CN110946116B - Preparation method of functionalized silk - Google Patents
Preparation method of functionalized silk Download PDFInfo
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- CN110946116B CN110946116B CN201911393162.5A CN201911393162A CN110946116B CN 110946116 B CN110946116 B CN 110946116B CN 201911393162 A CN201911393162 A CN 201911393162A CN 110946116 B CN110946116 B CN 110946116B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 241000255789 Bombyx mori Species 0.000 claims abstract description 72
- 108010022355 Fibroins Proteins 0.000 claims abstract description 59
- 239000002994 raw material Substances 0.000 claims abstract description 4
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 78
- 239000000243 solution Substances 0.000 claims description 65
- 238000000034 method Methods 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 38
- 239000007787 solid Substances 0.000 claims description 32
- 238000003756 stirring Methods 0.000 claims description 27
- 238000002156 mixing Methods 0.000 claims description 25
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 21
- 239000000843 powder Substances 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 20
- 102000004169 proteins and genes Human genes 0.000 claims description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 18
- 240000000249 Morus alba Species 0.000 claims description 16
- 235000008708 Morus alba Nutrition 0.000 claims description 16
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 16
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 claims description 16
- 238000009835 boiling Methods 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 13
- 238000001914 filtration Methods 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 239000003054 catalyst Substances 0.000 claims description 8
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 8
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 7
- UKVIEHSSVKSQBA-UHFFFAOYSA-N methane;palladium Chemical compound C.[Pd] UKVIEHSSVKSQBA-UHFFFAOYSA-N 0.000 claims description 7
- 229910017604 nitric acid Inorganic materials 0.000 claims description 7
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 claims description 7
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- 238000000502 dialysis Methods 0.000 description 8
- 238000002189 fluorescence spectrum Methods 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 210000004907 gland Anatomy 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- 241001671301 Morus nigra Species 0.000 description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000002657 fibrous material Substances 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/04—Silkworms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- Life Sciences & Earth Sciences (AREA)
- Polymers & Plastics (AREA)
- Chemical & Material Sciences (AREA)
- Animal Husbandry (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
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Abstract
The invention relates to a preparation method of functionalized silk, which comprises the following steps: (1) TPE-NO is prepared from TPE raw materials2(ii) a (2) TPE-NO2Preparation to obtain TPE-NH2(ii) a (3) TPE-NH2Preparing TPE-NCO; (4) connecting TPE-NCO to fibroin, and synthesizing to obtain TPE-SF fibroin; (5) feeding TPE-SF fibroin to silkworms to obtain silkworm cocoons; (6) and processing the silkworm cocoons into a fibroin solution to obtain the functionalized silk. The preparation method of the functionalized silk can obtain the required fluorescent silk, and the fluorescence does not disappear when the silk fibroin solution is processed.
Description
Technical Field
The invention relates to preparation of silk, in particular to a preparation method of functionalized silk.
Background
Silk has been used as a textile material for centuries for over 5000 years since now because of its excellent luster, hand and tensile strength. As a fiber material, silk is not limited to the clothing industry. Since silk is a natural biopolymer material with excellent mechanical properties, good biocompatibility and biodegradability, it has been widely used in the biomedical field. Silk can be dissolved to obtain silk fibroin solution, and materials such as fiber, film, gel and the like can be obtained after molding. In recent years, researchers carry out more in-depth research on the functionalization of silk, and various methods are adopted to modify and functionalize the silk, so that the characteristics of the silk are improved, and functions which the silk does not have are increased, and the silk can be applied to more fields.
The current method for functionalizing silk comprises genetic engineering, dyeing and feeding methods to prepare color fluorescent silk. Researchers have introduced fluorescent protein genes into silkworm bodies by using genetic engineering to obtain green, red and orange fluorescent silks. Although the method is feasible, the method has high cost, low efficiency and complex operation, so that the method is limited in practical application. In the early 20 th century, people add colored dye into silkworm food and finally obtain colored silkworm cocoons by a feeding method, which lays a foundation for producing functional silk by the feeding method later. However, in the prior art, when TPE is directly fed to silkworms, although fluorescent silk is available, when TPE is processed into a fibroin solution, fluorescence disappears.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a preparation method of functionalized silk, fluorescent silk is obtained by a feeding method, and fluorescence still exists after the fluorescent silk is processed into silk fibroin solution.
