A method for preparing a cholesteric liquid crystal with adjustable pitch based on lysozyme fibers

Abstract

The application discloses a method for preparing cholesteric liquid crystal with adjustable pitch based on lysozyme fibers, and belongs to the field of biological liquid crystals. The preparation method comprises the following steps: (1) uniformly dispersing lysozyme in water, adjusting pH, filtering, and obtaining a protein dispersion liquid; (2) stirring and heating the protein dispersion liquid obtained in step S1 to obtain a fibrous aggregate dispersion liquid; (3) shearing and dialyzing the fibrous aggregate dispersion liquid obtained in step S2 to obtain a short fiber dispersion liquid; (4) concentrating the short fiber dispersion liquid obtained in step S3 to obtain a short fiber concentrated liquid; and (5) centrifuging the short fiber concentrated liquid obtained in step S4, taking supernatant, and obtaining cholesteric liquid crystal with adjustable pitch. The cholesteric liquid crystal is prepared based on lysozyme fibrous aggregates for the first time, and the pitch of the cholesteric liquid crystal is regulated by changing pH value and ionic strength, so that a basis and a simple strategy are provided for producing cholesteric liquid crystal systems with target characteristics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological liquid crystals, and particularly relates to a method for preparing pitch-adjustable cholesteric liquid crystals based on lysozyme fibers. BACKGROUND

[0002] Chiral hierarchical structures self-assembled from nanoscale building blocks are ubiquitous in nature and play an important role in frontier fields such as biomedical engineering, bionics and energy science. Cholesteric liquid crystals are a kind of chiral structure, which can be self-assembled by liquid-liquid separation of concentrated rod-like colloidal particles, and with the increase of the concentration of the system, it changes from isotropic to nematic phase. This liquid-liquid phase separation is first elucidated by Onsager, which is due to the balance between the orientation-dependent repulsive volume (favorable for orientation order) and the orientation entropy (favorable for orientation disorder). According to this explanation and the direct influence of orientation on electrostatic repulsion, the electrostatic interaction between rod-like particles will have a key influence on the formation of isotropic / anisotropic phase and the pitch of cholesteric liquid crystals.

[0003] Compared with the widely reported self-assembled rod-like colloidal systems, the phase transition and pitch control of liquid crystals based on protein fibrous aggregates are still largely unknown. The lack of this knowledge seriously hinders the development and application of chiral nematic phase generated by protein fibrous aggregates, especially in the field of protein-related biological engineering, including biomimetic mineralization, bioadhesive materials and biomimetic cytoskeletal structures. Fibrous aggregates are anisotropic colloids that self-assemble by stacking the four-level structures of β-sheet-rich polypeptides through dense hydrogen bonds at the molecular level, with high persistence length and large aspect ratio at the single fibril level. Its unique polymorphism combined with controllable fiber structure provides an ideal platform for tracking phase transition behavior under different internal / external parameters and advancing our understanding of chiral nematic phase.

[0004] Lysozyme is prone to fibrillation in vivo and in vitro, and its mutation can further cause systemic amyloidosis. The isoelectric point of lysozyme is about 11.3, which makes it an ideal model for studying the influence of environmental parameters (especially pH) on the phase transition, tower-like morphology and periodic helical pitch of liquid crystals. Recently, in a closed system, cholesteric liquid crystals were successfully prepared using shortened β-lactoglobulin fibrous aggregates with a relatively narrow length distribution, and the appearance of cholesteric phase with helical twist orientation rods may be directly related to the inherent chirality of single fibrils. However, due to the complexity of protein fibrous aggregates, such as persistence length, periodic fluctuation, the number of assembly filaments in single fibrils, the difference of primary structure amino acid sequence and the change of surrounding microenvironment, etc., little is known about cholesteric liquid crystals based on lysozyme fibrous aggregates. SUMMARY

[0005] In view of the above problems, the present application first prepares a cholesteric liquid crystal based on lysozyme fibrous aggregates, and regulates the pitch by changing the pH and ionic strength.

