CN105056305A - Dermal substitute and preparation method thereof - Google Patents
Dermal substitute and preparation method thereof Download PDFInfo
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- CN105056305A CN105056305A CN201510556711.1A CN201510556711A CN105056305A CN 105056305 A CN105056305 A CN 105056305A CN 201510556711 A CN201510556711 A CN 201510556711A CN 105056305 A CN105056305 A CN 105056305A
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Abstract
The invention discloses a dermal substitute. The dermal substitute is characterized by taking silk protein as a main matrix and carboxyethyl-chitosan as a secondary matrix; mixing silk protein solution with carboxyethyl-chitosan, carrying out film formation, soaking with mixed aqueous solution of 1-(3-dimethylamino propyl)-3-ethyl carbodiimide and N-hydroxyl succinimide, and carrying out freeze drying, so that the dermal substitute is obtained. The dermal substitute has the functions of inhibiting formation of scars and promoting dermal tissue regeneration; and the preparation method of the dermal substitute is characterized in that physical crosslinking is carried out on silk protein molecules through evaporation of water vapour, operation is easy, energy is saved, and no pollution is produced.
Description
Technical field
The present invention relates to field of biomedical materials, especially relating to a kind of is main matrix with fibroin, and carboxyetbyl chitosan is dermal substitute of secondary substrate and preparation method thereof.
Background technology
The skin injury that the open wounds such as burning wound cause is common clinical and frequently-occurring disease, and in skin injury repair process, wound excessively healing can cause generation and the development of pathologic scar.Pathologic scar comprises hypertrophic cicatrix and keloid, normal with pruritus, disfeature, a series of serious complication such as disabled, sometimes even bring out malignant tumor, the serious life quality reducing patient.Therefore, the control of pathologic scar is difficult medical problem urgently to be resolved hurrily in the surgery such as burn, shaping always.
In recent years, along with deepening continuously of wound healing Mechanism Study, it is found that, cicatrization and dermal tissue lack degree and have high correlation, this theory have also been obtained evidence in clinical practice, as superficial burns wound surface is similar with normal skin after healing, and deep burn wound will form pathologic scar after healing.This theoretical prompting: the existence of dermal tissue can suppress generation and the development of cicatrix.Therefore, the dermal substitute material playing dermal tissue effect clinical for promoting deep dermis Wound Defect to heal and suppressing cicatrization.But, although existing dermal substitute material achieves certain effect, still do not reach the target suppressing pathologic scar to occur and develop.At present, the dermal substitute product that gone on the market can be divided into two classes: acellular dermal matrix and external synthesis polymeric stent.Acellular dermal matrix is the heterogeneous macromolecular scaffold material of the composition that obtained through de-cell process by people's cadaver skin, Corii Sus domestica, trees-Osima jacoti, Osima excavata, and its main component is collagen protein; External synthesis polymeric stent is by the polymeric stent of the external synthesis such as synthesis macromolecule (as polyglactin) or animal sources natural polymer (as collagen protein and/or glycosaminoglycans).
In process of clinical application, existing dermal substitute is Shortcomings part still.First, because processing technique is difficult to control, the quality of acellular dermal matrix class dermal substitute is unstable, may there is difference in quality between each production batch, and then affect its effect as dermal substitute wound repairing, and the serious consequences such as second operation may be caused; Secondly, there is the probability of biological pollution in acellular dermal matrix dermal substitute, because raw material people cadaver skin may carry virus, and these viruses in material processing not by effective deactivation, and cause virus disseminating, further, acellular dermal matrix belongs to allosome material, exists and causes body inflammatory reaction and immunoreactive risk; Again, external synthesis dermal substitute does not mate with organizing formation speed at the degradation speed of wound surface, often causes it to promote wound repair because degrading too fast and suppresses synulotic declines; In addition, the target that existing dermal substitute is suppressing the action effect in cicatrization to reach far away the regeneration of promotion functions class dermal tissue, trace it to its cause, dermal substitute destructurized and external synthesis dermal substitute in the course of processing is unrealized is affect the key factor that dermal substitute promotes dermal tissue regeneration on the bionical of dermal tissue.