In order to solve the technical problems, the invention adopts the technical scheme that:
a preparation method of functionalized silk comprises the following steps:
(1) TPE-NO is prepared from TPE raw materials2；
(2) TPE-NO2Preparation to obtain TPE-NH2；
(3) TPE-NH2Preparing TPE-NCO;
(4) connecting TPE-NCO to fibroin, and synthesizing to obtain TPE-SF fibroin;
(5) feeding TPE-SF fibroin to silkworms to obtain silkworm cocoons;
(6) and processing the silkworm cocoons into a fibroin solution to obtain the functionalized silk.
The invention has the beneficial effects that:
through the process design, the TPE is sequentially prepared into TPE-NO2、TPE-NH2，TPE-NH2And then converting into TPE-NCO, directly connecting the TPE-NCO on the fibroin to obtain TPE-SF fibroin, feeding the TPE-SF fibroin to silkworm food, enabling the TPE-SF fibroin to more easily enter silkworm glands to generate expected fluorescent silk, and enabling the fluorescent silk to still exist after the TPE-SF fibroin is processed into a fibroin solution.
Drawings
FIG. 1 is a fluorescence spectrum of silk obtained by feeding prepared TPE-SF silk protein to silkworms in the preparation method of functionalized silk according to the embodiment of the present invention;
FIG. 2 is a fluorescence spectrum of a silk fibroin solution obtained after silk obtained by feeding prepared TPE-SF silk protein to silkworms in the preparation method of functionalized silk according to the embodiment of the present invention is processed;
FIG. 3 is a fluorescence spectrum of silk obtained by directly feeding TPE to silkworms;
FIG. 4 is a fluorescence spectrum of a fibroin solution obtained by treating silk obtained by directly feeding TPE to silkworms.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key concept of the invention is as follows: TPE-SF fibroin is obtained by converting TPE into TPE-NCO and then connecting the TPE-NCO to the fibroin, and the TPE-SF fibroin can more easily enter silkworm glands when being fed to silkworm food.
The preparation method of the functionalized silk provided by the invention comprises the following steps:
(1) TPE-NO is prepared from TPE raw materials2；
(2) TPE-NO2Preparation to obtain TPE-NH2；
(3) TPE-NH2Preparing TPE-NCO;
(4) connecting TPE-NCO to fibroin, and synthesizing to obtain TPE-SF fibroin;
(5) feeding TPE-SF fibroin to silkworms to obtain silkworm cocoons;
(6) and processing the silkworm cocoons into a fibroin solution to obtain the functionalized silk.
From the above description, the beneficial effects of the present invention are:
through the process design, the TPE is sequentially prepared into TPE-NO2、TPE-NH2，TPE-NH2And then converting into TPE-NCO, directly connecting the TPE-NCO on the fibroin to obtain TPE-SF fibroin, feeding the TPE-SF fibroin to silkworm food, enabling the TPE-SF fibroin to more easily enter silkworm glands to generate expected fluorescent silk, and enabling the fluorescent silk to still exist after the TPE-SF fibroin is processed into a fibroin solution.
For the existing TPE silkworm feed, although fluorescent silk can be obtained, after the fluorescent silk is processed into fibroin solution, the fluorescence disappears, which indicates that the TPE silkworm feed is not connected by chemical bonds in the silkworm body and is only mixed commonly.
Further, the step (1) is specifically as follows: mixing TPE, acetic acid and dichloromethane, cooling the mixed solution to-15 ℃ under the condition of ice salt bath, then adding concentrated nitric acid into the mixed solution, and stirring to obtain TPE-NO2。
As can be seen from the above description, as a specific process example, TPE and acetic acid are mixed with 60-80ml of dichloromethane with the mass fraction of 99.5% according to the mass ratio of 1:4, 20-24mmol of commercially available concentrated nitric acid is adopted as the concentrated nitric acid, and the stirring can be carried out under the condition of-5 ℃ to 5 ℃ by adopting a magnetic stirring manner for 15-30 min.