[0006] The first object of the present application is to provide a method for preparing a cholesteric liquid crystal with adjustable pitch based on lysozyme fibrous aggregates, and to describe the transition behavior from nematic phase to cholesteric phase. The second object is to regulate the pitch by changing the pH and ionic strength, taking advantage of the high sensitivity of the cholesteric liquid crystal to changes in pH and ionic strength. The present application provides a basis and simple strategy for producing cholesteric liquid crystal systems with target characteristics.

[0007] The technical scheme adopted by the present application to achieve the above object is: a preparation method of a cholesteric liquid crystal with adjustable pitch, comprising the following specific steps:

[0008] S1, dispersion: uniformly dispersing lysozyme in water, adjusting the pH, filtering, and obtaining a protein dispersion;

[0009] S2, heat treatment: stirring and heating the protein dispersion obtained in step S1 to obtain a fibrous aggregate dispersion;

[0010] S3, shear purification: shearing and dialyzing the fibrous aggregate dispersion obtained in step S2 to obtain a short fiber dispersion;

[0011] S4, concentration: concentrating the short fiber dispersion obtained in step S3 to obtain a short fiber concentrate;

[0012] S5, centrifugation: centrifuging the short fiber concentrate obtained in step S4, taking the supernatant, and obtaining a cholesteric liquid crystal with adjustable pitch.

[0013] Further, the concentration of lysozyme dispersed in water in step S1 is 1-2% (w / v).

[0014] Further, the pH adjustment in step S1 is 1.5-2.5.

[0015] Further, the filtration in step S1 is filtration using a filter membrane with a pore size of 0.4-0.45 μm.

[0016] Further, the stirring and heating in step S2 is stirring and heating at 85-95℃ at a stirring speed of 200-400 r / min for 20-28 h.

[0017] Further, the shearing time in step S3 is 60-180 s.

[0018] Further, the dialysis in step S3 is dialysis in ultrapure water at pH 1.5-2.5 using a dialysis bag with a molecular weight cutoff of 90-100 kDa for 3-5 days.

[0019] Further, the length of the short fibers in the short fiber dispersion liquid in step S3 is 400-600 nm.

[0020] Further, the concentration in step S4 is concentrated in a polyethylene glycol solution with a mass concentration of 5-15% (w / v) using a dialysis bag with a molecular weight of 3-3.6 kDa for 24-36 h.

[0021] Further, the concentration of the short fiber concentrated solution in step S4 is 2.5-4% (w / v).

[0022] Further, the centrifugation in step S5 is centrifuged at 10,000-15,800 g for 10-30 min.

[0023] The present application provides a cholesteric liquid crystal with adjustable pitch prepared according to the above method.

[0024] The present application provides the application of the cholesteric liquid crystal with adjustable pitch in the preparation of optical instruments and the field of biological liquid crystals.

[0025] The present application also provides a pitch regulation method for the above cholesteric liquid crystal, which regulates the pitch of the cholesteric liquid crystal by changing the pH and ionic strength of the cholesteric liquid crystal.

[0026] Further, when the pitch of the cholesteric liquid crystal needs to be regulated to 14.3-29.0 μm, the pH needs to be adjusted to 2.0-2.8, and the ionic strength needs to be adjusted to 0-10 Mm.

[0027] Advantages:

[0028] 1. The present application first prepares a cholesteric liquid crystal based on lysozyme fibrous aggregates, which is simple and convenient.

[0029] 2. The cholesteric liquid crystal prepared by the present application can regulate the pitch by changing the pH value and ionic strength, which provides a basis and simple strategy for producing cholesteric liquid crystal systems with target characteristics. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is an atomic force microscope image of the lysozyme fibrous aggregate before shearing of the present application.

[0031] Figure 2 It is an atomic force microscope image of the lysozyme fibrous aggregate after shearing of the present application.

[0032] Figure 3 It is an atomic force microscope image of the bottom nematic phase of the lysozyme fibrous aggregate of the present application.

[0033] Figure 4Fragment length distribution plot for the bottom nematic phase of the lysozyme fibrous aggregate of the present invention.

