecule is one of 6-aza-2-thiothymine, 6-aza-2-thiouracil, 2-thiobarbituric acid, 4, 6-dimethyl-2-mercaptopyrimidine, 4-mercaptopyridine, 3-mercapto-1-propanol or hydroxysilane.
Further, the nano particles in the nano-colloid solution comprise one of gold, silver, silicon dioxide, polystyrene or polymethyl methacrylate, the form of the nano particles comprises one or more of nanospheres, nanorods, nanocubes and nano octahedrons, and the particle size of the nano particles is 13-80 nm.
Further, in the step S2, the volume ratio of the small molecule solution to the nano-gel solution is 1:5-5:1, the small molecule solution and the nano-gel solution are stirred at room temperature, the stirring speed is 200-1000rpm, and the stirring time is 4-12 hours.
Further, the rotation speed of the centrifugation in the step S3 is 3000-10000rpm, and the centrifugation time is 10-30 min.
Further, the number of washing in step S3 is 2-4, and the washing solvent is deionized water or PBS buffer solution.
A nanoparticle antifreeze agent containing a small molecule monolayer, which comprises the nanoparticle antifreeze agent prepared by using the preparation method of the nanoparticle antifreeze agent containing a small molecule monolayer.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention fully utilizes the interface size effect of nano particles and sulfydryl on small molecules to form stable covalent bonds, takes the nano particles as an inner core, couples a layer of compact hydroxyl-containing small molecule monomer substance which does not have frost resistance activity originally on the surface of the nano particles to form nano particles with exposed hydroxyl and densely arranged nano particles, so that the hydroxyl-containing small molecules are enriched on the surfaces of the nano particles to form nano particles with exposed hydroxyl and densely arranged nano particles, and the high-density hydroxyl and ice crystals form a large number of hydrogen bonds, so that the nano particles are better adsorbed on the surfaces of the ice crystals to achieve high-efficiency frost resistance activity;
2. simple and universal synthesis routes, shapes and sizes of gold, silver, silicon dioxide, polystyrene, polymethyl methacrylate and other nano particles are uniform and controllable; the micromolecules have wide sources and low price, and the coupling process of the micromolecules and the nanoparticles is simple and easy to operate;
3. compared with the antifreeze material reported at present, the antifreeze material has lower required concentration and less dosage, and can reduce the size of the ice crystal to about 20 percent of the original size only under nanomolar concentration.
Drawings
FIG. 1 is a schematic diagram of the principle of monolayer nanomaterials in inhibiting ice crystal growth;
FIG. 2 is a schematic diagram of UV-vis spectra before and after coupling of 5 small molecules with AuNPs in example;
FIG. 3 is an optical photograph and a graph of the change in size of ice crystals after 30min during the recrystallization of ice crystals for 5 different concentrations of small molecules in example;
FIG. 4 is an optical photograph and a graph of the dimensional change of ice crystals after 0, 10, 20 and 30min during the recrystallization of ice crystals for pure water, pure AuNPs and 5 kinds of monolayer nanomaterials in example;
fig. 5 is a bar graph comparing ice recrystallization inhibitory activity of 5 monolayer nanomaterials at different concentrations.
Detailed Description
The following description of the embodiments of the present application is provided by way of specific examples, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein. The present application is capable of other and different embodiments and its several details are capable of modifications and variations in various respects, all without departing from the spirit of the present application. It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.
Referring to fig. 1-5, the present invention provides the following technical solutions:
a nanoparticle antifreeze agent containing small molecule monolayers is shown in figure 1, the structure of the nanoparticle antifreeze agent comprises an inner core 1 with a spherical inner part, a compact small molecule layer 2 is coupled on the surface of the inner core 1, one end of the small molecule layer 2 contains hydroxyl to form a coating for the inner core 1, and the preparation method comprises the following steps:
step S1, preparing a micromolecule solution and a nanometer glue solution;
step S2, stirring and mixing the micromolecule solution prepared in the step S1 and the nanometer glue solution to obtain hydroxyl exposed nanoparticles which take the nanoparticles as cores and are coupled with micromolecule monomer substances on the surfaces of the nanoparticles;
and S3, centrifuging and washing the hydroxyl exposed nanoparticles obtained in the step S2 to remove uncoupled micromolecules, and obtaining the nanoparticle antifreeze agent.