Further, in the step (1), the solid condensate obtained after stirring is washed for at least one time by water, then the washed solid condensate is dried by adopting anhydrous sodium sulfate, and then the dried solid condensate is sequentially filtered, dried in vacuum and recrystallized to obtain TPE-NO2。
As is apparent from the above description, as a specific process example, the solidified material obtained after stirring may be washed three times with water, the content of anhydrous sodium sulfate is 5 to 20g, and the solidified material after vacuum drying may be recrystallized in methanol using methanol.
Further, the step (2) is specifically as follows: TPE-NO to be obtained2Dissolving the palladium catalyst and the carbon palladium catalyst in absolute ethyl alcohol respectively, adding hydrazine hydrate after stirring, refluxing and cooling to 25-40 ℃.
As can be seen from the above description, as a specific process example, 8-12% by weight of a carbon-palladium catalyst, TPE-NO, is used2Adding the powder and a carbon palladium catalyst into 80-120ml of absolute ethyl alcohol according to the mass ratio of 1:2-2.5, stirring at room temperature (25-40 ℃) for 15-30min, adding 4-6ml of hydrazine hydrate with the volume percentage concentration of 78-82%, and refluxing for 4-6 h.
Further, in the step (2), dichloromethane is added after cooling, and the solution added with dichloromethane is filtered through diatomite; removing the solvent from the solution to obtain a solid; the solid is dissolved in dichloromethane, washed at least once with water, then dried with anhydrous sodium sulfate, and the dried solid is sequentially filtered, evaporated under reduced pressure and recrystallized.
As can be seen from the above description, as a specific process example, 20-40ml of 99.5% by mass dichloromethane can be added after cooling to dilute the reaction product, the solid after removing the solvent is dissolved in 40-60ml of 99.5% by mass dichloromethane, and then washed with water three times, the content of anhydrous sodium sulfate is 5-20g, and recrystallization is carried out by using methanol as an option.
Further, the step (3) is specifically as follows: TPE-NH obtained in the step (2)2Dissolving triethylamine and the mixture in dichloromethane, stirring and cooling to 0 ℃ under the ice bath condition, adding triphosgene into the solution, and stirring until the solid completely disappears.
As can be seen from the above description, as a specific process example, 99% by weight triethylamine, TPE-NH, is used2Dissolving the mixture and triethylamine in a mass ratio of 1:1.5-2 in 40-60ml of dichloromethane with the mass percentage concentration of 99.5%, adding 1-2mmol of triphosgene with the mass percentage concentration of 99%, and stirring for 2-3h after adding the triphosgene.
Further, the step (4) is specifically as follows: and (4) adding silk protein powder into the solution obtained in the step (3), stirring, and washing and drying the stirred solution in sequence.
As can be seen from the above description, as a specific process example, the silk protein powder is a commercially available (e.g. Taobao shopping platform) silk protein powder with a concentration of 100%, 8-10g of silk protein powder can be added, and further, 0.01mol of azobisisobutyrimidazoline hydrochloride can be simultaneously added, and the azobisisobutyrimidazoline hydrochloride and the silk protein powder are stirred at 60-70 ℃ for 20-24 h.
Further, in the step (4), the stirred solution is washed at least once with water, then dried by adopting anhydrous sodium sulfate, and then the dried solid condensate is sequentially filtered and the solvent is removed.
As can be seen from the above description, as a specific process example, the stirred solution is washed three times with water, then added to 5-20g of anhydrous sodium sulfate, dried, and the solvent is removed under vacuum, at which time the TPE molecules are attached to the fibroin, called TPE-SF fibroin.
Further, the step (5) is specifically as follows: and (3) mixing the TPE-SF silk protein obtained in the step (4) with mulberry leaves to obtain a functional silkworm feed, feeding the mulberry leaves to silkworms at the 1-2 week old of silkworms, and feeding the mixture of the functional silkworm feed and the mulberry leaves to silkworms at the 3 week old of silkworms to obtain silkworm cocoons.