Summary of the invention
For the problems referred to above that prior art exists, the applicant provides a kind of dermal substitute and preparation method thereof.Dermal substitute of the present invention has the function suppressing cicatrization and promote dermal tissue regeneration, and the preparation method of this dermal substitute makes Silk Protein Molecules generation physical crosslinking by steam evaporation, simple to operate, energy-conservation, pollution-free.
Technical scheme of the present invention is as follows:
A kind of dermal substitute, described dermal substitute take fibroin as main matrix, and carboxyetbyl chitosan is secondary substrate; Wherein, the mass fraction of fibroin is 60% ~ 90%, and the mass fraction of carboxyetbyl chitosan is 10% ~ 40%.
The preparation method of described dermal substitute, comprises the steps:
(1) Bombyx bombycis is placed in Na
2cO
3boil in aqueous solution and boil 30min, then use deionized water rinse 3 times, obtained silkworm silk cellulose fiber; Then silkworm silk cellulose fiber is placed in lithium-bromide solution, dissolves under 37 DEG C of conditions, obtained fibroin lithium-bromide solution; Be finally, after dialysing 3 days in the dialysis cassette of 3500Da, obtain silk protein solution at molecular cut off by gained solution, regulate solution quality percentage concentration to be 5%;
(2) by silk protein solution obtained for step (1) and mass percentage concentration be 3% carboxyetbyl chitosan solution under 37 DEG C of conditions by volume 54:10 ~ 9:10 mix, in speed be 50rpm/min vortex oscillator on to vibrate 5min, to mix homogeneously, then centrifugal 3min under centrifugal force is 1500g, the bubble in removing solution;
(3) mixed liquor after centrifugal for step (2) is transferred in politef groove, the thickness of solution is 2mm, then this groove is positioned over 37 DEG C, air draught wind speed be 0.3 meter per second fume hood central authorities, standing time is 24h, formed fibroin/carboxyetbyl chitosan polymeric membrane;
(4) fibroin/carboxyetbyl chitosan polymeric membrane is immersed in 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide mixed aqueous solution, at 40 DEG C, places 8h; Gained polymeric membrane is soaked 30min in 300ml deionized water, and this step repeats 3 times, finally by gained polymeric membrane freezing 10min in 300ml liquid nitrogen solution;
(5) by the lyophilization in vacuum freeze drier of freezing polymeric membrane, vacuum lyophilization condition is: condenser temperature-55 DEG C, vacuum 3 ~ 5Pa, time 18h, obtained fibroin/carboxyetbyl chitosan composite, i.e. described dermal substitute.
Na described in step (1)
2cO
3concentration of aqueous solution is 0.01 ~ 1M; The concentration of described lithium-bromide solution is 1 ~ 20M.
In (3-the dimethylamino-propyl)-3-ethyl carbodiimide of 1-described in step (4) and N-hydroxy-succinamide mixed aqueous solution, the concentration ratio of 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide is 1:1, is 0.02M; The pH value of described mixed solution is 4.5.
The technique effect that the present invention is useful is:
(1) can there is physical crosslinking in the present invention's Silk Protein Molecules used, avoids and introduce chemical cross-linking agent in the process building dermal substitute, improves the biocompatibility of material; The repetition block of the glycine about containing 85% in Silk Protein Molecules structure, alanine and serine, these repeat form hydrogen bond between block and make to produce hydrophobic structure between Silk Protein Molecules inside or Silk Protein Molecules, this hydrophobic structure makes Silk Protein Molecules generation physical crosslinking, and avoids using chemical cross-linking agent.
(2) the present invention makes Silk Protein Molecules generation physical crosslinking by steam evaporation, the bionical performance of fibroin/carboxyetbyl chitosan composite to dermal tissue structure and mechanical property can be improved, the hardness of the fibroin prepared by reduction/carboxyetbyl chitosan composite, make it more close with dermal tissue in mechanical property, be conducive to improving dermal substitute and suppress cicatrization and the function promoting dermal tissue regeneration.; This Silk Protein Molecules self assembly can be promoted to form SilkI structure due to steam evaporation process and suppress the generation of SilkII structure, and mainly form SilkII structure in methanol aqueous solution intermediate filment molecule generation self assembly, i.e. β-sheet structure; When the physical crosslinking form of Silk Protein Molecules is mainly SilkI, fibroin base timbering material is better than based on the respective material of SilkII structure in pore structure and mechanical property.