The preparation method of the small molecule solution in the step S1 comprises the following steps: weighing 0.0010-0.0018g of micromolecule, dissolving in 1-10ml of deionized water, and fully stirring and mixing to obtain a micromolecule solution with the concentration of 10-100 mu M; the micromolecules in the micromolecule solution are monomer micromolecule substances, and the structural general formula of the monomer micromolecules is as follows: one end of the micromolecule contains hydroxyl, and the hydroxyl is connected with a coupling group through a carbon skeleton, wherein the hydroxyl end of the micromolecule can contain methyl or not contain methyl; the carbon skeleton has one of alkane, alkene, alkyne, aromatic ring or nitrogen-containing heterocycle; the coupling group structure is one of sulfydryl, carboxyl, amino or silane groups, and the micromolecule is one of 6-aza-2-thiothymine (ATT), 6-aza-2-thiouracil (DMTP), 2-thiobarbituric acid (TBA), 4, 6-dimethyl-2-mercaptopyrimidine (DMMP), 4-Mercaptopyridine (MP), 3-mercapto-1-propanol or hydroxysilane;
the preparation method of the nano-gel solution comprises the following steps: dispersing the nano particles in a water phase after centrifugal concentration to form a nano glue solution, wherein the concentration of the prepared nano glue solution is 0.1-10nM, and adjusting the pH of the prepared nano glue solution to 4.5-5.5 by using a 50-200mM NaOH solution; the nano particles in the nano glue solution comprise one of gold, silver, silicon dioxide, polystyrene or polymethyl methacrylate, the form of the nano particles comprises one or more of nanospheres, nanorods, nanocubes and nano octahedrons, and the particle size of the nano particles is 13-80 nm.
In addition, the volume ratio of the micromolecule solution to the nano-gel solution in the step S2 is 1:5-5:1, the micromolecule solution and the nano-gel solution are stirred at room temperature, the stirring speed is 200-; the rotation speed of the centrifugation in the step S3 is 3000-10000rpm, and the centrifugation time is 10-30 min; the washing times are 2-4 times, and the washing solvent is deionized water or PBS buffer solution.
Example 1
Preparation of nanoparticle antifreeze from gold nanoparticles prepared from HAuCl, 6-aza-2-thiothymine (ATT)4Synthesized by a citrate reduction method and a hydroxylamine hydrochloride reduction method, and the synthesis steps are as follows:
1. a citrate reduction method: in an Erlenmeyer flask, 98.9ml of deionized water and 1ml of 3% sodium citrate are added, heated to boiling at 400rpm, and then 0.1ml of 250mM HAuCl is added4Kept boilingStirring for 7min, and cooling in ice bath to obtain 15nm gold seeds;
2. and (3) a hydroxylamine hydrochloride reduction method: 10ml of 15nm gold seed was added with 9.8ml deionized water, 100mM hydroxylamine hydrochloride and 0.2ml of 1% sodium citrate, stirred at 400rpm for 5min, and then 47.1. mu.l of 250mM HAuCl was added4Continuously stirring for 1h to obtain 40nm gold particles;
3. preparing a gold nano-gel solution: dispersing 40nm gold particles in a water phase after centrifugal concentration to form a gold nano-gel solution, and then adding 100mM NaOH to adjust the pH of the gold nano-gel solution to 4.5-5.5, wherein the process conditions of centrifugal concentration are as follows: the centrifugation speed is 400rpm, and the centrifugation time is 20 min.
Preparing 10 mu M of 6-aza-2-thiothymine, adding 3mL of the 6-aza-2-thiothymine into 5mL of the gold nano-gel solution after pH adjustment, stirring at 400rpm for 12h at room temperature, centrifuging at 4000rpm, removing supernatant, adding deionized water, washing, resuspending, centrifuging, repeating the operation for 2 times, and finally adding deionized water to prepare 2nM, 4nM, 6nM and 8nM nano-particle antifreeze agents.