As can be seen from the above description, as a specific process example, mulberry leaves are commercially available (e.g., Taobao shopping platform), and preferably, existing mulberry leaf powder is purchased as a feeding material. The TPE-SF fibroin and the commercially available mulberry leaf powder can be mixed according to the mass ratio of 1:15-20, and the mixture is uniformly mixed to be used as functional silkworm food. When silkworms are 3 weeks old, the functional silkworm food and the mulberry leaves are mixed according to the mass ratio of 1:10-20 to be used as the silkworm food when the silkworms are 3 weeks old.
Further, the step (6) specifically comprises the following substeps in sequence:
(a) adding the silkworm cocoons obtained in the step (5) into a boiled sodium bicarbonate aqueous solution, maintaining the boiling for at least 10min, taking out the silks in the sodium bicarbonate aqueous solution, and repeating the operation of the substep (a) at least once;
(b) immersing silk into water of 60-70 ℃ for stirring, taking out the silk in the water, and repeating the substep (b) for at least one time;
(c) drying the silk to obtain degummed silk; and (3) dissolving the degummed silk in a lithium bromide solution, and then sequentially dialyzing and filtering the lithium bromide solution to obtain a fibroin solution.
As can be seen from the above description, as a specific process example, in the sub-step (a), 8-12g of sodium bicarbonate can be put into 1.8-2.2L of water, 8-12g of silkworm cocoon can be added after boiling, and the boiling state is maintained for 25-35min by continuously heating, and the above operations are repeated for three times; in the substep (b), the silk is immersed in distilled water with the temperature of 60-70 ℃, stirred for 15-30min, taken out and the operation is repeated for 3-5 times; in the substep (c), the degummed silk is dissolved in 9-10mol/L lithium bromide solution, then the solution is put into a dialysis bag for dialysis, and finally the dialyzed silk fibroin solution is filtered by gauze to obtain the final silk fibroin solution.
The underlying mechanism of the present application may, but does not include, the following.
The inventor researches and discovers that whether the dye can be taken up by silkworms or not is closely related to the hydrophilic and hydrophobic structures of the dyes, and the dye with moderate hydrophilicity and hydrophobicity can more easily enter the glands of the silkworms. The required substances are directly connected to the fibroin and then fed to the silkworm, so that the required substances can more easily enter the silkworm glands to generate the expected fluorescent silks.
Referring to fig. 1-2, an embodiment of the present invention is:
example 1
The preparation method of the functionalized silk of the embodiment comprises the following steps:
(1) and (3) mixing TPE and acetic acid according to a mass ratio of 1:4 and 60ml of dichloromethane with the mass concentration of 99.5 percent are respectively placed in a flask, the solution is cooled to-15 ℃ by using a ice salt bath, then 20mmol of commercial concentrated nitric acid is added, and the reaction solution is magnetically stirred for 15min at-5 ℃. Washing with water for three times, drying 5g anhydrous sodium sulfate, filtering, vacuum drying to obtain yellow residue, and recrystallizing the yellow residue in methanol to obtain TPE-NO as yellow powder2。
(2) Mixing the yellow powder obtained in the step (1) with a carbon palladium catalyst with the mass fraction of 10% according to the mass ratio of 1:2 in 80ml of absolute ethanol, stirring at room temperature for 15min, then adding 4ml of hydrazine hydrate with the volume concentration of 80%, refluxing for 4h, cooling to room temperature, adding 20ml of dichloromethane with the mass concentration of 99.5% to dilute the reaction, and then filtering through a layer of kieselguhr. The solvent was removed in vacuo to give a white solid which was dissolved in 40ml of 99.5% strength by mass dichloromethane and washed three times with water. Then dried over 5g anhydrous sodium sulfate, filtered, evaporated under reduced pressure and recrystallized from methanol to give a white solid.
(3) Mixing the white solid obtained in the step (2) and triethylamine with the mass concentration of 99% according to the mass ratio of 1: dissolving 1.5 in 40ml of dichloromethane with the mass concentration of 99.5%, stirring and cooling to 0 ℃ in an ice bath, adding 1mmol of triphosgene with the mass concentration of 99%, and stirring for 2 hours until white solids completely disappear to obtain TPE-NCO.