(3) introducing of carboxyetbyl chitosan molecule of the present invention can improve dermal fibroblast and capillary endothelium sticking and proliferation activity in the bracket; Glycosaminoglycan structures in the molecular structure of carboxyetbyl chitosan and acellular dermal matrix is similar, can play and biological action like glycosaminoglycan, namely by interacting with the cytokine in extracellular matrix, somatomedin etc., improve the bioavailability of these protein signal molecules, thus improve the biological activity of cell; Thus, in fibroin support, adhesion and the proliferation activity that carboxyetbyl chitosan can promote dermal fibroblast and capillary endothelium is introduced.
(4) the present invention adopts 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide concentration and N-hydroxy-succinamide to carry out crosslinking Treatment to fibroin/carboxyetbyl chitosan composite, carboxyetbyl chitosan molecule can be fixed in fibroin cross-linked network by this chemical crosslink technique, form interpenetrating networks, thus hinder the diffusion of enzyme in support network and activity, the biodegradation rate of the fibroin/carboxyetbyl chitosan that can slow down
(5) the present invention adopts vacuum lyophilization process to obtain fibroin/carboxyetbyl chitosan base dermal substitute, can keep its loose structure and mechanical characteristic by material under dry state.
(6) the present invention adopts fibroin as the main matrix preparing dermal substitute, and carboxyetbyl chitosan is the secondary substrate preparing dermal substitute, has regulating and controlling effect to fibroin self assembling process.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of the embodiment of the present invention 1 gained dermal substitute.
Fig. 2 is the scanning electron microscope (SEM) photograph of comparative example 1 gained pure silk albuminous coat of the present invention.
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention is specifically described.
Embodiment 1
A preparation method for dermal substitute, concrete steps are as follows:
(1) 10g Bombyx bombycis is placed in the 0.02MNa of 500ml
2cO
3boil in aqueous solution and boil 30min, then use deionized water rinse 3 times, obtained silkworm silk cellulose fiber; Then silkworm silk cellulose fiber is placed in the lithium-bromide solution of 10ml9.3M, dissolves under 37 DEG C of conditions, obtained fibroin lithium-bromide solution; Be finally, after dialysing 3 days in the dialysis cassette of 3500Da, obtain silk protein aqueous solution at molecular cut off by gained solution, regulate solution quality percentage concentration to be 5%;
(2) by silk protein solution obtained for step (1) and mass percentage concentration be 3% carboxyetbyl chitosan solution under 37 DEG C of conditions by volume 30:10 mix, in speed be 50rpm/min vortex oscillator on to vibrate 5min, to mix homogeneously, then centrifugal 3min under centrifugal force is 1500g, the bubble in removing solution;
(3) mixed liquor after centrifugal for step (2) is transferred in politef groove, the thickness of solution is 2mm, then this groove is positioned over 37 DEG C, air draught wind speed be 0.3 meter per second fume hood central authorities, standing time is 24h, formed fibroin/carboxyetbyl chitosan polymeric membrane;
(4) fibroin/carboxyetbyl chitosan polymeric membrane is immersed in 300ml1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide mixed aqueous solution that (solute 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide concentration is 0.02M, solute N-hydroxy-succinamide concentration is 0.02M, pH=4.5), 8h is placed at 40 DEG C; Gained polymeric membrane is soaked 30min in 300ml deionized water, and this step repeats 3 times, finally by gained polymeric membrane freezing 10min in 300ml liquid nitrogen solution;
(5) by the lyophilization in vacuum freeze drier of freezing polymeric membrane, vacuum lyophilization condition is: condenser temperature-55 DEG C, vacuum 5Pa, time 18h, obtained fibroin/carboxyetbyl chitosan composite, i.e. described dermal substitute.Wherein, the mass fraction of fibroin is 64%, and the mass fraction of carboxyetbyl chitosan is 36%.