Example 2
Preparation of nanoparticle antifreeze agents exemplified by gold nanoparticles prepared from HAuCl, 6-aza-2-thiouracil (DMTP)4Synthesized by a citrate reduction method and a hydroxylamine hydrochloride reduction method, and the synthesis steps are as follows:
1. a citrate reduction method: in an Erlenmeyer flask, 98.9ml of deionized water and 1ml of 3% sodium citrate are added, heated to boiling at 400rpm, and then 0.1ml of 250mM HAuCl is added4Keeping boiling and stirring for 7min, and cooling in ice bath to obtain 15nm gold seeds;
2. and (3) a hydroxylamine hydrochloride reduction method: 10ml of 15nm gold seed was added with 9.8ml deionized water, 100mM hydroxylamine hydrochloride and 0.2ml of 1% sodium citrate, stirred at 400rpm for 5min, and then 47.1. mu.l of 250mM HAuCl was added4Continuously stirring for 1h to obtain 40nm gold particles;
3. preparing a gold nano-gel solution: dispersing 40nm gold particles in a water phase after centrifugal concentration to form a gold nano-gel solution, and then adding 100mM NaOH to adjust the pH of the gold nano-gel solution to 4.5-5.5, wherein the process conditions of centrifugal concentration are as follows: the centrifugation speed is 400rpm, and the centrifugation time is 20 min.
Preparing 10 mu M of 6-aza-2-thiouracil (DMTP), adding 3mL of the DMTP into 5mL of the gold nano-gel solution after pH adjustment, stirring at 400rpm at room temperature for 12h, centrifuging at 4000rpm, removing supernatant, adding deionized water, washing, resuspending, centrifuging, repeating the operation for 2 times, and finally adding deionized water to prepare 2nM, 4nM, 6nM and 8nM nano-particle antifreeze agents.
Example 3
Preparation of nanoparticle antifreeze agent by using gold nanoparticles and 2-thiobarbituric acid (TBA), wherein the gold nanoparticles are prepared from HAuCl4Synthesized by a citrate reduction method and a hydroxylamine hydrochloride reduction method, and the synthesis steps are as follows:
1. a citrate reduction method: in an Erlenmeyer flask, 98.9ml of deionized water and 1ml of 3% sodium citrate are added, heated to boiling at 400rpm, and then 0.1ml of 250mM HAuCl is added4Keeping boiling and stirring for 7min, and cooling in ice bath to obtain 15nm gold seeds;
2. and (3) a hydroxylamine hydrochloride reduction method: 10ml of 15nm gold seed was added with 9.8ml deionized water, 100mM hydroxylamine hydrochloride and 0.2ml of 1% sodium citrate, stirred at 400rpm for 5min, and then 47.1. mu.l of 250mM HAuCl was added4Continuously stirring for 1h to obtain 40nm gold particles;
3. preparing a gold nano-gel solution: dispersing 40nm gold particles in a water phase after centrifugal concentration to form a gold nano-gel solution, and then adding 100mM NaOH to adjust the pH of the gold nano-gel solution to 4.5-5.5, wherein the process conditions of centrifugal concentration are as follows: the centrifugation speed is 400rpm, and the centrifugation time is 20 min.
Preparing 10 mu M of 2-thiobarbituric acid (TBA), adding 3mL of the 2-thiobarbituric acid into 5mL of the gold nano-gel solution after pH adjustment, stirring at 400rpm for 12h at room temperature, centrifuging at 4000rpm, removing supernatant, adding deionized water, washing, resuspending, centrifuging, repeating the operation for 2 times, and finally adding deionized water to prepare 2nM, 4nM, 6nM and 8nM nano-particle antifreeze agents.
Example 4
Preparation of nanoparticle antifreeze agents exemplified by gold nanoparticles prepared from HAuCl, 4, 6-dimethyl-2-mercaptopyrimidine (DMMP)4Synthesized by a citrate reduction method and a hydroxylamine hydrochloride reduction method, and the synthesis steps thereofComprises the following steps:
1. a citrate reduction method: in an Erlenmeyer flask, 98.9ml of deionized water and 1ml of 3% sodium citrate are added, heated to boiling at 400rpm, and then 0.1ml of 250mM HAuCl is added4Keeping boiling and stirring for 7min, and cooling in ice bath to obtain 15nm gold seeds;
2. and (3) a hydroxylamine hydrochloride reduction method: 10ml of 15nm gold seed was added with 9.8ml deionized water, 100mM hydroxylamine hydrochloride and 0.2ml of 1% sodium citrate, stirred at 400rpm for 5min, and then 47.1. mu.l of 250mM HAuCl was added4Continuously stirring for 1h to obtain 40nm gold particles;
3. preparing a gold nano-gel solution: dispersing 40nm gold particles in a water phase after centrifugal concentration to form a gold nano-gel solution, and then adding 100mM NaOH to adjust the pH of the gold nano-gel solution to 4.5-5.5, wherein the process conditions of centrifugal concentration are as follows: the centrifugation speed is 400rpm, and the centrifugation time is 20 min.