(4) Adding 8g of commercially available 100% silk protein powder into the solution obtained in the step (3), adding 0.01mol of azobisisobutyrimidazoline hydrochloride, stirring at 60 ℃ for 20 hours, washing the solution with water for three times, drying with 5g of anhydrous sodium sulfate, filtering, and removing the solvent under vacuum to obtain a white solid, wherein TPE molecules are connected to the silk protein, and the white solid is named TPE-SF silk protein.
(5) And (3) mixing the TPE-SF fibroin synthesized in the step (4) with commercially available mulberry leaf powder according to the mass ratio of 1:15, uniformly mixing the mixture to serve as a functional silkworm feed, eating common mulberry leaves by silkworms at 1 and 2 weeks of age, and mixing the functional silkworm feed and the mulberry leaves at a mass ratio of 1:10 mixing and feeding together to obtain silkworm cocoon.
(6) The method comprises the steps of putting 10g of sodium bicarbonate into 2L of water, boiling, then boiling 10g of silkworm cocoons for 30min in a boiling state, repeating the process for three times, then putting silk into distilled water at 60 ℃ and stirring for 15min, repeating the process for 3 times, then drying to obtain degummed silk, then dissolving the silk into 9mol/L lithium bromide solution, then putting the solution into a dialysis bag for dialysis, and finally filtering the dialyzed silk fibroin solution with gauze to obtain the final silk fibroin solution.
(7) And finally, collecting the fluorescence spectrum of the fibroin solution.
Example 2
The preparation method of the functionalized silk of the embodiment comprises the following steps:
(1) and (3) mixing TPE and acetic acid according to a mass ratio of 1:4 and 80ml of dichloromethane with the mass concentration of 99.5 percent are respectively placed in a flask, the solution is cooled to-15 ℃ by using a ice salt bath, then 24mmol of commercial concentrated nitric acid is added, and the reaction solution is magnetically stirred for 30min at the temperature of 5 ℃. Washing with water for three times, drying with 20g anhydrous sodium sulfate, filtering, vacuum drying to obtain yellow residue, and recrystallizing the yellow residue in methanol to obtain TPE-NO as yellow powder2。
(2) Mixing the yellow powder obtained in the step (1) with a carbon palladium catalyst with the mass fraction of 10% according to the mass ratio of 1: 2.5 is dissolved in 120ml of absolute ethanol, stirred at room temperature for 30min, then 6ml of hydrazine hydrate with the volume concentration of 80% is added, the mixture is refluxed for 6h, after the room temperature is cooled, 40ml of dichloromethane with the mass concentration of 99.5% is added to dilute the reaction, and then the reaction is filtered through a layer of kieselguhr. The solvent was removed in vacuo to give a white solid which was dissolved in 60ml of 99.5% strength by mass dichloromethane and washed three times with water. Then dried over 20g anhydrous sodium sulfate, filtered, evaporated under reduced pressure and recrystallized from methanol to give a white solid.
(3) Mixing the white solid obtained in the step (2) and triethylamine with the mass concentration of 99% according to the mass ratio of 1:2 is dissolved in 60ml dichloromethane with the mass concentration of 99.5 percent, stirred and cooled to 0 ℃ in an ice bath, then 2mmol triphosgene with the mass concentration of 99 percent is added, and stirred for 3 hours until white solid completely disappears, thus obtaining TPE-NCO.
(4) And (3) adding 10g of commercially available 100% silk protein powder into the solution obtained in the step (3), adding 0.01mol of azobisisobutyrimidazoline hydrochloride, stirring at 70 ℃ for 24 hours, washing the solution with water for three times, drying with 20g of anhydrous sodium sulfate, filtering, and removing the solvent in vacuum to obtain a white solid, wherein TPE molecules are connected to the silk protein, and the white solid is called TPE-SF silk protein.
(5) And (3) mixing the TPE-SF fibroin synthesized in the step (4) with commercially available mulberry leaf powder according to the mass ratio of 1: 20, uniformly mixing the mixture to serve as a functional silkworm feed, eating common mulberry leaves by silkworms at 1 and 2 weeks of age, and mixing the functional silkworm feed and the mulberry leaves at the mass ratio of 1: 20 mixing and feeding together to obtain silkworm cocoon.