[0034] Figure 5 Fragment height distribution plot for the bottom nematic phase of the lysozyme fibrous aggregate of the present invention.

[0035] Figure 6 Critical transition volume plot for the cholesteric liquid crystal of Example 1 of the present invention from the isotropic structure to the cholesteric structure.

[0036] Figure 7 Critical transition volume plot for the cholesteric liquid crystal of Example 2 of the present invention from the isotropic structure to the cholesteric structure.

[0037] Figure 8 Critical transition volume plot for the cholesteric liquid crystal of Example 3 of the present invention from the isotropic structure to the cholesteric structure.

[0038] Figure 9 Critical transition volume plot for the cholesteric liquid crystal of Example 4 of the present invention from the isotropic structure to the cholesteric structure.

[0039] Figure 10 Critical transition volume plot for the cholesteric liquid crystal of Example 5 of the present invention from the isotropic structure to the cholesteric structure.

[0040] Figure 11 Pitch plot for the cholesteric liquid crystal of Example 1 of the present invention.

[0041] Figure 12 Pitch plot for the cholesteric liquid crystal of Example 2 of the present invention.

[0042] Figure 13 Pitch plot for the cholesteric liquid crystal of Example 3 of the present invention.

[0043] Figure 14 Pitch plot for the cholesteric liquid crystal of Example 4 of the present invention.

[0044] Figure 15 Pitch plot for the cholesteric liquid crystal of Example 5 of the present invention.

[0045] Figure 16 Morphology of the cholesteric liquid crystal of Example 1 of the present invention.

[0046] Figure 17 Morphology of the cholesteric liquid crystal of Example 2 of the present invention.

[0047] Figure 18 Morphology of the cholesteric liquid crystal of Example 3 of the present invention.

[0048] Figure 19 Morphology of the cholesteric liquid crystal of Example 4 of the present invention.

[0049] Figure 20 The morphology of the sample obtained in Example 5 of the present application.

[0050] Figure 21 The morphology of the sample obtained in Comparative Example 1 of the present application.

[0051] Figure 22 The atomic force microscopy image of the lysozyme fibrous aggregate after shearing in Comparative Example 2 of the present application.

[0052] Figure 23 The morphology of the sample obtained in Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0053] The present application is further illustrated by the following specific examples:

[0054] Source of raw materials

[0055] Lysozyme from egg white, ~70000 U / mg, Sigma-Aldrich, CAS No.: 12650-88-3.

[0056] Example 1

[0057] S1, dispersion: lysozyme was uniformly dispersed in water at a mass concentration of 2% (w / v) and adjusted to pH 2.0, and filtered through a filter membrane with a pore size of 0.45 μm to obtain a protein dispersion;

[0058] S2, heat treatment: the protein dispersion obtained in step S1 was heated at 85°C with magnetic stirring at a speed of 200 r / min for 28 h to obtain a fibrous aggregate dispersion;

[0059] S3, shearing purification: the fibrous aggregate dispersion obtained in step S2 was sheared for 100 s, and dialyzed in ultrapure water at pH 2.0 using a dialysis bag with a molecular weight cut-off of 100 kDa for 4 days to obtain a short fiber dispersion with a length of about 580 nm;

[0060] S4, concentration: the short fiber dispersion obtained in step S3 was concentrated in a polyethylene glycol solution with a mass concentration of 7.5% (w / v) using a dialysis bag with a molecular weight cut-off of 3.6 kDa for 24 h to obtain a short fiber concentrate with a concentration of about 3.0% (w / v);

[0061] S5, centrifugation: the short fiber concentrate obtained in step S4 was centrifuged at 10000 g for 10 min, and the supernatant was placed in a sealed glass bottle and stored at 4°C to obtain a cholesteric liquid crystal.

[0062] S6, pitch regulation: the cholesteric liquid crystal obtained in step S5 was adjusted to pH 2.0 to obtain a cholesteric liquid crystal with a pH of 2.0 and an ionic strength of 0 mM.