Preparing 10 mu M of 4, 6-dimethyl-2-mercaptopyrimidine (DMMP), adding 3mL of the DMMP into 5mL of the gold nano-gel solution with the pH adjusted, stirring at 400rpm for 12h at room temperature, centrifuging at 4000rpm, removing supernatant, adding deionized water, washing, resuspending, centrifuging, repeating the operation for 2 times, and finally adding deionized water to prepare 2nM, 4nM, 6nM and 8nM nano-particle antifreeze agents.
Example 5
Preparation of nanoparticle antifreeze from gold nanoparticles prepared from HAuCl and 4-Mercaptopyridine (MP)4Synthesized by a citrate reduction method and a hydroxylamine hydrochloride reduction method, and the synthesis steps are as follows:
1. a citrate reduction method: in an Erlenmeyer flask, 98.9ml of deionized water and 1ml of 3% sodium citrate are added, heated to boiling at 400rpm, and then 0.1ml of 250mM HAuCl is added4Keeping boiling and stirring for 7min, and cooling in ice bath to obtain 15nm gold seeds;
2. and (3) a hydroxylamine hydrochloride reduction method: 10ml of 15nm gold seed was added with 9.8ml deionized water, 100mM hydroxylamine hydrochloride and 0.2ml of 1% sodium citrate, stirred at 400rpm for 5min, and then 47.1. mu.l of 250mM HAuCl was added4Continuously stirring for 1h to obtain 40nm gold particles;
3. preparing a gold nano-gel solution: dispersing 40nm gold particles in a water phase after centrifugal concentration to form a gold nano-gel solution, and then adding 100mM NaOH to adjust the pH of the gold nano-gel solution to 4.5-5.5, wherein the process conditions of centrifugal concentration are as follows: the centrifugation speed is 400rpm, and the centrifugation time is 20 min.
Preparing 10 mu M of 4-Mercaptopyridine (MP), adding 3mL of the 4-Mercaptopyridine (MP) into 5mL of gold nano-gel solution with the pH adjusted, stirring at the room temperature of 400rpm for 12h, centrifuging at 4000rpm, removing supernatant, adding deionized water, washing, resuspending and centrifuging, repeating the operation for 2 times, and finally adding deionized water to prepare 2nM, 4nM, 6nM and 8nM nano-particle antifreeze agents.
The wavelength of the maximum absorption peak before and after the coupling of the small molecules and the gold nanoparticles in the examples 1 to 5 is measured by a UV-vis spectrometer, and as shown in figure 2, the wavelength of the maximum absorption peak of the coupled nanoparticle antifreeze agent is increased.
In order to show the anti-freezing effect of the nano-particle anti-freezing agent prepared by the invention, the following experiment is carried out.
Experiment one:
using 8nM of the nanoparticle antifreeze prepared in examples 1-5 as an example, the following experiment was performed:
10 μ L of the nanoparticle antifreeze agent of 8nM as in examples 1-5, pure water and pure AuNPs solution were respectively dropped vertically from 1.6m height onto a glass plate precooled with liquid nitrogen to instantaneously form a circular ice wafer with a thickness of about 10 μm; then quickly transferring the ice crystals into a closed cold and hot table chamber at the temperature of minus 60 ℃, raising the temperature to minus 8 ℃ at the temperature rise rate of 15 ℃/min, finally maintaining the temperature for 30min, then taking a photo by using an optical microscope, selecting the average value of the 10 largest ice crystal sizes from the photo by using Image J software, comparing the average value with pure water and pure AuNPs solution, and observing the anti-freezing activity of the monolayer nano material to obtain the optical photo and the size change chart of the ice crystals after 0, 10, 20 and 30min in the process of recrystallization of the ice crystals of the pure water, the pure AuNPs and the 5 kinds of monolayer nano particle anti-freezing agents shown in the figure 4.