(6) The method comprises the steps of putting 10g of sodium bicarbonate into 2L of water, boiling, then boiling 10g of silkworm cocoons for 30min in a boiling state, repeating the process for three times, then putting silk into 70 ℃ distilled water, stirring for 30min, repeating the process for 5 times, then drying to obtain degummed silk, then dissolving the silk into 10mol/L lithium bromide solution, then putting the solution into a dialysis bag for dialysis, and finally filtering the dialyzed silk fibroin solution with gauze to obtain the final fibroin solution.
(7) And finally, collecting the fluorescence spectrum of the fibroin solution.
Example 3
The preparation method of the functionalized silk of the embodiment comprises the following steps:
(1) and (3) mixing TPE and acetic acid according to a mass ratio of 1:4 and 70ml of dichloromethane with the mass concentration of 99.5 percent are respectively placed in a flask, the solution is cooled to-15 ℃ by using a ice salt bath, then 22mmol of commercial concentrated nitric acid is added, and the reaction solution is magnetically stirred for 20min at the temperature of 0 ℃. Washing with water for three times, drying with 15g anhydrous sodium sulfate, filtering, vacuum drying to obtain yellow residue, and recrystallizing the yellow residue in methanol to obtain TPE-NO as yellow powder2。
(2) Mixing the yellow powder obtained in the step (1) with a carbon palladium catalyst with the mass fraction of 10% according to the mass ratio of 1: 202 was dissolved in 100ml of absolute ethanol, stirred at room temperature for 20min, then 5ml of 80% by volume hydrazine hydrate was added, refluxed for 5h, cooled at room temperature, and then diluted with 30ml of 99.5% by mass dichloromethane, and then filtered through a layer of celite. The solvent was removed in vacuo to give a white solid, which was dissolved in 50ml of 99.5% strength by mass dichloromethane and washed three times with water. Then dried over 10g anhydrous sodium sulfate, filtered, evaporated under reduced pressure and recrystallized from methanol to give a white solid.
(3) Mixing the white solid obtained in the step (2) and triethylamine with the mass concentration of 99% according to the mass ratio of 1: 1.8 is dissolved in 50ml dichloromethane with the mass concentration of 99.5 percent, stirred and cooled to 0 ℃ in an ice bath, 1.5mmol triphosgene with the mass concentration of 99 percent is added, and stirred for 2.5 hours until white solid completely disappears, thus obtaining TPE-NCO.
(4) And (3) adding 9g of commercially available 100% silk protein powder into the solution obtained in the step (3), adding 0.01mol of azobisisobutyrimidazoline hydrochloride, stirring at 65 ℃ for 22 hours, washing the solution with water for three times, drying with 10g of anhydrous sodium sulfate, filtering, and removing the solvent in vacuum to obtain a white solid, wherein TPE molecules are connected to the silk protein, and the white solid is called TPE-SF silk protein.
(5) And (3) mixing the TPE-SF fibroin synthesized in the step (4) with commercially available mulberry leaf powder according to the mass ratio of 1: 18, uniformly mixing the mixture to serve as a functional silkworm feed, eating common mulberry leaves by silkworms at 1 and 2 weeks of age, and mixing the functional silkworm feed and the mulberry leaves at a mass ratio of 1:15 mixing and feeding together to obtain silkworm cocoon.
(6) The method comprises the steps of putting 10g of sodium bicarbonate into 2L of water, boiling, then boiling 10g of silkworm cocoons for 30min in a boiling state, repeating the process for three times, then putting silk into 65 ℃ of distilled water, stirring for 18min, repeating the process for 4 times, then drying to obtain degummed silk, then dissolving the silk into 9.5mol/L of lithium bromide solution, then putting the solution into a dialysis bag for dialysis, and finally filtering the dialyzed silk fibroin solution with gauze to obtain the final fibroin solution.
(7) And finally, collecting the fluorescence spectrum of the fibroin solution.
Referring to fig. 1-4, it can be seen from fig. 1-2 that the TPE-SF silk protein of examples 1-3 can obtain the desired fluorescent silk from the cocoon after being fed to the silkworm (see fig. 1), and the fluorescence of the functionalized fluorescent silk still exists after the cocoon is processed into silk solution (see fig. 2); as can be seen from fig. 3-4, the TPE can obtain the desired fluorescent silk on the cocoons after being directly fed to silkworms (see fig. 3), but the fluorescence disappears after the cocoons are processed into silk solution (see fig. 4).