By the microstructure of dermal substitute described in sem observation embodiment 1, as shown in Figure 1.As seen from Figure 1, by the dermal substitute of steam evaporation process gained, its internal microstructure is the loose structure of UNICOM.
Embodiment 2
A preparation method for dermal substitute, concrete steps are as follows:
(1) 10g Bombyx bombycis is placed in the 0.02MNa of 500ml
2cO
3boil in aqueous solution and boil 30min, then use deionized water rinse 3 times, obtained silkworm silk cellulose fiber; Then silkworm silk cellulose fiber is placed in the lithium-bromide solution of 10ml9.3M, dissolves under 37 DEG C of conditions, obtained fibroin lithium-bromide solution; Be finally, after dialysing 3 days in the dialysis cassette of 3500Da, obtain silk protein aqueous solution at molecular cut off by gained solution, regulate solution quality percentage concentration to be 5%;
(2) by silk protein solution obtained for step (1) and mass percentage concentration be 3% carboxyetbyl chitosan solution under 37 DEG C of conditions by volume 54:10 mix, in speed be 50rpm/min vortex oscillator on to vibrate 5min, to mix homogeneously, then centrifugal 3min under centrifugal force is 1500g, the bubble in removing solution;
(3) mixed liquor after centrifugal for step (2) is transferred in politef groove, the thickness of solution is 2mm, then this groove is positioned over 37 DEG C, air draught wind speed be 0.3 meter per second fume hood central authorities, standing time is 24h, formed fibroin/carboxyetbyl chitosan polymeric membrane;
(4) fibroin/carboxyetbyl chitosan polymeric membrane is immersed in 300ml1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide mixed aqueous solution that (solute 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide concentration is 0.02M, solute N-hydroxy-succinamide concentration is 0.02M, pH=4.5), 8h is placed at 40 DEG C; Gained polymeric membrane is soaked 30min in 300ml deionized water, and this step repeats 3 times, finally by gained polymeric membrane freezing 10min in 300ml liquid nitrogen solution;
(5) by the lyophilization in vacuum freeze drier of freezing polymeric membrane, vacuum lyophilization condition is: condenser temperature-55 DEG C, vacuum 3Pa, time 18h, obtained fibroin/carboxyetbyl chitosan composite, i.e. described dermal substitute.Wherein, the mass fraction of fibroin is 90%, and the mass fraction of carboxyetbyl chitosan is 10%.
Embodiment 3
A preparation method for dermal substitute, concrete steps are as follows:
(1) 10g Bombyx bombycis is placed in the 0.05MNa of 500ml
2cO
3boil in aqueous solution and boil 30min, then use deionized water rinse 3 times, obtained silkworm silk cellulose fiber; Then silkworm silk cellulose fiber is placed in the lithium-bromide solution of 10ml15M, dissolves under 37 DEG C of conditions, obtained fibroin lithium-bromide solution; Be finally, after dialysing 3 days in the dialysis cassette of 3500Da, obtain silk protein aqueous solution at molecular cut off by gained solution, regulate solution quality percentage concentration to be 5%;
(2) by silk protein solution obtained for step (1) and mass percentage concentration be 3% carboxyetbyl chitosan solution under 37 DEG C of conditions by volume 9:10 mix, in speed be 50rpm/min vortex oscillator on to vibrate 5min, to mix homogeneously, then centrifugal 3min under centrifugal force is 1500g, the bubble in removing solution;
(3) mixed liquor after centrifugal for step (2) is transferred in politef groove, the thickness of solution is 2mm, then this groove is positioned over 37 DEG C, air draught wind speed be 0.3 meter per second fume hood central authorities, standing time is 24h, formed fibroin/carboxyetbyl chitosan polymeric membrane;
(4) fibroin/carboxyetbyl chitosan polymeric membrane is immersed in 300ml1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide mixed aqueous solution that (solute 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide concentration is 0.02M, solute N-hydroxy-succinamide concentration is 0.02M, pH=4.5), 8h is placed at 40 DEG C; Gained polymeric membrane is soaked 30min in 300ml deionized water, and this step repeats 3 times, finally by gained polymeric membrane freezing 10min in 300ml liquid nitrogen solution;
(5) by the lyophilization in vacuum freeze drier of freezing polymeric membrane, vacuum lyophilization condition is: condenser temperature-55 DEG C, vacuum 5Pa, time 18h, obtained fibroin/carboxyetbyl chitosan composite, i.e. described dermal substitute.Wherein, the mass fraction of fibroin is 60%, and the mass fraction of carboxyetbyl chitosan is 40%.