[0063] The cholesteric liquid crystal prepared in this example was used to measure the fiber length distribution and height distribution using an atomic force microscope; after the concentrated sample reached equilibrium, phase separation occurred macroscopically, 100 μL of sample was taken from the top and bottom phases, respectively, and the protein concentration was determined according to the weight change measured by a microbalance; the morphological characteristics and transformation behavior of the sample during phase transition were observed using a polarizing microscope, and the pitch was determined according to the distance between the two maximum bright bands of the cholesteric liquid crystal.

[0064] Example 2

[0065] S1, dispersion: lysozyme was uniformly dispersed in water at a mass concentration of 2% (w / v) and the pH was adjusted to 2.0, and then filtered through a filter membrane with a pore size of 0.45 μm to obtain a protein dispersion;

[0066] S2, heat treatment: the protein dispersion obtained in step S1 was heated at 90°C with magnetic stirring at a speed of 300 r / min for 24 h to obtain a fibrous aggregate dispersion;

[0067] S3, shear purification: the fibrous aggregate dispersion obtained in step S2 was sheared for 120 s, and then dialyzed in ultrapure water at pH 2.0 for 4 days using a dialysis bag with a molecular weight cut-off of 100 kDa to obtain a short fiber dispersion with a length of about 530 nm;

[0068] S4, concentration: the short fiber dispersion obtained in step S3 was concentrated in a 10% (w / v) polyethylene glycol solution using a dialysis bag with a molecular weight cut-off of 3.6 kDa for 24 h to obtain a short fiber concentrate with a concentration of 3.6% (w / v);

[0069] S5, centrifugation: the short fiber concentrate obtained in step S4 was centrifuged at 15800 g for 20 min, and the supernatant was placed in a sealed glass bottle and stored at 4°C to obtain a cholesteric liquid crystal.

[0070] S6, pitch regulation: the cholesteric liquid crystal obtained in step S5 was adjusted to a pH of 2.4 to obtain a cholesteric liquid crystal with a pH of 2.4 and an ionic strength of 0 mM.

[0071] The cholesteric liquid crystal prepared in this example was used to measure the fiber length distribution and height distribution using an atomic force microscope; after the concentrated sample reached equilibrium, phase separation occurred macroscopically, 100 μL of sample was taken from the top and bottom phases, respectively, and the protein concentration was determined according to the weight change measured by a microbalance; the morphological characteristics and transformation behavior of the sample during phase transition were observed using a polarizing microscope, and the pitch was determined according to the distance between the two maximum bright bands of the cholesteric liquid crystal.

[0072] Example 3

[0073] S1, dispersion: lysozyme was uniformly dispersed in water at a mass concentration of 2% (w / v) and adjusted to pH 2.0, filtered through a filter membrane with a pore size of 0.45 μm, and a protein dispersion liquid was obtained;

[0074] S2, heat treatment: the protein dispersion liquid obtained in step S1 was heated at 95°C with magnetic stirring at a speed of 300 r / min for 24 h, and a fibrous aggregate dispersion liquid was obtained;

[0075] S3, shear purification: the amyloid fibril dispersion liquid obtained in step S2 was sheared for 150 s, and dialyzed in ultrapure water at pH 2.0 for 5 days using a dialysis bag with a molecular weight cut-off of 100 kDa, and a short fiber dispersion liquid with a length of about 500 nm was obtained;

[0076] S4, concentration: the short fiber dispersion liquid obtained in step S3 was concentrated in a 15% (w / v) polyethylene glycol solution using a dialysis bag with a molecular weight cut-off of 3.6 kDa for 24 h, and a short fiber concentrate with a concentration of 3.6% (w / v) was obtained;

[0077] S5, centrifugation: the short fiber concentrate obtained in step S4 was centrifuged at 15800 g for 20 min, and the supernatant was placed in a sealed glass bottle and stored at 4°C, and a cholesteric liquid crystal was obtained.