Experiment two
Taking small molecules with different concentrations to perform the same test as the test condition, taking pictures by an optical microscope, selecting the size of 10 largest ice crystals by Image J software, and calculating the average value to obtain an optical picture and a size change chart of the ice crystals after 30min in the process of recrystallizing the ice crystals, wherein the optical picture and the size change chart are shown in figure 3.
Experiment three
The small-molecule nanoparticle antifreeze agents with different concentrations are taken to carry out a test with the same test condition, the size of the ice crystal obtained after 30min is compared with the size of the ice crystal obtained after 30min of pure water to obtain a bar-shaped contrast diagram of ice recrystallization inhibition activity of 5 monomolecular-layer nanoparticle antifreeze agents with different concentrations as shown in figure 5, and data shows that the small-molecule nanoparticle antifreeze agents are added into the solution to effectively inhibit the formation of larger ice crystals.
According to FIGS. 3 and 4, pure water, pure AuNPs solution, 10. mu.M pure 6-aza-2-thiothymine (ATT), 10. mu.M pure 6-aza-2-thiouracil (DMTP), 10. mu.M pure 2-thiobarbituric acid (TBA), 10. mu.M pure 4, 6-dimethyl-2-mercaptopyrimidine (DMMP), 10. mu.M pure 4-Mercaptopyridine (MP) have average maximum ice crystal sizes of 175. mu.m, 170. mu.m, 175. mu.m, 184. mu.m, 158. mu.m, 169. mu.m, respectively, after 30min, whereas 6-aza-2-thiothymine single-molecule nanoparticle antifreeze, 6-aza-2-thiouracil single-molecule nanoparticle antifreeze, 2-thiobarbituric acid single-molecule antifreeze, 4, the average maximum ice crystal sizes of the 6-dimethyl-2-mercaptopyrimidine monomolecular nanoparticle antifreeze agent and the 4-mercaptopyridine monomolecular nanoparticle antifreeze agent after 30min are 34 micrometers, 35 micrometers, 33 micrometers, 64 micrometers and 70 micrometers, which are respectively 19.43 percent, 20 percent, 18.86 percent, 36.57 percent and 40 percent of the ice crystal size of pure water, and simultaneously, the average maximum ice crystal sizes are also lower than the ice crystal size of a small molecular material after 30 min. Water in the process of freezing and storing biological solution such as cells can form ice crystals, and the ice crystals can be recrystallized to form larger ice crystals to damage the cells and the like; the antifreeze material is added into the solution to effectively inhibit the formation of larger ice crystals, and the micromolecule nanoparticle antifreeze agent provided by the invention can effectively preserve the activity of biological solutions such as cells and the like without being damaged.
According to the experimental results, the hydroxyl-containing micromolecular monomer substances which do not have the anti-freezing activity originally are enriched on the surfaces of the nanospheres to form the nanoparticles with exposed hydroxyl groups and densely arranged, and the growth and recrystallization of ice crystals can be effectively inhibited under the condition of low sol concentration. Meanwhile, through ingenious design of hydrophilic and hydrophobic groups in the small molecular monomer structure, namely, the small molecular monomer structure only contains hydroxyl, only contains methyl, simultaneously contains hydroxyl and methyl and does not contain hydroxyl and methyl, the hydroxyl plays a decisive role in the anti-freezing process and is irrelevant to the existence of the methyl. The size of the ice crystals was reduced by 80% compared to pure water or a solution of gold nanoparticles without small molecules coupled. And the recrystallization inhibition activity of ice is independent of the size of small molecules and the morphology of nanoparticles. By the invention, a compact single layer formed by hydroxyl-containing small molecules on a nanometer scale can realize a good anti-freezing effect. The antifreeze material has important significance for the increasing demand of cryopreservation of cells, tissues and embryos, has guiding significance for the design of novel antifreeze materials, and enriches the diversity of the existing cryoprotectants; the micromolecules have wide sources and low price, and the coupling process of the micromolecules and the nano particles is simple and easy to operate; compared with the antifreeze material reported at present, the antifreeze material has lower required concentration and less dosage, and can reduce the size of the ice crystal to about 20 percent of the original size only under nanomolar concentration.
In addition, simple and universal synthesis routes, shapes and sizes of gold, silver, silicon dioxide, polystyrene, polymethyl methacrylate and other nano particles are uniform and controllable, and the anti-freezing agent can be used for the small-molecule nano particles.