Comparative example 1
(1) 10g Bombyx bombycis is placed in the 0.02MNa of 500ml
2cO
3boil in aqueous solution and boil 30min, then use deionized water rinse 3 times, obtained silkworm silk cellulose fiber; Then silkworm silk cellulose fiber is placed in the lithium-bromide solution of 10ml9.3M, dissolves under 37 DEG C of conditions, obtained fibroin lithium-bromide solution; Be finally, after dialysing 3 days in the dialysis cassette of 3500Da, obtain silk protein aqueous solution at molecular cut off by gained solution, regulate solution quality percentage concentration to be 5%;
(2) solution that step (1) is obtained is transferred in politef groove, the thickness of solution is 2mm, then this groove is positioned over 37 DEG C, air draught wind speed is the fume hood central authorities of 0.3 meter per second, standing time is 24h, forms fibroin polymeric membrane;
(4) the fibroin polymeric membrane that step (2) is obtained soaks 30min, then freezing 10min in liquid nitrogen solution in 300ml deionized water;
(5) by the lyophilization in vacuum freeze drier of freezing polymeric membrane, vacuum lyophilization condition is: condenser temperature-55 DEG C, vacuum 5Pa, time 18h, obtained pure silk albuminous coat.
By the microstructure of sem observation comparative example gained pure silk albuminous coat, as shown in Figure 2.As seen from Figure 2, pure silk albuminous coat internal microstructure contains a large amount of layer structures, without remarkable loose structure.
Comparative example 2
(1) 10g Bombyx bombycis is placed in the 0.02MNa of 500ml
2cO
3boil in aqueous solution and boil 30min, then use deionized water rinse 3 times, obtained silkworm silk cellulose fiber; Then silkworm silk cellulose fiber is placed in the lithium-bromide solution of 10ml9.3M, dissolves under 37 DEG C of conditions, obtained fibroin lithium-bromide solution; Be finally, after dialysing 3 days in the dialysis cassette of 3500Da, obtain silk protein aqueous solution at molecular cut off by gained solution, regulate solution quality percentage concentration to be 5%;
(2) by silk protein solution obtained for step (1) and mass percentage concentration be 3% carboxyetbyl chitosan solution under 37 DEG C of conditions by volume 30:10 mix, in speed be 50rpm/min vortex oscillator on to vibrate 5min, to mix homogeneously, then centrifugal 3min under centrifugal force is 1500g, the bubble in removing solution;
(3) mixed liquor after centrifugal for step (2) is transferred in politef groove, the thickness of solution is 2mm, in 300ml liquid nitrogen solution after freezing 10min, lyophilization in vacuum freeze drier, condenser temperature-55 DEG C, vacuum 3 ~ 5Pa, time 18h, obtains fibroin/carboxyetbyl chitosan film;
(4) fibroin/carboxyetbyl chitosan film step (3) obtained is at methanol aqueous solution (V
methanol: V
water=1:1) middle immersion 1h, obtain the fibroin/carboxyetbyl chitosan film of methanol aqueous solution process;
(5) fibroin/carboxyetbyl chitosan film that will process through step (4) freezing 10min in 300ml liquid nitrogen solution; Then lyophilization in vacuum freeze drier, vacuum lyophilization condition is: condenser temperature-55 DEG C, vacuum 3 ~ 5Pa, time 18h, obtained fibroin/carboxyetbyl chitosan composite.