[0078] S6, pitch regulation: the cholesteric liquid crystal obtained in step S5 was adjusted to pH 2.8, and a cholesteric liquid crystal with a pH of 2.8 and an ionic strength of 0 mM was obtained.

[0079] The cholesteric liquid crystal prepared in this example was used to measure the fiber length distribution and height distribution using an atomic force microscope; after the concentrated sample reached equilibrium, phase separation occurred macroscopically, and 100 μL of sample was taken from the top and bottom, respectively, and the protein concentration was determined according to the weight change measured by a microbalance; the morphology and transformation behavior of the sample during phase transition were observed using a polarizing microscope, and the pitch was determined according to the distance between the two brightest bands of the cholesteric liquid crystal.

[0080] Example 4

[0081] S1, dispersion: lysozyme was uniformly dispersed in water at a mass concentration of 2% (w / v) and adjusted to pH 2.0, filtered through a filter membrane with a pore size of 0.45 μm, and a protein dispersion liquid was obtained;

[0082] S2, heat treatment: the protein dispersion liquid obtained in step S1 was heated at 90°C with magnetic stirring at a speed of 200 r / min for 28 h, and a fibrous aggregate dispersion liquid was obtained;

[0083] S3, shearing purification: the fibrous aggregate dispersion obtained in step S2 was sheared for 100 s, dialyzed in ultra-pure water at pH 2.0 using a dialysis bag with a molecular weight cut-off of 100 kDa for 4 days to obtain a short fiber dispersion with a length of about 580 nm;

[0084] S4, concentration: the short fiber dispersion obtained in step S3 was concentrated in a 10% (w / v) polyethylene glycol solution using a dialysis bag with a molecular weight cut-off of 3.6 kDa for 30 h to obtain a short fiber concentrate with a concentration of 4.0% (w / v);

[0085] S5, centrifugation: the short fiber concentrate obtained in step S4 was centrifuged at 15800 g for 10 min, and the supernatant was placed in a sealed glass bottle and stored at 4°C to obtain a cholesteric liquid crystal.

[0086] S6, pitch regulation: the cholesteric liquid crystal obtained in step S5 was adjusted to pH 2.0, and NaCl was added to obtain a cholesteric liquid crystal with a pH of 2.0 and an ionic strength of 2.5 mM.

[0087] The cholesteric liquid crystal prepared in this example was used to measure the fiber length distribution and height distribution using atomic force microscopy; after the concentrated sample reached equilibrium, phase separation occurred macroscopically, and 100 μL of sample was taken from the top and bottom, respectively, and the protein concentration was determined according to the weight change measured by the microbalance; the morphological characteristics and transition behavior of the sample during phase transition were observed using a polarizing microscope, and the pitch was determined according to the distance between the two brightest bands of the cholesteric liquid crystal.

[0088] Example 5

[0089] S1, dispersion: lysozyme was uniformly dispersed in water at a mass concentration of 2% (w / v) and adjusted to pH 2.0, and filtered through a filter membrane with a pore size of 0.45 μm to obtain a protein dispersion;

[0090] S2, heat treatment: the protein dispersion obtained in step S1 was heated at 95°C with magnetic stirring at a speed of 400 r / min for 24 h to obtain a fibrous aggregate dispersion;

[0091] S3, shearing purification: the fibrous aggregate dispersion obtained in step S2 was sheared for 180 s, dialyzed in ultra-pure water at pH 2.0 using a dialysis bag with a molecular weight cut-off of 100 kDa for 5 days to obtain a short fiber dispersion with a length of about 450 nm;

[0092] S4, concentration: the short fiber dispersion obtained in step S3 was concentrated in a 10% (w / v) polyethylene glycol solution using a dialysis bag with a molecular weight cut-off of 3.6 kDa for 24 h to obtain a short fiber concentrate with a concentration of 3.6% (w / v);

[0093] S5, centrifugation: centrifuge the short fiber concentrate obtained in step S4 at 10000g for 30min, take the supernatant and store in a sealed glass bottle at 4℃, and obtain the cholesteric liquid crystal.