In conclusion, the preparation method of the functionalized silk provided by the invention can obtain the required fluorescent silk, and the fluorescence does not disappear when the silk fibroin solution is processed.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (10)
1. A preparation method of functionalized silk is characterized by comprising the following steps:
(1) TPE-NO is prepared from TPE raw materials2；
(2) TPE-NO2Preparation to obtain TPE-NH2；
(3) TPE-NH2Preparing TPE-NCO;
(4) connecting TPE-NCO to fibroin, and synthesizing to obtain TPE-SF fibroin;
(5) feeding TPE-SF fibroin to silkworms to obtain silkworm cocoons;
(6) and processing the silkworm cocoons into a fibroin solution to obtain the functionalized silk.
2. The method for preparing functionalized silk according to claim 1, wherein the step (1) is specifically as follows: mixing TPE, acetic acid and dichloromethane, cooling the mixed solution to-15 ℃ under the condition of ice salt bath, and then adding the mixed solution into the mixed solutionAdding concentrated nitric acid into the solution, and stirring to obtain TPE-NO2。
3. The method for preparing functionalized silk according to claim 2, wherein in the step (1), the solidified substance obtained after stirring is washed at least once with water, then the washed solidified substance is dried by using anhydrous sodium sulfate, and then the dried solidified substance is sequentially filtered, vacuum-dried and recrystallized to obtain TPE-NO2。
4. The method for preparing functionalized silk according to claim 2 or 3, wherein the step (2) is specifically as follows: TPE-NO to be obtained2Dissolving the mixture and a carbon palladium catalyst in absolute ethyl alcohol respectively, adding hydrazine hydrate after stirring, refluxing, and then cooling to 25-40 ℃.
5. The method for preparing functionalized silk according to claim 4, wherein in the step (2), dichloromethane is added after cooling, and the solution after adding dichloromethane is filtered through diatomite; removing the solvent from the solution to obtain a solid; the solid is dissolved in dichloromethane, washed at least once with water, then dried with anhydrous sodium sulfate, and the dried solid is sequentially filtered, evaporated under reduced pressure and recrystallized.
6. The method for preparing functionalized silk according to claim 5, wherein the step (3) is specifically as follows: TPE-NH obtained in the step (2)2Dissolving triethylamine and the mixture in dichloromethane, stirring and cooling to 0 ℃ under the ice bath condition, adding triphosgene into the solution, and stirring until the solid completely disappears.
7. The method for preparing functionalized silk according to claim 6, wherein the step (4) is specifically as follows: and (4) adding silk protein powder into the solution obtained in the step (3), stirring, and washing and drying the stirred solution in sequence.
8. The method for preparing functionalized silk according to claim 7, wherein in the step (4), the stirred solution is washed with water at least once, then dried with anhydrous sodium sulfate, and then the dried solid condensate is sequentially filtered and the solvent is removed.
9. The method for preparing functionalized silk according to claim 7 or 8, wherein the step (5) is specifically as follows: and (3) mixing the TPE-SF silk protein obtained in the step (4) with mulberry leaves to obtain a functional silkworm feed, feeding the mulberry leaves to silkworms at the 1-2 week old of silkworms, and feeding the mixture of the functional silkworm feed and the mulberry leaves to silkworms at the 3 week old of silkworms to obtain silkworm cocoons.
10. The method for preparing functionalized silk according to claim 9, wherein the step (6) comprises the following substeps in sequence:
(a) adding the silkworm cocoons obtained in the step (5) into a boiled sodium bicarbonate aqueous solution, maintaining the boiling for at least 10min, taking out the silks in the sodium bicarbonate aqueous solution, and repeating the operation of the substep (a) at least once;
(b) immersing silk into water of 60-70 ℃ for stirring, taking out the silk in the water, and repeating the substep (b) for at least one time;
(c) drying the silk to obtain degummed silk; and (3) dissolving the degummed silk in a lithium bromide solution, and then sequentially dialyzing and filtering the lithium bromide solution to obtain a fibroin solution.
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