Test case:
(1) by observing dermal fibroblast and capillary endothelium sticking and breeding in embodiment 1 ~ 3 and comparative example 1 resulting materials, its biocompatibility and the performance as wound repair material thereof is investigated.The inoculum density of dermal fibroblast is 5 × 10
4/ cm
2, the inoculum density of endotheliocyte is 1 × 10
4/ cm
2, the adherence rate inoculating 1 day in the material with DNA content algoscopy quantitative assay two kinds of cells and the rate of increase cultivated after 3 days.Test result is shown in Table 1.
(2) by the mechanical property of GB/T1040.3-2006 testing example 1 ~ 3 and comparative example 2 resulting materials.Test result is shown in Table 1.
(3) carry out Degrading experiment by GB/T16886.13-2001 to embodiment 1 ~ 3 and comparative example 2 resulting materials, the temperature of selection is 37 DEG C, and in testing liquid, pancreas enzyme concentration is decided to be 0.2IU/ml, and degradation time is 7 days.Test result is shown in Table 1.
Table 1
As can be seen from institute's column data in table, compare with comparative example 2 with comparative example 1, the dermal substitute that embodiment obtains has good biocompatibility and cell compatibility, dermal tissue fibroblast, capillary endothelium sticking and breeding in the bracket can be promoted, its degradation speed lower than comparative example, and has the mechanical property more close with dermal tissue.These character of dermal substitute in embodiment are conducive to it and play promotion wound repair and suppress synulotic function, thus provide new material for solving pathologic scar.
Claims (4)
1. a dermal substitute, it is characterized in that described dermal substitute take fibroin as main matrix, carboxyetbyl chitosan is secondary substrate; Wherein, the mass fraction of fibroin is 60% ~ 90%, and the mass fraction of carboxyetbyl chitosan is 10% ~ 40%.
2. a preparation method for dermal substitute described in claim 1, is characterized in that comprising the steps:
(1) Bombyx bombycis is placed in Na
2cO
3boil in aqueous solution and boil 30min, then use deionized water rinse 3 times, obtained silkworm silk cellulose fiber; Then silkworm silk cellulose fiber is placed in lithium-bromide solution, dissolves under 37 DEG C of conditions, obtained fibroin lithium-bromide solution; Be finally, after dialysing 3 days in the dialysis cassette of 3500Da, obtain silk protein solution at molecular cut off by gained solution, regulate solution quality percentage concentration to be 5%;
(2) by silk protein solution obtained for step (1) and mass percentage concentration be 3% carboxyetbyl chitosan solution under 37 DEG C of conditions by volume 54:10 ~ 9:10 mix, in speed be 50rpm/min vortex oscillator on to vibrate 5min, to mix homogeneously, then centrifugal 3min under centrifugal force is 1500g, the bubble in removing solution;
(3) mixed liquor after centrifugal for step (2) is transferred in politef groove, the thickness of solution is 2mm, then this groove is positioned over 37 DEG C, air draught wind speed be 0.3 meter per second fume hood central authorities, standing time is 24h, formed fibroin/carboxyetbyl chitosan polymeric membrane;
(4) fibroin/carboxyetbyl chitosan polymeric membrane is immersed in 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide mixed aqueous solution, at 40 DEG C, places 8h; Gained polymeric membrane is soaked 30min in 300ml deionized water, and this step repeats 3 times, finally by gained polymeric membrane freezing 10min in 300ml liquid nitrogen solution;
(5) by the lyophilization in vacuum freeze drier of freezing polymeric membrane, vacuum lyophilization condition is: condenser temperature-55 DEG C, vacuum 3 ~ 5Pa, time 18h, obtained fibroin/carboxyetbyl chitosan composite, i.e. described dermal substitute.
3. preparation method according to claim 2, is characterized in that Na described in step (1)
2cO
3concentration of aqueous solution is 0.01 ~ 1M; The concentration of described lithium-bromide solution is 1 ~ 20M.
4. preparation method according to claim 2, the concentration ratio that it is characterized in that 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide in (3-the dimethylamino-propyl)-3-ethyl carbodiimide of 1-described in step (4) and N-hydroxy-succinamide mixed aqueous solution is 1:1, is 0.02M; The pH value of described mixed solution is 4.5.
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