[0094] S6, pitch regulation: adjust the pH of the cholesteric liquid crystal obtained in step S5 to 2.0, and add NaCl to obtain a cholesteric liquid crystal with a pH of 2.0 and an ionic strength of 10mM.

[0095] Take the cholesteric liquid crystal prepared in this example, use atomic force microscopy to determine the fiber length distribution and height distribution; after the concentrated sample reaches equilibrium, macroscopically phase separation occurs, 100μL of sample is taken from the top and bottom, respectively, and the protein concentration is determined according to the weight change measured by the microbalance; use polarized light microscope to observe the morphological characteristics and transformation behavior of the sample during phase transition, and measure the pitch according to the distance between the two maximum bright bands of the cholesteric liquid crystal.

[0096] Comparative Example 1

[0097] This comparative example is a comparison with Reference Example 1, where only the pH in step S6 is adjusted to 4.0, and the other steps and parameters remain unchanged.

[0098] The results show that the cholesteric liquid crystal cannot be formed, indicating that not any adjustment of pH can form a cholesteric liquid crystal with adjustable pitch.

[0099] Comparative Example 2

[0100] This comparative example is a comparison with Reference Example 1, where only the shear time in step S3 is adjusted to 30s to obtain a short fiber dispersion with a length greater than 1μm, and the other steps and parameters remain unchanged.

[0101] The results show that the cholesteric liquid crystal cannot be formed, indicating that not any length of short fiber dispersion can form a cholesteric liquid crystal.

[0102] The examples provided above are not intended to limit the scope covered by the present application, and the described steps are not intended to limit the order of execution. Those skilled in the art can make obvious improvements to the present application in combination with existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. A method for preparing cholesteric liquid crystal with adjustable pitch, characterized in that, The specific steps include the following: S1. Dispersion: Disperse lysozyme evenly in water, adjust the pH, filter, and obtain a protein dispersion; the concentration of lysozyme dispersed in water is 1~2%, w / v. Adjust the pH to 1.5~2.5; S2. Heat treatment: The protein dispersion obtained in step S1 is stirred and heated to obtain a fibrous aggregate dispersion; the stirring and heating is carried out at 85~95°C and at a speed of 200~400 r / min for 20~28 h. S3. Shearing and purification: The fibrous aggregate dispersion obtained in step S2 is sheared and dialyzed to obtain a short fiber dispersion; the shearing time is 60~180 s; the short fiber length in the short fiber dispersion is 400-600 nm. S4. Concentration: The short fiber dispersion obtained in step S3 is concentrated to obtain a short fiber concentrate; the concentration of the short fiber concentrate is 2.5~4%, w / v. S5. Centrifugation: Centrifuge the short fiber concentrate obtained in step S4, take the supernatant, and obtain cholesteric liquid crystal with adjustable pitch; the centrifugation is carried out at 10000~15800 g for 10~30 min.

2. The method according to claim 1, characterized in that, In step S1, the filtration is performed using a filter membrane with a pore size of 0.4~0.45 μm.

3. The method according to claim 1, characterized in that, In step S3, the dialysis is performed using a dialysis bag with a capacity of 90-100 kDa in ultrapure water at pH 1.5-2.5 for 3-5 days.

4. The method according to claim 1, characterized in that, In step S4, the concentration is carried out using a 3-3.6 kDa dialysis bag in a polyethylene glycol solution with a mass concentration of 5-15% w / v for 24-36 h.

5. The pitch-adjustable cholesteric liquid crystal prepared by the method of any one of claims 1 to 4.

6. The application of the pitch-adjustable cholesteric liquid crystal as described in claim 5 in the fields of optical instrument fabrication and bio-liquid crystals.

7. The pitch control method for cholesteric liquid crystal as described in claim 6, characterized in that, The pitch of the cholesteric liquid crystal can be controlled by changing its pH and ionic strength. When the pitch of the cholesteric liquid crystal needs to be adjusted to 14.3~29.0 μm, the pH needs to be adjusted to 2.0~2.8, and the ionic strength needs to be adjusted to 0~10 Mm.

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