The above-described embodiments are merely illustrative of the principles and utilities of the present patent application and are not intended to limit the present patent application. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of this patent application. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical concepts disclosed in the present application shall be covered by the claims of the present application.
Claims (10)
1. A preparation method of a nanoparticle antifreeze agent containing a small molecule monolayer is characterized by comprising the following steps:
step S1, preparing a micromolecule solution and a nanometer glue solution;
step S2, stirring and mixing the micromolecule solution prepared in the step S1 and the nanometer glue solution to obtain hydroxyl exposed nanoparticles which take the nanoparticles as cores and are coupled with micromolecule monomer substances on the surfaces of the nanoparticles;
and S3, centrifuging and washing the hydroxyl exposed nanoparticles obtained in the step S2 to remove uncoupled micromolecules, and obtaining the nanoparticle antifreeze agent.
2. The method for preparing the nanoparticle antifreeze agent containing the small molecule monolayer as claimed in claim 1, wherein the method comprises the following steps: the preparation method of the small molecule solution in the step S1 comprises the following steps: weighing 0.0010-0.0018g of micromolecule, dissolving in 1-10ml of deionized water, and fully stirring and mixing to obtain a micromolecule solution with the concentration of 10-100 mu M;
the preparation method of the nano-gel solution comprises the following steps: and (3) dispersing the nano particles in a water phase after centrifugal concentration to form a nano glue solution, wherein the concentration of the prepared nano glue solution is 0.1-10nM, and the pH of the prepared nano glue solution is adjusted to 4.5-5.5 by using a 50-200mM NaOH solution.
3. The method for preparing the nanoparticle antifreeze agent containing the small molecule monolayer as claimed in claim 2, wherein the method comprises the following steps: the micromolecules in the micromolecule solution are monomer micromolecule substances, and the structural general formula of the monomer micromolecules is as follows: one end of the hydroxyl-containing polymer contains hydroxyl, and the hydroxyl-containing polymer is connected with a coupling group through a carbon skeleton; the carbon skeleton has a structure of one of alkane, alkene, alkyne, aromatic ring or nitrogen-containing heterocycle; the structure of the coupling group is one of sulfydryl, carboxyl, amino or silane group.
4. The method for preparing the nanoparticle antifreeze agent containing the small molecule monolayer as claimed in claim 3, wherein the method comprises the following steps: the hydroxyl end of the small molecule contains a methyl group.
5. The method for preparing the nanoparticle antifreeze agent containing the small molecule monolayer as claimed in claim 3, wherein the method comprises the following steps: the small molecule is one of 6-aza-2-thiothymine, 6-aza-2-thiouracil, 2-thiobarbituric acid, 4, 6-dimethyl-2-mercaptopyrimidine, 4-mercaptopyridine, 3-mercapto-1-propanol or hydroxysilane.
6. The method for preparing the nanoparticle antifreeze agent containing the small molecule monolayer as claimed in claim 2, wherein the method comprises the following steps: the nano particles in the nano glue solution comprise one of gold, silver, silicon dioxide, polystyrene or polymethyl methacrylate, the form of the nano particles comprises one or more of nanospheres, nanorods, nanocubes and nano octahedrons, and the particle size of the nano particles is 13-80 nm.
7. The method for preparing the nanoparticle antifreeze agent containing the small molecule monolayer as claimed in claim 1, wherein the method comprises the following steps: the volume ratio of the micromolecule solution to the nanometer gel solution in the step S2 is 1:5-5:1, the micromolecule solution and the nanometer gel solution are stirred at room temperature, the stirring speed is 200-1000rpm, and the stirring time is 4-12 h.
8. The method for preparing the nanoparticle antifreeze agent containing the small molecule monolayer as claimed in claim 1, wherein the method comprises the following steps: the rotation speed of the centrifugation in the step S3 is 3000-10000rpm, and the centrifugation time is 10-30 min.
9. The method for preparing the nanoparticle antifreeze agent containing the small molecule monolayer as claimed in claim 1, wherein the method comprises the following steps: the number of washing in step S3 is 2-4, and the washing solvent is deionized water or PBS buffer solution.
10. A nanoparticle antifreeze agent containing a small molecule monolayer is characterized in that: a nanoparticle antifreeze agent prepared by using the method for preparing a nanoparticle antifreeze agent containing a small molecule monolayer according to any one of claims 1 to 9.
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